Trimetazidine (1-[2,3,4-trimethoxibenzyl]-piperazine, TMZ) has been described as an antiischemic agent both in experimental conditions (1,2) and in clinical trials. Myocardium of patients with stable exercise-induced angina (3) and during percutaneous coronary angioplasty (4) or coronary artery graft surgery (5) appeared to be protected. In particular, TMZ was shown to reduce angina symptoms and to improve exercise tolerance. In surgery patients, TMZ was reported to reduce the so-called reperfusion injury (5).
The precise mechanism of action of TMZ is not fully known. The apparent lack of hemodynamic effects and its ability to prevent the decrease in myocyte ATP, to reduce intracellular acidosis, and to protect against oxygen free radical toxicity (6-8) has led to suggestion that TMZ may be cardioprotective by modulating myocardial metabolism and oxygen free radical formation.
Postischemic myocardial dysfunction (“stunning”) has been postulated to be mediated by oxygen free radicals and/or depletion of myocardial ATP (9,10). Therefore, in the present study, we assessed the potential of TMZ to reduce the extent of postischemic myocardial stunning resulting from 15-min coronary artery occlusion followed by 3-h reperfusion in an open-chest dog model. Myocardial adenine nucleotides and plasma lipid peroxides were measured. We also investigated whether contractile reserve recruitment by inotropic stimulation (dobutamine infusion) might be facilitated in TMZ-treated dogs as compared with controls.
MATERIALS AND METHODS
All animals used were maintained in accordance with the guidelines of the Committee on the Care and Use of Laboratory Animals of the American Heart Association. Adult anglonormand dogs of both sexes weighing 18-22 kg were used.
Anesthesia and surgical procedures
Dogs were premedicated with subcutaneous morphine sulfate (0.5 mg/kg). Anesthesia was induced with thiopental (25 mg/kg intravenously, i.v.). After tracheal intubation, dogs were ventilated with constant-volume intermittent positive-pressure at a rate of 15 breaths/min with a mixture of 50% oxygen and 50% nitrogen with a SF4 respirator (Robert et Carrière, Paris, France). Anesthesia was maintained with 1.5-1% inspired halothane supplied by a Fluotec vaporizer. Expired gases (O2, CO2, halothane) were continuously analyzed with a Hewlett Packard analyzer (HP M1015B) to fit volume and percentage of inspired gases. Temperature was maintained between 37° and 38°C by a heating element incorporated under the operating table. Catheters were inserted in the femoral artery and vein for blood flow measurements, arterial blood pressure (BP) recording, and administration of intravenous fluid (Ringer's lactate solution 5 ml/kg/h) or drugs. ECG leads were placed subcutaneously for continuous monitoring. An aseptic thoracotomy was performed in the fifth left intercostal space; the heart was exposed and suspended in a pericardial cardle. A 6F micromanometer-tip catheter (Gaeltec Instrument, Scotland) was inserted into the left ventricle for measurement of left ventricular pressure (LVP) and its first derivative (dP/dt). The left atrial appendage was cannulated for injection of radioactive microspheres. The left anterior descending coronary (LAD) was isolated distal to its first diagonal branch, and a silk snare was passed around it for subsequent occlusion and reperfusion.
Drug administration and experimental protocol
Three days before the experimental day, dogs were randomly assigned to receive either a 3-day oral pretreatment (1 mg/kg/day) of TMZ or placebo. The drug administration protocol was blinded. Oral and intravenous forms were packed with the same randomization number. Tablets and intravenous flashes (containing either placebo or TMZ) were unrecognizable.
After instrumentation, animals were allowed to recover for 15 min before baseline recordings. Dogs then received a bolus of 0.5 mg/kg TMZ diluted in saline followed by infusion of 0.5 mg/h TMZ (diluted in 0.9% saline) for the next 4 h of the experiment. Control animals received equivalent doses of saline.
