EVIDENCE THAT MITOCHONDRIAL RESPIRATION IS A SOURCE OF POTENTIALLY TOXIC OXYGEN-FREE RADICALS IN INTACT RABBIT HEARTS SUBJECTED TO ISCHEMIAAND REFLOW

Previous in vitro studies have shown that isolated mitochondria can ge nerate oxygen radicals. However, whether a similar phenomenon can also occur in intact organs is unknown. In the present study, we tested th e hypothesis that resumption of mitochondrial respiration upon reperfu sion might be a mechanism of oxygen radical formation in postischemic hearts, and that treatment with inhibitors of mitochondrial respiratio n might prevent this phenomenon. Three groups of Langendorff-perfused rabbit hearts were subjected to 30 min of global ischemia at 37-degree s-C, followed by reflow. Throughout ischemia and early reperfusion the hearts received, respectively: (a) 5 mM KCl (controls), (b) 5 mm sodi um amobarbital (Amytal(TM), which blocks mitochondrial respiration at Site I, at the level of NADH dehydrogenase), and (c) 5 mM potassium cy anide (to block mitochondrial respiration distally, at the level of cy tochrome c oxidase). The hearts were then processed to directly evalua te oxygen radical generation by electron paramagnetic resonance spectr oscopy, or to measure oxygen radical-induced membrane lipid peroxidati on by malonyl dialdehyde (M-DA) content of subcellular fractions. Seve rity of ischemia, as assessed by P-31-nuclear magnetic resonance measu rements of cardiac ATP, phosphocreatine, and pH, was similar in all gr oups. Oxygen-centered free radical concentration averaged 3.84 +/- 0.5 4 muM in reperfused control hearts, and it was significantly reduced b y Amytal treatment (1.98 +/- 0.26; p < 0.05), but not by KCN (2.58 +/- 0.96 muM; p = not significant (NS)), consistent with oxygen radicals being formed in the mitochondrial respiratory chain at Site I. Membran e lipid peroxidation of reperfused hearts was also reduced by treatmen t with Amytal, but not with KCN. MDA content of the mitochondrial frac tion averaged 0.75 +/- 0.06 nM/mg protein in controls, 0.72 +/- 0.06 i n KCN-treated hearts, and 0.54 +/- 0.05 in Amytal-treated hearts (p < 0.05 versus both groups). Similarly, MDA content of lysosomal membrane fraction was 0.64 +/- 0.09 nm/mg protein in controls, 0.79 +/- 0.15 i n KCN-treated hearts, and 0.43 +/-0.06 in Amytal-treated hearts (p < 0 .05 versus both groups). Since the effects of Amytal are known to be r eversible, in a second series of experiments we investigated whether t ransient mitochondrial inhibition during the initial 10 min of reperfu sion was also associated with beneficial effects on subsequent recover y of cardiac function after wash-out of the drug. At the end of the ex periment, recovery of left ventricular end-diastolic and of developed pressure was significantly greater in those hearts that had been treat ed with Amytal during ischemia and early reflow, as compared to untrea ted hearts. In conclusion, our data demonstrate that in intact hearts electron flow through the respiratory chain may be an important source of oxygen radicals, which may form at the sites of interactions betwe en Fe-S clusters and ubiquinone, and that resumption of mitochondrial respiration upon reoxygenation might contribute to reperfusion injury.