Evidence that mitochondrial respiration is a source of potentially toxic oxygen free radicals in intact rabbit hearts subjected to ischemia and reflow

• Published in: The Journal of biological chemistry 1993-09, Vol.268 (25), p.18532-18541
• Main Authors: Ambrosio, G., Zweier, J.L., Duilio, C., Kuppusamy, P., Santoro, G., Elia, P.P., Tritto, I., Cirillo, P., Condorelli, M., Chiariello, M.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Amobarbital - pharmacology; Animals; Biological and medical sciences; Cell structures and functions; Electron Spin Resonance Spectroscopy; Female; Free Radicals; Fundamental and applied biological sciences. Psychology; Lipid Peroxidation; Magnetic Resonance Spectroscopy; Mitochondria and cell respiration; Mitochondria, Heart - drug effects; Mitochondria, Heart - metabolism; Molecular and cellular biology; Myocardial Ischemia - metabolism; Myocardial Reperfusion; Oxygen - metabolism; Oxygen Consumption - drug effects; Potassium Cyanide - pharmacology; Rabbits
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)46660-9

Previous in vitro studies have shown that isolated mitochondria can generate oxygen radicals. However, whether a similar phenomenon can also occur in intact organs is unknown. In the present study, we tested the hypothesis that resumption of mitochondrial respiration upon reperfusion might be a mechanism of oxygen radical formation in postischemic hearts, and that treatment with inhibitors of mitochondrial respiration might prevent this phenomenon. Three groups of Langendorff-perfused rabbit hearts were subjected to 30 min of global ischemia at 37 degrees C, followed by reflow. Throughout ischemia and early reperfusion the hearts received, respectively: (a) 5 mM KCl (controls), (b) 5 mM sodium amobarbital (Amytal, which blocks mitochondrial respiration at Site I, at the level of NADH dehydrogenase), and (c) 5 mM potassium cyanide (to block mitochondrial respiration distally, at the level of cytochrome c oxidase). The hearts were then processed to directly evaluate oxygen radical generation by electron paramagnetic resonance spectroscopy, or to measure oxygen radical-induced membrane lipid peroxidation by malonyl dialdehyde (MDA) content of subcellular fractions. Severity of ischemia, as assessed by 31P-nuclear magnetic resonance measurements of cardiac ATP, phosphocreatine, and pH, was similar in all groups. Oxygen-centered free radical concentration averaged 3.84 +/- 0.54 microM in reperfused control hearts, and it was significantly reduced by Amytal treatment (1.98 +/- 0.26; p < 0.05), but not by KCN (2.58 +/- 0.96 microM; p = not significant (NS)), consistent with oxygen radicals being formed in the mitochondrial respiratory chain at Site I. Membrane lipid peroxidation of reperfused hearts was also reduced by treatment with Amytal, but not with KCN. MDA content of the mitochondrial fraction averaged 0.75 +/- 0.06 nM/mg protein in controls, 0.72 +/- 0.06 in 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 in 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 reversible, in a second series of experiments we investigated whether transient mitochondrial inhibition during the initial 10 min of reperfusion was also associated with beneficial effects on subsequent recovery of cardiac function after wash-out of the drug.