ADVANCES IN SKELETAL MUSCLE

BIOLOGY IN HEALTH & DISEASE

PROGRAM AND ABSTRACT BOOK

WEDNESDAY MARCH 5 TO FRIDAY MARCH 7, 2014

EMERSON ALUMNI HALL & PUGH HALL UNIVERSITY OF FLORIDA, GAINESVILLE, FL USA

ConferenCe orGAnIZerS AnD SPeAKerS Scientific Organizers Leo Ferreira, Department of Applied Physiology & Kinesiology, University of Florida Andy Judge, Department of Physical Therapy, University of Florida Scott Powers, Department of Applied Physiology & Kinesiology, University of Florida Glenn Walter, Department of Physiology & Functional Genomics, University of Florida Sue Bodine, Department of Neurobiology, Physiology and Behavior, UC- Davis

Administrative Organizers Christa Stout, Department of Physical Therapy, University of Florida Ellen Esparolini, Department of Physical Therapy, University of Florida Melanie Blackburn, Department of Physical Therapy, University of Florida Laurie Bialosky, Department of Physical Therapy, University of Florida Speakers Christopher Adams, University of Iowa, USA Keith Baar, University of California, Davis, USA Marcas Bamman, University of Alabama, Birmingham, USA Peter Bialek, Pfizer, USA Sue Bodine, University of California, Davis, USA Tom Clanton, University of Florida, USA Ron Cohn, University of Toronto, Canada Bruce Damon, Vanderbilt University, USA Karyn Esser, University of Kentucky, USA Darin Falk, University of Florida, USA Bret Goodpaster, Sanford/Burnham Medical Research Institute, USA Paul Gregorevic, Baker IDI Heart and Diabetes Institute, Melbourne, Australia Denis Guttridge, The Ohio State University, USA Thomas Hawke, McMaster University, Canada Russ Hepple, McGill University, Canada David Hood, York University, Canada Troy Hornberger, University of Wisconsin, Madison, USA Andy Judge, University of Florida, USA Gordon Lynch, The University of Melbourne, Australia David Marcinek, University of Washington, USA Andrew Marks, Columbia University, USA Beth McNally, University of Chicago, USA Darrell Neufer, East Carolina University, USA Charlotte Peterson, University of Kentucky, USA Russ Price, Emory University, USA George Rodney, Baylor College of Medicine, USA Seward Rutkove, Beth Israel Deaconess Medical Center , USA Marco Sandri, Università di Padova, Italy Martin Schneider, University of Maryland, USA Melinda Sheffield-Moore, The University of Texas Medical Branch at Galveston, USA LaDora Thompson, University of Minnesota, USA Scott Trappe, Ball State University, USA Glenn Walter, University of Florida, USA Sam Wickline, Washington University, USA Zhen Yan, University of Virginia, USA Teresa Zimmers, Indiana University, USA

The Neuromuscular Plasticity Training Program NIH/NICHD T32 HD043730 Department of Physical Therapy UNIVERSITY of FLORIDA

The ConferenCe organizers graTefully aCknowledge The finanCial supporT of :

Office of Associate Dean of Research College of Health & Human Performance UNIVERSITY of FLORIDA

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WEDNESDAY, MARCH 5 Presidents Room, Emerson Alumni Hall, University of Florida Time 1:55pm Welcome and Opening Remarks Bernie Machen President, University of Florida

Michael G. Perri Dean, College of Public Health & Health Professions, University of Florida 2:10 Session Biomarkers of Muscle Disease Chair Krista Vandenborne 2:10-2:30 Beth McNally Genomic Biomarkers in Muscular Dystrophy

University of Chicago 2:30-2:50 Bruce Damon MRI Biomarkers of Inflammatory Myopathies

Vanderbilt University 2:50-3:10 Sam Wickline Nanomedicine Therapeutics for Muscular Dystrophy

Washington University 3:10-3:30 Glenn Walter Emerging Magnetic Resonance Biomarkers

University of Florida 3:30 -3:50 Peter Bialek Biomarker Discovery Related to Muscle Disease Pfizer Inc 3:50-4:10 Seward Rutkove Electrical Impedance Myography in Muscle Disease

Beth Israel Deaconess Medical Center

4:10 - 4:30 Coffee Break

Coffee, tea, and light snacks provided Session Cell Signaling Pathways Regulating Muscle

Atrophy Chair Sue Bodine

4:30-5:00 Marco Sandri Novel Signaling Pathways That Control Muscle Mass

Università di Padova 5:00-5:30 Ron Cohn Insights into the regulation of muscle homeostasis

University of Toronto gained from studying hibernation

5:30-6:00 Christopher Adams Using systems-based strategies to discover small University of Iowa molecule inhibitors of skeletal muscle atrophy

6:00-6:15 Martin Schneider Mathematical Modeling Separates Influx and Efflux

University of Maryland Contributions to Net Foxo1 Nuclear Movements Under Various Experimental Conditions in Skeletal Muscle Fibers

6:15-6:30 Juliane Campos Disrupted protein quality control in skeletal muscle

University of Sao Paulo atrophy: role of Beta2-adrenoceptor 6:30-6:45 Andy Judge Identification of novel FoxO target genes

University of Florida

7:00 – 10:00 Dinner - The speakers will be dining at the Swamp Restaurant and we

encourage all attendees to dine there also so that informal interactions can continue.

THURSDAY, MARCH 6 Presidents Room, Emerson Alumni Hall, University of Florida Time 7:40 am Coffee, Tea, and Pastries 8:00-9:00 Grant Workshop

Chair Tom Clanton 8:00-8:15 Bruce Damon Key Elements for a Successful R01 Application Vanderbilt University 8:15-8:30 Charlotte Peterson To Revise or Not Revise an Application and University of Kentucky Responding to Reviewers 8:30-8:45 Karyn Esser The Art of Flawless Packaging or How To Make Your

University of Kentucky Grant Application Bullet Proof 8:45-9:00 Open Panel Discussion 9:15 Session Sarcopenia and Cachexia: Muscle Loss and

Anabolic Resistance Chair Mike Reid

9:15-9:45 Denis Guttridge The role of the muscle microenvironment in cancer-

The Ohio State University induced muscle loss 9:45-10:15 Russ Price Mechanisms of Post-Transcriptional Control of Emory University Muscle Atrophy

10:15-10:45 Teresa Zimmers Stem cell pathways regulating muscle wasting in Indiana University cancer cachexia

10:45-11:15 Melinda Sheffield-Moore Skeletal muscle's mid-life crisis: metabolic mayhem or

The University of Texas controlled chaos in cancer and aging? Medical Branch at Galveston

11:15-11:40 Coffee Break Coffee, tea, and light snacks provided

Chair Andy Judge 11:40-12:10 pm Sue Bodine Skeletal Muscle Aging and the Development of

University of California, Anabolic Resistance Davis

12:10-12:40 Russ Hepple The Role of Denervation in Mitochondrial Functional

Mcgill University Alterations in Aging Human Muscle and its Implications for Therapeutic Intervention

12:40-12:55 Joseph McClung Muscle Paracrine Regulation of Endogenous

East Carolina University Progenitors for PAD Therapy - Roles for Tie2 and Angiopoietin-1.

12:55-1:40 Lunch Break Boxed lunches provided

Session Cell Signaling Pathways Regulating Muscle

Hypertrophy Chair David Criswell 1:40-2:10 Marcas Bamman Exercise is Regenerative Medicine: Muscle Regrowth

University of Alabama in Atrophied Humans Birmingham

2:10-2:40 Troy Hornberger The Role of Diacylglycerol Kinase Zeta in the

University of Wisconsin Mechanical Activation of mTOR Signaling and Madison Skeletal Muscle Hypertrophy

2:40-3:10 Keith Baar mTOR-independent control of protein synthesis after

University of California, resistance exercise Davis

3:10-3:40 Paul Gregorevic Activin-mediated signalling is a negative regulator of Baker IDI Heart and muscle mass

Diabetes Institute 3:40-4:10 Coffee Break

Coffee, tea, and light snacks provided

Session Muscle Diseases and Regeneration Chair Glenn Walter

4:10-4:40 Charlotte Peterson Evolving Role of Satellite Cells in Muscle Adaptation

University of Kentucky 4:40-5:10 Gordon Lynch Novel Treatment for Muscle Wasting Disorders

The University of Melbourne 5:10-5:25 Darin Falk Reassembly of the neuromuscular junction following

University of Florida gene therapy in Pompe disease

5:25-5:40 Thomas Hawke Diabetic Myopathy: A Therapeutic Role for Myostatin McMaster University

6:15 – 9:00 Poster Session and Reception

President A, Emerson Alumni Hall, University of Florida Participants with even numbered posters please stand with your poster between 7:00 - 8:00 Participants with odd numbered posters please stand with your poster between 8:00 - 9:00 FRIDAY, MARCH 7 Pugh Hall, University of Florida Time 7:45 am Coffee, Tea, and Pastries Session Control of Muscle Force Production

Chair Tom Clanton 8:00 - 8:30 Scott Trappe The Aging Athlete

Ball State University 8: 30 - 9:00 LaDora Thompson Age-related muscle contractility - Protein damage and

University of Minnesota expression 9:00-9:30 George Rodney NADPH oxidase as a regulator of skeletal muscle

Baylor College of Medicine function 9:30- 10:00 Andrew Marks The Role of Leaky Ryanodine Receptors In Disorders

Columbia University of Muscle 10:00-10:15 David Waning Cancer-induced bone destruction leads to skeletal

Indiana University muscle oxidative stress and weakness 10:15 - 10:45 Coffee Break

Coffee, tea, and light snacks provided

Session Muscle Functions Beyond Contraction Chair Leo Ferreira

10:45-11:15 Karyn Esser Understanding the muscle clock; what it is teaching us

University of Kentucky about muscle and systems homeostasis

11:15-11:45 Zhen Yan SNO-mediated protection in skeletal muscle University of Virginia

11:45-12:15pm Tom Clanton Acute regulation of skeletal muscle IL-6 in stressed

University of Florida muscle fibers 12:15-12:30 Katsuhiko Funai Obesity modulates insulin signaling and lipidome in

East Carolina University human primary myotubes 12:30-1:30 Lunch Break

Boxed lunches provided Session Mitochondria in Muscle Health and Disease Chair Scott Powers

1:30-2:00 David Hood Muscle mitochondrial biogenesis, beyond PGC-1α

York University 2:00-2:30 Darrell Neufer PDC and NNT Comprise an Energy Consuming Redox

East Carolina University Circuit: Implications for Cellular Redox Control 2:30-3:00 Bret Goodpaster The Role of Mitochondrial Energetics in the Loss of

Sanford/Burnham Medical Function With Aging Research Institute

3:00-3:30 David Marcinek Targeting Mitochondria to Reverse Skeletal Muscle University of Washington Dysfunction with Age 3:30-3:45 Espen Spangenburg Breast Cancer 1, early onset (BRCA1) gene expression

University of Maryland is critical for physiological and mitochondrial function of skeletal muscle

4:00 Farewell

ABSTRACTS/POSTERS Please refer to the number of your poster to locate the board where your poster can be displayed

Biomarkers of Muscle Disease

1. Lower Extremity Muscle Involvement in Duchenne muscular dystrophy and Collagen VI myopathy. Abhinandan Batra, Lott DJ, Willcocks RJ, Forbes SC, Triplett WT, Dastgir JS, Sweeney HL, Bonnemann CG, Byrne B, Rooney W, Senesac CR, Wang DJ, Vandenborne K, Walter GA

2. Metabolomic Analysis for Duchenne Muscular Dystrophy in the Mdx Mouse Model. Brittany Lee-McMullen, Chaevien Clendinen, Stephen Chrzanowski1, Ravneet Vohra, William Triplett, Sean Forbes, Arthur S. Edison, Krista Vandenborne, Glenn Walter

3. Examination of the Effects of Corticosteroid Treatment on Skeletal Muscles of Boys with DMD using Magnetic Resonance Imaging and Spectroscopy. Arpan I, Willcocks RJ, Forbes SC, Lott DJ, Senesac C, Triplett WT, Daniels MJ, Byrne BJ, Finanger EL, Finkel, RS Russman BS, Rooney WD, Wang DJ, Tennekoon GI, Walter GA, Sweeney HL, Vandenborne K.

4. Profiling of skeletal muscle using high-definition mass spectrometryJatin G. Burniston, Joanne B. Connolly, Heikki Kainulainen, Steven L. Britton and Lauren G. Koch

5. A 3’ UTR mutation in PLIN2 in a family with a novel distal myopathy causes toxic disruption of miR-590-3p binding. Kyung-Ah Cho, Steven E. Boyden, Genri Kawahara, Elicia Estrella, Matthew Alexander, Satomi Mitsuhashi, Hart G. W. Lidov, Randall D. Craver, Amparo Gutierrez, John D. England, Louis M. Kunkel, Peter B. Kang

6. Plethysmography as a noninvasive biomarker for the diagnosis and prognosis of MDC1A. Ajay Kumar, Thomas Mehuron and Mahasweta Girgenrath.

7. Magnetic Resonance Imaging Differences in Animal Models of Duchenne and Congenital Muscular Dystrophy. Ravneet Vohra, A Kumar, SC Forbes, A Batra, SM Chrzanowski, BA Lee, K Vandenborne, M Girgenrath, GA Walter

8. Arm muscle involvement in Duchenne muscular dystrophy: A quantitative MRI and MRS study. Willcocks, RJ, Forbes, SC, Lott, DJ, Senesac, CR, Nicholson, TR, Arora, H, Walter, GA, Vandenborne, K

9. Magnetic Resonance Imaging and Spectroscopy as a Biomarker for Muscle Injury. Stephen Chrzanowski, Brittany Lee-McMullen, Abhinandan Batra, Ravneet Vohra, Yanhua Deng, Huabei Jiang, Krista Vandenborne, Glenn Walter

10. In vivo measurements of mitochondrial respiratory capacity in skeletal muscle Terence E. Ryan, Chien-Te Lin, Patricia Brophy, Robert C. Hickner, Kevin K. McCully, P. Darrell Neufer

11. FNDC5 Expression in Skeletal Muscle in Chronic Heart Failure – Relevance of Inflammatory Cytokines. Volker Adams, Konstanze Gleitsmann, Norman Mangner, T Scott Bowen, Axel Linke

12. Effects of voluntary running during growing period on bone tissue in type 2 diabetic rats. Yuri Takamine, Takamasa Tsuzuki, Noriko Ichinoseki-Sekine, Toshinori Yoshihara, Hisashi Naito

Cell Signaling Pathways Regulating Muscle Atrophy

13. KLF-4: a novel target to prevent tumor-induced muscle wasting? Andrea Bonetto, Teresa A. Zimmers

14. Knockdown of ATG5 in the diaphragm reduces oxidative stress and protects against ventilator-induced diaphragm dysfunction. Ashley J. Smuder, Kurt J. Sollanek, W. Bradley Nelson, Kisuk Min, Erin E. Talbert and Scott K. Powers

15. Contractile Proteins are Key Targets for Lysine Specific Hypoacetylation and Hyperubiquitination during Muscle Atrophy. Daniel J Ryder, Beharry AW, Farnsworth CL, Silva JC, Judge AR

16. p53 and ATF4 Mediate Distinct and Additive Pathways to Skeletal Muscle Atrophy During Limb Immobilization. Daniel K. Fox, Kale S. Bongers, Scott M. Ebert, Michael C. Dyle, Steven A. Bullard, Jason M. Dierdorff, Steven D. Kunkel, and Christopher M. Adams

17. microRNA 208b is decreased in quadriceps muscles of COPD patients. Sharon Rosenberg, Danielle Barkema, Robert Sufit, Ravi Kalhan, Jacob I. Sznajder and Emilia Lecuona.

18. High CO2 levels lead to rodent skeletal muscle atrophy through the activation of AMP-activated protein kinase and the ubiquitin-proteasome pathway. Ariel Jaitovich, Ermelinda Ceco, Martin Angulo, Laura A Dada, Lynn Welch, Emilia Lecuona, Yuan Cheng, Galina Gusarova, Cam Patterson, Gustavo A. Nader and Jacob I Sznajder.

19. Recovery of diaphragm contractile function with JAK inhibition during mechanical ventilation is accompanied by reduced accumulation of mitochondrial STAT3. Ira J. Smith, Tarikere Gururaja, Guillermo L. Godinez, Baljit K. Singh, Kelly M. McCaughey, Raniel R. Alcantara, Melissa S. Ho, Yan Chen, Rajinder Singh, Esteban S. Masuda, Vanessa C. Taylor, Donald G. Payan, Taisei Kinoshita, and Todd M. Kinsella

20. Muscle glucocorticoid receptors and long-term alcohol abuse: Preliminary findings. Jakob L. Vingren, Bryon Adinoff, Anthony, A. Duplanty, Ronald, Budnar, JR., Hui Ying Luk, Hong Xiao & David W. Hill

21. Decreased nuclear CRTC contributes to the dexamethasone-induced reduction in PGC-1α expression in muscle cells. Rahnert JA, Zheng B, Woodworth-Hobbs ME, Hudson MB, Price SR

22. Disrupted protein quality control in skeletal muscle atrophy: role of 2-adrenoceptor. Campos, JC; Voltarelli VA; Bechara, LRG; Moreira, JBN; Brum, PC; Ferreira JCB

23. Skeletal Muscle Denervation Causes Skeletal Muscle Atrophy through a Pathway that Involves Both Gadd45a and HDAC4. Kale S. Bongers, Daniel K. Fox, Scott M. Ebert, Steven D. Kunkel, Michael C. Dyle, Steven A. Bullard, Jason M. Dierdorff, Christopher M. Adams

24. BDH1 overexpression in skeletal muscle enhances exercise capacity and protects against muscle wasting. Kristopher Chain, Vitor Lira, Rhianna Laker, Jarrod Call, Nic Greene, Mei Zhang, Zhen Yan

25. SMAD3 augments, and is essential for, FoxO3-induced MuRF-1 promoter activity. Lance M. Bollinger and Jeffrey J. Brault

26. Mathematical Modeling Separates Influx and Efflux Contributions to Net Foxo1 Nuclear Movements Under Various Experimental Conditions in Skeletal Muscle Fibers. Martin Schneider, Robert Wimmer, Yewei Liu and Bradford Peercy

27. MicroRNA-182 targets FoxO3 and attenuates glucocorticoid-induced atrophic signaling in muscle. Matthew B. Hudson, Myra E. Woodworth-Hobbs, Bin Zheng, Jill A. Freret, Harold A. Franch, and S. Russ Price

28. Inhibition of FoxO dependent translation prevents mechanical ventilation-induced reduction in protein synthesis. Michael P. Wiggs, Ashley J. Smuder, Kurt J. Sollanek, Kevin L. Shimkus, James D. Fluckey, Scott K. Powers

29. Docosahexaenoic acid attenuates palmitate-induced unfolded protein response in myotubes. Myra E. Woodworth-Hobbs, Matthew B. Hudson, Jill A. Rahnert, Bin Zheng, S. Russ Price

30. Antagonism of Myostatin/Activin Type IIB Receptor Signaling via a Novel Small Molecule. Robert D. Hyldahl, Ryan Matekel, Allen C. Parcell, David Bearss

31. The Changes of Muscle Morphology after Locomotor Training in a New Model of Spinal Cord Injury. Lim W, Baligand C, Ye F, Vohra RS, Ruhella A, Keener J

, Bose P, Thompson F, Walter G, Vandenborne K

Sarcopenia and Cachexia: Muscle Loss and Anabolic Resistance

32. Role of FoxO in diaphragm fiber atrophy during cancer cachexia. Adam W. Beharry, Roberts BM, Senf SM, Judge AR

33. Cancer-induced bone destruction leads to skeletal muscle oxidative stress and weakness. David L. Waning, Khalid S. Mohammad, Steven Reiken, Wenjun Xie, Daniel C. Andersson, Andrew R. Marks and Theresa A. Guise.

34. Aerobic Exercise as a Treatment for Frailty. Haiming Liu, Ted G. Graber, Lisa Ferguson-Stegall, LaDora V. Thompson

35. Gene polymorphisms Influence on exercise-induced changes of bone density and skeletal muscle mass In Japanese young women. Hiroyo Kondo, Hidemi Fujino, Fumiko Nagatomo, Akihiko Ishihara.

36. Relationships between Glycogen Content, Translational Signaling, Protein Synthesis, and Hypertrophy in Overloaded Fast-Twitch Skeletal Muscles of Young Adult and Aged Rats. Marcus M. Lawrence, Rengfei Shi, B. Clay Myers, Hoke B. Whitworth, William T. Mixon, and Scott E. Gordon.

37. Novel Model of Voluntary Resistance Training for Mice. Ted G. Graber, Katie Fandrey, LaDora V. Thompson

Cell Signaling Pathways Regulating Muscle Hypertrophy

38. Muscle growth effect of RNA-binding motif protein 3 (RBM3) is associated with binding to muscle-specific mRNAs and miRNAs. Amy Confides, Andrew Judge, and Esther Dupont-Versteegden

39. Lack of alpha-actinin-3 attenuates m-TOR signaling in human skeletal muscle after sprint exercise. Barbara Norman, Mona Esbjörnsson, Håkan Rundqvist, Ted Österlund, Eva Jansson

40. Putative role of REDD1 in the Activation of mTORC1 Following Resistance Exercise. Gordon BS, Steiner JL, Lang CH, Jefferson LS, Kimball SR

41. Ca2+/Calmodulin-Dependent Protein Kinase Kinase (CaMKK) is Sufficient, But Not Necessary, for Growth in Mouse Skeletal Muscle. Jeremie L.A. Ferey, J. Matthew Hinkley, Cheryl A.S. Smith, Jeffrey J. Brault and Carol A. Witczak

42. Mechanosensitivity may be enhanced in skeletal muscles of spinal cord injured (SCI) vs. able-bodied men (AB). Ceren Yarar-Fisher, C Scott Bickel, Samuel T Windham, Neil A Kelly, and Marcas M Bamman

43. Fiber type-specific satellite cell response to aerobic training in sedentary adults. Christopher S. Fry, Brian Noehren, Jyothi Mula, Margo F. Ubele, Philip A. Kern, Charlotte A. Peterson

44. Sprint exercise and myoblast proliferation. Heléne Fischer, Seher Alam, Barbara Norman, Håkan Rundqvist, Andreas Montelius, Mona Esbjörnsson, Eva Jansson

45. G protein coupled receptor 56 is a target gene of the PGC-1α4 isoform and regulates overload-induced muscle hypertrophy. J.P. White, C.D. Wrann, R.R. Rao, Z. Wu, D.J. Glass, and B.M. Spiegelman

46. Sepsis antagonizes muscle contraction-induced increases in mTOR signaling. Jennifer L. Steiner, G. Deiter, M Navaratnarajah, CH Lang

47. Resistance exercise training (RET) in ~70 y men causes adaptations in bulk and microvascular blood flow promoting maintenance of a bigger muscle mass - a paradigm for training effects on myofibrillar protein synthesis (MPS). Michael J Rennie

48. Examining the interindividual early hypertrophic response to resistance training. Michael Stec and Marcas M Bamman

49. The Acute Effect of Two Resistance Exercise Intensities with Equal Volume Load on Skeletal Muscle mRNA Expression of Insulin-like Growth Factor-1Ea (IGF-1Ea) and Mechano Growth Factor (MGF). Neil A. Schwarz, Mike Spillane, Sarah K. McKinley, Thomas L. Andre, Joshua J. Gann, & Darryn S. Willoughby

50. ICAM-1: A Novel Mechanism by which the Inflammatory Response Augments Myogenesis. Goh Q, Dearth CL, Awadia S, Garcia-Mata R, Corbett JT and FX Pizza

51. Lean Gain Is Enhanced by Administration of Leucine Pulses during Long-term Continuous Feeding. Teresa A. Davis, Claire Boutry, Samer W. El-Kadi, Agus Suryawan, Julia Steinhoff-Wagner, Barbara Stoll, Renan A Orellana, Hanh V. Nguyen, and Marta L. Fiorotto

52. Changes in HDAC expressions in response to acute heat stress in rat skeletal muscle. Toshinori Yoshihara, Ryo Kakigi, Takamasa Tsuzuki, YuriTakamine, Noriko Ichinoseki-Sekine, Hisashi Naito.

53. Myonuclear transcriptional output during muscle hypertrophy in the absence of satellite cell fusion. Tyler J. Kirby, Christopher S. Fry, Janna R. Jackson, Thomas Chaillou, Charlotte A. Peterson, and John J. McCarthy

Muscle Diseases and Regeneration

54. Generating a Gene Therapy Vector for Myotubular Myopathy. Angela L. McCall, Denise Cloutier, Jeffry Kelley, Meghan Soustek, Darin Falk, Nathalie Clément and Barry Byrne.

55. Estradiol Alters the Expression of Key Transcription Factors that Regulate Skeletal Muscle Regeneration. Brittany C. Collins, Tara L. Mader, Coco Le, Gordon L. Warren, and Dawn A. Lowe.

56. Changes in skeletal muscle structure and function following genetic inactivation of myostatin in rats. Christopher L Mendias, Evan B Lynch, Jonathan P Gumucio.

57. Neuronal and neuromuscular junction pathology in Pompe disease. Darin J. Falk, A. Gary Todd, Sooyeon Lee, Meghan S. Soustek, Robin Yoon, David D. Fuller, Lucia Notterpek, Barry J. Byrne.

58. Cellular and Extra-Cellular Responses to Mechanical Overload in Tissue Engineered Skeletal Muscle. DJ Player, HC Stobbs, NRW Martin, MP Lewis

59. Forelimb neuromuscular plasticity after cervical spinal cord injury in the rat. Gonzalez-Rothi EJ, Fitzpatrick G, Armstrong GT, Reier PJ, Lane MA, Fuller DD.

60. Satellite Cells Expand in Muscle of Pancreatic Cancer Patients. Erin E. Talbert, Mark Bloomston, Ericka Haverick, and Denis C. Guttridge

61. AAV9 improves hallmarks of neuromuscular junction deterioration and physiological function in Pompe disease. Todd A.G., McElroy, J.A., Grange R.W., Fuller D.D., Walter G.A., Byrne B.J. and Falk D.J.

62. Deficiency in Lipin 1-mediated Phosphatidic Acid Phosphohydrolase Activity is Associated with Rhabdomyolysis in Humans and Skeletal Myopathy in Mice. George G Schweitzer, Sara L Collier, Kyle S McCommis, Zhouji Chen, Kari T Chambers, Thurl E Harris, Alan Pestronk, Brian N Finck

63. Developing a New Resistance Running Wheel System for Mice. Rodden GR, Stylianos K, Doering JA, McMillan RP, Frisard MI, and Grange RW.

64. Performance on upper limb functional tests in control and DMD subjects. Arora, H, Willcocks, RJ, Lott, DJ, Senesac, CR, Forbes, SC, Bendixen, R.M., Harrington, A.T., Walter, GA & Vandenborne, K.

65. Myoplasticity-related gene expression in the diaphragm following cervical spinal cord injury. HH Ross, LC Gill, EJ Gonzalez-Rothi, AR Judge, DD Fuller.

66. Exercise training improves capillary architecture via enhancing VEGF/VEGFR and angiopoietins/Tie2 signaling pathways in type 2 diabetic muscle. Hidemi Fujino, Hiroyo Kondo, Shinichiro Murakami, Masayuki Tanaka, Miho Kanazashi, Fumiko Nagatomo, Akihiko Ishihara, and Roland R. Roy.

67. AAV9 improves lysosomal organization and diaphragmatic contractile function in Pompe disease. Jessica A. McElroy, A. Gary Todd, Bumsoo Ahn, David D Fuller, Barry J Byrne, Leonardo F Ferreira and Darin J Falk.

68. Antagonizing PPARγ Improves Muscle Fiber Force Production and Reduces Myosteatosis Following Chronic Rotator Cuff Tear. Gumucio, Jonathan P; Flood, Michael D; Roche, Stuart M; Mendias, Christopher L.

69. Muscle biopsy and HSk cell analysis for Pax7 and fusion protein, Kirrel. KH Myburgh, P Durcan, M van de Vyver, K Goetsch, CU Niesler.

70. Changes in Macrophage Phenotype and Induction of Epithelial-to-Mesenchymal Transition Genes Following Acute Achilles Tenotomy and Repair. Kristoffer B Sugg, Jonathan P Gumucio, Christopher L Mendias.

71. Glycogen Accumulation Varies By Fiber Type in the Pompe Diaphragm. Pascual LM, Elmallah MK, Falk DJ, Todd AG, Byrne BJ, Fuller DD.

72. Chronic insulin exposure results in transcriptional alternations in metabolic and myogenic genes in in vitro skeletal muscle. Mark C Turner, Darren J Player, Neil RW Martin, Mark P Lewis

73. Ankrd2 is a modulator of NF-kB mediated inflammatory responses in muscle. Verma N.K., Yamamoto D., Chemello F., Bang M.L., Lanfranchi G. and Bean C.

74. High intensity exercise improves skeletal muscle mitochondrial function corresponding with reduced fatigability in Parkinson's disease. NA Kelly, DR Moellering, CS Bickel, MP Ford, DG Standaert, and MM Bamman

75. Generating 3D neuromuscular junctions in vitro. Martin, N.R.W., Player, D.J., Lewis, M.P.

76. Recovery of altered neuromuscular junction morphology and muscle function in mdx mice after injury. Stephen J.P. Pratt, Sameer B. Shah, Christopher W. Ward, Joseph P. Stains and Richard M. Lovering

77. The expression and role of Notch in exercise-induced regenerating aged skeletal muscle. J Demick, M Lord, A Lyle, K Brown, M Keith, J Tkach, S Blanton, I Cooley, J Marino, R Howden and ST Arthur.

77b. Estrogen Alters Macrophage and Neutrophil Recruitment and Response Following Skeletal Muscle Injury. Tara L Mader, Coco Le, Dawn A Lowe, Gordon L Warren

78. Adoptive transfer of ischemia/reperfusion-conditioned macrophages enhances functional recovery of skeletal muscle after tourniquet-induced ischemia/reperfusion injury. Viktoriya Rybalko, David Hammers, Melissa Merscham-Banda, Roger P. Farrar

Control of Muscle Force Production

79. Adaptations in muscle fiber length, pennation angle, and curvature following an anterior cruciate ligament tear. Brian Noehren, Anders Andersen, Peter Hardy, and Bruce Damon

80. Mice lacking p47phox subunit of NADPH oxidase are protected from diaphragm dysfunction elicited by heart failure. Bumsoo Ahn, Gregory S. Frye, Adam W. Beharry, Jennifer. S. Moylan, Andrew R. Judge, Leonardo F. Ferreira.

81. Inactivity-Induced Decline in Single Muscle Fiber PowerOutput: The Role of Myosin Light Chain 3f.

Jong-Hee Kim and LaDora V. Thompson.

82. Inactivity-Induced Muscle Weakness: The Role of Myosin. Jong-Hee Kim and LaDora V. Thompson

83. Isolated human intercostal muscle fibers an in vitro skeletal muscle model. Karl Olsson, Joseph Bruton, Seher Alam, Håkan Westerblad, Thomas Gustafsson

84. AMP Deaminase overexpression improves skeletal muscle relaxation kinetics during high energy demands. PR Davis, CA Witczak, JJ Brault

85. A myocardial infarction rapidly induces diaphragm muscle weakness in mice. T Scott Bowen, Norman Mangner, Sarah Werner, Axel Linke, Volker Adams,

Muscle Functions Beyond Contraction

86. Levels of leukocyte mobilizing factors in skeletal muscle and circulating monocytes after an acute

bout of exercise in humans. Anna Strömberg, Karl Olsson, Jacomijn Dijksterhuis, Gunnar Schulte, Thomas

Gustafsson.

87. Early postnatal undernutrition programs the contractile and metabolic phenotype of the mouse plantaris with unanticipated consequences for physical activity. David P. Ferguson, C.J. Martinez, Brooks P. Scull, Ryan Fleischmann and Marta L. Fiorotto,

88. Aerobic Exercise Induces RAGE Shedding via ADAM10 in Human Skeletal Muscle. Abeer M. Mahmoud, Brian K. Blackburn, Karia Coleman, Jacob T. Mey, Vikram S. Somal, Thomas P. J. Solomon, Ciaran E. Fealy, Steven K. Malin, John P. Kirwan, Jacob M. Haus.

89. Skeletal muscle-derived EcSOD mitigates diabetic cardiomyopathy in streptozotocin-diabetic mice by reducing oxidative stress. Jarrod A. Call, Kristopher H. Chain, Kyle S. Martin, Vitor A. Lira, Mitsuharu Okutsu, Mei Zhang, Shayn M. Peirce-Cottler, Zhen Yan.

90. Activation of CaMKKα Signaling Stimulates the Pentose Phosphate Pathway in Mouse Skeletal Muscle. Jeremie L.A. Ferey, J. Matthew Hinkley, and Carol A. Witczak.

91. Intermittent hypoxia-induced disruption of skeletal muscle insulin signaling: A pilot study. Joseph S Marino, Paige Driver, Auburne Overton, Susan Arthur and Reuben Howden

92. Muscle Paracrine Regulation of Endogenous Progenitors for PAD Therapy-­‐Roles for Tie2 and Angiopoietin‐1. Joseph M. McClung, Mahroo Mofarrahi, Jessica Reinardy, Sarah Frazier, Sabah N.A. Hussain, Christopher D. Kontos

93. SLN-null mice cannot sustain muscle-based thermogenesis during cold exposure. Leslie A. Rowland, Naresh C Bal and Muthu Periasamy.

94. Kinetics of skeletal muscle cytokine mRNA responses in exertional vs. passive heat stroke. Michelle A. King, Deb A. Morse and Thomas L. Clanton

95. Sarcolipin mediates skeletal-muscle based nonshivering thermogenesis in mammals. Bal NC, Maurya SK, Saporiwala DH, Sahoo SK, Gupta SK, Shaikh SA, Pant M, Rowland LA, and Periasamy M.

96. Sarcolipin is the key regulator of diet-induced thermogenesis in the skeletal muscle. Santosh K Maurya, Naresh C Bal, Danesh H Sopariwala, Sana A Shaikh, and Muthu Periasamy.

97. Reduced Atg7 expression in muscle potentiates the metabolic effects of exercise against diet-induced obesity and insulin resistance. Vitor A. Lira, Jarrod A. Call, Rhianna C. Laker, Mei Zhang, Zhen Yan.

Mitochondria in Muscle Health and Disease

98. Effect of rapamycin on the mitochondrial profile in skeletal muscle of aged rats. Alexandra K. Gentilella, Linda M.-D. Nguyen, Kurtis A. Dickson, Anna-Maria Joseph, Dallas Khamiss, Drake Morgan, Christy Carter, and Peter J. Adhihetty

99. Changes in energy state acutely alter insulin sensitivity in healthy humans. Chien-Te Lin, Laura A. Gilliam, Patty M. Brophy, Angela H. Clark, Terence E. Ryan, Robert C. Hickner, and P. Darrell Neufer.

100. Accelerated lipid oxidation increases the rate of mitochondrial H2O2 production in skeletal muscle. Cody D. Smith, Chien-Te Lin, Kelsey H. Fisher-Wellman, Laura A. A. Gilliam, Lauren R. Reese, Cheryl A. Smith, Hyo Bum Kwak and Darrell Neufer

101. Protein Kinase A activity augments skeletal muscle mitochondrial bioenergetics. Daniel Stephen Lark, Lauren Rose Reese and P. Darrell Neufer

102. A Genome-wide Approach to Delineate Novel PPARδ Targets Involved in Muscle Fitness. Emily Y. Smith, Zhenji Gan, Rick Vega, Daniel P. Kelly

103. Effects of voluntary running and milk-protein supplements on skeletal muscle sirtuins in rats with elevated risk factors for metabolic disorders. Sanna Lensu1, Satu Pekkala2, Anne Mäkinen1, Juha J. Hulmi1, Anu Turpeinen3,Urho M. Kujala2, Lauren G. Koch4, Steven L. Britton4 and Heikki Kainulainen

104. Alternative NF-B and MyoD cooperatively regulate PGC-1 during myogenesis. Jonathan Shintaku and Denis C. Guttridge

105. Obesity modulates insulin signaling and lipidome in human primary myotubes. Christopher W Paran, Sanghee Park, Haowei Song, Joseph A Houmard, G Lynis Dohm, Heather A Lawson, John Turk, Katsuhiko Funai

106. Effects of exercise training and high fat diet on glycerol kinase protein content in skeletal muscle. Kazuhiko Higashida and Mitsuru Higuchi

107. Exercise training induces a diaphragmatic phenotype the resists apoptotic stimuli. Kurt J. Sollanek, Andreas N. Kavazis, Ashley J. Smuder, Michael P. Wiggs, Aaron B. Morton and Scott K. Powers.

108. Cancer chemotherapy impairs skeletal muscle mitochondrial function in non-tumor bearing tissue. Laura A. A. Gilliam, Daniel S. Lark, Kelsey H. Fisher-Wellman, Maria J. Torres,Lauren R. Reese, Brook L. Cathey, and P. Darrell Neufer

109. Rat model systems to explore the link between exercise capacity, complex disease, aging, and longevity. Lauren Gerard Koch and Steven L. Britton

110. 2-Hydroxyestradiol as a pro-oxidant regulator of muscle mitochondrial function. Lauren R. Reese, Daniel S. Lark, and P. Darrell Neufer

111. Effect of exercise on prostate cancer-induced changes in the mitochondrial profile of rodent skeletal muscle. Linda M.-D. Nguyen, Danielle J. McCullough, Lucas R. Goss, Michael A. Olson, Anna-Maria Joseph, Bradley J. Behnke, Peter J. Adhihetty

112. Mitochondrial capacity is decreased in skeletal muscle with estrogen depletion. Torres, MJ; Gilliam LAA; Neufer, PD

113. Is Mitochondrial COX Deficiency a Cause of Myofiber Atrophy in Humans? Martin Picard, D.Hom, Julie Murphy, Sally Spendiff, Russell T Hepple, Basil J Petrof, Douglas C Wallace, Douglass M Turnbull, Tanja Taivassalo

114. Curcumin evokes mitochondrial alterations and suppresses apoptosis in muscle and BAT of aged mice. Nicholas R. Wawrzyniak, Andrew Duarte, Linda M.-D. Nguyen, Anna-Maria Joseph, Andrew S. Layne, David S. Criswell, and Peter J. Adhihetty

115. Salt inducible kinase 1 is required for exercise-stimulated MEF2 activity in skeletal muscle. Randi Stewart and Rebecca Berdeaux

116. Protein S-nitrosylation protects against mitochondrial oxidative stress. Rebecca J. Wilson, Rhianna C. Laker and Zhen Yan

117. Detection of transient mitochondrial stress and damage in skeletal muscle following a single bout of exercise by a novel MitoTimer reporter gene. Rhianna C. Laker, Vitor A. Lira, Rebecca J. Wilson and Zhen Yan.

118. Elevated skeletal muscle mitochondrial quality control proteins are associated with higher mitochondrial respiration in young adults. Robert A. Standley, Giovanna Distefano, Frederico G.S. Toledo, Bret H. Goodpaster, John J. Dubé, Paul M. Coen

119. Loss of Adenine Nucleotide Translocase Alters Muscle Mitochondrial Function and Enhances Insulin Sensitivity. Ryan Morrow, Martin Picard, Meagan McManus, Gilles Gouspillou, Russell T. Hepple, Douglas C. Wallace

120. Mitochondria-targeted ROS scavenger improves post-ischemic recovery of cardiac function and attenuates mitochondrial abnormalities in aged rats. Nelson Escobales, Rebeca E. Nuñez, Rebecca Parodi-Rullan, Joshua R. Sacher, Erin M. Skoda, Sylvette Ayala-Peña, Peter Wipf, Walter Frontera, Sabzali Javadov

Biomarkers of Muscle Disease

1. Lower Extremity Muscle Involvement in Duchenne muscular dystrophy and

Collagen VI myopathy. Abhinandan Batra, Lott DJ, Willcocks RJ,

Forbes SC, Triplett WT, Dastgir JS, Sweeney HL, Bonnemann CG, Byrne B,

Rooney W, Senesac CR, Wang DJ, Vandenborne K, Walter GA

Department of Physiology & Functional Genomics, University of Florida

Muscular dystrophies are a group of genetic neuromuscular diseases characterized by progressive muscle weakness and degeneration. Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder caused by the absence of dystrophin in the cytoplasmic membrane of muscle fibers. Collagen VI myopathy (COL6) is a congenital muscular dystrophy caused by mutation of genes encoding for a structural protein on in the extracellular muscle matrix. Both DMD and COL6 are characterized by replacement of muscle by fatty and fibrotic tissue. In this study, we used MRI and strength tests to examine disease progression in these two patient populations. Method: MRIs of age matched Controls (n=7); COL6 (n=8) and DMD (n=8) subjects were acquired from the leg muscles at 3T. Muscles were analyzed for maximal cross sectional area (CSA) and contractile area. Strength was measured for the Knee Extensors (KE) and Plantar flexors (PF) using computerized isokinetic dynamometer and was normalized to contractile area (specific torque). Results: In the thigh, CSA and contractile area were lowest in COL6 and highest in controls. In the lower leg, children with DMD had highest CSA and contractile area for all muscles. Specific torque for both PF and KE was significantly lower for DMD and COL6 compared to controls (p<0.05), but was higher in COL6 than DMD (p<0.05). Conclusion: Col6 subjects have extensive leg muscle atrophy, while DMD subjects demonstrate hypertrophy of the calf muscles but atrophy of the thigh muscles.

Specific torque was impaired in both DMD and COL6, but the deficit was greater in DMD. These data suggest that factors beyond atrophy and muscle replacement with non-contractile tissue may contribute to weakness in these muscular dystrophies.

