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CARDIOVASCULAR

Cardioprotective Effect of Morphine and a Blocker of Glycogen Synthase Kinase 3β, SB216763 [3-(2,4-Dichlorophenyl)-4(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione], via Inhibition of the Mitochondrial Permeability Transition Pore

INSERM, Unité 841, Créteil, France; Université Paris 12, Facultéde Médecine, France (F.N.O., C.P.-M., R.A., R.Z., J.L.D.-R., A.B., D.M.); and Fédération de Cardiologie, Hôpital Henri Mondor, France (J.L.D.-R., A.B.)

Received February 13, 2008; accepted April 22, 2008.

	Abstract

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Morphine has been shown to protect the myocardium against ischemia-reperfusion injury through inhibition of glycogen synthase kinase-3β (GSK-3β). Given that GSK-3β is known to modulate the mitochondrial permeability transition pore (mPTP), we investigated the role of mPTP in the cardioprotective effect of morphine and the GSK-3β inhibitor SB216763 [SB; 3-(2,4-dichlorophenyl)-4(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione] during ischemia-reperfusion. Both morphine (0.3 mg/kg) and SB (0.6 mg/kg) reduced infarct size in a model of regional myocardial ischemia-reperfusion in rats (13 ± 1 and 14 ± 3% of the area at risk versus 33 ± 4% in controls; p < 0.05). Morphine and SB protected the ischemic myocardium against Ca²⁺-induced mPTP opening as demonstrated by the increased capacity of mitochondria to retain Ca²⁺ when they were isolated from the ischemic zone 10 min after the onset of reperfusion (59 ± 8 and 66 ± 3 versus 29.5 ± 6 nmol Ca²⁺/mg · protein, respectively; p < 0.05). This was associated with a restoration of mitochondrial oxidative phosphorylation parameters. In isolated adult rat cardiomyocytes subjected to anoxia-reoxygenation, morphine (2 µM), SB (3 µM), and the direct mPTP inhibitor cyclosporine A (3 µM) delayed mPTP opening as assessed by the calcein loading Co²⁺-quenching technique. This was accompanied by an increase in cell survival as measured by nuclear staining with propidium iodide. These in vitro effects of morphine on inhibition of mPTP opening during anoxia-reoxygenation were suppressed by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin (0.1 µM). These data indicate that the infarct-limiting effect of morphine and SB is linked by a cause-effect relationship, which leads to an increased mitochondrial resistance and inhibition of mPTP opening through the PI3-kinase pathway and subsequent inactivation of GSK-3β.

Recently, Gross et al. (2004) demonstrated that morphine and -selective opioids induced cardioprotection during reperfusion through a phosphatidylinositol-3 kinase (PI3-kinase) and molecular target of rapamycin pathway leading ultimately to inactivation of glycogen synthase kinase-3β (GSK-3β) by phosphorylation at Ser⁹ (Murphy, 2004). Given that inhibition of GSK-3β was involved in the mechanism by which pharmacological preconditioning prevents mPTP opening in cardiomyocytes (Juhaszova et al., 2004), a relevant hypothesis is that the cardioprotection afforded by opioids may be mediated by preventing mPTP opening via inhibition of GSK-3β. However, this mechanism has never been directly demonstrated at the mitochondrial level.

The first goal of this study then was to determine whether morphine and the selective GSK-3β inhibitor, SB216763 (SB) reduced myocardial ischemia-reperfusion injury by modulating mPTP opening. Subsequently, to establish whether this association was linked by a direct cause-effect relationship, we investigated the effects of morphine and SB in a model of rat cardiomyocytes in which anoxia-reoxygenation was used to induce mPTP opening.

	Materials and Methods

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All animal procedures used in this study were in strict accordance with the European Community Council Directive (86-609/87-848 EEC) and recommendations of the French Ministère de l'Agriculture.

Pharmacological Agents. Morphine hydrochloride (Renaudin, France) was dissolved in water, whereas wortmannin and SB were purchased from Tocris Cookson Inc. (Ellisville, MO) and were dissolved in a final concentration of 0.01% dimethyl sulfoxide.