Fifteen minutes after the saline or TMZ infusion was started, the LAD was occluded for 15 min and then reperfused for 3 h. After 125 min of reperfusion, dobutamine 10 μg/kg/min, was infused for 15 min and hemodynamics and regional contractility was measured again.
After myocardial biopsies and blood sampling and immediately before the dogs were killed, Unisperse blue pigment (Ciba-Geigy, 0.5 g/kg) was injected into the left atrial appendage for determination of in vivo area at risk (AAR). Under deep anesthesia, the hearts were then stopped by intracardiac injection of potassium chloride and excised. With this technique, nonischemic tissue appeared blue, whereas the previously ischemic myocardium (AAR) was unstained. The left ventricle was then sectioned from apex to base in transverse slices 8 mm thick parallel to the atrioventricular grove. AAR was measured by planimetry expressed as percent of left ventricle.
Blood sampling and biological determinations
For determination of malondialdehyde (MDA), a marker of lipid peroxidation, gluthatione peroxidase (GPX), and superoxide dismutase (SOD), venous blood was withdrawn in EDTA-containing tubes immediately before TMZ injection and 2, 30, and 180 min after reperfusion. Blood was centrifuged for 20 min at 4°C. The plasma removed from the centrifugate was frozen at -70°C for later analysis. Erythrocytes were washed three times with 0.9% NaCl solution for assays of SOD and GPX.
Assay for plasma lipoperoxides. Plasma lipoperoxides were hydrolyzed by bioling in phosphoric acid. MDA, one of the hydrolysis products, reacts with thiobarbituric acid (TBA) to form MDA-TBA adduct. Plasma proteins were precipitated with methanol and removed from the reaction mixture by centrifugation. The protein-free extracts was fractionated by high-performance liquid chromatography (HPLC) on a column of octadecyl silica gel to separate the MDA-TBA adduct from interfering chromogens. The MDA-TBA adduct was eluted from the column with methanol/phosphate buffer and quantified spectrophotometrically at 532 nm. Plasma lipoperoxide concentrations were computed by reference to a calibration curve prepared by assays of tetraethoxypropane, which underwent hydrolysis to liberate stoichiometric amounts of MDA (11).
SOD assay. The SOD assay uses xanthine and xanthine oxidase to generate superoxide radicals, which react with 3-(4-nitrophenol)-5-phenyltetrazolium chloride to form a red formazan dye. The superoxide activity is measured by the degree of inhibition of this reaction by spectrophotometry and expressed as SOD units per gram of hemoglobin (12).
GPX. We used the method described by Paglia and colleagues (13). GPX catalyzes the oxidation of glutathione by Cumene hydroperoxide. In the presence of glutathione reductase and NADPH, the oxidized glutathione is immediately converted to the reduced form with a concomitant oxidation of NADPH to NADP+. The decrease in adsorbance at 340 nm is measured and expressed as GPX units per gram of hemoglobin.
Determination of myocardial high-energy phosphate compounds
After 3-h of reperfusion, two transmural myocardial fragments (Tru-cut biopsy needle, Travenol Laboratories) were obtained in the ischemic and nonischemic regions at the site of implantation of ultrasonic crystals. Samples were immediately frozen in liquid nitrogen. Frozen biopsies were weighed, crushed to powder in liquid nitrogen, and homogenized with 0.6 n perchloric acid (7 ml/g frozen tissue). After 10-min centrifugation (1,000 g/4°C), the supernatant was neutralized with K2CO3 5 M. The neutralized extract was freed of potassium percholorate by centrifugation and separated by HPLC (14). Tissue levels were expressed as micromoles per gram of tissue wet weight. Total adenine nucleotide pool was calculated as the sum of ATP, ADP, and AMP content.
