The fundamental process controlling cardiac contraction is the interaction among actin and myosin filaments. Thus, elucidation of these interactions is essential for comprehending the factors that regulate cardiac contraction. To achieve this objective, it is planned to isolate the various proteins and study their physical-chemical and catalytic properties. Efforts will be concentrated initially on clarifying the substructure of cardiac myosin and defining the influence of the light chains on myosin function: by reversibly dissociating LC2, it is possible to monitor the regulatory effect of phosphorylated and unphosphorylated LC2 on actomyosin ATPase and binding. Attempts will be made to map the myosin molecule using anti-LCl and anti-LC2 antibodies, to identify their binding sites on myosin. In parallel experiments, myosin subfragments (LMM, S2, rod, HMN, S1) will be made from purified cardiac isomyosins to identify the region in myosin that retains the structural changes which give rise to cardiac myosin polymorphism. Subsequently, S1's from the two isomyosins will be assayed for ATPase activities to establish if the activity differences seen in the isoenzymes is retained or not and thus clarify the structural basis of these activity differences. Functional characterization of individual isomyosins with phosphorylated or unphosphorylated LC2 will involve also a study of filament formation and actin-binding studies using pure and regulated thin filaments i.e., actin complexed with troponin and tropomyosin. At the same time, myosin will be selectively removed from individual myocardial cells leaving behind intract Z-bands and the thin filament scaffolding. To these cells, control, LC2-deficient or phosphorylated myosin will be readded to establish: 1) whether myosin will reform functional thick filaments in such cells and 2) compare a physiological parameter such as velocity of shortening to untreated cells in order to further clarify the role of LC2. We feel that such a rigorous approach will provide a thorough understanding of myocardial contractility and its regulatory mechanisms at the molecular level.
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