In Vivo Coronary Occlusion-Reperfusion. Male Wistar rats (150–200 g) purchased from Janvier (Le Genest-St-Isle, France) were used for these experiments. The general surgical protocol and determination of infarct size have been described previously in detail (Obame et al., 2007). In all groups of rats, the coronary artery was occluded for 35 min and released for a 2-h reperfusion before mitochondrial functional assessment and infarct size measurement. Mitochondrial functional assessment was also made after a 10-min reperfusion because it was demonstrated that mPTP opens within the first min of reperfusion (Halestrap et al., 2004). In the sham group in which the surgical procedure was identical to others, the coronary artery was not occluded. In the other groups of treated rats, i.e., vehicle control, morphine (0.3 mg/kg) or SB (0.6 mg/kg) was administered separately as a 3-min infusion through the jugular vein at 5 min before reperfusion.

Infarct Size Determination. After completion of reperfusion, rats received heparin (1000 UI/kg) and were re-anesthetized with sodium pentobarbital (60 mg/kg). After thoracotomy, the coronary artery was re-occluded at the same place as previously used to induce myocardial infarction. Potassium chloride was used to induce cardiac arrest, and the hearts were excised. The ascending aorta was cannulated and perfused retrogradely (Langendorff apparatus, 100 mm Hg) with saline solution followed by 5% Evans blue. Infarct size was determined as the percentage of the area at risk (AAR). The left ventricle was cut from apex to base into six slices, which were weighed and incubated with 1% triphenyltetrazolium chloride (Sigma Chemical, Saint-Quentin Fallavier, France) in a pH 7.4 buffer for 20 min at 37°C. Slices were fixed overnight in 10% formaldehyde and then photographed with a digital camera mounted on a stereomicroscope. Using a computerized planimetric program (Scion Image; Scion Corporation, Frederick, MD), AAR and the infarcted zones were quantified. AAR was expressed as a percentage of the left ventricle weight, and infarct size was expressed as a percentage of AAR.

Isolation of Mitochondria. For measurement of respiratory parameters and mPTP opening, a cardiac mitochondrial fraction was prepared from hearts subjected to coronary artery-reperfusion as described previously (Obame et al., 2007; Zini et al., 2007). The AAR of the left ventricle of rats treated with either vehicle or morphine (0.3 mg/kg) or SB (0.6 mg/kg) and sacrificed after 10 min of reperfusion was rapidly minced in 15 ml of a cold buffer containing 220 mM mannitol, 70 mM sucrose, 10 mM HEPES, 1 mM EGTA, and 0.04 mM free fatty acid bovine serum albumin, pH 7.4, at 4°C. Homogenates were centrifuged at 1000g for 5 min, and supernatants were centrifuged at 10,000g for 10 min. The final pellets were resuspended in the homogenization buffer, including 0.1 mM EGTA without bovine serum albumin to obtain a protein concentration of approximately 15 mg/ml. Protein concentration was determined by the method of Lowry et al. (1951).

Isolation of Crude Particulate Fractions. For measurement of lipid peroxidation, a crude particulate fraction was used. Homogenates from the AAR of the left ventricle of rats were prepared as described previously. Homogenates were centrifuged at 600g for 5 min at 4°C. The supernatants were centrifuged at 100,000g for 1 h at 4°C. The resulting pellets considered as the total crude particulate fractions were resuspended in 300 µl of homogenization buffer supplemented with 0.3% Triton X-100 and immediately frozen at –80°C before thiobarbituric acid-reactive substance (TBAR) determination.

Mitochondrial Oxygen Consumption and mPTP Opening in Isolated Mitochondria. As described previously (Obame et al., 2007; Zini et al., 2007), oxygen consumption was measured with a Clark-type electrode (Hansatech, Eurosep, Cergy-St-Christophe, France). Mitochondria were incubated at 25°C in a respiration buffer containing sucrose (50 mM), KCl (100 mM), HEPES (10 mM), and KH₂PO₄ (5 mM), pH 7.4, at 25°C. Oxygen consumption was induced by the addition of 5 mM pyruvate/malate (state 4 or substrate-respiration rate). ATP synthesis was measured by adding 250 µM ADP (state 3 or ADP-stimulated respiration rate). The respiratory control ratio (state 3/state 4) and the ADP/O values (nanomoles of ADP consumed divided by the nanograms of atoms of oxygen used during state 3 respiration) were then determined.

