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(The FASEB Journal. 2006;20:269-276.)
© 2006 FASEB

Protective effect of melatonin against mitochondrial dysfunction associated with cardiac ischemia- reperfusion: role of cardiolipin

G. Petrosillo*, N. Di Venosa{dagger}, M. Pistolese*, G. Casanova*, E. Tiravanti{dagger}, G. Colantuono{dagger}, A. Federici{ddagger}, G. Paradies*,1 and F. M. Ruggiero*

* Department of Biochemistry and Molecular Biology and CNR Institute of Biomembranes and Bioenergetics,
{dagger} Department of Emergency and Transplantation, and
{ddagger} Department of Pharmacology and Human Physiology University of Bari, Bari Italy

1 Correspondence: E-mail: g.paradies{at}biologia.uniba.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reactive oxygen species (ROS) are considered an important factor in ischemia/reperfusion injury to cardiac myocytes. Mitochondrial respiration, mainly at the level of complex I and III, is an important source of ROS generation and hence a potential contributor of cardiac reperfusion injury. Appropriate antioxidant strategies could be particularly useful to limit this ROS generation and associated mitochondrial dysfunction. Melatonin has been shown to effectively protect against ischemic-reperfusion myocardial damage. The mechanism by which melatonin exerts this cardioprotective effect is not well established. In the present study we examined the effects of melatonin on various parameters of mitochondrial bioenergetics in a Langerdoff isolated perfused rat heart model. After isolation of mitochondria from control, ischemic-reperfused and melatonin-treated ischemic-reperfused rat heart, various bioenergetic parameters were evaluated such as rates of mitochondrial oxygen consumption, complex I and complex III activity, H2O2 production as well as the degree of lipid peroxidation, cardiolipin content, and cardiolipin oxidation. We found that reperfusion significantly altered all these mitochondrial parameters, while melatonin treatment had strong protective effect attenuating these alterations. This effect appears to be due, at least in part, to the preservation, by ROS attack, of the content and integrity of cardiolipin molecules which play a pivotal role in mitochondrial bioenergetics. Protection of mitochondrial dysfunction was associated with an improvement of post-ischemic hemodynamic function of the heart. Melatonin had also strong protective effect against oxidative alterations to complex I and III as well as to cardiolipin in isolated mitochondria.—Petrosillo, G., Di Venosa, N., Pistolese, M., Casanova, G., Tiravanti, E., Colantuono, G., Federici, A., Paradies, G., Ruggiero, F. M. Protective effect of melatonin against mitochondrial dysfunction associated with cardiac ischemia-reperfusion: role of cardiolipin.


Key Words: ROS • mitochondria • MPT


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THERE IS AMPLE EVIDENCE demonstrating that reactive oxygen species (ROS) play an important role in producing lethal cell injury associated with cardiac ischemia/reperfusion (1 2 3 4 5 6) . The mechanism for the enhanced ROS generation and cellular and subcellular target of ROS attack are not well established. One of the main sources of reactive oxygen species in cardiomyocytes during ischemia and early reperfusion may be a decrease in the electron transport chain in mitochondria, particularly at the level of complex I and III (7 8 9 10) . A major source of ROS production, mitochondria could be major targets of ROS attack. Given that ROS are a highly reactive and short-lived species, their effect should be greatest in an immediate area surrounding their locus of production. It is conceivable that mitochondrial membrane constituents, including the complexes of the respiratory chain and phospholipid constituents, could be the major target of ROS attack.

Cardiolipin, a phospholipid localized almost exclusively within the mitochondrial inner membrane, is particularly rich in unsaturated fatty acids. Thus, mitochondrial cardiolipin molecules are a possible early target of ROS attack, either because of their high content of unsaturated fatty acids or because of their location in the inner mitochondrial membrane near to the site of ROS production, mainly at the level of complex I and III of the respiratory chain (9 , 10) . This phospholipid plays a pivotal role in mitochondrial bioenergetics, optimizing the activity of the respiratory chain complexes as well as of anion carrier proteins (11 12 13) . More recently an involvement of cardiolipin in the execution phase of the apoptotic process has been suggested (14 15 16) .

Previous studies from this and other laboratories have shown that the activity of the respiratory chain complexes is reduced in mitochondria from ischemic/reperfused rat heart (17 18 19 20 21) . This decrease has been ascribed to ROS-induced cardiolipin oxidation. The impairment of mitochondrial complexes I, III, IV activity, due to ROS-induced cardiolipin damage may increase the electron leak from the electron transport chain, generating more superoxide anion 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.

