Project No. and Title: NC1131: Molecular Mechanisms Regulating Skeletal Muscle Growth and DifferentiationPeriod Covered: 10-2009 to 09-2010Date of Report: 3 Jan 2010Annual Meeting Dates: 17-Oct-2010 to 18-Oct-2009Participants:

R. E. Allen – University of ArizonaYong Soo Kim – University of HawaiiWilliam Dayton – University of MinnesotaMike White – University of MinnesotaPaul Mozdziak – North CarolinaShihuan Kuang – Purdue UniversityTerry Bradley – University of Rhode IslandJoshua Selsby – Iowa StatePenny Riggs – Texas A&M University, Texas AgriLife ResearchBradley Johnson – Texas TechDavid Gerrard – Virginia Polytechnic Institute and State UniversityHonglin Jiang – Virginia Polytechnic Institute and State UniversityGale Strasburg – Michigan State UniversityMarion Greaser – University of WisconsinMin Du – University of WyomingDebra Hammernick – Administrative AdvisorMark Mirando – NIFA Representative

Brief Summary of Minutes of Annual Meeting: Members not attending: Sally Johnson, Florida; Rod Hill, Idaho; J.E. Minton, Kansas; Michael Zeece, Nebraska; Sandra Velleman, Ohio; Michael Dodson, Washington State.

The annual meeting of the NC-1184 technical committee meeting was held at the Texas A&M University, College Station, TX, on October 17-18, 2010, and was hosted by Dr. Penny Rigges, Department of Animal Science. On October 17th the group was welcomed by Dr. Russell Cross, Interim Head of the Department of Animal Science. NIFA Representative, Dr. Mark Mirando, made brief remarks to the group regarding changes at NIFA, the USDA funding outlook, and status of funding RFA revisions. The business meeting was chaired by the Administrative Advisor, Dr. Deb Hamernik. Next year's annual meeting of the NC-1184 committee will be held at Virginia Tech. The group decided that the 2012 meeting will most likely be held at North Carolina State University, Paul Modziak, chair. The remainder of the day was filled by oral station reports summarizing each station's contributions to the objectives of the NC-1184 project. The meeting adjourned, and the group toured the department’s animal facilities and reconvened for a Texas barbeque.

I. Accomplishments

Objective 1. Characterize the signal transduction pathways that regulate skeletal muscle growth and differentiation.

The Florida Station reported on where activation and lineage marker expression patterns were examined in bovine satellite cells isolated from young and adult cattle. Immunofluorescent detection of Pax7, Myf5 and MyoD indicate subtle differences between rodent and bovine satellite cells (BSC). Greater than 90% of BSC contain nuclear Pax7 and Myf5 independent of donor age. A smaller population exhibits Pax7-only suggestive of a muscle stem cell. BSC cultures demonstrate an age-dependent delay in G0 exit and cell cycle reentry. Unlike rodent satellite cells, BSC become immunopositive for MyoD after entry into S-phase and DNA synthesis. Gene expression changes during the activation period were monitored by microarray using RNA isolated from BSC at 18 and 48 hr post-plating. Gene ontology analysis revealed up-regulation of genes associated with energy and mitochondrial biogenesis, chemotaxis and cell cycle progression among others. Future efforts will be focused on validation of differential gene expression in vitro and in vivo.

Researchers at the Arizona Station reported the participation of satellite cells in myogenesis and angiogenesis. Skeletal muscle regeneration is a multifaceted process requiring the spatial and temporal coordination of myogenesis as well as angiogenesis. While these processes are often studied independently, recent evidence from our lab has shown that the resident adult stem cell population within skeletal muscle, called satellite cells, begins secreting soluble growth factors likely to contribute to the proangiogenic response. Activation of satellite cells in regenerating muscle plays a critical role in the coordination of these two independent events as both the primary cell for myofiber replacement and as a contributor to revascularization. Several growth factors are secreted by satellite cells, many with proangiogenic properties, including the growth factors vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). The Arizona station showed VEGF is likely to play a role in this satellite cell mediated angiogenic response using an in vitro three dimensional microvascular fragment (MVF) construct both in co-culture with satellite cells as well as from satellite cell conditioned media (CM). The overall aim of their study was to investigate the role of HGF as a potential pro-angiogenic factor secreted by satellite cells during skeletal muscle regeneration. Results showed elevated HGF protein levels in the satellite cell conditioned media where, upon neutralization of this protein, the angiogenic effect was decreased in a dose dependent manner. This reduction in angiogenesis however was recovered on addition of recombinant HGF. A final verification was via infection of satellite cells with a HGFα/β shRNA lentivirus prior to conditioning media, which resulted in a decrease in HGF protein secretion and reduction in angiogenic effect of the CM. A role for HGF in satellite cell mediated angiogenesis is now becoming apparent; next, they mimiced an injury state of muscle by placing satellite cells in hypoxic environments. Interestingly, satellite cells decreased their proangiogenic effect when oxygen levels fell below a threshold level. This decrease in proangiogenic effect in the hypoxic environment appeared to be due to the decrease in HGF expression and protein secretion and was not compensated for by the increase in VEGF secretion also seen in the hypoxic response. Furthermore, the regulation of HGF in these hypoxic conditions appeared to be in part due to increased levels of hypoxia inducible factor (HIF), which are acting on the hypoxia response element (HRE) site found on the HGF promoter. Finally, this injury response was investigated as the effect of satellite cell mediated angiogenesis was examined in the disease state of muscular dystrophy. It has previously been shown that there is a

reduced number of satellite cells in dystrophic muscle and that the remaining satellite cells appear to have diminished myogenic capacity. Here, they observed a reduction in angiogenesis from media conditioned by satellite cells from dystrophic muscle compared to healthy muscle. While HGF gene expression and protein secretion increased in the dystrophic satellite cells, VEGF expression levels were reduced and thus may explain the reduction in angiogenesis. This suggests that the impaired myogenic properties of satellite cells observed in dystrophic muscle may also be accompanied by a diminished angiogenic potential as well. These results also indicate that these impaired satellite cells may contribute to the reduction in capillary density previously observed in dystrophic muscle and may serve as a potential site for therapeutic intervention.

Work at the Arizona Station is also targeted at studying the migration of satellite cells to sites of damage within muscle. This work has been focusing on one possible pathway that mediates chemotaxis. From early work in vitro, it was noted that expression of the chemokine receptor CXCR4 increased significantly during satellite cell activation. This receptor and its ligand, stromal derived factor 1 (SDF1), are known to mediate chemotaxis in other cell types but have not been described in myogenic cells. They examined the expression pattern of CXCR4 and SDF1 in cultured satellite cells as well as in damaged skeletal muscle. A significant increase of CXCR4 expression (message and protein) was detected shortly after satellite cell activation in vitro and it persisted through the proliferation period. Expression was down-regulated following differentiation, and it was not observed in cultured cells that did not express Pax7 and/or MyoD. CXCR4+ satellite cells were found in injured muscle by immunohistochemistry (IHC), and a higher density of CXCR4+ satellite cells was detected in tissue closer to injury site. Transwell assays in vitro indicated that satellite cell migrated in response to SDF1 signaling and the migration was blocked by AMD3100, an antagonist of the CXCR4 chemokine receptor. SDF1 signaling was detected in muscle injury site by IHC. Satellite cell migration showed dose-dependent responsiveness to SDF1 injected into uninjured muscle and this migration was significantly blocked by the systematic existing of AMD3100. Their results indicate that the chemotactic interaction of SDF1-CXCR4 plays an essential role in regulating satellite cell migration in muscle tissue. Overall, these studies strengthen the case for satellite cells as important mediators of the angiogenic response and their ability to move to and in regenerating muscle. Understanding these processes are important for conditions of muscle degeneration and weakness as seen with aged and dystrophic muscle.

The Indiana Station report consisted of additional work in satellite cell biology. These scientists argue phenotypic heterogeneities of satellite cells derived from slow and fast muscles. However, the intrinsic molecular mechanisms are unknown due to technical difficulties in isolating fiber type-specific satellite cells. As a result, these scientists are exploring the intrinsic molecular mechanisms that program the developmental fate of satellite cells and restrict them to a specific muscle fiber type. Using genome-wide gene expression approach, the Indiana Station is attempting to identify genes that uniquely regulate slow versus fast muscle development. Three levels of analysis have been used. First, they analyzed published GEO database and identified candidate genes differentially expressed in representative fast (EDL) and slow (SOL) muscles. Then, they isolated RNA from purified myofibers (excluding interstitial non-muscle cells) from EDL and SOL muscles, and conducted Affymetrix microarray analysis to identify genes differentially expressed in fast and slow myofibers. Finally, they extracted RNA from fresh-isolated satellite cells from EDL and SOL muscles, and subjected the RNA to Agilent two-color microarray. Comparing genes differentially expressed at whole muscle, mature muscle cell

(myofiber), and muscle progenitor cell (satellite cell) levels, they have identified candidate genes acting at different stages of development to regulate the formation of slow versus fast muscles. This group has confirmed the differential expression of several candidates and is investigating the function of these genes in myogenesis. Knowledge learned from this study may lead to novel strategies for regulating muscle fiber type that affects lean growth efficiency and meat quality, hence maintaining the sustainability and competitiveness of US meat industry.

The South Dakota and Ohio Stations reported of work with satellite cells but with a slightly different question in mind, how do hormones modulate muscle growth. Much of their efforts have been restricted to the effects that estradiol and testosterone have on the proliferation and differentiation of satellite cells from poultry. Parallel to the aforementioned studies, they are also exploring whether these hormones influence glypican or syndecan expression of the cells. To conduct these studies, considerable changes were made to the media formulation. In particular, most cell culture media contains phenol red, a pH indicator that has estrogenic properties. In addition to this, the basal medium we had been using for such studies (McCoy’s 5A) contains Bacto Peptone which also contains estrogenic activity. Therefore, they switched to phenol red-free DMEM/F12 as the basal medium and made a number of modifications to the amino acid and vitamin content, and used gelding serum in the plating medium because of the low levels of testosterone, estradiol and estrone. Results of their studies suggest that the addition of estradiol stimulates endogenous production of insulin-like growth factor (IGF) by satellite cells. The effects are biphasic in their effect on myogenic cells and corroborate findings that estradiol implants in feedlot steers increased IGF-I mRNA expression in skeletal muscle, mechanisms that clearly increase the productivity and growth potential of feedlot cattle.

As stated above, efforts of the Ohio Station are focused on understanding the mechanism of how the heparan sulfate family of proteoglycans may be involved in the regulation of muscle growth properties. Fibroblast growth factor 2 (FGF2) is a potent stimulator of muscle cell proliferation and a strong inhibitor of muscle cell differentiation. Heparan sulfate proteoglycans function as a low affinity receptor for FGF2 thus permitting a high affinity interaction of FGF2 with its receptor. Our research is focused on the heparan sulfate proteoglycan syndecan and glypican families. There are 6 glypican family members with only glypican-1 found in skeletal muscle. Glypican-1 is a heparan sulfate proteoglycan composed of a core protein and covalently attached glycosaminoglycan (GAG) chains and N-linked glycosylated (N-glycosylated) chains. The glypican-1 GAG chains are required for cell differentiation and responsiveness to FGF2. The role of the glypican-1 N-glycosylated chains in regulating cell activities has not been reported. The station reported they investigated the role of glypican-1 N-glycosylated chains and the interaction between N-glycosylated and GAG chains in turkey myogenic satellite cell proliferation, differentiation, and FGF2 responsiveness (with the South Dakota Station). The wild-type turkey glypican-1 and turkey glypican-1 with mutated GAG chain attachment sites were cloned into the pCMS-EGFP mammalian expression vector and were used as templates to generate glypican-1 N-glycosylated 1-chain and no-chain mutants with or without GAG chains by site-directed mutagenesis. The wild-type glypican-1 and all glypican-1 N-glycosylated 1-chain and no-chain mutants with or without GAG chains were transfected into turkey myogenic satellite cells. Cell proliferation, differentiation, and FGF2 responsiveness were measured. The overexpression of glypican-1 N-glycosylated 1-chain and no-chain mutants without GAG chains increased cell proliferation and differentiation compared with the wild-type glypican-1 but not the glypican-1 N-glycosylated mutants with GAG chains attached. Cells overexpressing glypican-1 N-glycosylated mutants with or without GAG chains increased cell responsiveness to

FGF2 compared with wild-type glypican-1. These data suggest that glypican-1 N-glycosylated chains and GAG chains are critical in regulating turkey myogenic satellite cell proliferation, differentiation, and responsiveness to FGF2, and may be related to muscle growth in turkeys.

