Reperfusion-induced modulation of cardiac mitochondrial function by free radicals and calcium

Reperfusion of an ischemic myocardium is associated with a number of deleterious events that are thought to contribute to myocardial dysfunction. Cellular events that predominate during reperfusion include calcium overload, mitochondrial dysfunction and enhanced production of free radicals. Cardiac mitochondria are believed to play an important role in the pathophysiology of reperfusion by contributing to calcium overload as well as free radical production. In an initial in vivo study, the effect of in vivo ischemia/reperfusion on mitochondrial function was investigated. The LAD coronary artery of male SD rats was ligated to induce ischemia, and reperfusion was initiated by removal of the ligature. Cardiac mitochondria were isolated from the affected area of the left ventricular free wall and were utilized for assays of ADP linked respiration as well as individual mitochondrial enzyme activities and western blot analysis of mitochondrial proteins. We were able to demonstrate that reperfusion was associated with a significant decline in ADP linked respiration. The mechanism of decline in overall mitochondrial respiration was due to selective inactivation of the redox sensitive Krebs cycle enzymes KGDH and aconitase. Reperfusion was also associated with a significant decline in activity of complex I of the electron transport chain. The mechanism of inactivation of complex I was further investigated in an in vitro setting by simulating conditions that predominate during ischemia and reperfusion such as pH changes and calcium overload. Results indicate that mitochondrial calcium regulates complex I activity by inducing an endogenous superoxide burst. This leads to a sulfhydryl modification of complex I resulting in its inactivation. In the presence of NADH, and after chelation of calcium, complex I is capable of self-reactivation. The findings of our in vivo and in vitro studies suggest a scenario in which calcium overload during reperfusion results in an endogenous superoxide burst at the complex I site. This radical burst potentially contributes to inactivation of redox sensitive enzymes during reperfusion. The inhibitory effect of calcium on complex I activity also suggest a regulatory effect of calcium on mitochondrial proton gradient, which in turn regulates mitochondrial calcium uptake.