It has been reported that the production of free radicals occurs during the first minutes after cardiac reperfusion (4 , 5 , 22) . Appropriate antioxidant strategies could be particularly useful to limit ROS production and ROS-induced alterations to mitochondrial structure and function and hence to protect ischemic reperfused myocardium. Thus, there is continued interest in defining and discovering new antioxidants or free radical scavengers of high potency, low toxicity, and easy permeability to cellular and subcellular compartments (23) . Melatonin, a hormonal product of the pineal gland, appears to fulfill most of these criteria. This compound has been shown to directly scavenge free radicals such as peroxynitrites, hydroxyl and peroxyl radicals (for review, see ref 24 ), while its lipophilic nature allows its rapid access to cellular and subcellular membranes compartments. Melatonin has been found to be effective in protecting against pathological states characterized by an increase in basal rate of ROS production (25) . Melatonin has been shown to effectively protect against ischemic-reperfusion myocardial damage (26 27 28 29 30 31) , although the mechanism by which this compound exerts the cardioprotective effect is not well established. Recent data suggest that some of the cell protective effect of melatonin may be produced through its action at the mitochondrial level.

In view of the above, we examined the hypothesis that the alterations to mitochondrial oxidative metabolism associated with ischemia-reperfusion may be prevented or attenuated by melatonin treatment of reperfused rat heart. For this purpose, we studied the effects of melatonin treatment of reperfused rat heart on various mitochondrial bioenergetic parameters such as oxygen consumption, respiratory chain complexes activity, mitochondrial ROS production rates. We measured mitochondrial lipid peroxidation, an indicator for oxidative stress and ROS production as well as the cardiolipin content and its degree of oxidation. Since ROS and mitochondrial damage play a role in contractile dysfunction associated with reperfusion of ischemic heart, we also tested the efficacy of melatonin to limit the impairment of ventricular mechanics in a reperfused heart model.

This study showed that the alterations to mitochondrial bioenergetic parameters associated with ischemia-reperfusion are significantly reduced by melatonin treatment. This effect appears to be due, at least in part, to the preservation of cardiolipin content and integrity. Protection of mitochondrial dysfunction was associated to an improvement of post-ischemic hemodynamic function of the heart.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal experiments
Male Wistar rats (250–300 g) were used throughout these studies. After intraperitoneally injection of heparin (1000 UI/Kg) and administration of thiopental (50 mg), hearts were removed, then placed in ice-cold Krebs-Henseleit buffer. The aorta was cannulated and the heart was perfused in retrograde fashion according to Langerdoff constant flow model (10 mL/min) with Krebs-Henseleit buffer, at 37°C, saturated with 95% O2/5% CO2. Hearts were placed in a water-jacketed chamber 37°C.

Fifteen hearts were used; five hearts were perfused for 60 min, at 37°C with a Krebs-Henseleit solution and used as control group. The other five hearts were perfused for 15 min, then subjected to global ischemia for 30 min followed by 15 min of reperfusion. The last five hearts were perfused for 15 min with a Krebs-Henseliet solution in the presence of 50 µM melatonin, then subjected to global ischemia for 30 min, followed by 15 min of reperfusion with Krebs-Henseleit solution in the presence of 50 µM melatonin.

Left ventricular isovolumic pressure was recorded by a strain-gauge pressure transducer (Hewlett-Packard Medical Electronic Division, Model 1280C, Waltham, MA, USA). The end-diastolic pressure (LVEDp) was adjusted to 5–10 mmHg.

Left ventricular pressure parameters were recorded just before ischemia and at the end of reperfusion. The following parameters were measured: left ventricular end-diastolic pressure LVEDp and developed pressure LVDp.

Isolation of mitochondria
Rat heart mitochondria were isolated in a medium of 250 mM sucrose, 10 mM Tris-HCl, 1 mM EGTA, pH 7.4, by differential centrifugation of heart homogenates essentially as described previously (20) . Mitochondria were resuspended in 250 mM sucrose, 10 mM Tris-HCl (pH 7.4) and stored in ice. The yield of mitochondrial proteins (mg/g heart wet wt) 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 dichlorofluorescin to the fluorescent dichlorofluorescein in the presence of horseradish peroxidase (32) . Rat heart mitochondria (0.5 mg protein) were suspended in 3 mL of a medium of 100 mM sucrose, 100 mM KCl, 5 mM Tris, pH 7.4, supplemented with 7.5 µg horseradish peroxidase and 1 µM dichlorofluoresein. The production of hydrogen peroxide was induced by addition of 5 mM malate + 2 mM pyruvate or 5 mM succinate as substrates (state 4). The amount of H2O2 produced was calculated by measuring the fluorescence changes upon addition of known amounts of H2O2.

