Introduction

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Calcium channel blockers are used in the treatment of arterial hypertension, chronic stable angina pectoris and cardiac arrhythmias (Braunwald, 1982; Boden et al., 1985; Krikler, 1987; Singh and Nademanee, 1987). Despite their desirable peripheral vasodilatory effects, antiischemic action and their ability to prevent intracellular calcium overload, the use of calcium channel blockers in patients with HF remains highly controversial. In patients with chronic HF, short-term therapy with the dihydropyridine compounds caused both hemodynamic and clinical deterioration (Elkayam et al., 1985). Long-term clinical trials with these compounds, and with diltiazem, in patients with a spectrum of cardiac diseases including unstable angina, myocardial infarction and HF showed either no clinical improvement or an increase in cardiovascular morbidity and mortality (Goldstein et al., 1991; Muller et al., 1984; The Multicenter Diltiazem Postinfarction Trial Research Group, 1988). Some controversy also exists with respect to the long-term use of calcium channel blockers even in patients with chronic stable angina and hypertension (Psaty et al., 1995). The mechanisms responsible for the potentially harmful effects of calcium channel blockers, particularly in the setting of chronic HF, include the depressant effect on cardiac function through a direct negative inotropic action on the myocardium, neurohumoral activation that can produce cellular damage and exacerbate the HF state and, in some instances, excessive hypotension and tachycardia (Packer 1989a). To circumvent the cardiodepressant effects of existing calcium channel blockers such as nifedipine, verapamil and diltiazem, new so-called "second generation" calcium channel blockers were developed (Packer 1989b). These compounds that include nitrendipine, felodipine, nisoldipine and amlodipine produced significant coronary and peripheral vasodilatory effects with less negative inotropic effects compared to first generation calcium antagonists (Packer 1989b; Freedman and Waters 1987). Nevertheless, long-term therapy using some of these compounds in patients with HF continued to show some harmful effects and clinical deterioration consistent with those seen with first generation compounds (Tan et al., 1987).

The term "calcium channel blockers" covers a diverse class of compounds often with substantial differences in action even among two formulations of the same compound. All of the calcium channel antagonists currently used in cardiovascular medicine, whether first or second generation, act primarily on what are known as L-type or long-lasting, high voltage activated calcium channels. This type of channel, however, represents only one of several recognized classes of calcium channels found in mammalian cells that include T-type channels. L-type calcium channels are characterized by long lasting openings, sensitivity to 1,4-dihydropyridines, activation at high voltage (Bers, 1991) and are present in high density in cardiac muscle cells. In contrast, T-type calcium channels are characterized by transient openings, insensitivity to dihydropyridines, activation at low voltages or more negative membrane potentials (Bers, 1991) and are present in relatively high density in vascular smooth muscle cells. Unlike L-type calcium channels, T-type calcium channels have not been identified with a characteristic blocker. Recent in vitro studies have shown that the new benzimidazolyl-substituted tetraline derivative, mibefradil (Ro 40-5967), selectively inhibits T-type calcium channels at concentrations that only partially block L-type channels (Mishra and Hermsmeyer, 1994; Mehrke et al., 1994). In both animal and in patients, mibefradil has been shown to be a potent vasodilator that appears to be devoid of negative inotropic effects (Clozel et al., 1991; Veniant et al., 1991a; Portegies et al., 1991), making it a promising therapeutic candidate for the treatment of chronic HF. We compared the acute hemodynamic effects of intravenous mibefradil to i.v. diltiazem in a canine model of chronic HF produced by multiple sequential intracoronary microembolizations. We hypothesized that mibefradil, being a potent vasodilator that is free of negative inotropic effects, will improve LV performance whereas diltiazem, at doses that decrease systemic pressure to the same degree as mibefradil will have little or no effect on global LV performance.

