Trimetazidine: In vitro influence on heart mitochondrial function

Trimetazidine: In vitro Mitochondrial Luc Demaison,

PhD, Elisabeth

Influence Funtlion

Fantini, PhD, Emmanuelle Sentex, and Pierre Athias, PhD

on Heart PhD, Alain

Grynberg,

PhD,

The mechanism of action of the antianginal trimetazidine (TMZ) remains largely unknown. In cultured rat ventricular myocytes in physiologic conditions, TMZ (5 x lo-* M) reduced the plateau potential level, the upstroke velocity, and the spontaneous action potential rate. When the cardiomyocytes were submitted to hypoxia (150 or 240 minutes) in a glucose-free medium, treatment with TMZ largely prevented the hypoxia-induced electromechanical alterations, i.e., the decrease in plateau amplitude, in resting membrane potential, in action potential duration, in rate, and in contractility. No hypoxiainduced arrhythmia was observed in the TMZtreated cells. Moreover, the lactate dehydrogenase leakage was significantly reduced in the TMZtreated cardiomyocytes (-58% and -36%, after 150 and 240 minutes of hypoxia, respectively). The drug was not efficient in reducing the hypoxiainduced decrease in adenosine triphosphate (ATP) content. The cellular ATP content was slightly lower in the TMZ-treated cells in normoxic conditions and

in hypoxic conditions, but only in the glucose-free medium. To investigate further the relation between TMZ and energy metabolism, the respiration pammeters were measured in heart mitochondria isolated from control and TMZ-treated mts (6 mg/kg/day, 7 days) with different substrates. This treatment resulted in a slight altemtion of pyruvate oxidation, which was obsewed in the absence and in the presence of TMZ (lo-* M) in the respiration medium. Conversely, a potent inhibition of palmitoylcamitine oxidation was measured when TMZ was added to the respimtion medium. Neither pretreatment of the rats, nor addition of TMZ to the medium affected the oxidation of glutamate or citrate. In conclusion, pretreatment of ventricular myocytes with TMZ resulted in an increased cell resistance to hypoxic stress, as was evident from electromechanical functions and lactate dehydrogenase leakage. This cytoprotective action of TMZ could be related to an effect of the drug in the fatty acid metabolism. (Am J Cardioll995; 76:318-378)

T

TMZ was reported to decrease the cardiac work under normoxic conditions and thus to favor energy preservation by limiting the fall in intracellular adenosine triphosphate (ATP) content due to ischemic conditions.8 Most of the data obtained suggest that TMZ exerts its anti-ischemic effect at the cellular level. For this reason, this study was focused on the effects of TMZ on the electromechanical and biochemical impairments induced in cultured rat ventricular myocytes by hypoxia and on respiration characteristics of isolated rat cardiac mitochondria.

rimetazidine (1-[2,3,4-trimethoxy-benzyl] piperazine HCl; TMZ) was introduced as an antianginal drug 25 years ago1 and was reported to exert beneficial effects on ischemic injury.2,3 In the myocardium, the occurrence of an hypoxic and/or ischemic event induces major functional disturbances (such as arrhythmias and contractile failure), which are most often associated with biochemical alterations in energy metabolisrnM and/or in membrane integrity, as shown by cytoplasmic enzyme leakage.7 In vitro experiments outlined the cardioprotective effect of TMZ, suggesting that the molecule may limit the high energy phosphate depletion* and interfere with calcium accumulation and/or intracellular acidosis.9 Experimental data with TMZ indicates a decrease of the intracellular accumulation of Ca2+ and Na+ and protection of cardiac cells against the accumulation of H+, thus limiting acidosis.9 Moreover, a reduction of the oxygen radical-mediated myocardial damage was suggested.*O More recently, TMZ was reported to inhibit the Na+,K+-pump in guinea l1 In the isolated rat heart, pig ventricular muscles. From I.N.R.A., Unit6 de Nutrition Lipidique, Physiologie, Faculti? de MBdecine, Diion, Address for reprints: Alain Grynberg, 21034 Diion, France.

