Trimetazidine Counteracts the Hepatic Injury Associated with Ischemia-Reperfusion by Preserving Mitochondrial Function AZIZ ELIMADI, ABDELLATIF SETTAF, DIDIER MORIN, ROSA SAPENA, FATIMA LAMCHOURI, YAHIA CHERRAH and JEAN-PAUL TILLEMENT

Recent studies suggest a crucial role played by mitochondria in the pathogenesis of ischemia-reperfusion injury. This study was conducted to clarify the role of trimetazidine, a cellular antiischemic agent, on mitochondria isolated from rat liver subjected to 120-min normothermic ischemia followed by 30-min reperfusion. Rats were divided into groups, pretreated with different doses of trimetazidine (5, 10 and 20 mg/kg/day) or saline and subjected to the ischemia-reperfusion process; another group served as the sham-operated controls. Alanine aminotransferase and aspartate aminotransferase activities and hepatocyte ATP content, bile flow and mitochondrial functions were assessed. Ischemia-reperfusion caused membrane leakage from hepatocytes and a decrease in ATP content and in bile flow. These effects were well correlated with alterations in mitochondrial function, namely, decrease in ATP synthesis, NAD(P)H level and mitochondrial membrane potential and generation of mitochondrial permeability transition. The pretreatment of rats with trimetazidine prevented these ischemia-reperfusion deleterious effects at both the cellular and mitochondrial level in a dose-dependent manner. It is concluded that trimetazidine at an optimal dosage of 10 mg/kg/day protects mitochondria against the deleterious effects of ischemia-reperfusion. This protective effect appears to be the key factor through which this drug exerts its cytoprotective activity. Trimetazidine [1-(2,3,4-trimethoxybenzyl)-piperazine dihydrochloride; Vastarel] has been described as a cellular anti-ischemic agent both in experimental conditions and in clinical trials (for review, see Harpey et al., 1989). By using isolated cardiac quiescent myocytes prepared from rat heart ventricles, Cruz et al. (1987) have shown that under anoxic conditions, trimetazidine improved the resistance of these cells to the effects of high concentrations of Ca. Their ATP content was maintained at almost the control value, and the K leakage was reduced. It has been shown using an experimental model of liver ischemia-reperfusion that pretreatment with trimetazidine limited the extent of the pathological dysfunctions, namely the increase in plasma membrane aminotransferases and the decrease in biliary flow and in hepatocyte ATP content (Tsimoyiannis et al., 1993). Moreover, Guarnieri and Muscari (1993) demonstrated that trimetazidine improved the functions of mitochondria isolated from hypertrophied perfused rat hearts. Recently, we have shown that trimetazidine protected isolated liver mitochondria against the deleterious effects of t-BH, a free radical generator, which when associated with Ca overload induced the MPT (Elimadi et al., 1997). Salducci et al. (1996) have also demonstrated, using the same mitochondrial preparation, that trimetazidine restored ATP synthesis previously decreased by CsA. In a clinical study, Detry (1993) has shown that trimetazidine administration significantly improved exercise tolerance, namely total work, duration of exercise and time to 1-mm ST-segment depression, without changing the ratezpressure product of patients with angina. Although its effects have been demonstrated, the mechanism or mechanisms of action of this drug are not fully elucidated. Because mitochondria are the cellular organelles most affected by ischemia-reperfusion, our objectives were to check the protective effect of trimetazidine on liver functions, to define its dose dependency and to gain insight into the mechanism of action of this drug. For this purpose, an experimental protocol associating normothermic ischemia and then reperfusion of rat liver in situ was used in which rats were pretreated with increasing doses of trimetazidine. At the end of the experiment, mitochondria were extracted and checked for respiratory control, membrane potential, resistance to induced swelling and NAD(P)H level. Received for publication December 17, 1997. 1 This work was supported by grants from the Réseau de Pharmacologie Clinique, the Ministère de l’Education Nationale (EA 427) and the Institut de Recherches Internationales Servier. ABBREVIATIONS: MPT, mitochondrial permeability transition; t-BH, t-butylhydroperoxide; CsA, cyclosporin A; RCR, respiratory control ratio; ASAT, aspartate aminotransferase; ALAT, alanine aminotransferase; Dc, mitochondrial membrane potential; ROS, reactive oxygen species. 