Determination of regional myocardial function
Two pairs of ultrasonic piezoelectric crystals (2.5-mm diameter), coupled to a sonomicrometer (Triton Technology, San Diego, CA, U.S.A.) were inserted into the subendocardium as described previously (15). One pair was located in the area supplied by the LAD and oriented perpendicular to the external long axis. The other pair was located in the myocardium supplied by the left circumflex coronary artery (LCX) and implanted parallel to the short axis of the heart. The crystals were connected to an ultrasonic amplifier, and the tracings were digitized through an eight-channel interface MacLab LCII computer. Regional myocardial function was assessed by continuous measurement of segment length between each pair of crystals as previously described (15,16). End-diastolic segment length (EDL) was measured immediately before the rapid increase in positive dP/dt (onset of isovolumic contraction), and end-systolic segment length (ESL) was measured at peak negative dP/dt. To minimize respiratory artefact, all measurements were performed at the end of expiration time as indicated by the tracheal pressure curve recording. Regional systolic shortening (RSS) was calculated as RSS (%) = (EDL-ESL)/EDL × 100 and expressed as percent of baseline RSS before coronary artery occlusion. At least three distinct (nonconsecutive beats) were averaged for each timepoint. Two independent investigators performed RSS measurements without knowing group assignment.
Determination of regional myocardial blood flow (RMBF)
RMBF was measured as previously described (16,17): 1 million microspheres (15-μm diameter) labeled with 141-Cerium, 45-Scandium, and 85-Strontium (New England Nuclear, Boston, MA, U.S.A.) were injected into the left atrium for 20 s, while a reference arterial blood flow sample was collected. Three measurements of RMBF were made in each dog after 10-min coronary occlusion and after 30- and 160-min reperfusion. RMBF was measured in the nonischemic and the central ischemic zones. Each region was subdivided into subendocardial and subepicardial halves. Myocardial pieces were weighed and counted in a γ-counter with selected energy windows. After correction of counts for background and cross-over, RMBF was expressed in milliliters per minute per gram of tissue (ml/min/g) and for collateral flow in percent of the flow in the nonischemic zone (17).
The following variables were recorded directly on the hard disk of a Macintosh LCII computer: ECG lead II and heart rate (HR), aortic pressure and LVP, dP/dt, the two length signals, and the pulmonary insufflation trace. In statistics, all values are mean ± SEM. Hemodynamic data correspond to the mean of three measurements. Comparison of the difference between baseline values and values obtained after reperfusion in the two groups were made by an analysis of variance for repeated measures (ANOVA), followed by Dunnett's t test.
Thirty dogs were randomized to receive either TMZ or placebo. One TMZ dog developed ventricular fibrillation (VF) and died immediately after coronary occlusion, whereas 5 dogs (3 TMZ dogs and 2 controls) developed VF at reperfusion. These 6 dogs were excluded from analysis. One TMZ dog was also excluded because of the total absence of cyanosis and dyskinesia during coronary occlusion. Therefore, data of 13 controls and 10 TMZ-treated dogs are reported.
At baseline (p < 0.06) and after TMZ bolus (p < 0.05), HR was lower in TMZ dogs. Occlusion of LAD in control dogs resulted in hemodynamic alterations typical of acute ischemia, with increased HR and decreased LV dP/dt without modification of arterial BP. In TMZ, dogs, coronary occlusion induced no change in HR and arterial BP, whereas LVdP/dt showed a significant decrease. During reperfusion and except for HR (Table 1), which was lower in TMZ dogs, the two groups were similar in particular for arterial BP and the arterial BP/HR product. Therefore, the only hemodynamic difference observed between the two groups of dogs was a lower HR in the TMZ group.
Platelet count, white and red blood cell counts, and hemoglobin level were similar in both groups. Baseline MDA level was significantly lower in TMZ dogs (Table 2) whereas GTX and SOD were similar in both groups. MDA increased during reperfusion in TMZ dogs; after 180 min, the difference between groups was not significant. SOD increased slightly in controls, but GTX was stable (Table 2).
Myocardial high-energy phosphate compounds
Adenine nucleotides in myocardial biopsy of the nonischemic zone were similar in both groups (Table 3). In the ischemic zone, as expected, there was significant loss of all types of nucleotides except for hypoxanthine in both groups and of AMP in TMZ dogs, for which the difference between ischemic and nonischemic zones was not significant.