Diion, and Laboratoire de France. PhD, I.N.R.A., 17 rue Sully,

METHODS Cultured cardiomyocytes: The myocytes were isolated and cultured according to published procedures.‘“l3 The ventricles were minced to small fragments, which were submitted to a proteolytic dissociation with trypsin (Difco). The proportion of myocytes in the cell suspension was increased by a 2-step selective adhesion procedure.14 The isolated myocytes were then seeded in 60-mm plastic tissue culture dishes (Falcon Primaria, BectonDickinson) at a density of 2 x lo6 cells per dish. The growth medium was composed of Ham’s F-10 basal medium supplemented with 10% fetal calf serum (Seromed), 10% human serum (1:l mix of A

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preparations, and the statistical treatment was the same as that described for lactate dehydrogenase. Mitochondria: Male Wistar rats (n = 20) received either 2 intraperitoneal injections of TMZ per day during 7 days (6 mg/kg/day) or only vehicle. The hearts were then excised and the mitochondria were isolated.22 Their oxygen consumption was measured polarographically at 37” C in KH2P04/Tris HCI (10 rm’t4 each, pH 7.4) containing sucrose (250 m&f), EDTA (2 mM), and 0.1% fatty acid-free albumin. 22 After addition of mitochondria (0.85 mg protein/ml) and substrate, state 3 respiration was initiated by addition of ADP (166 nmol). All the parameters (state 3 and state 4 respiration rates, respiratory control index, ADP/ oxygen, and rates of ATP production) were determined following the third addition of ADP. The reaction mixture contained malate (2.5 mM) and 1 of the following substrates: citrate (10 mM), glutamate (10 mM), pyruvate (10 n&f), or palmitoylcarnitine (25 CLM). Each measurement was made in these conditions and repeated in the presence of TMZ in the respiration medium (0.1 n&f). The results were expressed as mean 2 SEM and were submitted to a l-way analysis of variance.20

and 0 groups), 100 U/mL penicillin, and 100 p.g/mL streptomycin. l2 The cells were incubated at 37” C in a humidified atmosphere (5% CO2, 19% 02, and 76% N2). The growth medium was renewed 1 day after seeding, and every 2 days thereafter. The experiments were conducted on 5-day-old cultures that were treated with TMZ (stock solution in Puck’s saline). Additions of either TMZ (500 CLM) or vehicle in the control dishes were done 16 hours and 1 hour before and at the beginning of the experiment. Hypoxia and reoxygenation: The growth medium was replaced by glucose-free, Puck’s F balanced salt solution covered with a paraffin oil layer.13 Hypoxia was conducted in a 24-well device already described, l5 allowing the simultaneous exposure of 4 groups of 6 dishes (3 control and 3 TMZ-treated dishes) to 4 different experimental conditions: 4-hour normoxic control, 2.5-hour hypoxia, 4-hour hypoxia, and 2.5-hour hypoxia followed by 1.5-hour reoxygenation. Electrical and contractile act’tities: The culture dish was fitted in a gas-controlled microchamber16 on the stage of an inverted phase-contrast microscope (Leitz Diavert) at 36 + 0.1” C. The transmembrane potentials were recorded using 3 M KClfilled microfiber glass capillary microelectrodes, with 62 MR average resistance and connected to a Biologic VF-180 amplifier.17 The contractions were recorded by the use of an online video motion detector (Nestor) already described.18 The electrical and motion signals were acquired and analyzed using a PC/AT computer-based system. lactate dehydrogenase release: At the end of the experiment, the cells were harvested and homogenized, and lactate dehydrogenase activity was measured in the medium and the cell homogenate using a commercial kit (Biomerieux). The lactate dehydrogenase release was expressed as the percentage of the total lactate dehydrogenase activity.19 The whole experiment was repeated 3 times on 3 different culture preparations and the data were submitted to a 3-way analysis of variance20 with 3 fixed factors (TMZ, hypoxia condition, and culture preparation). Only the TMZ factor was considered in the result section. Adenine nucleotides: Adenine nucleotides were measured according to Jones.21 The cell layer was homogenized in 0.6 M perchloric acid, and ATP, adenosine diphosphate (ADP), and adenosine monophosphate (AMP) were determined by reverse-phase high-performance liquid chromatography using xanthine as internal standard. The whole experiment was repeated on 3 different culture 328

