Evidence That Mitochondrial Respiration Is a Source of Potentially ...

THEJOURNALOF B ~ L O G I C ACHEMISTRY L

Vol. 268, No. 25, Iesue of September 5, pp. 18532-1@541,1993 Printed in U.S. A.

0 1993 by The Amencan Society for Biochemistry and Molecular Biology, Inc.

Evidence That Mitochondrial Respiration Is a Source of Potentially Toxic Oxygen Free Radicals in IntactRabbit Hearts Subjected to Ischemia and Reflow’ (Received for publication, December 17, 1992, and in revised form, March 31, 1993)

Giuseppe AmbrosioS, Jay L. ZweierjT, Carlo Duilio, Periannan Kuppusamyj, Giuseppe Santoro, Pietro P. Elia, Isabella Tritto, Plinio Cirillo, Mario Condorelli, Massimo Chiariello, and John T. Flahertyj From the Department of Medicine, Division of Cardiology, Second School of Medicine, University of Naples, 80131 Naples, Italy and the $Department of Medicine, Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Previous in vitro studies have shown that isolated f 0.06 in Amytal-treatedhearts ( p < 0.06 versus both mitochondria can generateoxygen radicals. However, groups). Similarly, MDA content of lysosomal memwhether a similar phenomenon can also occur in intact brane fraction was0.64 f 0.09 nM/mg protein in conwe tested the trols, 0.79 C 0.16 in KCN-treated hearts, and 0.43 2 organs is unknown. In the present study, hypothesis that resumption of mitochondrial respira- 0.06 in Amytal-treated hearts ( p 0.06 versus both tion upon reperfusion might be a mechanism of oxygen groups). Since the effects of Amytal are known to be radicalformationin postischemic hearts,andthat reversible, ina second series of experiments we investreatment with inhibitors of mitochondrial respiration tigatedwhethertransient mitochondrial inhibition might prevent thisphenomenon. Three groups of Lan- during the initial10 min of reperfusion wasalso assogendorff-perfused rabbit hearts were subjected to 30 ciated with beneficial effects on subsequent recovery min of global ischemia at 37 “C, followed by reflow. of cardiac function after wash-out of the drug. At the Throughout ischemia and earlyreperfusion the hearts end of the experiment, recovery of left ventricularendreceived, respectively: (a)6 mMKC1 (controls), ( b ) 6 diastolic and of developed pressure was significantly mM sodium amobarbital (Amytal“, which blocks mito- greater in those hearts that had been treated with chondrial respiration at Site I, at the level of NADH Amytal during ischemia and earlyreflow, as compared dehydrogenase), and (c) 6 mM potassium cyanide (to to untreated hearts. In conclusion, our data demonblock mitochondrial respiration distally,at the level of strate that in intact hearts electron flow through the processed respiratory chain may be an important source of oxycytochrome c oxidase). The hearts were then to directly evaluate oxygen radical generationby elec- gen radicals, which may form at the sites of interactron paramagnetic resonancespectroscopy, or to meas- tions between Fe-S clusters and ubiquinone, and that ure oxygen radical-induced membrane lipid peroxida- resumption of mitochondrial respiration upon reoxytion by malonyl dialdehyde (MDA) content of subcel- genation might contribute to reperfusion injury. lular fractions. Severity of ischemia, as assessed by “P-nuclear magnetic resonance measurementsof cardiac ATP, phosphocreatine, and pH, was similar in all groups. Oxygen-centered freeradicalconcentration Restoration of flow after a period of ischemia is accomaveraged 3.84 f 0.64 PM in reperfused control hearts, panied by generation of large amounts of potentially toxic and it was significantly reduced by Amytal treatment oxygen free radicals (1-4), and it has been proposed that this (1.982 0.26; p < 0.06), but not by KCN (2.68 f 0.96 phenomenon may account for the occurrence of a specific PM; p = not significant (NS)), consistent with oxygen form of reperfusion-mediated tissue damage (5, 6). In reperradicals being formed in themitochondrial respiratory fused hearts, oxygen radicals can be generated by several chain at Site I. Membrane lipid peroxidationof reper- mechanisms, including the xanthinelxanthine oxidase reacfused hearts was also reduced by treatment withAmy- tion (7, 8), and the activity of NADPH oxidase and myelotal, but not with KCN. MDA content of the mitochon- peroxidase of activated leukocytes, which migrate into the drial fraction averaged 0.76 f 0.06 nM/mg protein in controls, 0.72 f 0.06 in KCN-treated hearts, and0.64 previouslyischemic area (9). Anotherpotential source of oxygen radicals is thought to be mitochondrial respiration * This work has been supported in part by Grant 91.00122.PF41 (10,ll). In the process of mitochondrial electron transport, oxygen from Consiglio Nazionale delle Ricerche (Progetto Finalizzato Prevenzione e Controllo dei Fattori di Malattia), by Specialized Center of is normally reduced t o water through several steps in which Research in Ischemic Heart Disease Grant P50HL 17655, by Grant hydrogen atomsact as electrondonors (Fig. 1). However, HL-38324 from the National Institutes of Health, and byNATO shown that oxygen can International Research Grant 0139/88. A preliminary account was studies on isolated mitochondria have presented at the 65th Meeting of the American Heart Association, also undergo 1-electron reduction, with formation of superand hydrogen peroxide (Hz02) (10-17). November 16-19,1992, New Orleans, LA. The costs of publication of oxide radicals (‘0;) have this article were defrayed in part by the payment of page charges. At leasttwo sites in the mitochondrial respiratory chain This article must therefore beherebymarked “advertisement” in beenidentifiedwhere oxygen radicals maybegenerated accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. NADH dehydrogenase (15, 16) and ubiquinone (17) (Fig. 1). $ To whom correspondence should be addressed Cattedra di Carderive from 1-electron At both sites,’ 0;formation appears to diologia, I1 Facolta’diMedicina,Via S. Pansini 5, 80131Naples, transfer through iron-sulfur clusters, with formation of seItaly. Tel.: 39-81-7462216; Fax: 39-81-7462229. miquinone, which eventually oxidizes yielding ’ 0;and then ll Established Investigatorof the American Heart Association.

