MECHANISM OF LENGTH DEPENDENT CONTRACTILE REGULATION

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The regulation of contraction in cardiac and skeletal muscle results from the complex interplay of multiple molecular processes. Our purpose is to determine the mechanism(s) by which changes in sarcomere length affects force in skeletal and cardiac muscle (Starling's Law). Exclusively thin filament-based mechanisms have been proposed, as have thick filament-based mechanisms which include a substantial modulatory role for crossbridge attachment. Other mechanisms, which are not exclusively thick or thin filament based, involve altered myofilament lattice spacing or charge density. To distinguish the relative contribution, if any, of these various mechanisms we will take advantage of newly developed methods for (i) inhibiting or modifying actomyosin interactions (metallofluorides, sulfhydryl reagents, cationic peptides, GTP) and (ii) activating skinned fibers without Ca2+). Activation without Ca2+ will enable us to directly separate Ca2+ from crossbridge effects on length regulation and activation, as well as effects due to Ca2+ binding to sites other than TnC, such as at myosin light chains. Since force will be inhibited in many experiments, we will use fluorescently labeled TnC to estimate the level of thin filament activation and will also monitor fiber stiffness to determine the degree, as well as the strength of crossbridge attachment. Exchange of troponin subunits between skeletal and cardiac muscle will be done to determine if the properties of thin filament regulatory proteins affect the length dependence of force-calcium relations and the kinetics of contraction. We will also determine the role that myosin properties play in determining the activation and length dependence of force and kinetics. Experiments will be done with both skinned skeletal and cardiac muscle; the steeper length dependence of force in cardiac muscle suggests that the underlying mechanisms of length regulation are even more potent. While no single experimental approach is perfect, the convergence of data to be obtained from the experiments which we propose, will enable us to reach strong conclusions about the underlying mechanism(s) by which this highly nonlinear, complex system of cooperative interactions between muscle proteins results in the length regulation of contractile activity.