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RESULTS Heart cell culture: Figure 1 shows the action potentials and the contractions continuously recorded before and after the administration of TMZ (5 x 10e4 M> in normal and hypoxic conditions. The TMZ treatment caused the action potential amplitude (Figure 1C) decrease due to a depression of the overshooting plateau potential level, whereas the resting membrane potential was not altered. The drug induced a strong decrease in upstroke velocity (compare Figure 1A and C, lower traces) and in the spontaneous rate. The action potential duration at 80% repolarization was, in turn, increased. The contraction contour was not affected by TMZ, which did not induce rhythm abnormalities. Hypoxia induced typical functional alterations.17 After 60 minutes of oxygen depletion and in the absence of TMZ (Figure lB), the spontaneous electrical activity persisted, whereas the cell motion was arrested. The main electrophysiologic effect of hypoxia was a drastic decrease in action potential duration. At the same time, a decrease in action potential amplitude was observed, which was mainly due to the decrease in plateau potential level. However, the Na+-dependent fast upstroke of the action potential remained unmodified (Figure 1 A and B, lower traces). Moreover, failure of cell-to-cell coupling and early 76

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postdepolarizations (Figure lB, arrows) were generated under hypoxic conditions. In TMZ-treated cells, the action potential and contraction contours after 60 minutes of hypoxia (Figure 1D) were not different from those recorded before the oxygen deprivation. The treatment with TMZ prevented the hypoxia-induced arrhythmias (compare with Figure 1B) and the fall in plateau amplitude. However, the TMZ-induced decrease in upstroke velocity (V,,) and in spontaneous rate observed in normoxia (Figure lC, lower trace) were slightly enhanced during hypoxia (Figure lD, lower trace). TMZ prevented also the hypoxia-induced shortening of action potential duration at 40% and 80% repolarization. Hypoxia induced significant lactate dehydrogenase release in cultured rat ventricular myocytes, which reached approximately 20% and 40% of total cell lactate dehydrogenase after 2.5 and 4 hours of hypoxia, respectively (Table I). In normoxie conditions, TMZ did not affect the lactate dehydrogenase leakage. Conversely, after 2.5 and 4 hours of hypoxia, the lactate dehydrogenase release was significantly (p
FIGURE 1. Action potential and contraction simultaneously recorded before (A, C) and after (B, D) 60 minutes of hypoxia in cultured rat ventricular myocytes. Upper traces: untreated cells (A, B). Lower traces: trimetaxidine (TMZ; 5 x 1 O-‘M) treated cells (C, D). For each row, all records were obtained from the same heart cell culture. Trace significance: ap: action potential; c: contraction; dv/ dk first derivative of the potential changes. B, note triggered activity initiated from early postdepolarixation (filled arrow) and failing intercellular electrotonic coupling (open arrows); these abnormalities were absent in D. CTRL = control; N2 = nitrogen.