2. Metabolomic Analysis for Duchenne Muscular Dystrophy in the Mdx Mouse

Model Brittany Lee-McMullen1, Chaevien

Clendinen3, Stephen Chrzanowski1, Ravneet Vohra1, William Triplett1, Sean

Forbes2, Arthur S. Edison3, Krista Vandenborne2, Glenn Walter1

1Department of Physiology and Functional Genomics, 2Department of Physical

Therapy, 3Department of Biochemistry and Molecular Biology, University of Florida

Duchenne Muscular Dystrophy (DMD) is an X-linked genetic disorder in which the absence of the dystrophin protein causes sarcolemma fragility resulting in progressive pathophysiology. Repeated bouts of muscle damage, degeneration, and regeneration result in the replacement of healthy muscle with non-uniform regions of fat and fibrosis until boys succumb to the disease in third decade of their life from cardiac and respiratory failure. The current FDA clinical outcome measure lacks sensitivity to detect cellular changes in such a heterogeneous disease. As a result, there is a dire need for biomarkers to track disease progression in DMD. We have found that changes in the proton transverse relaxation times (T2) are able to detect muscle damage and disease both in dystrophic mice (mdx) and humans. The relationship between MRI based measures of disease progression and metabolomic changes in the tissue and blood is currently unknown. Differences in the muscle and serum metabolome of mdx at two ages were determined using in vivo and ex vivo techniques. MRIs were used to calculate the T2 and score the heterogeneity seen within muscle groups using an in-house Matlab script. NMR was used to analyze the metabolite profiles in

vivo and ex vivo in intact muscles with High-Resolution Magic Angle Spinning (HR-MAS) NMR and on serum using solution 1H/13C NMR. Preliminary analysis has revealed metabolic differences between the two ages at different stages of the disease and tissues in mdx mice. Texture analysis showed differences in tissue heterogeneity between the two stages of disease. NMR results show that serum amino acid profiles differ between the two states of disease and were correlated with a determined phenotype from T2 and tissue heterogeneity. Metabolic profiles and MR properties of dystrophy can be used to characterize different stages in mdx mice with the potential of becoming a biomarker for DMD.

3. Examination of the Effects of

Corticosteroid Treatment on Skeletal Muscles of Boys with DMD using Magnetic Resonance Imaging and

Spectroscopy Arpan I1, Willcocks RJ1, Forbes SC1, Lott

DJ1, Senesac C1, Triplett WT1, Daniels MJ2, Byrne BJ3, Finanger EL4, Finkel, RS5

Russman BS4,6, Rooney WD4, Wang DJ5, Tennekoon GI5, Walter GA7, Sweeney HL8,

Vandenborne K1. 1Department of Physical Therapy, University

of Florida; 2Divison of Statistics and Scientific Computation, The University of

Texas at Austin; 3Department of Pediatrics and Molecular Genetics & Microbiology,

Powell Gene Therapy Center, University of Florida; 4Oregon Health & Science

University; 5The Children’s Hospital of Philadelphia; 6Shriners Hospital for

Children; 7Department of Physiology and Functional Genomics, University of Florida;

8Department of Physiology, University of Pennsylvania.

Examination of the Effects of Corticosteroid Treatment on Skeletal Muscles of Boys with DMD using Magnetic Resonance Imaging and Spectroscopy Objectives: This study utilized magnetic resonance imaging (MRI) and spectroscopy (MRS) to evaluate the

effects of corticosteroids on lower extremity muscles of boys with Duchenne muscular dystrophy (DMD) using three experimental designs: 1) a cross-sectional comparison of corticosteroid-treated and corticosteroid-naïve boys; 2) 1-year longitudinal progression of intramuscular fat fraction in corticosteroid-treated and corticosteroidnaive boys; and 3) initiation of corticosteroids on muscle T2 and intramuscular fat fraction in boys with DMD. Methods: T2 by MRI/MRS and fat fraction were measured in lower extremity muscles of fifteen boys with DMD (age: 5.0-6.9 years) on corticosteroids and fifteen corticosteroid naïve boys. The change in intramuscular fat fraction in 1 year with respect to baseline was measured in subset of boys from aim 1. Finally, MR data were collected from sixteen corticosteroid naïve boys with DMD (age: 5-8.9 years) at baseline, 3-months and 6-months. Five boys went on corticosteroids after initial assessment and the others served as natural history controls. Results: The cross-sectional comparisons between 5-6.9 year old boys with DMD on corticosteroid treatment and corticosteroid naïve boys demonstrated significantly lower muscle T2 and less fat deposition in lower extremity muscles of treatment group; suggesting reduced inflammation/damage and fat infiltration with corticosteroid treatment (p≤0.05). Boys on corticosteroids demonstrated significantly less change in intramuscular fat infiltration in 1-year (p≤0.05). Finally, both T2 by MRI and MRS detected the beneficial effects of corticosteroid treatment on leg muscles of boys with DMD as early as 3 months of drug initiation. Conclusions: The results of the study support the proposed role of corticosteroids in reducing inflammatory processes in skeletal muscles of boys with DMD. Overall, this study emphasizes the potential of MR as a biomarker for the quantification of early and subtle muscle changes caused by the disease process or therapeutic interventions in DMD.

4. Profiling of Skeletal Muscle Using High-Definition Mass Spectrometry

Jatin G. Burniston1, Joanne B. Connolly2, Heikki Kainulainen3, Steven L. Britton4 and

Lauren G. Koch4 1Research Institute for Sport and Exercise

Sciences, Liverpool John Moores University, Liverpool, L3 3AF, UK; 2Waters, Atlas Park, Manchester, UK; 3Department of

Biology of Physical Activity, University of Jyväskylä, Jyväskylä, Finland and

4Department of Anesthesiology, University of Michigan, Ann Arbor, MI

Accurate profiling of the skeletal muscle proteome is challenging but could bring a step increment in our understanding of disease processes. For instance, broad-scale profiling may discover candidate biomarkers at the protein level, which are amenable to pharmacological targeting. Here we report automated and time efficient (2 h per sample) profiling of muscle using ultra-performance liquid chromatography (LC) coupled directly with high-definition mass spectrometry (HDMSE). Soluble proteins extracted from rat gastrocnemius (n=10) were digested with trypsin and analysed in duplicate using a 90 min LC gradient. Protein identification and label-free quantitation were performed from HDMSE spectra, and used to assess the breadth and reliability of the technique. In total 1,514 proteins were identified. Of these, 811 had at least 3 unique peptides and were subsequently used to assess the reliability of LC-HDMSE label-free profiling. Proteins included in this subset encompass the entire complement of glycolytic, beta-oxidation and tricarboxylic acid enzymes. In addition, numerous components of the electron transport chain and protein kinases important in skeletal muscle regulation were detected. The dynamic range of protein abundances spanned 4 orders of magnitude and the correlation across technical replicates of 10 biological samples was R-squared = 0.9961 ± 0.0036 (95 % CI = 0.9940 - 0.9992). The coefficient of variation averaged 7.3 ± 6.7 % (95 % CI = 6.87 - 7.79 %), and 95 % (767 of 811) of proteins

exhibited a coefficient of variation of less than 20 %. Based on these data a sample size of n = 10 would be sufficient to detect a 30 % difference in protein abundance between 2 independent groups with a power of 80 %, α = 0.05. This level of accuracy was maintained across the top 767 most abundant skeletal muscle proteins and represents the most sophisticated profiling of skeletal muscle to date.

5. A 3’ UTR Mutation in PLIN2 in a Family

with a Novel Distal Myopathy Causes Toxic Disruption of miR-590-3p Binding Kyung-Ah Cho1,2, Steven E. Boyden3, Genri

Kawahara3, Elicia Estrella3, Matthew Alexander3, Satomi Mitsuhashi1, Hart G. W.

Lidov4, Randall D. Craver5, Amparo Gutierrez6, John D. England6, Louis M.

Kunkel3, Peter B. Kang1,2 1Department of Neurology, Boston

Children’s Hospital and Harvard Medical School, Boston, Massachusetts; 2Division of

Pediatric Neurology, Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida; 3Division of Genetics, Boston Children’s Hospital and

Harvard Medical School, Boston, Massachusetts; 4Department of Pathology,

Boston Children’s Hospital and Harvard Medical School, Boston, Massachusetts;

5Department of Pathology, Louisiana State University School of Medicine, New Orleans, Louisiana; 6Department of

Neurology, Louisiana State University School of Medicine, New Orleans, Louisiana

We examined a multi-generation family containing several members affected with an unclassified autosomal dominant distal myopathy/muscular dystrophy. High-density linkage analysis most strongly implicated an interval on chromosome 9p23-21, and whole genome sequencing of two affected patients identified a heterozygous c.*467C>T variant in the 3′ untranslated region (UTR) of PLIN2 as the only rare, exonic candidate mutation within the linkage peak. The mutation co-segregated with the phenotype in all available family members

and was absent from numerous polymorphism databases. Known muscle disease genes were excluded by linkage analysis, whole genome sequence data, and Sanger sequencing. PLIN2 encodes perilipin 2, a lipid droplet-associated protein highly expressed in skeletal muscle that regulates lipid metabolism and storage. The c.*467C>T mutation is located one nucleotide downstream from the seed sequence of miR-590-3p. This microRNA suppressed PLIN2 expression in wild type 293T and C2C12 cells, but the suppression was significantly reduced in PLIN2 mutant cell lines. Likewise, skeletal muscle tissue from an affected individual revealed increased expression of PLIN2 relative to a control. Furthermore, both patient muscle and C2C12 myoblast cells expressing mutant PLIN2, but not wild type PLIN2, demonstrated abnormal lipid accumulation accompanied by increased pro-apoptotic gene expression. ,Our findings indicate that the PLIN2 mutation likely exerts a toxic gain-of-function effect mediated by interference with binding of miR-590-3p in the PLIN2 3’ UTR, thereby implicating PLIN2 as an important regulator of human skeletal muscle function.

6. Plethysmography as a Noninvasive

Biomarker for the Diagnosis and Prognosis of MDC1A

Ajay Kumar, Thomas Mehuron and Mahasweta Girgenrath.

Department of Health Sciences, Boston University, Boston MA.

MDC1A is the second most prevalent form of the numerous congenital muscular dystrophies (CMD) that have been identified. Children afflicted with this disease have profound muscle weakness and hypotonia at or soon after birth, feeding difficulties, and usually never achieve independent ambulation. These children often die prematurely from failure to thrive or acute respiratory failure resulting from weak respiratory muscles and progressive scoliosis. Even though respiratory failure is

one of the major causes of mortality in patients with MDC1A, respiratory function has never been investigated in a laminin-α2 deficient disease model. Therefore, in this study we investigated the respiratory function of DyW mice, a model that closely resemble the human pathology, using the Buxco plethysmograph in accordance with the S.O.P provided by TREAT NMD. Each unrestrained, conscious mouse was placed in the Buxco chamber at a controlled temperature and humidity. Bronchoconstriction was estimated by the Enhanced Respiratory Pause (Penh) index, calculated by the formula Penh = (Te/RT -1) * PEF/PIF (peak inspiratory flow (PIF), peak expiratory flow (PEF), expiratory time (Te), and relaxation time (RT)). Increased Penh values are indicative of impaired respiratory function. We found that base line PenH values normalized to the body weight were twice as high in the DyW compared to their age-matched WT mice. While PenH values decreased in response to IGF-1 overexpression in DyW muscle, they were consistently higher than those recorded from WT mice. Most interestingly, we saw a sharp increase (approximately 2 fold or more) in PenH value over the baseline in both DyW and DyW+IGF-1, immediately prior to death. Correlating with increased PenH, cytospin analysis of BAL fluid retrieved from the lungs of these mice revealed a large increase in the presence of neutrophils, likely indicating increased inflammatory response in the lung. Thus, impaired respiratory function (particularly PenH values) correlates tightly with the severity of disease pathology. These findings strongly support the use of plethysmography as a non-invasive biomarker for MDC1A.

7. Magnetic Resonance Imaging Differences in Animal Models of

Duchenne and Congenital Muscular Dystrophy.

Ravneet Vohra1, A Kumar2, SC Forbes1, A Batra1, SM Chrzanowski3, BA Lee3, K

Vandenborne1, M Girgenrath2, GA Walter3 1Department of Physical Therapy, University of Florida; 2Department of Health Sciences,

Boston University; 3Department of Physiology and Functional Genomics,

University of Florida. Mutation in dystrophin gene causes duchenne muscular dystrophy (DMD) whereas defect in laminin- α2 (LAMA-2) gene causes congenital muscular dystrophy (MDC1A). Although several murine models have been developed to study the underlying mechanism and therapeutic interventions but mdx and Lama2Dy-w mice closely resembles the pathophysiological changes occurring in DMD and MDC1A respectively. Significant amount of apoptosis, fibrosis and inflammation have been observed in these mice using histological methods. In this study we used Magnetic resonance imaging (MRI), a non-invasive biomarker to detected differences in hindlimb muscles of animal model of DMD and MDC1A. Methods: MR measurements were performed on mdx (n=6, 2-3 months), LAMADyw (n=8, 2-3 months), WT (n=8, 2-3 months), mice within the AMRIS facility. All mice were imaged on a 4.7T Oxford Magnet with a Varian/Agilent operating system. T2-weighted single spin–echo images of the hind-limb muscles were acquired (TR 2,000 ms; TE 14 and 40 ms; field of view, 20X20 mm2; slice thickness, 1.0 mm; acquisition matrix size, 256 X 128; averages, 2) using a custom built solenoid coil. T2 values were derived using average signal intensity from anterior and posterior hind-limb muscles of each TE using OsiriX software. Results: mean muscle T2 of hind-limb muscles of mdx (30.30 ± 2.96 ms) mice was significantly (p<0.05) higher than WT (22.85 ± 0.43 ms) and LAMADyw (26.07 ± 2.10 ms) mice. Furthermore, Hindlimb (anterior and posterior compartment)

muscles mdx mice had significantly (p<0.05) greater volume than WT and LAMADyw mice. Conclusions: The results of this study indicate that similar to CMD patients, the mouse model, LAMADyw demonstrates stark muscle atrophy and elevated water T2 determined by MRI compared to control mice. In contrast, the mdx mouse was found to possess a greater muscle mass with T2 elevations significantly higher than that for control and LAMADyw mice. 8. Arm Muscle Involvement In Duchenne Muscular Dystrophy: A Quantitative MRI

and MRS Study Willcocks, RJ, Forbes, SC, Lott, DJ,

Senesac, CR, Nicholson, TR, Arora, H, Walter, GA, Vandenborne, K

Department of Physical Therapy, University of Florida, Gainesville, FL

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease that causes progressive proximalto-distal muscle weakness resulting in disability and premature mortality. Upper extremity weakness is a feature of DMD, and results in loss of the ability to feed oneself or perform self-care. Magnetic resonance imaging (MRI) and spectroscopy (MRS) have provided a great deal of insight into disease progression in the lower extremity, but little is known about muscle quality in the upper extremity in boys with DMD. Individuals with DMD are nonambulatory for a large part of their lives, so it is critical to understand how the disease affects the upper extremity muscles. The purpose of this study was to document changes in fat fraction (FF) and muscle transverse relaxation time (T2) in the arm muscles of boys with DMD and unaffected controls (CON). 12 boys with DMD (10.5 ± 3.1 yrs, 10 ambulatory) and 5 controls (11.6 ± 2.5 yrs) completed localized MRS measurements of FF and T2 (1H spectroscopy) in the biceps brachii (BB). Boys also completed MRI measurements of FF (3 point Dixon imaging) and T2 (T2

weighted imaging) in the BB, triceps brachii (TB), and deltoid (DEL). Compared with CON, boys with DMD had higher MRI T2 in all muscles studied (BB: 42.1 ± 11.3 ms vs 30.2 ± 2.4 ms, p=0.01, TB: 42.1 ± 7.0 vs 31.4 ± 0.9, p=0.001, DEL: 40.9 ± 5.0 vs 30.9 ± 0.8, p=0.02). MRS FF and T2 in the BB were also higher in DMD than CON (FF: 0.164 ± 0.169 vs 0.08 ± 0.005, p=0.003, T2: 30.2 ± 1.6 vs 25.8 ± 0.9, p=0.001). This is the first study to report disease-related changes in upper arm muscle quality in DMD measured using MRI and MRS. Both T2 and FF were higher in DMD than CON, and T2 was elevated even in young (7 yrs) boys whose clinical function was relatively unaffected.

9. Magnetic Resonance Imaging and Spectroscopy as a Biomarker for Muscle

Injury Stephen Chrzanowski, Brittany Lee-

McMullen, Abhinandan Batra, Ravneet Vohra, Yanhua Deng, Huabei Jiang, Krista Vandenborne, Glenn Walter

All authors are affiliated with the University of Florida

Magnetic Resonance Imaging and Spectroscopy as a Biomarker for Muscle Injury Background: Muscle damage, a common finding of many neuromuscular diseases, is currently assessed through mechanisms such as biopsy, serum biomarkers, and functional testing, each possessing inherent limitations. Magnetic Resonance Imaging and Spectroscopy (MRI and MRS, respectively) has recently been utilized to assess and measure muscle damage non-invasively. Here, we utilize a preclinical model to induce muscle damage and repair in vivo. Muscle damage/inflammation and regeneration were monitored in vivo using MR methods. Methods: Single hindlimbs of mice were casted for two weeks in a plantar flexed position. Following removal of the casts, mice were allowed to freely ambulate, and MRI and MRS data was collected for up to a week. T2 relaxation was calculated in

several lower leg muscle groups (Soleus, Gastrocnemius, Tibialis Anterior) using diffusion corrected T2 Spin Echo Imaging. Additionally, a STEAM acquisition sequence was utilized to collect 1H spectroscopic information with a 1x1x2 mm3 voxel placed within the soleus. Results: Prior to cast immobilization, MRI and MRS showed indistinguishable differences in T2 signal. Upon reambulation, both MRI and MRS demonstrated parallel trends of a delayed increase in T2 signal in only soleus of the casted leg peaking between two and three days of reambulation, followed by a return to baseline T2 signal by the end of the week. Neither the Tibialis Anterior nor the Gastrocnemius showed appreciable changes from baseline in the T2 signal prior to and during reambulation. Conclusion: Muscle damage and repair, as measured by MRI and MRS, is shown to be limited to specifically the soleus muscle in this immobilization-reambulation protocol. A well-defined time course of muscle damage and repair is observed through this experiment.

10. In Vivo Measurements of Mitochondrial Respiratory Capacity In

Skeletal Muscle Terence E. Ryan1,4, Chien-Te Lin1,4, Patricia Brophy2,3, Robert C. Hickner1,2,3,4, Kevin K.

McCully5, P. Darrell Neufer1,2,3,4 1East Carolina Diabetes and Obesity

Institute, 2Human Performance Laboratory, Departments of 3Kinesiology, and

4Physiology, East Carolina University 5Department of Kinesiology, University of

Georgia

We recently developed a non--‐invasive technique for measuring skeletal muscle mitochondrial respiratory capacity in humans using near infrared spectroscopy (NIRS). PURPOSE: To compare NIRS measurements of skeletal muscle mitochondrial respiratory capacity with the following current gold standard techniques: 31P--‐MRS in vivo and high--‐resolution respirometry (HRR) in permeabilized fibers.

METHODS: Two studies were performed. A NIRS probe was placed in the medial

gastrocnemius (31P--‐MRS study) or vastus lateralis (HRR study). NIRS was used to measure the recovery kinetics of muscle oxygen consumption after short duration voluntary exercise. The recovery kinetics of phosphocreatine (PCr) were also measured with 31P--‐MRS in the 31P--‐MRS study. Muscle biopsies were obtained from the vastus lateralis, manually separated in small fiber bundles, and permeabilized with saponin in the HRR study. Permeabilized fibers were assessed using standard titration protocols with substrates/inhibitors. RESULTS: In the 31P--‐MRS study, NIRS measured recovery kinetics were well correlated with PCr recovery kinetics (r =0.94, N=16, p < 0.0001). In the HRR study, NIRS recovery kinetics were strongly correlated with State 3 respiration with substrates supporting Complex I (r = 0.64, N=21, p < 0.01), Complex II (r = 0.74, N=21, p < 0.01), and Complex I+II (r = 0.69, N=21, p < 0.01). CONCLUSION: These data clearly indicate the validity of NIRS as a valuable tool for assessing skeletal muscle mitochondrial respiratory capacity in vivo. Given the reasonable cost of NIRS, this technique is ideal for studies involving human patient populations 11. FNDC5 Expression in Skeletal Muscle in Chronic Heart Failure – Relevance of

Inflammatory Cytokines Volker Adams, Konstanze Gleitsmann, Norman Mangner, T Scott Bowen, Axel

Linke University Leipzig– Heart Center Leipzig,

Leipzig, Germany Background: Chronic heart failure (CHF) is commonly associated with muscle atrophy. Irisin, a myokine proteolytically processed by the FNDC5 protein and suggested to be PGC-1α activated, modulates the browning of adipocytes and is related to muscle mass. In addition, skeletal muscle expression of FNDC5 correlates to exercise capacity in patients with CHF. Therefore, we

investigated whether skeletal muscle FNDC5 expression in CHF was reduced and if this was mediated by inflammatory cytokines and/or angiotensin II (Ang-II). Methods: Skeletal muscle FNDC5 mRNA/protein and PGC-1α mRNA expression were analyzed in: 1) Rats with CHF; 2) Mice injected with TNF-α (24h); 3) Mice infused with Ang-II (4 wk); 4) C2C12 myotubes exposed to recombinant cytokines or Ang-II. To elucidate the signal cascade of cascade of cytokine-induced FNDC5 expression in myotubes western blot analysis with phospho-specific antibodies or specific inhibitor were used. Results: CHF reduced (p<0.05) FNDC5 protein (1.3•}0.2 vs. 0.5•}0.1 arb. units) and PGC-1α mRNA expression (8.2•}1.5 vs. 4.7•}0.7 arb. units) compared to control, respectively. TNF-α and Ang-II reduced (p<0.05) FNDC5 protein expression by 28% and 45%, respectively. Incubation of myotubes for 24h with a TNF-α, IL-1s, and IFN- γ cocktail reduced (p<0.05) FNDC5 protein expression by 71%, whereas Ang-II had no effect. PGC-1α was linearly correlated to FNDC5 in all conditions. In C2C12 inflammatory cytokine activate the ERK1/2 pathway, but this activation did not result in FNDC5 expression. Conclusion: Skeletal muscle FNDC5 expression is reduced in CHF, which seems mediated by inflammatory cytokines, Ang-II, and/or PGC-1α. This suggests a reduced FNDC5 expression may act as a protective mechanism in catabolic states, by preserving energy homeostasis via slowing the browning of adipocytes. Further studies are necessary to identify the signal cascade for the cytokine-induced FNDC5 expression.

12. Effects of Voluntary Running During Growing Period On Bone Tissue In Type

2 Diabetic Rats Yuri Takamine1, Takamasa Tsuzuki1, Noriko

Ichinoseki-Sekine2, Toshinori Yoshihara1, Hisashi Naito1

1Juntendo University, 2The Open University of Japan

Introduction: Fragile bones have been reported in type 2 diabetic patients and could be related to abnormal glucose tolerance or an increase in body fat. Exercise during growing period contributes to diabetes prevention and inhibits body fat accumulation. We investigated the effects of voluntary running during the growing period on bone tissue in type 2 diabetic rats. Methods: Twenty-one 5-week-old male type 2 diabetic Otsuka-Long-Evans-Tokushima-Fatty (OLETF) rats were assigned to a sedentary(n=11)or voluntary exercise group(n=10). Rats in the exercise group were allowed access to a running wheel. At 24 weeks of age, glucose tolerance was measured by intraperitoneal glucose tolerance test in all rats. At 25 weeks, white adipose tissue and the right and left femur were removed. The maximum breaking force was determined using a three-point-bending test and normalized to bone weight, while bone volume and marrow-fat volume were measured histologically. Results: The daily running distance during the experimental period in the exercise group was 4.2±0.4 km/day. The body weight and white adipose tissue mass at 25 weeks of age were lower and glucose tolerance significantly higher (p < 0.05)in the exercise group, indicating that glucose tolerance was improved by exercise. There were no significant differences in relative maximum breaking force and bone volume between the groups and bone fragility was not observed in either group, although the marrow-fat volume was significantly lower (p < 0.05) in exercise group than sedentary group. Conclusion: The onset of diabetes and accumulation of marrow-fat were prevented by voluntary exercise during growing period.

Cell Signaling Pathways Regulating Muscle Atrophy

13. KLF-4: A Novel Target To Prevent

Tumor-Induced Muscle Wasting? Andrea Bonetto, Teresa A. Zimmers

Department of Surgery, Indiana University School of Medicine

Cachexia affects almost half of cancer patients and is responsible for body and muscle weight loss, fatigue and poor quality of life. We have shown that STAT3 is necessary and sufficient for muscle wasting induced by IL-6 and IL-6-dependent cancer cachexia. Among the STAT3 downstream targets, the transcription factor Kruppel-like factor 4 (KLF-4) has been associated with cell proliferation, differentiation and reprogramming of somatic cells into induced pluripotent stem cells. An essential role for KLF-4 in ERK5-dependent muscle cell fusion and differentiation has been described as well. Whether KLF-4 is also involved in regulating muscle growth in cancer cachexia is still unknown. Here we showed that KLF-4 is up-regulated in the skeletal muscle in experimental cancer cachexia and in disease states associated with muscle atrophy. Our microarray analysis revealed KLF-4 is increased in mice with IL-6-induced muscle wasting. KLF-4 protein levels were also up-regulated in the skeletal muscle of mice bearing the C26 tumor, a well-known experimental model of cancer cachexia. Elevated KLF-4 prevented myotube formation in C2C12 myoblast cultures, while overexpression of KLF-4 in myotubes for up to 72 hours induced a dedifferentiation-like process, associated with progressive condensation and shrinkage, cell detachment and myofiber atrophy. Gene profiling of C2C12 expressing KLF-4 from a recombinant adenovirus showed modulation of 510 probe sets (change >1.5 fold; p < 0.05). In conclusion, our findings suggest that enhanced expression of KLF4, together with its known activity of maintaining pluripotency in stem cells, might influence muscle wasting in cancer and in conditions

of elevated IL-6 by affecting differentiation. This hypothesis is further supported by recent data from Costelli and Guttridge groups implicating a role for muscle precursor cells in promoting abnormal muscle differentiation and cachexia. Future experiments are required to establish KLF-4 as a new target for the prevention of tumor-induced muscle atrophy.

14. Knockdown of ATG5 in the Diaphragm Reduces Oxidative Stress

and Protects Against Ventilator-Induced Diaphragm Dysfunction

Ashley J. Smuder, Kurt J. Sollanek, W. Bradley Nelson, Kisuk Min, Erin E. Talbert

and Scott K. Powers Department of Applied Physiology and

Kinesiology, University of Florida, Gainesville FL

Mechanical ventilation (MV) is a life-saving measure for patients in respiratory failure. However, prolonged MV results in diaphragm weakness due to both muscle fiber atrophy and contractile dysfunction. Specifically, this ventilator-induced diaphragm dysfunction (VIDD) is a major contributor in the failure to remove patients (i.e. wean) from the ventilator. Previous work from our laboratory shows that VIDD is mainly due to increased diaphragm proteolysis. In this regard, the calpain, capsase-3, autophagy and the ubiquitin-proteasome system are all activated in the diaphragm following prolonged MV. However, the overall contribution of autophagy to VIDD is unknown, and data suggest that increased autophagy may cause either protective or deleterious effects depending on the stimulus. Therefore, these experiments were designed to determine the effects of accelerated autophagy on the diaphragm during MV. To test this, we knocked down autophagy-related protein 5 (ATG5) in the diaphragm of rats and then subjected them to 12 hours of MV. Diaphragm transduction was achieved via direct intramuscular injection with AAV2/9-pTRUF12-ATG5-K130R. MV decreased

diaphragm muscle fiber size and force generation, but knockdown of ATG5 in the diaphragm prior to MV prevented these changes. In addition, knockdown of ATG5 prior to MV caused a significant reduction in MV-induced diaphragm lipid peroxidation (4-HNE modified protein conjugates) and mitochondrial ROS production. Also, transduction with ATG5 resulted in increased diaphragmatic cytosolic catalase content compared to mechanically ventilated control animals. Finally, our results show that a regulatory cross-talk between autophagy, calpain and caspase-3 exists. Specifically, knockdown of diaphragmatic ATG5 resulted in a reduction of the MV-induced increase in proteolytic activity of calpain and caspase-3. Therefore, our data indicate that MV-induced autophagy is detrimental to the diaphragm and that knocking down protein expression of ATG5 can protect against VIDD by reducing MV-induced diaphragmatic oxidative stress and proteolysis. 15. Contractile Proteins are Key Targets for Lysine Specific Hypoacetylation and

Hyperubiquitination During Muscle Atrophy

Daniel J Ryder1, Beharry AW1, Farnsworth2 CL, Silva JC2, Judge AR1

1Department of Physical Therapy, University of Florida, Gainesville FL; 2Cell Signaling

Technology, Danvers, MA Skeletal muscle atrophy is a consequence of several physiological conditions including muscle inactivity and aging. Lysine ubiquitination and subsequent degradation of skeletal muscle proteins through the ubiquitin proteasome pathway (UPP) is a key mechanism contributing to the loss of muscle proteins during these conditions. Another post-translational modification which may regulate UPP-mediated protein degradation is lysine acetylation. Previous data from our lab shows that inhibition of deacetylation via HDAC inhibitor treatment prevents muscle atrophy. However, little is known about the coordinated actions of

acetylation and ubiquitination during muscle atrophy. The purpose of this study was to delineate the global deposition of lysine acetylation and ubiquitination during disuse muscle atrophy. We hypothesized that decreased lysine acetylation would be paralleled by an increase in ubiquitination as muscle atrophy progressed over time. To induce muscle atrophy, rats were cast immobilized for 0, 2, 4 or 6 days and muscles harvested for identification and quantification of acetylated and ubiquitinated peptides via LC-MS/MS (LTQ Orbitrap) spectrometry. Of the 452 acetylated proteins identified in the AcetylScan® dataset, 41, 30 and 38 were found to be hypoacetylated and 8, 22, and 18 were found to be hyperacetylated at 2, 4 and 6 days of immobilization, respectively, compared to weight bearing controls. By comparison, there were 1,131 ubiquitinated proteins identified in the UbiScan® dataset, of which, 73, 117 and 86 were found to be hyperubiquitnated and 220, 321 and 215 were found to be hypoubiquitinated at 2, 4 and 6 days of immobilization, respectively, compared to weight bearing controls. Within the contractile/sarcomeric protein category there were 20 lysine sites which were concomitantly hypoacetylated and hyperubiquitinated during muscle atrophy, thus confirming our hypothesis. These findings provide the first proteome-wide identification of skeletal muscle proteins exhibiting changes in lysine acetylation and ubiquitination during atrophy. Supported by R01AR060209 (to A.R.J.)

16. p53 and ATF4 Mediate Distinct and Additive Pathways to Skeletal Muscle Atrophy During Limb Immobilization

Daniel K. Fox1, Kale S. Bongers1, Scott M. Ebert1, Michael C. Dyle1, Steven A.

Bullard1,2, Jason M. Dierdorff1, Steven D. Kunkel1, and Christopher M. Adams1,2 1Departments of Internal Medicine and

Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of

Medicine, The University of Iowa, Iowa City,

IA, and 2Iowa City Veterans Affairs Medical Center, Iowa City, IA.

Immobilization causes skeletal muscle atrophy via complex molecular mechanisms that are not well understood. To better understand these mechanisms, we investigated and compared the roles of p53 and ATF4, two transcription factors that mediate adaptations to a wide variety of cellular stresses. Previous studies established that muscle immobilization increases skeletal muscle ATF4 expression, which is sufficient to induce muscle fiber atrophy. However, skeletal muscles lacking ATF4 are only partially resistant to muscle atrophy during limb immobilization, indicating the existence of an ATF4-independent pathway. Here, we demonstrate that p53 mediates an ATF4-independent pathway to muscle atrophy during limb immobilization. Using mouse models, we show that limb immobilization increases p53 expression in skeletal muscle, and forced expression of p53 in skeletal muscle fibers is sufficient to induce muscle fiber atrophy. Conversely, mice lacking p53 expression in skeletal muscle fibers are partially resistant to immobilization-induced skeletal muscle atrophy, similar to mice lacking ATF4 expression in skeletal muscle fibers. Importantly, p53 promotes muscle fiber atrophy in the absence of ATF4, whereas ATF4 promotes muscle fiber atrophy in the absence of p53. Furthermore, forced expression of both p53 and ATF4 induces more muscle fiber atrophy than either p53 alone or ATF4 alone. In addition, skeletal muscle lacking both p53 and ATF4 is more resistant to immobilization-induced muscle atrophy than muscle lacking only p53 or ATF4. Collectively, these results demonstrate that p53 and ATF4 mediate distinct and additive pathways to skeletal muscle atrophy during limb immobilization.

17. MicroRNA 208b is Decreased In Quadriceps Muscles of COPD Patients

Sharon Rosenberg, Danielle Barkema, Robert Sufit, Ravi Kalhan, Jacob I. Sznajder

and Emilia Lecuona. Division of Pulmonary and Critical Care

Medicine, Northwestern University, Chicago.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the United States. COPD has multiple and variable systemic manifestations including skeletal muscle dysfunction which contribute to poor health status and mortality. MicroRNAs (miRs) are noncoding RNA of ~22 nucleotides that regulate gene expression by promoting mRNA degradation and inhibiting mRNA translation. Some muscle specific miRs participate in diseases including muscular dystrophy. We conducted a study to determine differentially miR expression in COPD. Five patients with severe COPD with mean age of 70, all males, mean FEV1 0.98 (34%) and five healthy controls with mean age of 54, all males, all with normal spirometry, participated in the study. Closed biopsies from the vastus lateralis were obtained with a side cut needle. Tissue samples for microarray analysis were immediately submerged in RNAlater for RNA stabilization and samples used for protein analysis by Western blotting were snap-frozen in liquid nitrogen. The miR array showed 26 miRs statistically differentially expressed with miR-208b being the main decreased miR (p<0.01). We confirmed the decreased in miR-208b in COPD vs healthy subjects by qPCR. We also found an increased expression of myostatin in quadriceps of COPD patients compared to healthy controls. In conclusion, patients with severe COPD have decreased expression of miR-208b and increased expression of myostatin, a demonstrated target for miR-208b, compared to healthy controls. The increased level of myostatin, a known negative regulator of muscle mass, could contribute to the muscle dysfunction found in these patients. Future research is

needed to understand the regulation of miR-208b and subsequently of myostatin in the muscle of COPD patients. Regulating specific miRs in the setting of muscle disease may provide new therapeutic targets to improve muscle function in patients with COPD who have skeletal muscle dysfunction. Supported by a 2012 Dixon Translational research Grant Initiative and HL 85534.

18. High CO2 Levels Lead To Rodent Skeletal Muscle Atrophy Through The Activation Of AMP-Activated Protein

Kinase And The Ubiquitin-Proteasome Pathway.

Ariel Jaitovich1, Ermelinda Ceco1, Martin Angulo1, Laura A Dada1, Lynn Welch1, Emilia Lecuona1, Yuan Cheng1, Galina

Gusarova1, Cam Patterson3, Gustavo A. Nader2 and Jacob I Sznajder1.

1Division of Pulmonary and Critical Care Medicine, Northwestern University, Chicago, IL, USA;2Department of

Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden;3McAllister

Heart Institute, University of North Carolina, Chapel Hill, NC

Skeletal muscle atrophy occurs in patients with respiratory diseases and is associated with impaired quality of life, since it affects both, muscle strength and endurance. Hypercapnia or elevated blood CO2 levels, occurs in patients with chronic lung diseases. It is well known that the ubiquitin-proteasome system mediates muscle atrophy. Specifically, increased activity of two ubiquitin ligases, muscle RING finger 1 (MuRF1) and muscle atrophy F-box (atrogin-1/MAFbx) has been shown in models of muscle atrophy. Also, mice lacking atrogin-1 and MuRF1 are resistant to muscle atrophy induced by denervation. In this study, we sought to understand the molecular mechanism by which chronic hypercapnia affects muscle function. We found that exposure of wild-type mice to high CO2 levels (akin of patients with COPD) resulted in reduced muscle weight,

smaller myofiber size, increased central nuclei and reduced grip strength. Moreover, high CO2 exposure induced the expression of the ubiquitin E3 ligase, MuRF1 in C2C12 myotubes and in vivo in the soleus muscle of mice exposed to high CO2. Silencing MuRF1 in vitro restored myotube diameter and Western blot analysis revealed LKB1-mediated AMPKα2 activation, which triggered phosphorylation and nuclear translocation of FoxO3a, resulting in an upregulation of MuRF1. Mutation of AMPK consensus phosphorylation sites on FoxO3a prevented high CO2-induced myotube atrophy. In addition, MuRF1-/- mice were protected from CO2-mediated muscle atrophy, further implicating the ubiquitin-proteasome pathway. In summary, we provide evidence that exposure to high CO2

levels activates the LKB1-AMPKα2-FoxO3a-MuRF1 catabolic pathway, which results in muscle atrophy which is of biologic and potentially clinical relevance for patients with lung diseases. Supported in part by HL-76139 and HL-85534.

19. Recovery of Diaphragm Contractile Function with JAK Inhibition During

Mechanical Ventilation is Accompanied by Reduced Accumulation of

Mitochondrial STAT3. Ira J. Smith, Tarikere Gururaja, Guillermo L.

Godinez, Baljit K. Singh, Kelly M. McCaughey, Raniel R. Alcantara, Melissa S. Ho, Yan Chen, Rajinder Singh, Esteban S. Masuda, Vanessa C. Taylor, Donald G.

Payan, Taisei Kinoshita, and Todd M. Kinsella

Rigel Pharmaceuticals, South San Francisco, California, USA

We recently reported that inhibition of the JAK/STAT3 signaling pathway during mechanical ventilation (MV) in rats prevented diaphragmatic mitochondrial dysfunction, oxidative stress, and weakness. Here we investigate potential mechanisms of JAK/STAT3 mediated mitochondrial dysfunction. Although STAT3 is best characterized for its role as a nuclear

transcription factor, recent findings have demonstrated that serine 727-phosphorylated STAT3 is imported into mitochondria, where it associates with complex I of the electron transport chain and modulates respiration. Since this association can lead to ROS production in certain contexts and ROS-related mechanisms are thought to play a prominent role in the development of ventilator induced diaphragm dysfunction, we analyzed the levels of serine 727-phosphorylated STAT3 in total cellular lysates. MV led to significant increases in the amount of phospho-STAT3S727 and this could be prevented by inhibition of JAK signaling, indicating that these events were coupled. Importantly, the increased abundance of phospho-STAT3 (serine 727 and/or tyrosine 705) induced by MV was accompanied by substantial increases in its accumulation within mitochondria and this was also prevented by treatment with a JAK inhibitor. GRIM-19, a component of complex I, is critical for the import of phospho-STAT3S727 into mitochondria and, like STAT3, its presence there has also been linked to increased ROS generation. GRIM-19 was readily detected within mitochondria but MV did not lead to additional increases of this protein. These findings implicate STAT3 translocation into mitochondria as an initiating event in mitochondrial dysfunction, oxidative stress, and contractile dysfunction during mechanical ventilation. 20. Muscle Glucocorticoid Receptors and Long-Term Alcohol Abuse: Preliminary

Findings 1Jakob L. Vingren, 2Bryon Adinoff, 1Anthony,

A. Duplanty, 1Ronald, Budnar, JR., 1Hui Ying Luk, 2Hong Xiao & 1David W. Hill

1University of North Texas, Denton, TX 2University of Texas Southwestern Medical

Center, Dallas, TX Long-term alcohol abuse can induce muscle atrophy as well as chronically elevated concentrations of cortisol. Alcohol induced muscle atrophy results from a persistent

negative protein balance due to increased muscle protein degradation combined with reduced protein synthesis, outcomes which correspond with effects of cortisol. Thus, dysfunction of the myoendocrine system might provide for a physiological mechanism underlying the negative effect of alcohol use on muscle tissue. The purpose of this study was to investigate the effect of long-term alcohol abuse on muscle glucocorticoid receptor (GR) content. Methods: Seven 4-week abstinent men with alcohol dependence and 9 healthy age-matched males (Control) were recruited for this study. A muscle biopsy sample was obtained from the vastus lateralis for determination of GR content. Participants also completed a Trier Social Stress Test (TSST; public speaking task) with blood samples obtained before, immediately after, and every 10 min after the TSST for 60 min for determination of HPA axis reactivity (cortisol concentration area under the curve). Chronic stress over the prior six months was assessed using the UCLA Chronic Stress Assessment. Results: GR content was not significantly different (p=0.14, n=15) between groups (alcohol-dependent: 0.95 ± 0.09 AU; Control: 1.03 ± 0.10 AU). When groups were combined there was a significant correlation (r=0.67, p=0.004, n=16) between GR content and the cortisol response to the TSST. There was also a significant correlation (r=0.64, p=0.007, n=16) between GR content and the UCLA measure of chronic stress. Conclusion: The preliminary findings suggest that long-term alcohol abuse does not result in reduced GR in men who are 4 weeks alcohol abstinent; although a near trending effect was observed. GR content did appear to be associated with chronic stress as well as HPA axis reactivity to a stressful public speaking task (TSST).

21. Decreased Nuclear CRTC Contributes to the Dexamethasone-Induced

Reduction In PGC-1α Expression In Muscle Cells

Rahnert JA1, Zheng B1, Woodworth-Hobbs ME1, Hudson MB1, Price SR1,2

1Emory University, Department of Medicine, Atlanta, GA, 2VA Medical Center, Atlanta,

GA Muscle wasting associated with chronic diseases, such as type I diabetes (DM), has been linked to decreased expression of the transcriptional coactivator PGC-1α. Overexpression of PGC-1α can reduce FoxO-mediated atrogene expression and counter muscle loss. Previous reports indicate that PGC-1α transcription is regulated by CREB in a process that requires CREB-regulated transcription coactivator (CRTC). We recently found that CRTC protein is reduced in skeletal muscle of diabetic rats whereas CREB is highly phosphorylated (i.e., activated). Nuclear translocation of CTRC is promoted by dephosphorylation via calcineurin (CnA) whereas nuclear export occurs by phosphorylation via the salt-inducible kinases (SIK). Our experiments tested the hypothesis that glucocorticoids decrease PGC-1α expression by reducing CRTC levels in the nucleus. L6 myotubes were treated with and without dexamethasone (Dex, 100nM) for 48hrs. Dexamethasone decreased PGC-1α protein and nuclear levels of CRTC. Dex decreased nuclear CnA and increased nuclear SIK, together supporting the observed reduction in nuclear CRTC levels. Over-expression of CRTC increased PGC-1α protein. To assess the role of CRTC on PGC-1α transcription, myotubes were transfected with a PGC-1α luciferase reporter plasmid (PGC-1α-Luc). Over-expression of CRTC increased PGC-1α-Luc activity. Dex decreased PGC-1α-Luc activity however deletion of the CRE binding site from PGC-Luc (PGC-1α- -Luc) prevented the response. Together, these data support the interaction of CRTC with phospho-CREB as a mechanism involved in maintaining PGC-

α transcription and expression level. Impaired CRTC function, due to reduced nuclear CRTC, may contribute to the loss of muscle mass arising during chronic diseases associated with elevated glucocorticoids. Supported by NIH RO1 DK95610, NIH T32 DK007656 and VA Merit X01BX001456 22. Disrupted Protein Quality Control In

Skeletal Muscle Atrophy: Role of 2- Adrenoceptor

Campos, JC1; Voltarelli VA2; Bechara, LRG1; Moreira, JBN3; Brum, PC2; Ferreira

JCB1 1Institute of Biomedical Sciences and

2School of Physical Education and Sport, University of Sao Paulo, Brazil, 3Norwegian

University of Science and Technology, Trondheim, Norway.