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Mitochondria and Reperfuswn Injury H&. It is estimated that under normal conditions 1-2% of oxygen utilized by mitochondria leads to the formation of superoxide radicals (11).This "physiologic" generation of oxygen radicals is normally inactivated by endogenous scavenger mechanisms present withinthe cells (5). However, data derived from i n vitro experiments suggest that oxygen radical generation can be greatly enhanced when mitochondrial respiration is stimulated underconditions of altered redox state and of decreased availability of ADP (10-17). Such conditions are likely to occur in hearts reperfused after ischemia. Formation of oxygen radicals might, in fact, drastically increase upon resumption of oxidative phosphorylation at the time of reflow, when oxygen is made again available to mitochondria that have accumulated large amounts of reducing equivalents, while decreasing their adenine nucleotide content, during the period of ischemia. In addition, decreased activity of oxygen radical scavenging enzymes may further aggravate the imbalance between generation and detoxification of oxygen radicals in postischemic hearts (18-20). It has therefore been proposed that mitochondrial respiration might be an important source of oxygen radicals and, hence, a potentialcontributor to reperfusion injury (5). This hypothesis is indirectly supported by the observation that a prominent generation of oxygen radicals could be induced in vitro upon reoxygenation of mitochondria isolated from hearts that had been subjected to ischemia (21). However, no data are currently available to demonstrate that this phenomenon can actually occur i n intact organs undergoing ischemia and reflow. The present study was therefore designed to test whether resumption of mitochondrial oxidative phosphorylation upon postischemic reflow can be a source of toxic oxygen radicals in isolated rabbit hearts. This experimental model has been previously characterized with respect to various aspects of oxygen radical toxicity (1,4, 22-25). In addition, it has the distinct advantage of allowing assessment of the contribution of mitochondrial respiration to oxygen radical generation without the confounding effects of two other major sources of free radicals, namely xanthine oxidase activity, which is very low or absent in rabbit hearts (26,27),and leukocytes, which are absent from the perfusion buffer. Oxygen radical formation was evaluated by electron paramagnetic resonance spectroscopy in untreated heartssubjected to ischemia and reflow and in hearts in which mitochondrial respiration duringearly reflow was blocked by administration of either sodium amobarbital (Amytal"', an inhibitor of NADH dehydrogenase; Ref. 28), or cyanide (which blocks electron flow at the level of cytochrome c oxidase; Ref. 28) (Fig. 1).Since lipid peroxidation is a major biochemical consequence of oxygen radical attack (25, 29, 30), we also investigated whether attempts to inhibit mitochondrial generation of oxygen radicals were accompanied by a concomitant effect on membrane lipid peroxidation of reperfused hearts. Finally, in another series of experiments we investigated whether transient inhibition of mitochondrial respiration during the early minutes of reflow would also reduce the development of reperfusion injury, as assessed by recovery of contractile function in postischemic hearts. MATERIALS AND METHODS

