Progressive deterioration of myocardial function during acute and/or chronic heart disease probably involves defects in Ca2+ uptake and release by the cardiac sarcoplasmic reticulum (SR). It is vital, therefore, to develop an understanding of how the SR is regulated. Accordingly, the studies proposed here will continue to examine and elucidate the role of phosphorylation/dephosphorylation processes of cardiac SR in regulating its calcium pump both under in vitro and in vivo conditions. Work from several laboratories including ours has shown that SR function is modulated by phospholamban. Phospholamban is a polymeric proteolipid, which is phosphorylated by three protein kinases (cAMP-dependent, Ca2+-calmodulin-dependent and Ca2+- phospholipid-dependent) at distinct sites. Phosphorylation by each protein kinase is associated with stimulation of the initial rates of Ca2+ transport. Recently, we have shown that the stimulatory effects of the protein kinases may be reversed by a protein phosphatase activity associated with cardiac SR. It is proposed here to further purify and characterize this enzymatic activity and determine its physiological role in the intact heart. Emphasis in this proposal is on characterization of the mechanism by which protein kinases and phosphatases regulate the ca2+ pump through phospholamban. Purified reconstituted systems will be used and the effect of phosphorylation/.dephosphorylation of each phosphorylatable site on phospholamban will be studied at the molecular level. Furthermore, the functional unit (monomer vs oligomer) of phospholamban will be determined using chemical crosslinkers. These in vitro studies will be complemented by studies in situ, in which phospholamban phosphorylation, specifically mediated by protein kinase C, will be studied in 32p i- perfused beating hearts. Changes in contractility, due to a1-adrenergic agonists and phorbolesters, will be correlated with changes in the phosphorylation of phospholamban, myofibrils and sarcolemma, all of which are known to be substrates for protein kinase C in vitro. Furthermore, changes in levels of phosphoinositide phosphorylation and the activation state of the phosphoinositide specific phospholipase C. The proposed research should provide important information concerning phosphorylation/dephosphorylation reactions as regulatory mechanisms for the mammalian myocardium.
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