The ubiquitin-proteasome system is the primary effector of the protein quality control (PQC), a process that can detect, repair and dispose cytotoxic proteins using multiple control mechanisms. Evidences suggest that _2-adrenoceptor agonists improve cardiac PQC by increasing ubiquitin-proteasome activity; however, little is known about this response on skeletal muscle

atrophy. Here we determined the role of 2- adrenoceptor agonist in regulating PQC and skeletal muscle mass/function in animal model of neurogenic muscle atrophy. We found that 14 days after sciatic nerve constriction (SNC) rats presented accumulation of ubiquitinated (181±34 vs. 100±8%) proteins along with increased levels of small chaperones ab-crystallin and HSP27 compared to sham animals. These changes were followed by a drastic reduction in both skeletal muscle mass (50%) and ex vivo functional capacity in SNC group. Of interest, a concomitant 14

days treatment with a 2-adrenoceptor agonist (clenbuterol 10 mg.kg-1.day-1) increased proteasome activity (23,7±4,6 vs. 9,2±0,7 uF.mg prot-1) and reduced protein ubiquitination (115±5 vs. 158±16%) in SNC rats. These changes were accompanied by

increased both skeletal muscle cross-sectional area and functional capacity.

Finally, mice lacking 2-adrenoceptor presented a more severe phenotype of PQC disruption and muscle dysfunction compared to wild-type littermates. Altogether, these findings suggest a new contribution of _2-adrenoceptor signalling pathway to the pathophysiology of skeletal muscle atrophy where _2-adrenoceptor activation contributes to a better PQC. These findings also strengthen the importance of PQC as a regulator of skeletal muscle atrophy.

23. Skeletal Muscle Denervation Causes

Skeletal Muscle Atrophy through a Pathway that Involves Both Gadd45a and

HDAC4 Kale S. Bongers1, Daniel K. Fox1, Scott M.

Ebert1, Steven D. Kunkel1, Michael C. Dyle1, Steven A. Bullard1,2, Jason M. Dierdorff1,

Christopher M. Adams1,2 1University of Iowa, Departments of

Molecular Physiology and Biophysics and Internal Medicine and Fraternal Order of Eagles Diabetes Research Center, Iowa

City, IA; 2Iowa City Veterans Affairs Medical Center, Iowa City, IA.

Denervation-induced skeletal muscle atrophy is a common secondary consequence of a variety of clinical conditions, including diabetes, amyotrophic lateral sclerosis, alcoholism, and Charcot-Marie-Tooth disease. However, despite its prevalence and severity, the molecular mechanisms responsible for denervation-induced muscle atrophy remain incompletely understood. To better understand the molecular pathogenesis of this common, debilitating condition, we investigated the regulation of growth arrest and DNA damage-inducible 45a (Gadd45a) in denervated skeletal muscle. Previous studies demonstrated that muscle denervation induces Gadd45a mRNA expression, which in turn increases Gadd45a, a small nuclear protein that is required for denervation-induced muscle

fiber atrophy. However, how denervation increases Gadd45a expression remained unknown. Our studies show that the lysine deacetylase HDAC4 is required for Gadd45a induction after muscle denervation. Conversely, HDAC4 overexpression increases skeletal muscle Gadd45a mRNA and causes muscle fiber atrophy in the absence of muscle denervation. Furthermore, Gadd45a mediates several important downstream effects of HDAC4, including induction of myogenin mRNA and, importantly, myofiber atrophy. Because Gadd45a is also a critical mediator of fasting-induced muscle atrophy, we next tested whether HDAC4 might regulate Gadd45a expression during fasting. Interestingly, however, HDAC4 is not required for Gadd45a mRNA induction or muscle atrophy after fasting. Additionally, the bZIP transcription factor ATF4, which is required for fasting-induced Gadd45a expression, does not contribute to denervation-induced Gadd45a expression or muscle atrophy. Taken together, these findings show that HDAC4 is an essential regulator of Gadd45a in muscle atrophy after denervation and identify Gadd45a as a convergence point for different upstream regulators during fasting- and denervation-induced muscle atrophy.

24. BDH1 Overexpression in Skeletal Muscle Enhances Exercise Capacity and

Protects Against Muscle Wasting Kristopher Chain1,5, Vitor Lira5, Rhianna Laker5, Jarrod Call5, Nic Greene5, Mei

Zhang2,5, Zhen Yan2,3,4,5 Departments of Biochemistry1, Medicine2,

Pharmacology3 and Physiology and Biological Physics4, Center for Skeletal Muscle Research at Robert M. Berne

Cardiovascular Research Center5, University of Virginia, Charlottesville, VA

Ketosis provides an alternative source of energy for skeletal muscle, particularly under the condition of severe fasting or diabetes. 3-hydroxybutyrate dehydrogenase, type 1 (BDH1) is a key

enzyme in the ketone body utilization pathway in skeletal muscle, and its expression is induced in response to endurance exercise training. We investigated the contribution of ketone body utilization to exercise at normal and ketotic conditions in transgenic mice overexpressing BDH1 in skeletal muscle (MTG). MTG mice and their wild type (WT) littermates were subjected to a treadmill running test to determine time to fatigue under conditions of a fed state, mild ketosis (following overnight fasting), or diabetic ketoacidosis (5 days post streptozotocin (STZ) injection at 200 mg/kg, i.p.). MTG mice had higher exercise capacity at the fed state (~15%, P<0.05), following overnight fasting (~30%, P<0.05) and 5 days after STZ injection (~60%, P<0.05) relative to WT. Hindlimb skeletal muscles (gastrocnemius, plantaris, tibialis anterior and extensor digitorum longus muscles) from MTG mice were completely protected from STZinduced loss of muscle mass compared to WT littermates. These findings indicate that enhanced BDH1 expression in skeletal muscle improves endurance exercise capacity, particularly under the ketotic states. Therefore, BDH1 may be a potential target to preserve muscle function and prevent muscle wasting under conditions of chronic ketosis

25. SMAD3 Augments, and is Essential for, FoxO3-Induced MuRF-1 Promoter

Activity Lance M. Bollinger1,2 and Jeffrey J. Brault1 1Departments of Kinesiology, Physiology, and Biochemistry and Molecular Biology,

East Carolina University 2Department of Kinesiology and Health

Promotion, University of Kentucky The transcriptional response to rapid muscle atrophy is driven in large part by the transcription factors FoxO3 and SMAD3. However, the combined effects of FoxO3 and SMAD3 on transcription of critical genes that activate protein degradation are unknown. The goal of this study was to

determine the whether SMAD3 and FoxO3 synergistically induce the expression of atrophy-related genes and whether such an effect is due to differential DNA binding. Methods: C2C12 myotubes were co-infected with wild-type FoxO3 and/or wild-type SMAD3 adenoviruses, and expression of muscle-specific ubiquitin ligases (MuRF-1 and Atrogin-1) and autophagic/lysosomal genes (LC3B, Gabarapl-1, ATG4B, and BECN1) was measured by RT-PCR. Wild-type and SMAD binding element mutant MuRF-1 promoter activity (luciferase reporters) and transcription factor-DNA binding (Chromatin Immunoprecipitation (ChIP)) were measured in human embryonic kidney (HEK293) cells co-transfected with plasmid DNA encoding for FoxO3, SMAD3, and/or MuRF-1 reporter. Results: In C2C12 myotubes, FoxO3 but not SMAD3 independently increased Atrogin-1, MuRF-1, LC3B, and Gabarapl-1 mRNA levels. FoxO3+SMAD3 increased gene expression (~50%, p<0.05) versus FoxO3 alone. While neither FoxO3 nor SMAD3 was sufficient to increase ATG4B or BECN1 mRNA levels, FoxO3+SMAD3 significantly increased mRNA levels of these genes (80% and 60%, p<0.05). FoxO3, but not SMAD3, independently increased MuRF-1 promoter activity (3.2-fold), and FoxO3+SMAD3 increased MuRF-1 promoter activity even further (6.2-fold, p<0.05). Mutation of two SMAD binding elements within 15 bp of the transcriptional start site attenuated FoxO3-induced MuRF-1 promoter activity by 60% (p<0.05). ChIP revealed that SMAD3 augments FoxO3 binding to DNA, while the SMAD binding element mutation greatly reduced FoxO3 binding to DNA. Conclusions: SMAD3 synergistically increases FoxO3-induced MuRF-1 gene transcription by augmenting FoxO3 binding to the proximal promoter region. Furthermore, SMAD3 is essential for optimal FoxO3-DNA binding and transcription of MuRF-1. Interrupting SMAD3-DNA interactions may be an effective method to curtail FoxO3-induced transcription of MuRF-1 during atrophic stimuli.

26. Mathematical Modeling Separates Influx and Efflux Contributions to Net

Foxo1 Nuclear Movements Under Various Experimental Conditions in

Skeletal Muscle Fibers Martin Schneider1, Robert Wimmer1, Yewei

Liu1 and Bradford Peercy2 1University of Maryland School of Medicine

and 2University of Maryland Baltimore County

Foxo family transcription factors contribute to muscle atrophy by promoting transcription of the ubiquitin ligases MuRF1 and MAFbx/atrogin1. Foxo transcriptional effectiveness is largely determined by its nuclear cytoplasmic distribution, with unphosphorylated Foxo1 transported into nuclei and phosphorylated Foxo1 transported out. We expressed the fluorescent fusion protein Foxo1-GFP in cultured adult mouse flexor digitorum brevis muscle fibers, and tracked the time course of nuclear (N) to cytoplasmic (C) mean pixel fluorescence in living fibers by confocal imaging. We previously showed that IGF1, which activates the Foxo kinase Akt/PKB, caused a marked and rapid decline in N/C, whereas inhibition of Akt caused a modest increase in N/C. We subsequently develop a 2 state mathematical model for Foxo1 nuclear cytoplasmic redistribution, where Foxo phosphorylation- dephosphorylation is assumed to be fast compared to nuclear influx and efflux. C is constant in muscle fibers due to the much larger cytoplasmic than nuclear volume. Analysis of N/C time courses reveals that IGF1 strongly increased unidirectional nuclear efflux, indicating similarly increased fractional phosphorylation of Foxo1-GFP within the nucleus. Unidirectional nuclear influx was decreased in IGF1, indicating decreased cytoplasmic fractional dephosphorylation of Foxo1. Inhibition of Akt increased Foxo1 unidirectional nuclear influx, consistent with block of Foxo1

cytoplasmic phosphorylation, but did not decrease Foxo1 unidirectional nuclear efflux, indicating that Akt may not be involved in Foxo1 nuclear efflux under control conditions. New experiments indicate that cultured fibers release IGF-like factors into the medium, maintaining low Foxo1-GFP N/C, and that repetitive electrical stimulation of muscle fibers can also lower Foxo1-GFP N/C. These new insights underline the power of monitoring and quantitative modelling of observed nuclear fluxes. Supported by NIH grant R01-AR056477

27. MicroRNA-182 Targets FoxO3 and Attenuates Glucocorticoid-Induced

atrophic signaling in muscle Matthew B. Hudson1, Myra E. Woodworth-Hobbs3, Bin Zheng1, Jill A. Freret1, Harold

A. Franch1, and S. Russ Price1,2 1Renal Division, School of Medicine, Emory

University, Atlanta, GA 30322, USA; 2Atlanta VA Medical Center, Decatur, GA; 3Nutrition and Health Sciences Graduate Program, Laney Graduate School, Emory

University, Atlanta, GA Skeletal muscle atrophy occurs in response to a variety of conditions including diabetes, chronic kidney disease, cancer, mechanical ventilation, and HIV/AIDS. Previous studies have demonstrated that activation of the Forkhead box O (FoxO) transcription factors results in skeletal muscle atrophy in patients, animals and cultured cells. The FoxO proteins cause muscle wasting by increasing the expression of components of the ubiquitin-proteasome and autophagy-lysosome proteolytic systems. To identify potential modulators of the atrophy process, an in silico target scan analysis of known microRNAs (miRs) was performed, and miR-182 was predicted to target the FoxO mRNAs. To test whether miR-182 regulates expression of the FoxOs, C2C12 myotubes were transfected with miR-182 and levels of FoxO1 and FoxO3 proteins were evaluated. miR-182 reduced the amount of FoxO3 but

not FoxO1. Treatment of C2C12 myotubes with dexamethasone (1 μM, 6 hr) to induce muscle atrophy decreased miR-182 expression by 63% (P<0.05). Transfection of miR-182 into myotubes prevented the glucocorticoid-induced upregulation of multiple FoxO3 target mRNAs including MAFbx/Atrogin-1, ATG12, Cathepsin L, and LC3. To determine if miR-182 is altered in an in vivo model of muscle atrophy, miR-182 was measured in the gastrocnemius muscle of rats with acute diabetes (3 d) induced by streptozotocin. miR-182 was decreased 44% (P<0.05) by diabetes. These data identify miR-182 as a new and important regulator of FoxO3-mediated signaling during muscle atrophy induced by catabolic disease states. Source of funding: NIH T32 DK007656 (M.B.H.) and NIH R01DK95610, AHA GRNT7660020, and VA Merit (S.R.P.)

28. Inhibition of FoxO Dependent Translation Prevents Mechanical

Ventilation-Induced Reduction in Protein Synthesis

Michael P. Wiggs1, Ashley J. Smuder1, Kurt J. Sollanek1, Kevin L. Shimkus2, James D.

Fluckey2, Scott K. Powers1 1University of Florida, Gainesville, FL,

2Texas A&M University, College Station, TX Mechanical ventilation (MV) is a life-saving intervention in patients suffering from respiratory failure. Unfortunately, prolonged MV promotes the rapid development of diaphragmatic atrophy due to an increase in proteolysis and a decrease in protein synthesis. In this regard, the FoxO family of transcription factors regulate MV-induced diaphragm proteolysis, however, emerging evidence suggest that this family of proteins may also be an important negative regulator of muscle protein synthesis. Purpose: To determine the role FoxO dependent transcription plays in the regulation of MV-induced decrease in diaphragmatic protein synthesis. Methods: Adult, female Sprague Dawley rats were divided into 12 hours of anaesthetized, spontaneous breathing (SB)

or 12 hours of MV. Within these groups, animals were divided into treatment with saline or diaphragm-specific overexpression of dominant negative FoxO using the AAV-9 vector. In vivo rate of diaphragmatic protein synthesis was measured by a primed-constant infusion of D25-Phe over the final 6 hours of the experimental protocol. Results: Inhibition of FoxO transcriptional activity was sufficient to prevent the MV-induced decrease in diaphragm protein synthesis in the myofibrillar, sarcoplasmic, and mitochondrial rich protein subfractions. Moreover, protection appears to be mediated via the Akt/mTOR signaling pathway. Conclusion: It is widely accepted that FoxO transcriptional activity can induce muscle atrophy by upregulating proteins critical in proteolysis; however this study demonstrates a dual role for the FoxO family through the regulation of muscle protein synthesis. Supported by the NIH R21 AR063805 (SKP).

29. Docosahexaenoic Acid Attenuates Palmitate-Induced Unfolded Protein

Response In Myotubes Myra E. Woodworth-Hobbs, Matthew B.

Hudson, Jill A. Rahnert, Bin Zheng, S. Russ Price

1Nutrition and Health Sciences Program, Graduate Division of Biological and

Biomedical Sciences, Emory University, Atlanta, GA

2Department of Medicine, Renal Division, Emory University, Atlanta, GA

3Atlanta VA Medical Center, Decatur, GA Accumulation of saturated fatty acids in skeletal muscle results in dysregulation of protein metabolism and muscle atrophy. In cultured myotubes, the saturated fatty acid palmitate (PA) stimulates protein degradation and decreases cell size, while co-treatment with the omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) prevents the response. There is evidence that PA also induces endoplasmic reticulum stress and activates the unfolded protein response (UPR) in myotubes,

resulting in decreased protein translation. This study tests whether DHA protects against palmitate-induced endoplasmic reticulum stress and activation of the UPR in muscle cells. C2C12 myotubes were treated with 500μM PA and/or 100μM DHA for 24h and protein markers of the UPR were evaluated by western analysis. PA induced activation of PKR-like endoplasmic reticulum kinase (PERK), as indicated by an increased phospho-PERK:total PERK ratio. PA also induced phosphorylation of (i.e. inactivated) eukaryotic initiation factor 2α (eIF2α) as well as increased the level of NF-E2 related factor 2 (Nrf2) protein. Co-treatment with DHA attenuated the effects of PA on PERK ratio and restored Nrf2 protein to control levels. However, eIF2α phosphorylation was similar in myotubes treated with PA and PA+DHA. These results indicate that DHA attenuates the effects of PA on activation of the UPR but does not restore global protein synthesis; this suggests that DHA maintains myotube diameter primarily by inhibiting palmitate-induced protein degradation.

30. Antagonism of Myostatin/Activin Type IIB Receptor Signaling via a Novel

Small Molecule Robert D. Hyldahl, Ryan Matekel, Allen C.

Parcell, David Bearss Brigham Young University, Provo, UT

Antagonism of the myostatin/activin receptor type IIB (ActRIIB) pathway represents a promising potential therapy for the muscular dystrophies and other muscle wasting disorders (i.e., cachexia or sarcopenia). Previous work has shown that antibody or soluble receptor-mediated myostatin or ActRIIB antagonism effectively attenuates muscle wasting and restores function in many of these conditions. We have developed a novel small molecule (SGI-1252) that interferes with ActRIIB ligand binding. In this study we determined how SGI-1252 treatment affected ActRIIB downstream signaling (phosphorylation of Smad2, Smad3, ERK1/2 and Akt) and

differentiation of C2C12 and human primary myoblasts. Multiplex analyses (Luminex) revealed that treatment of C2C12 myoblasts with SGI-1252 decreased phosphorylation of Smad2 (IC50 = 537.4 nM) and Smad3 (IC50 = 754.7 nM) in a dose dependent manner relative to myoblasts treated with the ActRIIB agonist myostatin. Treatment of human primary myoblasts with 500ng/ml of myostatin increased phosphorylation of Smad2 and Smad3 and concomitant treatment of myostatin and 1μm SGI-1252 reduced phosphorylation of Smad2 and Smad3 to control levels. Myostatin treatment had no effect on phosphorylation of ERK1/2; nevertheless, SGI-1252 treatment reduced ERK1/2 phosphorylation relative to control and myostatin treated cells. Neither myostatin nor SGI-1252 affected phosphorylation of Akt. Quantification of a differentiation index following 3 days of culture in a differentiation medium revealed that human primary myoblasts more effectively differentiated into multinucleated myotubes in the presence of 1μm SGI-1252. Furthermore, primary myoblasts that were differentiated for 4 days then treated for 2 days with 1μm SGI-1252 showed a significant increase in total myotube area and diameter relative to vehicle treated myotubes. Our in vitro findings suggest that SGI-1252 antagonizes downstream myostatin/ActRIIB signaling and improves myotube size and differentiation capacity. Collectively, these data indicate that SGI-1252 may be a viable candidate for further study pursuant to a treatment for muscle wasting disorders. 31. The Changes of Muscle Morphology

after Locomotor Training in a New Model of Spinal Cord Injury

Lim W1

,Baligand C2

,Ye F1

,Vohra RS1

,

Ruhella A1

,Keener J3

,Bose P3,4

,Thompson

F3,4

,Walter G2

,Vandenborne K1

Physical Therapy1

, Physiology and

Functional Genomics2

, University of Florida,

North Florida/South Georgia Veterans

Health System3

, Physiological Science and

Neurology4

, University of Florida, Gainesville, FL

Although animal models of contusion spinal cord injury (SCI) produce similarities with human SCI pathophysiology, animals often show more rapid improvement of muscle mass and locomotor function. To more closely represent the human condition of muscle atrophy and deficient locomotor function (Ye et al. 2013), in this study cast immobilization was used in combination with a severe contusion SCI model. In this model, we implemented locomotor training and examined the changes in muscle properties. Sixteen-week female Sprague-Dawley rats were assigned to 5 groups: Control (n=12), SCI (n=19), SCI+Treadmill training (SCI+TM, n=13), SCI+Cast immobilization (SCI+IMM, n=13), SCI+IMM+TM (n=13). SCI was induced at the T

8-9 level using the NYU impactor device

(10g, 50mm). Bilateral hindlimb cast immobilization and treadmill training were initiated at 8 days post-injury and maintained for 2 weeks. In vivo MRI of the lower leg was used to measure the maximal cross-sectional area (CSA

max) of triceps

surae (TS) muscle and in situ soleus force was measured 21 days post-injury. By twenty-one days post-injury, the CSA

max of

TS was decreased by 22% in SCI, 30% in SCI+TM, 33% in SCI+IMM, and 53% in SCI+IMM+TM compared with baseline. The absolute peak tetanic force (mN) in the soleus muscle was significantly decreased in all injured groups compared with controls, but no significant difference was observed among the injured groups. There was no significant difference in specific peak tetanic

force (N·g-1

) among all 5 groups. Severe contusion SCI showed more muscle atrophy than moderate model of SCI. Short term treadmill training after severe SCI did not induce a positive effect on muscle size and force. Cast immobilization resulted in increased muscle atrophy, even when

combined with treadmill training. Varying intensity, duration, and/or delay training initiation time post injury might be considered based on the severity of injury. Sarcopenia And Cachexia: Muscle Loss

And Anabolic Resistance

32. Role of FoxO in Diaphragm Fiber

Atrophy During Cancer Cachexia Adam W. Beharry, Roberts BM, Senf SM,

Judge AR Department of Physical Therapy, University

of Florida, Gainesville, FL Cancer cachexia is a devastating condition characterized by progressive weight loss due to significant skeletal muscle wasting, and is accountable for more than 20% of cancer deaths. Therefore understanding the molecular signaling pathways which cause muscle wasting during cancer is critically important. In a recent study comparing gene expression changes in skeletal muscle biopsies from pancreatic cancer patients with and without cachexia, the Forkhead BoxO (FoxO) transcription factor FoxO1 was identified as a cachexia-associated gene. Published work further demonstrates that FoxO is required for skeletal muscle atrophy of locomotor muscles in Lewis Lung Carcinoma and Sarcoma-180 tumor bearing mice, while unpublished data extends these findings to Colon-26 (C26) carcinoma. The diaphragm muscle also undergoes significant wasting in response to cancer, and is speculated to link the degree of cachexia with survival. Therefore in the current study we further determined the role of FoxO in driving the atrophy program in diaphragm muscles of C26 tumor-bearing mice. To block FoxO-dependent transcription in the diaphragm, we performed a single intrapleural injection of rAAV9-d.n.FoxO (or rAAV9-ev) prior to inoculation with C26. Twenty six days post-inoculation diaphragms were harvested. C26 caused a 32% decrease in diaphragm muscle fiber CSA in mice injected with rAAV9-ev (control, 1021 µm2 ± 91; C-26,

696 µm2 ± 81), which was abolished in the diaphragm of C-26 mice injected with rAAV9-d.n.FoxO (control, 1024 µm2 ± 82; C-26, 980 µm2 ± 50). Gene expression analyses in the diaphragm further demonstrated that FoxO was necessary for the cancer-induced upregulation of several proteasome components, including Psma2 and several ubiquitin E3 ligases, atrogin-1(Fbxo32), MuRF1 (Trim63), Ubr2 and Fbxo31. These findings therefore demonstrate that FoxO plays a critical role in driving cancer-induced muscle atrophy of locomotor and respiratory muscles, which further emphasizes FoxO as a key therapeutic target for the treatment of cancer cachexia. Supported by R01AR060209 (to A.R.J.).

33. Cancer-Induced Bone Destruction Leads To Skeletal Muscle Oxidative

Stress And Weakness David L. Waning1,2, Khalid S. Mohammad1,2, Steven Reiken3,4, Wenjun Xie3,4, Daniel C.

Andersson4, Andrew R. Marks3,4 and Theresa A. Guise1,2.

1Indiana University Simon Cancer Center and 2Department of Medicine, Indiana

University School of Medicine, Indianapolis, IN, 3The Clyde and Helen Wu Center for

Molecular Cardiology, 4Department of Physiology and Cellular Biophysics, College

of Physicians and Surgeons of Columbia University, New York, NY

Cancer-associated muscle weakness is an important paraneoplastic syndrome for which there is currently no treatment. Using a murine model of human breast cancer that is metastatic to bone (MDAMB-231) we show skeletal muscle oxidative stress and muscle dysfunction in mice with bone metastases. Skeletal muscle weakness occurs without direct involvement of tumor cells in muscle and the tumor-bone microenvironment is a critical determinant of muscle weakness because there was no muscle weakness in the absence of bone metastasis. Tetanic calcium (Ca2+), which directly determines the force of muscle

contraction, was reduced in mice with bone metastases (4.91±0.21 v. 2.28±0.28;p<0.0001). The ryanodine receptor/calcium release channel (RyR1) on the sarcoplasmic reticulum (SR), a key protein involved in skeletal muscle excitation-contraction coupling, was oxidized and depleted of the stabilizing subunit, calstabin1. Ex vivo contractility of the extensor digitorum longus (EDL) muscle showed a significant reduction in specific force in tumor mice (213.2kN/m2±16.6 v. 361.1kN/m2±9.6;p<0.001). Inhibiting the RyR1 mediated SR Ca2+ leak with a Rycal (S107) restored muscle force production (431.0kN/m2±19.4 v. 362.8kN/m2±7.2;p<0.0001) without affecting tumor burden. TGFβ is released in large quantities from bone during cancer-induced bone destruction and muscle from mice with bone metastases had increased SMAD3 phosphorylation. The degree of muscle weakness increased with increased bone destruction and inhibiting TGFβ or preventing bone resorption (using bisphosphonate therapy) reduced oxidative stress and restored muscle function (418.60kN/m2±15 v. 336.3kN/m2±28;p<0.001). C2C12 myotubes treated with TGFβ had elevated NADPH oxidase (Nox4) levels, oxidation of RyR1, reduced calstabin1 binding to the RyR1 complex and SR Ca2+ leak

-1 v.

1.44±1.3sparks/100μm.s-1;p<0.05). Our data show that TGFβ released during bone destruction due to bone metastases leads to oxidative stress in skeletal muscle causing SR Ca2+ leak and contributes to cancer-associated muscle weakness.

34. Aerobic Exercise as a Treatment for

Frailty Haiming Liu, Ted G. Graber, Lisa Ferguson-

Stegall, LaDora V. Thompson University of Minnesota Medical School,

Minneapolis, Minnesota Department of Physical Medicine and

Rehabilitation

Frailty is a clinical syndrome leading to increased morbidity, disability and mortality. We previously developed a frailty index to identify frail mice based on four clinically relevant frailty criteria: grip strength, walking speed, endurance score and physical activity. In the present study, we aimed to evaluate if aerobic exercise has potential to prevent and/or reverse frailty using the frailty index. In addition, a composite score, the Frailty Intervention Assessment Value (FIAV), was also developed to evaluate the efficacy of the treatment (aerobic exercise) to improve the Frailty Criteria at the individual mouse level by measuring the difference between the pre/post-intervention standardized criterion scores and the baseline mean of the cohort. Old (28 mo., n=11) and adult (6-8 mo., n=5) C57BL6 mice were singly housed with running wheels for 4 weeks (voluntary wheel running). Each mouse was tested on the four frailty criteria before and after the 4-week exercise period. Two old mice were identified as frail before the initiation of the exercise intervention; however, they were not frail following the exercise intervention. The rest of the mice were not frail either before or after the exercise period. The mean FIAV demonstrated marked improvement in both the adult (12SD) and the old (3SD) groups. The adult mice demonstrated a far greater positive response to the exercise (p < 0.001). Collectively, we conclude that the aerobic exercise reversed frailty and improved functional performance in old and adult mice. In addition, the FIAV proved to be a valuable tool to evaluate the treatment effects by quantifying the changes in frailty criteria at the individual mouse level.

35. Gene Polymorphisms Influence on Exercise-Induced Changes of Bone Density and Skeletal Muscle Mass In

Japanese Young Women Hiroyo Kondo1, Hidemi Fujino2, , Fumiko

Nagatomo3, Akihiko Ishihara3. 1Nagoya Women’s University, Nagoya, Japan: 2Kobe University, Kobe, Japan:

3Kyoto University, Kyoto, Japan. Locomotive syndrome is known to be a risk factor for osteoporosis and sarcopenia. Vitamin D receptor (VDR), estrogen receptor alpha (ESR1) and ciliary neurotrophic factor (CNTF) are good genetic candidates for a prime regulator of bone metabolism and skeletal muscle. The purpose of this study was to assess the interactive effects of habitual exercise, nutrient intake and gene polymorphisms, VDR, ESR1 and CNTF on bone density and skeletal muscle mass in young women. Two hundred and forty Japanese healthy young women (20-23 years old) were recruited in this study. Habitual exercise and nutrient intake were assessed using a questionnaire. Body fat and muscle mass were measured by the whole body impedance measurement method (InBody, Biospace). Bone mass was measured by quantitative ultrasound measurement (Lunar). The polymorphisms of ER1 at intron I (rs9340799), VDR (rs7975232) and CNTF (rs2510559) were genotyped using the TaqMan probe-based SNP method (Applied Biosystems). The subjects with habitual exercise intake were significantly higher bone density and skeletal muscle mass than those with non-habitual exercise. Calcium intake was correlated with skeletal muscle mass although there was not relationship between protein intake and muscle mass. Interestingly, the subjects with ER1 allele A and VDR allele C in the habitual exercise group had significantly higher bone density than those in the non-habitual exercise group. In Addition, the skeletal muscle mass in non-habitual exercise group with AA homozygote in CNTF was higher than that in TT homozygote. These results suggest that

habitual exercise and calcium intake are more important for bone mass metabolism and skeletal muscle mass in young women carrying C and A allele genotype in ER1, VDR and CNTF polymorphisms. Supported by Grants-in-Aid for Science Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology.

36. Relationships Between Glycogen Content, Translational Signaling, Protein

Synthesis, and Hypertrophy in Overloaded Fast-Twitch Skeletal Muscles

of Young Adult and Aged Rats Marcus M. Lawrence1, Rengfei Shi2, B. Clay

Myers2, Hoke B. Whitworth2, William T. Mixon2, and Scott E. Gordon1,2.

1Dept. of Kinesiology, University of North Carolina at Charlotte, Charlotte, NC and

2Dept.of Kinesiology and Dept. of Physiology, East Carolina University,

Greenville, NC. Our laboratory has previously shown a negative relationship between 5’-AMP-activated protein kinase (AMPK) activation and both translational signaling and hypertrophy in overloaded fast-twitch (FT) muscle in young adult (YA) and old (O) rats. To determine whether this phenomenon is related to glycogen content (GC; a negative regulator of AMPK activity), 6 YA (8-mo.) and 5 O (33-mo.) male Fisher344 x Brown Norway rats underwent a 7-day FT plantaris (PLT) muscle overload via unilateral gastrocnemius tenotomy. With PLT overload, the % increase in GC, % increase in mixed muscle fractional protein synthesis rate (FSR; radiolabeled phenylalanine incorporation), and wet weight hypertrophy were significantly (p ≤ 0.05) reduced with age. PLT AMPK phosphorylation (Thr172) and activation [phospho-acetyl CoA carboxylase (p-ACC; Ser79)] were significantly higher in O vs. YA regardless of loading status. Across both age groups, there were moderate negative relationships between % change in muscle GC and all measures of AMPK phosphorylation/activity (although not all were significant; range: r =

-0.61 to -0.42; p = 0.046 to 0.19). The % change in PLT GC with overload was positively, albeit not significantly, related to % hypertrophy (r = 0.55; p = 0.077) and % change in FSR (r = 0.53; p = 0.115). The % change in FSR was most strongly related to % change in ribosomal protein S6 phosphorylation (Ser235/236; r = 0.85; p = 0.002), % change in AMPK phosphorylation (Thr172; r = -0.65; p = 0.043), and % change in AMPK activation (r = -0.63; p = 0.05), and was non-significantly related to % hypertrophy (r = 0.59; p = 0.07). These data indicate that the greater translational signaling, FSR, and hypertrophic response in YA vs. O rat FT muscle with overload may be loosely related to a greater GC response and its potential suppression of AMPK.

37. Novel Model of Voluntary Resistance

Training for Mice Ted G. Graber, Katie Fandrey, LaDora V.

Thompson University of Minnesota Medical School,

Department of Physical Medicine and Rehabilitation, Minneapolis, Minnesota

55455 Sarcopenia, age-related loss of muscle mass and strength, contributes to frailty onset, loss of independence and reduced quality of life in the elderly. Resistance training is an accepted treatment that slows the progression of sarcopenia. Current hypertrophy models used to investigate cellular mechanisms in mice are less representative of voluntary human weight (resistance) training. Thus, the main purpose of this study was to produce a mouse exercise protocol that mimics human weight training as would be performed in the gymnasium. A successful model could then serve as a pre-clinical model of intervention/training synergy and to elucidate the mechanistic etiology underlying anabolic resistance in the elderly population. Two age cohorts of male C57BL/6 mice were used (12 and 28 months of age at study conclusion; n=20

and 24, respectively). The mice of each age were randomly separated into control and trained groups. The trained groups used progressive resistance (via a weight harness) and intensity (~4-10 RPM) on a custom motorized running wheel (possible speed from 1-10 RPM). The mice trained on a program similar to a human beginning workout regimen (4-5 sets/session, 3 sessions/week, for 10-14 weeks). The mice demonstrated significant improvement in overall neuromuscular function (rotarod), strength/endurance (inverted-cling grip test), training physiology (workload/power output per session), fat percentage (DEXA), muscle size (soleus mass), and force/power production (in vitro contractile physiology). There was evidence of anabolic resistance because some outcome measures in the elderly mice did not improve, or did not improve to the same degree as was observed in the adult group. Since the model produced results in the mice similar to what would be expected after humans engaged in a period of weight training, this validated protocol has the potential to serve as a valuable tool for future pre-clinical and mechanistic investigations.

Cell Signaling Pathways Regulating

Muscle Hypertrophy

38. Muscle Growth Effect of RNA-Binding

Motif Protein 3 (RBM3) is Associated With Binding To Muscle-Specific mRNAs

and miRNAs. Amy Confides1, Andrew Judge2, and Esther

Dupont-Versteegden1 1 College of Health Sciences, Department of

Rehabilitation Sciences, University of Kentucky, 40536

2 Department of Physical Therapy, University of Florida, 32610

RNA-binding motif protein-3 (RBM3) is a cold-inducible protein that has been suggested to play a role in regulating muscle size because of its differential expression during atrophy and hypertrophy. RBM3 has been shown to enhance global

protein synthesis and to inhibit apoptosis, potentially owing to its modulatory effect on miRNA biogenesis. The purpose of this study was to determine if over-expression of RBM3 affects muscle size and we hypothesized that increased RBM3 abundance is anabolic for skeletal muscle both under control or atrophy-inducing conditions. In addition, in an effort to identify mechanisms of action, we investigated which RNA species specifically bind to RBM3. In vitro: C2C12 myoblasts were transiently transfected with RBM3-YFP expressing plasmid and were differentiated into myotubes for 24 hours. In a subset of plates, dexamethasone (100 μM) was added for an additional 24 hours and myotubes were analyzed for size by measuring myotube diameter for RBM3-YFP transfected (green) and non-transfected cells. For in vivo studies, soleus muscles in the right leg of 15 month old rats were injected with RBM3 and GFP plasmid while the left leg received pCDNA and GFP plasmid after which the muscles were electroporated. Rats were either kept ambulatory (AMB, n=4) or were hind limb suspended (HS, n=4) for 14 days. Cross sectional area (CSA) of muscle fibers positive for RBM3 (green) was compared to non-transfected fibers. We performed RNA immunoprecipitation with RBM3 antibody to isolate all RNA species bound to RBM3 in C2C12 myoblasts. Microarray analysis was performed on the immunoprecipitated RNA. Results show that RBM3 positive myotubes were 30% larger in both control and dexamethasone treated myotubes compared to non-transfected cells. Cells transfected with an empty YFP vector did not show a change in myotube size. Also, CSA of soleus muscle fibers which over-expressed RBM3 was 17% and 20% larger than non-transfected fibers in AMB and HS rats, respectively; CSA from the left leg showed no difference between GFP-positive and -negative fibers. We further found that messages from muscle specific genes, such as MyoD, MEF2c, and myoglobin were enriched in the RNA that was immunoprecipitated with RBM3 antibody

and that the miRNA fraction was enriched 4- fold in immunoprecipitated samples. We conclude that RBM3 exerts an anabolic effect on muscle fibers under control as well as atrophy-inducing conditions and that RBM3 binding to muscle specific mRNAs and miRNAs may play a role in stabilizing these RNA components. This work was supported in part by a grant from the NIH/NIA (AG 028925).

39. Lack of Alpha-Actinin-3 Attenuates M-TOR Signaling In Human Skeletal

Muscle After Sprint Exercise Barbara Norman, Mona Esbjörnsson, Håkan Rundqvist, Ted Österlund, Eva

Jansson Karolinska Institutet, Stockholm, Sweden

Polymorphism (R577X) in the ACTN3 gene causes loss of α-actinin-3 protein in individuals with XX-genotype. The prevalence of the X-allele is lower in sprint/power oriented athletes compared with controls. Lack of α-actinin-3 is associated with smaller muscle mass which indicates that the protein may be involved in the modulation of hypertrophy signaling. α-Actinin-3 is a structural component of the Z-disc, where it acts as a tension sensor, and lack of α-actinin-3 has been shown to alter elastic properties of the sarcomeres. We hypothesized that mechanical sensing, important for hypertrophy signaling during muscle contraction, is affected by the altered properties of the Z-disc which may have implication for muscle growth. The aim of the study was therefore to elucidate if signaling via the AKT/mTOR pathway in response to exercise differs across ACTN3 genotypes. 21 healthy subjects with different ACTN3 genotype (4 RR, 11 RX and 7 XX) performed three bouts of 30-s sprint exercise with 20 min of rest in between. Muscle biopsy samples were obtained at rest and 140 min after the last sprint. Phosphorylation of Akt, mTOR, p70S6k, rpS6 and AMPK was analyzed by Western Blot. The exercise-induced

increase in phosphorylation of mTOR and p70S6k was smaller in XX than in RR/RX (p=0.03 and p=0.01 respectively) while there was no difference in the increase of rpS6 and Akt between the genotypes. AMPK phosphorylation level 140 min after exercise was not different from rest. The results indicate less pronounced activation of hypertrophy signaling in XX. The difference in the activation of factors that promote hypertrophy shown in the present study may explain the ACTN3 genotype-associated differences in muscle mass that have been reported in human and mouse skeletal muscle. Attenuated hypertrophy signaling may provide a mechanistic explanation for why lack of α-actinin-3 can be detrimental for sprint/power oriented athletes.

40. Putative Role of REDD1 in the Activation of mTORC1 Following

Resistance Exercise Gordon BS, Steiner JL, Lang CH, Jefferson

LS, Kimball SR Department of Cellular and Molecular

Physiology, Penn State College of Medicine, Hershey, PA 17033

Resistance exercise induces muscle hypertrophy by shifting protein metabolism in favor of net synthesis. Signaling through the mechanistic target of rapamycin complex 1 (mTORC1) regulates rates of protein synthesis in part through phosphorylation of p70S6K1 and 4E-BP1. Signaling through mTORC1 is necessary for the overloadinduced stimulation of muscle protein synthesis and subsequent hypertrophy. Regulated in Development and DNA Damage Response 1 (REDD1) is a repressor of mTORC1 signaling whose expression is downregulated following resistance exercise in humans. However, the contribution that REDD1 makes to the regulation of mTORC1 signaling following resistance exercise is unknown. The purpose of the present study was to investigate the role of REDD1 in regulating mTORC1 signaling following resistance exercise. Fasted C57BL/6 mice were

subjected to unilateral high frequency electrical stimulation of the sciatic nerve to induce eccentric contractions in the tibialis anterior. Thirty minutes post stimulation, phosphorylation of p70S6K1 (Thr389) was increased 619% despite no change in 4E-BP1 (Ser65) phosphorylation relative to the contralateral non-stimulated control leg. Four hours post stimulation, phosphorylation of p70S6K1 remained elevated while phosphorylation of 4E-BP1 increased 221% relative to the nonstimulated leg. REDD1 expression was inversely proportional to mTORC1 signaling and was reduced to 83% and 61% of the non-stimulated control value at 30 min and 4 h post stimulation, respectively. Feeding prior to stimulation repressed REDD1 protein expression 71% in non-stimulated muscle relative to fasted non-stimulated muscle concomitant with a 188% increase in 4E-BP1 phosphorylation 4 hours post stimulation. Similar to the effect in fasted mice, electrical stimulation in the fed condition both reduced REDD1 expression and enhanced 4E-BP1 phosphorylation compared to the non-stimulated leg. Overall, the results are consistent with a model in which resistance exercise induced repression of REDD1 expression acts in a permissive manner to promote mTORC1 activation. (supported by grants DK13499 (LSJ) and AA011290 (CHL))

41. Ca2+/Calmodulin-Dependent Protein

Kinase Kinase (CaMKK) is Sufficient, But Not Necessary, for Growth in Mouse

Skeletal Muscle Jeremie L.A. Ferey1-5, J. Matthew Hinkley1-5, Cheryl A.S. Smith1-5, Jeffrey J. Brault1-5 and

Carol A. Witczak1-5 Departments of 1Kinesiology, 2Physiology,

and 3Biochemistry & Molecular Biology, 4Brody School of Medicine, and the 5East Carolina University Diabetes & Obesity

Institute, East Carolina University, Greenville, NC 27834

Intracellular Ca2+ is a key regulator of skeletal muscle growth, yet the Ca2+-

sensitive proteins that regulate this process are largely unknown. Ca2+/calmodulin-

dependent protein kinase kinase

(CaMKK) is a Ca2+-activated, serine/threonine kinase; and previous work

has shown that CaMKK protein expression is increased in mouse muscle following 7 days of muscle overload (growth). To date, no studies have examined whether

CaMKK is a positive and/or crucial regulator of growth in skeletal muscle. AIMS: To determine if activation of

CaMKK signaling can stimulate muscle

growth, and if so whether CaMKK is essential for this process. METHODS AND RESULTS: Growth was induced in mouse plantaris muscle via unilateral ablation of synergist muscles (overload). Muscles were removed 1-, 3-, 7- or 10-days later, and immunoblots performed. Overload time-

dependently increased CaMKK protein levels (5-fold at day 1, up to 9-fold at day 10), demonstrating an early increase in

CaMKKduring muscle growth. To

determine if specific activation of CaMKK signaling is sufficient to stimulate muscle growth, mouse muscles were transfected with plasmid DNA containing constitutively

active CaMKK or empty vector using in vivo electroporation. Active

CaMKKexpression increased muscle weight (10-15%), protein content (~10%), protein synthesis rates (~2-fold); and stimulated key intracellular signaling proteins that regulate muscle protein synthesis (i.e. mTOR, p70S6K, 4E-BP1). These results demonstrate that activation of

CaMKKsignaling can stimulate muscle

growth. To determine if CaMKK is essential for growth, plantaris muscles from

CaMKK knockout mice were stimulated to hypertrophy via unilateral overload.