0022-3565/98/2861-0023$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 286, No. 1 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 286:23–28, 1998 23 at A PE T Jornals on July 8, 2017 jpet.asjournals.org D ow nladed from Materials and Methods Drug administration. Adult male Wistar rats, weighing 250 to 300 g (Janvier, le Genest-St-Isle, France), were used in this study. All animal procedures used are in strict accordance with the French agency’s policies (ministere de l’AgricuPture et de Pa Foret, authorization N° 00768) about animal experimentation. Animals were divided into five groups (15 rats in each). A nontreated group and three treated groups were subjected to 120 min of normothermic liver ischemia followed by a 30-min reperfusion protocol. Animals in the treated groups were randomly allocated to each trimetazidine (Servier Laboratories, Neuilly, France) pretreatment of 5 mg/kg (n 5 15), 10 mg/kg (n 5 15) or 20 mg/kg (n 5 15), whereas the nontreated group received the same quantity of saline solution. Trimetazidine was administered by intramuscular injection each day for 7 days before the induction of ischemia. Sham-operated group (n 5 15) received the same surgical procedure as the other groups without being subjected to the ischemia-reperfusion protocol. Trimetazidine solution was prepared daily; it was dissolved in saline [0.9% NaCl (w/v)] and appropriately warmed to body temperature before injection Surgical procedure. The technique of liver ischemia described by Nauta et al. (1989) was used in this study. The surgical procedure was performed 1 hr after the last drug or saline administration with the animals under general anesthesia using rectified ether. After section of the ligaments of the liver, hepatic normothermic ischemia was induced for 120 min by hilum clamping of the hepatic pedicles of segments I to V. To preclude the vascular congestion of the alimentary tract, the blood supply by the portal pedicles of segments VI and VII was not interrupted. During the period of ischemia, 0.5 ml of saline was given through the dorsal vein of the penis every 30 min to maintain hemodynamic stability. Bile was collected via the cannulation of the common bile duct with a fine catheter (Biotrol, Paris, France). Reperfusion was established by removal of the clamps. After a 30-min reperfusion, the animals were killed, and their livers were immediately removed; mitochondria were isolated according to the procedure described below. Liver function tests. Blood samples for measurement of ASAT and ALAT activities were collected after a 30-min reperfusion. Plasma enzymes activities were determined by enzymatic technique using a Boehringer-Mannheim (Mannheim, Germany) kit. The hepatic ATP content was determined by enzymatic procedure according to the method of Jaworec et al. (1974). Isolation of mitochondria. Rat liver mitochondria were isolated as described by Johnson and Lardy (1967). Briefly, after the rats were killed, liver was excised rapidly and placed in medium containing 250 mM sucrose, 10 mM Tris and 1 mM the chelator EGTA, pH 7.8, at 4°C. The tissue was scissor minced and homogenized on ice using a Teflon Potter homogenizer. The homogenate was centrifuged at 600 3 g for 10 min (Sorvall RC 28 S). The supernatant was centrifuged for 5 min at 15,000 3 g to obtain the mitochondrial pellet. The latter was washed with the same medium and centrifuged at 15,000 3 g for 5 min. Then, the resulting mitochondrial pellet was washed with the same medium from which the EGTA was omitted and centrifuged for 5 min at 15,000 3 g, resulting in a final pellet containing ;50 mg of protein/ml. The protein content was determined by the method of Lowry et al. (1951). The mitochondrial suspension was stored on ice before the assay of membrane potential, mitochondrial swelling, NAD(P)H level and mitochondrial respiration. Optical monitoring of mitochondrial membrane potential. Mitochondrial membrane potential (Dc) was evaluated from the uptake of rhodamine 123 (Interchim, Montlucon, France), which accumulates electrophoretically into energized mitochondria in response to their negative-inside membrane potential (Emaus et al., 1986). Then, 1800 ml of the phosphate buffer (250 mM sucrose, 5 mM KH2PO4, pH 7.2 at 25°C), 3 mM succinate and 0.3 mM rhodamine 123 were added to the cuvette, and the fluorescence scanning of the rhodamine 123 was monitored using a Perkin-Elmer SA (Courtaboeuf, France) LS 50B fluorescence spectrometer. After 30 sec, mitochondria (0.5 mg/ml) were added. The Dc was calculated according to the Nernst equation. Mitochondrial swelling measurements. Mitochondrial swelling was assessed by measuring the change in absorbance of the suspension at 520 nm by using a Hitachi (ScienceTec, les Ulis, France) model U-3000 spectrophotometer, according to the procedure described by Halestrap and Davidson (1990), with some modi-