Author + information
- Received June 7, 1996
- Revision received October 15, 1996
- Accepted October 18, 1996
- Published online February 1, 1997.
- Paul Mulder, PhDA,
- Vincent Richard, PhDA,
- Patricia Compagnon, BSA,
- Jean-Paul HenryA,
- Françoise LallemandA,
- Jean-Paul Clozel, PhDA,
- Robert Koen, MD, PhDA,
- Bertrand Macé, MDA and
- Christian Thuillez, MD, PhDA,*
- ↵*Dr. Christian Thuillez, Service de Pharmacologie, Centre Hospitalier Universitaire de Rouen, 76031 Rouen Cedex, France. E-mail: Christian.Thuillez@chu-rouen.fr.
Objectives. We sought to investigate the effects of mibefradil on survival, hemodynamic variables and cardiac remodeling in a rat model of chronic heart failure (HF) and to compare these effects with those of the angiotensin-converting enzyme (ACE) inhibitor cilazapril.
Background. The use of calcium channel blocking agents in chronic HF has been disappointing. Most studies have shown that these drugs have either no or even detrimental effects due in part to the negative inotropic effects they induce. Mibefradil is a calcium channel blocker that selectively blocks T channels and displays moderately negative inotropic properties only at high doses. Because T channels are upregulated in the hypertrophied heart and could mediate hypertrophic signals and increase arrhythmogenicity, blockade of these channels might be beneficial in chronic HF.
Methods. Rats were subjected to coronary artery ligation and 9 months of treatment with mibefradil (15 mg/kg body weight per day) or cilazapril (10 mg/kg per day) or no treatment. Survival and systolic blood pressure were assessed over the 9-month treatment period, after which cardiac hemodynamic variables and structure were determined.
Results. Mibefradil increased survival rate to the same extent as cilazapril (71% for mibefradil vs. 75% for cilazapril and 44% for no treatment). Mibefradil decreased systolic blood pressure, although to a lesser extent than cilazapril. Both treatments decreased left ventricular (LV) end-diastolic and central venous pressures, without any change in the first derivative of LV pressure over time or heart rate. Mibefradil decreased LV weight (although less than cilazapril) without affecting right ventricular weight. Finally, both drugs normalized LV collagen density.
Conclusions. Mibefradil in a rat model improved survival to the same extent as an ACE inhibitor, without impairing LV function, and was associated with a reduction in LV weight and fibrosis. Thus, mibefradil might be beneficial in the treatment of chronic HF.
(J Am Coll Cardiol 1997;29:416–21)
Calcium antagonists are widely used in the treatment of hypertension () and angina pectoris () because of their vasorelaxing properties and their favorable effects on myocardial oxygen supply and demand (). However, their use in chronic myocardial infarction has been disappointing, and clinical studies have shown variable results ([4–6]). Calcium channel antagonist use might be limited by the reflex increase in sympathetic tone, especially in the case of dihydropyridines ([7, 8]), or their negative inotropic effects (), which, in the case of verapamil or diltiazem, are enhanced in the presence of left ventricular (LV) dysfunction (). In addition, the dihydropyridine calcium channel blocking agent amlodipine seems to have favorable effects on mortality only in patients with nonischemic dilated cardiomyopathy ().
Mibefradil (Ro 40-5967) is a new calcium channel blocker that selectively blocks T-type calcium channels () and exerts moderate negative inotropic effects at high doses only ([9, 12]). Indeed, in rats with chronic heart failure (HF), mibefradil induces a dose-dependent decrease in arterial blood pressure and increases coronary blood flow without any detectable change in cardiac contractility (). Furthermore, mibefradil displays anti-ischemic properties similar to those of verapamil ([14, 15]), despite much more moderate effects on cardiac function (). This unique pharmacologic profile of mibefradil might be of interest in the treatment of chronic HF.