Surprisingly, muscles from CaMKK knockout mice exhibited significantly greater growth (~15%) compared to wild-type mice,

demonstrating that CaMKK is not essential for overload-induced muscle growth. CONCLUSION: Collectively, the results of this study demonstrate that activation of

CaMKK signaling is sufficient but not

necessary for growth in mouse skeletal muscle. SUPPORT: NIH R00AR056298, ECU start-up funds.

42. Mechanosensitivity May Be

Enhanced In Skeletal Muscles of Spinal Cord Injured (SCI) vs. Able-Bodied Men

(AB) Ceren Yarar-Fisher1,2, C Scott Bickel1,3, Samuel T Windham4, Neil A Kelly1,2, and

Marcas M Bamman1,2 1UAB Center for Exercise Medicine, and

Departments of 2Cell, Developmental and Integrative Biology, 3Physical Therapy, and

4Surgery, University of Alabama at Birmingham 35294.

Introduction: Neuromuscular electrical stimulation induced resistance training (NMES-RE) promotes robust muscle hypertrophy in individuals with spinal cord injury (SCI) and, in fact, we have found the relative muscle mass gains with NMES-RE in SCI exceed gains in able-bodied (AB) subjects undergoing traditional resistance training. The mechanisms by which these remarkable improvements occur in paralyzed muscle remain unclear. Objective: To determine the effects of an acute bout of NMES-RE on intracellular signaling pathways involved in translation initiation and mechanical loading induced muscle hypertrophy in 8 SCI vs. 9 AB men. Methods: All subjects performed ≈90 NMES-RE isometric contractions of the quadriceps at 30% maximum force with 100% activation of the vastus lateralis (VL). VL muscle biopsies were collected pre-, 10 min post-, and 60 min post-NMES-RE. Western blots were performed to determine the total and phosphorylated protein levels of Akt, Erk, FAK, GSK-3β, S6K1, 4EBP-1 and RPS6. Results: Main group effects revealed higher phosphorylation of Erk, FAK, GSK-3β, and RPS6 (p< 0.05) in SCI. Akt, Erk, and RPS6 phosphorylation robustly increased (p< 0.05) following NMES-RE in the SCI group only. Both groups demonstrated increased S6K1 phosphorylation following NMES-RE.

Conclusion: SCI muscle seems to be highly sensitive and more responsive than AB to acute NMES-RE even 22 years after the onset of injury. Heightened signaling associated with muscle mechanosensitivity and translation initiation in SCI muscle may be an attempted compensatory response to offset elevated protein degradation in atrophied SCI muscle.

43. Fiber Type-Specific Satellite Cell

Response to Aerobic Training in Sedentary Adults

Christopher S. Fry, Brian Noehren, Jyothi Mula, Margo F. Ubele, Philip A. Kern,

Charlotte A. Peterson University of Kentucky

In the current study, we sought to determine the effect of a traditional, 12-week aerobic training protocol on skeletal muscle fiber type distribution and satellite cell content in sedentary subjects. Muscle biopsies were obtained from the vastus lateralis (n=23 [6M; 17F]; BMI: 30.7±1.2 kg/m2) before and after 12 weeks of aerobic training performed on a cycle ergometer. Immunohistochemical analyses were used to quantify myosin heavy chain (MyHC) isoform expression, cross-sectional area (CSA) and satellite cell and myonuclear content. Following training, a decrease in MyHC hybrid type IIa/IIx fiber frequency occurred, with a concomitant increase in pure MyHC type IIa fibers. Pre-training fiber type correlated with BMI, and the change in fiber type following training was associated with improvements in maximal oxygen consumption. Twelve weeks of aerobic training also induced increases in mean CSA in both MyHC type I and IIa fibers. Satellite cell content was also increased following training, specifically in MyHC type I fibers, with no change in the number of satellite cells associated with MyHC type II fibers. With the increased satellite cell content following training, an increase in myonuclear number per fiber also occurred in MyHC type I fibers. Hypertrophy of MyHC type II fibers occurred

without detectable myonuclear addition, suggesting that mechanisms underlying growth in fast and slow fibers differ. These data provide intriguing evidence for a fiber type-specific role of satellite cells in muscle adaptation following aerobic training.

44. Sprint Exercise and Myoblast Proliferation

Heléne Fischer, Seher Alam, Barbara Norman, Håkan Rundqvist, Andreas

Montelius, Mona Esbjörnsson, Eva Jansson Department of Laboratory Medicine,

Division of Clinical Physiology, Karolinska Institutet, Stockholm, Sweden

Systemic factors as well as local intracellular factors seem to be important for proliferation and differentiation of SaCs. However, the influence of systemic and/or local factors on the function of the human SaCs is largely unknown. Hence the aim was to study proliferation of cultured SaCs /myoblasts in medium supplemented with serum from subjects performing sprint exercise in addition to the effect of sprint exercise on markers for proliferation in skeletal muscle tissue. Method: 18 subjects, 20 – 30 years old performed three 30 s all-out cycle sprints with 20 min rest in between each sprint. Sera were withdrawn from forearm vein at rest, 9 min after second and third sprint, and one and two hours after third sprint. SaCs were extracted from vastus lateralis biopsies from one young healthy male. Cells were cultured in medium containing 20% sera at different cell concentrations in triplicate and cell proliferation was measured with BrdU-ELISA kit. 14 subjects, 20-30 years, performed similar exercise as in the study above. Muscle biopsy samples were obtained at rest and 2 h after the third sprint. A global gene expression was conducted and special focus was paid to markers of cell proliferation and differentiation. Result: A significant decline was seen in cell proliferation rate in cells grown with sera obtained at rest, and up to two hours following the three bouts of sprint

exercise. In addition an increased gene expression of MIR-1 and CDKN1A (p21), both inhibiting myoblast proliferation, was observed in muscle tissue. Conclusion: Systemic factors may inhibit myoblast proliferation after sprint exercise but the observed increase in gene expression indicate that the local factors cannot be excluded.

45. G Protein Coupled Receptor 56 Is a Target Gene of The PGC-1α4 Isoform

And Regulates Overload-Induced Muscle Hypertrophy

J.P. White1, C.D. Wrann1, R.R. Rao1, Z. Wu2, D.J. Glass2, and B.M. Spiegelman1

1 Dana Farber Cancer Institute and Harvard Medical School, Department of Cell Biology,

Harvard University, USA 2 Novartis Institutes for Biomedical

Research, USA The PGC-1α isoform referred to as PGC-1α4 has recently been shown to promote muscle hypertrophy and attenuate muscle wasting. Gene array analysis has identified the G-protein coupled receptor 56 (GPR56) to be a transcriptional target of PGC-1α4. GPR56 is a newly defined adhesion receptor which can mediate both G protein coupled signalling and integrin function, although little in know about its function in skeletal muscle. We have found the anabolic effect of PGC-1α4 is, in part dependent on GPR56 signalling as knockdown of GPR56 attenuates PGC-1α4-induced muscle hypertrophy in vitro. Forced expression of GPR56 in primary myotubes results in mild hypertrophy. However, when forced expression of GPR56 is accompanied by the addition of its ligand, Collagen III, it drives robust myotube hypertrophy which is dependent on Gα12/13/Rho signalling. In vivo models of overload induced muscle hypertrophy increase GPR56 expression while GPR56 loss of function attenuates the hypertrophic response to mechanical overload. Lastly, we show muscle GPR56 expression is increased during human resistance

exercise. In conclusion, we have discovered a novel signaling pathway through GPR56 which regulates muscle hypertrophy associated with resistance/loading-type exercise.

46. Sepsis Antagonizes Muscle

Contraction-Induced Increases in mTOR Signaling

Jennifer L. Steiner, G. Deiter, M Navaratnarajah, CH Lang

Department of Cellular and Molecular Physiology, Penn State College of

Medicine, Hershey, PA, 17033. Skeletal muscle atrophy and weakness induced by sepsis or inflammation is due, at least in part, to a decrease in mTOR-mediated protein synthesis. Anabolic signaling to mTOR via nutrients and growth factors is impaired during sepsis preventing maximal stimulation of protein synthesis. Electrically stimulated muscle contraction is a potential clinical therapy to offset the sepsis-induced loss of muscle; however, whether the anabolic benefits of muscle contraction via mTOR signaling are maintained during sepsis is unknown. Cecal ligation and puncture was used to produce polymicrobial peritonitis in male C57BL/6 mice. Control animals (CON) underwent a similar procedure except the isolated cecum was neither ligated nor punctured. After 24 h, fasted mice were re-anesthetized and the right hindlimb was electrically stimulated via the sciatic nerve (10 sets of 6 contractions). The gastrocnemius muscle was collected 2 h after cessation of contractions. Sepsis increased mRNA expression of IL-6 (72-fold) and TNF-α (6.5-fold), while only TNF-α was increased by contraction in both CON (3-fold) and septic (1.6-fold) muscle. The contraction-induced phosphorylation of S6K1 Thr389 (67%), S6K1 Thr421/Ser424 (46%), ribosomal protein S6 (rpS6) Ser240/244 (54%) and 4E-BP1 Ser65 (85%) was blunted by sepsis. However, sepsis did not negatively modulate protein elongation, as phosphorylation of eEF2 Thr56 was similarly decreased by muscle

contraction in both CON and septic mice. mTOR-independent signaling appeared to be intact as phosphorylation of ERK Thr202/Tyr204 was similarly increased by muscle contraction in both CON and septic mice. Injection of the mTOR inhibitor, Torin2, in separate mice indicated contraction-induced increases in S6K1, rpS6, and 4E-BP1 were mTOR-mediated. These findings demonstrate that resistance to muscle contraction-induced anabolic signaling during sepsis is predominantly mTOR-dependent and that therapies to attenuate muscle atrophy may need to focus on restoring mTOR activation in response to anabolic stimuli.

47. Resistance Exercise Training (RET)

In ~70y Men Causes Adaptations In Bulk And Microvascular Blood Flow

Promoting Maintenance of a Bigger Muscle Mass - A Paradigm for Training

Effects on Myofibrillar Protein Synthesis (MPS).

Michael J Rennie, PhD FRSE, School of Life Sciences, University of Nottingham,

NG7 2RD. England. Confusion exists for many concerning MPS after RET exercise training. The confusion exists because despite reliable extant evidence (e.g. Tang et al, AJP 2009) which, when the exercise stimulus is sufficient shows a rise in fractional synthetic rate (FSR), but which wanes thereafter unless it is repeated within ~24 h or the %1RM is increased whereupon MPS will continue elevated with subsequent growth until some asymptotic maximum. Some coaches and athletes believe that while remaining strong they must be turning-over their muscle more rapidly and require more dietary-protein to maintain it. My collaborators and I studied 20 healthy men (10 untyrained 10 engaged in RET) ~72 y, BMI ~25 kg/m2 to investigate the effects of 20 w of RET on muscle mass (assessed by DXA, with conversion to true skeletal muscle (SM) mass after algorithms to “strip-out” non-SM lean tissue developed by S Heymsfield), rates of myofibrillar FSR

(MSR) in the post-absorptive and Glamin®-infused state, and both femoral arterial leg blood flow (FALBF; Doppler) and vastus lateralis microvascular blood volume (MBV) estimated by contrast-enhanced ultrasound (CEUS). The results showed a 39% rise in leg strength, small but significant increases (range -0.3-5%) in leg, appendicular and whole-body SM (ANOVA P<0.0001), but no difference in basal MSR at 0.47±0.05 %/h; RET did also not increase the elevation in MSR with AA feeding (to 0.072%/h). Nevertheless, whereas basal FALBF was unchanged by RET at 0.35±0.06 l/min, fed-state FALBF was significantly larger at 0.45±0.05 l/min; similarly, basal MBV was identical in untrained and RET men but MBV showed a 44% increase in response to feeding (P<0.001). The results demonstrate that with a bigger mass of muscle to maintain, the adaptive response is to retain the pre-training MSR but to service the bigger muscle mass by greater nutritive flow and AA delivery during feeding. I acknowledge my collaborators, principally K Sjøberg, J P Williams and S Heymsfield, the technical assistance of K Smith, B E Phillips, K Varadhan and M Limb and the financial support of The Dunhill Medical Trust.

48. Examining the Interindividual Early Hypertrophic Response to Resistance

Training Michael Stec and Marcas M Bamman

The University of Alabama at Birmingham

Resistance exercise training (RT) is the most effective stimulus for skeletal muscle regrowth from a number of muscle wasting diseases, as well as normal aging. However, the amount of muscle regrowth is highly variable between individuals, with some individuals having a poor hypertrophic response to long-term RT (i.e. nonresponders). In an effort to better elucidate the mechanisms regulating the diverse hypertrophic response to RT in humans, we are currently examining differences in mechanotransduction and

translation initiation signaling in whole tissue lysate and skeletal muscle satellite cells isolated from a cohort of sarcopenic, older (60-75 y) adults who performed RT for 4wks (3x/wk). Overall, our preliminary findings show that there was an early hypertrophic response among subjects in just 4wks of training, and that this response was quite variable between individuals (vastus lateralis muscle fiber area increased 3-35%). Satellite cells isolated from these subjects in the untrained, resting state were differentiated into myotubes and subjected to in vitro mechanical stretch. Protein synthesis following acute stretch was measured using the SUnSET technique, and mechanotransduction signaling is being assessed to determine if the mechanotransduction signaling and/or protein synthetic response of myotubes to acute mechanical stretch in vitro is predictive of an early myofiber hypertrophic response in vivo. Additionally, muscle tissue collected from subjects pre- and post-RT is being used to determine if changes in translational capacity (e.g. ribosome quantity) or putative mechanosensitive proteins (e.g. ERK, FAK) are related to the degree of hypertrophy in this relatively short training period. Overall, these experiments will help us better understand the early hypertrophic adaptations to short-term RT, as well as identify key molecular differences that may confer an advantage towards increasing muscle fiber size with only a few weeks of training.

49. The Acute Effect of Two Resistance Exercise Intensities with Equal Volume

Load on Skeletal Muscle mRNA Expression of Insulin-like Growth Factor-

1Ea (IGF-1Ea) and Mechano Growth Factor (MGF)

Neil A. Schwarz, Mike Spillane, Sarah K. McKinley, Thomas L. Andre, Joshua J.

Gann, & Darryn S. Willoughby Department of Health, Human Performance,

and Recreation, Baylor University, Box 97313, Waco, TX 76798, USA

The purpose of this study was to examine the acute effect of resistance exercise intensity on the mRNA expression of IGF-1Ea and MGF. In a randomized, uniform-balanced, cross-over design, 10 men [23.7 ± 0.9 years old (mean ± SE)] performed two separate lower-body resistance exercise sessions consisting of a lower-intensity protocol (50% of 1-RM) and a higher-intensity (80% of 1-RM) protocol with equal volume loads. Muscle samples were obtained at baseline, 45-min post-exercise (PE), 3-hr PE, 24-hr PE, and 48-hr PE. IGF-1Ea and MGF mRNA expression were determined using RT-PCR and normalized to β-actin. Two-way repeated-measures ANOVA were performed (p ≤ 0.05) with intensity and time as main effects. Additionally, paired-samples t tests were performed to compare delta scores (baseline expression subtracted from expression at each time point) at each time point between intensities. No significant main effects existed for intensity or time for IGF-1Ea and MGF (p > .05). However, a statistically significant interaction between intensity and time for IGF-1Ea, but not MGF, was observed (p < .05). A significant main effect for time was observed for IGF-1Ea mRNA expression for the higher-intensity session (24-hr PE > baseline; p < .05). Also, at 24-hr PE, IGF-1Ea mRNA expression was significantly greater after the higher-intensity session compared with the lower-intensity session (p < .01). No interaction between intensity and time was observed for MGF (p > .05). Delta scores were significantly greater for the higher-intensity session at 24-hr PE for IGF-1Ea (p < .05) and at 48-hr PE for MGF (p < .05). This study demonstrated differential responses of IGF-1Ea and MGF mRNA expression based on exercise intensity. Future research should determine if these differences exist at the translational level of expression and; furthermore, if these potential differences contribute to skeletal muscle training adaptations resulting from differing resistance exercise intensities.

50. ICAM-1: A Novel Mechanism by Which the Inflammatory Response

Augments Myogenesis Goh Q1, Dearth CL1, Awadia S2, Garcia-

Mata R2, Corbett JT1 and FX Pizza1 1Department of Kinesiology, University of

Toledo, Toledo, OH. 2Department of Biological Sciences, University of Toledo, Toledo, OH.

Recent investigations by our laboratory demonstrated that skeletal muscle cell expression of intercellular adhesion molecule-1 (ICAM-1), an important protein of the inflammatory response, augmented events of myogenesis in which myotubes are forming, adding nuclei, aligning, fusing, synthesizing proteins, and hypertrophying. Our current work extended these findings by incorporating pharmacological tools with an in vitro approach to discern underlying mechanisms associated with ICAM-1 mediated myogenesis. Antibody neutralization revealed the extracellular domain of ICAM-1 to be fundamentally important in augmenting homotypic adhesion of myoblasts, leading to myotube formation and myonuclear accretion. In contrast, inhibition of the cytoplasmic domain of ICAM-1 with a cell permeable peptide revealed its critical role in augmenting both myotube formation and myonuclear accretion, as well as myotube alignment and fusion, rates of protein synthesis, and overall myotube size. Peptide inhibition further demonstrated that the signal transducing function of the ICAM-1 cytoplasmic domain augmented events of myogenesis through mechanisms involving increased Rho GTPase (Rac1 and cdc42) and Akt/p70s6k activity. Taken together, our findings extend knowledge of the immunobiology of skeletal muscle cells by revealing a novel mechanism through which the inflammatory response facilitates myogenesis.

51. Lean Gain Is Enhanced by Administration of Leucine Pulses During

Long-term Continuous Feeding. Teresa A. Davis, Claire Boutry, Samer W. El-Kadi, Agus Suryawan, Julia Steinhoff-

Wagner, Barbara Stoll, Renan A Orellana, Hanh V. Nguyen, and Marta L. Fiorotto

USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor

College of Medicine, Houston, TX

Patients who are unable to feed orally can be nourished continuously by orogastric tube. We have previously shown in neonatal pigs that a leucine pulse administered during continuous feeding enhanced protein synthesis in skeletal muscle compared to continuous feeding alone for a 24 h period. Twenty-three piglets (7-d-old) were fed continuously a milk replacement formula via orogastric tube for 21 d with an additional parenteral infusion of either leucine (800 μmol•kg-1•h-1, LEU) or alanine (ALA) for 1 h every 4 h. At 21 d, the body weight was higher and the weight gain greater in LEU than in ALA pigs. The lean gain, determined by dual-energy X-ray absorptiometry (DEXA), was higher and the fat gain lower in the LEU than ALA group. Longissimus dorsi (LD), gastrocnemius and soleus muscles and kidney weights were heavier in LEU than ALA with no effect of treatment on liver, small intestine and heart weights. Fractional protein synthesis rate, phosphorylation of eukaryotic initiation factor (eIF) 4E binding protein 1 (4E-BP1), and formation of the active eIF4E•eIF4G complex in LD muscle were greater in the LEU than ALA group. In conclusion, administration of leucine pulses during 21 d of continuous feeding decreases fat gain and increases lean gain by stimulating the translation initiation pathway leading to protein synthesis in skeletal muscle of neonates. (NIH AR444474 and USDA/ARS 6250-51000-055)

52. Changes in HDAC Expressions in Response to Acute Heat Stress in Rat

Skeletal Muscle. Toshinori Yoshihara1,2, Ryo Kakigi3,

Takamasa Tsuzuki1,2, YuriTakamine1, Noriko Ichinoseki-Sekine1,4, Hisashi Naito1.

1School of Health and Sports Science, Juntendo University, Japan. 2JSPS

Research Fellow. 3School of Medicine, Juntendo University, Japan. 4Studies of

Living and Welfare, The Open University of Japan, Japan.

Purpose: To examine the effects of acute heat stress on histone deacetylases (HDACs) expression in rat skeletal muscle. Methods: Fourteen male Wistar rats (281.1± 6.6g) were divided into two groups: the non-heated group (CT, n=7)and the heated group(HS, n=7). Under anesthesia with pentobarbital sodium, both legs were immersed in hot water (41°C)for 30 min. The soleus and plantaris muscles in both legs were removed immediately after induction of heat stress. HDAC class I (HDAC1-3), II (HDAC4and5)and III (SIRT1)protein expressions in the muscles were determined by western blotting. Results: Cytoplasmic SIRT1 expression was significantly lower in both muscles in the HS than in the CT group (p<0.05). In the soleus muscle, nuclear HDAC1expressionwas significantly lower in the HS group than in the CT group. Although cytoplasmic HDAC2 expression in the HS group was significantly lower than that in the CT group, nuclear HDAC2 expression tended to be higher in the HS group (p=0.053). While, in the plantaris muscle, no significant change was observed in the cytoplasmic and nuclear HDAC 1 and 2 between the CT and HS groups. There were no significant differences in the level of HDAC3, 4 and 5 expressions in both the soleus and plantaris muscles between the two groups. Conclusion: Acute heat stress alters HDAC protein expression in the cytoplasm and nucleus in rat skeletal muscle; however, these alternations may be more sensitive in the soleus muscle.

53. Myonuclear Transcriptional Output During Muscle Hypertrophy in the Absence of Satellite Cell Fusion

Tyler J. Kirby1, Christopher S. Fry2, Janna R. Jackson2, Thomas Chaillou1, Charlotte A.

Peterson2, and John J. McCarthy1 1Department of Physiology, School of

Medicine, University of Kentucky 2Department of Rehabilitation Sciences, College of Health Sciences, University of

Kentucky, Lexington, KY 40536 We have previously demonstrated that acute hypertrophy of skeletal muscle can occur independently of satellite cell fusion; however, the compensatory mechanism that allows this to occur remains unidentified. Protein synthetic pathways and total protein content are similar between hypertrophied muscles with and without satellite cell fusion indicating that it does not appear to be driven by translational output. Furthermore, total RNA content is the same between muscles, even though DNA content is less in the absence of satellite cell fusion. Therefore, we postulate that in the absence of satellite cell fusion, individual myonuclei increase transcriptional output in order to maintain RNA levels and ultimately, growth of the muscle. Using our Pax7-DTA mouse strain, we conditionally depleted satellite cells upon administration of tamoxifen, which resulted in the absence of satellite cell fusion and an increase in myonuclear domain. Using this model, we hope to identify the mechanism with which the myonuclei of tamoxifen-treated mice “sense” an increase in myonuclear domain and respond by increasing transcriptional output. Preliminary analysis demonstrates that both the RNA:DNA ratio and RNA content per myonucleus is greater in tamoxifen-treated mice, which is accompanied by an increase in the overall length of the myonuclei. The significance of these nuclear morphometric changes are unclear, however we speculate that this may be indicative of increased transcriptional output, specifically ribosomal RNA. To test this hypothesis, we pulsed mice with 5-ethynyl-uridine to label newly

synthesized RNA, along with examining levels of pre-47s rRNA, following a period of muscle hypertrophy. Both 5-ethynyl-uridine and pre-47S levels appear to be greater in tamoxifen-treated mice, suggesting that in the absence of satellite cell fusion, resident myonuclei can increase in rRNA transcription in order to maintain hypertrophic growth.

Muscle Diseases and Regeneration

54. Generating a Gene Therapy Vector for Myotubular Myopathy

Angela L. McCall, Denise Cloutier, Jeffry Kelley, Meghan Soustek, Darin Falk, Nathalie Clément and Barry Byrne.

Department of Pediatrics, College of Medicine, University of Florida

X-linked myotubular myopathy (XLMTM) is caused by a deficiency in myotubularin 1 (MTM1), and affects 1 in 50,000 males. Patients with XLMTM often present with severe hypotonia, muscle weakness, and poor respiratory function, which lead to a life expectancy of one year. MTM1 plays a role in the regulation of endosomal trafficking and cytoskeletal organization by directly controlling the quantity of signaling molecules. Currently, patients with XLMTM receive palliative care as there is no FDA approved treatment. Initial studies have already demonstrated promise for adenoassociated virus (AAV)-based gene therapy strategies in treating MTM1 deficiency; however, cardiotoxicity due to overexpression of MTM1 has been observed in mice. A 1.3Kb region upstream of the human MTM1 gene has been identified as the endogenous human promoter (MTMPro). We confirmed the location of the promoter by measuring its ability to express a luciferase reporter (MTMPro-luciferase) in HEK293 cells. We next compared MTMPro expression levels to DES and the GAPDH promoter by luciferase activity assay in HEK293 cells and murine myoblasts. We found MTMProluciferase has similar expression as

GAPDH-Luciferase in HEK293 cells and expression compared to DES-Luciferase is cell type specific. 129SVE mice were administered an intramuscular injection of either rAAV2/8- or rAAV2/9- DES-MTM1 into the tibialis anterior. Western blot analysis and MTM1 activity assay revealed MTM1 was expressed and functional in injected muscle 1 month post-injection with no observed toxicity. We believe that using either the MTMPro or DES to drive MTM1 expression in an rAAV2/9 vector for XLMTM will result in therapeutic levels of gene expression without causing cellular toxicity.

55. Estradiol Alters the Expression of Key Transcription Factors that Regulate

Skeletal Muscle Regeneration Brittany C. Collins, Tara L. Mader, Coco Le,

Gordon L. Warren, and Dawn A. Lowe. University of Minnesota, Minneapolis, MN

Muscle regeneration is necessary for muscle to repair itself following injury. The regenerative capacity of muscle is partially due to the role of satellite cells and their ability to proliferate and differentiate into muscle fibers. Certain aspects of this myogenic response have been shown to be estrogen responsive. Moreover, we have previously shown that with estrogen deficiency the functional recovery of skeletal muscle is impaired. Purpose: To test the hypothesis that estrogen treatment would give a more robust myogenic response following injury compared to estrogen depletion. Methods: Adult, female C57BL/6 mice (n=40) were ovariectomized and then randomly assigned to no treatment or implantation of a 17-β estradiol time-release pellet. Three weeks following surgery, tibialis anterior (TA) muscles of both hindlimbs were injured via a freeze injury and mice then were allowed to recover for either 1, 2, 3, 4, 5, 7, or 10 days. Gene expression levels of key transcription factors involved in muscle regeneration were assessed in TA muscles by qPCR. Results: Pax7, a marker of satellite cells, was 48%

greater in estradiol-treated mice compared to estradiol-deficient mice 5, 7, and 10 days post injury (p≤0.05). MyoD1, an indicator of satellite cell activation, was not different between estradiol-treated and -deficient mice across all time points post injury (p≥0.06). Myogenin, a marker for muscle differentiation, was 84% greater in estradiol-treated mice compared to -deficient mice 7 days post injury (p≤0.01). Conclusion: Estradiol treatment to ovariectomized mice significantly increased transcription factors related to satellite cell expression and differentiation in response to injury in skeletal muscle. However, it is possible that signaling through myonuclei also contributes to the increase in differentiation signaling observed. We predict that estradiol is working through estrogen receptors in order to facilitate these signals in skeletal muscle and that the end result is improved functional recovery. 56. Changes in Skeletal Muscle Structure

and Function Following Genetic Inactivation of Myostatin in Rats

Christopher L Mendias, Evan B Lynch, Jonathan P Gumucio.

University of Michigan, Ann Arbor Myostatin (GDF-8) is a negative regulator of skeletal muscle mass. Inactivation of myostatin in mice causes increased muscle size and isometric force production (Po), but a decrease in specific force (sPo). There have been limited analyses of the impact of myostatin on the mechanics and biochemistry of muscles from organisms other than mice. We sought to evaluate the effect of myostatin deficiency on the structure and function of skeletal muscles from rats with a targeted inactivation of myostatin. We hypothesized that, compared to controls (MSTN∆/∆) rats in which the myostatin gene was inactivated using zinc-finger nucleases MSTN∆/∆) would exhibit an increase in muscle mass and Po, a reduction in sPo, and an increase in type II fibers. MSTN∆/∆ animals had a 21% increase in body mass, and a 37% and 45% increase in fibers per muscle and a 32% and a 19% increase in mass for the EDL and soleus,

respectively. EDL and soleus muscles from MSTN∆/∆ animals also displayed a 57% and 20% increase in Po while exhibiting a 31% and 21% increase in PCSA, respectively, which led to no substantive changes in sPo. EDL fiber area was increased in MSTN∆/∆ animals in all type II fibers and hybrid fibers. Soleus fiber area was not significantly increased in either type I or type IIA fibers, however in both the EDL and soleus, MSTN∆/∆ animals displayed a decrease in the percentage of type I and IIA/IIX fibers. MSTN∆/∆ animals had distinct expression profiles for several mRNAs and miRNAs involved in atrophy, ECM remodeling and lipid synthesis. The soleus muscles of MSTN∆/∆ rats had increased levels of phosphorylated GSK3β, IGF1R and mTOR compared to controls. The results from this study indicate there are species-specific consequences of myostatin inactivation, and further examination into the physiology of MSTN∆/∆ rats is warranted.

57. Neuronal and Neuromuscular

Junction Pathology in Pompe Disease Darin J. Falk, PhD1,2, A. Gary Todd1,2,

Sooyeon Lee3, Meghan S. Soustek1,2, Robin Yoon1, David D. Fuller4, Lucia Notterpek3, Barry J. Byrne1,2,

1Department of Pediatrics, 2Powell Gene Therapy Center, 3Department of

Neuroscience, 4Department of Physical Therapy, University of Florida, Gainesville,

FL 32610 Pompe disease is a neuromuscular disorder defined by lack of acid-alpha glucosidase (GAA) and characterized by the systemic depletion of glycogen resulting in ubiquitous glycogen accumulation. Respiratory and ambulatory dysfunction are prominent features in patients with Pompe yet the mechanism defining the development of muscle weakness is currently unclear. Transgenic animal models of Pompe disease mirroring the patient phenotype have been invaluable in mechanistic and therapeutic study. Here, we demonstrate significant pathogenesis in the neuromuscular junction (NMJ) of the

diaphragm and tibialis anterior muscle as a prominent feature of disease pathology. Post-synaptic defects including increase in motor endplate area and fragmentation were readily observed in Gaa knockout mice. Pre-synaptic neuropathic changes were also evident, demonstrated by significant reduction in neurofilament-heavy chain and increased G-ratio within the sciatic and phrenic nerve. Our data suggest for the first time that loss of NMJ integrity are primary contributors to the decline in respiratory and ambulatory function and arise from both pre- and post-synaptic pathology. These observations highlight the importance of systemic correction, particularly restoration of Gaa to skeletal muscle and the central nervous system for treatment of Pompe disease.

58. Cellular and Extra-Cellular Responses to Mechanical Overload in

Tissue Engineered Skeletal Muscle

DJ Player1, HC Stobbs

1, NRW Martin

1, MP

Lewis1 .

1Musculoskeletal Biology Research Group, School of Sport, Exercise and Health Sciences, Loughborough University,

Loughborough, Leicestershire, United Kingdom

Introduction: Mechanical forces play a pivotal role in both physiological and pathophysiological adaptation of skeletal muscle. Modelling situations of skeletal muscle overload with control at the cellular level, is problematic in vivo. This has led to the development of ex vivo or in vitro models, to investigate the biomechanical responses of skeletal muscle cells. These models do not account for the transmission of forces in three dimensions (3D) and often forbid the investigation of cell-matrix interactions. The aim of the current investigation was to examine the mechanical responses of tissue engineered cellular and a-cellular skeletal muscle constructs, to mechanical overload. Methods: Skeletal muscle constructs were

prepared and tethered to the t-CFM as previously described (Player et al., 2014). A-cellular constructs were mechanically overloaded to test strain (20, 40 and 60%) and velocity (0.72, 1.8 and 3.6 mm.s-1) components. Cellular and a-cellular constructs were overloaded at 20% strain to investigate cell-matrix differences and interactions. Results: A-cellular experiments revealed a differential stress relaxation (SR) response to the three strains applied, whereby greater SR responses were evident at higher (60%) and lower (20%) strains (no main effect, p>0.05). The velocity at which the strain was applied to a-cellular constructs, did not impact upon inherent tissue resistance, or SR (both p> 0.05). These data suggest strain has a more significant impact on extra-cellular matrix mechanics in this system. Tissue resistance was greater in a-cellular compared to cellular constructs (p = 0.024), with a greater SR response in a-cellular constructs (p = 0.1853), providing evidence for a cellular tensile resistance in response to the applied load. Conclusions: These data demonstrate the extra-cellular and cellular responses to overload in tissue engineered skeletal muscle. Furthermore, the model presented herein allows for the investigation of mechanical forces transmitted in 3D, an advancement on previously published systems.

59. Forelimb Neuromuscular Plasticity After Cervical Spinal Cord Injury in the

Rat Gonzalez-Rothi EJ1, Fitzpatrick G1,

Armstrong GT1, Reier PJ2, Lane MA2, Fuller DD1.

1Department of Physical Therapy,

3Department of Neuroscience, 2 McKnight

Brain Institute, University of Florida

Impaired upper extremity function is a major consequence of cervical spinal cord injury (cSCI). While some capacity for spontaneous recovery of the upper extremities has been demonstrated, the extent of this recovery is limited, and the

underlying mechanisms have not been clearly defined. Accordingly, we assessed both functional and muscular changes in the forelimbs following experimental cSCI (lateral C2 spinal cord hemisection - C2Hx). Gross forelimb motor function was assessed prior to, and weekly, up to 8-weeks post-injury using the limb-use asymmetry (cylinder) test and the Forelimb Locomotor Scale (FLS). Muscle wet weight was assessed and immunohistochemical techniques were used to quantify changes in muscle fiber cross sectional area (CSA) following C2Hx. At 1-week post-C2Hx, a dramatic reduction in ipsilateral forelimb use was observed 14% of pre-injury), average muscle muscle wet weight is reduced (81% of pre-injury), and average muscle fiber CSA is reduced (90% of pre-injury – all fiber types). By 8-weeks post-C2Hx, modest gains in ipsilateral forelimb use were evident (55% of pre-injury), average muscle wet weight was restored (100% of pre-injury), however no gains in average muscle fiber CSA were observed (89% of pre-injury). In parallel studies, we examined the neural circuitry innervating the forelimb using transysnaptic tracing methods. These studies identified a robust population of pre-motor interneurons associated with the forelimb motor circuitry that may represent a potential substrate by which the observed functional plasticity may occur. These results indicate considerable functional recovery and plasticity of the upper limbs following chronic cSCI. The pattern of forelimb recovery appears to parallel the previously documented recovery of ipsilateral respiratory (phrenic) motor output following C2Hx.

60. Satellite Cells Expand in Muscle of Pancreatic Cancer Patients

Erin E. Talbert1, Mark Bloomston1, Ericka Haverick1, and Denis C. Guttridge1,2.

1Arthur G. James Comprehensive Cancer

Center and the 2Department of Molecular Virology, Immunology, and Medical

Genetics, Translational Therapeutics Program, and The Ohio State

University, Columbus, OH, 43210, USA Cancer-induced weight loss, termed cachexia, is one of the most profound side effects of advanced cancer. Cancer cachexia results from losses in adipose tissue and skeletal muscle, and losses of skeletal muscle are associated with decreased patient survival and quality of life. Pancreatic cancer patients have amongst the highest incidences of cachexia, with perhaps one quarter of all pancreatic cancer deaths resulting from respiratory insufficiency associated with muscle wasting. Recent work from our laboratory has demonstrated that satellite cells expand in multiple mouse models of cancer cachexia and that the increase in the satellite cell protein Pax7 contributes to cancer-induced muscle wasting. To understand if a similar regulation occurs in humans, mononuclear cells were isolated from rectus abdominis muscle biopsies taken from pancreatic cancer patients undergoing tumor resection. CD31- and CD45-positive cells were excluded to eliminate blood vessel and bone-marrow associated stem cells, and the percentage of CD56+ cells was calculated from the remaining live-cell population. Our results demonstrate a significant relationship between the percentage of weight lost by pancreatic cancer patients and the number of satellite cells identified by FACS analysis. These data closely mirror our data generated from animal models of cachexia and suggest that dysfunctional muscle regeneration may contribute to muscle wasting in pancreatic cancer patients.

61. AAV9 Improves Hallmarks of Neuromuscular Junction Deterioration and Physiological Function in Pompe

Disease Todd A.G.1,2, McElroy, J.A.1,2, Grange R.W., Fuller D.D.3, Walter G.A.4, Byrne B.J.1,2 and

Falk D.J1,2. 1Department of Pediatrics, 2Powell Gene Therapy Center, 3Department of Physical Therapy, 4Department of Physiology and

Functional Genomics, University of Florida, Gainesville, FL 32610

Pompe disease is a glycogen storage disorder resulting from systemic loss of the glycogen metabolizing enzyme acid α-glucosidase (GAA). Affected patients and animal models characteristically present with progressive muscle weakness and ultimately succumb to cardiorespiratory failure. Recent evidence highlights both pre-and post- synaptic pathology are likely to contribute to reduced muscle activation and highlight important targets for therapeutic intervention. In this study we demonstrate significant elevated expression of acetylcholine receptor subunits within the tibialis anterior muscle (TA) is an early stage event in Pompe disease. Significant elevation of α- and γ-subunits are evident at 3 months of age in Gaa-/- (2.2 ± 0.36 and 2.3 ± 0.24 fold-change respectively relative to wild-type, p<0.0125) persisting through 6 months of age when elevation of the δ subunit also becomes significant (7.5 ± 2.0 fold change relative to wild- type p<0.0125). Elevated expression correlates with decreased functional performance demonstrated by significantly impaired peak torque generation in 6-month-old Gaa-/- mice compared to WT (10.9 ± 0.8 mN/g bodyweight (Gaa-/-): 15.5 ± 0.94 mN/g bodyweight (WT) p<0.001). Moreover, we demonstrate that early direct intramuscular injection within the TA of Gaa-/- mice with AAV9-CMV-GAA significantly increases muscle size (Gaa-/-+AAV9 42.15 ± 1.3mg; Gaa-/- 23.35 ± 1.4 mg), decreases the expression of α, δ and γ subunits, and improves torque generation (12.12 ± 2.071 mN/g bodyweight (Gaa-/-+AAV9)) of the

muscle to levels comparable to wild-type at 5 months post-injection. Our data supports AAV9 as a candidate serotype to improve physiological performance in Pompe disease and suggests early elevation in gene expression indicative of neuromuscular junction abnormalities can be corrected using AAV gene therapy.

62. Deficiency in Lipin 1-mediated Phosphatidic Acid Phosphohydrolase

Activity is Associated with Rhabdomyolysis in Humans and Skeletal

Myopathy in Mice George G Schweitzer1, Sara L Collier1,

Kyle S McCommis1, Zhouji Chen1, Kari T Chambers1, Thurl E Harris2, Alan

Pestronk1, Brian N Finck1 1Washington University in St. Louis School of Medicine, St. Louis, MO, 2University of Virginia, Department of Pharmacology,

Charlottesville, VA Mutations in the human gene encoding lipin 1 are a common cause of recurrent rhabdomyolysis, especially in children. The rhabdomyolysis episodes, in which skeletal muscle fibers undergo necrosis, are most commonly precipitated by fever. Lipin 1 is a bi-functional intracellular protein that regulates metabolism at two levels: a lipid phosphatase that dephosphorylates phosphatidic acid to form diacylglycerol (PA phosphohydrolase; PAP) and as a nuclear transcriptional coregulatory protein. However, the relationship of these functions to the causes of the episodic rhabdomyolysis are unknown. Using human lipin 1 cDNA as a template, various mutations associated with rhabdomyolysis in humans (L635P, R725H, and E766-S838_del) were cloned into expression vectors and transfected into HEK293 cells. Lipin 1 L635P and E766-S838_del proteins were not well-expressed. In contrast, lipin 1 R725H was well-expressed, retained transcriptional co-regulatory activity, but lacked PAP activity, likely due to the location of the non-conserved substitution in a key PAP regulatory domain. We also

developed mice expressing a truncated, hypomorphic lipin 1 protein that lacks PAP activity but that retains transcriptional regulatory function in a muscle-specific manner using Cre/LoxP methodology (MCK-Lpin1-/- mice). MCK-Lpin1-/- mice were overtly normal but muscle histochemistry showed an active and ongoing myopathy with necrotic and regenerating muscle fibers, and chronic pathology including many centrally nucleated muscle fibers. Quantitative RT-PCR detected increased expression of genes encoding macrophage markers, pro-inflammatory cytokines, as well as markers of necrosis and myofiber regeneration in MCK-Lpin1-/- muscle versus WT control. Compared to WT muscle, MCK-Lpin1-/- muscle also had increased PA and diacylglycerol content. We conclude that deficiency of lipin 1-mediated PAP activity can produce an active and chronic myopathy in mice in a myocyte-autonomous manner. The relationship between the active, ongoing myopathy in mice caused by the hypomorphic lipin 1 protein (lacking PAP activity) and the episodic rhabdomyolysis in children remains to be defined.

63. Developing a New Resistance Running Wheel System for Mice

Rodden GR, Stylianos K, Doering JA, McMillan RP, Frisard MI, and Grange RW.

