The thin filament regulatory proteins troponin (Tn) and tropomyosin (Tm) regulate skeletal and cardiac muscle force, the rate of shortening, and calcium sensitivity which are the ultimate expressions of the interaction between the contractile proteins myosin and actin Point, missense, and deletion mutations in troponin T and tropomyosin produce hypertrophic cardiomyopahties and alter cardiac muscle isometnc force, shortening velocity, and calcium sensitivity These changes are hypothesized to alter the cardiac function observed in patients expressing these myopathies which in turn are associated with increased incidence of heart failure and sudden cardiac death The mechanisms responsible for the changes in contractile function associated with these mutations are not understood It has been hypothesized that these proteins alter the the amount of force or displacement produced when crossbridges attach and cycle It has been hypothesized that they control the rate of cross bridge attachment, the duration of attachment, or the rate of crossbddge detachment Although recent reports have clarified the role of calcium in controlling the rate of strong cross bridge attachment, an increasing amount of data suggests that Tm and Tn also modulate maximal isometric force and unloaded shortening velocity both in normal and diseased (Hypertrophic Cardiomyopathy, HCM) muscle Further while HCM TnT and Tm mutations produce a variety of contractile defects, two common denominators in these diseases are an increased calcium sensitivity and decreased inhibition of contractility at low intracellular pH This proposars goal is to understand the mechanisms by which Tm and Tn control contractility and muscle mechanics in both health and disease The proposed experiments are designed to reveal how the three troponin subunits and tropomyosin communicate with each other and the specific aspects of contractile behavior (force, velocity of shortening, calcium sensitivity) each modifies They seek to clarify the role and mechanisms of change intracellular pH has on the mechanical behavior of the muscle and to characterize the how the activation of troponin units on the thin filament produce cooperative activation The mechanisms will be systemically examined by altering the Tn or Tm composition of regulated thin filaments and measuring contractile behavior in single molecule three bead optical trap studies, in single myofibrils, and single muscle muscle cells
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