More than 30 years ago, the antihypertensive and antianginal effect of the Ca2+-channel antagonists were first documented. Ca2+-channel antagonists were named after their interaction with voltage-dependent Ca2+ channels while blocking them.
Ca2+-channel antagonists are used because they reduce cardiac afterload and increase the coronary blood flow by decreasing the coronary vasotonus, and because they reduce heart rate by diminishing the activity of the sinus node, and decrease blood pressure by enhancing the renal sodium excretion (1-4).
Two types of voltage-dependent Ca2+ channels are described in cardiac and vascular myocytes: the L- and T-type Ca2+ channels (5,6). L-type Ca2+ channels are located in the transverse tubules (T-tubules) of the plasma membrane, which penetrate the cell's interior. In the atrial and ventricular myocytes, Ca2+ ions entering through L-type Ca2+ channels have several functions like the opening of intracellular Ca2+-release channels (ryanodine receptors), initiating contraction, pacemaker activity in the sinoatrial node, and atrioventricular conduction in the atrioventricular node (7). L-type Ca2+ channels are the most important voltage-dependent plasma membrane Ca2+ channels in the heart and vascular smooth muscle. They bind the Ca2+-channel blockers currently used in clinical practice including dihydropyridines (e.g., nifedipine), phenylalkylamines (e.g., verapamil), and benzodiazepines (e.g., diltiazem; 8,9).
T-type Ca2+ channels are found in a variety of tissues including the heart and vascular smooth muscle. Like L-type Ca2+ channels, T- type Ca2+ channels are opened by membrane depolarization. However, the gating of these channel types is different. T-type Ca2+ channels close and become inactive more rapidly than L-type Ca2+ channels. Thus, T stands for transient-opening, and L for long-lasting. Additionally, the threshold for T-type Ca2+-channel opening is lower than that of the L-type Ca2+ channel (10,11). These differences in gating, along with the absence of T-type Ca2+ channels in the T-tubules of the human heart, suggest important differences in the functions of T- and L-type Ca2+ channels (11,12).
T-type Ca2+ channels of the cardiac muscle can be blocked by mibefradil (Ro 40-5967), a recently developed Ca2+-channel antagonist of a new chemical class [substituted tetralin derivate (1S,2S)-2-[2-[[3-(2-benzimidazolyl)-propyl]-methylamino]-ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphthyl-methoxyacetate dihydrochloride)] with 3 times higher selectivity for T-type Ca2+ channels in comparison with L-type Ca2+ channels (13). Mibefradil as a pharmacologic tool to investigate Ca2+-channel effects also binds at higher concentrations to the verapamil-binding site of dihydropyridine-sensitive L-type Ca2+ channels in Chinese hamster ovary and in azygos vein cells of neonatal rats. In animal studies, a minor cardiodepressive effect of mibefradil was described (14-17). Mibefradil may be a new therapeutic agent in the treatment of coronary artery disease because of its vasodilating effects without the negative side effects of the formerly used therapeutic Ca2+-channel antagonists (18-20).
As both, T- and L-type Ca2+ channels have been described in guinea pig myocardium (5,6), in contrast to the human ventricle, our study investigated whether T-type Ca2+-channel blockade might reduce contractility similar to L-type Ca2+-channel blockade. As mibefradil exerts a higher potency for T-type Ca2+-channel-influx inhibition (13), differences in the negative inotropic potency could be an indication of T-type Ca2+-channel-mediated influences on contractility. Thus, the inotropic effect of mibefradil in isolated cardiomyocytes from guinea pig was compared with those of diltiazem. The inotropic action of increasing extracellular Ca2+ concentration was studied as well.
MATERIALS AND METHODS
Isolation and preparation of guinea pig cardiomyocytes
Guinea pigs (male, 200-250 g, n = 6) were heparinized (10 units/g) 10 min before anesthetization with sodium pentobarbital (50 mg/kg).