Mitochondrial Ca²⁺ retention was monitored as an index of mPTP opening. Mitochondria were loaded with increasing concentrations of Ca²⁺ until the load reached a threshold where mitochondria underwent a fast process of Ca²⁺ release due to mPTP opening. Cardiac mitochondria (1 mg/ml), energized with 5 mM pyruvate/malate, were incubated in the respiration buffer, including 1 µMCa²⁺ Green-5N fluorescent probe (Interchim, Montluçon, France). The reaction was started by the addition of successive 10 µMCa²⁺ pulses. After each addition, a rapid uptake was observed followed by a dynamic steady state corresponding to the equilibrium between the influx and the efflux of Ca²⁺. When maximal Ca²⁺-loading threshold was reached, this equilibrium was disrupted, and Ca²⁺ was released. The concentration of Ca²⁺ in the extramitochondrial medium was monitored by means of a Perkin-Elmer LS 50B spectrofluorimeter at excitation and emission wavelengths of 506 and 532 nm, respectively. The Ca²⁺ signal was calibrated by the addition to the medium of known amounts of Ca²⁺.

Assay of Lipid Peroxidation. Lipid peroxidation was assessed as the generation of TBARs, i.e., lipid peroxides, according to Ligeret et al. (2004) with some modifications. In brief, 500 µl of the crude particulate fraction (1.5 mg/ml) was added to 500 µl of a solution of trichloroacetic acid (3% w/v) and 500 µl of a solution of thiobarbituric acid (1% w/v). The mixture was heated at 90°C for 30 min and cooled on ice, and TBARs were extracted with 1 ml of butanol-1. The solution then was centrifuged at 3000 tr/min for 10 min. The butanol-1 phase (supernatant) was taken, and the generation of TBARs was determined by measuring the absorbance at 530 nm by means of a 530 spectrophotometer (Jasco, Nantes, France). TBARs concentrations were determined in triplicate using a molar extinction coefficient () of 156,000 M^–1 · cm^–1.

Isolation of Adult Cardiomyocytes. Ventricular cardiomyocytes were isolated from male Wistar rats by an enzymatic technique. The heart was retrogradely perfused for 10 min at 37°C with Krebs-Henseleit buffer bubbled with 95% O₂/5% CO₂ containing 120 mM NaCl, 25 mM NaHCO₃, 11 mM glucose, 10 mM butane-dione-monoxime, 4.7 mM KCl, 1.17 mM MgSO₄, 1.2 mM KH₂PO₄, and 1.8 mM CaCl₂, pH 7.4, to wash out blood. The heart then was perfused with the same medium in which Ca²⁺ was limited to 10 µM. After 5 min of perfusion, liberase (2 mg/100 ml, Blendzyme 3; Roche Diagnostics, Indianapolis, IN) was added to the buffer, and the heart was perfused for approximately 10 min. The heart was placed into a beaker in a buffer containing HEPES (25 mM), NaCl (130 mM), butane-dione-monoxime (10 mM), glucose (5 mM), KCl (4.5 mM), KH₂PO₄ (1.2 mM), and 2% bovine serum albumin, pH 7.4, at 37°C to stop the liberase activity. Ventricles were then cut into small fragments, and cells were isolated by stirring the tissue and successive aspirations of the fragments through a 10-ml pipette. After 5 min, the supernatant was removed, and the remaining tissue fragments were re-exposed to 20 ml of the same buffer. Two to three cycles were performed, and the cells released from the last supernatant were placed into an incubator at 37°C for sedimentation. The cells then were suspended in a buffer containing minimal essential medium (M-0518; Sigma) supplemented with penicillin and streptomycin (100 U–0.1 mg/ml), HEPES (25 mM), butane-dione-monoxime (10 mM), a mixture of insulin-transferrine-selenium (100 µl, I-3146; Sigma), L-glutamine (2 mM), and CaCl₂ (25 µM), pH 7.4, at 37°C, and Ca²⁺ was gradually added to 1.2 mM.

