The principal objective of this research is to learn how myocardial cells react to lethal and nonlethal ischemic injury and to determine what event, or series of events, dictates that the injury is irreversible. We propose to characterize the features of irreversible and reversible ischemic injury through direct biochemical, metabolic, and ultrastructural analysis of tissue injured by servere ischemia in vivo, or total ischemia in vitro. We have previously found that the onset of irreversibility is associated with: 1) virtual complete depletion of ATP and high energy phosphate plus cessation of glycolysis, and 2) the development of functional and ultrastructural evidence of disruption of the plasmalemma of the sarcolemma. The studies proposed are designed to better define the temporal and causal relationships between ATP and adenine nucleotide depletion, sarcolemmal damage, and the onset of lethal injury in ischemia. The hypothesis that ATP depletion is the ultimate cause of lethality in myocardial ischemia will be studied in models of total ischemia in vitro, anoxia in incubated slices of myocardium in vitro, and regional ischemia, in vivo. Using these models and various interventions which will either accelerate or delay ATP depletion, the relationship between tissue ATP content and the onset of functional and structural evidence of lethal injury, specifically including evidence of sarcolemmal disruption, will be assessed. The pathogenesis of plasmalemmal damage in ischemia will be assessed in intact myocardium with particular attention to toxic and osmotic effects of ischemia. In addition, we will test the hypothesis that alterations cytoskeletal proteins plus cell edema secondary to the smootic load of ischemia leads to the membrane disruption characteristic of irreversible ischemic injury. Particular attention will be paid to the cytoskeletal protein vinculin and other cytoskeletal proteins which are involved in the attachment of the sarcolemma to the underlying myofibrils. The effect of endogenous proteases or sarcolemmal and cytoskeletal proteins will be studied. The role of O2 derived metabolic products of leucocytes (free radicals) in the genesis of reperfusion injury will be tested in 40 minute and 3 hour temporary occlusions with and without critical stenosis of the occuluded artery. The relationship between the onset of sarcolemmal damage and the onset of defective cell volume regulation will be assessed in incubated slices of normal myocardium subjected to various periods of ischemia in the presence or absence of various inhibitors.
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