TABLE I Lactate

Dehydrogenase Release, Expressed Percentage of Total Activity, from Control and Trimetazidine-Treated Heart Cells in Culture

Control NC H2.5 H4

9.1 -t 1.08 18.5 '- 2.38 48.5 k 2.37

as

Treated

p Value

10.2 k 1.07 8.3 t 1.03 31.0 + 3.73

NS
Data ore mems f SEM. NC = normcwc control; HZ 5 = 2.5 hours of hypoxia; H4 = 4 hours of hypoxia. NS = difference not significant.

crease in cellular ATP content without major alteration in the ADP and AMP content (Figure 2). The data show that the TMZ treatment was not able to modify the evolution of adenine nucleotides during hypoxia. This was evident from the nonsignificant cross-interaction between the “TMZ” factor and the “hypoxia treatment” factor. Conversely, the analysis of variance revealed a significant overall TMZ effect resulting in a moderately decreased cellular ATP content, which can be observed even in normoxic controls (Figure 2). This indicated that the TMZ effect was not specifically related to hypoxia. Since these experiments were conducted in a glucose-free medium, the 4 hours of normoxic control was repeated with and without glucose in the bathing fluid. In the presence of glucose, the treatment by TMZ did not induce a significant decrease in cellular ATP content (Figure 3), suggesting that the TMZ-induced decrease in ATP mentioned above was due to interference between the drug and the substrate metabolism.

CTRL,

TM2

0

CTRL,

TMZ

500

200

ms

C

D 200

pM

TMZ

500

RM

60 min

N2

ms

“&&-J

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State III

_

250

*

0 200

State IV

ADP 100

6

0

3

4

ADPlO

0

0

Ctrl

=

TMZ

2

AMP 6

0 1.0

V ATP *

0.5

NC

H2.5

H4

0.0

FIGURE 2. Effect of trimetaxidine (TMZ) on adenine nucleotide content (w per dish) in cultured rat cardiomyocytes submii to 4 hours of nonnoxia (NC), 2.5 hours of hypoxia (H2.5), or 4 hours of hypoxia (H4) in a glucose-free medium. ANOVA is shown in inset: TMZ is the drug factor, C-l is the cross-intemdion between the drug factor and the hypoxia. N.S. = not signikmt. ADP = adenosine diphosphate; AMP = adenosine monophosphate.

Cit

-

0

Ctrl

~

TM2

5-

OFIGURE 3. Effect of trimetaxidine (TMZ) on the adenosine triphosphate (ATP) content (pg per dish) in cultured rat cardiomyocytes submii to 4 hours of normoxia in the presence of glucose (11 mM). Ctrl = control.

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Heart mitochondria: Figure 4 shows a comparison of the respiration parameters in mitochondria isolated from the hearts of either control or TMZtreated rats. When the mitochondria were supplied with citrate, glutamate, or palmitoylcarnitine, the respiration parameters (states 3 and 4 respiration rates, ADP/oxygen, and rate of ATP production) were not affected by the pretreatment of the rats with TMZ. Conversely, when the substrate was pyruvate, the chronic pretreatment of the rats with TMZ induced a moderate but significant reduction of the rates of oxygen consumption. The amplitude of this effect was the same for states 3 and 4 (- 11% and -12%, respectively), since the respiratory control index (state 3/state 4) was not significantly affected (data not shown). Moreover, the chronic

10 -

34B

Pear

FIGURE 4. Influence ofthe chronic treatment of rats with trimetaxidine (TMZ) on the respiration pammeters of the heart mitochondria with different substrates: citrate (Cit), glutamate (Glu), palmitoykamitine (Pear), or pyrwate (Pyr). (State 3 and state 4 respiration rates are in ng atoms 02/ min/mg protein; adenosine diphosphate/oxygen (ADP/O) in nmol ADP/ng atom 0,; and the rate of adenosine triphosphate (ATP) production (V,, in nmol/min/mg protein). *p <0.05; **p eo.01; ***p
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production rate (approximately -60%). However, the metabolic efficiency was not modified by the presence of TMZ, as shown by the unaltered ADP/oxygen ratio. Moreover, the respiratory control index was also reduced in the presence of TMZ (-43%) pointing out that state 3 is more sensitive to this effect than state 4.

State III 250

0 200

State IV

DISCUSSION

0

*** OH Im

4

ADP/O

100

0

Ctrl

=

TilK!