Finally, the cardiomyocytes were suspended in culture medium M199 (Invitrogen, Cergy-Pontoise, France). They were seeded on 35-mm Petri dishes precoated with 10 µg/ml sterilized laminin and incubated 2 h before being used. Cardiomyocytes were pretreated for 15 min at 37°C before anoxia-reoxygenation with vehicle, morphine (2 µM), morphine (2 µM) + wortmannin (0.1 µM), cyclosporine A (3 µM), or SB (3 µM).

Model of Anoxia-Reoxygenation. The cardiomyocytes were placed into a thermostated (37°C) chamber (Warner Instruments Inc., Hamden, CT), which was mounted on the stage of a IX81 Olympus microscope (Olympus, Tokyo, Japan), and perfused with Tyrode's solution (130 mM NaCl, 5 mM KCl, 10 mM HEPES, 1 mM MgCl₂, and 1.8 mM CaCl₂, pH 7.4, at 37°C) at a rate of 0.5 ml/min. The chamber was connected to a gas bottle diffusing a constant stream of O₂ (21%), N₂ (74%), and CO₂ (5%) maintaining a partial O₂ pressure of 21%. Oxygen in the perfusate was measured in the chamber using a fiber optic sensor system (Ocean Optics, Inc., Dunedin, FL). Myocytes were paced to beat by field stimulation (5 ms, 0.5 Hz).

To simulate ischemia, the perfusion was stopped, and cardiomyocytes were exposed for 30 min to a hypoxic medium maintaining a partial O₂ pressure of 2 to 3%. This medium was the Tyrode's solution (bubbled with 100% N₂) supplemented with 20 mM 2-deoxyglucose and subjected to a constant stream of N₂ (100%). At the end of the ischemic period, reoxygenation was induced by restoring rapidly the Tyrode's flow and 21% O₂ in the chamber.

Measurement of mPTP Opening in Adult Cardiomyocytes Subjected to Hypoxia-Reoxygenation. Direct assessment of mPTP opening in cardiomyocytes was made by means of the established loading procedure of the cells with calcein acetoxymethyl ester (calcein-AM) and CoCl₂ resulting in mitochondrial localization of calcein fluorescence (Petronilli et al., 1999, 2001; Katoh et al., 2002). Cells were loaded with 1 µM calcein-AM for 30 min at 37°C in 2 ml of M199, pH 7.4, supplemented with 1 mM CoCl₂. They were then washed free of calcein and CoCl₂ and incubated in the Tyrode's solution. To determine cell death, cells were co-loaded in some experiments with propidium iodide (5 µM), which permeates only the damaged cells. Cardiomyocytes were imaged with an Olympus IX-81 motorized inverted microscope equipped with a mercury lamp as a source of light for epifluorescence illumination and with a 12-bit cooled CC12 camera. For detection of calcein fluorescence, a 460 to 490-nm excitation and a 510-nm emission filter were used. Propidium iodide fluorescence was excited at 520 to 550 nm and recorded at 580 nm. Images were acquired every 60 s after an illumination time of 100 ms (calcein) and 1 s (propidium iodide) per image using a digital epifluorescence imaging software (Cell P; Olympus, Rungis, France).

Fluorescence was integrated over a region of interest (80 µm²) for each cardiomyocyte, and a fluorescence background corresponding to an area without cells was removed. For comparative purposes, the fluorescence intensity minus background was normalized according to the initial fluorescence value.

For each experiment, we calculated the response observed from a single cardiomyocyte and the global response by averaging the fluorescence changes obtained from all of the cardiomyocytes contained in a field (at least 20–25 cells). The time to mPTP opening (T_mPTP) was measured as the average reoxygenation time necessary to induce mPTP opening in the same field.

Statistical Analysis. Data are expressed as means ± S.E.M. Statistical significance was determined using a one-way analysis of variance followed by Scheffe's test. Significance was accepted when p < 0.05.

