The broad, long term objective of this project is to gain a better understanding of Ca ion homeostasis and energy metabolism as determinants of contractile function in normal hearts.
We aim to elucidate the role of these factors in cardiac pathophysiology through studies with isolated heart cells. The strength of contraction of the heart is controlled in large measure by the regulation of Ca fluxes; therefore, understanding how Ca fluxes are controlled is important. The specific focus of this application is to address three important questions relating to the control of Ca fluxes in adult rat heart cells. #1: Is all SR Ca released into the diad cleft? Much of the control of heart cell Ca fluxes occurs in the diad cleft. While Ca influx through the sarcolemma opens SR Ca release channels in the cleft, it is not yet even known if this is the major release pathway for SR Ca.
We aim to determine first whether or not Ca flux through the cleft is too slow to be the major pathway for SR Ca efflux, and second to measure the extent to which Ca release channels outside of the diad cleft are involved in excitation-contraction coupling. The first goal will be achieved by comparing the rates of detection of cellular Ca entry and SR Ca release by cytosolic fura-2. Since fura-2 itself may affect the fluxes under study, Ca fluxes measured with different levels of fura-2 will be extrapolated by modeling to the zero fura-2 condition. The second will use a new permeabilized myocyte preparation with intact (pinched off) t-tubules to measure the effect of inhibition of non-junctional Ca release channels on depolarization-induced SR Ca release. #2: What are the consequences of Na/Ca exchanger activation for cellular Ca influx and efflux? How Ca fluxes via Ca channels and by Na/Ca exchange interact to regulate SR Ca release in the diad cleft has been difficult to study, because conditions needed to measure Ca current eliminate Na/Ca exchange. We will establish the extent and rate of beat-dependent activation of the Na/Ca exchanger, and its Ca dependence. We will also use a new indirect measure of Ca channel activity using fura-2 quench by Mn to investigate whether or not Ca influx through the exchanger and through Ca channels is synergistic. #3: What can account for the increase in SR Ca pool size seen with intracellular chelators? We will test the hypotheses that intracellular chelators increase SR Ca pool size either by stimulating the rate of Ca uptake, by inhibiting SR Ca efflux, or uptake into the SR.
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