Progressive deterioration of myocardial function during congestive heart failure appears to involve defects in Ca2+-uptake and release by the cardiac sarcoplasmic reticulum (SR). Thus, it is vital to develop an understanding of how the SR is regulated. Accordingly, the studies proposed here will continue to examine and elucidate the role of phospholamban in the regulation of SR Ca2+-uptake and myocardial contractility. Phospholamban is a low Mr phosphoprotein, which can he phosphorylated by three protein kinases (cAMP-dependent, Ca2+-calmodulin- dependent, and Ca2+-phospholipid-dependent) in vitro. Phosphorylation by each protein kinase is associated with stimulation of Ca2+-uptake by the cardiac SR, and dephosphorylation by an endogenous phosphatase reverses the stimulatory effects of the protein kinases. Our hypothesis is that alterations in the levels of phospholamban or in specific amino acids in the phospholamban molecule reflect alterations in the SR Ca2+. ATPase activity and myocardial contractility. The studies proposed here have been designed to test this hypothesis by generating transgenic animal models with: a) increased levels of cardiac phospholamban; b) reduced levels of cardiac phospholamban; and c) cardiac phospholamban mutants. The effects of alterations in phospholamban levels or in its primary sequence on the SR Ca2+-ATPase activity will be correlated with alterations in cardiomyocyte function and calcium kinetics. These basic studies will be extended to include endomyocardial biopsy samples from patients with heart failure in order to correlate alterations in the phospholamban levels with alterations in cardiac function. The proposed experiments will provide an integrative approach to our understanding of the regulatory role of phospholamban in myocardial contractility and may lead to new therapeutic approaches in the treatment of congestive heart failure.
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