	Results

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Morphine and SB Reduce Infarct Size in Vivo. The AAR were not significantly different between groups of infarcted rats (46 ± 2, 45 ± 4, and 42 ± 4% in the control group receiving vehicle and in the groups receiving morphine and SB, respectively). As shown in Fig. 1, both morphine and SB significantly reduced myocardial infarct size compared with corresponding controls (13 ± 1, 14 ± 3, and 33 ± 4% AAR, respectively, p < 0.05).

View larger version (7K): [in this window] [in a new window]   Fig. 1. Effect of morphine and SB on myocardial infarct size expressed as a percentage of the AAR in regional ischemic (35 min), reperfused (2 h), and anesthetized rats. n = number of treated rats per group; *, p < 0.05 versus control.

Morphine and SB improved oxidative phosphorylation as demonstrated by increases in both respiratory control ratio and ADP/O values. This was due to the partial restoration of state 3 respiration rate promoted by the increase in the activity of the electron transport chain, which is evidenced in the presence of FCCP (Table 1). Indeed, morphine and SB restored (+87 and +76%, respectively) the FCCP-accelerated state 4 respiration rate altered by ischemia-reperfusion. The improvement of oxidative phosphorylation parameters was also observed when cardiac mitochondria were isolated after 2-h reperfusion (Table 1).

Cardiac Mitochondria Isolated from Morphine- or SB-Treated Rats Are Less Sensitive to mPTP Opening. As shown in Fig. 2, the amount of Ca²⁺ required to trigger mPTP opening in sham-operated rats reached 94 ± 7 nmol/mg mitochondrial proteins. Control ischemia-reperfusion significantly reduced the capacity of mitochondria to retain Ca²⁺ before mPTP opening, regardless of the duration of reperfusion (29.5 ± 6 and 27.8 ± 7 nmol/mg proteins for 10-min and 2-h reperfusion, respectively). Morphine significantly increased the resistance to mPTP opening to Ca²⁺ during reperfusion (59.7 ± 8.2 and 54.9 ± 6.8 nmol/mg protein for 10-min and 2-h reperfusion, respectively), whereas it did not modify this sensitivity when the drug was administrated to sham-operated rats (97.5 ± 3.9 nmol/mg protein). Like morphine, SB also significantly increased the resistance to mPTP opening to Ca²⁺ during reperfusion (66.5 ± 3.5 and 66.4 ± 8.7 nmol/mg protein for 10-min and 2-h reperfusion, respectively).

View larger version (31K): [in this window] [in a new window]   Fig. 2. Effect of morphine and SB on Ca2+-induced mPTP opening in mitochondria after myocardial ischemia (35 min) followed by either 10-min or 2-h reperfusion. Mitochondria were incubated at 25°C in the respiration buffer supplemented with 1 µMCa2+ Green-5N probe. Experiments were started by the addition of 5 mM pyruvate and 5 mM malate, and successive 10 µMCa2+ pulses were added (arrows). A, a typical experiment in mitochondria isolated from sham (a), ischemia-reperfused (b), and ischemia-reperfused morphine-treated hearts (c). Changes in Ca2+ levels were expressed in arbitrary units (AU) of fluorescence. B, morphine (M) and SB increased the capacity of mitochondria to retain Ca2+ during myocardial ischemia-reperfusion (I/R). Morphine and SB (not shown) were without effect in sham (S) animals. Hatched bars, 10-min reperfusion; black bars, 2-h reperfusion. Each value represents the mean ± S.E.M. of six independent experiments performed in triplicate. *, p < 0.05 versus sham; # p < 0.05 versus I/R.