The Hawaii Station reported of its work to understand the role of myostatin, a known regulator of muscle hypertrophy, which binds to activin type IIB (ActRIIB) receptor to initiate its signaling process. The soluble form of extracellular domain of ActRIIB (ActRIIB-ECD) suppresses Mstn activity. To examine whether similar response would occur in meat-producing animals, they initiated a project to produce recombinant ActRIIB-ECD in chickens and pigs.Coding sequences of chicken or pig ActRIIB-ECD were inserted into the pPICZA vector. Transformed colonies were screened for expression of recombinant proteins. Proteins were purified and Mstn-suppressing capacity was estimated using a pGL3-(CAGA)12 luciferase gene reporter assay. Clones expressing chicken ActRIIB-ECD were identified, and recombinant proteins were purified from culture media after induction for 2 days. N-glycosylation of the chicken ActRIIB-ECD was confirmed by SDS-PAGE analysis of the reaction products of the proteins with peptide-N-glycosidase F (PNGase F). Both glycosylated and N-deglycosylated chicken ActRIIB-ECD suppressed Mstn activity in an in vitro reporter gene assays. In an effort to continue their quest to understand myostatin’s mode of action, the Hawaii Station produced recombinant chicken Mstn prodoamin (chMSTNpro). The Mstn prodomain inhibits the activity of Mstn, suggesting a potential target for improving the efficiency of meat production by enhancing skeletal muscle growth. chMSTNpro gene was cloned, inserted into pMAL-c5X vector downstream of the maltose-binding protein (MBP) gene, and transformed into an E. coli strain (Top10F’). Colonies were screened for recombinant protein and proteins were purified by an amylose-resin affinity chromatography. Western blot analysis using anti-MBP and anti-chMSTNPro antibodies confirmed the expression of chMSTNPro. Affinity-purified chMSTNpro demonstrated Mstn-inhibitory activity in an in vitro reporter gene assay. Studies to show the bioactivity of the aforementioned is forthcoming.

Work to understand better the role of the growth hormone (GH)/IGF axes was reported by researchers from the Virginia Station. In particular, there has been a lot of confusion in the last ten years about the role of circulating IGF-I, or liver IGF-I, because most of the former is produced by the liver under the control of GH, in mediating the effect of GH on muscle growth. Whereas some knockout mouse studies suggested that GH stimulates muscle growth through either locally produced IGF-I (i.e., paracrine/autocrine IGF-I) or IGF-I-independent mechanisms, other similar studies argued that at least part of the growth-stimulating effect of GH is mediated by endocrine IGF-I from the liver. Prompted by this controversy, the Virignia Station has conducted studies to determine the role of liver- or muscle-produced IGF-I in the effect of GH on skeletal muscle in cattle. They found that GH administration to cattle caused a remarkable increase in serum IGF-I concentration, but this increase was not accompanied by increased phosphorylation of IGF-I receptor (IGF-IR) in skeletal muscle. These results suggest that the effect of exogenous GH on skeletal muscle growth in cattle may be not mediated by circulating IGF-I. The same study also showed that GH administration caused a marked increase in liver IGF-I mRNA expression, which explained the increase in serum IGF-I concentration, but had no effect on muscle IGF-I mRNA expression. This result and the earlier data that muscle IGF-IR phosphorylation was not increased together suggest that the effect of exogenous GH on skeletal muscle growth in cattle may be not mediated by skeletal muscle IGF-I either. They found that following GH administration, phosphorylation of signal transducer and activator of transcription 5 (STAT5), a well-established component of GH receptor (GHR) signaling, and mRNA

expression of cytokine inducible SH2-containing protein (CISH), a well-established GH target gene, were both increased in skeletal muscle. They also noticed that GHR mRNA was relatively abundant but IGF-I mRNA was barely detectable in skeletal muscle of cattle. These observations demonstrate that cattle skeletal muscle expresses GHR and is responsive to exogenous GH. To further determine if muscle IGF-I is involved in the effect of GH on muscle growth in cattle, we quantified the effect of GH on IGF-I mRNA expression in primary cattle myoblasts and myotubes. GH had no effect on IGF-I mRNA expression in either myoblasts or myotubes; GHR mRNA was expressed in both myoblasts and myotubes; GH increased CISH mRNA expression in myoblasts, but not in myotubes. These in vitro effects of GH on IGF-I and CISH mRNA expression were in general consistent with those in skeletal muscle. Overall, these in vitro and in vivo data suggest that the effect of exogenous GH on skeletal muscle growth in cattle is not mediated by either muscle IGF-I or liver IGF-I. In other words, these data suggest that GH stimulates skeletal muscle growth via IGF-I-independent mechanisms. To that end, they have determined the effects of GH on proliferation and fusion of cattle myoblasts into myotubes and the effects of GH on protein synthesis and degradation in cattle myotubes in vitro. Their data showed that GH increased protein synthesis in cattle myotubes but had no effect on proliferation or fusion of myoblasts, or protein degradation in myotubes. These in vitro data seem to support the hypothesis that exogenous GH stimulates skeletal muscle growth in cattle by stimulating protein synthesis through an IGF-I-independent mechanism.

The Wyoming Station reported on work centered on AMP-activated protein kinase (AMPK), a key regulator of energy metabolism. Emerging evidence also suggests that AMPK regulates cell differentiation, but the underlying mechanisms are unclear. Wingless Int-1 (Wnt)/-catenin signaling pathway regulates the differentiation of mesenchymal stem cells (MSC) through enhancing -catenin/T-cell transcription factor 1 (TCF) mediated transcription, and -catenin is a key mediator of the Wnt/-catenin signaling pathway. They hypothesized that AMPK regulates cell differentiation via altering -catenin expression, which involves phosphorylation of class IIa histone deacetylase 5 (HDAC5). In both C3H10T1/2 cells and mouse embryonic fibroblasts (MEF), AMPK activity was positively correlated with -catenin content. Chemical inhibition of HDAC5 increased -catenin mRNA expression. HDAC5 over-expression reduced and HDAC5 knockdown increased H3K9 acetylation and cellular -catenin content. HDAC5 formed a complex with myocyte enhancer factor-2 (MEF2) to down-regulate -catenin mRNA expression. AMPK phosphorylated HDAC5, which promoted HDAC5 exportation from the nucleus; mutation of two phosphorylation sites in HDAC5, Ser 259 and 498, abolished the regulatory role of AMPK on -catenin expression. In summary, AMPK promotes -catenin expression through phosphorylation of HDAC5, which reduces HDAC5 interaction with the -catenin promoter via MEF2. They further tested whether AMPK cross-talks with Wnt/-catenin signaling through phosphorylation of -catenin. C3H10T1/2 mesenchymal cells were used. Chemical inhibition of AMPK and the expression of a dominant negative AMPK decreased phosphorylation of β-catenin at Ser 552. The -catenin/TCF mediated transcription was correlated with AMPK activity. In vitro, pure AMPK phosphorylated -catenin at Ser 552 and the mutation of Ser 552 to Ala prevented such phosphorylation, which was further confirmed using -32P ATP autoradiography. In summary, AMPK phosphorylates -catenin at Ser 552, which stabilizes -catenin, enhances -catenin/TCF mediated transcription. Combining all above results, our data clearly show that AMPK cross-talk with Wnt/-catenin signaling pathway via enhancing the mRNA expression and protein stability of -catenin.

Because of AMPK’s role in modulating energy utilization, the Virginia Station postulated its role in postmortem metabolism, either directly or by establishing aberrant energy charge prior to slaughter. To that end, phosphocreatine (PCr) content of muscle was manipulated by feeding pigs the creatine analogue, β-guanidinopropionic acid (β-GPA). Pigs were fed a control diet or β-GPA supplemented diet (1 or 2 wk). At harvest, indicators of energy metabolism and glycolysis (ATP, pH, lactate) were similar among muscle from control and treatment animals; however, by 45 min, muscle from animals fed β-GPA had lower glucose, glucose-6 phosphate, and lactate, greater ATP, and increased pH. The decreased rate of glycolysis resulted in improved pork quality, as demonstrated by decreased reflectance and higher subjective color scores. In other studies at the same station, adult commercial pigs with AMPK γ3R200Q and CaRC mutations were used to evaluate the effect of chronic calcium exposure on glycogen storage and glycolysis capacities. The CaRC mutation confers increased sensitivity to agents that stimulate channel opening, thus resulting in enhanced calcium release into the cytosol of muscle. Wild type and γ3R200Q, CaRC, and γ3R200Q -CaRC mutant pigs were euthanized, and samples were immediately taken from the longissimus muscle. Western blotting was used to determine the proportion of phosphorylated (Thr172) to total AMPK protein. We also analyzed glycogen synthase (GS) and phosphorylase (GP) activity in longissimus muscle. Glycogen synthase activity was determined using the incorporation of [U-14C]glucose from UDP[U-14C] glucose into glycogen. Total phosphorylase was assessed in the direction of glycogen synthesis from [U-14C]glucose 1-phosphate (G1P). In the absence of effectors, the activities of these enzymes are governed by phosphorylation. The presence of activators (G6P for synthase and AMP for phosphorylase) can largely overcome the effects of phosphorylation. Therefore, GS activity was quantified both in the absence and presence (total activity) of G6P; activity is also represented as a ratio (-G6P/+G6P). GP activity was analyzed without and with AMP, and activity is also represented as a ratio (-AMP/+AMP). The γ3R200Q mutation dramatically affected GS activity. Interestingly, in the absence of G6P, GS activity is suppressed in γ3R200Q muscle, but not in γ3R200Q-CaRC mutant muscle. Yet, when G6P is included, GS activity is greatly increased in γ3R200Q muscle regardless of CaRC genotype. They expect that, in pigs with only the γ3R200Q

mutation, there is increased glucose transport due to elevated GLUT4 levels. This results in elevated intracellular G6P, which increases GS activity and thus explains elevated glycogen in this genotype. However, the γ3R200Q-CaRC genotype is curious because muscle exhibits ‘normal’ GS activity without G6P, but ‘mutant’ GS activity when G6P is present. They are currently investigating this apparent activated AMPK independent event.

