A five-year research plan is proposed with the long-term objective of elucidating the rate-limiting steps and routes of catabolism responsible for the selective degradation of specific contractile proteins in cardiac muscle cells. The proposed investigations at the cellular level are a logical extension of the whole animal research conducted during Years 01-05 of this project, which defined the relative importance of contractile protein synthesis and degradation during physiological growth of the left and right ventricles, and during experimental interventions leading to hypertrophy, regression from hypertrophy, and primary atrophy of the rabbit heart. In the proposed studies, the intracellular events responsible for contractile protein degradative processing will be examined in a well-characterized cell culture system that synthesizes and degrades pro- teins at rates comparable to those observed in vivo. Specific experiments are outlined to estimate rates of myosin heavy chain and light chain biosynthesis, accumulation and degradation in neonatal rat ventricular myocytes maintained under serum-free conditions. Particular emphasis will be placed on the effect of spontaneous contractile activity and contractile arrest on the rates of myosin subunit turnover. Pulse-chase biosynthetic labeling experiments will also be used to evaluate the kinetics of myosin subunit degradation, and to provide evidence for (or against) the existence of kinetic precursors of myofibrillar protein subunits. In vitro studies will also be performed to examine whether one form of spontaneous """"""""damage"""""""" to the essential myosin light chain of ventricular muscle alters its susceptibility to proteolysis by purified proteases, and to degradation by the ubiquitin-mediated, ATP-dependent proteolytic system. Finally, a mass microinjection system will be used to assess the rate-limiting steps and routes of catabolism of native and damaged ventricular myosin light chain 1 incorporated into living cardiac muscle cells. If successful, these studies will provide new and fundamental information regarding the manner in which specific cardiac contractile proteins are targeted and processed for intracellular destruction. These studies will help to define the steps in a physiologically important, metabolic pathway that is intimately involved in the structural adaptation and remodeling of both the neonatal and adult heart.
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