The long term goal of this project is to elucidate the pathogenesis of hereditary cardiomyopathy. Intracellular calcium overload due to changes in the calcium homeostatic mechanisms is a characteristic feature of the hereditary cardiomyopathy in Syrian hamster. It is our hypothesis that a primary defect in the regulation of the plasma membrane Ca2+ pump at the level of gene expression and/or protein phosphorylation can lead to altered Ca2+ hemostasis and overload. In order to test this hypothesis, we propose to characterize the defects in the Ca2+ pump regulation at gene and protein level by comparing the cardiac tissue or myocytes from control and myopathic animals. Specifically, we propose: 1. To study the regulation of Ca2+ pump gene expression. The mRNA level of Ca2+ pump in cultured myocytes will be determined under basal or stimulated conditions. The effect of various agents such as protein kinase C-activator (phorbol ester), protein kinase A activator (isoproterenol, cAMP), as well as Ca2+-ionophore (ionomycin) or hormones (insulin, and thyroid hormone) shall be tested either individually or in combination, using Northern analysis and results correlated with the Ca2+ ATPase activity assay. Furthermore, the promoter of the Ca2+ pump gene will be isolated and characterized by molecular cloning and DNA sequencing. 2. To detect altered RNA splicing by RNA-PCR analysis. Possible changes in the Ca2+ pump mRNA splicing pattern during the development of cardiomyopathy, will be determined by polymerase chain reaction (PCR) analysis. The effect of various stimulators (sp.
aim 1) on RNA splicing will also be studied in cultured myocytes. 3. To study the regulation of Ca2+ pump activity by kinase-mediated phosphorylation. The effect of various kinases such as protein kinase C, cAMP-dependent kinase, cGMP-dependent kinase or Ca2+/calmodulin-dependent kinase on Ca2+ pump phosphorylation will be examined in cardiac plasma membranes of the control and myopathic hamsters and results correlated with the Ca2+- ATPase activity assay. Similar analysis will be performed to examine the regulation of the sarcoplasmic reticulum calcium pump via phosphorylation of the phospholamban. Thus this project is not only relevant to the study of pathogenesis of cardiomyopathy but also may contribute to the understanding of mechanisms that regulate the Ca2+ pump protein and its gene expression.
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