The goal of this proposal is to investigate a novel hypothesis regarding myocardial stunning in which nitric oxide (NO) and oxygen radicals, which are generated during ischemia and reperfusion, damage the sarcoplasmic reticulum (SR) Ca2+-ATPase at the ATP binding site, and also inhibit the function of one of the key SR-bound glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), thereby interrupting the supply of glycolytic ATP to the SR Ca2+-ATPase. These effects on the SR result in an impairment in Ca2+ removal from the cytosol during early reperfusion, and contribute to a rise in [Ca2+]i, a central pathogenetic feature of stunning. This hypothesis follows from our previous results which demonstrate: (a) glycolysis must be intact during early reperfusion to prevent a marked rise in myocardial [Ca2+]i, (2) all of the glycolytic enzymes from aldolase to pyruvate kinase (PK) are bound to SR and are functionally coupled to the Ca2+-ATPase. Recent preliminary data suggest that SR Ca2+ pump function and SR-bound GAPDH activity are inhibited significantly in stunned myocardium. Studies with SR vesicles isolated from rabbit cardiac and skeletal muscle will determine whether the three enzymes proximal to aldolase in the glycolytic chain are bound to SR and coupled functionally to the other enzymes in the chain. SR isolated from ischemic/reperfused rabbit hearts will be examined for loss of Ca2+-ATPase activity and 45Ca transport function and inhibition of SR-bound glycolytic enzyme activity. The time course and reversibility of injury to these enzymes will be determined, the nature of the damage to the Ca2+-ATPase will be characterized, and the sources of oxidant injury will be determined using specific oxygen radical and NO scavengers and nitric oxide synthase (NOS) inhibitors. Electron paramagnetic resonance (EPR) spectrometry and spin traps will be used to verify oxidant inhibition. Immunogold electron microscopy will be used to identify anatomically the presence and possible loss of selected glycolytic enzymes from isolated SR. Isolated rat and rabbit cardiac myocytes will be studied to determine if (1) hypoxia/reoxygenation results in impaired SR Ca2+ uptake, (2) this impairment can be attributed in part to an interruption of glycolysis, and (3) it is related to cellular production of NO and/or oxygen radicals. To further define the role of superoxide and NO in these processes, the hearts of genetically engineered mice exhibiting overexpression of SOD or knockout of eNOS or nNOS will be examined for resistance to myocardial stunning, and resistance to SR Ca2+ pump and glycolytic inhibition. The proposed studies should lead to an improved understanding of functional compartmentation of ATP within the myocyte and also provide new information about mechanisms underlying myocardial stunning.