Department of Human Nutrition, Foods, and Exercise at Virginia Polytechnic Institute

and State University, Blacksburg, VA Running wheels with no imposed resistance are extensively used in rodent exercise studies. This approach is suitable for studying endurance but not resistance training adaptations. In addition, the amount of work performed is not determined. We designed a computer-controlled system to provide graded wheel resistance to calculate work (resistance-force x distance). Herein, we describe a pilot study to test our system. Eleven C57BL/6 mice had free access to running wheels. After initial system troubleshooting, mice were grouped

to run for 6 weeks at one of three resistance loads (% body mass (BM)): low resistance (LR; ~2%; n=3), moderate resistance (MR; ~16%; n= 6) or high resistance (HR; ~25%; n=2; excluded from statistical analyses below). After 6 weeks at constant load, there were no differences in BM or food intake between MR & LR. The MR group increased distance/day by 503% (1043±601 to 6392±1266 m/day; p= 0.0154) and work/day by 496% (46.6±26.8 to 278±55.9 J/day; p= 0.0155), while these variables did not change for the LR group (Distance: 12680±806 to 11853±2755 m/day (p= 0.7336); Work: 62.2±3.5 to 58.1±13.5 J/day (p= 0.7336)). Furthermore, 6-week total work for the MR group (8788±1372J) was greater than the LR group (3848±642J) (p=0.0140). However, during the 6-week training period, maximal hindlimb in-vivo plantar flexor torque did not change for either group. Absence of increased plantar flexor torque despite the increased work output for the MR group was surprising. Potential explanations are that sample sizes were small, the exercise stimulus and/or training duration were insufficient, or opposing endurance and strength signal pathways minimized strength adaptation. In a follow-up study, we will increase group size, imposed work load, study duration, and will explore both endurance and strength signaling pathways. Our ultimate aim for the system is to study the effects of resistance-like training in mouse disease models.

64. Performance on Upper Limb Functional Tests in Control and DMD

Subjects Arora, H1, Willcocks, RJ1, Lott, DJ1,

Senesac, CR1, Forbes, SC1, Bendixen, R.M.3, Harrington, A.T.4, Walter, GA2 &

Vandenborne, K1. 1Physical Therapy, 2Physiology and

Functional Genomics, University of Florida, Gainesville, Florida, 3Children’s Hospital of Philadelphia, Philadelphia, PA, 4School of

Health and Rehabilitation Sciences, University of Pittsburgh, PA

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder, caused by mutation in the dystrophin gene resulting in the lack of dystrophin protein. Weakness causes loss of ambulation in late childhood. A number of studies examined lower limb function, but few studies have examined upper limb function in nonambulatory boys with DMD. The objective of this study is to explore the potential of upper limb function tests for capturing functional deficits in DMD. Both DMD (n = 10) and control (n = 5) subjects were 5-14 years males. They performed four functional tests: Brooke scale, Performance of Upper Limb test (PUL), Motor Function Measure (MFM), and Jebsen-Taylor Hand Function Test (JTHFT). They also completed grip and pinch strength testing. The data were analyzed using Mann Whitney nonparametric tests. Significant differences between DMD and control subjects were found in grip strength, pinch, three point, and key pinch grips (p=0.018). There were significant differences found in Total PUL Score (p=0.005) and Total MFM Score (p=0.024). In PUL, lifting weights (p=0.024) and lifting heavy cans (p=0.009) and in Jebsen’s test, picking up objects (p = 0.029) and lifting full cans (p= 0.029) showed significant differences. No statistical differences were found in functional grade on Brooke scale. Upper limb functional and strength tests show functional level differences between control and DMD subjects, and might be useful for tracking disease progression over time in DMD subjects.

65. Myoplasticity-Related Gene Expression in the Diaphragm Following

Cervical Spinal Cord Injury HH Ross, LC Gill, EJ Gonzalez-Rothi, AR

Judge, DD Fuller. University of Florida, College of Public Health and Health

Professions A cervical (C2) hemilesion (C2Hx), which disrupts ipsilateral bulbospinal inputs to the phrenic nucleus, was used to study diaphragm plasticity after acute spinal cord injury. We hypothesized that C2Hx would result in rapid atrophy of the ipsilateral hemidiaphragm and increases in mRNA expression of proteolytic biomarkers. Diaphragm tissue was harvested from male Sprague-Dawley rats at 1 or 7 days following C2Hx. Histological analysis demonstrated a reduction in cross-sectional area (CSA) of type I and IIa fibers in the ipsilateral hemidiaphragm at 1 but not 7 days post-injury. Type IIb/x fibers, however, had reduced CSA at both 1 and 7 days. To examine the mechanisms of C2Hx-induced diaphragm atrophy, a targeted gene array was used to screen mRNA changes for genes associated with skeletal muscle myopathy and myogenesis; this was followed by qRT-PCR validation of single genes of interest. The gene arrays reflected changes in diaphragm gene expression that suggested profound myoplasticity is initiated immediately following C2Hx including activation of both proteolytic and myogenic pathways. qRT-PCR validation confirmed significant increases markers of atrophy (e.g. MuRF1, Atrogin-1), local proteolysis (e.g. Caspase 3, Calpain 3), myogenesis markers (e.g. Myogenin) and markers of neuromuscular junction plasticity (e.g. MuSK, Agrin). We conclude that an immediate myoplastic response occurs in the diaphragm after C2Hx with atrophy occurring in ipsilateral myofibers within 1 day.

66. Exercise Training Improves Capillary Architecture via Enhancing

VEGF/VEGFR and Angiopoietins/Tie2 Signaling Pathways in Type 2 Diabetic

Muscle Hidemi Fujino 1, Hiroyo Kondo2, Shinichiro

Murakami3, Masayuki Tanaka1, Miho Kanazashi1, Fumiko Nagatomo4, Akihiko

Ishihara4, and Roland R. Roy 5. 1Department of Rehabilitation Science,

Kobe University Graduate School of Health Sciences, Kobe, Japan; 2Department of Food Sciences and Nutrition, Nagoya Women’s University, Nagoya, Japan;

3Department of Physical Therapy, Himeji Dokkyo University, Himeji, Japan;

4Laboratory of Cell Biology and Life Science, Graduate School of Human and Environmental Studies, Kyoto University, Kyoto, Japan; 5 Brain Research Institute

and Department of Integrative Biology and Physiology, University of California, Los

Angeles, California Type 2 Diabetes (T2DM) is associated with impairment of angiogenesis such as a reduction of capillary formation in skeletal muscle. Exercise has been shown to be an effective non-pharmacological treatment in the prevention of diabetes-induced angio-complications. Therefore, this study determined the effects of exercise training on the three-dimensional capillary structural changes and the associated pro- and anti-angiogenic factors in the soleus muscle of T2DM rats. Male rats were studied: sedentary non-diabetic Wistar (Con), exercised non-diabetic control (Ex), sedentary Goto-Kakizaki diabetic (GK), and exercised GK (GK+Ex) groups. Rats in the Ex and GK+Ex were subjected to running on a treadmill for 3 weeks. Although there were no differences in the capillary-to-fiber ratio among the four groups, mean capillary volume and diameter were lower in the GK compared to all other groups. On the contrary, mean capillary volume and diameter were higher in the Ex and GK+Ex than in the Con and GK. In addition, mean fiber succinate dehydrogenase activity and PGC-1α levels were lower in the GK

compared to all other groups. Nevertheless, exercise training increased both of these measures in GK rats. Moreover, VEGF, Flk-1, and ANG-1 mRNA levels were lower in the GK than Con, and the mRNA levels of all angiogenic factors, except VEGF, were in general higher in the GK+Ex than all other groups. TSP-1 protein level, an anti-angiogenic factor, was higher, and VEGF protein level and VEGF-to-TSP-1 ratio, an indicator of angiogenesis, were lower in the GK than all other groups. Exercise increased VEGF protein levels and the VEGF-to-TSP-1 ratio in the GK rats. These results indicate that exercise training reduces skeletal muscle circulatory complications in T2DM via enhancing angiogenic signaling pathways.

67. AAV9 Improves Lysosomal

Organization and Diaphragmatic Contractile

Function In Pompe Disease Jessica A. McElroy1,2, A. Gary Todd1,2, Bumsoo Ahn, David D Fuller4, Barry J

Byrne1,2, Leonardo F Ferreira3 and Darin J

Falk1,2. 1Department of Pediatrics, 2Powell Gene

Therapy Center, 3Department of Health and Human Performance, 4Department of

Physical Therapy, University of Florida, Gainesville, FL 32610

Pompe disease is a neuromuscular disorder resulting from lysosomal storage of glycogen in individuals lacking the glycogen degrading enzyme alpha acid glucosidase (GAA). Progressive glycogen accumulation within the lysosome leads to muscle weakness and ultimately respiratory failure. The purpose of this study was to determine if AAV9-hGAA could restore myofibrillar diaphragmatic contractile function following intrathoracic administration in a murine model of Pompe disease. Myofibers were isolated from wild-type (WT) or Pompe (Gaa-/-) and AAV9-hGAA treated Gaa-/- mouse diaphragms at 6 months of age (5 months post injection). While no significant difference in the rate of tension

development was observed between groups the 20% decrease in maximal calcium-activated specific force usually observed in Gaa-/- mice (117.8 ± 8.4 kN/m2) was significantly improved to levels resembling wild-type following AAV9 treatment (AAV9-treated 143.7 ± 5.1 kN/m2; WT 147.9 ± 9.5 kN/m2). Biochemical analysis of diaphragm lysates revealed a significant increase in GAA enzyme levels compared to Gaa-/- and WT animals. Confocal microscopy confirmed both absence of lysosomal GAA and lysosomal aggregation in Gaa-/- single fibers and subsequent AAV9-mediated lysosomal hGAA targeting in treated animals. Our data suggest that AAV9-hGAA remediates the decreased myofibrillar force and restores proper lysosomal organization in a murine model of Pompe disease.

68. Antagonizing PPARγ Improves Muscle Fiber Force Production and Reduces Myosteatosis Following

Chronic Rotator Cuff Tear Gumucio, Jonathan P1,2; Flood, Michael D1; Roche, Stuart M1; Mendias, Christopher L1,2.

Department of 1Orthopaedic Surgery and 2Molecular & Integrative Physiology

University of Michigan, Ann Arbor, MI Following chronic tears, certain muscle groups develop an accumulation of ectopic fat within muscle fibers, as well as in plaques within the extracellular matrix. Muscle atrophy, fibrosis and inflammation often accompany this fat infiltration, and these changes are commonly referred to as "myosteatosis." As PPARγ is a potent inducer of adipogenesis, we tested the hypothesis that antagonizing PPARγ signaling would reduce myosteatosis and increase muscle fiber specific force production in a rat full-thickness severe rotator cuff tear model. Rats were subjected to a full thickness tenectomy of the supraspinatus and infraspinatus tendons and received either standard chow or chow infused with the PPARγ antagonist GW9662. Thirty days after tear, muscles were harvested and prepared for muscle

fiber contractility, histology, and RNA isolation. Compared with control rats, torn muscles from PPARγ antagonist rats exhibited a 15% increase in specific force production. Additionally, antagonizing PPARγ reduced the amount of lipid plaques, along with a downregulation of several mRNA transcripts involved in canonical muscle lipid metabolism, macrophage accumulation, atrophy, autophagy and fibrosis. While further studies are necessary, the results from the current work support PPARγ as a potential therapeutic target for the treatment of myosteatosis in chronic muscle tears.

69. Muscle Biopsy and Hsk Cell Analysis for Pax7 and Fusion Protein, Kirrel. KH Myburgh1, P Durcan1, M van de Vyver1,2, K Goetsch3, CU Niesler3.

1Stellenbosch University, Dept Physiological Sciences, 2Stellenbosch University, Dept

Medicine, Discipline of Biochemistry, School of Life Sciences, University of KwaZulu-

Natal, South Africa. Myoblast or satellite cell fusion is thought to be critical for skeletal muscle growth and regeneration following damage. The occurrence of somatic cell fusion in skeletal muscle offers a potential mechanism of delivering a desired therapy to skeletal muscle e.g. to cure genetic myopathies via cell therapy. Our understanding of the molecular mechanisms regulating muscle cell fusion in humans is currently rudimentary. We have studied fusion or the lack thereof in human subjects and preliminary investigations have started in cell culture. Human volunteers performed intense intermittent downhill running (DHR) for 60 minutes. Muscle biopsies of vastus lateralis were analysed for satellite cell (SC) proliferation (immuno-histochemistry) and the transcription and translation of a fusion protein, Kirrel (PCR; SDS PAGE and immuno-detection). Pax7+ SCs increased significantly 1 day after DHR and returned to baseline on day 2. It was unclear if this was due to fusion or other factors. We next

determined expression of the fusion protein Kirrel which is the human homolog of Kin of Irre (Kirre) and roughest (Rst) that are essential for muscle cell fusion in Drosophila. Two splice variants of Kirrel 1 were present in human skeletal muscle biopsies as well as a Kirrel 3 transcript. Preliminary data suggest changes in Kirrel 1 mRNA and protein in response to DHR. Kirrel protein was identified in HSk cells, but was not evenly distributed and did not appear to be membrane bound. In conclusion, we have established 2 models in which regulation of fusion could be investigated in detail, particularly the role fusion proteins. No previous studies on Kirrel in human muscle have been done.

70. Changes in Macrophage Phenotype and Induction of Epithelial-to-

Mesenchymal Transition Genes Following Acute

Achilles Tenotomy and Repair Kristoffer B Sugg, Jonathan P Gumucio,

Christopher L Mendias. University of Michigan.

Tendons are an extension of skeletal muscle extracellular matrix (ECM) that play an important role in transmitting forces generated within muscle fibers to allow for locomotion. Injuries to tendons can change the mechanical properties of this tissue and increase the susceptibility of muscle fibers to stretch-induced injury. In this way, tendons and muscles are functionally linked, and despite the importance of tendon to the overall function of the musculoskeletal system, relatively little is known about the cellular and molecular mechanisms that control its growth, maintenance and repair. In many injured tissues, the repair process is orchestrated by two types of cells, macrophages and fibroblasts. Macrophages, which have both proinflammatory (M1) and antiinflammatory (M2) phenotypes, can directly participate in tissue remodeling and direct the response of other cells through the secretion of cytokines and growth factors. Fibroblasts,

which have a well-described role in synthesizing and repairing damaged ECM, have been shown to arise from epithelial cells in other organ systems. This process is triggered via activation of epithelial-to-mesenchymal transition (EMT) signaling programs. Most tendons are surrounded by sheets of epithelial cells, and these tissue layers could provide a source of fibroblasts to repair injured tendon tissue. To gain greater insight into the biology of tendon repair, we performed a tenotomy and repair in Achilles tendons of adult rats and determined changes in macrophage phenotype and EMT-related genes over a four-week time course. M1 macrophages rapidly appear in three days following tear and repair, while M2 macrophages accumulate more slowly and become the predominant macrophage phenotype by four weeks. Additionally, expression of EMT-related genes correlate temporally with other fibrotic programs in tendon. The results from this study suggest that changes in macrophage phenotype and activation of EMT-related genes may contribute to the degradation and subsequent repair of injured musculoskeletal tissue.

71. Glycogen Accumulation Varies By Fiber Type in the Pompe Diaphragm

Pascual LM, Elmallah MK, Falk DJ, Todd AG, Byrne BJ, Fuller DD.

University of Florida Physical Therapy Department

Pompe disease is a form of muscular dystrophy due to lysosomal glycogen storage caused by deficiency of acid a-glucosidase (GAA). Glycogen accumulation in diaphragm myofibers is a hallmark of this disease and likely contributes to breathing insufficiency. It is currently unknown if histopathology in the Pompe diaphragm varies by myofiber type. However, models of diaphragm myofiber recruitment suggest that slow fibers are the primary contributors to normal “tidal breathing” and fast fibers are recruited primarily during high intensity tasks (e.g., coughing). Thus, the purpose of

these preliminary studies was to use a murine model of Pompe disease, the Gaa-/- mouse, to compare the pathological appearance of Type I, IIa, and IIx/b myofibers. We hypothesize that Type I fibers, being more metabolically active, will show greater relative glycogen accumulation and histopathology. Following anesthesia, the diaphragms of female Gaa-/- and 129SVE mice (12-18 mo) were harvested, frozen and sectioned at 7 μm. Type I and Type IIa myofibers were then immunohistochemically identified using standard procedures. Alternate tissue sections were stained with the periodic acid-Schiff (PAS) method to recognize glycogen. Preliminary results indicate that fiber type distribution is similar between 129SVE and Gaa-/- mice, and that Type I fibers are larger in Gaa-/- mice. PAS staining was negative in 129SVE tissues, but positive PAS staining could be observed in Gaa-/- myofibers. Type IIa and IIx/b fibers, however, were not always PAS positive in Gaa-/- tissues. In contrast, Type I fibers were always PAS positive in Gaa-/- diaphragms with histological evidence of extensive glycogen accumulation. These initial results are consistent with the hypothesis that diaphragm myofibers with the highest metabolic activity are most susceptible to glycogen accumulation in the Pompe diaphragm.

72. Chronic Insulin Exposure Results in

Transcriptional Alternations In Metabolic And Myogenic Genes in In Vitro Skeletal

Muscle. Mark C Turner1, Darren J Player1, Neil RW

Martin1, Mark P Lewis1 1Loughborough University, Loughborough,

Leicestershire, United Kingdom Introduction: Skeletal muscle is a major site of insulin stimulated glucose disposal. Furthermore, insulin regulates the transcription of a number of metabolically important genes, which are subsequently impaired within insulin resistant individuals’. Cell culture models using pure myogenic

cells can provide a controlled environment to investigate the effects of insulin exposure upon transcriptional changes. Therefore, the purpose of these experiments was to investigate the effects of chronic insulin exposure upon pre- and post-mitotic skeletal muscle in vitro. Methods: C2C12 myoblast were grown until confluent, and either differentiated into myotubes before being incubated with either a control media (DMEM + 2% Horse serumserum (HS)), or insulin supplemented media (DMEM + 2% HS and 100nM Insulin), or were differentiated in the presence of the above medias. The media was changed twice daily for 72 hours as previously described (3). RNA was extracted to analyse mRNA expression of Hexokinase II (HKII), GLUT4, Peroxisome proliferator-activated receptor coactivator (PGC)-1α, and Myogenin (MyoG) by qRT-PCR. Results: There was no effect of chronic insulin exposure upon GLUT4 mRNA expression in C2C12 myotubes (P>0.05), although it was reduced in insulin exposed differentiating myoblasts (P=0.051). In addition HKII expression was impaired in differentiating myoblasts exposed to insulin (P<0.05). PGC-1α expression was not effected by condition in myotubes (P>0.05) however was different between conditions in myoblasts (P<0.05). MyoG expression was reduced in myoblasts treated with insulin (P<0.05), but not within myotubes (P>0.05). Conclusions: Chronic insulin exposure negatively impacts upon key metabolic genes in regenerating of skeletal muscle, whilst it appears not to affect these same genes in differentiated skeletal muscle. These findings support in vivo research, which suggests a mechanistic defect within peripheral tissue of insulin resistant individuals at the transcriptional level.

73. Ankrd2 is a Modulator of NF-Kb Mediated Inflammatory Responses in

Muscle Verma N.K.1, Yamamoto D.2, Chemello F. 1,

Bang M.L.2,3, Lanfranchi G.1 and Bean C.1

1University of Padova, Department of Biology, Padova, Italy; 2Institute of Genetic

and Biomedical Research, Milan Unit, National Research

Council (IRGB-CNR), Milan, Italy; 3Humanitas Clinical and Research Center,

Rozzano (Milan), Italy.

Skeletal muscle may change its structural and functional properties in accordance to enviromental conditions. Among the most intriguing candidates for a role in the adaptive response of skeletal muscle are mechanically sensitive proteins displaying dynamic distributions within cells. Many studies show that Adaptive responses of skeletal muscle regulate the nuclear shuttling of the sarcomeric protein Ankrd2 that can transduce different stimuli into specific adaptations by interacting with both structural and regulatory proteins. In a genome-wide expression study on Ankrd2 knockout or overexpressing primary proliferating or differentiating myoblasts, we found an inverse correlation between Ankrd2 levels and the expression of pro-inflammatory genes and identified Ankrd2 as a potent repressor of inflammatory responses through direct interaction with the NF-kB repressor subunit p50. In particular, we identified Gsk3ß as a novel direct target of the p50:Ankrd2 repressosome dimer and found that the recruitment of p50 by Ankrd2 is dependent on Akt2-mediated phosphorylation of Ankrd2 upon oxidative stress during myogenic differentiation. Surprisingly, the absence of Ankrd2 in slow muscle negatively affected the expression of cytokines and key calcineurin-dependent genes associated with the slow-twitch muscle program. Thus, our findings support a model in which alterations in Ankrd2 protein and phosphorylation levels modulate the balance between physiological and

pathological inflammatory responses in muscle.

74. High Intensity Exercise Improves Skeletal Muscle Mitochondrial Function

Corresponding With Reduced Fatigability in Parkinson's Disease

NA Kelly1, DR Moellering1, CS Bickel1, MP Ford1, DG Standaert1, and MM Bamman1

1The University of Alabama at Birmingham, Birmingham, Alabama, USA

Objective: Parkinson’s disease (PD) is a debilitating, neurodegenerative disease that manifests as disrupted motor behavior (bradykinesia, tremor, postural instability, rigidity) which ultimately reduces physical activity and weight-bearing ambulation. Deconditioning as a result of PD often leads to weakness, low muscle power, and fatigability. We hypothesized that a high-intensity exercise training prescription which simultaneously challenges strength, power, balance, and endurance would improve muscle function and fatigability in PD patients. Methods: Participants (n=12, 65±6 y, Hoehn & Yahr 2-3) exercised 3 d/wk x 16 wk. Fatigue was assessed in three domains (patient perception, cardiorespiratory fatigue, and neuromuscular fatigue) and skeletal muscle biopsies were analyzed for changes in muscle fiber size, type distribution (I, IIa, IIx), and mitochondrial function. Results: Improvements were noted in the Fatigue Severity Scale, UPDRS motor score, voluntary strength (57%), and power (49%) (p<0.05). Muscle fiber size (type I: 14%; type II: 36%) and mitochondrial complex I activity (48%) increased (p<0.05), and fiber type distribution shifted toward the more oxidative, fatigue-resistant type IIa phenotype (IIa from 39% to 55%; IIx from 12% to 2% of total myofibers). In conclusion, PD patients are capable of, and responsive to, high-intensity exercise training with improvements in skeletal muscle phenotype, functionality, and fatigability.

75. Generating 3D Neuromuscular Junctions In Vitro

Martin, N.R.W., Player, D.J., Lewis, M.P. 3D skeletal muscle constructs provide a test bed for physiological experimentation in a controlled environment. Such models could be advanced by the addition of motoneurons in an attempt to develop neuromuscular junctions (NMJs) which would offer the potential for more advanced neuromuscular testing, to both advance our understanding of the underlying physiology of the NMJ, and also allow clinical testing to be undertaken. In our laboratory, we have developed methods for generating tissue engineered skeletal muscle constructs which can in turn be co-cultured alongside motoneurons. By using immuno-histochemical techniques it is possible to visualise these motoneurons interacting with myotubes, and also show evidence of immature NMJ formation by the presence of co-localised pre- and post-synaptic proteins. Furthermore, real-time PCR can be used to help understand potential changes in maturation of the myotubes e.g. myosin heavy chain transcripts, and in the transcription of NMJ proteins e.g. Ach-R. Novel techniques allowing functional data to be obtained from these constructs can also be generated in order to give an insight into how the addition of motoneurons to in vitro skeletal muscle affects its physiology and phenotype. Developing models such of those described here greatly benefits the field of skeletal muscle physiology in health and disease. Neuromuscular pathologies, NMJ development and neuromuscular adaptation to increased/decreased use can be investigated in a controlled environment, with the ability to generate numerous experimental repeats and measure functional, biochemical and molecular outputs without many of the ethical constraints associated with in vivo experimentation.

76. Recovery of Altered Neuromuscular Junction Morphology and Muscle Function in Mdx Mice After Injury

Stephen J.P. Pratt1, Sameer B. Shah2, Christopher W. Ward3, Joseph P. Stains1

and Richard M. Lovering1 University of Maryland School of Medicine,

Department of Orthopaedics1 University of California San Diego,

Departments of Orthopaedic Surgery and Bioengineering2

University of Maryland School of Nursing3 Duchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by the absence of dystrophin, a structural protein found in striated muscle fibers. Considerable attention has been dedicated to studying myofiber damage and muscle plasticity, but less attention to damage after contraction-induced injury at or near the nerve terminal. The neuromuscular junction (NMJ) in mdx mice (murine model of DMD) has significantly altered morphology compared to healthy (wild type, WT) mice. We previously reported these changes are further exacerbated 24 hrs following eccentric injury; however, it is not known if they occur immediately after injury due to mechanical disruption or are the result of post-synaptic reorganization. It is also not known if the increase in disruption resolves during the course of whole muscle recovery. We induced injury to the quadriceps muscles of WT and mdx mice using an established in vivo model and followed the animals until full functional recovery. NMJ morphology and neuromuscular transmission failure (NTF) rates were assessed at 5 time points after injury: Day 0 (immediately after injury) and Days 1, 7, 14 and 21. Injury resulted in a significant loss of maximal torque in WT (39 ± 6 %) and mdx (76 ± 8 %) quadriceps, with recovery by Day 7 (WT) or Day 21 (mdx). Significant changes in NMJ morphology and NTF were found only in mdx following injury, and only at Days 0 and 1. The data indicate that eccentric contraction-induced injury can induce immediate changes at the NMJ, disrupting both morphology and NMJ

function. The lack of parallel recovery in muscle force production with NMJ morphology/function over the entire recovery period suggests that disruption of NMJ in loss of muscle function is limited to the early time points after injury. Supported by NIH grant 1R01AR059179 to RML 77. The Expression and Role of Notch in

Exercise-Induced Regenerating Aged Skeletal Muscle

J Demick, M Lord, A Lyle, K Brown, M Keith, J Tkach, S Blanton, I Cooley, J

Marino, R Howden and ST Arthur. The Laboratory of Systems Physiology, Dept. of Kinesiology, The University of North Carolina-Charlotte, Charlotte, NC

Notch signaling is critical for skeletal muscle regeneration and has been suggested to contribute to the poor regenerative response in aged skeletal muscle. Downhill running (DHR) is an injurious exercise model that is representative of daily activity, has a high external validity, and involves multiple active biological systems. Little is known about the effects of injurious exercise on Notch expression in aged skeletal muscle or on the role of Notch in aged muscle repair. Purpose: 1) To determine Notch signaling in aged skeletal muscle exposed to downhill running and 2) to determine if inhibiting Notch affects muscle repair in aged skeletal muscle exposed to downhill running. Methods: Aged male C57B/J6 mice (20-25 mo old) were divided into no exercise and exercise groups. The exercise group performed one bout of injurious DHR at 10m/min, -15% grade until exhaustion. Starting 24h post-exercise and continued every 24h until day of euthanasia (4D, 5D, & 6D), Notch inhibitor (gamma secretase inhibitor X; L-685,458) or PBS control was injected into the left and right gastrocnemius respectively. Immunofluorescence was performed using antibodies directed to Notch signaling markers and co-stained with myogenic marker, MyoD. Results: DHR induced significant injury in both gamma

secretase (4D:P=<0.001; 5D:P=<0.001; and 6D: P=<0.001) and PBS-treated (4D:P=0.016; 5D: P = 0.004) muscles. Relative to PBS, gamma secretase-treated muscles experienced a two-fold increase in muscle injury at 4D-6D-post-exercise (P< 0.001). In addition, preliminary findings report a possible two fold increase in Active Notch in PBS-treated muscles at 5D post-exercise. Conclusion: DHR may increase Notch signaling in aged gastrocnemius and inhibiting Notch signaling may delay repair of aged muscle.

77b Estrogen Alters Macrophage and Neutrophil Recruitment and Response

Following Skeletal Muscle Injury Tara L Mader1, Coco Le1, Dawn A Lowe1,

Gordon L Warren2 University of Minnesota1, Georgia State

University2 Inflammation is a necessary component for skeletal muscle to recover from injury; alterations of this response can lead to prolonged or incomplete recovery which can have devastating effects. Certain aspects of the inflammatory response have been shown to be estrogen responsive. Furthermore, when estrogen is lacking, skeletal muscle recovery from injury is impaired. The purpose of this study was to determine the extent to which estradiol modulates the recruitment and response of neutrophils and macrophages following skeletal muscle injury. Adult female C57BL/6 mice were ovariectomized and then randomly assigned to implantation of a 17-β estradiol time-release or placebo pellet. Three weeks following surgery, tibialis anterior (TA) muscles were freeze injured and mice were allowed to recover for either 1, 2, 3, or 4 days. Gene expression levels for chemokines related to recruitment of macrophages and neutrophils, and cell surface markers for these inflammatory cells, were assessed using qPCR. Expression of markers for recruitment and activation of monocytes and macrophages (Spp1/MCP-1), recruitment of neutrophils

(Cxcl1/Cxcl5), pro-inflammatory macrophages (CD68), anti-inflammatory macrophages (CD163/CD206), and neutrophils (Ly6g) were affected by estradiol treatment although the marker for total leukocytes (Mac-1) was not. Compared to estradioldeficient mice: Spp1 expression was upregulated by 1.6 fold at 3-4 days (p≤0.023) in estradiol-treated mice; Cxcl5 and Cxcl1 expression was upregulated by 2.5-3.2 fold at 1-2 days (p≤0.014) in estradiol-treated mice; CD163 expression was upregulated by 2-5 fold (p≤0.032) at 1-4 days in estradiol-treated mice; and Ly6g expression was upregulated by 9.5-18 fold at 1-4 days (p≤0.008) in estradiol-treated mice. The results of this study indicate that the presence of estrogen significantly affects the early inflammatory response to injury in skeletal muscle. When estradiol is present, the neutrophil response is more robust and there is a shift to a more anti-inflammatory macrophage subpopulation than when estradiol is lacking.

78. Adoptive Transfer of Ischemia/ Reperfusion-Conditioned Macrophages

Enhances Functional Recovery of Skeletal Muscle After Tourniquet-

Induced Ischemia/Reperfusion Injury

Viktoriya Rybalko, David Hammers, Melissa Merscham-Banda, Roger P. Farrar

University of Texas at Austin

Tourniquet-induced ischemia reperfusion (TK-I/R) is severe skeletal muscle injury with extensive pathophysiology. Detrimental consequences of ischemia-reperfusion (I/R) injury to skeletal muscle involve prolonged post-reperfusion recovery periods, functional impairments and loss of muscle function. Routine use of tourniquets in clinic and in the military makes TK-induced muscle injury a serious clinical problem. The etiology of I/R injury in skeletal muscle is complex and is mediated by the global tissue energy depletion, overproduction of tissue damaging reactive oxygen species, excessive inflammation, vascular damage

and tissue necrosis. Search for pharmacological strategies to protect skeletal muscle from ultrastructural and functional damage is ongoing. We have shown that adoptive macrophage transfer of I/R-conditioned macrophages significantly enhances skeletal muscle regeneration 14 days after TK-I/R injury (as evidenced by 15% increase in functional recovery of maximal force over saline injection). At the time of transfer, macrophages exhibit several features of M2-type macrophages characterized by reduced expression of inflammatory and up-regulated expression of pro-regenerative/anti-inflammatory genes. The significant upregulation of IGF-I and transient up-regulation of IL-10 gene expression may mediate beneficial effects on muscle regeneration. Studies are underway to identify key mediators responsible for improved skeletal muscle recovery after TK-I/R injury. AMP-activated protein kinase (AMPK) activity is inhibited due to a number of pathophysiological conditions. We previously show that AMPKα1 promotes myogenesis through increasing myogenin expression, but the role of AMPK in muscle regeneration remains unclear. The objective is to define the role of AMPKα1 catalytic subunit in muscle regeneration. Satellite cell density was lower in AMPKα1 knockout (KO) compared to wild-type (WT) mice, and maintained lower after cardiotoxin (CTX) injection to induce muscle injury. Transcriptions of Pax7, MyoD, Myf5, and myogenin were attenuated in AMPKα1 KO mice. As a consequence, the regeneration of damaged muscle fibers was retarded in AMPKα1 KO mice compared to WT mice. To discern whether the impaired muscle regeneration is due to reduced density or myogenic differentiation of satellite cells, we used AMPKα1 conditional KO mice. Despite of no difference in satellite cell density, acute AMPKα1 KO just before muscle injury hampered muscle regeneration, showing that AMPKα1 promotes muscle regeneration at least partially through enhancing myogenic differentiation of satellite cells. In summary, absence of

AMPKα1 impairs muscle regeneration, which could be due to reduced satellite cell density and/or decreased myogenic differentiation of activated satellite cells. Thus, AMPK is likely a convenient drug target to facilitate muscle regeneration, considering the wide availability of anti-diabetic drugs known to activate AMPK. (Support by NIH 1R01HD067449)

Control of Muscle Force Production

79. Adaptations in Muscle Fiber Length, Pennation Angle, and Curvature

Following an Anterior Cruciate Ligament Tear

Brian Noehren, Anders Andersen, Peter Hardy, and Bruce Damon

University of Kentucky Quadriceps muscle weakness is common among individuals who have torn their anterior cruciate ligament (ACL). Unfortunately this weakness persists even after surgical reconstruction of the ACL. To date, little is known of the adaptations to muscle fiber architecture that may explain some of the reduction in muscle strength. We hypothesized that following an ACL tear the injured limb would have a smaller pennation angle, fiber length, and curvature. Seven males (22±3 yrs old) scheduled to undergo an ACL reconstruction participated in this study. Diffusion tensor magnetic resonance imaging was performed on both the injured and non-injured thighs. Muscle fiber bundles were tracked by integrating the first eigenvector of the diffusion tensor, starting from seed points on the aponeurosis of fiber insertion, until either the fiber exited the side or top of the selected slices. From these tracts we then calculated the pennation angle, fiber length and curvature. Between limb differences were made with a paired t-test. The injured limb had a significantly smaller pennation angle (p=0.01, injured 17±1.1, non-injured 21.2±3.4 degrees), fiber length (p=0.002, injured 34±3.8, non-injured 44.5±7.4 mm) and higher curvature (p=0.01, injured 6.8±

0.9, non-injured 5.2±1.0 m-1). These results indicate that even prior to the surgery, significant reorganization of the muscle is already occurring. The reduction in fiber length in particular would have a large effect on the physiological cross sectional area and thus the muscle’s ability to generate force. The increase in fiber curvature could cause fibers to traverse the muscle’s thickness with a shorter overall length. These preliminary results suggest that pre- operative interventions may be needed to be implemented sooner and be of greater intensity to maintain muscle structure.

80. Mice Lacking P47phox Subunit of NADPH Oxidase Are Protected From

Diaphragm Dysfunction Elicited By Heart Failure

Bumsoo Ahn1, Gregory S. Frye1, Adam W. Beharry2, Jennifer. S. Moylan3, Andrew R.

Judge2, Leonardo F. Ferreira1

Department of Applied Physiology and Kinesiology, University of Florida1;

Department of Physical Therapy, University of Florida2; Department of Physiology,

University of Kentucky3 Patients with chronic heart failure (CHF) suffer from shortness of breath and have increased circulating inflammatory cytokines. In CHF diaphragm, inflammatory cytokines may activate NADPH oxidases (NOX) and promote oxidant production that causes weakness. Specific NOX isoforms require p47phox for enzyme activation. Therefore, we hypothesized that p47phox

knockout (KO) mice would be protected from CHF-induced increases in oxidants and contractile dysfunction. We caused myocardial infarction via ligation of the left anterior descending coronary artery to induce CHF in mice with p47phox KO and wild-type genetic controls (C57BL/6J) and performed experiments 14-16 weeks post-surgery. We confirmed CHF based on 40-50% decrease in left ventricle fractional shortening for both WT and p47phox KO mice (P < 0.05). Extracellular oxidants were increased in CHF WT diaphragm (P < 0.05; n = 4-5/group), whereas the oxidants were unchanged in the diaphragm of CHF p47phox

knockout mice. CHF decreased diaphragm isometric maximal tetanic force in WT mice (P < 0.05; n = 8-9/group). However, diaphragm maximal tetanic force was not different in Sham and CHF p47phox knockout mice. Furthermore, we observed that CHF impaired diaphragm isotonic contractile properties in WT (P < 0.05; n = 8-9/group) but not in p47phox knockout mice. Together, our findings suggest that p47phox is required for increased oxidants and contractile dysfunction in CHF diaphragm. Thus, p47phox-dependent NOX may be a therapeutic target for CHF-induced diaphragm contractile dysfunction.

81. Inactivity-Induced Decline in Single Muscle Fiber Power Output: The Role of

Myosin Light Chain 3f

Jong-Hee Kim*1,2 and LaDora V. Thompson2

1Department of Health and

Human Performance, University of Houston, Houston, TX,

2Department of Physical Medicine and Rehabilitation

University of Minnesota, Minneapolis, MN Muscle power is a key contractile parameter representing work output per unit time. Muscle power is significantly reduced with inactivity, and this deterioration is associated with impaired force-generating capacity and/or shortening velocity under loaded muscle contraction conditions. We recently demonstrated that increasing the relative myosin light chain 3f (MLC3f) protein content attenuates inactivity-induced decline in velocity under unloaded conditions in myosin heavy chain (MHC) type IIB fibers. Based on these findings, it is possible that increasing MLC3f content maybe a countermeasure to combat inactivity-induced alterations in power output. Therefore, the purpose of this study was to investigate whether (1) inactivity-induced reduction in power output is associated with a reduction in MLC3f protein content; and (2) increasing MLC3f protein content attenuates inactivity-induced reduction in power by increasing velocity under loaded conditions. Fischer-344 rats were randomly

assigned to control (CON), non-weight bearing (NWB), or NWB plus rAd-MLC3f treated (NWM). NWB and NWM rats were hindlimb unloaded (HU) for 7 days. For NWM rats, the optimal recombinant

adenovirus (rAd)-MLC3f solution (1×1012

ifu/ml; 375 or 500µl at four-day after HU) was injected into the semimembranosus muscles to increase MLC3f protein content. We determined peak power (PP), velocity at PP, and velocity and power under selected loaded conditions (10-90% Po). MHC and MLC isoforms were identified by SDS-PAGE and silver-staining techniques. PP (39.10±3.15µN·FL/s), %MLC3f (8.13±0.81%), and MLC3f/MLC2f (0.17±0.02) in NWB were significantly lower than that in CON (69.68±5.61µN·FL/s, 11.17±0.82%, 0.25±0.02), respectively. Increasing MLC3f content (+52% in %MLC3f; +37% in MLC3f/MLC2f) in NWM, however, did not affect PP. Currently, the velocity at PP and the velocities and power output under selected loads are being analyzed to identify if the increased MLC3f content influences power-velocity parameters. At the moment, increasing MLC3f content to attenuate the reduction in power output associated with inactivity is not effective.

82. Inactivity-Induced Muscle Weakness: The Role of Myosin

Jong-Hee Kim*12 and LaDora V. Thompson1

1Department of Physical Medicine and Rehabilitation University of Minnesota,

Minneapolis, MN, 2Department of Health

and Human Performance University of Houston, Houston, TX

Muscle weakness is associated with inactivity such as immobilization, denervation and non-weightbearing. The underlying mechanisms responsible for muscle weakness (i.e., specific force; peak force/CSA) under these conditions, however, are not well understood. In a previous study we showed that the decline

in specific force in myosin heavy chain (MHC) type II fibers was associated with a reduction in the fraction of myosin in the strong-binding structural state. These reductions imply altered myosin ‘quality’. Hence, we tested the hypothesis that inactivity-induced weakness is due to both a decrease in myosin quality (i.e., force per MHC in half sarcomere and fraction of myosin strong-binding during contraction) and quantity (i.e., MHC content per half sarcomere and MHC to actin ratio) in MHC type II fibers. Fisher-344 rats were assigned to weightbearing control (CON) or non-weightbearing (NWB). The NWB rats were hindlimb unloaded for two weeks. Permeabilized single fibers were teased from the semimembranosus muscle and evaluated for diameter, force, and MHC content. MHC isoform and MHC to actin ratio in each fiber were determined by gel electrophoresis and silver-staining techniques. The fraction of myosin strong binding during contraction was evaluated using electron paramagnetic resonance (EPR) spectroscopy. Following 2 weeks of NWB, specific force was reduced by 38% in MHC type IIB fibers and by 18% in MHC type IIXB fibers. MHC content per half-sarcomere and force per MHC in half-sarcomere were reduced by 21% and 52%, respectively. EPR analyses showed that the fraction of myosin strong binding during contraction was 34% lower in fibers from NWB rats (20.4±0.5%) compared to fibers from CON (31.2±1.0%) rats. These results suggest that myosin quality and quantity collectively contribute to muscle weakness associated with inactivity conditions.

83. Isolated Human Intercostal Muscle Fibers as an In Vitro Skeletal Muscle

Model. Karl Olsson1, Joseph Bruton2, Seher Alam1,

Håkan Westerblad2, Thomas Gustafsson1 1Department of Laboratory Medicine,

2Department of Physiology and

Pharmacology, Karolinska Institutet,

Stockholm, Sweden

Background. Changes in skeletal muscle intracellular Ca2+ handling have been demonstrated in a number of diseases, including heart failure and critical illness, and are suggested to contribute to the disease process. While skeletal muscle Ca2+ handling has been extensively studied in animal models, studies in humans are sparse. In addition, the in vitro studies performed on human muscle cells have been performed using myotubes, not intact muscle fibers. In the present study we established a human adult muscle fiber in vitro model and compared intact fibers vs myotubes in the analysis of human skeletal muscle Ca2+ handling and force production. Methods. Single adult muscle fibers were isolated from human intercostal muscle obtained during thoracotomy performed as part of the treatment for non-small-cellular lung carcinoma. Myoblasts isolated from the vastus lateralis muscle of healthy subjects were differentiated to myotubes. We measured changes in intracellular free Ca2+ ([Ca2+]i) in myotubes and muscle fibers in response to electrical stimulation. In addition, force production was analyzed in muscle fibers. Immunocytochemistry for RYR1 and DHPR was performed in muscle fibers and myotubes. Results. The isolated human single muscle fibers all responded to electrical stimulation by an increase in [Ca2+]i and they all contracted. The maximum tetanic force that fibers generated was 450-­‐500 kN/m2 before induction of fatigue. After fatigue was induced tetanic force fell by about 20-­‐40% and [Ca2+]i fell by about 70%. In comparison, a majority of human myotobes responded to electrical stimulation by an increase in [Ca2+]i, but no contraction was observed in any of the myotubes. The Ca2+ transient decay phase was markedly longer in the myotubes than in the adult muscle fibers. The colocalisation of the Ca2+ handling proteins RYR1 and DHPR was less in the myotubes compared to that in adult muscle fibers. Conclusion. Our results show marked differences in intracellular Ca2+ handling and contractile capability

between adult fibers and differentiated myotubes of human muscle. Thus, the differentiated myotube is not a good model for studies of Ca2+-­‐dependent functions in adult human muscle.

