Recent evidence suggests that in myocardium sodium dependent calcium exchange is operative during reperfusion and that the exchange, and the potential ramifications of cellular calcium loading, are critically dependent on the transmembrane sodium gradient. The purpose of this proposal is to investigate this hypothesis by utilizing two parallel experimental strategies and methodologies: 1) continuously monitoring intracellular sodium activity by ion selective electrodes in papillary muscles during hypoxia-reoxygenation or during calcium deprivation-calcium reflow; 2) continuously measuring intracellular calcium via microinjected aequorin during hypoxia-reoxygenation or calcium deprivation-calcium reflow. The proposed experiments will first define the factors which primarily alter the sodium gradient (during the deprivation periods and during reflow), and then apply the same perturbations while measuring calcium. Intracellular sodium activity will be monitored in quiescent superfused ferret papillary muscles; while in parallel experiments intracellular calcium will be monitored in beating ferret papillary muscles. In these experiments the changes in intracellular calcium will be analyzed in terms of the changes in the previously defined sodium gradient. The long term objectives will be divided into five parts: in part one the factors influencing intracellular sodium during hypoxia will be defined; in part two we will measure sodium activity during reoxygenation and will investigate methods to alter the gradient; in part three we will assay the sodium gradient with similar strategy during a calcium free period and subsequent calcium reflow; in part four we will measure intracellular calcium during hypoxia and reoxygenation in response to the previously defined changes in the sodium gradient; in part five intracellular calcium will be measured during calcium free perfusion followed by calcium reflow, in a manner and with a strategy as noted above. The results of these parallel experiments are designed to be complimentary and allow for interpretation of the mechanisms responsible for transarcolemmal sodium-calcium exchange during reperfusion.
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