2

0 1 .o

V ATP

0.5

0.0

Cit

Pear

Glu

W

FIGURE 5: Influence of the presence of trimetazidine (TMZ) in the medium on the respiration parameters of the heati mitochondria with different substrates: citrate (Cit), glutamate (Glu), palmitoylcamitine (Pear), or pyruvate (Pyr). (State 3 and state 4 respiration rates are in ng atoms 02/ min/mg protein; adenosine diphosphate/oxygen (ADP/O) in nmol ADP/ng atom 0,; and the rate of adenosine triphosphate (ATP) production (V,,W in nmol/min/mg protein). *p eo.05; **p
pretreatment resulted in a reduced rate of ATP production when pyruvate was used as substrate. A second set of investigations was focused on the direct effect of TMZ on mitochondria, by comparing the presence and absence of the drug (100 CLM) in the respiration medium (Figure 5). The respiration parameters were not affected by TMZ when citrate or glutamate were supplied as substrate. Interestingly, when TMZ was present in the respiration medium, neither the oxidation of pyruvate nor its metabolic efficiency in energy production was altered. On the contrary, the oxidation of palmitoylcarnitine was strongly and significantly influenced by the presence of TMZ in the respiration medium, which resulted in a large decrease in states 3 and 4 respiration rates (approxi‘mately -60% and -3O%, respectively) and in ATP

TMZ has been reported to exert a pronounced beneficial effect on the ischemic myocardium,3,23 although no alteration of coronary flow could be observed in isolated working hearts.” The data reported here indicate that TMZ (at a concentration of 500 l&f in the bathing fluid) can exert a pronounced protective effect on isolated cardiomyocytes in culture during hypoxia. Under physiologic conditions, TMZ caused a decline in the plateau potential level, suggesting a depressing effect on the calcium permeability. Consistently, Kiyosue et a125 demonstrated that TMZ was able to decrease the peak amplitude of calcium current, to block the slow inward current, and to decrease the intracellular calcium concentration. Moreover, the upstroke velocity (V,,) is decreased in the presence of TMZ, which indicated clearly a block of the fast inward sodium current. In this connection, TMZ was recently reported to alter the fluidity of the outer leaflet of the plasma membrane in human platelets and erythrocyte ghosts.26 TMZ also decreased the spontaneous rate. Lavanchy et al8 reported also the negative chronotropic effects of TMZ, at high concentrations, in isolated rat hearts. This negative chronotropic response in culture may result from the assumed calcium antagonistic action of the drug, since in this model the spontaneous rate is dependent on external calcium and sensitive to calcium antagonist drugs.27Js TMZ treatment led to a lengthening of the overall action potential duration. This observation would suggest an additional influence of TMZ on the repolarizing potassium conductance, which has been also evoked by Kiyosue et al. 25 In turn, TMZ had no major effect on the contraction parameters. In substratefree conditions, but in absence of TMZ in the bath, hypoxia induced a decline in plateau potential level and in action potential duration and rate, accompanied by contractile failure and rhythm abnormalities. The data presented here show that the treatment of cells with TMZ strongly reduced these deleterious functional effects of hypoxia: the shortening of AP duration and the hypoxia-induced arrhythmias were prevented, and the contraction was maintained.