The heart was quickly excised, and the aorta was cannulated to the base of a Langendorff column (height, 1 m) maintained at 37°C. After an initial 5-min perfusion period with a Ca2+-free Tyrode's solution (contains in mM: glucose, 10; HEPES-NaOH, 10; KCl, 4; MgCl2, 1; NaH2PO4, 0.33; and NaCl, 135), the heart was perfused for 10 min with the collagenase-protease enzyme solution (CLS II: 111 U/ml; Seromed, Berlin, Germany; Protease XIV: 0.35 U/ml; Sigma, Deisenhofen, Germany), followed by 5-min perfusion with low-Ca2+ (0.2 mM) Tyrode's solution. The ventricle was separated from atria, and cells were mechanically dispersed in Tyrode's with low-Ca2+ concentration (0.2 mM). After the cells rested for 30 min at room temperature, Ca2+ was added to a final concentration of 2 mM. After that, cells can be used for the measuring contraction amplitude.
Only rod-shaped cells with distinct sarcomeres in 2 mM Ca2+ and no signs of granulation, cells that have no spontaneous contractions when not electrically stimulated in 2 mM Ca2+, and cells contracting in line with their long axis were used for experiments.
Measurement of contraction
Experiments were performed at 32°C in Tyrode's solution containing (in mM): CaCl2, 2; NaCl, 120; KCl, 5.4; MgCl2, 1; NaHCO3, 22.6; NaH2PO4, 0.42; glucose, 5; ascorbic acid, 0.3; and EDTA, 0.05 (adjusted at pH 7.4, and carbogen gassed). About 400 μl of cell suspension (held at room temperature) were placed on the stage of an inverted microscope (Diaphot 300, Nikon, Japan). The myocytes spontaneously attached to the glass coverslip forming the floor of the chamber (12 mm in diameter). After 5 min of resting, the cells were superfused with prewarmed (32°C) Tyrode's solution (0.2 ml/min) and electrically stimulated to contract by using platinum electrodes running through the length of the chamber. Cell contraction was performed by field stimulation with 0.2-2 Hz with a pulse duration of 2 ms.
The image of the contracting cell was detected by a one-dimensional high-speed camera with a time resolution of 4 ms. A one-line image analyzer detects the cell edges and measures their distance during contraction. The output of the analyzer provides an analog signal proportional to the cell length. This signal was stored in a computer and evaluated by special software (SI, Heidelberg, Germany). The following cell-contraction parameters were determined: maximal cell-contraction amplitude Lmax (μm) and diastolic cell length Ldia (μm). For experiments, concentrations of inotropes were increased cumulatively until there was no further increase in contraction amplitude or until toxic signs like large decreases in resting cell length or phasic contractions were observed. Dose ranges used were 2-7.5 mM for Ca2+, 1-100 μM for mibefradil, and 1-100 μM for diltiazem.
Mibefradil (Ro 40-5967) was obtained from Hoffmann-La Roche AG (Basel, Switzerland), and diltiazem from Gödecke (Freiburg, Germany). The chemical structures of the used Ca2+-channel antagonists are presented in Fig. 1. Mibefradil and diltiazem were prepared as stock solutions of 10 mM in H2O. Only bidistilled water was used in these experiments. The final concentrations of the inotropes were reached by dilution with 2 mM Ca2+-containing Tyrode's solution. All other chemicals were of analytic grade or the best grade commercially available.
Results are presented as mean ± SEM. Statistical significance was determined by Student's t test or one-way analysis of variance (ANOVA). The level of statistical significance was set at a probability of 0.05.
EC50 is the drug concentration needed to achieve the halfmaximal effectiveness.
Diastolic call length of ventricular myocytes
The stable diastolic cell length of a continuous contracting cardiomyocyte is a criterion of the viability of the cell (21). In this study, the diastolic length of the cells and stable contraction amplitude at a stimulation rate of 0.5 Hz in 2 mM Ca2+ were observed during all experiments. The average diastolic cell length of myocytes from guinea pig ventricle (n = 12) at basal conditions (2 mM Ca2+, 0.5 Hz) was 107.9 ± 8.8 μm (mean ± SEM). This is in good agreement with previous studies (22). There was no significant trend toward longer or shorter diastolic cell length for cumulative concentration-response experiments with diltiazem. For the following concentrations from 0, 1, 10, to 100 μM diltiazem, the values (n = 6) obtained for the diastolic cell lengths were 108.8 ± 11, 106 ± 10, 104.8 ± 12, and 104.5 ± 14 μm (see Fig. 2). For concentration-response curves with mibefradil (0, 1, 10, and 100 μM), the corresponding values (n = 6) were 107.2 ± 9, 103 ± 9, 102.4 ± 8, and 100.8 ± 4 μm (see Fig. 2). Statistical analysis showed no significant differences between diastolic cell lengths for either diltiazem or mibefradil.
