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

Giuseppe Ambrosio, Jay L. Zweier, Carlo Duilio, Periannan Kuppusamy, Giuseppe Santoro, Pietro P. Elia, Isabella Tritto, Plinio Cirillo, Mario Condorelli, Massimo Chiariello, John T. Flaherty

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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 °C, followed by reflow. Throughout ischemia and early reperfusion the hearts received, respectively: (a) 5 mM KCl (controls), (b) 5 mM sodium amobarbital (AmytalTM, which blocks mitochondria! 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 μM 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 μM; 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. At the end of the experiment, recovery of left ventricular enddiastolic and of developed pressure was significantly greater in those hearts that had been treated with Amytal during ischemia and early reflow, as compared to untreated 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 between Fe-S clusters and ubiquinone, and that resumption of mitochondrial respiration upon reoxygenation might contribute to reperfusion injury.

Original languageEnglish (US)
Pages (from-to)18532-18541
Number of pages10
JournalJournal of Biological Chemistry
Issue number25
StatePublished - Sep 5 1993

ASJC Scopus subject areas

  • Biochemistry


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