	Materials and Methods

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The animal model. The canine model of chronic HF used in the present study was previously described in detail (Sabbah et al., 1991). In this experimental preparation, chronic LV dysfunction and failure are produced by multiple sequential intracoronary embolizations with polystyrene Latex microspheres (70-102 µm in diameter) which lead to loss of viable myocardium and decrease in ejection fraction. The model manifests many of the sequelae of HF seen in humans, including marked and sustained depression of LV systolic and diastolic function, reduced cardiac output, increased LV filling pressures, increased systemic vascular resistance and enhanced activity of the sympathetic nervous system (Sabbah et al., 1991). In the present study, eight healthy mongrel dogs, weighing between 20 and 31 kg, underwent coronary microembolizations to produce HF. Embolizations were performed 1 to 3 wk apart and were discontinued when LV ejection fraction, determined angiographically, was 35%. Microembolizations were performed during cardiac catheterization under general anesthesia and sterile conditions. The anesthetic regimen used in the present study consisted of a combination of i.v. injections of oxymorphone (0.22 mg/kg), diazepam (0.17 mg/kg) and sodium pentobarbital (150-250 mg to effect). This anesthesia regimen was previously shown to be effective in preventing the tachycardia, hypertension and myocardial depression seen with pentobarbital alone and does not have a significant effect on global LV function when compared to the conscious state (Sabbah et al., 1994). Studies were performed at an average of 3 mo after the last coronary microembolization. At the time of the study, LV ejection fraction was 29 ± 1%. The study was approved by the institution Care of Experimental Animals Committee and conformed to the "Position of the American Heart Association on Research Animal Use" and the guiding principles of the American Physiological Society.

Study protocol. Each of three compounds namely, mibefradil, diltiazem and normal saline (as a placebo control), were studied in each dog on 3 separate days. A minimum of 6 days was allowed between each drug intervention to ensure that the drug was completely washed out. Diltiazem has a half life of 2 to 8 hr (Leier and Boudoulas, 1997) and mibefradil has a mean elimination half life of 17 to 25 hr (Giles, 1997). The study was not blinded but in all instances, the order of administration of the drugs was randomized. Mibefradil, available in powder form, was dissolved in normal saline. Mibefradil was administered intravenously as a 100 µg/kg bolus (total volume 10 ml) followed by a continuous i.v. infusion of 6 µg/kg/min for 15 min (total volume 30 ml). Diltiazem was available in injectable form (5 mg/ml) and was also diluted in normal saline. Diltiazem was also administered i.v. as a 100 µg/kg bolus followed by a continuous i.v. infusion of 4 µg/kg/min for 15 min. The total volume of injectate of diltiazem (after dilution) was similar to that of mibefradil. Normal saline was administered i.v. in equivalent volumes and using the identical protocol as mibefradil and diltiazem. The doses of mibefradil and diltiazem were chosen to produce, on average, a 5 to 10% reduction of mean aortic pressure. The use in this study of a reduction of blood pressure for dose determination of both diltiazem and mibefradil is consistent with previous investigations (Porter et al., 1983; Su et al., 1994; Clozel et al., 1989, 1991; Veniant et al., 1991a). The choice of 15 min for continuous intravenous infusion of diltiazem or mibefradil was based on previous studies in dogs (Su et al., 1994). The chemical structure of mibefradil and diltiazem are shown in figure 1. 

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Left, Chemical structure of diltiazem (2S-cis)-3-(acetyloxy)-5-[2-dimethylamino)ethyl]-2,3-dihydro-2-94-methoxyphenyl)-1,5-benzothiazepin-4(5H)-one. Right, Chemical structure of mibefradil (1S,2S)-2-[2-[3-(2-benzimidazolyl) propyl]methylamino]-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtylet methoxyacetate dihydrochloride.