Isolated Heart Preparation Female New Zealand White rabbits (1.5-2.0 kg) were heparinized and anesthetized with intraperitoneal pentobarbital. The heartswere removed, the ascending aorta was cannulated, and retrograde perfusion was started under constant pressure (80 mm Hg) with perfusate containing 117 mM sodium chloride, 6.0 mM potassium chloride, 3.0 mM calcium chloride, 1.0 mM magnesium sulfate, 0.5 mM EDTA, 16.7 mM glucose, and 24 mM sodium bicarbonate, pH 7.4. The perfusate

was equilibrated at 37 "C with a 95/5% mixture of 0 2 and COS,and not recirculated. The hearts were paced at 175 beats/min by means of a wick electrode (containing saturated KCl) connected to a Grass SD-9 stimulator. To assess contractilefunction,a latex balloon, connected to a Statham P23Db transducer, was inserted into the left ventricular cavity through the mitral opening and secured with a ligature that included the left atrial remnants. The balloon was initially inflated with saline to produce an end-diastolic pressure of 10 mm Hg, which is on the plateau of the end-diastolic volume/endsystolic pressure curve for this preparation. Coronary flow wasmeasured by aspiration of perfusate overflow. Experimental Protocol After equilibration, the hearts were subjected to 30 min of global ischemia at 37 "C, followed by reperfusion. At the time of ischemia, the heartswere divided into threegroups which received,respectively: (i) 5 mM KC1 (control hearts); (ii) 5 mM Amytal (to block mitochondrial NADH dehydrogenase; Ref. 281, plus 5 mM KCk (iii) 5 mM potassium cyanide (to block mitochondrial cytochrome c oxidase; Ref. 28). Five mM KC1 was added to groups I and I1 hearts, to control for the additional 5 mmol/liter K+ ions given to KCN-treated hearts (group 111). Global ischemia was induced by interrupting the aortic inflow. The drugs were administered by intracoronary infusion during the initial 5 min of ischemia via a side arm in the perfusion line, at the rate of 0.5 ml/min, which corresponded to 95% inhibition of mitochondrial respiration (48, 54). In spite of this fact, oxygen radical production was reduced only about 50% by Amytal treatment, even though two other major sources of oxygen radicals are minimally present or absent in our model (i.e., xanthine oxidase and neutrophils). This finding indicates that mitochondrial respiration is not the only mechanism of oxygen radical formation in our model. Cyclooxygenase and microsomal oxidases are otherpotential sources of oxygen radicals in theheart (66, 67). In addition, recent experiments by Vandeplassche et al. (68,69) have documented histochemically that a specific form of NADH oxidase associated with mitochondria (but unrelated to oxidative phosphorylation) may become activated in hearts subjected to ischemia and reperfusion. Interestingly, the activity of this enzyme is known to be unaffected by mitochondrial inhibitors, and it is actually expected to be stimulated when the mitochondrial respiratory chainis blocked (70). Therefore, this metabolic pathway might have remained operative in spite of Amytal administration, and in fact it might be speculated that thedrug, by preventing NADH and oxygen from being utilized in the oxidative phosphorylation, might have actually increased the amount of oxygen radicals generated by way of NADH oxidase, as well as by microsomes (67, 71) and/or other sources. A similar mechanism might also explain the finding in the present study of lipid peroxidation being more pronounced in the microsomal fraction of Amytal-treated hearts. Again, it is possible that microsomal oxidase activity may be more pronounced when 0,utilization is diverted from the mitochondrial respiration process. The present study hascertainlimitations. One obvious limitation is represented by the fact that our experimental conditions do not allow to perform a detailed characterization of the relationship between mitochondrial respiration and oxygen radical production, which can be optimally performed on isolated mitochondria, and not in intact beating hearts. Another possibility is that the beneficial effects of Amytal that we observed might have been unrelated to inhibition of mitochondrial respiration. However, Amytal has been previously used to specifically block mitochondrial respiration in intact hearts (48, 49, 54). In fact, detailed analysis of the relationship between mitochondrial respiratory ratesand Amytal concentrations indicates that in the perfused heart this drug affects only a single component (i.e.NADH dehydrogenase) (48). In addition, our in uitro experiments documented that Amytal was devoid of direct scavenging effects on superoxide radicals. Finally, the 31PNMR data allowed us to confirm that Amytal had no effects on the severity of ischemia. Another factor to be considered when interpreting

