Regulation of Glycolytic Flux in Ischemic Preconditioning

Exact adjustment of the Embden-Meyerhof pathway (EMP) is an important issue in ischemic preconditioning (IP) because an attenuated ischemic lactate accumulation contributes to myocardial protection. However, precise mechanisms of glycolytic flux and its regulation in IP remain to be elucidated. In open chest pigs, IP was achieved by two cycles of 10-min coronary artery occlusion and 30-min reperfusion prior to a 45-min index ischemia and 120-min reperfusion. Myocardial contents in glycolytic intermediates were assessed by high performance liquid chromatographic analysis of serial myocardial biopsies under control conditions and IP. Detailed time courses of metabolite contents allow an in-depth description of EMP regulation during index ischemia using metabolic control analysis. IP reduced myocardial infarct size (control, 90.0 3.1 versus 5.05 2.1%; p < 0.001) and attenuated myocardial lactate accumulation (end-ischemic contents, 31.9 4.47 versus 10.3 1.26 mol/wet weight, p < 0.0001), whereby a decrease in anaerobic glycolytic flux by at least 70% could constantly be observed throughout index ischemia. By calculation of flux:metabolite co-responses, the mechanisms of glycolytic regulation were investigated. The continuous deceleration of EMP flux in control myocadium could neither be explained on the basis of substrate availability nor be attributed to regulatory “key enzymes,” as multisite regulation was employed for flux adjustment. In myocardium subjected to IP, an even pronounced deceleration of EMP flux during index ischemia was observed. Again, the adjustment of EMP flux was because of multisite modulation without any evidence for flux limitation by substrate availability or a key enzyme. However, IP changed the regulatory properties of most EMP enzymes, and some of these patterns could not be explained on the basis of substrate kinetics. Instead, other regulatory mechanisms, which have previously not yet been described for EMP enzymes, must be considered. These altered biochemical properties of the EMP enzymes have not yet been described. Myocardial survival in states of supply/demand imbalance critically depends on cellular energy status (1). To limit energy deficit in conditions of energy shortage, e.g. hypoxia and ischemia, myocardial energy production switches from the preferential use of fatty acids to carbohydrates, thereby allowing maintenance of adequate ATP synthesis when decreased oxygen availability becomes the limiting factor (2–5). However, in zeroflow ischemia, experimental analysis has shown increased glycolysis to be a double-edged sword, as the accumulation of glycolytic end products (6, 7) outweighs the potential benefits of increased ATP synthesis. In accordance with this paradigm, myocardial protection by prior exposure to ischemic preconditioning (IP) employs a limitation in myocardial energy deficit (8, 9). Because IP tremendously reduces ischemic myocardial energy demands (8), energy deficit is largely decreased at even reduced rates of anaerobic glycolytic ATP formation in zero-flow ischemia. There is good evidence that this attenuation in ischemic lactate accumulation represents an important mechanism whereby IP myocardium better withstands the challenges of sustained ischemia. Although a decrease in anaerobic glycolytic flux is a consistent finding in myocardium protected by ischemic preconditioning (10–12), the precise mechanism used to adjust anaerobic glycolytic flux still remains unclear. The elucidation of this adaptive mechanism, which is not merely of theoretical interest, was the aim of our study. To analyze the regulation of metabolic pathways, traditional concepts mostly imply that control over a pathway is achieved by action on a single pathway enzyme, which is often assumed to be a nonequilibrium step near the beginning of the pathway subjected to feedback inhibition (13, 14). However, because the rise of metabolic control analysis (MCA) (15) there is growing evidence that these comfortable, time honored concepts may mislead more than enlighten. Hence, the tools of MCA were used to obtain a state-of-the-art analysis of glycolytic regulation in ischemic myocardium protected by preceding ischemic preconditioning.