Decrease in Mitochondrial Complex I Activity in Ischemic/Reperfused Rat HeartInvolvement of Reactive Oxygen Species and Cardiolipin

From the Department of Biochemistry and Molecular Biology and CNR Institute of Biomembranes and Bioenergetics (G. Paradies, G. Petrosillo, M.P., F.M.R.), Department of Emergency and Transplantation (N.D.V.), and Department of Pharmacology and Human Physiology (A.F.), University of Bari, Italy.

	Abstract

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Reactive oxygen species (ROS) are considered an important factor in ischemia/reperfusion injury to cardiac myocytes. Mitochondrial respiration is an important source of ROS production and hence a potential contributor to cardiac reperfusion injury. In this study, we have examined the effect of ischemia and ischemia followed by reperfusion of rat hearts on various parameters related to mitochondrial function, such as complex I activity, oxygen consumption, ROS production, and cardiolipin content. The activity of complex I was reduced by 25% and 48% in mitochondria isolated from ischemic and reperfused rat heart, respectively, compared with the controls. These changes in complex I activity were associated with parallel changes in state 3 respiration. The capacity of mitochondria to produce H₂O₂ increased on reperfusion. The mitochondrial content of cardiolipin, which is required for optimal activity of complex I, decreased by 28% and 50% as function of ischemia and reperfusion, respectively. The lower complex I activity in mitochondria from reperfused rat heart could be completely restored to the level of normal heart by exogenous added cardiolipin. This effect of cardiolipin could not be replaced by other phospholipids nor by peroxidized cardiolipin. It is proposed that the defect in complex I activity in ischemic/reperfused rat heart could be ascribed to a ROS-induced cardiolipin damage. These findings may provide an explanation for some of the factors responsible for myocardial reperfusion injury.

Key Words: complex I  cardiolipin  reactive oxygen species  mitochondria  ischemia/reperfusion

	Introduction

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It is generally acknowledged that reactive oxygen species (ROS) play an important role in producing lethal cell injury associated with cardiac ischemia and reperfusion.^1–3 Experimental evidence has shown that during the first minutes after cardiac postischemic reperfusion, a burst of ROS generation occurs.^4–6 Oxygen radicals can be generated by several mechanisms, including the xanthine/xanthine oxidase reaction^7–10 and the activity of NADPH oxidase.¹¹ Another potential source of oxygen radicals is thought to be the mitochondrial respiratory chain.¹² Because of the abundance of mitochondria in cardiac myocytes, mitochondrial electron transport might be an important subcellular source of ROS and hence a potential contributor to heart reperfusion injury.¹³

Peroxidation of membrane lipid components has been hypothesized to be a major mechanism of oxygen free radical toxicity, resulting in generalized impairment of membrane function. Cardiolipin, a phospholipid of unusual structure localized almost exclusively within the mitochondrial inner membrane, is particularly rich in unsaturated fatty acids (90% represented by linoleic acid). Thus, mitochondrial cardiolipin molecules are a possible and early target of oxygen-free radical attack, either because of their high content of unsaturated fatty acids or because of their location in the inner mitochondrial membrane, near the site of ROS production, mainly at the level of complex I¹⁴ and complex III¹⁵ of the mitochondrial respiratory chain. This phospholipid plays an important role in mitochondrial bioenergetics, optimizing the activity of key mitochondrial inner membrane proteins, including several anion carriers and some electron transport complexes.^16–18 It seems likely that mitochondrial oxidative stress may lead to cardiolipin modification and hence to loss of mitochondrial enzyme function. This condition is likely to be encountered on reperfusion of ischemic cardiac tissue. In this regard, recent results from this laboratory have demonstrated that mitochondrial-mediated ROS generation affects the activity of cytochrome c oxidase via oxidative damage of cardiolipin.¹⁹ These results have been useful to explain the molecular basis of the decline in the cytochrome c oxidase activity, observed in mitochondria isolated from animals in certain physiopathological conditions, such as different thyroid hormones status,^20,21 aging,^22–24 and ischemia/reperfusion,^25,26 which are characterized by an increase in the basal rate of ROS production.