Hemodynamic and angiographic measurements. Aortic and LV pressures were measured with the catheter-tip micromanometer. Aortic pressure was measured during pullback of the micromanometer catheter from the LV cavity to the ascending aorta. The LV peak rate of change of pressure during isovolumic contraction (peak +dP/dt) and relaxation (peak dP/dt) were derived from analog differentiation of the LV pressure waveform. Cardiac output was measured in duplicate using the thermodilution method in conjunction with the Swan-Ganz catheter. Systemic vascular resistance was calculated as mean aortic pressure times 80 divided by cardiac output. In all instances, ventriculograms were performed after completing the hemodynamic measurements. Left ventriculograms were obtained with the dog placed on its right side during the injection of 20 ml of contrast material (Reno-M-60, Squibb, New Brunswick, NJ) and were recorded on 35 mm cine at 30 frames/sec. Correction for image magnification was made with a radiopaque calibrated grid placed on the level of the LV. Left ventricular end-systolic and end-diastolic volumes were measured using the area-length method. LV ejection fraction was calculated as the ratio of the difference between LV end-diastolic and end-systolic volume to end-diastolic volume times 100 (Sabbah et al., 1991, 1994). Extrasystolic and postextrasystolic beats were excluded from all analyses.

Data analysis. To ensure that the hemodynamic and angiographic parameters at baseline were similar between all three interventions (mibefradil, diltiazem and normal saline), comparisons among all three groups were made using one-way analysis of variance. For this test the level of significance was set at  = 0.05. For each of the three interventions (mibefradil, diltiazem and saline), measurements obtained at baseline, 5, 10 and 15 min after bolus drug administration were examined using repeated measures analysis of variance. For this test, the level of significance was also set at  = 0.05. If significance was attained, pairwise comparisons between measurements obtained at baseline and those obtained at 5, 10 and 15 min were performed using the Student-Newman-Keuls test. For this test, P  .05 was considered significant. To test for drug effect, the change () in any one variable from baseline to 5, 10 and 15 min was calculated. Comparisons of  values at 5, 10 and 15 min between saline mibefradil or diltiazem and between mibefradil and diltiazem were made using Student's paired t test. P  .05 was considered significant. All data are reported as the mean ± S.E.M.

	Results

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There were no significant differences in any of the hemodynamic or angiographic variables at baseline between diltiazem, mibefradil and saline (table 1). Intravenous administration of saline had no effect on any of the hemodynamic or angiographic measures of global LV performance. Similarly, i.v. saline had no effect on mean aortic pressure but caused a slight, yet significant, reduction in heart rate at 10 and 15 min after bolus administration (table 1).

Effects of i.v. diltiazem. The hemodynamic and angiographic effects of i.v. diltiazem are shown in table 1. Diltiazem produced a significant reduction of mean aortic pressure and an increase of heart rate. It had no effect on LV end-diastolic pressure but tended to increase cardiac output. The observed increase in cardiac output reached statistical significance only at 10 min after bolus administration of diltiazem. The trend toward an increase in cardiac output was associated with a modest but significant decline of systemic vascular resistance. The decline in systemic vascular resistance was statistically significant at 5, 10 and 15 min after bolus administration of diltiazem. Intravenous administration of diltiazem had no effect on measures of LV systolic and diastolic function namely, peak LV +dP/dt and dP/dt, LV end-systolic volume and LV ejection fraction (table 1). Comparison of the  at 5, 10 and 15 min relative to baseline between normal saline and diltiazem are shown in figures 2 and 3 and the statistical probabilities are shown in table 2. The  heart rate and  cardiac output increased significantly with diltiazem compared to saline and the  mean arterial pressure and  systemic vascular resistance decreased. There were no significant differences, however, between saline and diltiazem with respect to  LV end-diastolic pressure,  peak LV +dP/dt,  peak LV dP/dt,  LV end-systolic volume and  LV ejection fraction.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Change () from baseline (time = 0) of mean aortic pressure (mAoP), Heart Rate (HR), left ventricular end-diastolic pressure (LVEDP) and systemic vascular resistance (SVR) at 5, 10 and 15 min of treatment with mibefradil (circles), diltiazem (squares) and normal saline (triangles). * P < .05 mibefradil vs. diltiazem.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Change () from baseline (time = 0) of left ventricular (LV) peak +dP/dt, peak dP/dt and stroke volume (SV) at 5, 10 and 15 min of treatment with mibefradil (circles), diltiazem (squares) and normal saline (triangles). Change of LV ejection fraction (EF) is depicted at 15 min of treatment. * P < .05 mibefradil vs. diltiazem.

