The overall goal of this research is to elucidate detailed mechanisms by which Ca2+, intermolecular cooperation, and protein phosphorylations regulate contraction of striated muscles. Experiments will test specific hypotheses regarding the regulation of tension, the rate of tension development (k(tr)), and shortening velocity (V(max)) in skinned cardiac myocytes and skeletal muscle fibers. Mechanisms of these effects will be studied by altering the phosphorylation states and subunit composition of endogenous contractile and regulatory proteins, in some cases replacing them with truncated or mutant proteins having altered functional properties. (1) The hypothesis that strong binding cross- bridges cooperatively activate tension and k(tr) in myocardium will be studied by performing mechanical measurements in the presence of N- ethylmaleimide conjugated myosin S-1 (NEM-S1), which strongly binds actin. (2) The hypothesis that myosin LC2 and C-protein modulate the availability of cross-bridges to actin will be tested by recording X-ray equatorial reflections from single skinned fibers to assess changes in distribution of cross-bridge mass between thick and thin filaments following extractions of LC2 or C-protein. (3) The hypothesis that Ca2+ modulates contraction by a direct effect upon the thick filament will be explored by measuring Ca2+ sensitivities of tension and k(tr) when the thick and thin filaments are independently activated by changing their protein subunit compositions. The possibility that thick filament-linked effects are mediated by Ca2+ binding to myosin will be assessed by replacing endogenous LC2 with a mutant LC2 having reduced binding affinity for divalent cations. (4) The roles of strong binding cross- bridges and Ca2+ in modulating kinetic transitions in the cross-bridge interaction cycle will be studied by characterizing tension transients following photorelease of ADP and P(i) from chemically caged compounds. (5) The molecular basis for reduced V(max) at low levels of activation will be studied using NEM-S1 to probe the role of thin filament activation by strongly bound cross-bridges and by replacing endogenous C-protein with truncated forms to test the idea that C-protein contributes to a mechanical load that slows shortening. (6) Functional roles of phosphorylations of troponin I and C-protein by beta adrenergic agonists and of LC2 by a Ca2+/calmodulin dependent kinase will be studied in skinned cardiac myocytes. The results of this study should provide information about mechanisms by which contractile state changes in myocardium and mechanisms underlying functional deficits in diseased hearts.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Physiology Study Section (PHY)
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University of Wisconsin Madison
Schools of Medicine
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