The long-term goal of our research is to document the molecular mechanisms of contraction in the striated muscle system; in particular, we will study the mechanism of pathogenesis in hypertrophic cardiomyopathy (HCM). To this end, we will characterize the elementary steps of the cross-bridge cycle in a system in which active tension can be measured. We will also characterize force and velocity using the single molecule technique. Our research involves selectively removing the thin filament from cardiac muscle fibers (myocardium) by gelsolin treatment, and sequentially reconstituting the thin filament with G-actin and the regulatory proteins tropomyosin (Tm) and troponin (Tn). The degree of reconstitution is assessed by isometric tension, SDS-PAGE, and electron microscopy. This reconstitution technique is used to establish the structure- function relationships of particular domains within actin. The following are our specific aims: (1) to test the hypothesis that the hydrophobic interaction between actin and myosin is significant for force generation, by using an actin mutant in which one of the hydrophobic amino acid residues known to interact with myosin is altered; (2) to test the hypothesis that hypertrophic cardiomyopathy (HCM) mutants of actin significantly diminish force/cross-bridge, and that that the effect of temperature on isometric tension significantly diminishes in mutants; (3) to test the hypothesis that the negatively charged N-terminal residues of actin are essential for its strong interaction with myosin in the presence of Tm and Tn, and that cooperativity and Ca2+ sensitivity increase as the negative charge is increased. We assess cross-bridge kinetics using the sinusoidal and step- analysis methods, and analyze concomitant tension transients in terms of three exponential processes. When the effects of ATP, ADP, and phosphate on the cross-bridge rate constants are studied together with the ATP hydrolysis rate, the 8 kinetic constants that characterize the elementary steps of the cross-bridge model which consists of six states can be deduced. This information is then used to determine the force per cross- bridge. Step analysis is used to study the strain sensitivity of the kinetic constants, and the in-vitro motility assay to study the force/cross-bridge. Our goal is to document the mechanism of activation by using thin filament-reconstituted myocardium, and to determine the functions of specific actin domains. Because it has been hypothesized that the actin-myosin interface is impaired in three HCM mutants, our studies should provide insight into the pathogenic mechanisms that underlie these HCM mutations. PROJECT NARRATIVE: The proposed research will advance an understanding of the molecular mechanism of muscle contraction, in particular the pathogenic mechanism of myocardial diseases. The particular method to be used (reconstitution of the thin filament in cardiac muscle fibers) is ideal for establishing the structure-function relationships of domains within protein components of the thin filament, in the case of this particular proposal, within actin. Our research is aimed at uncovering the molecular basis of the mechanism of contraction, and the pathogenic mechanisms at work in familial hypertrophic cardiomyopathy (HCM), a condition that causes sudden cardiac death in healthy adults; in the near future, the method developed and the knowledge acquired will also be used in efforts to elucidate other pathogenic mechanisms of skeletal and cardiac muscle diseases. ? ? ?
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