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heart mitochondria displaying the same characteristics. Since TMZ did not affect the oxidation of citrate, its effect could be located either to the citrate synthase activity or to the beta-oxidation pathway (including the inner membrane palmitoylcarnitine translocator, the palmitoylcarnitinecoenzyme A transferase II, or the enzymes involved in beta oxidation itself). A mechanism related to a citrate synthase inhibition would probably have resulted in a modified pyruvate oxidation, and the results obtained in this study mainly argue for an interference of TMZ with the betaoxidation pathway. Conversely, 7 days of pretreatment of the rats with TMZ resulted in heart mitochondria that displayed a slightly reduced pyruvate oxidizing capacity, although TMZ was not present during the measurements. The reasons for this decrease cannot be explained from the present results. If the above hypothesis as to the effect of TMZ on beta oxidation could be extrapolated to the in vivo situation, the l-week pretreatment may have induced a l-week in vivo inhibition of fatty acid oxidation, which could, in turn, affect some enzyme activities (pyruvate dehydrogenase, for instance) or some specific pools (coenzyme A or carnitine or their acylated derivatives). Further, this TMZinduced decrease in pyruvate oxidation was very weak, compared with that noticed in fatty acid oxidation in the presence of TMZ. This suggests that TMZ may favor the utilization of nonlipidic substrates at the detriment of fatty acid oxidation, which could be considered as a “preischemic” metabolic switchb5 This is in agreement with the data obtained on the adenine nucleotide pools in cultured cardiomyocytes: when glucose was not supplied, TMZ induced a slight but significant ATP decrease, which was similar in normoxia and in hypoxia. This decrease, which could be related to a lowered fatty acid oxidation, was not observed in the presence of glucose in the incubation medium, indicating that exogenous glucose was sufficient to satisfy the energy requirement of the cells in these conditions. In hypoxic conditions, the isolated cardiomyocytes in culture may display up to 50% lactate dehydrogenase release without major alteration of their membrane integrity.17 Moreover, when these cells are submitted to hypoxia in the presence of glucose, lactate dehydrogenase leakage is avoided. The hypoxia-induced lactate dehydrogenase leakage in culture cardiomyocytes (before they reach the state of necrosis) could thus be considered as an adaptation phenomenon, which contributes to

However, the effects of TMZ observed in normoxie conditions (mainly the potassium-blocking action and the decreased calcium entry) were also observed during hypoxia. The hypoxia-induced rhythm disturbances being mainly caused by early postdepolarizations and favored by the action potential shortening in our cell culture model, it seems reasonable to assume that the antiarrhythmic action of TMZ was due to its faculty to maintain action potential duration and hence to prolong the refractory period. Also, TMZ tended to block inward sodium current and thus behaved like a local anesthetic antiarrhythmic compound.29T30 The cytoprotective effect of TMZ was also evident from a large decrease of lactate dehydrogenase leakage in both short-term (2.5 hours) and long-term (4 hours) hypoxia. These 2 conditions correspond in this model to “reactivable cells” and “no-more reactivable cells,” respectively.i7 Hypoxia resulted in a large decrease of the cellular ATP content. Using isolated arrested rat hearts maintained in a cold cardioplegic solution, Aussedat et aP1 suggested that TMZ may improve ATP preservation. In the cellular model used in this study, TMZ was not able to slow the hypoxiainduced decrease in ATP content. The cytoprotective effect of TMZ evident from the lactate dehydrogenase release during hypoxia should not be attributed to a slackening of energy loss. However, the results of adenine nucleotide screening suggest that the pretreatment with TMZ could have induced a small global decrease in cellular ATP content, which can be observed in normoxia as well as in hypoxia. This moderate TMZ effect in the normoxic conditions could be related to a differential behavior toward the absence of oxidizable substrate, since the cardiomyocytes analyzed for their response to TMZ were maintained in glucosefree medium throughout the experimental protoc01.l~ In agreement with this hypothesis, this TMZ effect on ATP content was not observed when the cardiomyocytes were incubated in similar normoxic conditions but in glucose-containing bathing fluid. These results appear consistent with the data reported by Lavanchy et a1.8 In working hearts submitted to global ischemia and reperfusion, the treatment with TMZ did not result in a significantly higher ATP leve1.24 The results obtained with isolated cardiac mitochondria indicated that TMZ is a rapid and potent inhibitor of palmitoylcarnitine oxidation. This appears to be a direct effect of the presence of the drug during the course of respiration. Pretreatment of the rats in vivo with TMZ did not result in 36B

