Ischemic heart disease is a major health problem. Thus, understanding of the mechanisms of cellular damage is crucial to better prevention and treatment. While the cause of ischemic injury is probably multi-factorial, damage to the major energy-producing system (mitochondria) of the heart may play a critical role in progression to irreversible injury.
The specific aims of this proposal are to test the following hypotheses: 1. Abnormally high levels of inorganic phosphate induces an atractyloside-sensitive efflux of adenine nucleotides from mitochondria during myocardial ischemia. 2. The loss of mitochondrial adenine nucleotides is reversible. To test the validity of hypothesis 1, studies will be designed to determine if the loss of adenine nucleotides is an artifact of isolation. First, the effect of ischemia on the adenine nucleotide content of the mitochondria will be evaluated using different techniques of mitochondrial isolation. Three methods of cell disruption will be tested: mechanical, enzymatic, and mechanical and enzymatic. In each case, the following variables will be evaluated: duration of mechanical disruption or enzymatic digestion, volume of the whole tissue homogenate, and type of isolation buffer used (sucrose vs KC1). Secondly, mitochondria from normal heart muscle will be incubated in the 15,000 g supernatant of an ischemic heart homogenate to test for the presence of an agent which may cause the loss of mitochondrial adenine nucleotides following homogenization. Thirdly, since adenine nucleotide efflux may occur during isolation via the adenine nucleotide translocase, the possible inhibition of efflux by the addition of atractyloside to the isolation buffers will be evaluated. To test whether the loss of adenine nucleotide from mitochondria is due to elevated phosphate levels, the rate and extent of nucleotide efflux will be determined for different phosphate concentrations in the heart. The phosphate content will be varied by varying the rate of coronary flow. Moreover, it will be determined if adenine nucleotide efflux can occur from normal heart mitochondria incubated in vitro under conditions simulating the ischemic heart cell. Finally, hypothesis 1 will be tested by determining whether atractyloside infused into the heart prior to the ischemia can prevent adenine nucleotide loss from the mitochondria. Hypothesis 2 will be tested by determining the mitochondrial adenine nucleotide content after ischemia and after ischemia followed by reperfusion. The isolated perfused rat heart will be used throughout this study. Ischemia will be produced by cross-clamping the aorta.
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