Reperfusion injury is an important phenomenon leading to irreversible cell death after myocardial ischemia. During ischemia, anoxia and tissue acidosis develop. ATP declines leading to perturbation of ion gradients, depolarization, loss of contraction, and cellular uncoupling. Despite these changes, cell viability may be maintained for long periods. However, as oxygen and normal pH are restored, a reperfusion injury occurs that worsens cell damage and precipitates tissue necrosis. Recent work from our laboratories shows that the return of normal pH contributes substantially to reperfusion injury. Lethal cell injury was prevented by reperfusion with acidotic buffer. Dimethylamiloride, an inhibitor of the Na+ + exchanger, blocked the rise of intracellular pH(pH-i) after reperfusion and also prevented reperfusion injury both to cultured myocytes and to blood-perfused myocardium. Dimethylamiloride also improved recovery of contractile function after reperfusion. These new findings form the basis for this application. Our method of approach utilizes models of ischemia/reperfusion injury in cultured adult rabbit cardiac myocytes and isolated blood-perfused rabbit papillary muscles. In cultured cells under conditions simulating ischemia/reperfusion, structure, function and viability will be assessed using multiparameter digitized video microscopy (MDVM) and laser scanning confocal microscopy (LSCM). Parameter specific fluorescent probes will be used to assess cell viability, cytosolic and mitochondrial ions (H+, Ca2+, Na+, Mg2+), mitochondrial membrane potential, and lysosomal integrity and pH. Other metabolic parameters will be monitored by conventional biochemical assay of whole cultures. In papillary muscles, contractile function, electrical potentials, cell viability, and intracellular ions will be assessed during recovery from ischemic stress using electrophysiologic techniques and LSCM. Our experiments will evaluate competing hypotheses that link lethal reperfusion injury: (1) to pH-i-dependent processes directly; (2) to Ca+=activated processes, which are secondarily linked to changes of pH-i; (3) to mitochondrial failure; and (4) to osmotically induced cell swelling and sarcolemmal failure. A specific practical goal of this project is development of strategies for delivering cytoprotective agents to ischemic myocardium without first causing lethal reperfusion injury. The project will provide fundamental new information concerning mechanisms underlying cell death and contractile dysfunction resulting from reperfusion of ischemic myocardium. The results also will provide a scientific basis for effective new clinical strategies to reduce the extent of tissue necrosis in heart attacks.
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