Myocardial ischemia is accompanied by accelerated phospholipid catabolism resulting in alterations in the chemical constituents of the sarcolemmal membrane which precipitate electrophysiologic dysfunction and myocytic cell death. We have previously demonstrated that plasmalogens are the predominant phospholipid constituents of myocardial sarcolemma and that myocardium contains a novel class of calcium-independent phospholipases A2 which selectively hydrolyze plasmalogen substrate. During the current grant interval we have demonstrated that: 1) sarcolemmal phospholipids are selectively hydrolyzed during myocyte injury; 2) the activity of membrane associated plasmalogen selective phospholipase A2 increases over 9-fold during 15min of myocardial ischemia; and 3) ATP activates calcium- independent plasmalogen selective phospholipase A2 activity in ischemic, but not in control myocardial cytosol. Collectively, these results demonstrate that profound alterations in phospholipid metabolism selectively occur in the electrically active membrane of myocyte which are likely mediated by calcium-independent plasmalogen selective phospholipase A2. Accordingly, the overall goal of the proposed research is the identification of the biochemical mechanisms which result in the activation of these novel phospholipase during myocardial ischemia. Purification of the polypeptide catalyzing membrane associated phospholipase A2 will identify salient physical, structural and kinetic characteristics of this important enzyme. Factors responsible for the induction of membrane associated phospholipase A2 activity during ischemia will be identified and purified. The potential significance of the direct association of cytosolic calcium-independent phospholipase A2 with phosphofructokinase will be delineated and the relationship of this complex to ATP induced activation of cytosolic phospholipase A2 during ischemia will be examined. Cytosolic and membrane bound calcium-independent phospholipase activities will be cloned and their primary sequences will be determined. Critical differences in the stereoelectronic conformation of plasmalogen and diacyl phospholipids will be delineated to assess their role as determinants of the kinetic specificities of these phospholipases A2. Through utilization of this multidisciplinary approach the detailed biochemical mechanisms responsible for the activation of these novel phospholipases will be identified. Collectively, the proposed research will identify critical structural and kinetic characteristics of these novel enzymic targets whose pharmacologic manipulation can potentially prevent lethal ventricular dysrhythmias and attenuate cell death during myocardial ischemia.
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