Reversible redox-dependent modulation of mitochondrial aconitase and proteolytic activity during in vivo cardiac ischemia͞reperfusion Anne-Laure Bulteau*†, Kathleen C. Lundberg*, Masao Ikeda-Saito‡, Grazia Isaya§, and Luke I. Szweda*¶ *Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106-4970; ‡Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira, Aoba-ku, Sendai 980-8577, Japan; and §Departments of Pediatric and Adolescent Medicine and Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905 Communicated by E. R. Stadtman, National Institutes of Health, Bethesda, MD, February 23, 2005 (received for review June 23, 2004) Prooxidents can induce reversible inhibition or irreversible inacti- derangements in cardiac function (7–9). Nevertheless, reper- vation and degradation of the mitochondrial enzyme aconitase. fusion is associated with functional alterations and, in many Cardiac ischemia͞reperfusion is associated with an increase in cases, further loss in myocardial function (7–9). It has long mitochondrial free radical production. In the current study, the been recognized that reoxygenation of myocardial tissue re- effects of reperfusion-induced production of prooxidants on mi- sults in an increase in the production of oxygen-based free tochondrial aconitase and proteolytic activity were determined to radical species (7–9). Based on their reactive properties, these assess whether alterations represented a regulated response to species have been hypothesized to play a role in the loss of changes in redox status or oxidative damage. Evidence is provided cardiac function during reperfusion. Mitochondria, a major that ATP-dependent proteolytic activity increased during early subcellular source of oxygen radicals during reperfusion, reperfusion followed by a time-dependent reduction in activity to exhibit decrements in activity attributed, in part, to oxidative control levels. These alterations in proteolytic activity paralleled an modifications to key metabolic enzymes (10). Aconitase and increase and subsequent decrease in the level of oxidatively ␣-ketoglutarate dehydrogenase, which are known to be sus- modified protein. In vitro data supports a role for prooxidants in ceptible to oxidative inactivation in vitro (11–19) have been the activation of ATP-dependent proteolytic activity. Despite inhi- reported to decline in activity during in vivo cardiac ischemia͞ bition during early periods of reperfusion, aconitase was not reperfusion (20). degraded under the conditions of these experiments. Aconitase Loss in aconitase activity is commonly used as a biomarker of activity exhibited a decline in activity followed by reactivation oxidative damage due to the susceptibility of the enzyme’s during cardiac reperfusion. Loss and regain in activity involved [4Fe-4S]2ϩ cubane cluster to oxidative disassembly (16–19). reversible sulfhydryl modification. Aconitase was found to asso- Nevertheless, we have recently shown that, when mitochondria ciate with the iron binding protein frataxin exclusively during are exposed to H2O2 in vitro, aconitase can undergo oxidative reperfusion. In vitro, frataxin has been shown to protect aconitase inhibition followed by reactivation upon resolution of the oxi- ؉ from [4Fe-4S]2 cluster disassembly, irreversible inactivation, and, dative stress (15). If the oxidative stress is prolonged or of potentially, degradation. Thus, the response of mitochondrial sufficient magnitude, reversible modulation of aconitase activity aconitase and ATP-dependent proteolytic activity to reperfusion- progresses to irreversible inactivation and ATP-dependent deg- induced prooxidant production appears to be a regulated event radation (15). Thus aconitase is capable of participating in redox that would be expected to reduce irreparable damage to the regulation and is itself a potential target of oxidative damage. mitochondria. The metabolic fate of aconitase depends on (i) the relative rates of mitochondrial free radical production and removal, (ii) the ighly reactive oxygen derived free radicals, such as super- ability of components to reverse redox-dependent modifications; •Ϫ Hoxide anion (O2 ) and the prooxidant hydrogen peroxide and (iii) mitochondrial capacity for protein degradation. In the (H2O2), can interact with a variety of cellular components, present study, we investigated the effects of in vivo cardiac altering both structure and function (1). Although evidence for ischemia and varying durations of reperfusion on mitochondrial these reactions has long been sought as indication of free proteolytic capacity and the level and activity of aconitase to radical involvement in degenerative disorders, recent evidence assess the