AAR and RMBF
AAR represented 33 ± 2 and 29 ± 3% of the left ventricle in control and TMZ dogs, respectively. Both groups showed markedly reduced subendocardial and subepicardial blood flow in the ischemic zone after LAD occlusion (Table 4). There was no significant difference between groups. During reperfusion, blood flow in the subendocardium of the previously ischemic region represented ≈70% of the flow in the nonischemic region, demonstrating adequate reduction of blood flow during reduced regional function.
The TMZ bolus caused no alteration in regional segment contractility. After coronary occlusion, contractility decreased similarly in both groups (Fig. 1). During reperfusion, myocardial stunning developed in both groups. In addition, we noted a significant linear relation between subendocardial blood flow during ischemia (reflecting the severity of ischemia) and contractility during ischemia: r2 = 0.50, p < 0.01 in controls and r2 = 0.62, p < 0.005 in TMZ dogs. In contrast, we noted no correlation between the severity of stunning and RMBF during ischemia (or during reperfusion) and between the severity of stunning and myocardial adenine nucleotides.
After 15 min of dobutamine infusion (10 μg/kg/min), stunning decreased only slightly (nonsignificantly) in both groups, whereas contractility in the nonischemic zone as well as LVdP/dt augmented significantly in both groups (Table 5). These augmentations were more pronounced in TMZ dogs since the increases in HR and BP were significant only in these dogs and not in controls. Finally, there was no correlation between the degree of regional dysfunction and the response to inotropic stimulation.
We examined the effects of TMZ on hemodynamics, RMBF, and RSS during 15-min coronary artery occlusion followed by 3-h reperfusion. At that time, we investigated whether dobutamine infusion might limit or reverse the stunning phenomenon. We also examined whether TMZ may influence lipid peroxide formation during the course of ischemic-reperfusion sequence and/or may prevent, at least in part, the loss in high-energy phosphate myocardial content. In contrast to previous clinical studies (3-5), in the present study TMZ affected HR significantly, whereas BP, rate-pressure product, dP/dt, RMBF, and contractility were not different as compared with that of control dogs during both ischemia and reperfusion. In accordance with results of previous studies (6-8), TMZ significantly reduced chronic lipid peroxide formation. During dobutamine infusion, hemodynamics of TMZ-treated dogs were significantly different as compared with that of controls, with greater increase in HR, BP, and dP/dt. Whereas contractility augmented significantly during dobutamine infusion in both groups in the nonischemic myocardium, myocardial stunning in the postischemic myocardium was not significantly affected in either group.
TMZ and anesthesia
Whatever its pharmacological properties in various systems, TMZ did not limit or prevent myocardial stunning in the present study. In a porcine model, neither did Koning and associates observe any protective effect of TMZ on contractile function (18).
Previous experimental studies have shown the importance of the model in investigating myocardial stunning. Whether body temperature is controlled (19) and whether chest closure is performed before coronary occlusion (20), the level of oxygen concentration in inspired gases (21) and the type of anesthesia (22) may affect accuracy and reproducibility of measurements or myocardial function itself. In view of the present data, an important question therefore is whether any model-related factor influenced the results.
Owing to their hemodynamic impact, anesthetic drugs are probably among the main factors which could, in a given experimental model and by either their nature or the dosage, influence the experiments. Barbiturates, the most frequently used anesthetic drugs in experimental cardiology, have marked chronotropic positive effect (23) and are cardiodepressive, especially when given as bolus (24,25). They accumulate in various tissues and, being released secondarily, they can induce overdosage in experiments lasting several hours.
In contrast, halothane, the main anesthetic agent used in the present study, permits steady anesthesia (26). Halothane has been reported to increase myocardial blood flow and to protect the ischemic myocardium by reducing oxygen consumption, probably because of inotropic and chronotropic negative effect and reduction of catecholamine secretion due to the absence of painful stimuli (24,27). Cardiodepressant effect is observed only when the halothane dosage is much higher than that used in the present study (27-29).