The obesity epidemic is becoming increasingly serious. To that end, the Wyoming Station keyed on the fact that obesity induces low-grade inflammation, which inhibits AMPK via a tumor necrosis factor (TNF) induced, and protein phosphatase 2C (PP2C) mediated, dephosphorylation, which has been demonstrated in our previous studies in fetal skeletal muscle. In addition, AMPK is also regulated by adipokines such as leptin, adiponectin, and resistin, and cytokines, such as interleukin-6. Furthermore, AMPK is activated during exercise. Therefore, obesity, inflammation, exercise, and likely other physiological factors that alter AMPK activity affect the differentiation of multi-potent cells through cross-talk between AMPK and Wnt/-catenin signaling. Due to the critical role of Wnt/-catenin signaling in cell differentiation and tissue development, such cross-talk between AMPK and -catenin signaling likely has important physiological and developmental implications, including exerting long-term effects on the properties of skeletal muscle and other tissues.

Researchers at the North Carolina State station are studying the phenotypical differences between muscle fibers. Coenzyme Q(10) (CoQ(10)) is a major component of the mitochondrial oxidative phosphorylation process, and it significantly contributes to the production of cellular energy in the form of ATP, a characterisitic of muscle fiber type. The objective of their studies was to determine the relationship between whole-tissue CoQ(10) content, mitochondrial CoQ(10) content, mitochondrial protein, and muscle phenotype in turkeys. Four specialized muscles (anterior latissimus dorsi, ALD; posterior latissimus dorsi, PLD; pectoralis major, PM, and biceps femoris, BF) were evaluated in 9- and 20-week-old turkey toms. The amount of muscle mitochondrial protein was determined using the Bradford assay and CoQ(10) content was measured using HPLC-UV. The amount of mitochondrial protein relative to total protein was significantly lower at 9 compared to 20 wks of age. All ALD fibers stained positive for anti-slow (S35) MyHC antibody. The PLD and PM muscle fibers revealed no staining for slow myosin heavy chain (S35 MyHC), whereas half of BF muscle fibers exhibited staining for S35 MyHC at 9 weeks and 70% at 20 weeks of age. The succinate dehydrogenase (SDH) staining data revealed that SDH significantly increases in ALD and BF muscles and significantly decreases in PLD and PM muscles with age. The study reveals age-related decreases in mitochondrial CoQ(10) content in muscles with fast/glycolytic profile, and demonstrates that muscles with a slow/oxidative phenotypic profile contain a higher proportion of CoQ(10) than muscles with a fast/glycolytic phenotypic profile. Next, they investigated the effect of Coenzyme Q analog (MitoQ(10)) on oxidative phenotype and adipogenesis in myotubes derived from turkey PM and ALD muscles. Myotubes cultures were subjected to: fusion media alone, fusion media+125nM MitoQ(10), and 500nM MitoQ(10). Lipid accumulation was visualized by Oil Red O staining and quantified by measuring optical density of extracted lipid at 500nm. Quantitative Real-Time PCR was utilized to quantify the expression levels of peroxisome proliferator-activated receptor (PPARγ) and PPARγ co-activator-1α (PGC-1α). MitoQ(10) treatment resulted in the highest lipid accumulation in PM myotubes. MitoQ(10) up-regulated genes controlling oxidative mitochondrial biogenesis and adipogenesis in PM myotube cultures. In contrast, MitoQ(10) had a limited effect on adipogenesis and down-regulated oxidative metabolism in ALD myotube cultures. Differential response to MitoQ(10) treatment may be dependent on the cellular redox state. MitoQ(10) likely controls a range of metabolic pathways through its differential regulation of gene expression levels in myotubes derived from fast-glycolytic and slow-oxidative muscles.

Insulin-like growth factor binding protein (IGFBP)-3 suppresses proliferation of numerous cell types, including myogenic cells, via both insulin-like growth factor (IGF)-dependent and IGF-independent mechanisms; however, the mechanism of IGF-independent suppression of proliferation is not clearly defined. In some non-muscle cells, binding of IGFBP-3 to the low-density lipoprotein receptor-related protein (LRP) -1/activated α2M receptor is reportedly required for IGFBP-3 to inhibit proliferation. Additionally, decorin hs been shown to suppress myogenic cell proliferation by interacting with the LRP-1 receptor. These findings suggest that binding to this receptor also may be required for IGFBP-3 to suppress proliferation of cultured myogenic cells. In order to investigate the role of the LRP-1 receptor in suppression of myogenic cell proliferation by IGFBP-3, the Minnesota station examined the effect of receptor-associated protein (RAP), an LRP-1 receptor antagonist, on IGFBP-3 inhibition of L6 myogenic cell proliferation. Treatment with RAP results in a 37% decrease (p < 0.05) in the ability of IGFBP-3 to inhibit L6 cell proliferation. In L6 cells subjected to LRP-1 siRNA treatment for 48 h (LRP-1-silenced), LRP-1 mRNA levels are reduced by greater than 80% compared to control cultures treated with nonsense siRNA (mock silenced). Additionally, the 85-kDa, transmembrane, subunit

of LRP-1 is undetectable in Western immunoblots of total protein lysates from LRP-1-silenced cells. Even though LRP-1 mRNA and protein levels were dramatically reduced in LRP-1-silenced L6 cells as compared to mock-silenced controls, IGFPB-3 suppressed proliferation rate to the same extent in both LRP-1-silenced and mock-silenced cultures. These results strongly suggest that, in contrast to data obtained for non-muscle cell lines, the LRP-1 receptor is not required in order for IGFBP-3 to suppress proliferation of myogenic cells.

Finally, the Indiana Station reported on work with a gene found in sheep known for excessive muscle growth. Delta-like 1 homolog (Dlk1) is an imprinted gene encoding a transmembrane protein whose increased expression occurs in Callipyge sheep. However, the mechanisms by which Dlk1 regulates skeletal muscle plasticity remain unknown. Here they combined conditional gene knockout and over-expression analyses to investigate the role of Dlk1 in mouse muscle development, regeneration and myogenic stem cells (satellite cells). Genetic ablation of Dlk1 in the myogenic lineage resulted in reduced body weight and skeletal muscle mass due to reductions in myofiber numbers and myosin heavy chain IIB (Myh4) gene expression. In addition, muscle-specific Dlk1 ablation led to postnatal growth retardation and impaired muscle regeneration, associated with augmented myogenic inhibitory signaling mediated by NF-B and inflammatory cytokines. To examine the role of Dlk1 in satellite cells, we analyzed the proliferation, self-renewal and differentiation of satellite cells cultured on their native host myofibers. We showed that ablation of Dlk1 inhibits the expression of the myogenic regulatory transcription factor MyoD, and facilitated the self-renewal of activated satellite cells. Conversely, Dlk1 over-expression inhibited the proliferation and enhanced differentiation of cultured myoblasts. As Dlk1 is expressed at low levels in satellite cells but its expression rapidly increases upon myogenic differentiation in vitro and in regenerating muscles in vivo, our results suggest a model in which Dlk1 expressed by nascent or regenerating myofibers non-cell autonomously promotes the differentiation of their neighbor satellite cells and therefore leads to muscle hypertrophy. Interestingly, Dlk1 mutation resulted in robust up-regulation of a Notch signaling reporter activity in vivo, suggesting that Dlk1 is a negative regulator of Notch signaling. Ongoing studies are examining this possibility. We have also established Dlk1conditional over-expression mouse model to investigate if Dlk1 over-expression enhances muscle growth, and, if so, the underlying molecular mechanism.

Objective 2. Determine molecular mechanisms that control gene expression in skeletal muscle.

The Wisconsin Station reported that several years ago they discovered a mutation in rats that dramatically affects the alternative splicing of titin. The mutation has been mapped to an obscure RNA binding protein that has a single RNA binding motif, two zinc finger domains, and an arginine-serine rich region (RS). Specific antibodies have been raised against the full length expressed protein, and staining of cultured heart and skeletal muscle cells shows the protein is localized in the nucleus. Verification that this gene controls titin splicing has been achieved using cultured cardiomyocytes from homozygote mutants that have been infected with adenoviruses containing the splice factor construct. Infected cells express the wild type titin isoform, while cells infected with a GFP construct contain only the high molecular weight titin associated with the mutant. Skeletal muscles from wild type rats undergo developmental changes in isoform expression where the fetal-neonatal titins (3.6-3.7 MDa size) become reduced in size over the next few weeks and months. The extent of the size change varies considerably

between different muscles. In contrast the titin from homozygous mutants remains at approximately 3.8 MDa in all muscles and at all postnatal ages.

The Texas station summarized their efforts to identify networks of genes in cattle that are critical for production of consistently tender and highly palatable beef, and for the effectiveness of electrical stimulation of carcasses. Their second objective is to integrate gene expression phenotype (microarray data) with SNP array data to refine QTL, and identify distinct genetic contributions that distinguish Bos indicus from Bos taurus cattle to influence tenderness and other meat quality traits. Considerable emphasis has been placed on identifying regions of the genome that harbor genes for traits that directly impact the consumer, such as marbling and tenderness. For mapping genes associated with production efficiency and nutrient utilization, a resource population of multiple F2 families of Nellore-Angus, Brahman-Angus, and Brahman-Hereford was generated at the Research Station in McGregor, TX. DNA has been collected for all animals, and phenotype has been scored for multiple characteristics including disposition, feed intake, age at puberty, and carcass and meat traits of steers. The first phase of the project has been completed. Forty-eight samples were selected from split carcasses with equal numbers representative of high or low Warner-Bratzler shear force measurements and electrical stimulation or no treatment groups. Single channel microarray hybridizations were conducted on Agilent bovine microarrays. The second phase of the project including data analysis and follow-up experiments are underway.

The objective of the Michigan Station was to quantify expression of four genes responsible for Ca2+-regulation in turkey skeletal muscle as a function of heat stress: α and β ryanodine receptors (RYRs), the sarcoplasmic reticulum (SR) Ca2+-pump (SERCA1), and the SR Ca2+-storage protein calsequestrin (CASQ1). Two genetic lines of turkeys were utilized: a growth-selected commercial line and a random-bred control line. Market-weight birds were subjected to one of five heat stress treatments: 0d, 1d, 3D, or 5D; or 7d heat followed by 7d ambient temperature. Breast muscle samples were harvested and classified as “normal” or “PSE” based on high marinade uptake/low cook loss, or low marinade uptake/high cook loss, respectively. TaqMan quantitative real-time PCR assays were developed to quantify expression of the four genes. β-actin expression was not altered by line, stress, or meat quality status and was used as an endogenous control. The results indicate that heat treatment significantly (p<0.05) enhanced expression of αRYR, βRYR and CASQ1 in normal muscle samples from both lines. Conversely, mRNA abundance from these genes was significantly (p<0.05) reduced in PSE meat samples from both lines. Expression of SERCA1 in both normal and PSE samples from both lines was unchanged or trending downward with stress. Our results suggest that birds within both genetic lines exhibit differential responses to heat stress resulting in altered Ca2+ homeostasis which may, in part, be responsible for observed differences in meat quality.

With the aim of developing a new tool for the study of turkey growth and development at the muscle transcriptome level, a 6K oligonucleotide microarray was constructed by the Michigan Station: the Turkey Skeletal Muscle Long Oligo (TSKMLO) microarray. Skeletal muscle samples were collected at three critical stages in muscle development: 18-day embryo (hyperplasia), 1-day post-hatch (hypertrophy), and 16-week (market age) from two genetic lines of turkeys: RBC2, a line maintained without selection pressure, and F, a line selected from the RBC2 line for increased 16-week body weight. Oligonucleotides were designed from sequences obtained from skeletal muscle cDNA libraries from the three developmental stages. Several unique controls, including mismatch and distance controls and scrambled sequences, were

designed for 30 genes. Quality control hybridizations were completed, confirming the validity and repeatability of the array. Control features were evaluated across two larger experiments comparing developmental stage within genetic line or genetic line within each developmental stage, totaling 70 arrays. Mismatch and scrambled sequences appeared to be useful controls of specific hybridization for most genes. In addition, quantitative real-time RT-PCR confirmed microarray results.