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the metabolic switch. According to this hypothesis, the TMZ-induced reduction of lactate dehydrogenase leakage in cardiomyocytes could be related to the metabolic effect of TMZ in mitochondria, since the drug may lead the cells to a prehypoxic metabolic switch characterized by a decreased oxygen cost of energy synthesis. TMZ, at high concentrations, has negative chronotropic and inotropic effects in isolated rat hearts.* In isolated cardiomyocytes, we also observed negative chronotropic effects. The protective effect of TMZ in isolated ventricular cells could thus be due to a mechanical decreased cell activity (eventually related to the metabolic alteration discussed above). This should be attributed neither to a Ca2+ antagonistic effect nor to a Ca2+ blocking activity-associated decrease of cellular activity,32 since TMZ is devoid of detectable Ca2+ blocking activity.9 In conclusion, the effect of TMZ should be mediated through a mechanism that could bring the cells either to a reduced activity state independent of Ca2+ homoeostasis, or to a “normoxic” metabolic switch. This study outlined the possibility that TMZ may induce a lowered utilization of lipidic substrates for energy production, but other mechanisms, such as a direct effect on membrane preservation or a local anesthetic-like effect, cannot be excluded. 1. Schmitt H. Toxio~logical

and Pharmacological Investigation of 404s. OrICans: Laboratories setier, 1964. 2. Dalla-Volta S, Maraglino G, Della-Valentina P, Desideri A. Comparison of trimetazidme with nifedipioe in effort angina: a double-blind cross-over study. CaWc Dmgs 77~ 1990;4:853-860. 3. Liiersa C, Honore E, Adamantidii M, Rouet E, Dupois B. Anti-ischemic effect of trimetazidioe-enzymatic and electric response in a model of in vitro myocardial ischemia. Cnrdiovasc Dogs Ther 1990$80%309. 4. Farbe JL, Chien KR, Mittnacht S. The pathogenesis of irreversible cell injury in ischemia.Aim JParhd 1981;102:271-281. 5. Opie LH. Myocardial metabolism in ischemia. In: Heusch G, ed. Pathophysiology and Rational Pharmaootherapy of Myocardial Ischemia. Darmstadt: Steinkopf Verlag, 1990:37-57. 6. Reimer KA, Jennings RB. Myocardial &hernia, hypoxia and infarction. In: Fozzard HA, Haber E, Jennings RB, Katz A, Morgan H, eds. The heart and cardiovascular system, vol. 2. New York: Raven Press, 1991:1875-1973. 7.Apple FS. Acute myocardial infarction and coronary reperfusion. Serum markers for the 1990s. Am J Clin Pathol1992$?217-226. 8. Lavanchy N, Martin J, Rossi A. Anti-ischemic effect of trimetazidine: 31-PNMR spectmswpy in the isolated rat heart. Arch Int Phamacodyn 1987@6:97110. 9. Renaud JF. Internal pH, Na+, and Caz+ regulation by trimetazidme during cardiac cell necrosis. Ca&wax Drug 7% 1988;1:677+86. 10. Guamieri C, Mwcari C. Beneficial effects of trimetidme on mitochondrial fimction and superoxide prodwtion in the cardiac muscle of monocrotalinetreated rats. Biochem Pharmmol1988,37:4685-&%

11. Hiitome I, fshiko R, Tanaka Y, Kosaka H, Hasegawa J, Yoshida A, Kotake H, Mashiba H, Arita M. Trimetazkime inhibits Na+,K+-ATPase activity, and overdrive hyperpolarization in guinea-pig ventricular muscles. Ew J Phunnacol1991;195:381-388. 12. Gtynberg A, Athias P, Degois M. Effect of change in growth environment on cultured myocardial cells investigated in a standardized medium. In V?tm Cell Dev Bid 1986;22:4446. 13. Athias P, Grynberg A. Electrophysiological studies on heart cells in cultore. In: Pin A, ed. Heart Cell in Cultures, vol. 1. Boca Raton, FL: CRC Press, 1987:1&158. 14. Blonde1 B, Roijen I, Cheneval JP. Heart cells in culture: a simple method for increasing the proportion of myoblasts. &wie&~ 1971;27:356-358, 15. Chevalier A, Demaison L, Gryxberg A, Athias P. Influence of the phosphw lipid polyunsaturated fatty acid composition on some metabolic disorders induced in rat cardiomywytes by hypoxia and reoxygenation. .I MO/ Cell Cardiol 1990,22:1177-1186. 16. Fantini E, Athias P, Courtois M, Grynberg A. A simple gas flow chamber for cultured cell eledrophysiology in a controlled atmosphere. ppiigers Arch Era JPhyd 1987;409,632-634. 17. Fantini E, Athias P, Courtois