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 mM pyruvate + 5 mM malate or 5 mM succinate. After 2 min. state 3 respiration was induced by the addition of 0.5 mM 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 mM phosphate buffer pH 7.2. The assay mixture contained 3 mM sodium azide, 1.2 µM antimycin A, 50 µM decylubiquinone and 50 mM 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 µM NADH. The reaction was measured by following the rotenone-sensitive decrease in absorbance of NADH at 340 nm with a diode array spectrophotometer. The activity was calculated using an extinction-coefficient of 6.22 mM–1 x cm–1 for NADH. The specific activity of the enzyme is expressed as nmol of NADH oxidized/min/mg of mitochondrial protein.

Complex III activity
The complex III activity (decylubiquinol/ferricytochrome c oxidoreductase) was measured in mitochondrial particles prepared by sonicating 1 mg of rat heart mitochondria dissolved in 1 mL of 50 mM phosphate buffer pH 7.2. The assay mixture contained 3 mM sodium azide, 1.5 µM rotenone, 50 µM ferricytochrome c, and 50 mM phosphate buffer pH 7.2. The sample (10 µg) was added to 3 mL of the assay mixture and the reaction was started by the addition of 30 µM of decylubiquinol. The reaction was measured with a diode array spectrophotometer by following the increase in reduced cytochrome c absorbance at 550-540 nm. The activity was calculated using an extinction-coefficient of 19.1 mM–1x cm–1.

The specific activity of the enzyme is expressed as nanomoles of cytochrome c reduced/min/mg of mitochondrial particles.

Decylubiquinol was synthesized by reduction of decylubiqionone (10 µM) with NaBH4 in 2 mL of 1:1 ethano/H2O mixture (v/v, pH 2). The ubiquinol formed was extracted twice with 1 mL of diethylether/isooctane 2:1 (v/v). The combined organic phases were washed with 2 mL of 2 M NaCl and evaporated to dryness at room temperature under a stream of nitrogen. The residue was dissolved in ethanol and the resulting light yellow solution was acidified with 10 µL of 0.1 M HCl and stored at –20°C.

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 (33) . Lipid extraction was carried out on ice immediate after the preparation of mitochondria in the presence of BHT and under nitrogen atmosphere. Phospholipids were separated by the HPLC method described previously (34) 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, v/v) and solvent B, hexane/2-propanol/water (6: 8: 1.4, v/v/v). The percentage of solvent B in solvent A was increased in 15 min from 0% to 100%. Flow rate was 1 mL/min and detection at 206 nm. The peak of cardiolipin was identified by comparison with the retention time of standard cardiolipin and rechromatographed by TLC.

Lipid and cardiolipin peroxidation
Lipid peroxidation was estimated by the appearance of conjugated dienes as follows. Lipids were extracted from mitochondria by the Bligh e Dyer procedure (33) . Lipid extracts from 4 mg of mitochondrial membrane, were dissolved in 2.5 mL of chloroform: methanol (1: 1) and the absorption spectra were followed between 210 and 310 nm (35) with a Perkin-Elmer Lambda 3B spectrophotometer.

Peroxidized cardiolipin was identified by normal-phase HPLC, as described above, with UV detection at 235 nm, indicative of conjugated dienes (35 , 36) . The resulting peak was rechromatographed by TLC and used as standard.

Statistical analysis
The results have been expressed as mean ±SE and their statistical significance was determined by the Student’s t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The degree of mitochondrial membranes lipid peroxidation can be assayed by recording the increase in absorbance of extracted membrane lipids at 233 nm. Figure 1 shows absorbance values obtained from mitochondrial lipid extracts of control, ischemic-reperfused, and melatonin-treated reperfused rat heart. Mitochondria from reperfused heart exhibited a large increase in the level of lipid peroxidation compared with the control heart. Melatonin had a strong protective effect reducing the degree of the mitochondrial lipid peroxidation.


