The principal objective of this research is to learn how myocardial cells react to lethal and non-lethal ischemic injury and to determine what events, 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 severe or moderate ischemia, and by evaluating pharmacologic interventions which might alter the cell's response to injury. We have previously found that early changes occurring in severely ischemic myocardium concomitant with the onset of irreversibility, include: 1) marked loss of ATP and total adenine nucleotides, 2) failure of cell volume regulation, and 3) (with reperfusion) massive influx of calcium associated with mitochondrial calcification and destruction of the contractile apparatus. The latter phenomenon is associated with structural breaks in the plasmalemma of the sarcolemma. The experiments proposed are designed to better define, in vivo and in vitro, the relationship between adenine nucleotide depletion, irreversible injury, cardiac rigor, loss of cell volume regulation, and to evaluate the capacity of reversibly and irreversibly injured myocardium to resynthesize ATP. The mechanism of cell volume regulation will be studied in vitro, using thin slices of heart muscle injured in vivo, or in vitro with anoxia or metabolic inhibitors. The effects of reperfusion with normal or low Ca21 solutions on calcium influx and structural damage will be evaluated. The features of reversible and irreversible injury in moderately ischemic myocardium will be compared and contrasted with the biologic features already known for severely ischemic myocardium. We will study the mechanism of the protective effects of pharmacologic agents such as verapamil, and determine whether such agents prevent the ATP depletion and cell volume defects associated with irreversible injury. A spontaneously active myocyte tissue culture model will be used for a direct study of membrane permeability and electrical activity after specific types of injury. The results of these studies will be compared and contrasted to those found in adult myocardial cells after in vivo or in vitro injury.
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