Recent experiments have also shown ([16, 17]) that T-type calcium channels mediate the responses to various hypertrophic signals. Thus, it is possible that they may represent a trigger for the development of cardiac hypertrophy in various pathophysiologic situations. Furthermore, cardiac hypertrophy is associated with an increased T-channel activity in myocytes ([18–20]). The presence of these channels in hypertrophied hearts may make such hearts more prone to spontaneous action potentials and increase arrhythmogenicity (). Thus, blockade of these channels might have a beneficial effect on cardiac hypertrophy and arrhythmias in pathologic situations such as chronic HF.
Thus, the goals of our study were to investigate the effects of the calcium channel blocker mibefradil on survival, systemic and cardiac hemodynamic variables and cardiac structure in a rat model of chronic HF and to compare these effects with those of an angiotensin-converting enzyme (ACE) inhibitor.
1.1 Animals and treatment.
Myocardial infarction was produced in 10-week old male Wistar rats by left coronary artery ligation. For this purpose, animals were anesthetized with 50 mg/kg body weight of sodium methohexital intraperitoneally. The trachea was intubated, and the lungs were mechanically ventilated with room air supplemented by low flow oxygen. After a left thoracotomy, the proximal left coronary artery was occluded by a suture to induce myocardial infarction. Sham-operated rats were subjected to the same protocol, except that the snare was not tied. Fifteen minutes after occlusion, the chest was closed in three layers (ribs, muscles and skin). A plastic catheter connected to a 5-ml syringe was placed in the chest before sewing and was used to remove air from the chest after closure. The rats were allowed to recover from anesthesia (usually within 15 min), after which they were returned to their cages. The 24-h postoperative mortality rate for this method was ∼20% for the infarction group.
Seven days after ligation, infarcted rats were randomly assigned to three treatment groups: no treatment (n = 68) or treatment with the calcium antagonist mibefradil (n = 59, 15 mg/kg per day [Hoffmann-La Roche Ltd. Basel, Switzerland]) or the ACE inhibitor cilazapril (n = 59, 10 mg/kg per day [Hoffmann-La Roche]). Twenty sham-operated rats were used as control animals. All treatments were given as food admix in normal rat chow. Rats were weighed every week, and their food intake was measured to allow adjustment of the drug concentrations in the chow.
1.2 Study design.
The survival study began 7 days after operation to exclude early mortality caused by the procedure or acute myocardial infarction and was designed to last 9 months. During the treatment period, cages were inspected daily for dead animals. The body weight of the dead rats was recorded, and the chest was opened to search for signs of cardiac rupture or pulmonary infection. The heart was then removed, cleaned, weighed and immersed in Bouin solution for subsequent determination of infarct size.
1.3 Hemodynamic assessment.
Systolic blood pressure (plethysmography) and heart rate were determined in conscious rats just before the start of the treatment (i.e., 7 days after the surgical procedure) and after 30, 90, 180 and 270 days of treatment. After 270 days, the surviving rats were anesthetized with thiopental (50 mg/kg intraperitoneally). The right carotid artery and right external jugular vein were cannulated with 2F micromanometer-tipped catheters (SPR 407, Millar Instruments) that were advanced into the aorta and thoracic vena cava, respectively, to record arterial and central venous pressures. The aortic catheter was then advanced into the left ventricle to record LV pressure and its maximal rate of increase (dP/dt max). Rats were then killed by an overdose of thiopental before removal of the heart.
1.4 Cardiac morphometry.
Infarct size and cardiac collagen density were measured in histologic sections by image analysis as described previously (). Infarct size was determined in all rats (i.e., those spontaneously deceased and those killed after 9 months).
1.5 Measurement of plasma renin and plasma catecholamines.
Ten days before completion of the study, the surviving animals were anesthetized with ether, and venous blood samples (1.25 ml) were collected in prechilled tubes containing EDTA (10 mmol/L final concentration). Tubes were immediately centrifuged at 3,000gfor 8 min and stored at −80°C for determination of plasma renin activity () and catecholamine concentrations (high performance liquid chromatography).
1.6 Statistical analysis.
All results, except survival, are given as mean value ± SEM. Survival curves were compared by the log-rank test and the Mantel-Haenszel procedure (). Intergroup differences were evaluated by analysis of variance (ANOVA) followed, if ANOVA revealed significant differences, by a Tukey test for multiple comparisons. Differences were considered significant at p < 0.05.