Figure 1
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  		Figure 1. Absorbance values of the conjugated dienes of mitochondrial lipids extracted from control, reperfused and melatonin-treated reperfused rat heart. The conjugated dienes spectra were recorded as described in Materials and Methods. Each value represents the mean ± SE of 5 separate experiments. *P < 0.05 vs. control; **P < 0.01 vs. reperfused.

Respiratory activities of mitochondria isolated from control, ischemic-reperfused and melatonin-treated reperfused rat heart, measured in the presence of pyruvate + malate or succinate as substrates and ADP to stimulate respiration (state 3) are reported in Table 1 . The rate of state 3 respiration was markedly decreased in mitochondria isolated from reperfused rat heart, while melatonin treatment significantly attenuated this decrease. Almost similar results were obtained with succinate as substrate. State 4 rates of respiration were slightly decreased in all these preparations of mitochondria. The respiratory control ratio (RCR), an index of mitochondrial membrane integrity, was also decreased in mitochondria from reperfused rat heart, while melatonin limited this decrease.


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  		Table 1. Respiratory activities in mitochondria isolated from control, reperfused, and melatonin-treated reperfused rat hearta

Complex I and complex III activities were measured in all these preparations of mitochondria. As shown in Fig. 2 , mitochondria from reperfused rat heart exhibited a marked decrease in the activity of both these enzyme complexes, compared with the control heart. Melatonin-reperfusion had a protective effect and actually attenuated the decline in the complex I and complex III activity.


Figure 2
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  		Figure 2. Complex I and complex III activity in mitochondria isolated from control, reperfused and melatonin-treated reperfused rat heart. Complex I (a) and complex III (b) activities were measured as described in Materials and Methods. Each value represents the mean ± SE of 5 separate experiments. *P < 0.01 vs. control; **P < 0.01 vs. reperfused.

The addition of the respiratory substrates pyruvate + malate or succinate to aerobic mitochondria results in a generation of H2O2 (7 , 37 , 19) . The capacity of mitochondria isolated from control, ischemic-reperfused and melatonin-reperfused rat heart to generate oxygen radical in the presence of pyruvate + malate or succinate was evaluated. As shown in Fig. 3 , the basal rate of H2O2 production was significantly enhanced in mitochondria from ischemic-reperfused rat heart, respiring with pyruvate + malate or succinate. This H2O2 production was largely decreased in mitochondria from melatonin-treated reperfused rat heart.


Figure 3
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  		Figure 3. H2O2 production in mitochondria isolated from control, reperfused and melatonin-treated reperfused rat heart. The H2O2 formation was induced by the addition of 2 mM pyruvate + 5 mM malate (a) or 5 mM succinate (b) and measured as described in Materials and Methods. Each value represents the mean ± SE of 5 different experiments. *P < 0.01 vs. control; **P < 0.05 vs. reperfused.

The effect of melatonin on mitochondrial cardiolipin content was tested. The content of mitochondrial cardiolipin was measured by a very sensitive HPLC technique set up in our laboratory with a detection limit of 0.5 nmol/sample. As reported in Fig. 4 the content of cardiolipin was dramatically reduced in mitochondria from ischemic-reperfused rat heart compared with the control heart. Melatonin treatment significantly prevented this cardiolipin loss.


Figure 4
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  		Figure 4. Cardiolipin content in mitochondria isolated from control, reperfused and melatonin-treated 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 5 different experiments. *P < 0.01 vs. control; **P < 0.01 vs. reperfused.

We also measured the content of peroxidized cardiolipin in these preparations of mitochondria. The content of peroxidized cardiolipin was measured by an HPLC method based on the absorbance at 233 nm, indicative of the formation of conjugated dienes. As shown in Fig. 5 the content of peroxidized cardiolipin was enhanced in mitochondria isolated from ischemic-reperfused heart compared with control heart. Melatonin treatment largely reduced the content of the peroxidized cardiolipin.


Figure 5
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  		Figure 5. Relative content of peroxidized cardiolipin in mitochondria from control, reperfused and melatonin-treated reperfused rat heart. Mitochondrial content of peroxidized cardiolipin was determined by the HPLC technique described in the Materials and Methods. The content of peroxidized cardiolipin is expressed as peak area (at 235 nm) per mg of phospholipids and the peak area of the control is assumed as unit. Each value represents the mean ±SE obtained from 5 different experiments. *P < 0.05 vs. control; **P << 0.05 vs. reperfused.