progression of and relationships between specific indicates that these processes also participate in the regulation oxidative events. We present evidence that mitochondrial acon- of cellular function (1, 2). This finding is exemplified by the itase and proteolytic activities are reversibly altered during BIOCHEMISTRY discovery of enzymatic systems, such as thioredoxin reduc- cardiac reperfusion and discuss the potential regulatory role tase͞thioredoxin (3), glutaredoxin (4), methione sulfoxide these processes play in the mitochondrial response to oxidative reductase (5), and sulfiredoxin (6), which catalyze reversal of stress. oxidative modifications to protein and restoration of protein function. Additionally, receptor- and enzyme-mediated sys- Materials and Methods tems exist that catalyze the production of free radicals in In Vivo Model of Coronary Occlusion͞Reperfusion and Isolation of response to changes in extracellular and intracellular factors Cardiac Mitochondria. As described ref. 20, Sprague–Dawley rats (1, 2). It is therefore important that free radicals produced were anesthetized and ventilated, and, after midline thoracot- during physiological and pathophysiological conditions be omy and pericardiectomy, a ligature was placed around the left investigated not simply for their potential to carry out dam- aging processes but also to induce appropriate alterations in response to changes in cellular homeostasis. †Present address: Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement, EA ͞ 3106͞IFR 117, Universite´Paris 7–Denis Diderot, Couloir 23-33, 1er E´ tage, 2, Place Jussieu, Reduction and or cessation of blood flow to myocardial 75251 Paris Cedex 05, France. tissue, termed ischemia, occurs primarily as a result of forma- ¶To whom correspondence should be sent at the present address: Free Radical Biology and tion of atherosclerotic lesions in the coronary arteries. Reso- Aging Research Program, Oklahoma Medical Research Foundation, 825 Northeast 13th lution of the ischemic event by reintroduction of blood flow is Street, Oklahoma City, OK 73104. E-mail: [email protected]. the sole means for preventing cell death and irreparable © 2005 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0501519102 PNAS ͉ April 26, 2005 ͉ vol. 102 ͉ no. 17 ͉ 5987–5991 Downloaded by guest on September 29, 2021 anterior descending coronary artery (LAD) close to its origin con). Western blot analyses were performed by using 5.0 ␮gof to induce coronary occlusion. Reflow was initiated by releas- mitochondrial protein per lane and primary antibody against ing the ligature. For each experimental condition tested, five dinitrophenylhydrazone. rats were used. Experiments consisted of 0–90 min of sham- operated perfusion; 30 min of LAD occlusion; and 30 min of EPR Spectroscopy. Mitochondria isolated after each experimen- LAD occlusion, followed by 5, 15, 30, or 60 min of reperfusion. tal protocol were placed in quartz EPR tubes (30 mg͞ml) and After each experimental protocol, hearts were removed and frozen by immersion in liquid nitrogen. EPR spectra were immediately rinsed in ice-cold homogenization buffer (210 recorded by using a Bruker ESP300 spectrometer operating at mM mannitol͞70 mM sucrose͞1.0 mM EDTA͞5.0 mM Mops, 9.45 GHz with a 10-G field modulation at 100 kHz. Measure- pH 7.4), and mitochondria were isolated by differential cen- ments were carried out at 10 K by use of an Oxford liquid He trifugation as described in ref. 15. flow cryostat. The microwave frequency was monitored by a frequency counter (model HP-5350, Hewlett–Packard) and Treatment of Isolated Mitochondria with a H2O2 Generating System. the magnetic field strength was determined by an NMR Isolated control mitochondria were diluted to 0.25 mg͞ml in gaussmeter (model ER-035M, Bruker, Billerica, MA). Each 125 mM KCl͞5.0 mM KH2PO4, pH 7.25, and incubated with a spectrum represents an average of 10 scans. H2O2 generating system for 30 min. As described in ref. 15, glucose͞glucose oxidase was used to generate a steady-state Results level of 100 ␮MH2O2. Reperfusion-Induced Alterations in Mitochondrial Oxidatively Modi- fied Protein and Protease Activity. Reperfusion is associated with Assay of Aconitase. As described in ref. 15, mitochondria were a rapid and transient increase in the production of oxygen diluted to 0.05 mg͞ml in 25 mM KH2PO4, pH 7.25, containing radicals (7–9). By using dinitrophenylhydrazine-reactive car- 0.05% Triton X-100. Aconitase activity was assayed as the rate bonyl groups as an index, evidence for oxidative modification of NADPϩ reduction (340 nm) by isocitrate dehydrogenase to mitochondrial proteins was sought. As shown in Fig. 1A, the upon addition of 1.0