In congestive heart failure, pronounced changes in cellular Ca2+ handling and altered cAMP-dependent phosphorylation are among the primary alterations thought to underlie the prolonged action potentials and contractile defects that predispose the failing heart to fatal electrical events and pump failure. Important changes in excitation-contraction coupling include a reduction in the Ca2+ content of the sarcoplasmic reticulum (SR), due to decreased activity of the sarcoplasmic reticulum Ca2+ ATPase (SERCA2a), and an increase in sarcolemmal Na+/Ca2+ exchange (NCX). As a consequence, triggered Ca2+ release is depressed and the action potential profile is markedly altered through changes in Ca2+-dependent feedback on sarcolemmal ion channels and transporters. These include altered Ca2+-dependent inactivation of the L-type Ca2+ current and changes in the magnitude and direction of the electrogenic NCX current. These key components of excitation-contraction coupling are modulated by Ca2+- and phosphorylation-dependent mechanisms. However, the molecular details of such regulation in normal cells, let alone in heart failure, remain obscure, and completely undefined in intact myocytes. The main goal of this application is to investigate the structural determinants of L-type Ca2+ channel and NCX protein regulation in adult myocytes using a novel reverse engineering approach, capitalizing on our recent advances in adenoviral technology. L-type Ca2+ channels and NCX proteins genetically engineered to be distinguishable from their native counterparts will be expressed in cardiac myocytes using new viral vectors. Expression in the context of the native cardiac cell, with all of its accessory proteins, regulatory pathways and spatial organization intact, will allow us, for the first time, to investigate the molecular determinants of excitation contraction coupling, phosphorylation, and Ca2+-dependent allosteric regulation that have not been attainable before. Elucidation of the molecular details of the regulation of cardiac excitation-contraction coupling will help us to fulfill our long term goal of restoring normal contractile function to the failing heart.
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