Global normothermic ischemia followed by reperfusion lead to a significant decline in the hemodynamic function in the control heart (Fig. 6 ). Pretreatment of hearts with melatonin significantly improved functional recovery of the heart during reperfusion as reflected in the greater LVDp and lower LVEDp.


Figure 6
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  		Figure 6. Changes in left ventricle end-diastolic pressure (LVEDp) and left ventricle developed pressure (LVDp=systolic minus end-diastolic pressure), in two groups of 5 isolated and perfused rat hearts at the end of reperfusion. Each value represents the mean ±SE obtained from 5 different experiments. *P < 0.001 vs. reperfused in absence of melatonin.

To see whether the action of melatonin in protecting against cardiac dysfunction associated with ischemia/reperfusion was at the level of mitochondria, we performed in vitro experiments on the effects of melatonin on isolated mitochondria. We previously showed that rat heart mitochondria exposed to free radical generating system tert-butilhydroperoxide (t-BuOOH)/Cu2+ undergo lipid peroxidation (38) . As shown in Fig. 7 , treatment of heart mitochondria with t-BuOOH resulted in a marked loss in the activity of complex I and complex III, which was completely prevented by melatonin. Similarly, the content of cardiolipin was decreased in t-BuOOH treated mitochondria while the addition of melatonin totally prevented this decrease (Fig. 8 ).


Figure 7
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  		Figure 7. Lipid peroxidation-induced loss in the complex I and complex III activity in rat heart mitochondria and prevention by melatonin. Lipid peroxidation in isolated mitochondria was induced by the addition of 5 µM CuCl2 and 100 µM t-BuOOH in the standard reaction medium at 37°C. After 30 min. of incubation the reaction was stopped by the addition of 1 mM EDTA. Where present, 10 µM melatonin was added at the beginning of incubation. Complex I (a) and complex III (b) activities were measured as described in Materials and Methods. Each value represents the mean ± SE of 5 separate experiments. *P < 0.01 vs. control; **P < 0.01 vs. t-BuOOH.


Figure 8
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  		Figure 8. Lipid peroxidation-induced loss in cardiolipin content in rat heart mitochondria and prevention by melatonin. Lipid peroxidation in isolated mitochondria was induced as described in the legend of Fig. 7 . Where present 10 µM melatonin was added at the beginning of the incubation. Mitochondrial cardiolipin content was determined by the HPLC technique as described in Materials and Methods. Each value represents the mean ± SE obtained from 5 different experiments. *P < 0.01 vs. control; **P < 0.01 vs. reperfused


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It is generally acknowledged that ischemic-reperfusion injury to cardiac myocytes is mediated, among other factors, by an overproduction of oxygen free radicals. These oxygen radicals can be generated by several mechanisms, including the xanthine oxidase reaction (39) , the activity of NADPH oxidase (40) , and mitochondrial respiration (41 , 42) . Experimental evidences have shown that mitochondrial ROS generation occurs in part during ischemia (6) and more abundantly during early reperfusion (1 2 3 4 5) . Appropriate antioxidant strategies could be particularly useful to limit ROS production and hence to protect ischemic/reperfused myocardium.

Several recent publications present evidence that melatonin has significant protective action against the cardiac damage and altered physiology that occur during ischemia-reperfusion injury (27 28 29 30 , 43) . This protective effect of melatonin has been also demonstrated in ischemia and reperfusion of rat liver (44) . In line with these data, our results indicate that melatonin, at a pharmacological concentration of 50 µM, strongly protect against ischemic- reperfusion myocardial damage. This protective effect of melatonin appears to be produced through its action at mitochondrial level as also suggested by the protective effect of this compound against free radical-induced peroxidative damage on isolated mitochondria (see Figs. 7 , 8 ). Melatonin protects against the alterations to various mitochondrial bioenergetic parameters associated with ischemia-reperfusion. Specifically, we found that melatonin reperfusion of ischemic heart significantly lowered the degree of mitochondrial lipid peroxidation, counteracted the reduction in state 3 respiration and the associated decrease in the respiratory control ratio and prevented the loss in complex I and complex III activities. In addition, melatonin largely prevented the increase in the H2O2 production, the loss in cardiolipin content and the increase in its level of peroxidation. The protection afforded by melatonin-reperfusion on all these mitochondrial bioenergetics parameters was associated with the protection observed on the heart contractility.