Effect of Anoxia-Reoxygenation on mPTP Opening in Adult Cardiomyocytes. Figure 3 illustrated the changes in fluorescence of mitochondria in cardiomyocytes loaded with calcein in the presence of Co²⁺ and subjected to anoxia-reoxygenation. As shown in Fig. 3A, reoxygenation induced a large decrease in calcein fluorescence corresponding to mPTP opening, but the kinetics of this phenomenon was different from one cardiomyocyte to another. Two types of mPTP opening were observed. Globally, in 80% of the cardiomyocytes analyzed, a complete drop in calcein fluorescence occurred rapidly (2 min) with a T_mPTP after reoxygenation of approximately 25 min in the example of Fig. 3B (cell 1). In the other 20% of cardiomyocytes, a slow but constant decrease in calcein fluorescence was observed before the rapid drop of fluorescence (Fig. 3B, cell 2). As cyclosporine A significantly increased from 18 ± 10 to 45 ± 6%, the percentage of cardiomyocytes that exhibited this slow process of drop in calcein fluorescence, one can suppose that it could be also attributed to anoxia-reoxygenation-induced mPTP opening. Nevertheless, for simplification and clarity in expression of the data, we considered the rapid and complete drop in calcein fluorescence as the definite T_mPTP in each cardiomyocyte, and the average of T_mPTP for all cardiomyocytes investigated in one field in each experiment was calculated and compared between treatments.

View larger version (22K): [in this window] [in a new window]   Fig. 3. Typical recordings of changes in calcein fluorescence intensity in isolated cardiomyocytes subjected to anoxia-reoxygenation. Cardiomyocytes were co-loaded with calcein-AM and CoCl2, and images were collected at 1-min intervals. After a 15-min normoxic period and 30-min hypoxia (omitted for clarity, no calcein change being observed), reoxygenation was induced. The initial fluorescence intensities measured in single cardiomyocytes were normalized to 100%. A, each trace (n = 20) represents normalized change of calcein obtained in a single cardiomyocyte. B, two profiles of mPTP opening during reoxygenation were recorded. In cell 1, a rapid decrease in calcein fluorescence was observed. In cell 2, this rapid decrease was preceded by a gradual decrease in calcein fluorescence.

Effects of Morphine and SB on mPTP Opening Induced by Anoxia-Reoxygenation in Cardiomyocytes. As shown in Figs. 4 and 5, both morphine (2 µM) and SB216763 (3 µM) increased mean T_mPTP by 51 (41.9 ± 2.6 min) and 67% (57.7 ± 8.0 min) from their respective control values (27.7 ± 3.3 and 34.5 ± 3.3 min, all p < 0.05). For comparison, cyclosporine A (2 µM), which is known to inhibit mPTP opening through a direct mechanism, also increased mean T_mPTP by 62% from 23.3 ± 4.4 to 37.8 ± 2.7 min (p < 0.05) (Fig. 5). In the same conditions, the selective and irreversible PI3-kinase inhibitor, wortmannin (0.1 µM), abolished the effect of morphine on mPTP opening (mean T_mPTP 29.5 ± 5.9 versus 22.5 ± 3.2 min before and after wortmannin, respectively). To analyze whether mPTP opening contributes to cell death during reoxygenation, the cardiomyocytes were also co-loaded with calcein and propidium iodide. As shown in Fig. 4, the decrease in calcein fluorescence observed during reoxygenation was associated with concomitant plasma membrane permeabilization, as attested to by the increase in nuclear staining with propidium iodide, a marker of cell death.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Time course of mPTP opening and cell death in isolated cardiomyocytes subjected to anoxia-reoxygenation in the absence or in the presence of morphine. Cardiomyocytes were co-loaded with calcein-AM, CoCl2 and propidium iodide and pretreated with or without morphine. Images were collected at 1-min intervals. After a 15-min normoxic period and 30-min hypoxia (omitted for clarity, no calcein change being observed), reoxygenation was induced. The initial fluorescence intensities measured in single myocytes were normalized to 100%, and the global response was calculated by averaging 20 to 25 traces obtained from single cardiomyocytes under the same conditions, and TmPTP was determined.

View larger version (11K): [in this window] [in a new window]   Fig. 5. Morphine and SB delayed mPTP opening in isolated cardiomyocytes subjected to anoxia-reoxygenation. Cardiomyocytes were co-loaded with calcein-AM and CoCl2 and were pretreated without (C, for control) or with morphine (M), morphine + wortmannin (M+W), cyclosporine A (CsA), or SB. They were subjected to the protocol of anoxia-reoxygenation, and TmPTP was calculated. Each value is the mean ± S.E.M. of four to seven experiments performed with different isolated preparations of cardiomyocytes, each including the measure of mPTP opening in 20 to 25 cells as shown in Fig. 3. *, p < 0.05 versus corresponding control value.

