SUMMARY. Studies in ischemic canine left ventricle have shown that the depletion of membrane phospholipids is a critical event in the development of a sarcolemmal calcium permeability defect and associated irreversible myocyte injury. The mechanism of phospholipid loss is unclear, but may be due to the activation of endogenous phospholipases. Since arachidonic acid is a fatty acid found almost entirely in phospholipid, increases in arachidonate provide evidence for increased phospholipase activity. The present study was designed to examine the temporal relationship of the accumulation of free arachidonate with the onset of phospholipid depletion during fixed ligation of the left anterior descending coronary artery in canine myocardium. The following results were demonstrated in ischemic canine myocardium: (1) the accumulation of unesterified arachidonate is minimal during 10-30 minutes of ischemia, but is significantly increased after prolonging the duration of ischemia to 1-3 hours; (2) significant increases in arachidonate precede the development of a significant decrease in total phospholipid content; (3) the decrease in the arachidonate content of phosphatidylcholine is accompanied by similar decreases in all of the fatty acyl moieties; (4) the arachidonate content of lysophosphatidylcholine and diacylglycerol are unchanged during myocardial ischemia; (5) there is evidence of a deacylarion-reacylation cycle in phosphatidylcholine prior to the accumulation of free arachidonate; (6) the fatty acyl specificity of the lysophosphatidylcholine acyltransferase corresponds to the pattern of fatty acyl remodeling of phosphatidylcholine during early myocardial ischemia. These data suggest that the accumulation of arachidonate may be a more sensitive measure of phospholipid degradation than the decrease in total phospholipid content in ischemic canine myocardium. It is postulated that the defective reacylation of arachidonate into phosphatidylcholine may contribute to the net loss of membrane phospholipid during myocardial ischemia. (Circ Res 54: 313-322, 1984)

The effects of adrenergic stimulation on the rates of protein synthesis, degradation, and accumulation were examined in primary cultures of neonatal rat heart cells. Treatment of myocardial cells with norepinephrine increased total cellular protein content and the rate of incorporation of radiolabeled tyrosine into trichloroacetic acid insoluble protein. alpha 1-Adrenergic, but not alpha 2- or beta-adrenergic blockade, inhibited these norepinephrine induced increases. The rate of protein synthesis estimated from the kinetics of equilibrium labeling and from combined equilibrium and pulse labeling was increased by norepinephrine stimulation, whereas protein degradation estimated by release of previously incorporated radiolabeled tyrosine or in pulse-chase experiments was unaffected. To determine whether alpha 1-adrenergic stimulation produced similar effects on the turnover of myofibrillar proteins, rates of synthesis and degradation were estimated for a myofibrillar-enriched protein fraction and for myosin heavy chain and actin. Norepinephrine treatment produced increases in the synthesis of myofibrillar protein without significantly altering degradation rates. These experiments suggest that alpha 1-adrenergic stimulation increases myocardial cell protein content by accelerating protein synthesis.

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Previous studies have suggested that the accumulation of free arachidonic acid may be of major importance in the pathophysiology of myocardial ischemia. The purpose of the present study was to determine if the release of arachidonic acid from myocardial cells was more dependent on the extent of ATP depletion than on the inhibition of fatty acid oxidation. In addition, these studies were designed to determine if arachidonic acid release only occurred when ATP was depleted beyond a critical threshold level. To examine the relationship between arachidonic acid release and ATP depletion, cultured myocardial cells from neonatal rat hearts were labeled with [3H]arachidonate and [14C]palmitate. In response to ATP depletion with various metabolic inhibitors, [3H]arachidonic acid and [14C]palmitic acid were released from phospholipids. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid were the major esterified sources of the arachidonate. The release of both fatty acids was related to the extent of ATP depletion and not whether a glycolytic or respiratory inhibitor was utilized. Various combinations and doses of metabolic inhibitors were used, and experimental conditions that produced a greater than 75% decrease in ATP content were associated with the accumulation of arachidonic acid. These results suggest that an ATP-dependent step may be linked to the accumulation of arachidonic acid during myocardial ATP depletion. It is suggested that myocardial cells may release arachidonic acid directly in response to ATP depletion.

The present study utilized a cultured myocardial cell