Complex I, also known as NADH-ubiquinone oxidoreductase, is a multisubunit integral membrane complex of the mitochondrial electron transport chain that catalyzes electron transfer from NADH to ubiquinone. Results from different laboratories have shown that cardiolipin is specifically required for electron transfer in complex I of the mitochondrial respiratory chain.^27,28 In fact, the presence of this phospholipid has been shown to be an absolute necessity for regenerating enzyme activity from phospholipid-depleted preparation of complex I.²⁹ The involvement of cardiolipin in the function of complex I is also supported by the results of our recent study showing inactivation of this enzyme complex by micromolar concentrations of nonylacridine, a compound that interacts specifically with cardiolipin.³⁰

Complex I is considered an important site of superoxide anion generation in mitochondria.¹⁴ The formation of superoxide occurs via the transfer of a free electron to molecular oxygen. This reaction occurs at specific sites of the electron transport chain, which resides in the inner mitochondrial membrane. Complex I produces most of the superoxide, which is then scavenged by the mitochondrial enzyme Mn-superoxide dismutase to produce H₂O₂. Thus, a defect of complex I activity can be considered a potential source of ROS in heart ischemia/reperfusion.

Previous results from this and other laboratories have shown a decline in complexes III and IV activity in mitochondria isolated from ischemic and ischemic/reperfused rat heart.^25,26,31 This decline was attributed to a loss of mitochondrial cardiolipin, a phospholipid that is needed for full functioning of these enzyme complexes.^17,18 We recently reported that mitochondrial-mediated ROS production affects the complex I activity via cardiolipin peroxidation in beef-heart submitochondrial particles.³⁰ Thus, it is likely that oxidative damage to cardiolipin may affect the activity of mitochondrial complex I in ischemic and ischemic/reperfused rat heart as well. This possibility was explored in the present investigation. We have also evaluated other changes induced by heart ischemia/reperfusion related to mitochondrial function, such as oxygen consumption, mitochondrial ROS production rates, and cardiolipin content.

	Materials and Methods

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Preparation and Perfusion of Isolated Rat Heart
Male Wistar rats (250 to 300 g) were used throughout the studies. After intraperitoneal injection of heparin (1000 U/kg) and administration of thiopental (50 mg), hearts were removed and then placed in ice-cold Krebs-Henseleit buffer. The aorta was cannulated, and the heart was perfused in retrograde fashion according to Langendorff with Krebs-Henseleit buffer at 37°C, saturated with 95% O₂ and 5% CO₂. Hearts were placed in a water-jacketed chamber at 37°C, and perfusion pressure was maintained at 60 mm Hg. One group of hearts was subjected to 30 minutes of global ischemia at 37°C, and another group of hearts was subjected to 30 minutes of global ischemia followed by 15 minutes of reperfusion. In control perfusions, the ischemic period was replaced by an equal period of flow-through perfusion.

Isolation of Mitochondria
Rat heart mitochondria were isolated in a medium of 250 mmol/L sucrose, 10 mmol/L Tris-HCl, and 1 mmol/L EGTA, pH 7.4, by differential centrifugation of heart homogenates, essentially as described previously.³² Mitochondria were resuspended in 250 mmol/L sucrose and 10 mmol/L Tris-HCl (pH 7.4) and stored in ice. The yield of mitochondrial proteins (mg/g heart wet weight) within each group of animals was consistent, suggesting minimal variation in the preparations of the mitochondrial fraction. Mitochondrial protein concentration was measured by the biuret method using serum albumin as standard.

