The long term goal of this project is to elucidate the pathogenesis of hereditary cardiomyopathy in human. Intracellular Ca 2+ overload and spontaneous myocytolysis are characteristics of the cardiomyopathy in Syrian hamsters. In this animal model, they have shown previously that a simultaneous reduction of the expression of plasma membrane Ca 2+ pump (PMCA) and sarcoplasmic reticulum Ca 2+ pump (SERCA) may be involved in myocyte death. They have also obtained preliminary evidence that apoptosis occurs in myopathic heart, which is accompanied by altered expression of the genes involved in cell cycle (and apoptosis). Based on these evidence, they hypothesize that this cardiomyopathy involves altered Ca 2+ homeostasis which leads to aberrant cell cycle gene expression, cell cycle activation, and apoptosis. They propose: 1. To study the coordinated regulation of PMCA and SERCA genes. PMCA and SERCA genes will be separately transferred into rat adult cardiomyocyte in culture by using a novel adenovirus system. The effect of the exogenous gene transfer on [Ca 2+ ]i homeostasis and on the expression of endogenous PMCA, SERCA, and Ca 2+ channels will be examined. Furthermore, the effect of [Ca 2+ ]i on PMCA and SERCA gene expression will be studied in normal, uninfected myocyte. 2. To study the role of Ca2+ in apoptosis and to test the protective effect of Ca2+ pump. Apoptosis will be induced in cultured myocytes using SERCA inhibitor thapsigargin (Tg) and the expression of cell cycle regulatory genes (c-myc, cyclin A, p53, p21 and bcl-2) will be assayed. The protection of myocyte from Tg-induced apoptosis will be tested by overexpression of PMCA via gene transfer. 3. To study the molecular mechanism of apoptosis in cardiomyopathy. They will examine the development of apoptosis in myopathic heart using in situ terminal deoxynucleotidyl transferase staining. They will determine altered mRNA and protein expression of the cell cycle regulators in isolated heart as well as myocyte. The results will be verified in myopathic heart by in situ hybridization and immunostaining of these markers. DNA synthesis activity will be determined by BrdU labeling of myocytes during disease development. Intervention of the cardiomyopathy will be tested by treatment with calcium antagonist, and heart tissue sections examined for the prevention of lesion development and for the restoration of normal expression pattern of the cell cycle regulators. Thus this project is not only relevant to the study of pathogenesis of cardiomyopathy but also may contribute to the understanding of mechanisms that control apoptosis.
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