View this table: [in this window] [in a new window]   TABLE 2 Probability values for changes from baseline () at 5, 10 and 15 min between normal saline (control) and mibefradil or diltiazem

Effects of i.v. mibefradil. The hemodynamic and angiographic effects of i.v. mibefradil are shown in table 1. As with diltiazem, mibefradil also produced a significant decrease of mean aortic pressure and an increase of heart rate. Mibefradil tended to decrease LV end-diastolic pressure but the reduction did not reach statistical significance. Cardiac output increased significantly at 5, 10 and 15 min after bolus injection of mibefradil and was associated with a substantial decline in systemic vascular resistance. In contrast to diltiazem, i.v. mibefradil produced a significant improvement in global indexes of LV systolic function namely an increase in peak LV +dP/dt, LV end-systolic volume and LV ejection fraction. This improvement was evident at 5, 10 and 15 min after bolus administration (table 1). Peak LV dP/dt also tended to increase with i.v. mibefradil but the increase did not reach statistical significance. The acute hemodynamic improvements elicited by i.v. mibefradil persisted, albeit to a lesser extent, for up to 60 min after intravenous bolus administration of the drug (table 1). Comparison of  at 5, 10 and 15 min between normal saline and mibefradil are shown in figures 2 and 3 and the statistical probabilities are shown in table 2. Compared to saline, diltiazem resulted in an increase in  heart rate and a decrease in  mean aortic pressure and LV end-diastolic pressure. Intravenous mibefradil also produced significant increases in  cardiac output and decreases in  systemic vascular resistance compared to saline. Unlike diltiazem, mibefradil, when compared to saline, produced significant increases in  peak LV +dP/dt,  peak LV dP/dt and  LV ejection fraction and a significant decrease in  LV end-systolic volume.

Comparison of mibefradil and diltiazem. Comparison of  at 5, 10 and 15 min relative to baseline between mibefradil and diltiazem are shown in figures 2 and 3 and the statistical probabilities are shown in table 3. There were no differences between mibefradil and diltiazem with respect to  heart rate and  mean aortic pressure. At all time points (5, 10 and 15 min), mibefradil tended to lower LV end-diastolic pressure to a greater extent than diltiazem but the difference did not reach statistical significance (fig. 2). Mibefradil produced significantly larger increases in cardiac output, stroke volume, peak LV +dP/dt and peak LV dP/dt compared to diltiazem. Finally, i.v. mibefradil produced a significantly greater increase of LV ejection fraction and a greater decrease of LV end-systolic volume compared to diltiazem.

View this table: [in this window] [in a new window]   TABLE 3 Probability values for changes from baseline () at 5, 10 and 15 min between mibefradil and diltiazem

	Discussion

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The results of the study indicate that short-term i.v. administration of mibefradil, a predominant T-type calcium channel antagonist, leads to an overall improvement of LV function in dogs with chronic heart failure produced by intracoronary microembolization. The hemodynamic response to mibefradil is characterized by a decrease of systemic vascular resistance, an increase of cardiac output and a marked improvement in indexes of LV function as evidenced by significant increases of LV peak +dP/dt, peak dP/dt and ejection fraction. However, the short-term i.v. administration of diltiazem, an L-type calcium channel antagonist, resulted in a limited improvement of cardiac output and systemic vascular resistance but had no effects on indexes of global LV function including ejection fraction. The pronounced hemodynamic benefits of mibefradil over diltiazem were observed despite the fact that both compounds elicited nearly identical alterations in mean arterial blood pressure and heart rate. The observed contrasting hemodynamic actions of these two distinctly different calcium channel antagonists support the potential beneficial role of selective T-type calcium channel blockade for the treatment of HF.

