Developmentally regulated and structurally distinct protein isoforms self-asssemble into thick and thin filaments in the cardiac sarcomere. The properties and interactions of these filaments will be investigated through biophysical and biochemical methods to comprehend the interplay of dynamic forces that control and produce cardiac contraction. The role of the regulatory light chain, LC2, in cardiac myosin ATPase will be further analyzed and its relationship to C-protein determined using HMM to complement the work done with myosin. Actin- activated ATPase assays will also be performed using rat and dog heart myosin subfragments to delineate the kinetic parameters that differentiate these myosin isoforms. Pure and regulated actin will be used in binding experiments with S1 and HMM and in kinetic analysis of ATP hydrolysis to clarify the factors involved and the mechanism that regulate actomyosin interactions in the heart. These studies will be executed under a number of ionic conditions and varying concentrations of calcium, as a function of LC2 and troponin phosphorylation. To provide the structural correlates to the results obtained from activity and binding measurements, the structure of cardiac myosin will be probed by monoclonal antibodies raised against the hinge and the functional domains in S1. Such an approach will permit a direct test of the involvement of the S2 region in force generation in the myocardium and identification of the actin-binding, light chain- binding and ATPase domains within the myosin heads. Also, cardiac TnT (the tropomyosin-binding subunit of troponin (will be isolated, its shape determined by electron microscopy and cleaved into discrete fragments, T1 and T2, either by a-chymotrypsin or cyanogen bromide. The functional characteristics of each fragment with respect to tropomyosin and other troponin subunits will be analyzed from the crystalline and paracrystalline aggregtes they generate. Furthermore, the reasons for the observed impairment in regulation in myopathic hamsters will be studied by analyzing the structure of the regulatory factor complex with particular attention to the integrity of TnT and the ability of troponin to bind calcium. Removal of cardiac LC2 by the myopathic hamster protease was shown to destabilize thick filament structure. This, together with the results from analysis of the regulatory factor function may provide a molecular explanation for cardiomyopathy.
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