Determination of Mitochondrial H₂O₂ Production
The rate of mitochondrial hydrogen peroxide production was estimated by measuring the linear fluorescence increase induced by H₂O₂ oxidation of dichlorofluorescein to the fluorescent dichlorofluorescein in the presence of horseradish peroxidase.³³ Rat heart mitochondria (0.3 mg protein) were suspended in 2.5 mL of a medium of 100 mmol/L sucrose, 100 mmol/L KCl, and 5 mmol/L Tris, pH 7.4, supplemented with 7.5 µg horseradish peroxidase and 1 µmol/L dichlorofluorescein. The production of hydrogen peroxide was induced by addition of 5 mmol/L malate plus 2 mmol/L pyruvate as substrates (state 4). The amount of H₂O₂ produced was calculated by measuring the fluorescence changes on addition of known amounts of H₂O₂. Alternatively, mitochondrial H₂O₂ production was measured using 1 µmol/L scopoletin plus 1 µmol/L horseradish peroxidase at 365 nm for excitation and 450 nm for emission.³⁴

Mitochondrial Oxygen Consumption
Mitochondrial ADP-dependent state 3 respiration was measured polarographically with an oxygen electrode at 25°C. Respiration was initiated by the addition of 2 mmol/L pyruvate plus 5 mmol/L malate. After 2 minutes, state 3 respiration was induced by the addition of 0.5 mmol/L ADP.

Complex I Activity
The complex I (NADH-CoQ reductase) activity was measured in mitochondrial particles prepared by sonicating, under nitrogen atmosphere, 1 mg of rat heart mitochondria dissolved in 1 mL of 50 mmol/L phosphate buffer, pH 7.2. The assay mixture contained 3 mmol/L sodium azide, 1.2 µmol/L antimycin A, 50 µmol/L decylubiquinone, and 50 mmol/L phosphate buffer, pH 7.2. The mitochondrial sample (50 µg) was added to 3 mL of the assay mixture, and the reaction was started by the addition of 60 µmol/L NADH. The reaction was measured by following the decrease in absorbance of NADH at 340 nm with a diode-array spectrophotometer. The activity was calculated using an extinction coefficient of 6.22 mmol/L^-1xcm^-1 for NADH. The specific activity of the enzyme is expressed as nanomole of NADH oxidized per minute per milligram of SMP.

Analysis of Cardiolipin in Mitochondrial Membranes
Cardiolipin was analyzed by high-pressure liquid chromatography (HPLC) using a Hewlett Packard series 1100 gradient liquid chromatograph. Lipids from heart mitochondria were extracted with chloroform/methanol by the procedure of Bligh and Dyer.³⁵ Lipid extraction was carried out on ice immediately after the preparation of mitochondria in the presence of BHT and under nitrogen atmosphere. Phospholipids were separated by the HPLC method previously described³⁶ with an Lichrosorb Si60 column (4.6x250 mm). The chromatographic system was programmed for gradient elution using two mobile phases: solvent A, hexane/2-propanol (6:8, vol/vol), and solvent B, hexane/2-propanol/water (6:8:1.4, vol/vol/vol). The percentage of solvent B in solvent A was increased in 15 minutes from 0% to 100%. Flow rate was 1 mL/min, and detection was at 206 nm. The peak of cardiolipin was identified by comparison with the retention time of standard cardiolipin and rechromatographed by thin-layer chromatography.

Preparation and Analysis of Peroxidized Cardiolipin
Bovine heart cardiolipin was autoxidized overnight in a thin film at 37°C.³⁷ Peroxidized cardiolipin was identified by normal-phase HPLC, as described above, with UV detection at 235 nm, indicative of conjugated dienes.^37,38 The resulting peak was rechromatographed by thin-layer chromatography and used as standard.

Preparation of Liposomes
Liposomes (small unilamellar vesicles) were prepared by sonicating 1.7 mg of phospholipids in 1 mL of incubation medium of 25 mmol/L phosphate buffer (pH 6.7) with the microtip probe of a Branson sonifier (model 250) at 40 W for six cycles of 2.5 minutes in an ice bath under N₂ stream.

