This research proposal deals primarily with the processes which mediate electromechanical coupling in heart muscle. Specifically, experiments are proposed which will probe processes of Ca++ entry, Ca++ release and reuptake in the myocardial cell. Experimental work will proceed along six major directions: The first two projects deal with measurements of Ca-dye signal in single isolated cells and in intact ventricular trabeculae. Ca++ signals, as well as intrinsic optical indicators of SR activity, will be measured under voltage clamp conditions in different hearts with well known ultrastructural differences. In this way we hope to establish the contribution of various Ca++ transporting systems as well as the role of the SR in the Ca++ release and reuptake cycle. The third project deals directly with Ca++ current through the Ca++ channel. Single cell studies as well as clamp studies on isolated patches of myocardial membranes in both mammalian and frog heart are planned to identify whether the Isi channel is truly different in frog and mammalian heart, or if the difference is related to the presence of intracellular Ca++ stores in mammalian heart. The fourth project deals with experiments which probe the molecular mechanisms which mediate the relaxant effect of adrenaline on the heart. Specific experiments are planned to determine the role of Na+ pump stimulation and cyclic AMP in mediating this process. Light-sensitive caged cAMP and ion-selective Na+ and K+ electrodes will be used to test the alternate two schemes. The fifth project will probe the kinetics of myocardial contraction by using light-sensitive """"""""caged"""""""" compounds such as EGTA, Ca++, cAMP and organic Ca++ antagonists. The use of these agents will make it possible to do step changes of ionic or drug concentrations to probe the kinetics of the Ca++ channel and development of tension. Performing such experiments on hearts with different ultrastructural components will provide structure-function information regarding the pathway of the Ca++ cycle and the source of activator Ca++. In the sixth project we shall continue our studies on the role of (Ca)i and releasable Ca++ stores in generation of pacemaker current in SA nodal cell. We hope that these studies will provide a better understanding of the processes which control myocardial excitation and contraction.
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