Myocardial ischemia often results in prolonged dysfunction of the hearth (stunned myocardium"""""""") or complete loss of contractile function (irreversible cell death). Thus understanding of the mechanisms of cellular damage is crucial to better prevention and treatment of ischemic heart disease. While the cause of ischemic to the heart is probably multifactorial, damage to mitochondria (mito) of the heart may play a critical role progression to irreversible injury. The broad goals of this project are to understand the mechanism of adenine nucleotide (AdN) loss from the mito compartment of the heart cell during ischemia; and to determine interventions that may be used to prevent this loss and to enhance recovery of mito AdN during the postischemic period.
Specific Aim 1 : To determine to what extent the loss AdN from the mito during myocardial ischemia can be inhibited by atractyloside, phenylsuccinate, and butylmalonate. To determine this, an isolated heart system will be used. The hearts will be perfused at several concentrations of the agents. After 30 min of ischemia, mito AdN will be determined. Dose response curves will be generated for each agent.
Specific Aim 2 : To determine if extramitochondrial AdN, inorganic phosphate, {H+} and lactate levels may modulate efflux of AdN from heart mito. The rates of efflux will be determined in the presence of varying concentrations of the agents described.
Specific Aim 3 : To determine if the loss of mitochondrial AdN is reversible upon reperfusion. The left circumflex artery will be ligated. After a specific time (to be determined) the ligature will be removed and the animals will be allowed to recover. Mito will be isolated from affected and unaffected regions before, at the end of the ischemic period, and at four times following reperfusion (6 hours, 24 hours, 48 hours and 1 week). The mito will be assayed for AdN. In addition, mito isolated from normal and ischemic hearts will be studied to determine the conditions necessary to observe net accumulation of AdN.
Specific Aim 4 : To better understand the roles of the adenine nucleotide translocase and decarboxylate carrier in phosphate- induced efflux (and uptake) of AdN from (into) heart mito. To accomplish this, the adenine nucleotide translocase and decarboxylate carrier will be isolated from native mito membranes. The purified carriers will be reconstituted into phospholipid vesicles. AdN transport will be studied in vesicles that have been reconstituted with either the adenine nucleotide translocase alone, the decarboxylate carrier alone, and both carriers together.
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