Fig. 1illustrates the survival curves for the three treatment groups. Within the first 135 days, the rate of death in the mibefradil and cilazapril groups was similar to that of the untreated group. After 135 days, mortality was higher in the untreated group than in either treatment group. After 9 months, the survival rate of the mibefradil (71%, p < 0.05) and cilazapril groups (75%, p < 0.05) was significantly higher than that of the untreated group (44%). The beneficial effect of both treatments was also illustrated by the increase in mean survival time from 200 days in the untreated group to 230 and 238 days in the mibefradil- and cilazapril-treated groups, respectively.
Survival was also analyzed with regard to infarct size. In rats with a moderate infarct (<40% of the left ventricle), both mibefradil and cilazapril significantly improved the survival rate at 9 months (no treatment 64%, mibefradil 92% [p < 0.05], cilazapril 97% [p < 0.05]). In contrast, in animals with a large infarct (>40% of the left ventricle), neither mibefradil nor cilazapril improved the survival rate (no treatment 34%, mibefradil 35%, cilazapril 39%).
2.2 Infarct size.
Four rats (two untreated, one mibefradil treated, one cilazapril treated) were excluded from the study for an infarct size <10% of the left ventricle. In addition, determination of infarct size was not possible in two rats that died, (one cilazapril treated, one mibefradil-treated), and they were excluded from the study.
Infarct size in rats that died spontaneously and those killed after 270 days was identical in both treatment groups (both 40 ± 2%) and the untreated group (42 ± 2%). Furthermore, randomization resulted in a balanced distribution of infarct sizes (Fig. 2).
2.3 Hemodynamic measurements in conscious rats.
Fig. 3shows the evolution of systolic blood pressure and heart rate determined in conscious surviving rats in the four different groups during the 9-month study period. Seven days after coronary artery ligation, systolic blood pressure in untreated rats was significantly lower than that in sham-operated rats, with no significant change in heart rate. Moreover, at this time (i.e., immediately before the onset of treatment), systolic blood pressure in the mibefradil- and cilazapril-treated groups was similar and was not different from that in the untreated rats. Compared with untreated rats, mibefradil induced a significant decrease in systolic blood pressure after 30 treatment days (−9 mm Hg, p < 0.05). This effect was similar after 90 treatment days (−7 mm Hg), after which it slightly increased with time (−15 and −29 mm Hg at 180 and 270 treatment days, respectively). Mibefradil did not significantly modify heart rate at any time.
Compared with the untreated group, the ACE inhibitor cilazapril also decreased systolic blood pressure (−30 mm Hg at day 30, p < 0.05), and this effect persisted over time (−30, −33 and −39 mm Hg at days 90, 180 and 270, respectively). Furthermore, the decrease in systolic blood pressure induced by cilazapril was always significantly more marked than that induced by mibefradil. Cilazapril did not significantly modify heart rate at any time.
2.4 Hemodynamic measurements in anesthetized rats.
Table 1summarizes cardiac hemodynamic variables and central venous pressure measured after 9 months in surviving anesthetized animals. Compared with sham-operated animals, chronic HF significantly decreased LV systolic pressure and LV dP/dt max and significantly increased both LV end-diastolic and central venous pressures. Compared with untreated rats, mibefradil reduced LV systolic pressure (−20%, p < 0.05), LV end-diastolic pressure (−45%, p < 0.05) and central venous pressure (−22%, p < 0.05), without affecting LV dP/dt max. Cilazapril also reduced LV systolic, LV end-diastolic and central venous pressures without affecting LV dP/dt max. Finally, all long-term hemodynamic changes induced by either mibefradil or cilazapril were present both in moderate (<40% of the LV) and large infarcts (>40% of the LV (Table 1).