84. AMP Deaminase Overexpression Improves Skeletal Muscle Relaxation

Kinetics During High Energy Demands.

PR Davis1,2

, CA Witczak1,2,3,4

, JJ Brault1,2,3,4

East Carolina Diabetes and Obesity

Institute1, Departments of Kinesiology

2,

Physiology3, Biochemistry and Molecular

Biology4, East Carolina University,

Greenville, NC 27834 .

During intense skeletal muscle contractions, ATP consumption outpaces ATP re-

synthesis resulting in an increase in free

ADP and Pi and a concomitant reduction in

the free energy (ΔG) of ATP hydrolysis. Removal of AMP by AMP Deaminase (AMPD) (AMP ↔ IMP + NH3) favors ATP

production and ADP clearance via adenylate kinase (ADP + ADP ↔ ATP + AMP) thereby protecting the energetic state. The purpose of this study was to determine if AMPD overexpression could improve force production or contraction kinetics of skeletal muscle under high energy demands, indicating an improvement in energetics. Methods: Solei of adult male CD-1 mice were transfected with empty vector or AMPD3 plasmid by in vivo electroporation. Seven days later, solei were removed and electrically stimulated for twitch characteristics and then either kept at resting length or fatigued (150 Hz, 167 ms train, 2 tetani/s) over 60 seconds in oxygenated Krebs-Henseleit bicarbonate buffer at 37°C. Force was recorded via a Dual-Mode Muscle Lever System (Aurora Scientific Inc.). Solei were then snap-frozen, and adenine nucleotides and IMP were measured from PCA extracts via UPLC. Results: Adenine nucleotides were no different between AMPD3 and control solei in resting and contracted conditions. Twitch characteristics (max force, ½Relaxation

Time, time to max) were no different between AMPD3 and control solei. During tetanic contractions, both AMPD3 and control solei exhibited similar fatigue patterns and significant accumulation of IMP (p<0.0001). However, there was no effect of AMPD3 on IMP accumulation. Regression analysis showed a significantly shorter ½RT in AMPD3 transfected solei compared to controls (p<0.001). Conclusions: These data suggest that AMPD overexpression improves muscle function during high energy demands. It is likely that shorter ½RT in AMPD treated solei resulted from additional buffering of the ΔG of ATP hydrolysis which protects SERCA function.

85. A Myocardial Infarction Rapidly Induces Diaphragm Muscle Weakness in

Mice T Scott Bowen, Norman Mangner, Sarah

Werner, Axel Linke, Volker Adams, Department of Cardiology, Leipzig University – Heart Center, Leipzig

Introduction: Chronic heart failure (CHF) induced by myocardial infarction (MI) results in diaphragm muscle weakness, with increased oxidants directly implicated. It remains unknown, however, whether diaphragm muscle function is impaired immediately following MI and if increased oxidants may be involved in this response. Methods: Ligation of the left coronary artery to induce MI (n=10) or sham operation (n=10) was performed on 8 wk old C57BL/6 mice. Three days later, in vitro isometric force of diaphragm muscle fiber bundles was assessed. Enzyme activities (spectrophotometric assays) and protein expression (western blot) were subsequently assessed in muscle homogenates. Results: Histology confirmed a left ventricular infarct size of 57±1% (mean±SE). MI depressed (p<0.05) specific force compared to sham between the frequencies of 80-300 Hz, with maximal tetanic force reduced by 20% (25±1 vs. 20±1 N/cm2). Western blot analyses

revealed, however, expression of contractile proteins did not differ between groups. Compared to sham, enzyme activity was increased (p<0.05) for NADPH oxidase (45%) and xanthine oxidase (33%) following MI, which correlated to the reduction in maximal force (R2 = 0.50 and 0.43, respectively; both p<0.05). Protein carbonyls, however, were not different between sham and MI groups (2.78±0.53 vs. 2.47±0.33, respectively). Although antioxidant enzyme activities were similar between groups, glutathione peroxidase and catalase were inversely correlated to the maximal force observed in the MI group (R2 = 0.60 and 0.74, respectively; both p<0.05). No correlations were observed in the sham group.

Muscle Functions Beyond Contraction

86. Levels of Leukocyte Mobilizing

Factors in Skeletal Muscle and Circulating Monocytes After An Acute

Bout of Exercise in Humans Anna Strömberg1, Karl Olsson1, Jacomijn Dijksterhuis2, Gunnar Schulte2, Thomas Gustafsson1

, 1Department of Laboratory Medicine, 2Department of Physiology and

Pharmacology, Karolinska Institutet, Stockholm, Sweden

Human myoblasts have been demonstrated to secrete the chemokines fractalkine, MDC and MCP-1, all factors chemotactic for monocytes/ macrophages. In addition, macrophages have been suggested to be crucial for skeletal muscle remodeling and regeneration. In the present study the aim was to investigate the circulating level of monocytes following an acute bout of exercise, and the skeletal muscle expression of factors hypothesized to be involved in mobilizing monocytes. A. Healthy males performed 1 h of cycle exercise. Muscle biopsies from the m. vastus lateralis of both legs were obtained before and 2 h after the exercise bout. Blood samples for flow cytometric measure of monocyte subpopulations and ELISA

measurements of plasma protein levels were retrieved before, directly after, 30 minutes after and 2 h after the exercise bout. B. In a different study, healthy males and females performed a similar 1 h bout of cycle exercise with muscle biopsies obtained up to 24 hours post the exercise bout. Results. ICAM-1, E-selectin and VCAM-1 mRNA levels and fractalkine protein levels were increased in the skeletal muscle tissue 2 h post exercise. All factors were shown to localize to the skeletal muscle endothelium using immunofluorescence. The circulating level of monocytes increased robustly after the exercise bout. The plasma level of G-CSF was significantly increased directly after the bout when adjusted for plasma albumin concentration. Conclusion. The present results indicate that exercise induces increased number of monocytes and prepares the skeletal muscle tissue for recruiting cells from the circulation.

87. Early Postnatal Undernutrition Programs the Contractile and Metabolic Phenotype of the Mouse Plantaris with

Unanticipated Consequences for Physical Activity

David P. Ferguson1, C.J. Martinez2, Brooks P. Scull1, Ryan Fleischmann1 and Marta L.

Fiorotto1, 1Children’s Nutritional Research Center,

Baylor College of Medicine, Houston, TX, 77030, 2Southwestern

University, Georgetown, TX, 78626

Nutrition during critical periods of development can program permanent changes in physiology and metabolism increasing the risk for the development of chronic diseases in adulthood. We demonstrated that a limited postnatal episode of undernutrition permanently reduces skeletal muscle mass. Our objective was to determine if postnatal undernutrition also programs changes in muscle composition with potential consequences for physical activity. Newborn mice were suckled by dams fed

either a protein-restricted (PR; 8% protein) or a control (CON; 20% protein) diet. Pups were weaned to the CON diet and housed with locked (SEDentary) or weighted running wheels (EX). Wheel and cage activities were monitored for 3 wks after which plantaris contractile and metabolic compositions were characterized from myosin heavy chain (MHC) isoform and succinate dehydrogenase (SDH) histochemistry. Additional mice were subjected to maximal treadmill testing. Muscles from recovered SED PR mice were smaller and had a substantially more oxidative phenotype than those of SED CON. Wheel running in CON pups increased the proportion of oxidative fibers and reduced glycolytic fibers; intermediate and IIX fibers were unchanged. SDH staining in EX PR mice was unchanged from SED PR, whereas the changes in MHC were similar to those of EX CON mice, but of smaller magnitude. Wheel running activity in PR mice was reduced by more than 50% compared to CON pups (P<0.001) although cage ambulatory activity was similar. Time to exhaustion following forced treadmill running was shorter in PR mice (CON: 1726 ± 65; PR: 816 ± 63 seconds; p<0.0001), and their blood lactate levels were higher at exhaustion (P<0.01). Thus, early postnatal nutrition programs permanent changes in the mass, and metabolic and contractile properties of skeletal muscle. However, the differences did not have the anticipated consequences for physical activity suggesting that other mechanisms regulate physical activity in addition to skeletal muscle properties.

88. Aerobic Exercise Induces RAGE Shedding via ADAM10 in Human Skeletal

Muscle Abeer M. Mahmoud, Brian K. Blackburn, Karia Coleman, Jacob T. Mey, Vikram S. Somal, Thomas P. J. Solomon, Ciaran E. Fealy, Steven K. Malin, John P. Kirwan,

Jacob M. Haus. University of Illinois at Chicago

Advanced glycation endproducts (AGEs) mediate inflammation and oxidative stress through receptor for AGEs (RAGE). RAGE is a cell surface receptor with an extracellular ligand binding domain that can be cleaved by metalloproteinases (MMPs) into soluble form (sRAGE). We demonstrated previously that aerobic exercise (AE) training reduces skeletal muscle (SkM) RAGE expression in obese (OB) insulin resistant adults and increases circulating sRAGE levels comparable to lean healthy controls (LHC). We sought to investigate the role of MMPs such as ADAM10 and MMP9 and their regulators, tissue inhibitors of MMPs (TIMPs), as mediators of AE induced receptor shedding in human SkM. Muscle biopsies were performed on 8 LHC (Age 36±4, BMI 22.2±1.0 kg/m2) and 22 OB subjects (Age 65±1, BMI 34.1±0.7 kg/m2) before and after OB only) a 12 wk AE intervention (5d/wk, 60 min/d, 85% HRmax). SkM protein was immunoblotted for ADAM10, MMP9, TIMP1 and TIMP3. At baseline, LHC subjects demonstrated greater protein expression of ADAM10 (LHC: 0.79±0.11; OB: 0.50±0.06 AU, p=0.02) and MMP9 (LHC: 1.28±0.24; OB: 0.87±0.18 AU, p=0.09), while TIMP1 (LHC:0.45±0.07; OB: 0.66±0.09 AU, p=0.05) and TIMP3 (LHC: 0.43±0.08; OB: 0.70±0.17 AU, p=0.09) were reduced compared to OB. Following AE, ADAM10 and MMP-9 protein expression increased by 68.9±43.3% (p=0.13) and 187.3±92.9% (p=0.004), respectively. AE also reduced protein expression of TIMP-1 (-15±11.8%, p=0.03) and TIMP-3 (-116.6%±56.4, p=0.02) in OB subjects. Moreover, we identified a physical association (via immunoprecipitation) between ADAM10 and RAGE which motivated further inquiry into mechanisms of ADAM10 induced RAGE shedding. Human primary SkM cell cultures were used to examine sRAGE appearance in the presence or absence of siRNA directed ADAM10 knock down. ADAM10 silencing revealed a 1.8±0.09 fold decline in sRAGE release relative to control (p<0.001). Collectively, these data suggest ADAM10 is an effector in SkM RAGE

shedding whose activity may be mediated by AE.

89. Skeletal Muscle-Derived EcSOD

Mitigates Diabetic Cardiomyopathy in Streptozotocin-Diabetic Mice by

Reducing Oxidative Stress. Jarrod A. Call1,4, Kristopher H. Chain1,4,

Kyle S. Martin5, Vitor A. Lira1,4, Mitsuharu Okutsu1,4, Mei Zhang1,4, Shayn M. Peirce-Cottler5, Zhen Yan1,2,3,4,. 1Departments of Medicine, 2Pharmacology, and 3Molecular

Physiology and Biological Physics, 4Center for Skeletal Muscle Research at

Robert M. Berne Cardiovascular Research Center, 5Department of Biomedical Engineering, University of Virginia,

Charlottesville, VA 22908, USA Diabetic cardiomyopathy (DCM) is a manifestation of diabetes mellitus leading to left ventricular dysfunction independent of hypertension and atherosclerosis. Ventricular dysfunction is the phenotypic endpoint caused in part by oxidative stress. First lines of defense against oxidative stress are endogenous antioxidant enzymes, such as extracellular superoxide dismutase (EcSOD). EcSOD is present in many tissues, importantly in skeletal muscle, of which the expression increases in response to endurance exercise training. Skeletal muscle collectively represents a potentially important source of EcSOD to correct oxidative stress in peripheral tissues/organs like the heart, and this is supported by our observation that exercise training increases the levels of EcSOD protein in the heart, independent of transcription. We hypothesized that skeletal muscle-derived EcSOD is sufficient to protect against streptozotocin (STZ)-induced DCM by redistributing to the heart via the circulation. To test this hypothesis, we used transgenic mice with skeletal muscle specific overexpression of EcSOD (TG). TG mice demonstrate 10-fold more EcSOD in blood, and 5-fold more in the heart, independent of transcription. Intraperitoneal STZ injection-induced type-1

diabetes led to impaired cardiac function and significant cardiac hypertrophy and fibrosis in WT mice (WT-STZ), but not in TG mice (TG-STZ). Importantly, oxidative stress was evident in the hearts of WT-STZ mice, which was significantly reduced in TG-STZ mice, and TG-STZ mice displayed reduced calcineurin-dependent signaling leading to cardiac hypertrophy, as well as reduced p38MAPK activation and induction of inflammatory cytokines. We conclude that skeletal muscle-derived EcSOD is sufficient to mitigate STZ-induced DCM through attenuation of hyperglycemia-induced oxidative stress.

90. Activation of CaMKKα Signaling Stimulates the Pentose Phosphate Pathway in Mouse Skeletal Muscle.

Jeremie L.A. Ferey1-5

, J. Matthew Hinkley1-5

,

and Carol A. Witczak1-5. Departments of

1

Kinesiology, 2

Physiology, and 3

Biochemistry

& Molecular Biology, 4

Brody School of

Medicine, and the 5

East Carolina University

Diabetes & Obesity Institute, East Carolina

University, Greenville, NC 27834

Ca2+

/calmodulin-dependent protein kinase

kinase alpha (CaMKKα) is a Ca2+

-activated, serine/threonine kinase, and work from our lab and others has suggested a key role for CaMKKα in the regulation of skeletal muscle glucose and protein metabolism. In mouse muscle, constitutively active CaMKKα expression for 2 weeks increased glucose uptake and muscle mass, suggesting that the glucose may be fueling the energetic and/or biosynthetic demands of growth. Unfortunately, the link(s) connecting CaMKKα to these processes is currently unknown. The pentose phosphate pathway is a metabolic pathway that links glucose to growth-dependent processes, as it metabolizes glucose to produce both NADPH for reductive biosynthesis reactions, and pentose monosaccharides for nucleotide synthesis. To date, there are no studies that have examined whether CaMKKα signaling regulates the pentose

phosphate pathway. Therefore, the purpose of this study was to determine if chronic activation of CaMKKα signaling stimulates the pentose phosphate pathway in mouse skeletal muscle. To selectively and specifically activate CaMKKα signaling in skeletal muscle, tibialis anterior muscles from female, 6-8 week old CD-1 mice were transfected with plasmid DNA (100 ug) containing constitutively active CaMKKα or empty vector using in vivo electroporation/muscle gene transfer. Two weeks later, muscles were collected and sent for global biochemical profile analysis (i.e. metabolomics) to assess metabolic byproducts of the pentose phosphate pathway. The results showed that expression of constitutively active CaMKKα significantly increased levels of ribulose (1.57-fold), ribose (1.48-fold), ribitol (1.41-fold), arabitol (1.32-fold), xylulose (1.68-fold) and sedoheptulose-7-phosphate (2.15-fold), while ribulose-5-phosphate/xylulose-5-phosphate and ribose-5-phosphate trended towards being increased (1.33 fold, p=0.06; and 1.31-fold, p=0.25; respectively). Collectively, these data show that chronic activation of CaMKKα signaling stimulates the pentose phosphate pathway in mouse muscle, suggesting that the pentose phosphate pathway may be a key metabolic pathway linking skeletal muscle glucose and protein metabolism.

91. Intermittent Hypoxia-Induced Disruption of Skeletal Muscle Insulin

Signaling: A Pilot Study Joseph S Marino, Paige Driver, Auburne

Overton, Susan Arthur and Reuben Howden University of North Carolina at Charlotte

18 million American adults suffer from obstructive sleep apnea (OSA), which is diagnosed by experiencing 5 or more hypoxic/hypercapnic episodes per hour while sleeping. This causes intermittent periods of reduced oxygen supply to the body causing oxidative stress. Epidemiological and clinical studies demonstrate an increased risk of insulin

resistance in OSA patients independent of obesity, suggesting that OSA is a risk factor for insulin resistance and type 2 diabetes. The mechanisms underpinning sleep apnea-induced insulin resistance however, are poorly defined. Skeletal muscle accounts for a large portion of blood glucose disposal. Thus skeletal muscle insulin resistance is a primary contributor to hyperglycemia, a prerequisite to type 2 diabetes. The objective of this pilot study is to begin to define the cellular and molecular events that orchestrate skeletal muscle insulin resistance upon exposure to intermittent hypoxic episodes. C2C12 myotubes were exposed to normal culture conditions (control; 21% O2 / 5% CO2), or 6 minute cycles of mild hypoxia (14% O2 / and 5% CO2) or severe hypoxia (5% O2 / 5% CO2). Following 4 or 8 hours of intermittent hypoxic exposure, myotubes were treated with vehicle or insulin for 15 minutes. Cell lysates were used to analyze changes in protein expression of insulin signaling molecules as well as changes in oxidative stress. We hypothesize that insulin resistance and oxidative stress will increase in a hypoxic dose and time dependent manner. Data from these pilot experiments will guide mechanistic studies aimed at preserving insulin resistance in skeletal muscle exposed to prolonged episodes of intermittent hypoxia.

92. Muscle Paracrine Regulation of Endogenous Progenitors for PAD

Therapy-­‐Roles for Tie2 and

Angiopoietin-­‐1. Joseph M. McClung1,2, Mahroo Mofarrahi3,4,

Jessica Reinardy7, Sarah Frazier7, Sabah N.A. Hussain3,4,5, Christopher D. Kontos2,7**,

1Department of Physiology, East Carolina Diabetes and Obesity Institute, Brody School of Medicine, Greenville, NC

2Department of Medicine, Division of Cardiology, and 7Department of

Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA 3Meakins Christie Laboratories and

5Department of Anatomy and Cell Biology,

McGill University, Montreal, Quebec, Canada; 4Critical Care and Respiratory

Divisions, Royal Victoria Hospital, McGill University Health Centre, Montréal, Québec, Canada, 5Royal Military College, Saint Jean

sur Richelieu, Quebec, Canada

A primary barrier to a substantial breakthrough in progenitor cell therapy for PAD remains a relative lack of knowledge of the local environmental signals that promote progenitor cell differentiation into muscle or other vascular cells. Among the early genes expressed in response to skeletal muscle injury are several angiogenic growth factors, including the angiopoietins (Ang-­‐1, Ang-­‐2), and their cognate receptors. Viral expression of Ang-­‐1 in limb muscle rapidly restored muscle force production after snake venom-­‐induced muscle injury in vivo, and was characterized by increased density of both eMyHC+ myofibers and capillaries. Exogenous Ang-­‐1 enhanced myotube formation and increased the expression of myogenin, MyoD, and MyHC in vitro. Differentiating skeletal muscle cells also expressed Ang-­‐ 1 in vitro, and released Ang-­‐1 in paracrine fashion into the culture media. Co-­‐culture of C2C12 myoblasts and C3H-­‐ 10T1/2 cells (an established mesenchymal progenitor line) in the presence of either hypoxic muscle cell conditioned medium or recombinant Ang-­‐1 increased cell differentiation/fusion in vitro and was partially blocked by exogenous expression of the extracellular domain of the Ang-­‐1 cognate receptor Tie2 (ExTEK). Tie2-­‐Cre;ROSA26mTmG mice demonstrated heterogeneous GFP expression in regenerating muscle, an effect recapitulated in centralized nuclei using IF with the Tie2 antibody (Ab33). Primary cell isolates from BL6 mice demonstrated Tie2 expression and Tie2-­‐ Cre;ROSA26mTmG isolates partially transitioned from mT expression (Red) to GFP expression (mG) during myotube formation. Stable knockdown of Tie2 in either mesenchymal progenitors or

C2C12 cells during co-­‐culture differentially altered myotube formation, demonstrating a potential role for progenitor and myoblast expressed Tie-­‐2/Ang-­‐1 paracrine/autocrine signaling in the ischemic limb. These studies provide important insights into potential mechanisms of crosstalk between blood vessels, progenitor cells, and muscle cells in ischemic skeletal muscle tissue, and may lead to the refinement of existing therapies for the treatment of PAD.

93. SLN-null Mice Cannot Sustain

Muscle-Based Thermogenesis During Cold

Exposure. Leslie A. Rowland, Naresh C Bal and Muthu

Periasamy Department of Physiology and Cell Biology,

The Ohio State University, The synchronized recruitment of thermogenic mechanisms is essential for the survival of endothermic homeotherms, including humans, during cold exposure. While brown adipose tissue (BAT) is a significant contributor to thermogenesis, BAT-deficient mice are able to adapt to cold, demonstrating the presence of other mechanisms. We have previously shown that Sarcolipin (SLN), a regulator of the sarcoplasmic reticulum Ca2+ transport ATPase (SERCA), is an essential contributor to cold-induced thermogenesis. Recently, we found that mice deficient in both UCP1 and SLN (double knockout, DKO) are severely intolerant to acute cold exposure. Though the mechanism of heat production by UCP1 is well understood, the mechanistic details of SLN-based thermogenesis remain to be elucidated. Here, to define the physiological and molecular mechanisms involved in SLN-based thermogenesis, we challenged wild-type (WT) and SLN-knockout (SLN-KO) mice to a long-term cold exposure. We found SLN-KO mice can readily adapt to gradual cold exposure and maintain normal core body temperature. However, SLN-KO

mice exhibited increased oxygen consumption compared to WT littermates and had greater physical activity and food intake. Interestingly, examination of skeletal muscles from cold-adapted SLN-KO mice showed a reduction in markers of oxidative metabolism and an increased dependence on glycolytic metabolism, suggesting a reduced content of oxidative fibers in the SLN-KO muscle. Moreover, protein expression analysis showed increased UCP1 expression in BAT and inguinal white adipose tissue, and histological examination of these fat depots showed increased depletion of fat stores from cold-adapted SLN-KO mice. Together, these data show that SLN-KO mice must enhance UCP1-based thermogenesis in order to compensate for the loss of SLN-mediated thermogenesis. Furthermore, the relative lack of oxidative fibers in SLN-KO muscle after cold adaptation demonstrates that SLN-KO mice have a diminished reliance on muscle-based thermogenesis.

94. Kinetics of Skeletal Muscle Cytokine mRNA Responses in Exertional vs.

Passive Heat Stroke

Michelle A. King, 1Deb A. Morse,

1 and

Thomas L. Clanton1. 1Department of Applied

Physiology and Kinesiology, University of Florida, Gainesville, Florida

Previously, it has been demonstrated that stressors such as hyperthermia, exhaustive exercise, and endotoxin induce unique cytokine response profiles in skeletal muscle and blood. Exertional heat stroke

(EHS) serves as a model whereby these three stimuli place simultaneous demands on the innate immune system. To explore this we determined the kinetics of cytokine mRNA expression in skeletal muscles, post EHS, and compared these responses to passive heat stroke (PHS), naïve controls (NC) and exercise sham controls (EX). Gene expression was evaluated using qRT-PCR in the diaphragm (DIA) and soleus (SOL) muscles, 30 min and 3 h after treatment. For both EHS and PHS,

peak values for IL-6 mRNA were reached at 30 min. In PHS, DIA up-regulation of IL-6 mRNA was sustained for 3 h. Similarly, in EHS and PHS, IL-10 mRNA expression peaked at 30 min but lessened by 3 h. In PHS there was no elevation in TNFα or IL-1β mRNA expression at any time point. Dissimilarly, in EHS both IL-1β and TNFα were elevated at 30 min and attenuated by 3 hr. Interestingly, SOCS3 mRNA (a downstream response to IL-6 and IL-10 receptor activation and JAK/STAT3 signaling) was greatly elevated at 30 min in SOL, implying cytokine-induced signaling events in EHS began early in the exposure period. We conclude that although cytokine profiles in muscle for IL-6 and IL-10 are similar in response to both EHS and PHS, EHS induces a unique early response of TNFα and IL-1β which is absent in PHS. Based upon creatine kinase elevations in the serum, post EHS, we speculate this reflects inflammatory processes caused by ongoing muscle injury.

95. Sarcolipin Mediates Skeletal-Muscle Based Nonshivering Thermogenesis In

Mammals. Bal NC, Maurya SK, Saporiwala DH, Sahoo SK, Gupta SK, Shaikh SA, Pant M, Rowland

LA, and Periasamy M. Department of Physiology and Cell Biology, College of

Medicine, The Ohio State University, Columbus, OH 43210, United States

Sarcolipin (SLN) is a novel regulator of Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) pump and it is expressed exclusively in striated muscles. In-vitro studies suggested that SLN can increase heat production by uncoupling SERCA-mediated ATP hydrolysis from Ca2+ transport but its role in muscle physiology was not been established. To demonstrate that SLN is the basis for muscle-based nonshivering thermogenesis in vivo, we challenged SLN-/- mice to acute cold (4°C). Here we show that SLN-/- mice without interscapular Brown Adipose Tissue (iBAT)

failed to maintain core body temperature (Tc, 37°C) and died when exposed to acute cold (4°C), whereas wildtype (WT) mice without iBAT were able to maintain Tc. Overexpression of SLN in the null background fully restored muscle-based thermogenesis, suggesting that SLN is the basis for SERCA-mediated heat production. We found that curare mediated blockade of shivering in WT mice without iBAT did not significantly compromise thermogenesis indicating existence of non-shivering thermogenesis (NST) in muscle. Next, we utilized dantrolene (a chemical that inhibits Ryanodine receptor (RyR1)-mediated Ca2+ leak), to test if Ca2+-leak from the SR is involved in the recruitment of the SLN-mediated thermogenesis. Our data showing that dantrolene-treatment impairs thermogenic ability of WT mice without worsening Tc maintenance in SLN-/- mice suggest SR Ca2+-leak may be the mechanism to activate SLN- mediated NST. These data collectively suggest that SLN is an important mediator of muscle- based NST and may play even greater role in mammals that have minimal amounts of BAT. Analyzing the existence of BAT and skeletal muscle, suggest that muscle may be the first site of cold-induced thermogenesis in the evolution of vertebrate endothermic homeothermy.

96. Sarcolipin is the Key Regulator of Diet-Induced Thermogenesis in the

Skeletal Muscle Santosh K Maurya, Naresh C Bal, Danesh

H Sopariwala, Sana A Shaikh, and Muthu Periasamy. Department of

Physiology and Cell Biology, The Ohio State University, Columbus, OH.

Sarcolipin (SLN), a novel regulator of the Sarco-endoplasmic reticulum calcium ATPase (SERCA) pump. SLN causes futile Ca2+ cycling leading to increased ATP hydrolysis by SERCA pump. We found that loss of SLN predisposes mice to obesity; whereas WT mice showed significant upregulation of SLN (3-4 folds) in certain muscles after 12 weeks of high

fat diet (HFD) feeding. To study the mechanistic details of SLN-mediated diet-induced thermogenesis (DIT) we further exploited a new mouse model which overexpresses SLN specifically in skeletal muscle (SlnOE mice). We tested two hypotheses: 1. whether skeletal muscle-specific overexpression of SLN can provide protection against obesity, and 2. if, β-adrenergic signaling is involved in SLN-mediated DIT in skeletal muscle. Results show that despite an increased energy intake, high fat diet (HFD) fed SlnOE mice remained leaner characterized by decreased body fat content (30.0% less) and fat-droplet accumulation in tissues. SlnOE mice exhibited better glucose tolerance, lower fasting blood glucose, cholesterol and triglycerides than WT. Metabolic measurements by indirect calorimetry showed that HFD fed SlnOE mice exhibit increased O2 consumption and heat production. Further, SlnOE mice displayed lower RER than WT mice, which implies that increased rate of fat utilization in SlnOE mice. Next, we examined whether SLN-mediated DIT can be activated by adrenergic signaling pathway in whole animals by administering formoterol, β2- adrenergic receptor specific agonist. Formoterol treatment increases oxygen consumption and fatty acid utilization in SlnOE mice compared to WT mice. These data imply that SLN-mediated DIT could be actively recruited and amplified by strategies including adrenergic stimulation. Taken together our results provide mechanistic insight for SLN-based muscle diet-induced thermogenesis which could offer translational value for the control of obesity.

97. Reduced Atg7 Expression in Muscle Potentiates the Metabolic Effects of

Exercise Against Diet-Induced Obesity and Insulin Resistance

Vitor A. Lira1,2, Jarrod A. Call2, Rhianna C. Laker2, Mei Zhang2, Zhen Yan2,3,4,5,, Department of Health and Human Physiology, Obesity Research and

Educational Initiative, Fraternity Order of Eagles Diabetes Research Center, University of Iowa, Iowa City, IA1.

Departments of Medicine2, Pharmacology3, Molecular Physiology and Biological Physics4, Center for Skeletal Muscle

Research at Robert M. Berne Cardiovascular Research Center5,

University of Virginia School of Medicine, Charlottesville, VA.

Autophagy is a catabolic process for clearance of aggregate proteins and damaged organelles. Skeletal muscle autophagy is induced by exercise training and is essential for metabolic adaptations. However, recent findings show that reduced expression of Atg7 protects against diet-induced obesity and insulin resistance despite causing a severe skeletal muscle phenotype. To gain further insight into this apparently paradoxical finding, we generated muscle-specific Atg7 knockout (Atg7 MKO) mice by

crossing floxed Atg7 (Atg7fl/fl) mice with MCK-Cre mice. Atg7 MKO mice had normal exercise capacity, glucose tolerance and presented a slight reduction in body weight (~5%) at 12-14 weeks of age. After 12 weeks on high-fat diet (HFD, 60% calories from fat), Atg7 MKO mice displayed substantial limitations in basal autophagy flux in muscle (i.e., high p62 accumulation and blocked conversion of LC3-I into LC3-II), but gained weight and developed insulin resistance at the same rate as their wild-type littermates. However, voluntary running in Atg7 MKO mice resulted in a significant reduction in body weight gain and alleviated glucose intolerance caused by HFD (assessed via glucose tolerance test and insulin tolerance test). Surprisingly, ~40% of Atg7 MKO mice that

exercised died suddenly between 8 and 12 weeks of intervention (20-26 weeks of age) possibly due to partial deletion of the Atg7 gene in the heart. No early deaths were observed in Atg7 MKO mice on HFD that remained sedentary throughout the study. These findings indicate that reduced Atg7 gene expression in mouse muscle potentiates the metabolic benefits of exercise. Still, our results do not support a safe use of chronic inhibition of Atg7 as therapeutics for metabolic diseases.

Mitochondria In Muscle Health and

Disease

98. Effect of Rapamycin on the Mitochondrial Profile in Skeletal Muscle

of Aged Rats Alexandra K. Gentilella1, Linda M.-D.

Nguyen1, Kurtis A. Dickson1, Anna-Maria Joseph2, Dallas Khamiss2, Drake Morgan3,

Christy Carter2, and Peter J. Adhihetty1 1Dept. of Applied Physiology and

Kinesiology and the Center for Exercise Science, 2Dept. of Aging and Geriatric

Research, 3Dept. of Psychiatry, University of Florida, Gainesville, FL.

Rapamycin is a drug widely used clinically for its immunosuppressant and antiproliferative/chemotherapeutic roperties. Recently, it has also been shown to extend lifespan which may, in part, be due to rapamycin-induced modifications in mitochondrial metabolism but the specific underlying mitochondrial mechanisms are currently debatable and unclear. Thus, to address whether rapamycin influences the mitochondrial profile in skeletal muscle (quadriceps), we administered (IP) rapamycin (R; 1mg/kg IP; 3X/week; 5weeks) in young (YR; 6m; Fisher 344; n=6) and old (OR; 25m) animals, and compared these to controls (YC, OC; 1mg/kg saline). Interestingly, rapamycin seemed to have differential effects in O and Y muscle as it did not alter overall mitochondrial content in Y or O muscle, but did significantly reduce (31%) ETC complexes (I, II and IV) in the O,

while Y tissue was unaffected. Additionally, rapamycin tended to increase (32 %) the key mitochondrial regulator, PGC-1α, the mitochondrial chaperone HSP70 (10%), and the mitochondrial marker, cytochrome c (2.2-fold), in Y but not O muscle. Thus, rapamycin treatment seems to have distinct age-associated effects on mitochondrial regulation with modest mitochondrial biogenesis induction in young and mitochondrial dysfunction old muscle.

99. Changes in Energy State Acutely

Alter Insulin Sensitivity in Healthy Humans

Chien-Te Lin1,3, Laura A. Gilliam1,3, Patty M. Brophy1,3, Angela H. Clark1, Terence E. Ryan1,3, Robert C. Hickner1,2,3, and P.

Darrell Neufer1,2,3. East Carolina Diabetes and Obesity

Institute1, Departments of Kinesiology2 and Physiology3, East Carolina University,

Greenville, NC 27834 We recently found in rodents that both acute and chronic energy oversupply by high fat diet (HFD) increases skeletal muscle mitochondrial H2O2 emission potential (mEH2O2) and decreases insulin sensitivity (SI). Both of these deleterious effects of HFD were attenuated by simultaneously inducing a mild increase in energy expenditure. To determine the impact of acute and short-term chronic changes in energy status on mEH2O2 and SI in humans, ten healthy sedentary young male subjects (24.9±1.9 yr, BMI=25.2±0.7, SI=4.73±0.41), consumed a 60% HFD for 18 days. Intravenous glucose tolerance tests and skeletal muscle biopsies were performed in both fasted and fed states (8h after HFD meal) on Days 0, 6, 12, and 18. Mild exercise (1h at 50% HRmax twice/d) was performed from days 13-17. Muscle (permeabilized fiber bundles) mitochondrial respiratory capacity was unaltered by HFD or mild exercise. mEH2O2 increased (40-46%) and SI decreased (31-37%, P<0.05) in response to each HFD meal, but returned to baseline after overnight fasting. Prior

daily exercise blunted both the mEH2O2 and SI response to HFD meal. SI correlated with mEH2O2 across all metabolic states (R2=0.09, P<0.05). These data provide evidence that SI fluctuates in response to acute changes in whole body energy state during HFD feeding/fasting cycles, and that governance of mEH2O2 may be a primary factor regulating SI in skeletal muscle. Supported by NIH DK074825.

100. Accelerated Lipid Oxidation

Increases the Rate of Mitochondrial H2O2 Production in Skeletal Muscle

Cody D. Smith1,2, Chien-Te Lin1, Kelsey H. Fisher-Wellman1,3, Laura A. A. Gilliam1,2,

Lauren R. Reese1,2, Cheryl A. Smith1,3, Hyo Bum Kwak1,3,and Darrell Neufer1,2,3 Institution and contact information:

East Carolina Diabetes and Obesity Institute1, Departments of Physiology2 and

Kinesiology3, East Carolina University, Greenville, NC 27834

Increasing lipid oxidation flux has been suggested as a therapeutic strategy to treat insulin resistance and diabetes. However, mice overexpressing peroxisome proliferator-activated receptor-α in skeletal muscle (MCK-PPAR) are characterized by elevated lipid oxidation but reduced glucose and insulin tolerance. To explore the potential mechanism(s) underlying the development of insulin resistance, aspects of mitochondrial function were assessed in permeabilized muscle fiber bundles (PmFbs) prepared from red and white gastrocnemius muscle (RG/WG) of MCK-PPAR and wild-type (WT) mice. Maximal ADP-stimulated O2 consumption (mean±SEM) was higher (P<0.05) in PmFbs from MCK-PPAR (RG: 137.4±15.8; WG: 53.4±4.8 pmol O2/sec/mg dry wt) vs WT (RG: 83.1±5.0; WG: 12.9±1.1) during lipid-supported respiration. Mitochondrial membrane potential was >20% higher in PmFbs from MCK-PPAR WG during lipid-supported basal respiration, which is associated with greater rates of H2O2

production (MCK-PPAR RG: 16.3±1.1, WG:

10.1±0.7; WT RG: 9.8±1.7, WG: 3.3±1.2 pmol H2O2/min/mg dry wt). Interestingly, the ratio of reduced:oxidized glutathione (GSH:GSSG) was not different in MCK-PPAR mixed-fiber-type skeletal muscle; however, total glutathione concentration was 18% higher in MCK-PPAR mice. This may be a compensatory adaptation in MCK-PPAR skeletal muscle to balance the increased rate of H2O2 production due to accelerated lipid oxidation. Collectively, these data suggest that elevated flux through fatty-acid oxidation increases mitochondrial H2O2 emission, a factor that may contribute to development of insulin resistance.

101. Protein Kinase A Activity Augments

Skeletal Muscle Mitochondrial Bioenergetics

Daniel Stephen Lark 1,2, Lauren Rose Reese 1,3 and P. Darrell Neufer 1,2,3

1East Carolina Diabetes and Obesity Institute, Departments of 2Kinesiology and

3Physiology, East Carolina University Recent work has provided evidence that intramitochondrial cAMP-dependent signaling can augment mitochondrial respiratory capacity, although the mechanism is unknown. Complex I plays a critical role in determining the kinetics of the overall electron transport system and is also a key site of oxidant production. The purpose of this study was to determine whether mitochondrial protein kinase A-signaling alters either the respiratory kinetics or oxidant production. Permeabilized myofibers were prepared from white (WG) and red (RG) gastrocnemius muscle of 4h fasted, 10 week old male C57BL6/NJ mice (n=5). All experiments were performed in the absence or presence of H89 (10 μM), a potent PKA inhibitor. In WG, PKA inhibition decreased (P<0.05) the Km (-57%) and Vmax (-51%) for pyruvate, and the Km (-86%) and Vmax (-59%) for ADP, suggesting allosteric regulation by phosphorylation at multiple sites within the ETS. PKA inhibition also

decreased H2O2 emission (-75%) and production (-53%) at complex I induced by succinate-supported reverse electron flow, but did not affect H2O2 scavenging. In RG, PKA inhibition did not affect the Km but did decrease Vmax (-34%) for pyruvate, as well as the Km (-85%) and Vmax (-60%) for ADP. Furthermore, in RG, PKA inhibition decreased H2O2 emission (-53%) but not production or scavenging. These findings reveal that acute PKA inhibition alters mitochondrial respiratory kinetics, increasing the sensitivity to respiratory substrates but decreasing maximal respiratory capacity and H2O2 production and therefore suggest that PKA-dependent phosphorylation plays an important role in the regulation of both mitochondrial energy production and redox signaling.

102. A Genome-wide Approach to Delineate Novel PPARδ Targets Involved

in Muscle Fitness Emily Y. Smith, Zhenji Gan, Rick Vega,

Daniel P. Kelly Sanford Burnham Medical Research

Institute

The nuclear receptor PPARδ is a key regulator of skeletal muscle fuel metabolism. Muscle specific PPARδ transgenic mice exhibit many of the metabolic benefits of exercise in the absence of training, including enhanced capacity for muscle glucose and fatty acid oxidation, an increase in slow-twitch muscle fibers, and supernormal endurance exercise performance. We recently discovered a novel mechanism whereby PPARδ regulates lactate dehydrogenase b (Ldhb) gene transcription through cooperation with AMP-activated protein kinase (AMPK) and the transcription factor MEF2A. As an initial step to determine whether this PPARδ/AMPK/MEF2A cooperative mechanism is relevant to a broader array of muscle targets, we conducted unbiased genome-wide surveys of PPARδ binding sites in primary human myotubes in the

absence and presence of AICAR, a pharmacological AMPK activator. These studies identified 432 and 682 PPARδ occupation peaks in vehicle- and AICAR-treated myotubes, respectively. The PPARδ binding sites included many known PPAR target genes involved in cellular lipid metabolism. Interestingly, HOMER motif enrichment analysis revealed a significant number of peaks containing MEF2 sites only in AICAR treated myotubes. The long term goal of these studies is to define novel gene targets and pathways involved in improving muscle fuel metabolism and function relevant to a variety of metabolic and muscle diseases.

103. Effects of Voluntary Running and Milk-Protein Supplements on Skeletal Muscle Sirtuins in Rats with Elevated Risk Factors for Metabolic Disorders

Sanna Lensu1, Satu Pekkala2, Anne Mäkinen1, Juha J. Hulmi1, Anu

Turpeinen3,Urho M. Kujala2, Lauren G. Koch4, Steven L. Britton4 and Heikki

Kainulainen1 1Department of Biology of Physical Activity

and 2Department of Health Sciences, University of Jyväskylä, Jyväskylä, Finland,

3Valio Ltd, R&D, Helsinki, Finland and 4Department of Anesthesiology, University of Michigan, Ann Arbor, Michigan, USA.

Epidemiological studies indicate that exercise and consumption of milk proteins associate with a lower risk of metabolic disorders and cardiovascular diseases. These improvements are partially caused by improved aerobic capacity of skeletal muscles. Sirtuins (SIRT1-7) are important regulators of energy metabolism e.g. by inducing mitochondrial biogenesis and by regulating enzyme activities in metabolic pathways. We studied the effects of long-term milk protein supplements (whey protein; WP, and milk protein product containing carbohydrates; PD) and exercise (running wheel; RW) for 23 weeks on skeletal muscle sirtuins in low-capacity runner (LCR) rats. LCR rats are selectively

bred for low aerobic exercise capacity and have elevated risk factors for metabolic syndrome and cardiovascular diseases. Rats supplemented with whey and free access to running wheels (WP+RW) ran significantly less during the first 5-16 weeks of the intervention compared to RW and PD+RW groups. For total running distances run at 23 weeks, the WP+WR, RW, and PD+RW groups recorded distances of 495, 740, and 932 km, respectively. All groups with the access on running wheels improved similarly for maximal treadmill running capacity compared to non-running groups (sedentary control; SED). For plantaris muscle, the largest changes in sirtuin protein expression were in mitochondrial sirtuins SIRT3-5. Running increased by 250% (p<0.01) SIRT3 expression compared to SED rats. Protein supplements per se did not increase SIRT3 expression but when combined with exercise, increased by 400% (p<0.001). Increased SIRT4 expression was observed in RW and WP+RW groups (p<0.001). While running alone did not increase SIRT5 expression, all other groups increased significantly (p<0.001). SIRT6, which inhibits glycolysis, exhibited decreased expression in RW and WP+RW (p<0.01 and< 0.05, respectively) and increased expression in PD (p<0.001). SIRT1 and SIRT7 showed more modest differences. In conclusion, these results suggest that sirtuin activity coordinates an increased oxidation of lipids for energy use in response to exercise and protein supplementation.