The goal of a subsequent study by the Michigan Station was to identify genes associated with turkey muscle growth throughout development. To achieve this goal, gene expression was evaluated utilizing a 6K turkey skeletal muscle-specific long oligonucleotide (TSKMLO) microarray. Skeletal muscle samples were collected at three critical stages in muscle development: 18d embryo (hyperplasia), 1d post-hatch (shift from myoblast-mediated growth to satellite cell-modulated growth by hypertrophy), and 16wk (market age) from two genetic lines: a randombred control line (RBC2) maintained without selection pressure, and a line selected from the RBC2 line for increased 16wk body weight (F). Hybridizations were performed in two experiments: Experiment 1 (40 arrays) directly compared the developmental stages within genetic line, while Experiment 2 (30 arrays) directly compared the two lines within each stage. After statistical analysis, 3474 genes were found to be differentially expressed (false discovery rate; FDR < 0.001) by overall effect of development, while 16 genes were found to be differentially expressed (FDR < 0.10) by overall effect of genetic line. Ingenuity Pathways Analysis grouped annotated genes into networks, functions, and canonical pathways. The expression of twenty-eight genes involved in extracellular matrix regulation, cell death/apoptosis, and calcium signaling/muscle function, as well as genes with miscellaneous function was confirmed by qPCR. This study uncovered novel genes important in turkey muscle growth and development. Future experiments will focus further on several of these candidate genes and the expression and mechanism of action of their protein products.

Skeletal muscle development is a complex process involving the coordinated expression of thousands of genes.  The aim of this study by the Michigan Station was to identify differentially expressed genes in longissimus dorsi (LD) muscle of pigs at 40 and 70 days (d) of gestation (developmental stages encompassing primary and secondary fiber formation) in Yorkshire-Landrace (YL) crossbred pigs and Piau pigs (a naturalized Brazilian breed), two breed types that differ in muscularity.  Fetuses were obtained from gilts at each gestational age (n = 3 YL; n = 4 Piau), and transcriptional profiling was performed using the Pigoligoarray microarray containing 20,400 oligonucleotides.  A total of 486 oligonucleotides were differentially expressed (FC ≥ 1.5; FDR ≤ 0.05) between 40 and 70 d gestation in either YL or Piau pigs, and a total of 1,300 oligonucleotides were differentially expressed (FC ≥ 1.5; FDR ≤ 0.05) between YL and Piau pigs at either age.  Gene ontology annotation and pathway analyses determined functional classifications for differentially expressed genes and revealed breed type-specific developmental expression patterns.  Thirteen genes were selected for confirmation by qRT-PCR analyses and expression patterns for most of these genes were confirmed, providing further insight into the roles of these genes in pig muscle development.  This study revealed both developmental and breed type-specific patterns of gene expression in fetal pig skeletal muscle including genes not previously associated with myogenesis, and this information can contribute to future pig genetic improvement efforts.

The Arizona Station is examining the role of environmental influences on skeletal muscle growth, metabolism and physiology. Heat-stressed cattle also exhibit alterations in glucose

metabolism by increasing glucose disposal (rate of glucose entry into cells). Evidence indicates that beef and lactating dairy cattle experiencing heat stress undergo dramatic changes in energy and nutrient metabolism that may affect both their heat tolerance and production parameters, such as lean tissue accretion or milk yield. Because a large proportion of an animal’s mass is comprised of skeletal muscle, any alteration in skeletal muscle metabolism and function can have a profound impact on whole-animal energy metabolism and nutrient homeostasis especially during periods of stress. They have initiated a series of studies aimed at understanding how hyperthermia influences the set points of several metabolic pathways within skeletal muscle. For example, skeletal muscle PDK4 increases with heat stress and while abundance of PDH protein did not differ across treatments, phosphorylation state of the PDH complex was affected by treatment. Phosphorylated PDH-E1 was increased in soleus and tended to increase in TA following 1 and 2 bouts of heat. Data suggests a decrease in glucose oxidation within type 1 skeletal muscle following a heat load. Bioenergetic changes occurring in response to heat stress could be due to heat-induced mitochondria damage. Antioxidant mRNA abundances (GPX1 and 8, Nqo1 and catalase) were greater in type I compared to type II skeletal muscle and this difference was even greater following a single heat load, but was reduced following a 2nd heat exposure. Changes in gene expression are consistent with impaired cellular energy status, perhaps due to mitochondrial dysfunction, in oxidative compared to glycolytic muscle fibers during hyperthermia. These data suggest a greater level of oxidative stress is occurring within type I compared to type II skeletal muscle after a single heat load. Taken together, our studies in ruminants and rodents show that heat stress alters skeletal muscle gene expression in a manner that affects skeletal muscle metabolism and function, which may be important for whole-body metabolism and overall physiological adaptation to hyperthermia.

The underlying cause of Duchenne muscular dystrophy is the absence of a functional dystrophin protein. This protein is responsible for linking cytoskeletal actin to the cell membrane and ultimately to the extracellular matrix through the dystrophin-glycoprotein complex (DGC). DMD is modeled by the mdx mouse, which is also dystrophin deficient. Utrophin is a dystrophin-related protein that can interact with both the actin cytoskeleton and the DGC. In animal models, utrophin replacement has shown great promise as a therapeutic strategy for DMD, however, a practical means of delivery in human patients is lacking. It has been previously shown that PGC-1α increases utrophin expression, as well as expression of oxidative proteins, which may be of additional benefit to dystrophic muscle. Further, transgenic PGC-1α over-expression reduced eccentric injury in mdx mice. As dystrophin deficiency results in developmental abnormalities potentially corrected by transgenic PGC-1α over-expression, the Iowa Station reported that it is imperative to determine the extent to which PGC-1α over-expression induced postnatally blunts acute eccentric injury in dystrophin-deficient muscle. Neonatal mdx mice were injected in the left hind limb with null AAV6 and in the right hind limb with an AAV6 driving expression of PGC-1alpha (1x10^11 gc). At six weeks of age, animals ran downhill (-17 degrees, 10 m/min, 60 min) and were sacrificed three days later. PGC-1α over-expression reduced total damaged area by 50% (p<0.05). More specifically, the areas of hypercontracted cells, immune cell infiltration, and missing cells were reduced by 90%, 60%, and 66%, respectively (p<0.05). Centralized nuclei were reduced by 25% (p<0.05). Given that DMD patients are sensitive to eccentric injury, the degree of protection conferred to dystrophic muscle in this investigation justifies further exploration of PGC-1alpha as a therapy for DMD.

The Iowa Station determined the extent to which dystrophin deficiency impacts the muscle proteome. We performed two dimensional differential in-gel electrophoresis (2D-DIGE) (pH 4-

7) on gastrocnemi taken from 6 week old male mice (n=6 mdx; n=6 C57). Of a total of 2200 spots detected, 41 spots (representing unique isoforms or modified proteins) were found to be differentially expressed (p<0.1; 24 spots with p<0.05). Of these, 30 spots were increased and 11 were decreased in mdx compared to control. Using mass spectroscopy and MALDI-TOF, the identity of the proteins represented was confirmed in 36 spots. These proteins represent a variety of cell functions including apoptosis, mitochondrial function, and cell growth and differentiation. Further analysis of these proteins may allow for the identification of novel pathways and lead to the development of innovative strategies to minimize disease progression.

In other work, the Iowa Station elected to investigate microRNAs, a subclass of RNA that generally reduces gene product expression through transcript degradation and/or inhibition of translation. To better understand downstream consequences of dystrophin deficiency and regulation of protein expression, they measured microRNA expression in dystrophic skeletal muscle. Our hypothesis was that dystrophin deficiency would cause a change in expressed microRNAs compared to control muscle. Purified total RNA from six week old, male gastrocnemii (n=4 mdx; n=4 C57) were analyzed with miRCURY™ LNA arrays (Exiqon, Denmark). We identified 37 microRNAs that were differentially expressed (p<0.0001). Of these, 17 were down-regulated and 20 were up-regulated, including mir-206, in mdx mice compared to controls. Evaluation of the genes these microRNAs are predicted to regulate revealed an array of processes that are potential candidates for miRNA regulation. Therefore, these results may help to identify novel pathways that contribute to disease pathology. They are currently in the process of linking microRNA expression to alterations in protein expression.

The Nebraska Station reported on two-dimensional electrophoretic separations of muscle membrane proteins. The hydrophobic nature of SR membrane proteins presents strong challenges to characterize and study the physiological function of the proteins. Protein hydrophobicity and size contribute to limited resolution in traditional 2-D electrophoretic methods. We have developed a non-denaturing electrophoresis system based on solublization of membrane proteins with other surfactants. Selective dissection of membrane protein complexes has been achieved and high molecular weight membrane native complexes identified. Specifically, three high molecular weight complexes were identified using the Blue Native-SDS-PAGE 2D technique . Complex 1 at about 1.5 mDa) was found to be composed of myosin heavy chain dimer. Complex 2) at about 1.2 mDa was composed of fast twitch Ca ATPase , amylo-1,6-glucosidase, and phosphorylase b, and complex 3) at about 275 kDa, was composed of ATP synthase, calsequestrin and creatine kinase. These data demonstrate the capability of the electrophoresis technique to identify complexes found under native conditions.

Objective 3. Characterize mechanisms of protein assembly and degradation in skeletal muscle

The Nebraska Station reported on characterizing skeletal muscle proteolysis using HPLC followed by MS/MS sequencing of bovine skeletal muscle extracts of carnosine and other low mass peptides. The procedure involved extraction of pulverized muscle with a Tris buffered solution and differential sedimentation centrifugation to clarify the sample. Aliquots of the extract were centrifuged on 5 kDa or 30kDa spin filters and then dried. Samples were taken up in 0.02% TFA and analyzed by RP-HPLC. Fractions of the separation were collected, dried, and analyzed using newly developed LC/MS/MS methods. The protocol has identified numerous fragments of myofibrillar, sarcoplasmic, and SR proteins.

Troponin (Tn) mediated regulation of contraction involves calcium binding to regulatory domain of TnC resulting in a conformational change that exposes a high affinity binding site for the regulatory domain of TnI. Subsequently, the movement of the regulatory domain of TnI from actin-Tm to TnC switches the thin filament from the B to the C state. In previous work by the Indiana Station using Tn exchange kinetics, data suggest that the movement of TnI from actin-Tm to TnC is mediated by both calcium and rigor crossbridges. To further characterize this movement, the Indiana Station developed a Fluorescence Resonance Energy Transfer (FRET) probe to monitor the distance between the regulatory domains of TnI and TnC. Single cysteine mutants of skeletal TnI (amino acid 134C) and TnC (S10C) were developed by mutagenesis and labeled with a donor and acceptor respectively. Steady state fluorescence was used to monitor the donor intensity as a function of calcium, actin-Tm, and rigor S1 binding to actin-Tm. The calculated distances under the different conditions for Tn are 51Å at low and 44Å at high calcium. With Tn bound to actin-Tm, the distances are 56Å at low and 46Å at high calcium while addition of saturating rigor S1 to actin-Tm gave distances of 52Å at low and 44Å at high calcium. Thus, in Tn alone, calcium decreases the distances between the regulatory domains while binding of Tn to actin-Tm moves TnI further away from TnC at low calcium and addition of calcium moves TnI closer to TnC but not the same extent as in Tn alone. Rigor S1 moves TnI closer to TnC at both low and high calcium, and only in the presence of both rigor S1 and calcium is the distance between the regulatory domains the same as observed in Tn alone. To further investigate the calcium sensitivity of the distance change, donor emission was measured as a function of calcium for Tn alone, actin-Tm-Tn and actin-Tm-Tn-S1. The calcium sensitivity of donor emission was greatest with actin-Tm-Tn-S1 and least with actin-Tm-Tn.

