actin binding properties of cardiac myosin binding protein-c
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immunoreactive changes. To this aim cryosections from rabbit,mouse and rat hearts were incubated with Snitrosylglutathione(GSNO). Tm immunoreactivity was increased by GSNO. Thiseffect showed a bell-shaped dose-dependency with largerchanges detected 0.5 mM GSNO. At this concentration GSNOeffect was time dependent. The GSNO-induced increase in Tmimmunofluorescence was prevented by preincubation with (i)dithiothreitol, a reducing agent, suggesting the occurrence ofreversible modifications of cysteinyl residues; (ii) mercuricchloride, which specifically hydrolyzed S–NO bonds, suggest-ing the occurrence of Tm nitrosylation. In parallel, anti-Tmimmunoblot analyses revealed the formation of an additionalband in GSNO-treated samples with an apparent Mr of 42 kDathat disappeared under reducing conditions. In conclusion,tropomyosin is covalently modified by both ROS and RNS.
Keywords: Contractile proteins; Oxidative stress; Myocardialdysfunction
doi:10.1016/j.yjmcc.2007.03.273
Alpha skeletal actin expression in dedifferentiatingcardiomyocytesR.B. Driesen1, F.K. Verheyen1, C. Chaponnier3, E. Blaauw2,F.A. Babiker2, M.H. Lenders1, M. Borgers1, F.C.S. Ramaekers1.1Department of Molecular Cell Biology, University of Maas-tricht, University of Geneva, The Netherlands. 2Department ofPhysiology, University ofMaastricht, University of Geneva, TheNetherlands. 3Department of Pathology and Immunology,University of Geneva, The Netherlands
Actin isoform expression during cardiomyocyte differentia-tion is characterized by a downregulation of the “embryonic”alpha smooth muscle actin followed by an upregulation of alphaskeletal actin (αSKA) and a subsequent predominant expressionof alpha cardiac actin (αCAA) at the end of the myofibrillogen-esis process. Our objective was to provide evidence for the re-expression of αSKA during cardiomyocyte dedifferentiation indifferent models of hibernating myocardium. The expression ofαCAA and αSKA was studied on samples of goat atria ofsustained atrial fibrillation, on left ventricle samples of rabbitssuffering from pressure and volume overload and on dediffer-entiating adult rabbit ventricular cardiomyocytes in culture. Apatchy expression pattern ofαSKAwas observed in goats during16 weeks of atrial fibrillation together with a progressiveaccumulation of glycogen. Comparable staining patterns werefound in rabbit left ventricular tissue subjected to pressure andvolume overload. Dedifferentiation of adult rabbit ventricularmyocytes in vitro revealed a loss of myofilaments and theformation of αCAA positive stress fibers. A diffuse expressionof αSKA at the periphery of the cells progressing towards thecell centre was observed. The αSKA expression in adult dedif-ferentiating cardiomyocytes in combination with PAS positiveglycogen offers an additional tool in the evaluation of hiber-nating myocardium.
Keywords: Cardiomyocytes; Cardiac remodeling; Heart failure
doi:10.1016/j.yjmcc.2007.03.274
Actin binding properties of cardiac myosin bindingprotein-CInna N. Rybakova, Richard L. Moss. Department of Physiology,University of Wisconsin Medical School, Madison, WI, USA
The cardiac isoform of myosin binding protein-C (cMyBP-C) has been of particular interest because of its link to familialhypertrophic cardiomyopathy, a major cause of sudden death.While both structural and regulatory roles have been proposedfor cMyBP-C, its interactions with myosin and sarcomericproteins remain obscure. It is known that MyBP-C mayinteract with actin filaments. However, neither the location ofthe binding site on MyBP-C nor its actin binding propertieshas been clearly determined. To address these issues, weexpressed full-length mouse cMyBP-C in a baculovirusexpression system. The recombinant cMyBP-C was taggedwith the FLAG epitope and purified using Anti-FLAG M2-agarose. The purified cMYBP-C existed predominantly as ahighly soluble monomer in solution. Incubation of purifiedcMyBP-C with F-actin followed by high-speed sedimentationresulted in its cosedimentation with the F-actin pellet.Virtually no cMyBP-C sedimented in the absence of actin,indicating binding interaction between actin filaments andcMyBP-C. We further quantitatively characterized the actinbinding properties of cMyBP-C by high-speed cosedimenta-tion analysis of fixed amount of F-actin and increasingconcentrations of cMyBP-C. In order to determine how muchof the cMyBP-C is involved in interaction with actin, weexpressed few recombinant proteins encoding the segments ofcMyBP-C sequence, and further characterized their actin-binding properties. Our results suggest that cMyBP-C maymodulate cardiac contractility by interacting with thinfilaments.
Keywords: Cardiomyopathies; Contractile proteins; Proteins
doi:10.1016/j.yjmcc.2007.03.275
Restoring force is lower in human donor and hcm myocytescompared with ratAnita C. Hoskins, Adam M. Jacques*, Steven B. Marston*,Jonathan C. Kentish. Cardiovascular Division, King's CollegeLondon, UK and *NHLI, London, UK
The restoring force that causes isolated myocytes to re-lengthen after contraction is thought to be largely due to themyofilament protein, titin. Rodent ventricle possesses the N2Btitin isoform, whereas human ventricle contains approximatelyequal amounts of the N2B and N2BA isoforms. The N2BAisoform is less stiff, and so would be expected to exert a smallerrestoring force. We examined this using human tissue from 3
S119ABSTRACTS / Journal of Molecular and Cellular Cardiology 42 (2007) S102–S124