2.5 Left ventricular morphology.
Table 2shows LV and right ventricular (RV) weight and collagen density in the noninfarcted LV free wall in animals killed after 270 treatment days. Compared with sham-operated rats, chronic HF increased LV and RV weight and LV collagen density. Compared with untreated rats, mibefradil moderately reduced LV weight and more markedly decreased collagen density, without affecting RV weight. Cilazapril markedly reduced LV and RV weight and collagen density.
2.6 Renin and catecholamines.
Table 2shows plasma renin activity and plasma norepinephrine and epinephrine concentrations determined in surviving animals. After 9 months, chronic HF tended to increase plasma renin activity and norepinephrine and epinephrine concentrations but not significantly. In mibefradil-treated rats, plasma renin activity was increased, whereas norepinephrine and epinephrine levels were not modified. Cilazapril markedly increased plasma renin activity and reduced norepinephrine and epinephrine levels, although the latter was not significant.
The present study, performed in a rat model of chronic HF, shows that long-term administration of the calcium channel blocker mibefradil improved survival to the same extent as the ACE inhibitor cilazapril. This improved survival was associated with a reduction in both arterial and central venous pressures and a normalization of LV end-diastolic pressure, with no sign of negative inotropic effects. In addition, mibefradil moderately decreased LV weight and normalized collagen density.
3.1 Survival and hemodynamic variables.
The main finding of this study is that the calcium channel blocker mibefradil induced a marked increase in survival rate, close to that induced by the ACE inhibitor cilazapril. This beneficial effect differentiates mibefradil from other calcium channel blockers that have been shown to have no or a detrimental effect on survival in humans ([1, 5, 24]) or in experimental models of chronic HF ().
In the present study, only one calcium antagonist was tested. In light of the major differences between the various classes of calcium channel blockers, it would have been useful to compare mibefradil with other, structurally unrelated calcium channel blockers. However, our initial goal was to investigate the effect of mibefradil itself. Because there was no certainty as to study outcome, we considered it more reasonable and practical to first test whether mibefradil was efficacious in the present model before comparing it with other calcium antagonists. Furthermore, comparison of different drugs is difficult, not only with regard to which drugs to compare, but also which doses to use in the comparison. Ideally, comparisons between drugs require full dose–response curves, which was not possible in the present model. Nevertheless, comparisons between mibefradil and other calcium antagonists will require further investigation.
In our study, survival at 9 months was nearly identical in the cilazapril- and mibefradil-treated groups. We limited the treatment period to 9 months because of the high mortality rate that has been observed in similar models after 9 months (). Had we used a longer treatment period we would have performed the final hemodynamic and morphometric measurements in only a small subgroup of rats with only moderate cardiac dysfunction. Also, we wanted to compare mibefradil with an ACE inhibitor, and this latter class of drugs has been shown to improve survival after 9 months of treatment ([21, 26–28]). Thus, whether mortality may differ between mibefradil and ACE inhibitor therapy >9 months is not known and requires further investigation.
The hemodynamic effects of mibefradil, illustrated by the significant and persistent hypotensive effect (although less marked than that induced by the ACE inhibitor), probably contributes to the improved survival. Indeed, ACE inhibitors improve survival only when associated with a significant decrease in blood pressure (). In the case of mibefradil, the decrease in afterload is not associated with tachycardia but possibly because of the direct effect of mibefradil on sinoatrial conduction (). Alternatively, this lack of tachycardia could indicate that there is no activation of the sympathetic nervous system secondary to vasodilation, in contrast to the observed effects of dihydropyridine calcium channels blockers ([7, 30]) or hydralazine (). The slight decrease in plasma norepinephrine confirms the improvement in hemodynamic status without sympathetic stimulation. Finally, the vasorelaxing properties of mibefradil are apparently not associated with marked negative inotropic effects, as suggested by the lack of effect on LV dP/dt max. Such moderate negative inotropic effects have been described in other experimental models of ischemia and chronic HF ([9, 10, 13, 15]) and contrast with the more marked negative inotropic effects of other calcium antagonists, such as diltiazem and verapamil ([9, 10, 15]).
