The long-term goal of this project is to study the role of calcium transmembrane flux in maintaining normal cardiac structure and function. The spontaneous myocardial necrosis in hereditary cardiomyopathic Syrian hamster is accompanied by intracellular myocardial calcium overload. Based on preliminary findings suggesting depressed activity in the cardiac sarcolemmal Ca2+ pumping ATPase together with evidence of alteration in sarcolemmal membrane lipids in the myopathic hamster, we hypothesize that defects in this enzyme or in its regulation lead to decreased rate of calcium efflux at the plasma membrane level, resulting in calcium overload and then myocytolysis. In order to test this hypothesis, we propose: 1. To study alterations in both Ca2+ fluxes and plasma membrane lipids during the development of cardiomyopathy. Ca2+ efflux will be assessed by measuring the sarcolemmal Ca2+ pumping ATPase activity. Ca2+ influx through the voltage dependent Ca2+ channel will be assessed by using ligand binding assay. Membrane phospholipid and fatty acid composition will be analyzed by thin layer chromatography and gas-liquid chromatography. 2. To test for structural and functional defects in Ca2+ pumping ATPase. The enzyme will be purified by Calmodulin affinity chromatography and its biochemical characteristics determined. In addition, the kinetic properties of the enzyme as well as the effects of phosphatidylinositol lipids or calmodulin will be studied. The alterations in protein sequence will be studied by peptide mapping analysis using V8 protease and Cleveland digestion method. 3. To test for abnormal regulation of the Ca2+ pumping ATPase by membrane lipids. Ca2+ transport in reconstituted liposomes will be studied by using purified ATPase from human erythrocytes and sarcolemmal membrane lipids from both control and myopathic hamsters. Thus this project is not only relevant to the study of pathogenesis of cardiomyopathy but also may contribute to the understanding of the molecular properties of the plasma membrane calcium pump.
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