Stunned myocardium exhibits prolonged dysfunction following a brief period of ischemia, despite the absence of necrosis. This proposal will focus on the pathogenesis of myocardial stunning by examining the role of glycolysis in maintaining Ca2+ homeostasis. Results from recent experiments in our laboratory suggest that glycolytically derived ATP is essential for functional recovery of the post-ischemic rabbit heart by helping to prevent cellular Ca overload during early reperfusion. Hearts made globally ischemic for 20 min recovered significantly less function when they were reperfused with iodoacetate (IAA) to block glycolytic ATP production, despite free availability of oxygen and pyruvate for oxidative metabolism. Transient reperfusion with low Ca2+ prevented the functional deterioration of IAA treated hearts, and NMR measurements of the Ca2+ indicator 19F-BAPTA indicated a marked rise in cytosolic Ca2+ ([Ca2+]i) during reperfusion with IAA. Studies will be done in the perfused globally stunned rabbit heart and the intact open chest dog with regionally stunned myocardium. Simultaneous measurements will be made of mechanical function, oxygen consumption, high energy phosphate content P-31 NMR spectroscopy and unidirectional ATP flux from inorganic phosphate and ADP (by saturation transfer).
Aim #1 will determine whether enhancement of glycolysis can reduce stunning and whether the need for glycolysis during early reperfusion can be demonstrated in-vivo in the dog.
Aim #2 will provide further evidence that IAA exerts its effect through interference with Ca2+ homeostasis by determining whether IAA's effect can be modified by the level of [Ca2+]i at the end ischemia or the rate of Ca2+ entry during early reperfusion. [Ca2+]i levels will be measured in parallel groups of hearts by F-19 NMR. In some experiments, the time course of [Ca2+]i will be measured during reperfusion using aequorin micro-injected into superficial cardiomyocytes.
Aim #3 will determine whether scavenging of oxygen radicals during reperfusion reduced the harmful effect of IAA, thereby implicating an interaction between free radicals and Ca2+ overload in the pathogenesis of stunning.
Aim #4 will determine whether glycolysis is enhanced (using C-13 NMR) and/or oxidative metabolism depressed during early reperfusion in stunned myocardium.
Aim #5 will determine whether the detrimental effect of IAA can be modified by the type of substrate used for oxidative metabolism (pyruvate vs short chain fatty acids vs long chain fatty acids).