104. Alternative NF-B and MyoD

Cooperatively Regulate PGC-1 During Myogenesis

Jonathan Shintaku1,2 and Denis C. Guttridge1

1Department of Molecular Virology, Immunology, and Medical Genetics,

2Molecular, Cellular, and Developmental Biology Graduate Program, The Ohio State

University Dysfunction in skeletal muscle metabolism

and NF-B signaling are commonly

associated with diseases such as obesity

and diabetes. NF-B dysregulation and low

expression of PGC-1, a master regulator of oxidative metabolism and mitochondrial biogenesis, have been identified as risk factors for development of these diseases.

In contrast, mice over-expressing PGC-1 are able to run further and endure higher

workloads. Understanding how PGC-1 expression is regulated will offer insight into skeletal muscle metabolism and may help elucidate mechanisms of metabolic disease

pathogenesis. PGC-1 transcription is best known to be induced during myogenesis, but there are few reports characterizing how these processes are linked. Recently, the

alternative NF-B transcription factor RelB was identified as directly binding the PGC-

1 gene during differentiation to induce transcription. We sought to determine

whether PGC-1 expression is directly regulated by myogenic transcription factors and how this may be coordinated with

alternative NF-B to synergistically regulate skeletal muscle metabolism. We therefore initially focused on identifying myogenic transcription factors that could regulate

PGC-1 transcription. Bioinformatics and ChIP analyses determined that, during skeletal muscle differentiation, the myogenic transcription factor MyoD binds several

conserved sites along the PGC-1 locus.

This binding is important for PGC-1 expression both during myogenesis and in differentiated skeletal muscle. This is in

sharp contrast to PGC-1 which contains very few conserved MyoD binding sites and,

unlike PGC-1, is transcriptionally induced by stimuli such as exercise and cold.

Crosstalk between alternative NF-B and

MyoD in regulating PGC-1 was assessed by inhibiting either RelB or its upstream

kinase activator IKK. Knockdown of either

factor reduced MyoD binding at the PGC-1 gene locus. Furthermore, knockdown of MyoD reduced the binding of RelB, thus identifying novel crosstalk between

alternative NF-B and MyoD in regulating muscle oxidative metabolism.

105. Obesity Modulates Insulin Signaling and Lipidome in Human Primary

Myotubes Christopher W Paran1, Sanghee Park1, Haowei Song2, Joseph A Houmard1, G Lynis Dohm1, Heather A Lawson2, John

Turk2, Katsuhiko Funai1,2 East Carolina Diabetes & Obesity Institute1, Washington University School of Medicine2

Aberrant lipid metabolism in muscle is probably directly relevant to the pathogenesis of muscle insulin resistance. The exact identity of lipotoxic agents that promote human diabetes remains elusive. No studies have comprehensively examined lipid constituents of muscle cells from lean (LN) and obese (OB) humans, but such comparisons are facilitated by recent technological advances in mass spectrometry-based lipidomics. The amount of tissue contained in standard human muscle biopsies is insufficient for comprehensive lipid analyses, but Human Skeletal Muscle Cells (HSkMCs), primary cells isolated from muscle biopsy samples, can proliferate ex vivo and differentiate into myotubes. These cells appear to maintain some metabolic properties observed in vivo. Importantly, HSkMCs from insulin resistant individuals remained insulin resistant in vitro. Lipidomic analyses in LN and OB HSkMCs revealed measurable differences between multiple species of lipids between the groups. Some lipid species that have been implicated to be lipotoxic, such as 16:0/16:0-diacylglycerol and 16:0-ceramide were altered. In addition, multiple species of phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and lyso-phosphatidic acid were different between LN and OB HSkMCs. Because such differences were observed 4 wk ex vivo culture of HSkMCs, it is likely that they reflect long-lived changes in expression of genes involved in lipid metabolism. In turn, these changes in gene expression might promote insulin resistance in these cells. As there are multiple enzymes and pathways that can generate most lipid species, we conducted RNA-seq transcriptomic profiling

of the HSkMCs to identify enzymes responsible for the differences in lipid milieu. We identified 26 lipid-related genes that tended to be different between LN and OB HSkMCs and propose that aberrant muscle lipidome, and potentially pathogenesis of muscle insulin resistance, are due to changes in expression of these genes.

106. Effects of Exercise Training and High Fat Diet on Glycerol Kinase Protein

Content in Skeletal Muscle Kazuhiko Higashida and Mitsuru Higuchi

Faculty of Sport Sciences, Waseda University, Japan

Skeletal muscle stores considerable amounts of triglyceride. It is well known that chronic exercise training and obesity both increase intramuscular triglycerides. However, there is little information about the regulation of lipid synthesis in skeletal muscle, which depends on the provision of exogenous fatty acids and the availability of glycerol-3-phosphate for esterification. Glycerol kinase catalyzes the transfer of a phosphate group to form glycerol-3-phosphate. Although liver and kidney are thought to be the only tissues that express significant glycerol kinase activity, recent studies indicate that glycerol kinase activity is functional in skeletal muscle. Therefore, the purpose of this study was to examine the effects of exercise and high fat feeding on glycerol kinase expression in skeletal muscle. Four-week-old male Wistar rats were exercised by swimming for 2 h/ day, 5 days/week for 4 weeks. Another set of rats were fed high fat diet (50% calories from fat) for 4 weeks. Glycerol kinase protein content in epitrochlearis muscle was increased by about 2-fold after exercise training. Incubation with 0.5 mM 5-aminoimidazole-4-carboxamide ribonucleoside for 6 h significantly increased glycerol kinase mRNA expression in epitrochlearis muscle. Consistent with previous report, the level of glycerol kinase protein content was elevated in adipose tissue of rats fed high fat diet.

Furthermore, there was a significant increase in glycerol kinase protein content in epitrochlearis muscle in response to 4-week high fat diet. These data suggest that exercise training and high fat diet feeding both increase glycerol kinase protein content, and activation of AMP-dependent protein kinase is involved in exercise-induced increase in glycerol kinase in skeletal muscle.

107. Exercise Training Induces a Diaphragmatic Phenotype that Resists

Apoptotic Stimuli Kurt J. Sollanek1, Andreas N. Kavazis2, Ashley J. Smuder1, Michael P. Wiggs1, Aaron B. Morton1 and Scott K. Powers1. 1Department of Applied Physiology and

Kinesiology, University of Florida, Gainesville, Florida; 2 School of Kinesiology,

Auburn University, Auburn, Alabama. Mechanical ventilation (MV) is a life-saving intervention for patients in respiratory failure. Unfortunately, MV leads to rapid diaphragmatic atrophy and contractile dysfunction, collectively termed ventilator-induced diaphragm dysfunction (VIDD). Recent evidence reveals that endurance exercise training performed prior to MV results in protection against VIDD. Currently, the mechanism(s) mediating this protection are unclear, however adaptations within the mitochondria may be responsible. PURPOSE: These experiments investigated alterations in the diaphragmatic mitochondrial phenotype following endurance exercise training. METHODS: Female Sprague Dawley rats (~4 months) were assigned to a control (CON) or endurance exercise-training (EXS) group. EXS animals were exposed to treadmill running during a 5 day habituation period, followed by a two week training protocol (30m/min at 0°incline, 60 mins/day). Animals were sacrificed 24 hours after their last training session. Subsequently, diaphragm mitochondria were isolated and functional measurements were made including respiratory control ratio (RCR;

State 3/4 respiration), mitochondrial permeability transition pore assessment (mtPTP) and an apoptotic protein release assay. Mitochondrial samples were also probed for protein levels of endogenous antioxidants via western blotting. RESULTS: EXS does not change RCR values in diaphragm mitochondria (CON: 6.3±0.2 vs. EXS: 6.4±0.4, P>0.05). However, EXS does alter mtPTP opening kinetics resulting in a lower absolute Vmax (CON: 0.077±0.0.006 abs/min vs. EXS: 0.059±0.0.005 abs/min, P<0.05) and an increased time until reaching Vmax (CON: 615±36 sec vs. EXS: 767±23 sec, P<0.05). Additionally, EXS decreases the release of cytochrome c from mitochondria treated with pro-apoptotic stimuli (P≤0.05). Potential mediators for these changes may be the result of increased antioxidant expression (SOD2, Catalase, P<0.05). CONCLUSION: Exercise training results in adaptations to the diaphragmatic mitochondrial phenotype which allows them to resist pro-apoptotic stimuli. These adaptations may be responsible for the exercise-induced protection against VIDD. Supported by the NIH R01 AR064189 awarded to SKP and an alumni fellowship awarded to KJS.

108. Cancer Chemotherapy Impairs Skeletal Muscle Mitochondrial Function

in Non-Tumor Bearing Tissue Laura A. A. Gilliam1,3, Daniel S. Lark1,2, Kelsey H. Fisher-Wellman1,2, Maria J. Torres1,2,Lauren R. Reese1,3, Brook L.

Cathey1,3, and P. Darrell Neufer1,2,3 East Carolina Diabetes and Obesity

Institute1, Departments of Kinesiology2 and Physiology3

East Carolina University, Greenville, NC Fatigue and muscle weakness are chronic side effects in breast cancer patients undergoing chemotherapy. Our previous findings show the chemotherapeutic agent doxorubicin (DOX) causes muscle weakness and impairs skeletal muscle mitochondrial function, suggesting mitochondria in non-tumor-bearing tissue

can be targeted by cancer chemotherapy. We hypothesize that the combined effect of cancer and chemotherapy compromises mitochondrial respiratory control and increases reactive oxygen species (ROS) production, leading to a decrease in muscle mitochondrial function and ultimately impaired contractile function. Using an immunocompetent syngeneic breast cancer model, EO771 cells were implanted into the mammary fat pad of ovariectomized C57/BL6 mice (TB), and a subset received a single injection of DOX (20 mg/kg). Compared to controls, maximal ADP-stimulated O2 consumption was decreased during respiration supported by Complex I (-17.1 ± 7.8 %, p<0.01) and Complex II (-11.9 ± 9.9 %, p<0.05, n=9/group) in permeabilized fiber bundles (PmFB) from the soleus muscle of TB + DOX mice. PmFB from both TB (121 ± 38 %) and DOX (360 ± 66 %) treatment alone displayed increased rates of mitochondrial H2O2 emitting potential. Compared to controls, cancer chemotherapy lowered maximal isometric tetanic force (TB + DOX: 37.6 ± 2.8 N/cm2; CTRL: 43.5 ± 1.1 N/cm2). Preliminary data suggest targeted overexpression of mitochondrial catalase in muscle is protective of the cancer chemotherapy-induced mitochondrial decline in respiration and attenuates the elevation in ROS. Our findings indicate cancer chemotherapy causes mitochondrial dysfunction, impairing respiration and elevating ROS, representing an underlying cause of cancer chemotherapy-induced muscle dysfunction. Supported by F32-AR061946 and R01-DK0796907.

109. Rat Model Systems to Explore the

Link Between Exercise Capacity, Complex Disease, Aging, and Longevity Lauren Gerard Koch and Steven L. Britton

Department of Anesthesiology, University of Michigan, Ann Arbor, MI.

Exercise capacity can be divided into two components: 1) an intrinsic (inborn) component that operates in the sedentary

state, and 2) an extrinsic (response) component that follows as an adaptation accrued from all physical activity above the sedentary state. To explore the genetic and functional connection between low exercise capacity and increased morbidity and mortality, we developed via selective breeding, low and high rat lines for both the inborn and response elements. Both contrasting model systems use the genetically heterogeneous (N:NIH) rats as founder populations. For the Intrinsic Model: Selection was based upon the maximal distance run using a speed-ramped treadmill protocol in the non-trained condition. The founder population averaged 355 m of running until exhausted. After 28 generations of selection (n=11,422) the Low Capacity Runners (LCR) averaged 236 m and the High Capacity Runners (HCR) averaged 1,960 m run at exhaustion. For the Response Model: Selection was based on gain in maximal distance run after 8 weeks of moderate treadmill training. For the founder population, exercise training produced a 140 m gain in exercise capacity. After 15 generations of selection (n = 3,114), High Response Trainers (HRT) improved 223 m while Low Response Trainers (LRT) declined -65 m given the same absolute training environment. Through collaborative studies, we find the LCR, relative to the HCR, have a decreased lifespan, and demonstrate increased susceptibility for numerous complex diseases including fatty liver disease, Alzheimer's-like neurodegeneration, and inducible cancer, whereas HCR retain higher physical activity levels, VO2max, and lean body mass with age. Relative to HRT, LRT fail to increase VO2max and to display cardiac remodeling, and skeletal muscle angiogenesis with training. Compared to inbreds, selected models are mechanistic based, maintain genetic complexity, derive from a known pedigree, and are better suited for translational studies at all levels of biological organization.

110. 2-Hydroxyestradiol as a Pro-Oxidant Regulator of Muscle Mitochondrial

Function Lauren R. Reese 1,3, Daniel S. Lark 1,2, and

P. Darrell Neufer 1,2,3 East Carolina Diabetes and Obesity

Institute1, Departments of Kinesiology2 and Physiology3, East Carolina University,

Greenville, NC 27834 Estrogens exert numerous effects on mitochondria, but the effect of metabolites like 2-Hydroxyestradiol (2-HE) are poorly understood. Previous work has suggested that 2-HE is capable of generating oxidant species, but the exact species and the potential effects of this oxidant production on mitochondrial function are unknown. In a series of in vitro assays using the Amplex Red detection system and in the absence of superoxide dismutase (SOD), 2-HE at 0.2, 1.0 and 5.0 μM concentrations was found to generate a dose-dependent, linear rate of H2O2 that was abrogated by inclusion of catalase. Under the same conditions, 2-Methoxyestradiol, a metabolite of 2-HE, did not generate detectable amounts of H2O2. Peroxidases have been implicated in 2-HE derived oxidant production. However, high-resolution respirometric experiments revealed that 2-HE dose-dependently consumed O2 even in the absence of horseradish peroxidase, an enzyme required for H2O2 detection via Amplex Red, providing additional evidence that 2-HE generates H2O2 directly. Finally, addition of 2-HE to actively phosphorylating mitochondria in permeabilized fiber bundles (PmFB) from the gastrocnemius of mice resulted in a dose-dependent decrease in maximal ADP-stimulated O2 consumption (0.2 μM 2-HE, -9.0 ±3.4%; 1 μM 2-HE -20.8 ±6.7%, P<0.05; and 5 μM 2-HE, -26.8 ± 9.4%, P<0.05, n=8/group). These data demonstrate that 2-HE generates H2O2 non-enzymatically at rates sufficient to negatively regulate oxidative phosphorylation. As both estrogen metabolism and redox biology regulate cellular homeostasis, these findings raise the possibility that 2-HE may serve as an

estrogen-derived, redox-dependent mitochondrial signaling molecule.

111. Effect of Exercise on Prostate Cancer-Induced Changes in the

Mitochondrial Profile of Rodent Skeletal Muscle

Linda M.-D. Nguyen1, Danielle J. McCullough1, Lucas R. Goss2, Michael A. Olson3, Anna-Maria Joseph2, Bradley J.

Behnke1, Peter J. Adhihetty1 1Dept. of Applied Physiology and

Kinesiology and the Center for Exercise Science, 2Dept. of Biology, 3Dept. of

Chemistry, 4Dept. of Aging and Geriatric Research University of Florida, Gainesville

Cancer afflicts numerous primary tissues and is often associated with secondary skeletal muscle atrophy and wasting, known as cachexia. Mitochondrial abnormalities contribute to muscle atrophy/dysfunction in various diseases, and experimental perturbations (i.e. exercise) augmenting mitochondrial content tend to improve muscle but whether this occurs in prostate cancer is unknown. We used an orthotopic prostate cancer model, injecting AT-1 adenocarcinoma cells (tumor-bearing; TB) or saline (non-tumor bearing; NTB) into the prostate of young (5 mo) rats, and assigned a sedentary (Sed) or exercise (7 weeks; Ex) protocol (n=5-10/group). Exercise did not alter tumor size (4.12 g TB-Sed; 4.3 g TB-Ex), nor was there tumor-associated atrophy in muscles (SOL, EDL, Pl, TA). As expected, training induced mitochondrial biogenesis by increasing (P<0.05) COX activity, cytochrome c, and electron transport complexes (30%, 3.3-fold, 1.8-fold, respectively) in NTB animals while the TB group had a significantly blunted response. Interestingly, tumor presence per se did not alter mitochondrial content/regulation but tended to reduce (~25%) mitochondrial respiration and elevate (~2.5-fold) mitochondrial free radical production, and exercise did not evoke improvements. Our data suggests prostate cancer impairs exercise-induced

mitochondrial biogenesis and impairs mitochondrial function in skeletal muscle.

112. Mitochondrial Capacity Is Decreased In Skeletal Muscle with

Estrogen Depletion Torres, MJ1,2; Gilliam LAA1,3; Neufer, PD1,2,3

East Carolina Diabetes and Obesity Research Institute1, Dept. Kinesiology2 and

Physiology3, East Carolina University, Greenville, NC 27834

The onset of menopause dramatically increases a woman’s risk to develop cardiovascular disease and type-2 diabetes. Menopause coincides with a significant decline in the production of ovarian hormones, particularly 17β-estradiol (E2), a key regulator of energy and glucose homeostasis in numerous peripheral tissues. Skeletal muscle is responsible for ~80% of insulin-stimulated glucose uptake, and mitochondrial function in muscle has been linked to the control of insulin sensitivity. However, the underlying mechanism(s) by which E2 regulates insulin sensitivity and mitochondrial function in skeletal muscle remains unresolved. Using a short-term ovariectomized female mouse model (C57BL/6, 2 week-OVX), we determined the impact of E2 depletion on mitochondrial respiratory function in skeletal muscle. State 3 Complex Iand Complex II-linked respiration was decreased (-26 ± 7 %, p<0.05) in permeabilized fiber bundles from red gastrocnemius of OVX mice compared to normally cycling females (CTRL). Following E2 depletion citrate synthase activity was reduced (CTRL: 86 ± 6; OVX: 59 ± 4 μmol/min/mg protein, p<0.05), suggesting a decrease in mitochondrial content. The cellular redox environment, as indexed by the GSH/GSSG ratio, shifted to a more oxidized state with E2 depletion, primarily reflected by an increase in the total GSSG concentration (24 ± 6 %, p<0.05). Our results highlight the protective effects of E2 on mitochondrial function and redox state in skeletal muscle, and provide a potential mechanism for the pro-diabetogenic effects of menopause.

113. Is Mitochondrial COX Deficiency a Cause of Myofiber Atrophy in Humans? Martin Picard, D.Hom, Julie Murphy, Sally Spendiff, Russell T Hepple, Basil J Petrof, Douglas C Wallace, Douglass M Turnbull,

Tanja Taivassalo The Center for Mitochondrial and

Epigenomic Medicine, The Children’s Hospital of Philadelphia and University of

Pennsylvania, Colket Translational Research Building, Philadelphia,

Pennsylvania

Skeletal muscle fibers undergo hypertrophy and atrophy in response to mechanical and metabolic stressors, and it has been suggested that mitochondrial cytochrome c oxidase (COX) deficiency is a cause of muscle fiber atrophy. However, the validity of this notion in human skeletal muscle has not been robustly established, nor have potential underlying molecular mechanisms been established. We evaluated the relationship between increasing severity of COX deficiency (normal, intermediate or completely deficient) and myofiber size histologically, under various conditions. These included quadriceps biopsies of patients with defined single or multiple mtDNA deletions, mtDNA point mutations, and diaphragm biopsies after mechanical ventilation where rapid muscle atrophy occurs. COX negative cells captured by laser microdissection contained high-levels of abnormal mtDNA species, but were generally either of similar or greater size than COX normal fibers. Strikingly, COX-intermediate (containing a mixture of normal and deficient mitochondria) fibers were atrophied by ~40% in the diaphragm. One possible explanation is that the loss of respiratory chain function causes transient atrophy, along with a shift towards glycolytic metabolism akin to the Warburg effect, which eventually restores normal fiber size despite complete COX deficiency. However, COX negative myofibers did not show up regulation of glycolytic histological markers, and gene expression for key glycolytic enzymes in homogenates was actually decreased (P < 0.01), thus refuting this

hypothesis. Tracking individual myofibers along their length in longitudinal orientation of the muscle convincingly demonstrates the absence of a relationship between segmental COX deficiency, glycolytic metabolism and myofiber atrophy. Finally, we performed transcriptomic studies in human cybrid cell lines with increasing mtDNA mutation load causing COX deficiency. This revealed that transcriptional and chromatin remodeling are maximally induced when an equal mixture of normal and mutant mtDNA coexist within cells. Thus, intermediate levels of respiratory chain deficiency may impact myofiber size through transcriptional deregulation, but human myofibers with complete mitochondrial compromise can maintain normal size.

114. Curcumin Evokes Mitochondrial

Alterations and Suppresses Apoptosis in Muscle and BAT of Aged Mice

Nicholas R. Wawrzyniak1, Andrew Duarte1, Linda M.-D. Nguyen1, Anna-Maria Joseph2, Andrew S. Layne1, David S. Criswell1, and

Peter J. Adhihetty1 1Department of Applied Physiology &

Kinesiology – Center for Exercise Science, 2Department of Aging & Geriatric Research,

University of Florida, Gainesville FL

Curcumin, a polyphenol found in the spice turmeric, is shown to have antioxidant and anti-inflammatory properties in multiple tissues, but whether it alters mitochondrial biogenesis/apoptotic pathways is unknown. The aging process is associated with impaired mitochondrial function and elevated mitochondrial apoptotic susceptibility. Potential pharmacological and/or neutraceutical therapeutic interventions capable of improving mitochondrial function, like curcumin, have been postulated to delay this process. Thus, we investigated whether short-term (21d) dietary curcumin supplementation (5% diet) altered mitochondrial biogenesis in muscle and brown adipose tissue (BAT) of aged mice (24 month; C57BL/6) compared to

control diet mice (n=4-6/group). While curcumin supplementation increased the mitochondrial content markers cytochrome c (14%) and COX Vb (26%) and enhanced the mitochondrial regulators Tfam (25%) and NRF-1 (14%) in BAT, it suppressed and/or caused no change in these mitochondrial indices in muscle. In contrast, curcumin treatment evoked significant decreases (P<0.05) in the pro-apoptotic BAX protein in both BAT (20%) and muscle (55%). Our data indicate short-term curcumin treatment in aged mice causes tissue-specific mitochondrial biogenesis adaptations in BAT and muscle while potentially suppressing mitochondrial apoptotic susceptibility in both tissues.

115. Salt Inducible Kinase 1 Is Required for Exercise-Stimulated MEF2 Activity in

Skeletal Muscle Randi Stewart and Rebecca Berdeaux

Graduate School of Biomedical Sciences, UT Health, Houston, TX

Myocyte enhancer factor 2 (MEF2) transcription factors are well characterized regulators of skeletal muscle regeneration and exercise adaptation that can activate slow twitch muscle fiber genetic programs. The transcriptional activity of MEF2 transcription factors is inhibited by class II histone deacetylases (HDAC4, 5, 7, and 9). We previously demonstrated that salt inducible kinase I (SIK1) can de-repress MEF2 activity through phosphorylation and subsequent nuclear export of HDAC5. Other kinases, including calmodulin-dependent protein kinase II (CaMKII) and protein kinase D (PKD), have also been shown to promote MEF2-dependent transcription by phosphorylation of class II HDACs. The extent to which SIK1 is responsible for individual class II HDAC regulation in different physiological contexts remains unclear. We hypothesized that SIK1 activation of MEF2 activity may occur in specific contexts, either in certain fiber types or in response to particular types of stimuli. To test this hypothesis, we generated mice

lacking the kinase domain of Sik1 in all tissues (Sik1-knockout, SIK1-KO) and crossed these mice with MEF2-LacZ reporter mice. Although exercise endurance was similar between wildtype and SIK1-KO mice, we found that loss of full length SIK1 impaired acute exercise-induced MEF2 dependent transcription in quadriceps muscles. Anatomical analysis indicates that the impaired MEF2 activity we observe likely occurs predominantly in white, fast twitch myofibers. Interestingly, we also observe reduced Mef2c, Pgc1α, and Pdk4 mRNA levels in gastrocnemius muscles isolated from un-exercised SIK1-KO mice, suggesting reduced mitochondrial content or function. Since MEF2 isoforms differentially affect not only skeletal muscle metabolism but also skeletal muscle development and regeneration, it will be important to identify the specific MEF2 isoform(s) mis-regulated in SIK1-KO muscles. Furthermore, identification of the fiber type(s) in which SIK1 is required for MEF2 activity and determination of the pattern of MEF2 target gene activation may provide insight into the specific functions of this kinase in skeletal muscle.

116. Protein S-Nitrosylation Protects Against Mitochondrial Oxidative Stress Rebecca J. Wilson1,5, Rhianna C. Laker2

and Zhen Yan1,2,3,4,5 Departments of Biochemistry1, Medicine2, Pharmacology3, Molecular Physiology and

Biological Physics4 and the Center for Skeletal Muscle Research at Robert M.

Berne Cardiovascular Research Center5, at the University of Virginia School of

Medicine, Charlottesville, VA. Protein S-nitrosylation (SNO) is a reversible post-translational modification involved in cell signaling and protection of critical protein thiols against irreversible oxidative stress. We have observed hyper-SNO of cysteine residues of ~300 proteins involved in calcium handling, contractility and muscle metabolism 6 hours after an acute bout of exercise in mouse skeletal muscle. This

was in parallel with a transient increase in mitochondrial oxidative stress, detected by our newly developed oxidative stress-sensitive, fluorescent mitochondrial reporter gene, pMitoTimer. To test the hypothesis that NO-mediated S-nitrosylation protects mitochondria from oxidative stress, we treated MitoTimer-transfected C2C12 myoblasts with an NO donor and confirmed that NO donor protects against antimycin A induced mitochondrial stress. To test this hypothesis in vivo, we used mice heterozygous for S-nitrosoglutathione reductase (GSNOR+/-), which have increased basal protein SNO compared to wild type (WT). We transfected pMitoTimer into the flexor digitorum brevis (FDB) muscle using electric pulse mediated somatic gene transfer. Ten days later, WT and GSNOR+/- mice were subjected to an acute bout of treadmill running (5% incline, 13.5-19 m/min, 75 min), and FDB muscles were assessed by confocal microscopy at 6 hours post-exercise. FDB muscles from WT mice displayed a shift of MitoTimer fluorescence towards red with increased pure red puncta of possibly degenerated mitochondria. In contrast, GSNOR+/- mice did not show these changes following exercise. These findings suggest that increased protein SNO protects mitochondria against oxidative stress and damage, which may play an important role in protecting mitochondrial function and exercise-induced adaptation. 117. Detection of Transient Mitochondrial

Stress and Damage in Skeletal Muscle Following a Single Bout of Exercise by a

Novel Mitotimer Reporter Gene Rhianna C. Laker, Vitor A. Lira, Rebecca J.

Wilson and Zhen Yan. Departments of Medicine and Center for Skeletal Muscle Research at Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine,

Charlottesville, VA

Mitochondrial health is important for skeletal muscle function and is improved by exercise

training. We developed a mitochondrial reporter gene, pMitoTimer, which encodes a fluorescent protein targeted to the mitochondria. MitoTimer protein fluoresces green when newly synthesized and shifts fluorescent spectrum to red when oxidized. To ascertain the impact of an acute bout of exercise in mice on mitochondrial oxidative stress and damage, we performed electric pulse-mediated somatic gene transfer of pMitoTimer in the flexor digitorum brevis (FDB) muscle of one hindlimb. The contralateral limb was transfected with a similar reporter gene that targets to the endoplasmic reticulum (ER or sarcoplamic reticulum in skeletal muscle), called pERTimer. The mice were subjected to a single bout of treadmill running (5% incline, 13.5-19 m/min, 75 min). We performed whole mount confocal microscopy for the transfected FDB muscles in unexercised controls and at 0, 3, 6, 12 and 24 hours post exercise. There was a significant transient shift of the MitoTimer fluorescence red:green ratio towards red at 6 and 12 hours following exercise. This was accompanied by an increase in the number of pure red puncta, which were of mitochondrial origin and possibly represented completely degenerated mitochondria targeted by lysosomes for removal from the cell. Importantly, there was no change in fluorescence when we targeted this reporter protein to the sarcoplasmic reticulum. These findings suggest that an acute bout of exercise causes transient mitochondrial oxidative stress and damage during recovery. This may play an important role in the adaptive process for removal of damaged mitochondria and therefore improving the health of the existing mitochondrial population.

118. Elevated Skeletal Muscle Mitochondrial Quality Control Proteins

Are Associated With Higher Mitochondrial Respiration In Young

Adults Robert A. Standley1, Giovanna Distefano2,

Frederico G.S. Toledo2, Bret H. Goodpaster1, John J. Dubé2, Paul M. Coen1

1Translational Research Institute for Metabolism and Diabetes-Florida Hospital,

Orlando, FL; 2University of Pittsburgh, Pittsburgh, PA

Both acute and chronic aerobic exercise increases the expression and content of mitochondrial quality control proteins in human muscle. However, it is not known if these changes are associated with improvements in mitochondrial performance. This study sought to examine the relationship between mitochondrial quality control proteins and respiration in muscle from young active and sedentary lean and obese adults. Vastus lateralis biopsies were obtained from young active (YA: n=10, 27±1y, 62.5±4.2ml/kg/min), and sedentary lean (LS: n=9, 25±1y, 45.7±1.4ml/kg/min) and obese (OS: n=11, 29±1y, 29.9±1.8ml/kg/min) adults. Individuals were considered active if they participated in ≥5 structured exercise sessions per week. Mitochondrial content (OXPHOS) and the expression of proteins that regulate mitochondrial quality, including fission (Fis1 and DRP1), fusion (Mfn2 and OPA1), autophagy (beclin-1) and mitophagy (Bnip3) were measured by western blot. Mitochondrial respiratory characteristics were determined in permeabilized myofibers by high-resolution respirometry. Aerobic fitness (VO2 peak) was determined by a graded exercise test. Mitochondrial quality control proteins Fis1, DRP1, Mfn2, OPA1 and beclin-1 were elevated in the YA compared to OS (P<0.05) with no difference in Bnip3 (P>0.05). OXPHOS II-SDHB was lower in the YL compared to YA (P<0.05), while total OXPHOS was not different between the groups (P>0.05). Maximal coupled mitochondrial respiration was lower in the LS and OS compared to YA (YA vs.

LS and OS, 294.20 vs. 220.30 and 152.42 pmol O2/sec*mgDW, P<0.05). In addition, mitochondrial fission proteins Fis1 and DRP1 and fusion protein Mfn2 tended to be associated with maximal coupled mitochondrial respiration (Fis1, r=0.43 P=0.053; DRP1, r=0.37 P=0.06; Mfn2, r=0.39 P=0.07). In conclusion, regular physical activity increases mitochondrial quality control protein levels, which is associated with improved mitochondrial respiration independent of markers of mitochondrial content. These findings suggest the quality control mechanisms to increase mitochondria turnover play an important role in exercise-induced improvements in mitochondria performance.

119. Loss of Adenine Nucleotide

Translocase Alters Muscle Mitochondrial Function and Enhances Insulin

Sensitivity Ryan Morrow, Martin Picard, Meagan

McManus, Gilles Gouspillou, Russell T. Hepple, Douglas C. Wallace

Children’s Hospital of Philadelphia Whether mitochondrial dysfunction is a cause or consequence of type II diabetes is still an important question that has yet to be resolved. The adenine nucleotide translocase (ANT1) is a mitochondrial protein that exchanges cytosolic ADP for ATP produced by oxidative phosphorylation. Previous studies have shown that mice deficient in ANT1 have reduced muscle mitochondrial function and develop myopathy and cardiomyopathy. Our goal was to study the relationship of mitochondrial energetic function to insulin sensitivity by using Ant1 -/- and Ant1 +/+ mice fed a normal and high fat diet. The deletion of Ant1 resulted in a striking hyperproliferation of mitochondria in the gastrocnemius muscle as determined by electron microscopy, increased mitochondrial DNA (mtDNA) copy number, and histological staining for PGC-1α. In addition, there was a concomitant 3-fold and 7-fold increase in the mitochondrial

enzymes citrate synthase (CS), succinate dehydrogenase (SDH) and cytochrome c oxidase (COX) in the Ant1 -/- red and white gastrocnemius, respectively. This was accompanied by a muscle fiber type shift, with more myofibers in Ant1 -/- staining for oxidative myosin heavy chain isoforms IIa and IIx. Next we measured oxygen consumption and reactive oxygen species (ROS) production in permeabilized myofibers from red and white gastrocnemius muscle. Normalized to muscle mass, the Ant1 -/- mouse had a 50-150% increase in state II respiration in addition to an increase in ROS production 2-4 times that of control. Finally, Ant1 -/- mice showed signs of improved insulin sensitivity. Ant1 -/- mice were found to be significantly more glucose tolerant then the controls and had a 65% and 16% increase in glucose infusion rate and rate of glucose disposal, respectively, during a hyperinsulinemic-euglycemic clamp. Thus, in this model mitochondrial energy transfer deficiency results in hyperproliferation of mitochondria and reddening of skeletal muscle in conjunction with enhanced insulin sensitivity.

120. Mitochondria-Targeted ROS Scavenger Improves Post-Ischemic Recovery of Cardiac Function and

Attenuates Mitochondrial Abnormalities in Aged Rats

Nelson Escobales1, Rebeca E. Nuñez1, Rebecca Parodi-Rullan1, Joshua R.

Sacher4, Erin M. Skoda4, Sylvette Ayala-Peña2, Peter Wipf4, Walter Frontera1,3,

Sabzali Javadov1 1 Department of Physiology and 2 Department of Pharmacology and

Toxicology, School of Medicine, University of Puerto Rico, San Juan, PR; 3 Department

of PM&R, Vanderbilt University School of Medicine, Nashville, TN; 4 Department of

Chemistry, University of Pittsburgh, Pittsburgh, PA

Mitochondria-generated reactive oxygen species (ROS) are one of the factors that

play a crucial role in the pathogenesis of aging and age-related diseases. In this study, we evaluated the effect of XJB-5-131 (XJB), a mitochondria-targeted ROS scavenger, on the resistance of hearts to ischemia-reperfusion (IR)-induced oxidative stress in aged rats. Male adult (5-month old, n=17) and aged (29-month old, n=19) Fischer Brown Norway (F344/BN) rats were randomly assigned to the following groups: adult (A), adult+XJB (AX), aged (O), and aged+XJB (OX). XJB was administered 3 times per week (3 mg/kg body weight, IP) for four weeks. After treatments, excised hearts were perfused using the Langendorff technique for 30 min, followed by 25-min global ischemia and 60-min reperfusion. Cardiac function was continuously monitored. XJB improved post-ischemic recovery of aged hearts as evidenced by increases of left ventricular developed-pressure (O: 10.2±1.8% vs. OX: 28.7±4.9% of pre-ischemia, P<0.02) and rate pressure product (O: 14.1±1.5% vs. 36.9±3.1% of pre-ischemia, P<0.001) compared to the untreated aged-matched group. Likewise, the rate of left ventricle (LV) pressure rise (dP/dt max) and decrease (dP/dt min) at the end of reperfusion were 2.79 (P<0.023) and 1.89 (P<0.039) folds higher, respectively, for XJB-treated aged rats than for untreated animals. State 3 respiration rate of mitochondria isolated from XJB-treated aged hearts at complexes I, II and IV were 57% (P<0.015), 25% (P<0.016) and 28% (P<0.027), respectively, higher than controls. Ca2+-induced swelling, an indicator of permeability transition pore opening, was reduced in mitochondria from XJB-treated aged rats. These findings indicate that XJB improves mitochondrial function and enhances the resistance of aged rat hearts to IR-induced oxidative stress. This study underlines the importance of mitochondrial-derived ROS in aging-induced cardiac dysfunction and suggests that targeting of mitochondrial ROS may serve as an effective therapeutic approach to protect the aged heart against IR injury.

13th Biennial Advances in Skeletal Muscle Biology in Health and Diseases Conference

Participant List

Christopher M. Adams

Associate Professor University of Iowa 726 N. Van Buren Iowa City IA 52245 United States (319) 331-2955 christopher-adams@uiowa.edu

Volker Adams

Heart Center Leipzig Struempellstrasse 39 Leipzig 04289 Germany +49 341-8651671 adav@medizin.uni-leipzig.de

Peter Adhihetty

University of Florida PO Box 118205 Gainesville FL 32611 United States padhihetty@hhp.ufl.edu

Shakeel Ahmed

Ph.D Student University of Florida- PHHP 5318 D SW 91st Terr. Gainesville FL 32608 United States (352) 339-8520 shakeel81@ufl.edu

Bumsoo Ahn

Graduate Student University of Florida 156 Florida Gym Stadium Rd. Gainesville FL 32611 United States (352) 294-1739 ahnb@hhp.ufl.edu

Umar Alabasi

RSD Student University of Florida- PHHP 4455 SW 34th St., #TT242 Gainesville FL 32608 United States (412) 251-2399 ua1406@ufl.edu

Lindsey Anderson

Doctoral Candidate University of Southern California 1650 Amberwood Dr. #C South Pasadena CA 91030 United States (559) 859-3166 lindseja@usc.edu

Harneet Arora

University of Florida 3520 SW 20th Ave. #24 Gainesville FL 32607 United States (913) 299-7605 harora@phhp.ufl.edu

Ishu Arpan

Graduate Student University of Florida 326 University Village S. #2 Gainesville FL 32603 United States ishuarpanthakur@ufl.edu

Susan T. Arthur

UNC-Charlotte 9201 University City Blvd. Charlotte NC 28223 United States (704) 687-0856 (704) 687-0930 sarthur8@uncc.edu

Ernie Au

Indiana University Purdue University Indianapolis 310 W. Michigan St. # 235 Indianapolis IN 46202 United States (516) 606-3352 ernau@iupui.edu

Keith Baar

University of California Davis One Shields Ave. 181 Briggs Hall Davis CA 95616 United States (530) 752-3367 kbaar@ucdavis.edu

Naresh C. Bal

Research Scientist The Ohio State University 1280 Old Henderson Square Columbus OH 43220 United States (614) 208-1574 bal.8@osu.edu

Alexander Balaez

Student University of Florida/Malcom Randall VAMC 720 SW 34th St. # K-117 Gainesville FL 32607 United States (786) 376-3621 abalaez27@ufl.edu

Marcas M Bamman

Professor and Director University of Alabama at Birmingham UAB Center for Exercise Medicine MCLM 966A 1720 2nd Ave South Birmingham AL 35294 United States (205) 975-9042 mbamman@uab.edu

Joseph Bass

University of Nottingham MRC-ARUK Centre of Excellence for Musculoskeletal Entry Medicine, School of Medicine Royal Derby Hospital Centre Derby Derbyshire De22 3dt United Kingdom mzxjb1@nottingham.ac.uk

Abhinandan Batra

Student University of Florida 999 SW 16th Ave. # 110 Gainesville FL 32601 United States (352) 870-0046 abhinandanbatra@phhp.ufl.edu

Ranjan Batra

Research Fellow Mayo Clinic 4500 San Pablo Rd. Birdsall Building Leonard Petrucelli Lab Jacksonville FL 32224 United States (352) 273-8155 ranjanbatra9@yahoo.com

Luke Beggs

Doctoral Candidate University of Florida 4302 NW 26th Ter Gainesville FL 32605 United States lbeggs@hhp.ufl.edu

Adam Beharry

University of Florida 10000 SW 52nd Ave. Unit E26 Gainesville FL 32608 United States beharrya1@phhp.ufl.edu

Rebecca Berdeaux

Assistant Professor Univ. of Texas Houston University of Texas Medical School 6431 Fannin St., Mse R366 Houston TX 77030 United States (713) 500-5653 rebecca.berdeaux@uth.tmc.edu

Peter Bialek

Pfizer 200 Cambridge Park Dr. Cambridge MA 02140 Peter.Bialek@pfizer.com

Sue C. Bodine

Professor University of California, Davis One Shields Ave. 196 Briggs Hall Davis CA 95616 United States (530) 304-9539 (530) 752-5582 scbodine@ucdavis.edu

Lance Bollinger

Assistant Professor University of Kentucky 201 Seaton Bldg Lexington KY 40506 United States (859) 257-7904 lance.bollinger@uky.edu

Eric Bombardier

Post-Doc University of Waterloo 200 University Ave. W. Waterloo Ontario N2L 3G1 Canada (519) 885-1211 ext. 35791 ebombard@uwaterloo.ca

Andrea Bonetto

Assistant Research Professor IU School of Medicine 980 W. Walnut St. R3-C540 Indianapolis IN 46202 United States (317) 278-8030 abonetto@iupui.edu

Kale S. Bongers

University of Iowa 540H EMRB 500 Newton Rd. Iowa City IA 52242 United States kale-bongers@uiowa.edu

Matthew Borkowski

Aurora Scientific Inc. 360 Industrial Pkwy. S. Unit 4 Aurora Ontario L4g 3v7 Canada (905) 727-5161 ext. 26 (905) 713-6882 mattb@aurorascientific.com

T. Scott Bowen

Postdoctoral Research Fellow University of Leipzig - Heart Center Department of Internal Medicine and Cardiology StrüMpellstraßE. 39 Leipzig 04289 Germany +49 341-8651 bows@med.uni-leipzig.de

Jeffrey J. Brault

Assistant Professor East Carolina University BSOM Physiology 6N98 600 Moye Blvd. Greenville NC 27834 United States (252) 744-1225 braultj@ecu.edu

Matthew Brook

Nottingham University School of Graduate Entry Medicine Royal Derby Hospital Uttoxeter Rd. Derby De22 3dt United Kingdom mzxmb2@nottingham.ac.uk

Ronald G. Budnar

Graduate Student University of North Texas 107 S. Bonnie Brae St. # D Denton TX 76201 United States (972) 804-0042 ronald.budnar@unt.edu

Jatin George Burniston

Reader of Molecular Physiology Liverpool John Moores University Byrom St. Tom Reilly Building Liverpool L3 3af United Kingdom +44 0151-9046265 +44 0151-9046283 j.burniston@ljmu.ac.uk

Jarrod A. Call

University of Virginia 114 Old Fifth Cir. Charlottesville VA 22903 United States (740) 361-4955 jac4mt@virginia.edu

Juliane Cruz Campos

University of Sao Paulo Av. Professor Lineu Prestes, 2415 Sao Paulo Sao Paulo 05588-000 Brazil +55 11-30910931 jucruzcampos@gmail.com