	Discussion

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As previously reported by Gross et al. (2004), morphine and SB administered at reperfusion protected the myocardium against ischemia-reperfusion injury in rats, and we demonstrated that this cardioprotective effect is associated with an inhibition of mPTP opening in a model of anoxia-reoxygenation in isolated adult cardiomyocytes. This was associated with an improvement in mitochondrial respiratory function and enhancement of mitochondrial resistance to Ca²⁺ overload at the onset and after 2-h reperfusion. It must be noted that morphine administered to sham-operated rats did not modify the sensitivity of mPTP opening to Ca²⁺ overload in contrast to the direct mPTP inhibitor, cyclosporine A (Argaud et al., 2004). These results indicate that the cardioprotective effect of morphine was not due to a direct action of the drug on mPTP but was rather linked to modification(s) of mitochondrial conditions, which make them more resistant to mPTP opening. Given that SB, a selective inhibitor of GSK-3β, exhibited exactly the same cardioprotective profile as morphine in both in vivo and in vitro models used in this study, one can suggest that the infarct-limiting effect of morphine is linked in a cause-effect relationship to the reinforcement of mitochondrial resistance via a GSK-3β-dependent mechanism.

Measurement of oxygen consumption confirmed the protective effect of morphine and SB in vivo as they improved the respiration of mitochondria subjected to ischemia-reperfusion and their capacity to synthesize ATP. Furthermore, using the uncoupling agent FCCP, we showed that the improvement of the rate of ATP synthesis was caused by the restoration of the activity of the electron transport chain. The mechanism by which morphine exerts this effect is currently unknown, but a plausible hypothesis is that morphine might reduce a factor that favors both the electron transfer chain inhibition and mPTP opening during reperfusion. Reactive oxygen species production is probably such a factor (Solaini and Harris, 2005; Zini et al., 2007), and ROS, which are produced during the first minutes of reperfusion, are well known to be involved in myocardial injury. The present results showing that morphine inhibits lipid peroxidation support the notion that the inhibition of reactive oxygen species generation is involved in the cardioprotective effect of morphine.

Altogether, these observations strongly suggest an essential role of inhibition of mPTP opening in the prevention of reperfusion injury by morphine, confirming the data obtained with specific inhibitors of mPTP, such as cyclosporine A and sangliferin-A (Hausenloy et al., 2004; Shanmuganathan et al., 2005), and its role in pre- and postconditioning cardioprotection (Javadov et al., 2003; Argaud et al., 2005).

Because in vivo experiments demonstrating that mPTP opening was influenced by morphine and SB did not allow us to establish definitely a cause-effect relationship between the infarct-limiting effect of these drugs and the prevention of mPTP opening (indeed, any cardioprotective agent could produce the same results), we decided to investigate the effect of morphine in a model of isolated cardiomyocytes mimicking ischemia-reperfusion and measuring mPTP opening directly as a function of time. This model consists of beating adult cardiomyocytes loaded with calcein, a relevant marker to detect mPTP opening, subjected to 30 min of severe hypoxia in the presence of a metabolic blocker. The advantage of this model is to match optimally the conditions encountered during ischemia-reperfusion contrary to other protocols, such as permeabilization of the cellular membrane in the presence of high Ca²⁺ concentration or exposure of cells to pro-oxidant agents, such as H₂O₂ (Park et al., 2006) or generation of ROS generated by laser illumination (Juhaszova et al., 2004; Kadono et al., 2006). It should be noted that neither cell death nor mPTP opening could be obtained in the absence of electrical stimulation highlighting its crucial role in this model.