3.2 Neurohumoral assessment.
In the untreated rats with chronic HF that survived for 9 months, we observed only slight increases in plasma renin activity and plasma catecholamine levels, possibly because rats with high activation of these two systems died during the study. After 9 months, mibefradil-treated rats had slightly increased plasma renin activity compared with that in untreated rats, suggesting only moderate activation of the circulating renin–angiotensin system. Again, it is not possible to determine whether the increase in plasma renin activity results from the direct effect of a calcium antagonist or is due to the selection of rats with high plasma renin levels, secondary to the marked effect on survival. Nonetheless, this finding suggests that combined treatment with mibefradil and drugs that interfere with the renin–angiotensin system might have additive beneficial effects to those of the calcium antagonist administered alone.
3.3 Left ventricular structure.
Long-term treatment with mibefradil reduced LV weight as well as interstitial collagen density in the noninfarcted LV. However, the effect of mibefradil on LV ventricular weight was modest and much less marked than that induced by the ACE inhibitor. Furthermore, in contrast to the ACE inhibitor, mibefradil did not affect RV weight. Thus, it is unlikely that the effect of mibefradil on hypertrophy is the sole mechanism of the increased survival.
The effect of mibefradil on LV hypertrophy may be related to the drug-induced decrease in afterload, which is probably associated with a decrease in ventricular wall stress. These hemodynamic changes may thus counteract the potentially deleterious effects of the neurohumoral activation in chronic HF ([26, 32]). Alternatively, the decrease in LV weight might be related to the role played by T-type calcium channels in the development of myocyte hypertrophy. Indeed, T channels, which are overexpressed in feline hypertrophied left ventricular myocytes () and cardiomyopathic hamster hearts (), have been shown to mediate calcium entry in response to various mitogenic stimuli, such as angiotensin II (), endothelin-1 () or platelet-derived growth factor (). In addition, overexpression of T channels may make hypertrophied hearts more prone to spontaneous action potentials and increase arrhythmogenicity in partially depolarized hypertrophied myocardium (). Moreover, T channels are present at the level of adrenal cells and may mediate aldosterone secretion in response to potassium ions ([34–36]). Because aldosterone has been shown () to have a major influence on the development of cardiac fibrosis, it is possible that blockade of these channels may indirectly affect fibrosis through decreased aldosterone secretion. Thus, by directly preventing cardiac hypertrophy or arrhythmias, or both, T-channel blockade by mibefradil might contribute to the increased survival, independent of the hemodynamic effects.
Long-term treatment with the calcium antagonist decreased two indexes of cardiac preload: LV end-diastolic and central venous pressure. This decrease in preload has not been observed after short-term administration of mibefradil () or after other calcium antagonists in similar models of chronic HF ([37, 38]). Despite a smaller effect on afterload, the effect of mibefradil on preload was similar to that of the ACE inhibitor. Thus, the beneficial effects of mibefradil on preload, which were not observed after short-term administration, must be partly related to a direct cardiac effect, independent of the decrease in blood pressure. For example, the decrease in myocardial collagen density with mibefradil could lead to increased ventricular compliance and myocardial relaxation, which could contribute to a direct effect on preload.
Conclusions.Our study in a rat model of chronic HF showed that long-term treatment with the calcium antagonist mibefradil improved survival to the same extent as an ACE inhibitor. This improvement probably occurs as a result of the effects of the drug on cardiac preload and afterload, as well as on cardiac hypertrophy and possibly on arrhythmias, in the absence of marked negative inotropic properties. Our results suggest that mibefradil might be beneficial in the treatment of ischemic chronic HF.
We thank Eliane Abdelhouab and Marie-Française Gonzales for excellent technical assistance and J. Ménard, MD, PhD for renin assays.
☆ This work was supported by a grant from Roche, Neuilly, France. Drs. Koen and Clozel have financial relationships with Hoffmann-La Roche Ltd., Basel, Switzerland.
- angiotensin-converting enzyme
- analysis of variance
- heart failure
- left ventricular
- LV dP/dt
- first derivative of left ventricular pressure over time
- right ventricular
- Received June 7, 1996.
- Revision received October 15, 1996.
- Accepted October 18, 1996.
- The American College of Cardiology
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