Effects of mibefradil and diltiazem on contractility
To determine the inotropic effects of the blockade of voltage-dependent Ca2+ channels in contracting guinea pig ventricular myocytes, cumulative concentration-response curves were performed for different classes of Ca2+-channel blockers.
Changes of the contraction amplitude of the cell as a result of pharmacologic intervention were shown in micrometers of cell shortening and percentage shortening from basal conditions. Cumulative concentration-response curves were performed with the selective L-type Ca2+-channel antagonist diltiazem (0, 1, 10, and 100 μM) and the novel developed T-type Ca2+-channel blocker mibefradil (0, 1, 10, and 100 μM), which interacts in high concentrations with the verapamil-binding site of the dihydropyridine receptor as well (23). Experiments were performed with guinea pig ventricular cardiomyocytes because of the coexistence of L- and T-type Ca2+ channels in guinea pig heart (5,6). Concentration-response curves were only used if the inotropic effect could be fully reversed on washout. Original registrations of myocyte contractions are shown in Figs. 3 and 4. Contraction amplitude decreased in response to increasing concentrations of diltiazem (Fig. 3) and mibefradil (Fig. 4). The upper parts of Figs. 3 and 4 show a representative single contraction amplitude under basal conditions (2 mM Ca2+, 0.5 Hz, control) in comparison with the reduced contraction amplitude of the same cell after treatment with 100 μM diltiazem (Fig. 3A) or 10 μM mibefradil (Fig. 4A). Parts B of Figs. 3 and 4 show a representative original registration of a cumulative concentration-response curve experiment for diltiazem (Fig. 3B) and mibefradil (Fig. 4B). The values for cell shortening during contraction under basal conditions (2 mM Ca2+, 0.5 Hz) of six cells from six guinea pigs were 11.9 ± 1.5 μm for diltiazem and 11.2 ± 1.8 μm for mibefradil. Thus, the basal cell shortening was similar in both groups studied.
The stepwise addition of 1, 10, and 100 μM diltiazem (n = 6) or mibefradil (n = 6) decreased the amplitude of cell contraction significantly. The mean values ± SEM for diltiazem were 10.94 ± 2.3 (92 ± 12%), 9.45 ± 1.9 (79 ± 9%), and 4.22 ± 0.9 μm (35 ± 3%). The corresponding values for the stepwise addition of 1, 10, and 100 μM mibefradil were 8.71 ± 0.4 (78 ± 9%), 3.98 ± 0.2 (36 ± 5%), and 2.64 ± 0.3 μm (24 ± 4%), respectively (see Fig. 5). Both mibefradil and diltiazem reduced cell contraction in a concentration-dependent manner. However, mibefradil exerted significant negative inotropic effects at lower concentrations compared with diltiazem (<1 μM). Concentrations >100 μM of mibefradil or diltiazem almost completely abolished contraction is isolated guinea pig cardiomyocytes. The concentration at which the contraction amplitude of guinea pig cardiomyocytes was reduced by 50% (EC50) was 31.6 μM for diltiazem and 6.3 μM for mibefradil, indicating that the T-type Ca2+-channel blocker mibefradil is more potent in reducing cell length in guinea pig cardiac myocytes in comparison with the L-type Ca2+-channel antagonist diltiazem. The maximal negative inotropic effect of diltiazem (100 μM) in contracting guinea pig cardiomyocytes (n = 6) was 35 ± 2.6% of the basal contraction amplitude or 4.22 ± 0.2 μm. The corresponding values (mean ± SEM) for 100 μM mibefradil were 24 ± 3.6% of basal values or 2.6 ± 0.3 μm.
Effect of increasing extracellular calcium on contractility
The inotropic effect of increasing concentrations of extracellular Ca2+ on continuously contracting guinea pig cardiomyocytes was studied to determine the maximal positive inotropic effects of cell contraction and to demonstrate functional integrity after isolation procedure. The ability of the cells to respond to high extracellular Ca2+ concentrations and to recover completely after washout was used as a further criterion for cell viability (24). This (initial) challenge with high extracellular Ca2+ concentrations served not only as a test of the Ca2+ ability of the cell, but also as a control of inotropic response.