Analysis of the stability of the Tn complex using dilution and unlabeled TnC exchange suggested that the conservative cysteine mutations in TnC (C99S) and TnI (C49S and C65S) to generate the monocysteine mutants weaken the structural stability of the complex. This potentially complicates the interpretation of the FRET data. Mutation of these cysteines to alanines resulted in a more stable complex. Analysis of the distances using the alanine constructs shows the same trends in the distance changes under the various conditions, but the absolute magnitude of the distance is less for the alanine constructs.

These data show that rigor crossbridges move the regulatory domain of TnI closer to the regulatory domain of TnC by 1 - 3 Å and increase the calcium sensitivity of the regulatory interaction. Thus, part of the mechanism by which crossbridges facilitate the B to C state transition is by moving the regulatory domain of TnI from actin-Tm and towards the regulatory domain of TnC. The current generation of FRET probes has not proven useful for application in myofibrils since either the stability of the complex for the serine constructs results in loss of the acceptor during manipulation or, for the alanine constructs, the donor alone is sensitive to strong crossbridges. Further development with different donor/acceptor dyes is needed to address these problems.

Previous work by the Indiana Station showed that the B to C state equilibrium is more favored towards the C state at low calcium for cardiac (c) Tn than skeletal (s) Tn as shown by higher ATPase activity and a faster Tn exchange rate. At high calcium, the B to C state equilibrium is less favored for cTn than sTn as evidenced lower magnitude of the effect of calcium on Tn exchange in the non-overlap region. Some disease-associated mutations in cTnI result in decreased inhibition of ATPase and increased diastolic pressure suggesting that the B to C state equilibrium at low calcium is more favored towards the C state. To test this hypothesis, the

R145G human cardiac (hc) TnI mutant was made, reconstituted into hcTn and exchanged into myofibrils. The ATPase activity and hcTn exchange kinetics were characterized and compared to wt hcTn. The pCa – ATPase activity relationship showed that the R145G mutant had higher pCa 9 ATPase activity, was more calcium sensitive, and had similar cooperativity of calcium activation of activity as wt hcTn. The kinetics of Tn exchange showed that the R145G mutation had a faster exchange rate at low calcium than the wt hcTn. Interestingly, at high calcium, the R145G mutant had a faster exchange rate. These data suggest that this mutation results in weaker binding of Tn to actin-Tm mediated by a weaker interaction of the regulatory domain of TnI. This modulation of the TnI – actin-Tm interaction results in weaker inhibition of ATPase activity and a greater effect of calcium on Tn exchange kinetics. Overall, these data support a minimum two-step model for the B to C state transition that includes the TnI – actin-Tm equilibrium and the calcium-dependent TnI – TnC equilibrium as suggested by Yang et al., Biophys. J. 97:183.

Myosin binding protein C (MyBPC) is a multi-domain protein associated with the thick filaments of striated muscle. While both structural and regulatory roles have been proposed for MyBPC, its interactions with other sarcomeric proteins remain obscure. The current study by the Wisconsin Station was designed to examine the actin-binding properties of MyBPC and to define MyBPC domain regions involved in actin interaction. Here, we have expressed full-length mouse cardiac MyBPC (cMyBPC) in a baculovirus system and show that purified cMyBPC binds actin filaments with an affinity of 4.3 + 1.1 μM and a 1:1 molar ratio with regard to actin protomer. The actin binding by cMyBPC is independent of protein phosphorylation status and is not significantly affected by the presence of tropomyosin and troponin on the actin filament. In addition, cMyBPC-actin interaction is not modulated by calmodulin. In order to determine the region of cMyBPC that is responsible for its interaction with actin, we have expressed and characterized five recombinant proteins encoding fragments of cMyBPC sequence. Recombinant N-terminal fragments such as C0-C1, C0-C4 and C0-C5 cosediment with actin in a linear, non-saturable manner. At the same time, MyBPC fragments lacking either the C0-C1 or C0-C4 region bind F-actin with essentially the same properties as full-length protein. Together, our results indicate that cMyBPC interacts with actin via a single, moderate affinity site localized to the C-terminal region of the protein. In contrast, certain basic regions of the N-terminal domains of MyBPC may act as small polycations and therefore bind actin via non-specific electrostatic interactions.

Although androgenic and estrogenic steroids are widely used to enhance muscle growth and increase feed efficiency in feedlot cattle, their mechanism of action is not well understood. While in vivo studies indicate that E2 and TBA, a synthetic androgen, affect muscle protein synthesis and/or degradation, in vitro results are inconsistent. Therefore, the scientists at the Minnesota station examined the effects of E2 and TBA treatment on protein synthesis and degradation rates in fused bovine satellite cell (BSC) cultures. Additionally, in order to learn more about the mechanisms involved in E2- and TBA-enhanced muscle growth, they examined the effects of compounds that interfere with binding of E2, TBA or IGF-I to their respective receptors on E2- and TBA-induced alterations in protein synthesis and degradation rates in BSC cultures. Treatment of fused BSC cultures with E2 results in a concentration-dependent increase in protein synthesis rate and a decrease in protein degradation rate. A pure estrogen antagonist suppresses E2 -induced alterations in protein synthesis and degradation in fused BSC cultures. Although the G-protein coupled receptor (GPR)-30 is involved in the E2-stimulated increase in muscle IGF-1 mRNA level, the GPR30 agonist, G1, does not affect either synthesis or degradation rate

establishing that GPR30 does not play a role in E2-induced alterations in protein synthesis or degradation. JB1, a competitive inhibitor of IGF-I binding to the Type 1 insulin-like growth factor receptor (IGFR-1), suppresses E2-induced alterations in protein synthesis and degradation. In summary our data show that E2 treatment directly alters both protein synthesis and degradation rates in fused BSC cultures via mechanisms involving both the classical ER and IGFR-1. Treatment of fused BSC cultures with TBA results in a concentration-dependent increase in protein synthesis rate and a decrease in degradation rate, establishing that TBA directly affects these parameters. Flutamide, a compound that prevents androgen binding to the androgen receptor, suppresses TBA -induced alterations in protein synthesis and degradation in fused BSC cultures, indicating the androgen receptor is involved. JB1, a competitive inhibitor of IGF-I binding to the IGFR-1, suppresses TBA-induced alterations in protein synthesis and degradation indicating that this receptor also is involved in the actions of TBA on both synthesis and degradation. In summary, their data show that TBA acts directly to alter both protein synthesis and degradation rates in fused BSC cultures via mechanisms involving both the androgen receptor and IGFR-1

II. Impact Statements

New knowledge that will be disseminated by publication in peer reviewed journals. Graduate students and post-doctoral fellows are being educated. Characterization of genes that contribute to tenderness despite electrical stimulation will

contribute to development of improved selection strategies for the beef cattle industry. Creation and assessment of the TSKMLO array provides a valuable resource for the

study of gene expression changes related to turkey muscle growth and development. Several novel genes were identified which play critical roles in muscle cell growth and

development. Established that over-expression of select genes prevents acute muscle injury in

dystrophic mice. Established that early dystrophin-deficiency has a significant impact on muscle protein

expression. Established early dystrophin-deficiency alters micro-RNA expression. Demonstration of a biological function of the proteoglycan N-glycosylated chains for a

heparan sulfate proteoglycan. Showed N-glycosylated chains affect satellite cell properties critical for muscle growth. Discovery of gene products involved in calcium homeostasis. The ability to separate membrane proteins in the native state. Development of methods to characterize and identify low mass peptides. Characterization of alternative splicing in skeletal muscles.

III. Publications

a. Published

Abel, E.L., J.M. Angel, P.K. Riggs, L. Langfield, H.-H. Lo, M.D. Person, Y.C. Awasthi, L.E. Wang, S.S. Strom, Q. Wei, and J. DiGiovanni. 2010. Gsta4, a modifier of

susceptibility to skin tumor development in mice and humans. J. Natl Cancer Inst. 102:1663-1675.

Alexander L.S., A. Mahajan, J. Odle, K.L. Flann, R.P. Rhoads and C.H. Stahl. 2010. Dietary P restriction decreases stem cell proliferation and subsequent growth potential in the neonatal pig. Journal of Nutrition 140:477-82.

Anderson, K.E., P.E. Mozdziak and J.N. Petitte. 2010. The impact of scheduled cage cleaning on older hens (Gallus gallus). Lab. Anim. (NY). 39(7):210-5.

Bernabucci, U, N. Lacetera, L.H. Baumgard, R.P. Rhoads, B. Ronchi and A. Nardone. 2010. Metabolic and hormonal adaptations to heat stress in ruminants. Animal 4(7):1167-1183.

Chen, J., M.V. Dodson and Z. Jiang. 2010. Cellular and molecular comparison of redifferentiation of intramuscular- and subcutaneous- adipocyte derived progeny cells. International Journal of Biological Science 6(1):80-88.

Crawford, S.M., Moeller, S.J., Zerby, H.N., Irvin, K.M., Kuber, P.S., Velleman, S.G., and Leeds, T.D. 2010. Effects of cooked temperature on pork tenderness and relationships among muscle physiology and pork quality traits in loins from Landrace and Berkshire swine. Meat Sci. 84:607-612.

Depreux, F.F.S, A.L. Grant, C.A. Bidwell and D.E. Gerrard. 2010. Molecular cloning and characterization of porcine calcineurin-alpha subunit delta isoform expression in skeletal muscle. J Anim Sci. 88(2):562-71.

Dilger, A.C., M.E. Spurlock, A.L. Grant and D.E. Gerrard. 2010. Myostatin null mice respond differently to dietary-induced and genetic obesity. Anim Sci J. 81(5):586-93.

Dodson, M.V. G.J. Hausman, L.L. Guan, M. Du, T.P. Rasmussen, S.P. Poulos, P. Mir, W.G. Bergen, M.E. Fernyhough, D.C. McFarland, R.P. Rhoads, B. Soret, J.M. Reecy, S.G. Velleman, and Z. Jiang. 2010. Skeletal muscle stem cells from animals I. Basic cell biology. International Journal of Biological Sciences, 6:465-474.

Dodson, M.V. G.J. Hausman, L.L. Guan, M. Du, T.P. Rasmussen, S.P. Poulos, P. Mir, W.G. Bergen, M.E. Fernyhough, D.C. McFarland, R.P. Rhoads, B. Soret, J.M. Reecy, S.G. Velleman and Z. Jiang. 2010. Skeletal muscle stem cells from animals I. Basic cell biology. Int. J. Biol. Sci. 6:465-74.

Dodson, M.V. G.J. Hausman, L.L. Guan, M. Du, T.P. Rasmussen, S.P. Poulos, P. Mir, W.G. Bergen, M.E. Fernyhough, D.C. McFarland, R.P. Rhoads, B. Soret, J.M. Reecy, S.G. Velleman and Z. Jiang. 2010. Skeletal muscle stem cells from animals I. Basic cell biology. International Journal of Biological Science 6(5):465-474.