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18. Comtois M, Khatami S, Fantini E, Athias P, Grynberg A. Polyunsaturated fatty acids in cultured cardiomyaytes: effect on physiology and fi-adrencceptor function. Am J Physiol1992:262zH451-H456. 19. Grynberg A, Fantini E, Athias P, Degois M, Guenot L, Comtois M, Khatami S. Modification of the n-6/n-3 fatty acid ratio in the phospholipids of rat ventricular myocytes in culture by the use of synthetic media: functional and biochemical consequences in normoxic and hypoxic conditions. J h401 Cell cardi 1988;20%63-874. 20. Dagnelie P. Th&xies et m&hodes statistiques, vol 2. Grembloux, Belgium: Presses Agmnomiques de Gembloux, 1975. 21. Jones DP. Determination of pyridme dinucleotides in cell extracts by high-performance liquid chromatography. J Chmmatcgr 1981;225:446-l49. 22. Sordhal LA, Crow C, BlaUcck ZR, Schwartz A. The mitochondrion. Methods Phalmacol1971;1:~7-2%.

23. Belcher P, Drake-Holland AJ, Hynd JW, Noble MIM. Trimetaziame reduces myocardial infarct size, relative area at risk, after temporary coronary artery occlusion in the rabbit. BrJPhamaco11992;107%5P. 24. Hugtenburg JG, Jap TJW, Mathy MJ, Van Heiningen PNM, Bohnenn VA, Heijnis JB, Boddedke HWGM, Van Zwieten PA. Cardioprotective effect of trimetazidine and nifedipine in guinea pig hearts subjected to ischaemia. Amh Int Phatmmdyn Ther 1989;300:186-208. 25. Kiyosue T, Nakamwa S, Arita M. Effects of trimetazidioe on action potentials and membrane currents of guinea-pig ventricular myocytes. J Mel cell cardio[ 1986$8:1301-1911. 26. Devynck MA, Sang KHLQ, Jotim Y, Mazeaud M. Acute membrane effects of trimetazidme in human platelets. Ew J phnrmacd 1993;245zlO~llO. 27. Athias P, Groz B, Klepping J. Determination of the ionic basis of the spontaneous activity of cultured rat heart cells using microinjection technique. Biol Cell 1980,37:183-188. 28. De la Chapelle-Groz B, Athias P. Gentamicin causes fast depression of action potential and contraction in cultured cardiies. Ew J Phmmacol 1988;152:111-120. 29. Reiser HJ, Sullivan ME. Antiarrhythmic drug therapy: new drugs and changing concepts. Fed Pmc 1986,45:2X%2212. 30. Frumin H, Kerin NZ, Rubenfire M. Classification of antiarrhythmic drugs. J Clin Phamacol1989;29:387-394. 31. Aussedat J, Ray A, Kay L, Verdys M, Harpey C, Rossi A. Improvement

of long-term preservation of isolated arrested rat hearts: beneficial effect of the anti-ischemic agent trimetazidioe. J Canliovasc Phamcol1993;21:1%135. 32. Patmore L, Duncan GP. Effects of calcium channel antagonists and facilitators on beating of primary cultures of embryonic chick. Br J Phamawl1988;95: 771-776.

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