Molecular Characterization of Cardiomyopathy Mutations in Human Cardiac Myosin

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DESCRIPTION (provided by applicant): Although more than 200 mutations located in the motor domain of the human ??cardiac myosin heavy chain (MyHC) cause hypertrophic or dilated cardiomyopathy (HCM or DCM), their molecular effects on the myosin molecule remain elusive. Most studies to determine the functional impact of these mutations on myosin have used non-human myosins and most have not used cardiac myosins. Leinwand and colleagues have recently developed a system for producing recombinant human cardiac myosin motors and we will exploit this system for the first systematic study of the effects HCM and DCM mutations in human ??cardiac myosin. Since the majority of mutations that cause disease are located in the myosin motor, we will focus our studies there. We selected for study 10 mutations (5 HCM and 5 DCM), both because of their largely severe clinical phenotypes and because of their locations within the myosin motor domain. We propose to analyze the biochemical properties of the mutations in solution and their biomechanical properties using in vitro motility and laser trap single molecule assays. We hypothesize that mutations that lead to increased force production lead to HCM while those that lead to decreased force production lead to DCM, consistent with recent suggestions. Mechanisms that lead to decreased or increased force production by myosin can be varied. The inherent force-producing capability of the motor, for example, could be increased or decreased by mutations that change the spring constant of the elastic element of the motor. On the other hand, the force- producing capability could be changed in either direction by changes in the duty ratio of the myosin (the fraction of the ATPase cycle that the head is strongly bound to actin). An increase in the duty ratio, for example, would result in more heads bound to actin in a force-producing state at any moment, leading to an overall increase in force. Furthermore, the duty ratio can be changed in more than one way. For example, the ADP release rate (which determines the strongly-bound state time), the ATP hydrolysis step (which defines the recovery stroke time) or the Pi release (the rate of entry into the strongly bound state) can be affected and result in a change in the duty ratio. Thus, it is essential to begin to characterize different mutations, hypothesized to affect different mechanistic aspects of the motor. These studies will begin the process of linking the fundamental changes in motor function to the eventual different clinical outcomes. Therefore, we will determine the biochemical and biomechanical parameters of wild type human a- and b-cardiac myosin and mutant ? -myosin motors with respect to their steady-state and transient kinetic properties, velocities at ~zero load and under loaded conditions, their duty ratios, their stroke sizes, and the maximum force they produce upon interacting with actin.