The major objective is to evaluate the role of the depletion of membrane phospholipids in reperfusion injury. Ischemia, cardioplegic arrest and reperfusion in the pig heart model will be studied, estimating individual and total phospholipids, phosphatidic acid and arachidonic acid in the heart. Phospholipid metabolism will be further examined by estimating the enzymes of the de-novo pathway, including phospholipase A2, phospholipase C, diglyceride lipase, diglyceride kinase and lysophosphatidylcholine acyltransferase, lysophospholipase and fatty acyl CoA synthetase. Since phospholipds can be degraded by various pathways, the relative significance of these pathways will be compared using specific inhibitors. The major pathway of phospholipid metabolism and turnover, the """"""""deacylation-reacylation pathway"""""""", will be studied by comparing the intermediary metabolic products with the corresponding enzymes of the de-novo pathway. Phospholid turnover and arachidonic acid metabolism will be further evaluated by using isotopic arachidonic acid during ischemia and reperfusion and quantitating the incorporation of radioactivity from the arachidonate into the phospholipid pool. Since Ca++ entry during ischemia and reperfusion is a common event, and Ca++ is known to stimulate both phospholipase A and C, the role of calcium on lipase activation will be examined using calcium-calmodulin antagonists in the perfusion system. Phospholipid degradation will be correlated with Ca++ influx by simultaneously estimating the influx of Ca++ with tracer elements. The results of these experiments will be correlated with the measurements of myocardial preservation: high-energy phosphate levels (ATP, CP), myocardial contractility and compliance and CK activity. Phospholipid degradation potentiates reperfusion injury by disturbing metabolic pathways or by generating harmful products of arachidonic acid metabolism, and the above experiments will lead us to determine the relative significance of such mechanisms. This research will focus attention on the clinical events surrounding reperfusion and cardioplegic preservation during open-heart surgery. The interventions proposed (e.g., use of calcium-calmodulin antagonists or specific inhibitors of metabolic pathways) would have direct clinical relevance to improve myocardial preservation during infarct reperfusion and routine open-heart surgery.
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