NMR STUDIES OF SODIUM HOMEOSTASIS IN ISCHEMIC MYOCARDIUM

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Previous studies have demonstrated that intracellular Cs2+ overload plays an important role in myocardial ischemic injury, and that an important determinant of Ca2+ homeostasis is the control of intracellular Na+ (Na+). However, Na+ homeostasis during ischemia is still poorly understood. The proposed project will employ a unique combination of state-of-the-art NMR techniques to obtain a comprehensive assessment of how Na+I is controlled in the myocardium, during ischemia and hypoxia. Using the intact perfused rat heart model, interleaved 23Na and 31P NMR spectra will monitor changes in Na+I and cellular energy. In the same preparations, 87Rb NMR spectroscopy will be used to monitor Rb+ uptake and Na+/K+ ATPase activity. Using 7Li NMR spectroscopy, Li+ uptake will be measured to monitor voltage- gated Na+ channel activity. This methodology not only measures net Na+ accumulation, but is designed to also provide simultaneous information concerning how changes in unidirectional Na+ fluxes induce it. The following specific aims will be addresses: 1. To measure sarcolemmal Na+/K+ ATPase activity during control normoxic perfusion, hypoxia, and low-flow ischemia. The experiments will determine whether Na+ extrusion decreases or increases under the latter two conditions. Also, to examine Na+/K+ ATP activity in the context of the various metabolic modulators such as Na+, cellular energy status, pH, etc., in order to assess how functional alteration may occur during ischemia/hypoxia. 2. To determine the rate of unidirectional Na+ influx, during control normoxic perfusion, hypoxia, and low-flow ischemia, by using the Na+ and Na+ extrusion rate measurements. These measurements will determine if unidirectional Na+ influx is altered under the various conditions, and in particular, if it is downregulated during low-flow ischemia, as compared to control normoxia and hypoxia. Also, to compare these Na+ influx alterations with those obtained from measurement of Li+ uptake, which has specificity for Na+ channel activity. 3. To measure the rates of Na+ influx which occur via Na+/H+ exchange, during control normoxic perfusion, hypoxia, and low- flow ischemia, and to assess the importance of this mechanism in contributing to Na+ accumulation under various ischemic/hypoxic conditions. 4. To measure the rates of unidirectional Na+ influx which occur via Na+-HCO3- cotransport, during control normoxic perfusion, hypoxia, and low-flow ischemia, and to assess the importance of this mechanism in contributing to Na+ accumulation under various ischemic/hypoxic conditions. Also, to assess the possible interaction of this pH regulatory mechanism with Na+/H+ exchange (whether inhibition of one stimulates the other). 5. To measure the rate of Na+ influx which occur via non- inactivating Na+ channel current, during control, normoxia, hypoxia, and low-flow ischemia, and to assess the importance of this mechanism in contributing to Na+ accumulation under various ischemic/hypoxic conditions.