Ermelinda Ceco

Post Doctoral Fellow Northwestern University 240 E. Huron Mcgaw M-410 Chicago IL 60611 United States (312) 714-2440 ermelinda.ceco@northwestern.edu

Kristopher Chain

University of Virginia 1504 Villa Terr. Unit E. Charlottesville VA 22903 United States khc7aw@virginia.edu

Konstantinos Charizanis

Post-Doc University of Florida 4027 SW 28th Terr. Gainesville FL 32608 United States quijotes@ufl.edu

Juan C. Chavez

Director, Experimental Medicine Biogen Idec 14 Cambridge Center Cambridge MA 02142 United States juan.chavez@biogenidec.com

Xiaobin Chen

476 Farrish Cir. Charlottesville VA 22903 (434) 218-9558 xc4w@eservices.virginia.edu

Kyungah Cho

Postdoctoral Fellow University of Florida College of Medicine 1600 SW Archer Rd. Room R1-175 Gainesville FL 32610 United States kyungahcho@ufl.edu

Stephen Chrzanowski

MD-PhD Candidate University of Florida 12935 SW 3rd Pl. Newberry FL 32669 United States (216) 906-2508 stevechrzanowski@ufl.edu

Nele Cielen

Catholic University of Leuven Watertorenlaan 13 Zaventem 1930 Belgium +32 479-911934 nele.cielen@med.kuleuven.be

Thomas L. Clanton

Professor University of Florida 1864 Stadium Rd. 100 FLG Gainesville FL 32611 United States (352) 294-1712 (352) 392-5262 tclanton@hhp.ufl.edu

Paul M. Coen

Investigator Translational Research Institute for Metabolism and Diabetes - Florida Hospital 301 E. Princeton St. Orlando FL 32804 United States (407) 303-1306 (407) 303-1306 paul.coen@flhosp.org

Ronald D. Cohn

Chief, Clinical & Metabolic Genetics The Hospital for Sick Children 555 University Ave. Toronto Ontario M5G 1X8 Canada (416) 813-7654 ext. 228780 ronald.cohn@sickkids.ca

Brittany Charlotte Collins

University of Minnesota 420 Washington St. SE MCB 7-212 Minneapolis MN 55455 United States (612) 625-5022 bcc@ddt.biochem.umn.edu

Amy Lynne Confides

Research Analyst University of Kentucky 700 Riverwood Ln. Lexington KY 40514 United States (859) 797-2818 alferry2@uky.edu

Leslie Consitt

Assistant Professor Ohio University 228 Irvine Hall Biomedical Sciences Ohio University Athens OH 45701 United States (252) 367-9228 consitt@ohio.edu

Robin L. Cooper

Faculty University of Kentucky Department of Biology 675 Rose St. Lexington KY 40506-0225 United States (859) 559-7600 rlcoop1@uky.edu

David S. Criswell

Associate Professor University of Florida Department of APK P.O. Box 118205 Gainesville FL 32611 United States (352) 294-1721 dcriswell@hhp.ufl.edu

Bruce M. Damon

Associate Professor Vanderbilt University Aa-1105 MCN 1161 21st Ave. S. Nashville TN 37232 United States (615) 322-8355 bruce.damon@vanderbilt.edu

Srinivasan Dasarathy

Staff Cleveland Clinic 391 E. St. Andrews Dr. Highland Heights OH 44143 United States (216) 444-2980 (216) 445-3889 dasaras@ccf.org

Patrick Davis

East Carolina University Brody SOM Physiology 600 Moye Blvd. Greenville NC 27834 United States (252) 744-5383 davisp11@students.ecu.edu

Teresa A. Davis

Professor Baylor College of Medicine 1100 Bates St. Suite 9066, BCM-320 Children’s. Nutrition Research Center Houston TX 77581 United States (713) 798-7169 (713) 798-7171 tdavis@bcm.edu

Colleen Deane

Metabolic & Molecular Physiology Research Group School of Graduate Entry Medicine and Health University of Nottingham Royal Derby Hospital, Derby Derbyshire De22 3dt United Kingdom cdeane@bournemouth.ac.uk

Haydar Demirel

Professor University of Hacettepe Turkey +90(533) 772-7156 haydar.demirel@gmail.com

Steve Dodd

Professor University of Florida 8627 SW 42nd Pl. Gainesville FL 32608 United States (352) 294-1711 sdodd@hhp.ufl.edu

Amy Donate

Postdoctoral Associate University of Florida-College of Medicine 1600 SW Archer Rd. Gainesville FL 32610 United States pacakc@peds.ufl.edu

Anthony A. Duplanty

University of North Texas 1810 Scripture St. Denton TX 76201 United States (940) 595-4696 anthony.duplanty@gmail.com

Esther E Dupont-Versteegden

University of Kentucky 900 S Limestone CTW 204L Lexington KY 40536-0200 United States (859) 218-0592 (859) 323-6003 eedupo2@uky.edu

Michael C. Dyle

University of Iowa 540h EMRB Iowa City IA 52242 United States (316) 335-6746 michael-dyle@uiowa.edu

Scott M. Ebert

University of Iowa 500 Newton Rd. 540h EMRB Iowa City IA 52242 United States scott-ebert@uiowa.edu

Mai Elmallah

University of Florida Po Box 100296 1600 SW Archer Road Gainesville FL 32610 United States elmallah@peds.ufl.edu

Erin Epstein

Graduate Student Auburn University 650 Dekalb St. Unit 1335 Auburn AL 36830 United States eee0007@auburn.edu

Karyn Esser

Professor University of Kentucky 800 Rose St. MS567 College of Medicine Lexington KY 40536 United States (859) 323-8107 (859) 323-1070 karyn.esser@uky.edu

Val Andrew Fajardo

PhD Candidate University of Waterloo 83 First Ave. Cambridge Ontario N1s2b4 Canada (226) 339-8581 vafajard@uwaterloo.ca

Darin J. Falk

Assistant Professor University of Florida 1200 Newell Dr. Po Box 100296 Gainesville FL 32610 United States falkd@ufl.edu

Roger P. Farrar

Professor University of Texas Bellmont 822J 2100 San Jacinto Austin TX 78712 United States 512- 4718621 (512) 471-0948 rfarrar@austin.utexas.edu

Jeremie Ferey

PhD Candidate East Carolina University 1616 Treybrooke Cir. Greenville NC 27834 United States (540) 558-8868 fereyj12@students.ecu.edu

Brian Ferguson

Kinesiology Auburn 1174 Northlake Dr. Auburn AL 36832 United States (559) 908-2028 bsf0003@tigermail.auburn.edu

David Ferguson

Post Doctoral Fellow Baylor College of Medicine 1100 Bates Houston TX 77030 United States (713) 798-7051 (713) 798-7171 david.ferguson@bcm.edu

Leonardo Ferreira

Assistant Professor University of Florida 1864 Stadium Rd., Rm 100 Gainesville FL 32653 United States ferreira@hhp.ufl.edu

Marta Fiorotto

Baylor College of Medicine 3706 Nottingham Houston TX 77005 United States (713) 664-1306 (713) 798-7171 martaf@bcm.tmc.edu

Heléne Fischer

PhD, Associate Professor Karolinska Institutet Clinical Physiology, Lab Med, C1:82 Karolinska Inst. Huddinge Hospital Stockholm 184 73 Sweden +46 85858677 ext. 1 helene.fischer@ki.se

Gordon Fisher

Assistant Professor University of Alabama at Birmingham Education Building 202 901 13th St. S. Birmingham AL 35294 United States grdnfs@uab.edu

Sean Forbes

University of Florida Box 100154, UFHSC Gainesville FL 32610-0154 United States (352) 273-6111 scforbes@ufl.edu

Daniel Fox

University of Iowa 540H EMRB 500 Newton Rd. Iowa City IA 52245 United States (319) 455-6212 daniel-k-fox@uiowa.edu

Christopher Fry

Post-Doctoral Scholar College of Health Sciences University of Kentucky 900 S. Limestone St. Wethington Building Lexington KY 40536 United States (859) 218-1783 christopher.fry@uky.edu

Xing Fu

Washington State University 100 Dairy Rd. Animals Sciences Clark Hall 116 Pullman WA 99163 United States (509) 715-9866 xing.fu@wsu.edu

Hidemi Fujino

Kobe University 7-10-2 Tomogaoka Kobe 654-0142 Japan fujino@phoenix.kobe-u.ac.jp

David D. Fuller

Physical Therapy University of Florida 1225 Center Dr PO Box 100154 Gainesville FL 32610 United States ddf@phhp.ufl.edu

Katsuhiko Funai

Assistant Professor East Carolina University 115 Heart Dr. ECHI Rm 4107 Greenville NC 27834 United States (252) 737-4684 (252) 737-4689 funaik@ecu.edu

Tim Gavin

Professor Purdue University 800 W. Stadium Ave. West Lafayette IN 47907 United States (765) 494-3179 gavin1@purdue.edu

Ghislaine Gayan-Ramirez

KU Leuven O&N 1 bus 706 Herestraat 49 Leuven 3000 Belgium 32 16330193 ghislaine.gayan-ramirez@med.kuleuven.be

Alexandra Gentilella

University of Florida PO Box 118205 Gainesville FL 32611 United States agentilella92@gmail.com

Laura Gilliam

Post Doctoral Scholar East Carolina University 600 Moye Blvd. BSOM- Physiology 6N98 Greenville NC 27834 United States (252) 744-2764 gilliaml@ecu.edu

Mahasweta Girgenrath

Assistant Professor Boston University 635 Commonwealth Ave. Boston MA 02215 United States 617 353 ext. 2737 swetag@bu.edu

Qingnian Goh

Doctoral Research Fellow University of Toledo 2801 W. Bancroft Hh 2503, Ms 119 Toledo OH 43606 United States 419 530 ext. 5955 qgoh@rockets.utoledo.edu

Elisa J. Gonzalez-Rothi

Postdoctoral Fellow University of Florida 1149 S. Newell Dr. McKnight Brain Institute, Rm. L1-129 Po Box 100154, UFHSC Gainesville FL 32610 United States (352) 392-7873 elisagon@phhp.ufl.edu

Bret Goodpaster

Senior Investigator and Professor Translational Research Institute for Metabolism and Diabetes 301 E. Princeton St. Orlando FL 32804 United States (412) 901-9309 bret.goodpaster@flhosp.org

Susana Gordo

Senior Scientist Pfizer 200 Cambridgepark Dr. T3002-14 Cambridge MA 20140 United States (617) 665-5289 susana.gordo@pfizer.com

Bradley Scott Gordon

Post Doctoral Scholar Penn State Hershey Medical School 15 Brandywine Hershey PA 17033 United States (570) 350-4841 bsg11@psu.edu

Scott Gordon

Professor and Chairperson UNC Charlotte 9201 University City Blvd. Charlotte NC 28223 United States (704) 687-0855 scott.gordon@uncc.edu

Theodore Graber

PhD Candidate, Research Fellow University of Minnesota 2001 SE 6th Street McGuire Translational Research Facility Stem Cell Institute-Thompson Lab Minneapolis MN 55455 United States (651) 276-4117 (612) 624-2436 grab0170@umn.edu

Robert Grange

Professor Virginia Tech ILSB 23, Room 1029 1981 Kraft Drive Blacksburg VA 24061 United States (540) 231-2725 rgrange@vt.edu

Paul Gregorevic

Baker IDI Heart & Diabetes Institute PO Box 6492 St Kilda Road Central Melbourne Victoria 8008 Australia +61 3-85321224 +61 3-85321100 paul.gregorevic@bakeridi.edu.au

Stephanie Greufe

Post-Doc University of Florida 1864 Stadium Rd. Gainesville FL 32611 United States segreufe@ufl.edu

Theresa A. Guise

Professor of Medicine and Pharmacology Indiana University - School of Medicine 980 W. Walnut St. Room #C132 Indianapolis IN 46202 United States (317) 278-6014 (317) 278-2912 tguise@iu.edu

Jonathan Gumucio

PhD Student University of Michigan 109 Zina Pitcher Pl. BSRB 2278 Ann Arbor MI 48109 United States (734) 764-4206 (734) 647-0003 jgumucio@umich.edu

Thomas Gustafsson

MD, PhD, Assoc Prof Karolinksa Institutet C1-88 Department of Laboratory Medicine Stockholm 14186 Sweden +46 8-58586777 +46 8-7748082 thomas.gustafsson@ki.se

Denis Guttridge

Professor Ohio State University Medical Center 460 W. 12th Ave. BRT Room 520 Columbus OH 43210 United States (614) 688-4507 denis.guttridge@osumc.edu

Renzhi Han

Assistant Professor Loyola University Chicago 2160 S. 1st Ave. Building 102, Room 4676 Maywood IL 60153 United States (708) 216-6348 renhan@lumc.edu

Justin P. Hardee

University of South Carolina 223 Ravenel St. Columbia SC 29205 United States (850) 449-0088 hardee3@email.sc.edu

Tamara Beth Harris

Chief, Interdisciplinary Studies of Aging National Institute on Aging Laboratory of Epidemiology and Population Sciences 7201 Wisconsin Ave., Ste. 3c-309 Bethesda MD 20814 United States (301) 496-1178 (301) 496-4006 tamara.harris@nih.hhs.gov

Jacob M. Haus

Assistant Professor University of Illinois at Chicago 1919 W Taylor Street Rm 650 Chicago IL 60612 United States (312) 413-1913 hausj@uic.edu

Thomas Hawke

Associate Professor McMaster University, Dept. of Pathology & Molecular Medicine 1280 Main St. W. 4N65 Health Sciences Center Hamilton Ontario L8S 4L8 Canada (905) 525-9104 ext. 22372 hawke@mcmaster.ca

Russ Hepple

McGill University Department of Critical Care Royal Victoria Hospital 687 Pine Ave. W. Montreal Quebec H3a1a1 Canada (514) 589-3210 russell.hepple@mcgill.ca

Kazuhiko Higashida

Research Associate Waseda University 2-579-15, Mikajima Tokorozawa City Saitama 359-1192 Japan khigashida@aoni.waseda.jp

David A. Hood

York University 4700 Keele St. Toronto Ont M3j 1p3 Canada dhood@yorku.ca

Troy A. Hornberger

Associate Professor University of Wisconsin - Madison 2015 Linden Dr. Madison WI 53706 United States (608) 890-2175 thornb1@svm.vetmed.wisc.edu

Matthew Hudson

Postdoctoral Fellow Emory University 1639 Pierce Dr. WMB RM 3327 Atlanta GA 30322 United States (404) 757-3424 mbhudso@emory.edu

Hayden W. Hyatt

Graduate Student Auburn University 211 W. Longleaf Dr. # 1110 Auburn AL 36832 United States (205) 706-8634 hwh0001@auburn.edu

Robert D. Hyldahl

Assistant Professor Brigham Young University 106 Smith Fieldhouse Brigham Young University Provo UT 84602 United States (801) 422-1237 robhyldahl@byu.edu

Sajid Ibrahim

University of Karachi H#42 Alwajid Town,Metroville S.i.t.E. Karachi Karachi Sindh 74200 Pakistan 92021 32214320 sajidibrahim91@gmail.com

Noriko Ichinoseki-Sekine

Ph.D The Open University of Japan 2-11 Wakaba Mihama-Ku Chiba Chiba 2618586 Japan +81 43-2984118 noriko.sekine@ouj.ac.jp

Catherine Jankowski

Associate Professor University of Colorado College of Nursing Mail Stop C288-19 13120 E. 19th Ave. Aurora CO 80045 United States (303) 724-7383 (303) 724-8560 catherine.jankowski@ucdenver.edu

Sabzali Javadov

Associate Professor University of Puerto Rico, School of Medicine A-674, Medical Sciences Campus PO Box 365067 San Juan PR 00936-5067 United States (787) 758-2525 ext. 2909 sabzali.javadov@upr.edu

Casey John

Appalachian State University ASU Box 32071 Boone NC 28608 United States johnc@appstate.edu

Andrew Judge

University of Florida 1225 Center Dr. HPNP Building 1142 Gainesville FL 32653 United States (352) 231-9441 arjudge@phhp.ufl.edu

Heikki Kainulainen

University of Jyväskylä PO Box 35 Viveca Jyväskylä Fi-40014 Finland +358 50-3302901 heikki.kainulainen@jyu.fi

Peter Kang

Chief, Division of Pediatric Neurology University of Florida-College of Medicine 1600 SW Archer Rd. Gainesville FL 32610 United States (352) 273-8920 pbkang@ufl.edu

Neil A Kelly

University of Alabama at Brimingham 1918 University Blvd. 934 McCallum Bldg. Birmingham AL 35294-0005 United States (205) 996-7936 nakelly@uab.edu

Jong-Hee Kim

3855 Holman St. Garrison 104 Houston TX 77204 United States jkim77@central.uh.edu

Yuho Kim

Graduate Student Syracuse University 204 Suburban Park Dr. # 3 Manlius NY 13104 United States (315) 887-0788 ykim105@syr.edu

Scot R. Kimball

Professor Penn State College of Medicine Physiology, H166 500 University Dr. Hershey PA 17033 United States (717) 531-8970 (717) 531-7667 skimball@psu.edu

Michelle Ann King

Doctoral Student University of Florida 3813 SW 34th Street # 57 Gainesville FL 32608 United States 815 441 making13@ufl.edu

Tyler Kirby

Graduate Research Assistant, Physiology University of Kentucky 900 S. Limestone St. Wethington Building Lexington KY 40536 United States (859) 218-1783 tyler.kirby@uky.edu

Lauren Gerard Koch

Associate Professor University of Michigan 109 Zina Pitcher Pl. 2021 Biomedical Science Research Building Ann Arbor MI 48109 United States (734) 615-5969 (734) 615-1722 lgkoch@med.umich.edu

Hiroyo Kondo

Associate Professor Nagoya Wemens University 3-40, Shioji-Syo, Mizuho-Ku, Nagoya, Aichi 467-8610 Japan +81 90-34570621 +81 52-8529449 kondohiroyo@gmail.com

Rhianna Laker

University of Virginia 409 Lane Rd Mr4 6041a Charlottesville VA 22903 United States (434) 422-2125 rcl6n@virginia.edu

Daniel Lark

East Carolina University BSOM-Physiology 600 Moye Blvd. Greenville NC 27834 United States (252) 744-2764 larkd08@students.ecu.edu

Marcus Lawrence

UNC Charlotte 9201 University City Blvd. Charlotte NC 28223 United States mlawre18@uncc.edu

Andrew Layne

University of Florida 3944 NW 29th Ln. Gainesville FL 32606 United States (423) 276-5227 8 andrew.layne@ufl.edu

Emilia Lecuona

Research Associate Professor Northwest University 240 E. Huron McGaw M331 Chicago IL 60611 (312) 503-5397 e-lecuona@northwestern.edu

Brittany Lee-McMullen

Graduate Student University of Florida Newell Dr Gainesville FL 32608 United States (720) 839-8491 balm22@ufl.edu

Steven Lewis

818 Foxpointe Cir. Delray Beach FL 33445 United States (617) 596-5097 stevenkla@me.com

Wootaek Lim

PhD Student University of Florida 306 Diamond Village Apt 4 Gainesville FL 32603 United States wootaek@phhp.ufl.edu

Chien-Te Lin

Research Assistant Professor East Carolina University 600 Moye Blvd. BSOM- Physiology 6N98 Greenville NC 27834 United States (252) 744-2764 linch@ecu.edu

Vitor Agnew Lira

Assistant Professor The University of Iowa 225 S. Grand Ave. 432 Fh Iowa City IA 52242 United States (319) 335-6966 (319) 335-6669 vitor-lira@uiowa.edu

Haiming Liu

University of Minnesota 2001 6th St. SE Stem Cell Institute Thompson Lab Minneapolis MN 55455 United States liux0770@umn.edu

Donovan Lott

University of Florida 1225 Center Drive PO Box 100154 Gainesville, FL 32610-0154 United States (352) 273-9226 djlottpt@phhp.ufl.edu

Richard M. Lovering

University of Maryland School of Medicine Orthopaedics, 100 Penn St. AHB Room 540 Baltimore MD 21201 United States (410) 409-1077 rlovering@som.umaryland.edu

Hui Ying Luk

University of North Texas 1316 Margie St. # A2 Denton TX 76201 United States (607) 279-7276 hyluk85@gmail.com

Gordon S. Lynch

Head of Department, Professor of Physiology Department of Physiology Melbourne Medical School The University of Melbourne Grattan St. Melbourne Victoria 3010 Australia +61 3-83440065 +61 3-83445818 gsl@unimelb.edu.au

Tara L. Mader

University of Minnesota 420 Washington St. S.E. MCB 7-212 Minneapolis MN 55455 United States (612) 625-5022 mede0016@umn.edu

Zana Rafiq Majeed

University of Kentucky 407 Marquis Ave., #. 206 Lexington KY 40502 United States (859) 489-5745 zana.majeed@uky.edu

David J. Marcinek

Associate Professor University of Washington Brotman 142 850 Republican St. Seattle WA 98125 United States (206) 221-6785 dmarc@uw.edu

Joseph Marino

Assistant Professor UNC Charlotte 9201 University City Blvd. Charlotte NC 28223 United States (704) 687-0863 jmarin10@uncc.edu

Andrew R. Marks

Professor and Chair of Physiology Columbia University 1150 St. Nicholas Ave. New York NY 10032 United States (212) 851-5340 arm42@columbia.edu

Danny Martin

University of Florida Po Box 100154 Gainesville FL 32610 United States 352 273 ext. 6105 dmartin@phhp.ufl.edu

Neil Richard Martin

Post-Doctoral Research Associate Loughborough University School of Sport, Exercise and Health Sciences Sir John Beckwith Building Ashby Rd. Loughborough Leicestershire Le11 1jw United Kingdom +44 01509-226315 n.r.w.martin@lboro.ac.uk

Jeevendra Martyn

Professor Massachusetts General Hospital Harvard Medical School 55 Fruit St. Grb 444 Boston MA 02114 United States (617) 726-8807 jmartyn@mgh.harvard.edu

Santosh Kumar Maurya

Post Doctoral Researcher The Ohio State University 102 Hamilton Hall 1645 Neil Ave. Columbus OH 43210 United States (614) 688-4635 maurya.2@osu.edu

Angela L. McCall

Graduate Student University of Florida PO Box 100296 Gainesville FL 32610 United States gatorgirl980@ufl.edu

John McCarthy

Research Faculty, Physiology University of Kentucky 900 South Limestone St. Wethington Building Lexington KY 40536 United States (859) 323-4730 jjmcca2@email.uky.edu

Joe McClung

Assistant Professor East Carolina University, Brody Medical Center 604 White Horse Dr. Greenville NC 27834 United States mcclungj@ecu.edu

Jessica Mcelroy

Sr. Biological Scientist University of Florida 1015 NW 21st Ave. Gainesville FL 32608 United States (407) 920-4068 jessicaamcelroy@peds.ufl.edu

Graham Ripley McGinnis

Doctoral Candidate Auburn University 301 Wire Rd. Auburn AL 36849 United States (919) 604-0671 grm0006@auburn.edu

Elizabeth McNally

Professor/director of the Institute for Cardiovascular Research The University of Chicago 5841 S. Maryland, Mc6088 Chicago IL 60637 United States emmcnall@uchicago.edu

Christopher Mendias

Assistant Professor University of Michigan 109 Zina Pitcher Pl. BSRB 2017 Ann Arbor MI 48109 United States (734) 764-3250 cmendias@umich.edu

Edward Merritt

Assistant Professor Appalachian State University 111 Rivers St. Box 32071 Boone NC 28608 United States (512) 779-8039 merrittek@appstate.edu

Ronald A. Meyer

Professor Michigan State University Dept. of Physiology 2201 Bps Bldg. 567 Wilson Rd. East Lansing MI 48824 United States (517) 884-5130 (517) 355-5125 meyerr@msu.edu

Apoorva Mohan

Graduate Student University of Florida 2777,SW Archer Rd., # P70 Gainesville FL 32608 United States (434) 249-3518 apoorva@ufl.edu

Ryan M. Morrow

Postdoctoral fellow Children's Hospital of Philadelphia 3501 Civic Center Blvd. Room 6100 Philadelphia PA 19104 United States (267) 425-3068 morrowr1@email.chop.edu

Aaron B. Morton

Doctoral Student University of Florida 6400 SW 20th Ave. # 69 Gainesville FL 32607 United States (832) 477-6342 aaronbmorton@hhp.ufl.edu

Larry Mulcahy

Applications Scientist IonOptix LLC 309 Hillside St. Milton MA 02186 United States (617) 714-4352 (617) 698-3553 larry@ionoptix.com

Kathryn H. Myburgh

Stellenbosch University Mike De Vries Building Room 2013 Merriman Ave. Cnr Bosman Str Stellenbosch 7600 South Africa +27 82-5611775 +27 21-8083145 khm@sun.ac.za

Brad Nelson

Assistant Professor Ohio Dominican University 1216 Sunbury Rodad Columbus OH 43219 United States 80131953 nelsonb@ohiodominican.edu

P. Darrell Neufer

Professor East Carolina University ECHI - 4th Floor 115 Heart Dr. Greenville NC 27858 United States 252 744 ext. 2780 neuferp@ecu.edu

Linda M.-D. Nguyen

University of Florida Po Box 118206 Gainesville FL 32611 United States (352) 294-1736 linda.nguyen@ufl.edu

Brian Noehren

Faculty Health Sciences - Rehabilitation Science University of Kentucky Division of Pt 900 S. Limestone, Rm 204 Lexington KY 40536 United States (859) 218-1783 b.noehren@uky.edu

Barbara Norman

Karolinska Institutet Karolinska Univ. Hospital, Huddinge Clinical Physiology Stockholm 14186 Sweden 46 70755485 barbara.norman@ki.se

Catherine Maeve O'Connell

University College Cork 2337 SW Archer Rd. # 3010 Gainesville FL 32608 United States (352) 278-7119 108536881@umail.ucc.ie

Karl Olsson

Karolinska Institute Karolinska University Hospital Huddinge Division of Clinical Physiology Department of Laboratory Medicine Stockholm 141 86 Sweden karl.olsson@stud.ki.se

Christina Pacak

Assistant Professor University of Florida-College of Medicine 1600 SW Archer Rd. Gainesville FL 32610 United States pacakc@peds.ufl.edu

Christopher William Paran

Research Scientist East Carolina University 405 Brighton Park #. 4 Greenville NC 27834 United States (737) 252-4684 paranc@ecu.edu

Lauren Michele Pascual

Student University of Florida Physical Therapy Department 1013 SW 7th Ave. #. 204 Gainesville FL 32601 United States (305) 878-9962 lpascual@ufl.edu

Justin Percival

Assistant Professor University of Miami Miller School of Medicine Pharmacology, R-189 1600 NW 10th Ave. Miami FL 33136 United States j.percival@med.miami.edu

Charlotte A. Peterson

Professor and Associate Dean for Research University of Kentucky 900 S. Limestone Ctw 105 Lexington KY 40536 United States (859) 218-0476 cpete4@uky.edu

Bethan Phillips

Research Fellow University of Nottingham Division of Graduate Entry Medicine and Health School of Medicine Royal Derby Hospital De22 3dt United Kingdom +44 7515-412197 beth.phillips@nottingham.ac.uk

Martin Picard

Postdoctoral Fellow University of Pennsylvania and Children's Hospital of Philadelphia 3501 Civic Center Blvd. Colket Translational Building, Room 6100 Philadelphia PA 19104 United States (619) 301-4401 martinpicard@mac.com

Darren Player

Loughborough University SSEHS Clyde Williams Building Loughborough Le11 3tu United Kingdom d.player@lboro.ac.uk

Scott K. Powers

Professor University of Florida 8824 SW 113 Ave. Gainesville FL 32608 United States (352) 294-1713 spowers@hhp.ufl.edu

Stephen J.P. Pratt

Student University of Maryland School of Medicine Allied Health Building Rm 540 100 Penn St. Baltimore MD 21201 United States (410) 706-1421 sjpratt@g.coastal.edu

Russ Price

Associate Vice Chair for Research, Professor of Medicine and Physiology Emory University School of Medicine 3319a Woodruff Memorial Bldg Department of Medicine, Renal Division 1639 Pierce Dr. Atlanta GA 30322 United States (404) 727-3934 russ.price@emory.edu

Jill Rahnert

Postdoctoral Fellow Emory University 101 Woodruff Cir. WMB Rm 3327 Atlanta GA 30322 United States (404) 727-3424 jill.rahnert.freret@emory.edu

Lauren Rose Reese

Doctoral Scholar East Carolina University 600 Moye Blvd. BSOM- Physiology 6N98 Greenville NC 27834 United States (719) 314-9461 reesel13@students.ecu.edu

Michael B. Reid

Dean and Professor University of Florida College of Health and Human Performance Gainesburger FL 32611 United States (352) 294-1614 michael.reid@ufl.edu

Michael J Rennie

School of Life Sciences University of Nottingham School of Life Sciences Queens Medical Centre Nottingham NG7 2RD United Kingdom +44 0776-6662383 michael.rennie@nottingham.ac.uk

Brandon M. Roberts

Graduate Student University of Florida 4014 SW 21st Rd. Gainesville FL 32608 United States (386) 937-4537 brob21@ufl.edu

Greg Rodden

Virginia Tech ILSB 23, Room 1029 1981 Kraft Drive Blacksburg VA 24061 United States (540) 231-2725 grodd91@vt.edu

Rachael May Rodney

Short Term Scholar University of Florida 2250 Shealy Dr. PO Box 110910 Gainesville FL 32608 United States (352) 392-1981 ext. 220 (352) 392-1913 krueger@ufl.edu

Heather H. Ross

Research Assistant Professor University of Florida 1225 Center Drive Department of Physical Therapy PO Box 100154 Gainesville FL 32605 United States hhrunc@phhp.ufl.edu

Leslie Rowland

The Ohio State University 108 Hamilton Hall 1645 Neil Ave. Columbus OH 43210 United States (330) 692-3610 rowland.134@osu.edu

Seward Rutkove

330 Brookline Ave. Boston MA 02215 United States srutkove@bidmc.harvard.edu

Terence Ryan

Post Doctoral Scholar East Carolina University 600 Moye Blvd BSOM- Physiology 6N98 Greenville NC 27834 United States (252) 744-2764 ryant@ecu.edu

Viktoriya Rybalko

Ph.D. Student University of Texas at Austin 2900 Sunridge Dr. #1625 Austin TX 78741 United States (207) 745-2815 viktoriya.rybalko@gmail.com

Dan Ryder

Scientist University of Florida 677 NW 29th Ave. Gainesville FL 32609 United States (713) 502-2660 ryder.dan@gmail.com

Marco Sandri

Professor University of Padova Via Orus 2 Padova 35129 Italy marco.sandri@unipd.it

Martin F. Schneider

Professor University of Maryland School of Medicine 108 N. Greene St. Baltimore MD 21201 United States (410) 706-7812 (410) 706-8297 mschneid@umaryland.edu

Neil Andrew Schwarz

Baylor University 1401 Bagby Ave. # 6 Waco TX 76706 United States (504) 909-6502 neil_schwarz@baylor.edu

George Schweitzer

Post Doc Wasington University 660 S. Euclid St Louis MO 63110 United States (314) 221-7957 gschweit@dom.wustl.edu

Claudia Senesac

Clinical Associate Professor University of Florida Physical Therapy Department PO Box 100154 Gainesville FL 32610 United States (352) 273-6453 csenesac@phhp.ufl.edu

Sarah Senf

University of Florida 1225 Center Dr. HPNP Building 1142 Gainesville FL 32653 United States (352) 231-9318 smsenf@ufl.edu

Andy Shanely

Appalachian State University 600 Laureate Way Kannapolis NC 28081 United States (704) 340-3976 shanelyra@appstate.edu

Melinda Sheffield-Moore

Professor of Medicine University of Texas Medical Branch 301 University Blvd. Galveston TX 77555-1074 United States (409) 772-8707 (409) 747-1938 melmoore@utmb.edu

Jonathan Shintaku

Graduate Research Associate The Ohio State University Biomedical Research Tower, 536 460 W. 12th Ave. Columbus OH 43210 United States (559) 709-8237 jonathan.shintaku@gmail.com

Barbara K. Smith

Research Assistant Professor University of Florida PO Box 100154 Gainesville FL 32610-0154 United States bksmith@phhp.ufl.edu

Cheryl Smith

Research Associate East Carolina University 600 Moye Blvd. BSOM- Physiology 6N98 Greenville NC 27834 United States (252) 744-2764 smithch@ecu.edu

Cody D. Smith

Doctoral Candidate East Carolina University 600 Moye Blvd. BSOM- Physiology Greenville NC 27834 United States (252) 744-2764 smithco11@students.ecu.edu

Emily Smith

Postdoctoral Fellow Sanford Burnham Medical Research Institute 6400 Sanger Rd. Orlando FL 32827 United States (407) 745-2000 ext. 6102 esmith@sanfordburnham.org

Ira J. Smith

Senior Scientist Rigel Pharmaceuticals, Inc 681 Cedar St. # 6 San Carlos CA 94070 United States (650) 624-1212 ismith@rigel.com

Ashley J. Smuder

Post-Doctoral Fellow University of Florida PO Box 118206 Gainesville FL 32611 United States asmuder@ufl.edu

Kurt J. Sollanek

University of Florida Center for Exercise Science PO Box 118206 Gainesville FL 32611 United States (352) 294-1744 ksollanek@hhp.ufl.edu

Espen E. Spangenburg

Associate Professor University of Maryland School of Public Health Dept of Kinesiology College Park MD 20742 United States (301) 405-2483 espen@umd.edu

Robert Standley

Research Scientist - Post Doctoral Translational Research Institute for Metabolism and Diabetes 2045 Shroud St. #210 Orlando FL 32814 United States robert.standleyjr@flhosp.org

Michael J. Stec

The University of Alabama at Birmingham 1918 University Blvd. MCLM 936 Birmingham AL 35294 United States (205) 996-7936 mstec@uab.edu

Jennifer Steiner

Post Doctoral Scholar Penn State Hershey 15 Brandywine Hershey PA 17033 United States jls2tc@virginia.edu

Randi Stewart

Student Graduate School of Biomedical Sciences 1435 Nashua St. Houston TX 77008 United States (713) 500-5697 randi.stewart@uth.tmc.edu

Anna Strömberg

PhD candidate Karolinska Institutet C1:82 Division of Clinical Physiology Karolinska University Hospital Stockholm 141 86 Sweden +46 739-724334 anna.stromberg@ki.se

Carsten Struebing

Sanofi-Aventis Deutschland Industriepark Hoechst H8245 Frankfurt 65926 Germany -69 30583985 carsten.struebing@sanofi.com

Kristoffer Sugg

University of Michigan 109 Zina Pitcher Pl. BSRB 2278 Ann Arbor MI 48109 United States (734) 764-4206 krisugg@med.umich.edu

Marius Sumandea

Eli Lilly and Co Lilly Research Laboratories Lilly Corporate Center Indianapolis IN 46225 United States sumandeamp@lilly.com

Maurice Swanson

Professor University of Florida 2033 Mowry Rd. Cancer Genetics Research Complex Gainesville FL 32610 United States (352) 273-8076 (352) 273-8284 mswanson@ufl.edu

Yuri Takamine

Juntendo University 1-1 Hiragagakuendai Inzai Chiba 2701695 Japan +81 476-981001 +81 476-981030 takamine.yuri@gmail.com

Erin Talbert

Ohio State University 460 W. 12th Ave. Columbus OH 43016 United States (765) 860-5072 erin.talbert@osumc.edu

Ladora V. Thompson

Professor University of Minnesota Program in Physical Therapy 420 Delaware St. SE Mmc 388 Minneapolis MN 55455 United States (612) 626-5271 thomp067@umn.edu

A. Gary Todd

Post Doc University of Florida 3700 Windmeadows Blvd. # 173 Gainesville FL 32608 United States (571) 366-0581 gary.todd@ufl.edu

Maria Torres

East Carolina University BSOM Physiology 600 Moye Blvd Greenville NC 27834 United States (252) 744-2764 torresm12@students.ecu.edu

Scott Trappe

Professor and Director Ball State University Human Performance Laboratory Ball State University Muncie IN 47342 United States strappe@bsu.edu

A. Russell Tupling

University of Waterloo Department of Kinesiology 200 University Ave. W. Waterloo Ontario N2L 3G1 Canada (519) 888-4567 ext. 33652 (519) 746-6776 rtupling@uwaterloo.ca

Mark C. Turner

PhD Student Loughborough University Clyde Williams Building Ashby Rd. Loughborough Leicestershire Le11 3tu United Kingdom +44 1509-226351 m.c.turner@lboro.ac.uk

Krista Vandenborne

Professor and Chair University of Florida Department of Physical Therapy 1225 Center Dr.; HPNP 1142 PO Box 100154 Gainesville FL 32610 United States (352) 273-6085 kvandenb@phhp.ufl.edu

Narendra Kumar Kumar Verma

Marie Curie Researcher University of Padova, Italy Via Ognissanti,32 Primo 1; INT 8 Padova Padova 35121 Italy (049) 827-6368 (049) 827-6280 narendrakumar.verma@studenti.unipd.it

Jakob Langberg Vingren

Associate Professor University of North Texas 1921 Chestnut St. Peb 209, Khpr Denton TX 76203 United States 940 565 ext. 3899 jakob.vingren@unt.edu

Kalyan C. Vinnakota

Research Assistant Professor University of Michigan 2800 Plymouth Rd. NCRC, Building 10, Room A123 Ann Arbor MI 48109 United States (734) 763-8059 kalyanv@umich.edu

Ravneet S. Vohra

Post Doc University of Florida 3700 Windmeadows Blvd. Apt CC 287 Gainesville FL 32608 United States (352) 682-7254 ravneet@ufl.edu

Glenn Walter

University of Florida PO BOX 100274 Department of Physiology Gainesville FL 32610 United States glennw@ufl.edu

Xiaonan H. Wang

Assistant Professor Emory University WMB 338C, Renal Medicine 1639 Pierce Dr. Atlanta GA 30322 United States 404 727 ext. 1798 404 727 ext. 3425 xwang03@emory.edu

Amal A. Wanigatunga

PhD Student University of Florida 2635 SW 35th Pl., #401 Gainesville FL 32608 United States (352) 256-1684 asiri@ufl.edu

David Waning

Assistant Research Professor Indiana University - School of Medicine 980 W. Walnut St. Room #C132 Indianapolis IN 46202 United States (317) 278-6020 (317) 278-2912 dwaning@iu.edu

Gordon L. Warren

Professor Georgia State University PO Box 4019 Physical Therapy Atlanta GA 30302 United States (404) 413-1255 (404) 413-1230 gwarren@gsu.edu

Nicholas R. Wawrzyniak

University of Florida PO Box 118206 Gainesville FL 32611 United States (352) 294-1736 n.wawrzyniak@ufl.edu

John Robert Wells

University of Gloucestershire Priory House, Rowley Cam Dursley Gloucestershire Gl11 5nt United Kingdom jrwells2007@gmail.com

James White

Postdoc Fellow Dana Farber Cancer Institute Harvard Medical School 450 Brookline Ave. CLSB 11115 Boston MA 02215 United States (671) 429-4281 james_white1@dfci.harvard.edu

Sarah White

University of Florida 3870 SW 20th Ave. #. 1609 Gainesville FL 32607 United States (352) 246-1577 s.white@ufl.edu

Samuel Wickline

Professor of Medicine, Biomedical Engineering, Physics Washington University Campus Box 8215 660 S. Euclid Ave. Saint Louis MO 63110-1010 United States (314) 583-6840 (314) 454-5602 wicklines@aol.com

Charles G. Widmer

Associate Professor University of Florida Box 100444, JHMHSC College of Dentistry Gainesville FL 32610-0444 United States (352) 273-5696 (352) 846-0459 widmer@dental.ufl.edu

Michael P. Wiggs

Post-Doc University of Florida 100 Florida Gym Po Box 118205 Gainesville FL 32611 United States mwiggs@ufl.edu

Rebecca Willcocks

Postdoctoral Associate University of Florida Department of Physical Therapy UFHSC PO Box 100154 Gainesville FL 32610 United States (352) 219-8051 rw254@phhp.ufl.edu

Rebecca June Wilson

Graduate Student University of Virginia 1203 Wertland St. #9 Charlottesville VA 22903 United States (703) 477-8709 rjw7jc@virginia.edu

Robert Wiseman

Professor Michigan State University 567 Wilson Rd. Department of Physiology East Lansing MI 48824 United States (517) 884-5132 (517) 355-5125 rwiseman@msu.edu

Carol A. Witczak

East Carolina University 600 Moye Blvd. Brody, 6N-98 Greenville NC 27834 United States (252) 744-1224 witczakc@ecu.edu

Tracey L. Woodlief

Post-Doc Translational Research Institute-Florida Hosp. 301 E. Princeton St. Orlando FL 32804 United States (252) 412-5019 tracey.woodlief@flhosp.org

Myra Woodworth-Hobbs

Emory University 1512 Druid Oaks NE Atlanta GA 30329 United States mewoodw@emory.edu

Zhen Yan

Associate Professor University of Virginia 409 Lane Road, MR4 6041A Charlottesville VA 22901 United States (434) 982-4477 zhen.yan@virginia.edu

Ceren Yarar-Fisher

PT, PhD University of Alabama at Birmingham 996 McCallum Health Sciences Building Birmingham AL 35294 United States cyarar@uab.edu

Fan Ye

Post-Doc University of Florida 305 Diamond Village # 5 Gainesville FL 32603 United States (352) 215-6551 evan1979@phhp.ufl.edu

Toshinori Yoshihara

1-1 Hiragagakuendai Inzai 2701695 Japan +81 476-981001 ext. 312 +81 476-981030 basket0514@gmail.com

Ali Murat Zergeroglu

Professor Ankara University School of Medicine Sport Medicine Department Cebeci Ankara 06590 Turkey +90 533-7193100 +90 312-5622001 zerger@medicine.ankara.edu.tr

Xiao-Ling Zhong

Indiana University School of Medicine 980 W. Walnut St. R3-C518 Indianapolis IN 46202 United States (317) 278-8030 xzhong@iu.edu

Teresa A. Zimmers

Associate Professor Indiana University School of Medicine 980 W. Walnut St. R3-C518 Indianapolis IN 46074 United States (317) 278-7289 zimmerst@iu.edu

Kevin Zwetsloot

Assistant Professor Appalachian State University ASU Box 32071 111 Rivers St. Boone NC 28608 United States (828) 262-7281 (828) 262-3138 zwetslootka@appstate.edu