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the results of the present study is the experimental model. It is well known that production of oxygen radicals increases with increasing the oxygen tension to which isolated mitochondria are exposed in uitro (13, 67, 71). Therefore, it might be hypothesized that the high oxygen tension of crystalloid perfusate might have enhanced this phenomenon in our animal model, whereas in vivo other mechanisms might account for a larger fraction of oxygen radical generation. Against this speculation, however, is thefact that, inspite of higher oxygen tension, 02 carrying capacity of crystalloid perfusates is actually lower than thatof blood. Furthermore, in intact hearts intracellular O2tension ut the mitochondrialleuel is markedly lower than in the vascular lumen (72), and presumably similar both in uiuo, and in in uitro perfused hearts. Another potential concern relates to possible artifacts in the measurement of free radical concentrations or of lipid peroxidation products. In the present study, free radicals were measured by direct EPR spectroscopy of frozen tissue. It has been reported that oxygen-centered radicals may be artifactually generated during preparation of the sample for this type of assay (24, 73, 74). However, we have previously characterized this process and demonstrated that this problem can be minimized through appropriate sample handling (24). Mechanical processing of tissue samples was shown to result in the formation of alkyl radicals (R') through cleavage of covalent bonds. In the presence of oxygen, these R' radicals react to form ROO'. Thus, artifactual formation of oxygencentered signals can be minimized by reducing to a minimum the mechanical fracturing of the tissue and by rigorously maintaining the sample under anaerobic conditions throughout the procedure. By this approach we observed that the results obtained in intacthearts were identical to those achieved by EPR spectroscopy of cardiac specimens directly analyzed without tissue grinding, i.e. as obtained by needle biopsy or by high speed drill biopsy (24, 59). Furthermore, all features of reperfusion-induced oxygen radical generation, including time course, oxygen dependence, response to scavengers, role of iron, were consistently observed, in a similar fashion, when oxygen radicals were measured either by direct EPR spectroscopy of frozen tissue or by indirect spin-trap methodology (which does not require tissue processing) (1, 2, 4, 24, 75, 76). Thus, with proper careand controls, valid measurements of free radicals can be performed by direct EPR spectroscopy. In fact, this technique may offer some advantages over the spin-trap methodology when investigating the specific issues of the present study. On the one hand, perfusion of the heart with spin-traps does not allow rapid and complete cell permeation with the agent, and therefore oxygen radicals produced immediately upon reflowby an intracellular mechanism (such as mitochondria) might go undectected. On the other hand,only direct EPR spectroscopy of frozen tissuecandetect changes in the redox state of components of the mitochondrial respiratory chain that are not oxygen-centered, namely ubiquinone and Fe-S centers. Chemical measurements of tissue concentrations of MDA also have certain limitations. Artifactual increases in MDA content may bedue to chemical interference from other TBAreactive substances present in the sample or to subsequent auto-oxidation causing degradation of lipid peroxides during sample handling. These problems can be reduced by proper assay procedures (34, 35, 64, 77), and in fact MDA is widely utilized as a marker of lipid peroxidation both in uiuo and in perfused hearts (25, 38, 39, 78, 79). On theother hand, measurement of MDA content may underestimate the actual degree of lipid peroxidation, since MDA is formed only when lipids containing three or more double bonds are oxidized.

