Our goal is to understand the factors which regulate Ca movements in cardiac muscle, particularly as they relate to excitation-contraction (E-C) coupling and the control of cardiac force development. Several techniques will be used to answer related questions at different levels of Ca movement. Extracellular Ca microelectrodes will be used to study both the Ca influx which occurs with each beat (phasic Ca-o depletion) and the net cellular Ca gains and losses which occur in non-steady-state conditions (cumulative Ca-o depletion). Intracellular Ca microelectrodes will be used to study the regulation of intracellular Ca activity (and Ca compartmentalization). Isolated sarcolemmal vesicles will be used to study Ca binding and transport activities of the sarcolemma. Changes in contractile force, dF/dt and action potentials will also be monitored in most experiments with isolated muscle preparations. The individual approaches above will provide valuable insights by themselves and the combination of techniques will increase the depth of conclusions and help to build a clearer more comprehensive picture of cardiac Ca regulation. With the central theme of cardiac muscle cellular Ca regulation specific studies are planned to evaluate: 1) the relative roles of Ca influx and SR Ca release in the activation of the myofilaments; 2) the role of Na-Ca exchange in Ca influx (and efflux) in relation to Ca current as well as Na-H exchange (and Ca efflux routes); 3) the effects of rest duration and stimulation frequency on Ca influx and cellular Ca loading; 4) the extent and localization of intracellular Ca (and Na) compartmentalization; 5) the mechanism of ryanodine action (as compared to caffeine); 6) fundamental bases for differences in E-C coupling mechanisms in different cardiac tissues (i.e. rat vs. rabbit); 7) the role of sarcolemmal Ca binding, the asymmetry of binding and transport sites in physiologically and pharmacologically induced changes in contractile function; 8) how Ca-i is handled during anoxic or ischemic incidents. Conclusions to some of these studies will rely on several of the techniques above. The results of the planned studies should increase our understanding of the regulation of basic cardiac muscle function in normal as well as pharmacologically and pathologically altered states.
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