Preparation of Peroxidized Liposomes
Liposomes were peroxidized using the Fe²⁺-ADP ascorbic acid method. Briefly, aliquots of liposomes (1.7 mg of phospholipids) were dispersed in 1 mL of oxygenated buffer containing 20 µmol/L Fe²⁺ and 120 µmol/L ADP. The peroxidation reaction was initiated by adding 200 µmol/L ascorbic acid. Incubation was carried out at 37°C in a shaking water bath for 6 minutes. The extent of lipid peroxidation was monitored by conjugated dienes formation, as described by Buege and Aust.³⁹

Fusion of Liposomes With Mitochondria
The liposomes-mitochondrial membrane fusion was carried out essentially as described by Hackenbrock and Chazotte,⁴⁰ with some modifications.⁴¹ Briefly, 1 mL of freshly sonicated liposomes in phosphate buffer pH 6.7 was added to 1 mg of mitochondria at 30°C with constant stirring. After 40 minutes of incubation, phospholipid-enriched mitochondria were centrifuged at 10 000g for 20 minutes to remove the phospholipid excess. The mitochondrial pellet was then washed and resuspended in 250 mmol/L sucrose and 10 mmol/L Tris, pH 7.4.

Statistical Analysis
Results are expressed as mean±SE. Statistical significances were determined by the Student’s t test.

	Results

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The activity of complex I was measured in mitochondria isolated from control, ischemic, and ischemic/reperfused rat heart (). In ischemic heart, mitochondrial complex I activity was decreased by 25% compared with the normal heart. In ischemic/reperfused heart, the decrease of complex I activity was much more pronounced, reaching 48% compared with control heart.

fig.ommitted   Figure 1. Activity of complex I in mitochondria isolated from control, ischemic, and ischemic/reperfused rat heart. The complex I activity was measured as described in Materials and Methods. Each value represents the mean±SE of 4 separate experiments. *P<0.05 vs control.

Respiratory activities of mitochondria isolated from control, ischemic, and reperfused rat heart, measured in presence of pyruvate and malate as substrates and ADP to stimulate respiration (state 3), are reported in . Mitochondria from ischemic heart exhibited a 24% and those from reperfused heart a 48% reduction in the rate of state 3 respiration compared with the control value.

fig.ommitted   Figure 2. State 3 respiration in mitochondria isolated from control, ischemic, and reperfused rat heart. Mitochondrial respiratory activity was measured as described in Materials and Methods. Each value represents the mean±SE of 4 separate experiments. *P<0.05 vs control.

The site of the ischemia and ischemia/reperfusion damage to complex I was approached in subsequent experiments. As mentioned above, cardiolipin is specifically required for full activity of complex I. Thus, it is possible that ROS-induced oxidative damage to mitochondrial cardiolipin may be responsible for the observed defect in complex I activity in ischemia and ischemia/reperfusion rat heart. The content of cardiolipin was determined in mitochondria isolated from normal, ischemic, and ischemic/reperfused rat hearts by a very sensitive HPLC technique set up in our laboratory.³⁶ As shown in , the content of cardiolipin decreased by 28% and 50% in mitochondria from ischemic and ischemic/reperfused rat hearts, respectively, compared with the values obtained with mitochondria isolated from control hearts.

fig.ommitted Figure 3. Cardiolipin content in mitochondria isolated from control, ischemic, and ischemic/reperfused rat heart. Mitochondrial cardiolipin content was determined by the HPLC technique as described in Materials and Methods. Each value represents the mean±SE obtained from 4 different experiments. *P<0.05, **P<0.01 vs control.