Function and significance of T-type calcium channels and their blockade. Voltage and use-dependent blockade of L-type calcium channels have been extensively investigated for a variety of organic calcium entry blockers. In contrast, little is known about T-type calcium channels in the myocardium in both physiological and pathophysiological states. It is not known whether T-type calcium currents have a role in ventricular myocytes. The contribution of T-type calcium currents to the net membrane current before and during the action potential in ventricular myocytes appears to be very small but may possibly be involved in trigger release of calcium from internal pools (Morad and Cleemann 1987). An example of physiological significance of T-type calcium currents is their known contribution to the pacemaker depolarization of cardiac sino-atrial cells (Hagiwara et al., 1988). T-type calcium channels have been demonstrated to exist in a variety of vascular smooth muscle cells in relatively high density (Hagiwara et al., 1988). T-type calcium channels are thought to be permanently in the inactivated state but could recover from inactivation during hyperpolarization evoked by neurotransmitters (Ganitkevich and Isenberg, 1991). This observation suggests the possibility that increased sympathetic drive may be one possible trigger for activation of T-type calcium channels in heart failure. T-type calcium channels are seen largely in fetal and neonatal cardiac muscle cells and in vascular smooth muscle cells but infrequently in normal adult ventricular cardiomyocytes. Recent studies have shown that these calcium currents are expressed in hypertrophied adult feline LV myocytes and their density increased significantly in ventricular myocytes of cardiomyopathic hamsters (Nuss and Houser 1993; Sen and Smith 1994).

Previous studies with mibefradil. Several studies in animal models and in patients have demonstrated that mibefradil is devoid of negative inotropic effects compared to calcium channel blockers that act primarily on L-type calcium channels. Dose response studies in conscious normotensive rats showed that mibefradil had little or no negative inotropic effects compared to verapamil, diltiazem or amlodipine even though all four compounds produced similar reductions in mean arterial pressure (Veniant et al., 1991b). In rats with chronic myocardial infarction produced by coronary artery ligation, i.v. mibefradil had no effect on peak LV +dP/dt whereas diltiazem reduced peak LV +dP/dt in a dose-dependent manner (Veniant et al., 1991a). In patients with chronic stable angina pectoris, mibefradil was shown to be safe and effective as an antiischemic agent and, at the same time, devoid of negative inotropic effects (Portegies et al., 1991). Mibefradil's antianginal effects may be partially due to its apparent selectivity or preference for the coronary vasculature (Bian and Hermsmeyer, 1993).

Limitations of the study. A limitation of our study is that the results describe the effects of acute therapy with mibefradil when chronic therapy is indeed the ultimate target. Although it is tempting to use our results to imply a beneficial effect of mibefradil in the long-term treatment of HF, such an extrapolation is not warranted without further studies. Another possible limitation of the present study is the lack of measurement of plasma norepinephrine concentration before and after the administration of mibefradil, diltiazem and normal saline. In conscious dogs with HF produced by rapid ventricular pacing, i.v. infusion of mibefradil increased plasma noradrenaline level by 38% whereas infusion of diltiazem increased noradrenaline by 120% (Su et al., 1994). In patients with LV ejection fraction <40%, i.v. mibefradil was associated with a near 30% increase in plasma norepinephrine concentration but this increase was, in general, well within the limits of the analytical method used and considerably lower than that seen with i.v. infusion of dihydropyradine derivatives such as nicardipine and nisoldipine (Rousseau et al., 1984). Thus, even if some increase in sympathetic activation did take place in our study it is unlikely to have masked a drug-induced myocardial depression during mibefradil infusion given the abnormalities of cardiac beta-adrenergic receptor pathway in HF that are also manifested in this animal model (Gengo et al., 1992).

	Conclusions

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In conclusion, our results indicate that short-term i.v. mibefradil, a predominant T-type calcium channel antagonist that also partially blocks L-type calcium channels, improves LV function in dogs with chronic HF. This is in contrast to diltiazem, a prototypical selective L-type calcium channel blocker, which had no effect on LV function despite having a near identical effect on systemic blood pressure and heart rate as mibefradil. The beneficial effects of mibefradil compared to diltiazem appear to be a consequence of T-type calcium channel blockade resulting in a vasodilatory response that is free of negative inotropy. Chronic, long-term studies are needed to further establish the potential usefulness of T-type calcium channel antagonists as adjuncts for the long-term treatment of patients with HF.