Lipid peroxidation is considered a major mechanism of oxygen free radicals attack and an indicator for oxidative stress and ROS production. This process initiates with oxygen radical attack to double bonds of polyunsaturated fatty acids leading to formation of conjugated dienes, which absorb at 233 nm. Several studies demonstrated the protective effect of melatonin on lipid peroxidation induced by oxidative stress in mitochondrial membrane (27 , 31 , 45) . In line with this, our results demonstrate that melatonin treatment of reperfused rat heart results in a lower degree of mitochondrial lipid peroxidation.

The results reported in Table 1 show a significantly lower state 3 respiration rate in mitochondria isolated from reperfused heart compared with control, while state 4 rates of respiration were slightly decreased. This decline in state 3 respiration could be attenuated by melatonin treatment. Alterations of state 3 and 4 respiration determine changes in the respiratory control ratio (RCR). A decrease in the RCR is observed in mitochondria isolated from reperfused rat heart, which is diagnostic of extensive mitochondrial damage. This decrease was largely attenuated by melatonin treatment indicating that this compound had a protective effect on the integrity and on the function of the mitochondria.

Complex I and complex III are considered the main producers of superoxide anion in mitochondria (7 8 9 10) . The formation of superoxide occurs via the transfer of free electrons to molecular oxygen. This reaction occurs at specific sites of the electron transport chain, which resides in the inner mitochondrial membrane. A defect in the activity of both these respiratory complexes can be considered a potential source of ROS production. We found that heart ischemia-reperfusion is associated with a defect in mitochondrial complex I and complex III activity that accounts for an enhanced H2O2 production in mitochondria (19 , 20) . Melatonin-reperfusion is associated with a lower mitochondrial production of H2O2 compared with that found with ischemia-reperfusion.

Cardiolipin is emerging as an important factor in the regulation of mitochondrial bioenergetics in that it interacts with several vital inner membrane proteins, including anion carriers and respiratory chain complexes (11 12 13 , 46 , 47) . It has also been reported that this phospholipid is specifically required for the electron transfer in complex I and complex III of the mitochondrial respiratory chain (10 , 20 , 48 49 50) . In addition, an involvement of cardiolipin in higher order organization of these components of the respiratory chain in a supercomplex in the mitochondrial inner membrane, has been proposed (51) Thus, changes in the mitochondrial content of cardiolipin, due to alterations of one of the enzymatic steps involved in its biosynthetic process (52 , 53) or as consequence of oxidative damage by ROS attack, may affect the activity of complex I and complex III. As reported in Fig. 4 , a marked decrease in the cardiolipin content is observed in mitochondria isolated from reperfused rat heart, which was largely prevented by melatonin. These changes in the cardiolipin content are due to ROS-induced cardiolipin peroxidation as shown by changes in the level of conjugated dienes. The preservation of mitochondrial cardiolipin content results in a reduced alteration of complex I and complex III activity and hence, in a lower production of mitochondrial oxygen radicals production, with subsequent attenuation of mitochondrial oxidative damage and an improvement of the mechanical function of the heart.

The lipophilicity of melatonin allows it to accumulate in the inner mitochondrial membrane where it scavenges reactive species generated during respiration. Thus, in addition to preventing cardiolipin peroxidation, melatonin may also protect directly mitochondrial respiratory chain complexes from oxidative damage and this may also contribute to protect mitochondrial function.

Mitochondrial permeability transition (MPT) is considered an important factor in ischemia-reperfusion injury and subsequently a possible target of cardioprotection (54) . ROS generation is considered, in addition to other factors, a key event in pore opening (55 , 56) . Thus, a lower mitochondrial ROS production, in melatonin treated reperfused rat heart, may delay or attenuate the probability of MPT and this may also contribute to limit mitochondrial dysfunction and to prevent heart damage. The possibility that melatonin may direct interact with and inhibit MPT cannot be excluded (57) .

In conclusion, our results demonstrate that melatonin, at pharmacological concentration, strongly protects against the alterations to mitochondrial oxidative metabolism, thus limiting cardiac reperfusion injury. The mechanism of protection is likely to be due, at least in part, to the antioxidant efficacy of melatonin preventing cardiolipin peroxidation. Therapeutic use of melatonin may provide a useful strategy for the treatment of cardiac ischemia- reperfusion injury as well as for the wide range of diseases with mitochondrial oxidative damage in their etiology.


   ACKNOWLEDGMENTS
 
This study was supported by a grant from the National Research Project PRIN "Bioenergetics and Membrane Transport" (MIUR) Italy 2003–2005

Received for publication July 21, 2005. Accepted for publication October 10, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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