To asses that the decrease in the cardiolipin content observed in ischemic/reperfused rat heart could be attributable to cardiolipin peroxidation because of the ROS attack to its double bonds, the content of peroxidized cardiolipin was measured in mitochondria by an HPLC method based on the absorbance at 235 nm, indicative of the formation of conjugated dienes.^37,38 As shown in , an increase in the content of peroxidized cardiolipin was observed in mitochondria from ischemic rat heart, which was more pronounced on reperfusion compared with control heart.

fig.ommitted   Figure 4. Relative content of peroxidized cardiolipin in mitochondria from ischemic and ischemic/reperfused rat heart. Mitochondrial content of peroxidized cardiolipin was determined by the HPLC technique described in Materials and Methods. The content of peroxidized cardiolipin is expressed as peak area (at 235 nm) per milligram of phospholipids, and the peak area of the control is assumed as a unit. Each value represents the mean±SE obtained from 4 different experiments. *P<0.05 vs control.

The changes in the cardiolipin content observed in mitochondria from ischemic and reperfused rat heart paralleled the changes in the complex I activity, thus suggesting a possible involvement of cardiolipin in this effect. To demonstrate this more directly, we investigated whether addition of exogenous cardiolipin to mitochondria from reperfused heart was able to reverse the observed defect of complex I activity. Because cardiolipin is poorly permeable to mitochondrial membrane, a previously reported method of fusion of vesicular lipids with mitochondrial membrane was used to enrich the inner mitochondrial membrane with cardiolipin.^40,41 We have recently shown that fusion of mitochondria from reperfused rat heart, which contains a lower level of cardiolipin, with phosphatidylcholine/cardiolipin (PC/CL) liposomes resulted in a significant enrichment in the mitochondrial cardiolipin content.³¹ Using this procedure, we studied the effect of fusion of mitochondria from control and ischemic/reperfused heart with liposomes composed of various phospholipids PC, PC/CL, and PC/phosphatidylethanolamine (PE) on the activity of complex I. The results of these experiments are reported in . As shown above, mitochondria from reperfused rat heart exhibited a 48% decline in complex I activity compared with control rats (see also ). This lower activity of complex I was almost completely restored to the level of control rats after fusion of mitochondria from reperfused rat heart with liposomes containing cardiolipin, whereas PC or PC/PE liposomes could not replace cardiolipin in this effect. Interestingly, no restoration was obtained with liposomes containing peroxidized CL, thus suggesting that integral molecules of cardiolipin are required for the reactivation of complex I. Fusion of mitochondria from control rats with PC/CL liposomes had practically no effect on the complex I activity, indicating that the liposome-mitochondrial membrane procedure used in our experiments did not affect the activity of this enzyme complex in control mitochondria ().

fig.ommitted   Figure 5. Decreased complex I activity in mitochondria isolated from ischemic/reperfused rat heart and specific reactivation by cardiolipin liposomes. The fusion of mitochondria with liposomes composed of various phospholipids was carried out as described in Materials and Methods. PC/CL liposomes (4:1 molar ratio) and PC/PE liposomes (1:1 molar ratio). Control and ischemic/reperfused mitochondria were treated in the same manner as the liposome-treated mitochondria but in the absence of liposomes. Each value represents the mean±SE of 5 separate experiments. *P<0.05 vs control; **P<0.05 vs ischemic/reperfused heart.

It has been shown that addition of pyruvate plus malate to aerobic mitochondria causes detectable H₂O₂ production, which arises from superoxide anion formed at the level of complex I.^14,42 Mitochondria from control, ischemic, and reperfused rat heart were investigated for their capacity to generate oxygen radicals in the presence of pyruvate plus malate. As shown in , the basal rate of H₂O₂ production was enhanced by 14% and 32% in mitochondria isolated from ischemic and ischemic/reperfused heart, respectively, compared with control heart. Similar results were obtained using glutamate plus malate as substrates (results not shown).

fig.ommitted Figure 6. H2O2 production in mitochondria isolated from control, ischemic, and ischemic/reperfused rat heart. The H2O2 formation was induced by the addition of 2 mmol/L pyruvate plus 5 mmol/L malate and measured as described in Materials and Methods. Each value represents the mean±SE of 4 different experiments. *P<0.05 vs control.