This latter phenomenon might explain why the protective effects of Amytal on MDA accumulation are of a smaller magnitude as compared to the inhibitory effects we observed on oxygen radical production. In conclusion, reversible inhibition by Amytal of electron transport at Site I during early reflow can reduce oxygen radical production, decrease membrane lipid peroxidation, and at the same time improve functional recovery of rabbit hearts subjected to ischemia and reperfusion. Taken together, these lines of evidence demonstrate that oxidative phosphorylation is asource of oxygen radicals in intact hearts and suggest that resumption of mitochondrial respiration upon postischemic reperfusion might be an important contributor to thepathogenesis of reperfusion injury. Acknowledgments-Weacknowledge the technicalassistance of Uriah Lee Shang, Koenraad Vandegaar, and Annalisa Scognamiglio. REFERENCES 1. Zweier, J. L., Flaherty, J. T., and Weisfeldt, M. L. (1987) Proc. Natl. Acad. Sci. U.S. A. 8 4 , 1404-1407 2. Garlick, P. B., Davies, M. J., Hearse, D. J, and Slater, T. F. (1987) Circ. Res. 61,757-760 3. Bolli, R., Patel, B. S., Jeroudi, M. O., Lai, E. K., and McCay, P. B. (1988) J. Clin. Inuest. 8 2 , 476-485 4. Ambrosio, G.. Zweier.. J. L.,. and Flahertv, J. T. (1991) J. Mol. Cell Cardiol. 23,1359-i374 5. Hess, M. L., and Manson, N. H. (1984) J. Mol. Cell CardwL 16,969-978 6. Becker, L. C., and Ambrosio, G. (1987) Prog. Cardwuasc. Dis. 30.23-41 7. McCord, J. M. (1985) N. Engl. J. Med. 312,159-163 8. Chambers, D. E., Parks, D. A., Patterson, G., Roy, R., McCord, J., Yoshida, S., Parmley, L. F., and Downey, J. M. (1985) J. MOL Cell. Cardwl. 17, 145-152 9. Fantone, J. C., and Ward, P. C. (1982) Am. J. Pathol. 107,394-418 10. Chance, B., Sies, H., and Boveris, A. (1979) Physiol. Reu. 69! 527-605 11. Turrens, J. F., and McCord, J. M. (1990) in ,Clinical Ischemrc Syndromes: Mechanisms and Consequencesof %sue In ury (Zelenock,G. B., D'Alecy, L. G., Fantone, J. C., 111, Shlafer, M., and Stanley, J. C., e&) pp. 203212, C. V. Mosby Co., St. Louis, MO 12. Loschen, G., Floh6, L., and Chance, B. (1971) FEBS Lett. 18,261-264 13. Boveris, A., and Chance, B. (1973) Biochem. J. 134,707-716 14. Lozhen, G., Azzi, A,, Richter, C., and Floh6, L. (1974) FEBS Lett. 4 2 , s IZ

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