The aim of the proposed research is to elucidate detailed mechanisms by which Ca2+ regulates the contractile properties of cardiac and skeletal muscles. This will involve measurements of changes in the Ca2+ sensitivities of tension, shortening velocity and stiffness in skinned muscle preparations in response to experimental alterations and natural variations in the subunit composition of contractile and regulatory proteins. Specific alterations will include protelytic modifications of troponin(Tn)- T and tropomyosin within the thin filament to test the hypotheses (i) that these proteins are essential to molecular cooperativity within the thin filament, and (ii) that as a result of interactions of adjacent Tm molecules, a population of long-lived cross-bridges is present at low (Ca2+) which gives rise to an internal load that results in decreases in tension and velocity as shortening proceeds. The relative importance of various Tn subunits in conferring Ca2+ sensitivity to the thin filament will be determined by measuring tension-pCa relationships in muscle preparations containing hybrids of subunits from cardiac and skeletal Tn. The importance of various isoforms of cardiac myosin heavy chains in modulating mechanical properties and in the Ca2+ regulation of these properties will be investigated in myocardium from normal adult rats and from pregnant or senescent rats which express novel isoforms. An hypothesis that Ca2+ has a direct effect on myosin to facilitate cross-bridge attachment will be tested in experiments in which the thick and thin filaments will be modified to allow their independent activation with Ca2+. Finally, the regulatory roles of the phosphorylation of myosin light chain-2 and TnI will be determined. In each of these experiments, the protein content of the cells will be determined by ultrasensitive SDS-PAGE and isoelectric focusing procedures. Single cell preparations of cardiac and skeletal muscle will be used to avoid possible distorting effects due to protein inhomogeneity among the cells of a multicellular preparation or non-uniform responses of these cells to the extraction and recombination procedures. Careful attention will be given to monitoring and control of sarcomere length to prevent distortion of physiological results. These experiments should provide new information regarding the mechanisms of regulation of the interaction of myosin with actin and provide rigorous tests of novel hypotheses for specific mechanisms of the actions of Ca2. in myocardium from normal pregnant and senescent animals.
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