	Discussion

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ROS have been implicated in the cardiac tissue injury that follows ischemia and reperfusion. The mechanisms for the enhanced ROS generation and the cellular and subcellular targets of ROS attack are not well established. Mitochondria consume >90% of the oxygen used by the cell, and the mitochondrial respiratory chain generates a continuous flux of oxygen radicals. It has been estimated that 2% of the oxygen reacting with the respiratory chain leads to formation of superoxide radical. Subsequent dismutation of superoxide anion generates H₂O₂, which in turn can lead to production of OH^·. Ischemia may cause mitochondrial alterations that would favor oxygen radical production when the oxygen concentration is reestablished by reperfusion and oxidative phosphorylation resumes. In this respect, in vitro studies have demonstrated that mitochondrial production of oxygen radicals mostly occurs during state 4 respiration, because oxygen concentration increases and the level of reduced one-electron donors in the respiratory chain is concomitantly increased due to a decreased availability of ADP.¹² A similar situation is likely to be encountered during ischemia. When ischemic tissue is reoxygenated, electron transport through the respiratory chain is impaired because of depletion of ADP during ischemia, and this leads to a burst of ROS generation during the first minutes of reoxygenation.^4–6,13

It is conceivable that mitochondrial-mediated ROS generation leads to primary reaction and damage in the immediate area surrounding where these ROS are produced, given that they are a highly reactive and short-lived species. Therefore, as major sources of ROS production, mitochondria also could be major targets of ROS attack. The effects of ROS should be greatest at the level of mitochondrial membrane constituents, including the complexes of the respiratory chain and phospholipid constituents particularly rich in unsaturated fatty acids, such as cardiolipin. Previous studies from this and other laboratories have shown that the activity of complexes III and IV is reduced in mitochondria from ischemic and ischemic/reperfused rat hearts.^25,26,31 In the present study, we demonstrate that the activity of complex I is reduced by 25% in mitochondria from ischemic heart and by 48% in those from ischemic/reperfused heart compared with control heart. A defect in complex I activity in mitochondria isolated from hypoxic/reoxygenated rat heart has been already reported.⁴³ This defect has been explained on the basis of an increase in the Ca²⁺ uptake during reoxygenation by unknown mechanism. Interestingly, Ca²⁺ overload has been shown to stimulate ROS production.⁴⁴

Complex I is considered an important factor in the regulation of mitochondrial respiration. A decrease in the mitochondrial complex I activity, as observed in mitochondria from ischemic and ischemic/reperfused rat heart, should be associated with a decline in mitochondrial respiration. The results reported  clearly demonstrate that mitochondria from ischemic and reperfused rat heart exhibit lower rate of state 3 respiration compared with control heart. These changes in state 3 respiration are quantitatively related to changes in complex I activity, thus suggesting that the lowered complex I activity is probably the most important and rate-determining step responsible for the alteration to the mitochondrial oxidative metabolism in ischemic and reperfused rat heart.

Complex I is considered an important source of oxygen radicals in mitochondria,^45–48 although the mechanism responsible for this production is not well established. The lower activity of complex I can account for the enhanced production of H₂O₂ observed in mitochondria isolated from ischemic and ischemic/reperfused rat heart, supplemented with pyruvate plus malate . This finding is consistent with the results reported by others showing that mitochondrial electron transport complex I is a potential source of oxygen free radical in intact heart subjected to ischemia and reflow¹³ during ischemia⁴⁹ and in the failing myocardium.⁵⁰

Cardiolipin has a particularly important function in mitochondrial bioenergetics in that it interacts with several major inner membrane proteins, including anion carriers and complexes of the respiratory chain,^16–18 even if its precise mechanism of action is still not well understood. It has been reported that cardiolipin is specifically required for electron transfer in complex I of the mitochondrial respiratory chain.^27–29 Additional evidence for the cardiolipin involvement in the complex I functioning comes from our recent finding showing that nonylacridine orange, a compound that interacts specifically with cardiolipin,⁵¹ inactivates the complex I activity in SMP and that added cardiolipin fully prevented this inactivation.³⁰