Our data show that reoxygenation induced the release of calcein from mitochondria according to two different kinetic profiles, with a rapid release associated with cell death (as demonstrated by propidium iodide staining), preceded or not by a slow gradual decrease in calcein fluorescence that is not detrimental to cell viability (no propidium iodide staining of the cells occurred). These two phenomena are both delayed by cyclosporine A, indicating that they are due to mPTP opening, and suggest that they correspond to the transient (reversible) and long-lasting openings (irreversible) of the pore as described previously (Hüser and Blatter, 1999; Sharov et al., 2007). Our study reveals for the first time that morphine and SB are as efficient as cyclosporine A in preventing mPTP opening. Although the drugs delayed but did not block the mPTP opening, we might question whether delaying opening is beneficial physiologically inasmuch as the opening of the pore is the injurious event. We have no answer to this question presently; however, our results clearly demonstrate that this effect is associated with a concomitant inhibition of cell death in accordance with the infarct-limiting effect of the drugs observed in vivo.

We further found that the PI3-kinase inhibitor, wortmannin, suppressed the mPTP inhibiting effect of morphine and that SB mimicked the effect of morphine by reducing infarct size and delaying mPTP opening in isolated cardiomyocytes. This confirms that GSK-3β inhibition plays a critical role in the cardioprotective effect of morphine (Gross et al., 2004) and demonstrates a close link between GSK-3β inactivation and mPTP inhibition in the action of these drugs, reinforcing the recent data of Gross et al. (2008) on delayed cardioprotection. However, the exact mechanism by which GSK-3β inactivation leads to mPTP inhibition remains to be determined. A recent study showed that the myocardial protection afforded by ischemic preconditioning in the presence of erythropoietin might be due to the interaction of the inactivated form of GSK-3β with adenine nucleotide translocase, a component of mPTP (Zoratti and Szabò, 1995) decreasing the affinity of cyclophilin D to the adenine nucleotide translocase and subsequent inhibition of mPTP opening (Nishihara et al., 2007).

Given that GSK-3β inactivation appears to be a general mechanism involved in cardioprotection afforded by different agents, such as bradykinin (Park et al., 2006), adenosine receptor agonists (Förster et al., 2006), erythropoietin (Nishihara et al., 2006), and ischemic preconditioning (Tong et al., 2002), the binding of the inactivated form of GSK-3β to the adenine nucleotide translocase may be also responsible for mPTP inhibition by morphine (Nishihara et al., 2007). Moreover, GSK-3β inhibition was shown to reduce the production of ROS by mitochondria (Juhaszova et al., 2004).

In summary, our data demonstrate that the cardioprotective effect of morphine observed in vitro in a model of anoxia-reperfusion on isolated cardiomyocytes isolated from adult rats is related to inhibition of mPTP opening through the PI3-kinase pathway and inactivation of GSK-3β. Based on our data obtained in vivo and in vitro, we demonstrate that the infarct-limiting effect of morphine and SB is linked to a cause-effect relationship to the reinforcement of mitochondrial resistance via a GSK-3β-dependent mechanism.

	Footnotes

 
This work was supported by Grant 2004004775 from the Fondation de France.

F.N.O. was supported by the Ministère de la Recherche et de la Technologie.

C.P.-M. was supported by the Fondation Lefoulon Delalande-Institut de France.

R.A. was supported by a doctoral grant from Région Ile-de-France.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.108.138008.

ABBREVIATIONS: mPTP, mitochondrial permeability transition pore; AAR, area at risk; calcein-AM, calcein acetoxymethyl ester; FCCP, carbonyl cyanide 4-trifluoromethoxyphenylhydrazone; ROS, reactive oxygen species; GSK-3β, glycogen synthase kinase-3β; PI3-kinase, phosphatidyl-inositol 3-kinase; I/R, ischemia-reperfusion; SB216763 (SB), 3-(2,4-dichlorophenyl)-4(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione; TBAR, thiobarbituric acid-reactive substance; T_mPTP, time for mitochondrial permeability transition pore opening.

¹ Both authors contributed equally to this work.

Address correspondence to: Dr. Alain Berdeaux, INSERM U 841, équipe 3, Faculté de Médecine de Créteil, 8 rue du Général Sarrail, F-94010 Créteil, France. E-mail: alain.berdeaux{at}inserm.fr

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