It is noteworthy that, given the severity of myocardial ischemia (as evaluated by the residual flow in the ischemic region), the degree of stunning during reperfusion in both groups was lower than expected and lower than in studies in which barbiturates were used: stunning represented 60-70% of baseline contractility after 2-h reperfusion in the present study, whereas 0-40% was expected based on results of previous studies, for a comparable level of ischemia (19,20,30,31). Triana and co-workers reported that in conscious dogs the recovery of contractility after 15-min ischemia followed by reperfusion was markedly greater than that in pentobarbital-anesthetized dogs (19). Heyndrickx and colleagues reported that systolic wall thickening was ≈70% of baseline after 5- or 15-min of coronary occlusion and 3-h reflow in conscious dogs (32), a result similar to our present data. Whereas further studies are certainly needed to compare conscious and halothane-anesthetized dogs, the present data and results of several previous studies (33-35) suggest that the type of anesthesia likely interferes with the phenomenon of myocardial stunning.
The lack of reversibility of contractile dysfunction in both groups during dobutamine infusion is surprising. We selected a dobutamine dosage known to produce significant inotropic stimulation without inducing major chronotropic, arrhythmogenic, or vascular effects. Significant alterations in HR, preload, and afterload influence contractile function. A dosage of 10 μg/kg/min was thus theoretically adequate to obtain stable hemodynamic condition after 15-min infusion. Insufficient dosage of dobutamine may nervetheless explain our data; e.g., Hashimoto and co-workers, who infused dobutamine at a dosage of 24 μg/kg/min, increased rate-pressure product by 70% and completely reversed contractile dysfunction (30). However, the complete lack of effect of dobutamine on the stunned myocardium in our study is unclear since dobutamine infusion resulted in significant increase of dP/dt max and contractility in the reperfused zone in both groups, which demonstrates a significant inotropic effect in both groups whatever the baseline HR. In TMZ dogs only, dobutamine infusion also resulted in increased HR and BP (suggesting a greater response in TMZ dogs) but without modifying the degree of stunning. Therefore, there was a “significant recruitment” in both groups during dobutamine infusion, without alteration in the degree of stunning. One possibility, in a model of moderate stunning (60-70% of baseline contractility), is that either no contractile reserve exists to be recruited or some unknown factor prevents its recruitment. A role for halothane in that context is not impossible and should be tested in further studies. Finally, although we do not know whether inotropic stimulation may reverse stunning in conscious dogs, at least in part, the concept of contractile reserve recruitment should be reexamined with the idea that it is model dependent and may be influenced by the type of anesthesia.
Stunning, TMZ, and reactive oxygen species
In the present study, MDA level, a marker of lipid peroxidation, was significantly lower in TMZ-treated dogs as compared with controls. Previous studies in which spin trapping agents and electron paramagnetic resonance spectroscopy were used (36,37) showed similar data. The fact that stunning was not modified by TMZ suggests that antioxidant properties of TMZ were not sufficient to reduce stunning and may also suggest that free radicals are not as important in the present model as in the barbiturate-anesthesia model.
Stunning, TMZ, and adenine nucleotide pool
TMZ has also been proposed to preserve myocardial adenine nucleotide levels. Lavanchy and associates, using 31phosphorus nuclear magnetic resonance spectroscopy showed evidence of accelerated reconstitution of ATP during reperfusion of isolated rat hearts (2). We also noted considerable depletion of adenine nucleotide pool in both groups, in accordance with results of previous studies (38,39). However this cannot completely explain the persistent and parallel contractile dysfunction in our two groups of dogs, in particular during dobutamine administration. Taegtmeyer and colleagues showed that no direct correlation exists between the tissue content of ATP and cardiac function and that low levels of ATP do not exclude functional recovery (40). In the present study, we noted no correlation between ATP level and the degree of myocardial dysfunction.
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