Our preliminary studies show that reoxygenation of single cardiac myocytes after brief or prolonged hypoxia causes functional abnormalities analogous to those seen in reperfused myocardium: prolonged relaxation, manifestations of calcium overload and, after long hypoxic periods, cell destruction. To study the mechanism of these changes, single myocytes, under direct observation, will be made hypoxic (PO2<0.02 torr) using the Laminar counterflow Barrier Well technique developed by the Sponsor and the Candidate. Changes in intracellular calcium will be measured using the fluorescent probe indo-1, intracellular pH using the new probe SNARF-1, and intracellular sodium using the new fluorescent crown ether SBFI. Mechanical shortening and membrane potential/currents will be measured simultaneously. By manipulating extracellular pH and sodium, intracellular pH (by varying PCO2) and phosphate (by pipette dialysis) we will test the hypothesis that post-hypoxic delayed relaxation is due to altered myofilament function, determine whether it can be accounted for by changes in intracellular pH and Pi, and test the hypothesis that sodium-calcium exchange, possibly linked to sodium-hydrogen exchange, is critical in the calcium-mediated destruction of cells reoxygenated after prolonged hypoxia.
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