Dodson, M.V., G.J. Hausman, L.L. Guan, M. Du, T.P. Rasmussen, S.P Poulos, P. Mir, W.G. Bergen, M.E. Fernyhough, D.C. McFarland, R.P. Rhoads, B. Soret, J.M. Reecy, S.G. Velleman and Z. Jiang. 2010. Lipid metabolism, adipocyte depot physiology and utilization of meat animals as experimental models for metabolic research. International Journal of Biological Science 6(7):682-690.

Dodson, M.V., G.J. Hausman, L.L. Guan, M. Du, T.P. Rasmussen, S.P. Poulos, P. Mir, W.G. Bergen, M.E. Fernyhough, D.C. McFarland, R.P. Rhoads, B. Soret, J.M. Reecy,

S.G. Velleman, and Z. Jiang. 2010. Skeletal muscle stem cells from animals I. Basic cell biology. Int. J. Biol. Sci. 6:465-474.

Dodson, M.V., G.J. Hausman, L.L. Guan, M. Du, T.P. Rasmussen, S.P Poulos, P. Mir, W.G. Bergen, M.E. Fernyhough, D.C. McFarland, R.P. Rhoads, B. Soret, J.M. Reecy, S.G. Velleman and Z. Jiang. Lipid metabolism, adipocyte depot physiology and utilization of meat animals as experimental models for metabolic research. International Journal of Biological Science 6(7):682-690.

Dodson, M.V., J.L. Vierck, G.J. Hausman, L.L. Guan, M.E. Fernyhough, S.P. Poulos, P. Mir and Z. Jiang. 2010. Examination of adipose-specific PPAR moieties. Biochemical and Biophysical Research Communications 394:241-243.

Dodson, M.V., L.L. Guan, M.E. Fernyhough, P.S. Mir, L. Bucci, D.C. McFarland, J. Novakofski, J.M. Reecy, K.M. Ajuwon, D.P. Thompson, G.J. Hausman, M. Benson, W.G. Bergen, and Z. Jiang. 2010. Perspectives on the formation of an interdisciplinary research team. Biochem. Biophys. Res. Commun. 391:1155-1157.

Dodson, M.V., Z. Jiang, J. Chen, G.J. Hausman, L.L. Guan, J. Novakofski, D. Thompson, C. Lorenzen, M.E. Fernyhough, P. Mir and J. Reecy. 2010. Allied industry approaches to alter intramuscular fat content and composition in beef animals. Journal of Food Science 75:R1-8.

Dodson, M.V., L.L. Guan, M.E. Fernyhough, P.S. Mir, L. Bucci, D.C. McFarland, J. Novakofsky, J.M. Reecy, K.M Ajuwon, D.P. Thompson, G.J. Hausman, W.G. Bergen, M. Benson and Z. Jiang. 2010. Perspectives on the formation of an interdisciplinary research team. Biochemical Biophysical Research Communications 391:1155-1157.

Du, M., J.D. Yin, and M.J. Zhu. 2010. Cellular signaling pathways regulating adipogenesis and marbling of skeletal muscle. Meat Sci., 86:103-109.

Du, M., J.F. Tong, J.X. Zhao, K.R. Underwood, M.J. Zhu, S.P. Ford, and P.W. Nathanielsz. 2010. Fetal programming of skeletal muscle development in ruminant animals. Journal of Animal Science, 88: E51-60.

Du, M., X. Yan, J.F. Tong, J.X. Zhao, and M.J. Zhu. 2010. Maternal obesity, inflammation and fetal skeletal muscle development. Biology of Reproduction, 82: 4-12.

Fernyhough, M.E., L.R. Bucci, J. Feliciano and M.V. Dodson. 2010. The effects of nutritional supplements on the proliferation and differentiation of muscle-derived stem cells. International Journal of Stem Cells 3:63-67.

Gillespie M.A., F. Le Grand, A. Scime, S. Kuang, J. von Maltzahn, V. Seale, A. Cuenda, J.A. Ranish and M.A. Rudnicki. 2009. p38gamma-dependent gene silencing restricts entry into the myogenic differentiation program. J Cell Biol. 187:991-1005.

Hausman, G.J., M.V. Dodson, K. Ajuwon, M. Azain, K.M. Barnes, L.L. Guan, Z. Jiang, S.P. Poulos, R.D. Sainz, S. Smith, M. Spurlock, J. Novakofski, M.E. Fernyhough and W.G. Bergen. 2009. Board Invited Review: The biology and regulation of preadipocytes and adipocytes in meat animals. Journal of Animal Science 87:1218-1246 

Hawkridge, A.M., R.B. Wysocky, J.N. Petitte, K.E. Anderson, P.E. Mozdziak, O.J. Fletcher, J.M. Horowitz and D.C. Muddiman. 2010. Measuring the intra-individual variability of the plasma proteome in the chicken model of spontaneous ovarian adenocarcinoma. Anal Bioanal Chem. 398(2):737-49.

He, M.L., R. Sharma, P. Mir, E. Okine and M.V. Dodson. 2010. Feed withdrawl regimens can abate lipodystrophy, but glucose tolerance is associated with appropriate diameter of retroperitoneal adipocytes in rats. Nutrition Research 30:125-133.

Huang, Y., X. Yan, M.J. Zhu, R.J. McCormick, S.P. Ford, P.W. Nathanielsz, and M. Du. (2010). Enhanced transforming growth factor (TGF-) signaling and fibrogenesis in fetal skeletal muscle of obese sheep at late gestation. American Journal of Physiology-Endocrinology and Metabolism, 298: E1254-E1260.

Jin, W., M.V. Dodson, S.S. Moore, J.A. Basarb and L.L. Guan. 2010. Characterization of microRNA expression in bovine subcutaneous fat tissues: A potential regulatory mechanism of subcutaneous adipose tissue development. BMC Molecular Biology 11:29-37. 

Kamanga-Sollo, E., M.E. White, M.R. Hathaway, W.J. Weber, and W.R. Dayton. 2010. Effect of trenbolone acetate on protein synthesis and degradation rates in fused bovine satellite cell cultures. Domest. Anim. Endrocrinol. 39(1):54-62.

Kandadi, M. R., P.K. Rajanna, M.K. Unnikrishnan, S.P. Boddu, Y.N. Hua, J. Li, M. Du, J. Ren, and N. Sreejayan. (2010). 2-(3,4-Dihydro-2H-pyrrolium-1-yl)-3-oxoindan-1-olate (DHPO), a novel, synthetic small molecule that alleviates insulin resistance and lipid abnormalities. Biochemical Pharmacology, 79: 623-631.

Kochan, K.J., R.N. Vaughn, T.S. Amen, C.A. Abbey, J.O. Sanders, D.K. Lunt, A.D. Herring, J.E. Sawyer, C.A. Gill, and P.K. Riggs. 2009. Expression of mitochondrial respiratory complex genes in liver tissue of cattle with different feed efficiency phenotypes. Proc. Assoc. Advmt Anim. Breed Genet. 18:175-178.

Lee, R.S, S.B. Tikunova, K.P. Kline, H.G. Zot, D.R. Swartz, J.A. Rall, and J.P. Davis. 2010. Effect of the Ca2+ binding properties of troponin c on the rate of skeletal muscle force redevelopment. Am. J. Physiol. 299: C1091 - C1099.

Li, F., J. Yin, J. Ni, L. Liu, H. Zhang, and M. Du. (2010). Chloride intracellular channel 5 modulates adipocyte accumulation in skeletal muscle by inhibiting preadipocytes differentiation. Journal of Cellular Biochemistry, 110: 1013-1021.

Michelizzi, V.N., M.V. Dodson, Z. Pan, J.A. Michal, D.J. McLean, J.E. Womack and Z. Jiang. 2010. Water buffalo genome science comes of age. International Journal of Biological Science 6:333-349.

Mittal A., S. Bhatnagar, A. Kumar, P.K. Paul, S. Kuang and A. Kumar. 2010. Genetic ablation of TWEAK augments regeneration and post-injury growth of skeletal muscle in mice. Am J Pathol. 177(4):1732-42.

Nierobisz, L.S., N.G. Hentz, J.V. Felts and P.E. Mozdziak. 2010. Fiber phenotype and coenzyme Q ₁₀ content in turkey skeletal muscles. Cells Tissues Organs 192(6):382-94.

O’Brien M.D., R.P. Rhoads, S.R. Sanders, G.C. Duff, and L.H. Baumgard. 2010. Metabolic adaptations to heat stress in growing cattle. Domestic Animal Endocrinology 38:86-94.

Poulos, S.P., M.V. Dodson and G.J. Hausman. 2010. Cell line models for differentiation: Preadipocytes and adipocytes. Experimental Biology and Medicine 235:1185-1193.

Ranasinghesagara, J., T.M. Nath, S.J. Wells, A.D. Weaver, D.E. Gerrard, and G. Yao. 2010. Imaging optical diffuse reflectance in beef muscles for tenderness prediction Meat Sci. 84(3):413-21.

Rhoads, R., M.E. Fernyhough, S.G. Velleman, D.C. McFarland, X. Liu, G.J. Hausman and M.V. Dodson. 2009. Invited review: Extrinsic regulation of domestic animal-derived satellite cells II. Domestic Animal Endocrinology 36:111-126

Riggs, P. K., and M. Rønne. 2009. Fragile sites in domestic animal chromosomes: Molecular insights and challenges. Cytogenet. Genome Res. 126:97–109.

S.B. Lee, Y.S. Kim, M.Y. Oh, I.H Jeong, K.B. Seong and H.J. Jin. 2010. Improving rainbow trout (Oncorhynchus mykiss) growth by treatment with a fish (Paralichthys olivaceus) myostatin prodomain expressed in soluble forms in E.coli. 2010. Aquaculture 302:270-278.

Satterfield, M.C., G. Song, K.J. Kochan, P.K. Riggs, R.M. Simmons, C.G. Elsik, D.L. Adelson, F.W. Bazer, H. Zhou, and T.E. Spencer. 2009. Discovery of candidate genes and pathways in the endometrium regulating ovine blastocyst growth and conceptus elongation. Physiol Genomics 39:85-99.

Shen, Q.W. and D.R. Swartz. 2010. Influence of salt and pyrophosphate on bovine fast and slow myosin S1 dissociation from actin. Meat Sci. 84:364-370.

Song, Y., K.E. Nestor, D.C. McFarland, and S.G. Velleman. 2010. Effect of glypican-1 covalently attached chains on turkey myogenic satellite cell proliferation, differentiation, and fibroblast growth factor 2 responsiveness. Poultry Science 89:123-134.

Song, Y., Nestor, K.E., McFarland, D.C., and Velleman, S.G. 2010. Effect of glypican-1 covalently attached chains on turkey myogenic satellite cell proliferation, differentiation, and fibroblast growth factor 2. Poult. Sci. 89:123-134.

Sporer, K.R.B., W. Chiang, R.J. Tempelman, C.W. Ernst, K.M. Reed, S.G. Velleman, and G.M. Strasburg. 2010. Characterization of a 6K oligonucleotide turkey skeletal muscle microarray. Animal Genetics doi:10.111/j.1365-2052.

Tikunova, S.B., Liu, B., Swindle, N., Little, S.C., Gomes, A.V., Swartz, D.R. and J.P. Davis 2010. Effect of calcium-sensitizing mutations on calcium binding and exchange with troponin C in increasingly complex biochemical systems. Biochemstry. 49:1 1975-1984.

Underwood, K.R., J.F. Tong, P.L. Price, A.J. Roberts, E.E. Grings, B.W. Hess, W.J. Means, and M. Du. (2010). Gestational nutrition affects growth, adipose tissue deposition, and tenderness in steers. Meat Science, 86:588-593.

