Two major problems which complicate all in vitro studies of cardiac protein metabolism are the heterogeneity of cell type in available preparations (i.e., fibroblasts, endothelial cells) and the short duration of viability of preparations obtained from adult animals (e.g., perfused hearts, papillary muscles). The former results in an ambiguity of interpretation of results (i.e., is the observed result due to myocytenon myocyte cells) while the latter prevents studies on important long lived cardiac proteins of interest (e.g. myosin). For these reasons the specific goals of the proposal are a) to develop techniques and the methodology requisite to maintain pure cultures of calcium tolerant adult rabbit-ventricular myocytes in culture for periods upto 7-14 days; b) to comprehensively characterize protein and amino acid metabolism in this preparation as compared to presently available data of these processes obtained in other commonly used in vitro rabbit cardiac preparations; and c) only after adequate characterization studies have been performed, to initiate studies of protein turnover and lysosomal protease metabolism in this preparation. Preliminary methodology for culture of adult rabbit myocytes has been defined by the P.I. This methodology allows for stable culture of a relatively pure adult myocyte preparation for 4 days. Techniques to eliminate all fibroblast contamination in the culture, prolong culture viability to 7-14 days and characterize protein and amino acid-metabolism in this preparation are presented in this proposal. We believe such a newly developed preparation must be comprehensively characterized prior to initiating experiments of current interest on specific aspects of protein metabolism. Thus, although some such experiments are proposed, the bulk of this proposal defines methodology for improving culture technique and documenting a """"""""steady state"""""""" in the preparation as regards protein and amino acid metabolism.
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