Inhibition of glyceraldehyde-3-phosphate dehydrogenase in post-ischaemic myocardium

Objective: Myocardial reperfusion following brief periods of ischaemia is associated with prolonged, reversible periods of metabolic dysfunction. As the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is inhibited in vitro by reactive oxygen species, we hypothesized that production of reactive oxygen species during reperfusion would lead to inhibition of GAPDH in post-ischaemic myocardium. Methods: Anaesthetized closed-chest dogs were subjected to 20 min balloon occlusion of the left anterior descending coronary artery. Biopsy samples were taken after 3 and 24 h of reperfusion, to determine the activity of GAPDH and the concentrations of glycolytic intermediates in post-ischaemic and remote, non-ischaemic territories. Results: A significant reduction in GAPDH activity was observed in post-ischaemic relative to remote tissue after 3 h reperfusion (4.8+/-0.5 vs. 2.9+/-0.2 mu mol/min/mg protein; P <0.01). Western blotting revealed no reduction in the levels of GAPDH protein. Analysis of enzyme kinetics showed the loss of activity to be associated with decreased V-max (5.9+/-0.5 vs. 3.2+/-0.2 mu mol/min/mg protein; P <0.01) with no significant change in the K-m for glyceraldehyde-3-phosphate (GAP). Incubation of the inhibited enzyme under both mild and strong reducing conditions failed to reactivate the enzyme. The acute reduction in enzyme activity in post-ischaemic tissue was accompanied by regional differences in glycolytic intermediates, notably a twofold accumulation of GAP (P <0.05), and a reduction in the glucose metabolic rate (GMR) determined by positron emission tomography and [F-18]2-fluorodeoxyglucose. By 24 h reperfusion, no regional differences in GAPDH activity, reaction V-max or K-m, GAP concentrations or GMR were detectable. Conclusions: These results suggest that inhibition of GAPDH activity may represent an important point at which glycolysis is limited during reperfusion, and further, that the mechanisms of enzyme inhibition do not involve simple oxidation or S-thiolation of critical active site thiol groups.