Velleman, S.G., Coy, C.S., and Nestor, K.E. 2010. The influence of age on maternal inheritance of breast muscle morphology in turkeys. Poult. Sci. 89:876-882.

Velleman, S.G., Nestor, K.E., Coy, C.S., Harford, I., and Anthony, N.B. 2010. Effect of posthatch feed restriction on broiler breast muscle development and muscle transcriptional regulatory factor gene and heparan sulfate proteoglycan expression. Int. J. Poult. Sci. 9:417-425.

Velleman, S.G., X. Zhang, C.S. Coy, Y. Song, and D.C. McFarland. 2010. Changes in satellite cell proliferation and differentiation during turkey muscle development. Poultry Science 89:709-715.

Velleman, S.G., Zhang, X., Coy, C.S., Song, Y., and McFarland, D.C. 2010. Changes in satellite cell proliferation and differentiation during turkey muscle development. Poult. Sci. 89:709-715.

Wu, M., J. Hall, R.M. Akers and H. Jiang. 2010. Effect of feeding level on serum insulin-like growth factor I (IGF-I) response to growth hormone injection. Journal of Endocrinology 206:37–45.

Xiao, S., W.G. Zhang, Y. Yang, C.W. Ma, D.U. Ahn, X. Li, J.K. Lei, and M. Du. (2010). Changes of hormone-sensitive lipase (HSL), adipose tissue triglyceride lipase (ATGL) and free fatty acids in subcutaneous adipose tissues throughout the ripening process of dry-cured ham. Food Chemistry, 121: 191-195.

Yamada M., Tatsumi R., Yamanouchi K., Hosoyama T., Shiratsuchi S., Sato A., Mizunoya W., Ikeuchi Y., Furuse M., Allen R.E. 2010. High concentrations of HGF inhibit skeletal muscle satellite cell proliferation in vitro by inducing expression of myostatin: a possible mechanism for reestablishing satellite cell quiescence in vivo. Am J Physiol Cell Physiol. 298:C465-76.

Yamazaki, M., Q. Shen, and D.R. Swartz. 2010. Characterization of bovine fast and slow myosin subfragment 1 and myofibril tripolyphosphatase activity. Meat Sci. 85:446-452.

Yan, X., M. J. Zhu, W. Xu, J. F. Tong, S.P. Ford, P.W. Nathanielsz, and M. Du. (2010). Up-regulation of TLR4/NF-κB signaling is associated with enhanced adipogenesis and insulin resistance in fetal skeletal muscle of obese sheep at late gestation. Endocrinology, 151: 380-387.

Yang, Y., C. J. Zhao, S. Xiao, H. Zhan, M. Du, C. X. Wu, and C.W. Ma. (2010). Lipids deposition, composition and oxidative stability of subcutaneous adipose tissue and Longissimus dorsi muscle in Guizhou mini-pig at different developmental stages. Meat Science, 84: 684-690.

Yue, T., J. Yin, F. Li, D. Li, and M. Du. (2010). High glucose induces differentiation and adipogenesis in porcine muscle satellite cells via mTOR. BMB Reports, 43: 140-145.

Z. Li, B. Zhao, Y.S. Kim, C.Y. Hu and J. Yang. 2010. Administration of a mutated myostatin propeptide to neonatal mice significantly enhances skeletal muscle growth. Molecular Reproduction and Development 77:76-82.

Zhao, J.X., W.F. Yue, M.J. Zhu, N. Sreejayan, and M. Du. (2010). AMP-activated protein kinase (AMPK) cross-talks with canonical Wnt Signaling via phosphorylation

of β-catenin at Ser 552. Biochemical and Biophysical Research Communications, 395:146-151.

Zhao, J.X., X. Yan, J.F. Tong, W.J. Means, R. J. McCormick, M.J. Zhu, and M. Du. (2010). Mouse AMPK3 R225Q mutation affecting its growth performance when fed a high energy diet. Journal of Animal Science, 88: 1332-1340.

Zhao, Y.M., U. Basu, M. Zhou, M.V. Dodson, J.A. Basarb and L.L. Guan. 2010. Proteome difference associated with different fat formation in the bovine subcutaneous fat tissues. Proteome Science 8:14-24.

b. In press, accepted or submitted

Dodson M.V., G.J. Hausman, L.L. Guan, M. Du, T.P. Rasmussen, S.P. Poulos, P. Mir, W.G. Bergen, M.E. Fernyhough, D.C. McFarland, R.P. Rhoads, B. Soret, J.M. Reecy, S.G. Velleman and Z. Jiang. Lipid metabolism, adipocyte depot physiology and utilization of meat animals as experimental models for metabolic research. Int. J. Biol. Sci. (accepted).

Dodson, M.V., G. J. Hausman, L. L. Guan, M. Du, T. P. Rasmussen, S. P. Poulos, P. Mir, W. G. Bergen, M.E. Fernyhough, D. C. McFarland, R. P. Rhoads, B. Soret, J. M. Reecy, S. G. Velleman, and Z. Jiang. Skeletal muscle stem cells from animals I. Basic cell biology. International Journal of Biological Sciences (in press).

Du, M., J.X. Zhao, X. Yan, Y. Huang, L.V. Nicodemus, W. Yue, R.J. McCormick, and M.J. Zhu. Fetal muscle development, mesenchymal multipotent cell differentiation and associated signaling pathways. Journal of Animal Science, (accepted).

Guo, W., S. Bharmal, K. Esbona, and M. Greaser. Titin diversity - alternative splicing gone wild. J. Biomed. Biotechnol. (in press). PMID: 20339475.

Huang, Y., X. Yan, J. X. Zhao, M. J. Zhu, R. J. McCormick, S. P. Ford, P. W. Nathanielsz, J. Ren, and M. Du. Maternal obesity induces fibrosis in fetal myocardium of sheep. American Journal of Physiology-Endocrinology and Metabolism, (accepted).

Kamanga-Sollo, E., M.E. White, M.R. Hathaway, W.J. Weber and W.R. Dayton. Effect of estradiol-17β on protein synthesis and degradation rates in fused bovine satellite cell cultures. Domest. Anim. Endrocrinol. (in press).

Kim, K.H., Y.S. Kim and J.Z. Yang. Skeletal muscle hypertrophic effect of clenbuterol is additive to the hypertrophic effect of myostatin suppression. Muscle & Nerve (in press).

Morine K.J., L.T. Bish, J.T. Selsby, J.A. Gazzara, K. Pendrak, M.M. Sleeper, E.R. Barton, S.J. Lee, and H.L. Sweeney. Activin IIB receptor blockade attenuates dystrophic pathology in a mouse model of Duchenne muscular dystrophy. Muscle Nerve, (in press).

Morris CA, J.T. Selsby, L.D. Morris K. Pendrak and H.L. Sweeney. Bowman Birk inhibitor attenuates dystrophic pathology in mdx mice. Journal of Applied Physiology, (in press).

Nierobisz, L.S., D.C. McFarland, M.P. Murphy, and P.E. Mozdziak. MitoQ10 influences expression of genes involved in adipogenesis and oxidative metabolism in myotube cultures (submitted).

Pampusch, M.S., E. Kamanga-Sollo, M.R. Hathaway, M.E. White and W.R. Dayton. Low-density lipoprotein-related receptor protein (LRP)-1 is not required for insulin-like growth factor binding protein (IGFBP)-3 to suppress L6 myogenic cell proliferation. Domest. Anim. Endrocrinol. (submitted)

Selsby J.T, K. Pendrak, M. Zadel, Z. Tian, J. Pham, T. Carver, P. Acosta, E.R. Barton and H.L. Sweeney. Leupeptin based inhibitors do not improve the mdx phenotype. American Journal of Physiology – Regulatory, Integrative and Comparative Physiology, (in press).

Selsby J.T. Increased catalase expression improves muscle function in mdx mice. Experimental Physiology (London), (in press).

Sollero, B.P., S.E.F. Guimarães, V.D. Rilington, R.J. Tempelman, N.E. Raney, J.P. Steibel, J.D. Guimarães, P.S. Lopes, M.S. Lopes and C.W. Ernst. Transcriptional profiling during fetal skeletal muscle development of Piau and Yorkshire-Landrace crossbred pigs. Animal Genetics. (accepted).

Song, Yan, Douglas C. McFarland, Sandra G. Velleman. Effect of syndecan-4 covalently attached chains on turkey satellite cell proliferation, differentiation, and responsiveness to fibroblast growth factor 2. Development, Growth and Differentiation (in press).

Sporer K.R.B., W. Chiang, R.J. Tempelman, C.W. Ernst, K.M. Reed, S.G. Velleman, and G.M. Strasburg. Characterization of a 6K oligonucleotide turkey skeletal muscle microarray. Animal Genetics. no. doi: 10.1111/j.1365-2052.2010.02085.x. (in press)

Turdi, S., X.J. Fan, J. Li, J.X. Zhao, A.F. Huff, M. Du, and J. Ren. AMP-activated protein kinase deficiency exacerbates aging-induced myocardial contractile dysfunction. Aging Cell, (in press).

Waddell J.N., P. Zhang, Y. Wen, S.K. Gupta, A. Yevtodiyenko, J.V. Schmidt, C.A. Bidwell, A. Kumar, and S. Kuang. Dlk1 is necessary for proper skeletal muscle development and regeneration. Plos ONE. (in press).

Yan, X., Y. Huang, H. Wang, M. Du, B.W. Hess, S.P. Ford, P.W. Nathanielsz, and M.J. Zhu. Maternal obesity induces sustained inflammation in fetal and offspring large intestine of sheep. Inflammatory Bowel Diseases, (accepted).

Zhao D., A. Pond, B. Watkins, D.E. Gerrard, Y. Wen, S. Kuang and K. Hannon. 2010. Peripheral endocannabinoids regulate skeletal muscle development and maintenance. European Journal Translational Myology - Basic Applied Myology 20(4): (in press).

Zhu, M.J., Y. Ma, N.M. Long, M. Du, and S.P. Ford. Maternal obesity markedly increases placental fatty acid transporter expression and fetal blood triglycerides at mid-gestation in the ewe. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, (in press).

c. Invited presentations and abstracts

Abel E.L., J.M. Angel, P.K. Riggs, L. Langfield, A. Jiang, S. Carbajal, H-H. Lo, M. Person, Y. Awasthi, L-E. Wang, S. Strom, Q. Wei, and J. DiGiovanni. 2010. Gsta4 modifies susceptibility to skin tumor development in mice and humans. Proc Amer Assoc Cancer Res, Apr 17-21, Washington, DC, #4210.

Dixon, J., D. Gardan-Salmon, S. Lonergan, and J.T. Selsby. Analysis of dystrophic muscle by two dimensional differential in-gel electrophoresis. FASEB, Anaheim, April, 2010.

Du, M. 2010. Effect of maternal nutrient on carcass composition and meat quality of beef cattle. 2010 ADSA-CSAS-ASAS Joint Annual Meeting, Denver, CO, July 11-15.

Du, M. Cell signaling pathways regulating adipogenesis. China Agricultural University, Beijing, China, Nov. 3, 2010.

Du, M. Cellular signaling pathways affecting adipogenesis and marbling of skeletal muscle. International Congress of Meat Science and Technology (ICOMST), Jeju, South Korea, August 16-20, 2010.

Du, M. Maternal nutrition and growth characteristics of farm animals. American Society of Animal Scientists Annual Meeting, Denver, Colorado. July 11 to 14, 2010.

