The long-term objective of this research is to prompt the reassessment of the risk/benefit ratio of the use of cardiac glycosides for the treatment of heart failure in the light of the newly appreciated effects of these drugs on the regulation of cardiac growth-related genes and their potential effects on cardiac growth. The immediate goal is to characterize the signal transducing pathways that lead to these gene regulatory effects and are activated by the interaction of a cardiac glycoside with the cardiac sarcolemmal Na+/K+-ATPase. The specific studies proposed here are designed to determine the role of a specialized pool of Na+/K+- ATPase that is located within the sarcolemmal caveolae microdomains in the initiation of these signaling events, and in the classical function of this enzyme as an ion pump. Experiments of Specific Aim 1 are designed to establish the normal distributions of Na subunits and related signaling proteins; e.g., Src and epidermal growth factor receptor (EGFR), among caveolae microdomains and other cellular compartments, prepared by detergent-free and nondenaturing fractionation procedures, from cultured rat cardiac myocytes and adult rat heart ventricles. Studies of Specific Aim 2 are designed to test the hypothesis suggested by our preliminary studies that the cardiac glycoside-complexed Na+/K+-ATPase exerts its signal transducing function by regulating the traffics of Src, EGFR, and related signaling proteins between caveolae, the bulk plasma membrane, and the cytosol. Studies of Specific Aim 3 will test the hypothesis that in keeping with the specialized role of caveolar Na+/K+-ATPase in signal transduction, the ion pumping and catalytic activities of the caveolar Na+/K+-ATPase also differ from those of the enzyme in the bulk plasma membrane. These studies will clarify how cardiac function is controlled by Na in its dual roles as an energy transducing ion pump and a signal transducing protein complex that regulates neighboring proteins and enzymes to communicate with the organized intracellular signaling cascades, the contractile machinery, the mitochondria, and the nucleus.
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