The content of cardiolipin in the inner mitochondrial membrane may change either as a consequence of an alteration of one of the enzymatic steps involved in its biosynthetic process^52,53 or as a consequence of oxidative damage by ROS. In fact, cardiolipin, because of its high content of unsaturated fatty acids (90% represented by linoleic acid) and because of its location in the mitochondrial membrane near the site of oxygen radical production (mainly at the level of complexes I and III), is particularly susceptible to peroxidative attack by oxyradicals. This situation is likely to occur on reperfusion of ischemic heart, characterized by an increased production of ROS. Accordingly, our results demonstrate a pronounced loss in the cardiolipin content in mitochondria isolated from ischemic/reperfused rat heart, associated with an increase in peroxidized cardiolipin content () compared with control hearts.

The changes in complex I activity, observed in mitochondria from ischemic/reperfused rat heart, are quantitatively related to changes in the mitochondrial cardiolipin content. On this basis, it is reasonable to assume that the molecular basis for the ischemia/reperfusion defect in complex I activity can be mainly ascribed to a loss in mitochondrial content of cardiolipin, which is required for the optimal function of this enzyme complex. This assumption is also supported by the results of our recent study showing a close correlation between loss of cardiolipin content in submitochondrial particles and loss in complex I activity.³⁰

More firm evidence for the involvement of cardiolipin in the loss of complex I activity, observed in mitochondria isolated from ischemic/reperfused hearts, comes from the results of the experiments reported in . These results demonstrate that exogenously added cardiolipin to mitochondria from reperfused rat heart resulted in almost complete restoration of the normal activity of this enzyme complex. This effect of cardiolipin could not be replaced by other phospholipids, such as PC and PE, nor by peroxidized cardiolipin. These results clearly indicate that the defect in the complex I activity in mitochondria from reperfused heart could be mainly ascribed to a loss of cardiolipin content, attributable to ROS attack to double bonds of its fatty acid constituents.

In addition to the defect in mitochondrial complex I activity (present study), we have recently reported a defect in mitochondrial complex III and IV in ischemic/reperfused rat heart, similarly attributable to ROS-induced cardiolipin damage.^25,31 Thus, a common mechanism seems to be involved in the defect of these three mitochondrial respiratory complex activities in ischemia/reperfusion. These data appear particularly interesting in light of recent findings in the literature indicating the association of complexes I, III, and IV in a supercomplex in the mitochondrial inner membrane. Cardiolipin seems to play a central role in higher-order organization of these components of the respiratory chain.⁵⁴

Complex I exhibits a lower activity compared with the other respiratory complexes; therefore, it is considered an important factor in the regulation of oxidative phosphorylation. This enzyme complex is also considered the main site of oxygen radical production in mitochondria. Thus, the impairment of mitochondrial complex I activity, in addition to that of complexes III and IV previously reported,^25,31 attributable to ROS-induced cardiolipin damage may increase the electron leak from the electron transport chain, generating more superoxide radical and perpetuating a cycle of oxygen-radical–induced damage, which ultimately leads to a decrease in oxidative phosphorylation and to heart failure on reperfusion (see ). The pattern of results presented here may prove useful in elucidating the molecular mechanism of ROS-induced alterations to mitochondrial structure and function, which could be responsible for contractile defect of ischemic heart on reperfusion and in the development of appropriate treatment strategies.

fig.ommitted Figure 7. Schematic diagram of the role of ROS and cardiolipin in mitochondrial dysfunction associated with heart ischemia/reperfusion.

　

	Acknowledgments

 
This study was supported by a grant from the National Research Project PRIN "Bioenergetics and Membrane Transport," MURST, Italy, 2001–2003. We thank Annamaria Papagni for expert technical assistance.

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