Du, M. Maternal nutrition, fetal adipogenesis and offspring body composition. International Mini-Symposium on Lipogenesis and Lipid Metabolism in livestock, Beijing, China, Nov. 4 and 5, 2010.

Du, M. Maternal obesity, inflammation and fetal skeletal muscle development. 2010 Gordon Research Conference on Musculoskeletal Biology and Bioengineering, Proctor Academy, Andover, NH, August 1-6, 2010.

Du, M. Pre-natal muscle development affects beef composition and quality. American Society of Animal Scientists Annual Meeting, Denver, Colorado. July 11 to 14, 2010.

Du, M., M.J. Zhu, and S.P. Ford. 2010. Impact of maternal nutrition in farm animal species on growth characteristics of their offspring. 2010 ADSA-CSAS-ASAS Joint Annual Meeting, Denver, CO, July 11-15.

Gardan-Salmon, D., E.R. Fritz, D. Nettleton, J.M. Reecy, and J.T. Selsby. Differentially expressed microRNAs in dystrophin-deficient muscle. FASEB, Anaheim, April, 2010.

Guan, L.L., W. Jin, M.V. Dodson, S.S. Moore and J.A. Basarb. 2010. MicroRNA expression in bovine subcutaneous fat tissues: A potential regulatory mechanism of subcutaneous adipose tissue development. International Plant and Animal Genome Conference, San Diego, CA. #W136.

Guan, L.L., Y.-M. Zhao, U. Basu, W. Jin, M.V. Dodson and J.A. Basarb. 2010. Association of annexin-1 protein with fat accumulation in bovine subcutaneous adipose tissues. International Society of Animal Genetics (ISAG), 32nd Conference; July 26-30; Edinburgh, Scotland.

Guo, W., J.M. Pleitner, K.W. Saupe, T.A. Hacker, and M.L. Greaser. 2010. Characterization of a mutant rat model with altered titin isoform expression. Biophys. J. 98: Suppl 1, 553a-554a.

Guo, W., J.M. Pleitner, K.W. Saupe, T.A. Hacker, and M.L. Greaser. 2010. Characterization of a mutant rat model with altered titin isoform expression. Biophys J. 98: Suppl 1, 553a-554a.

Huang, Y., X. Yan, M.J. Zhu, J. Ren, R.J. McCormick, S.P. Ford, and M. Du. 2010. Maternal obesity induces fibrosis in fetal ventricular muscle of sheep. NIH, NCRR Third Biennial National IDeA Symposium of Biomedical Research Excellence (NISBRE), Bethesda, Maryland, June 16-18.

Huang, Y., X. Yan, M.J. Zhu, R.J. McCormick, S.P. Ford, P.W. Nathanielsz, and M. Du. 2010. Enhanced transforming growth factor β (TGF-β) signaling and fibrogenesis in ovine fetal skeletal muscle of obese dams at late gestation. 2010 ADSA-CSAS-ASAS Joint Annual Meeting, Denver, CO, July 11-15.

Huang, Y., X. Yan, M.J. Zhu, S.P. Ford, P.W. Nathanielsz, and M. Du. 2010. Enhanced transforming growth factor (TGF-) signaling and fibrogenesis in fetal skeletal muscle of obese sheep at late gestation. Experimental Biology (The American Physiological Society) 2010, Anaheim, CA. April 24-28, 2010.

Hudson, B.D., C.G. Hidalgo, J. Bogomolovas, Y. Zhu, B. Anderson, M. Greaser, S. Labeit and H.L. Granzier. 2010. PKC Phosphorylation of titin's PEVK element--A novel and conserved pathway for modulating myocardial stiffness, Biophys. J. 98, Issue 3, Suppl. 1, 756a.

Ing N.H., D.W. Forrest, P.K. Riggs, D.D. Varner, and T.H. Welsh, Jr. 2010. Dexamethasone acutely alters gene expression in stallion testes resulting in impaired steroidigenesis. Animal Repro. Sci. 121S:S143-S144.

Kamanga-Sollo, E., M.E. White, M.R. Hathaway, W.J. Weber and W.R. Dayton, W.R. (2010) Effect of estradiol-17β on protein synthesis and degradation rates in fused bovine satellite cell cutures. J. Anim. Sci. 88,(E-Suppl. 2):634.

Meyer, A.M., J.S. Caton, M. Du, and B.W. Hess. 2010. Supplementation of Ruminally Undegradable Protein to Maintain Essential Amino Acid Supply During Nutrient Restriction Alters Circulating Essential Amino Acids of Beef Cows in Early to Mid-Gestation. 4th Grazing Livestock Nutrition Conference, Estes Park, CO. July 9-10, 2010.

Mir, P. and M.V. Dodson. 2010. Adipose tissue characteristics and control of metabolic syndrome. 15th International Congress of Clinical Nutrition. Sept 19-22; Ain El Sokhna (Palmera), Egypt. 

Nicodemus, L.V., K.R. Underwood J.F. Tong, P.L. Price, E.E. Grings, B.W. Hess, W.J. Means, and M. Du. 2010. Effect of Maternal Nutritional Status on Muscle Development and Carcass Characteristics in Heifer Progeny. 2010 ADSA-CSAS-ASAS Joint Annual Meeting, Denver, CO, July 11-15.

Pleitner, J. and M.L. Greaser 2010. Titin isoform size is not correlated with thin filament length in rat skeletal muscle. Biophys. J. 98: Suppl 1, 544a.

Riggs, P.K. Multidisciplinary approach for functional genomics analyses through integration of genome-wide genotypes, multiple phenotypes and global gene expression data. 2009. The National Academies Keck Futures Initiative on Synthetic Biology – Building on Natures Inspiration. Irvine, CA, November 19-22, Poster #48.

Sanders, S.R., K.L. Flann, M. Yamazaki, L.H. Baumgard and R.P. Rhoads. 2010. Alteration in skeletal muscle antioxidant defense gene expression profile following multiple exposures to hyperthermia. FASEB J. 24:1001.11.

Selsby, J.T. and D. Gardan-Salmon. Postnatal PGC-1 alpha gene transfer attenuates acute injury in mdx mice. FASEB, Anaheim, April, 2010.

Selsby, J.T., D. Gardan-Salmon, and L. Gealow. Postnatal PGC-1alpha over-expression reduces acute injury in mdx mice. New Directions in Muscle Biology. Ottawa, May, 2010.

Skrzypek M.V., R.P. Rhoads, S.R. Sanders, K. Flann, L. Cole, J.W. Perfield, M.R. Waldron, and L.H. Baumgard. 2010. Effects of heat stress on insulin action in lactating Holstein cows. J. Dairy Sci. 93. E-Suppl. 1:868.

Song, Y, D.C. McFarland, and S.G. Velleman. 2010. The role of syndecan-4 cytoplasmic domain in muscle growth and development. Poultry Science 89, Suppl. 1:198.

Sporer, K.R.B., H.R. Zhou, Y. Malila, A.M. Booren, J.E. Linz, and G.M. Strasburg. Effects of Heat Stress on Expression of Ryanodine Receptors, SERCA1, and Calsequestrin in Turkey Breast Muscle from Two Genetic Lines. Plant and Animal Genome XVIII Conference, San Diego, CA. January 9-13, 2010.

Tong, J.F., X. Yan, J.X. Zhao, M.J. Zhu, P.W. Nathanielsz, and M. Du. 2010. Maternal metformin administration reverses impaired development in offspring skeletal muscle of mice fed a high fat diet. Society for Gynecologic Investigation Annual Conference, Orlando, Florida, March 24-27, 2010.

Tong, J.F., X. Yan, J.X. Zhao, M.J. Zhu, P.W. Nathanielsz, and M. Du. 2010. Maternal metformin administration reverses impaired development in offspring skeletal muscle (SM) of mice fed a high fat diet. Reproductive Sciences, 17 (3), 160A.

Tousley, C.B., N.M. Long, S.P. Ford, W.J. Means, B.W. Hess, and M. Du. 2010. Nutrient restriction from early to mid-gestation in the cow increases adipocyte size of offspring at slaughter. American Society of Animal Scientists Annual Meeting, Denver, CO, July 11 to 15, 2010.

Wang, H., J. Zhao, Y. Huang, X. Yan, A. Meyer, M. Du, K. Vonnahme, L. Reynolds, J. Caton and M.J. Zhu. 2010. Excessive maternal selenium intake induces inflammatory response in the ovine fetal gut. 2010 ADSA-CSAS-ASAS Joint Annual Meeting, Denver, CO, July 11-15.

Yan, X., M. Nijland, T. McDonald, S. P. Pereira, M. J. Zhu, P. W. Nathanielsz, and M. Du. 2010. Regulation of mitochondrial biogenesis in fetal baboon skeletal muscle (SM) by maternal nutrient reduction at mid-gestation (G). 58th Annual Meeting of the Society for Gynecologic Investigation. Miami Beach, Florida, March 16-19.

Yan, X., M. Nijland, T. McDonald, S.P. Pereira, M.J. Zhu, P.W. Nathanielsz, and M. Du. 2010. Maternal over-nutrition is associated with chronic inflammation and decreased mitochondrial number in fetal baboon skeletal muscle (SM) at late-gestation. 58th Annual Meeting of the Society for Gynecologic Investigation. Miami Beach, Florida, March 16-19.

Yan, X., M.J. Zhu, A. Dey, S.P. Ford, P.W. Nathanielsz, and M. Du. 2010. Maternal obesity affects microRNA expression in fetal muscle at mid-gestation. Experimental Biology (The American Physiological Society) 2010, Anaheim, CA. April 24-28, 2010.

Yan, X., Y. Huang, M.J. Zhu, N.M. Long, A.B. Uthlaut, R.J. McCormick, S.P. Ford, P.W. Nathanielsz, and M. Du. 2010. Lipid accumulation and fibrosis in skeletal muscle of offspring born to obese dams. 2010 ADSA-CSAS-ASAS Joint Annual Meeting, Denver, CO, July 11-15.

Yue, W.F., J. Zhao, M.J. Zhu, and M. Du. 2010. Hedgehog signaling mediates adipogenesis in C3H10T1/2 cells via down-regulation of COUP-TFII. 2010 ADSA-CSAS-ASAS Joint Annual Meeting, Denver, CO, July 11-15.

Zhao, J., J. Hu, M.J. Zhu, W.J. Means, and M. Du. 2010. Trenbolone regulates myogenic differentiation via inducing androgen receptor and β-catenin interaction in muscle-derived stem cells of cattle. 2010 ADSA-CSAS-ASAS Joint Annual Meeting, Denver, CO, July 11-15.

Zhao, J.X., M.J. Zhu, S. Nair, and M. Du. 2010. AMP-activated protein kinase (AMPK) phosphorylates -catenin at Ser 552 and enhances -catenin/TCF mediated transcription. Nuclear Receptors: Development, Physiology and Disease, Keystone Symposia, Keystone, CO, March 21 to 26, 2010.

d. Book chapters

NAKFI IDR Team 9. IDR Team Summary: How do we maximally capitalize on the promise of synthetic biology? 2010. In: Synthetic Biology:Building on Nature’s Inspiration. The National Academies Press, Washington, D.C., pp. 77-82.

Kuang S. and M.A. Rudnicki. 2010. Muscle Stem Cells. In: Giordano A, Galderisi U (Eds) Cell Cycle Regulation and Differentiation in Cardiovascular and Neural Systems. Humana Press - Springer, NJ. pp. 105-120. ISBN: 978-1-60327-152-3.