Figure 6A shows a sample trace of myocyte contraction under control conditions (2 mM Ca2+, 0.5 Hz) and the increased contraction amplitude after increasing the extracellular Ca2+ concentration to 7.5 mM in the same cell. Figure 6B shows a cumulative concentration-response curve for five cells from five guinea pigs. The mean values ± SEM of cell shortening after increasing extracellular Ca2+ (2, 3, 4, 5, and 7.5 mM) were 7.1 ± 0.28 (100%), 10.3 ± 0.6 (145 ± 3%), 11.9 ± 1.1 (168 ± 11%), 13.3 ± 1.6 (187 ± 15%), and 15.1 ± 1.1 μm (213 ± 14%), respectively. The maximal positive inotropic effect of 7.5 mM Ca2+ in guinea pig cardiomyocytes (n = 5) was +213 ± 14%. The concentration of extracellular Ca2+ at which the contraction amplitude of guinea pig cardiomyocytes was increased by 50% (EC50) was 3.1 mM.
As guinea pig ventricular cardiomyocytes contain both T- and L-type Ca2+ channels (6), they are suitable to study the effect of Ca2+ influx via T-type Ca2+ channels on contractility in comparison to L-type Ca2+-channel influx. T-type Ca2+ channels may be blocked with mibefradil. Studies in vascular smooth muscle cells exerted a 3 times higher affinity of mibefradil for T- compared with L-type Ca2+ channels (13). For comparison, diltiazem was studied. In our study, both T- and L-type Ca2+-channel blockade reduced contractility in isolated electrically driven guinea pig cardiomyocytes in a concentration-dependent manner with the same effectiveness (24). However, the T-type Ca2+-channel blocker mibefradil was more potent in reducing contractility in guinea pig cardiomyocytes than was diltiazem.
The use of isolated cardiac myocytes in our study allowed the detection and characterization of cell physiology and its pharmacologic interventions separately from the extracellular matrix and other nonmuscular influences. Harding et al. (25) reported similar inotropic effects for maximally activating Ca2+ for human papillary muscles and cells, whereas papillary muscles were less sensitive to the stimulatory effects of isoprenaline than cells taken from the same hearts. In comparison, these differences between cells and papillary muscles were not seen in rabbit hearts. In addition, Wang et al. (26) found that the negative load-contractility relation observed in multicellular myocardial preparations was preserved at the level of the isolated myocyte in pigs. Thus, species differences and disease states may be observed. Further to demonstrate the functional integrity of the contracting cells after the cell-isolation procedure, the maximal positive inotropic effect of cell contraction was determined with increasing concentrations of extracellular Ca2+ after a completely recovery of contractile activity after washout. The maximal contraction amplitude of the cell was judged to be reached when there was no increase in contraction amplitude or when signs of toxicity (such as phasic contractions) were seen. In previous studies, the positive inotropic effect of extracellular Ca2+ was characterized in myocardial cells from rat and rabbit cardiac cells (27). There was an increase of 425% for rat cells and 287% for rabbit cells at a maximal Ca2+ concentration of 8 mM (basal, 1 mM). Thus the inotropic effect elicited by increasing concentrations of extracellular Ca2+ reported in this study corresponds to previous reports. Our results showed a good agreement in the EC50 values with other observations (27) as well. As a further criterion for the viability of the cells, the stability of the diastolic cell length during all experiments was observed (see Fig. 2). There was no significant trend toward longer or shorter diastolic cell lengths for cumulative concentration-response experiments for mibefradil and diltiazem. The average diastolic cell length of the guinea pig cell in these experiments was in good agreement with previous studies (24).
Our study investigated the comparison of the inotropic effect of the selective T-type Ca2+-channel blocker mibefradil and the L-type Ca2+-channel blocker diltiazem in isolated guinea pig cardiomyocytes. Mibefradil and diltiazem concentration dependently inhibit the transsarcolemmal Ca2+ influx during contraction and reduce this way the contractility of guinea pig ventricular cardiomyocytes. Mibefradil acts more potent than diltiazem, as shown in the evaluation of the EC50 values that describe the half-maximal effect of the drugs. The EC50 value for mibefradil in this study was 6.3 μM in comparison to the EC50 value for diltiazem of 31.6 μM. These findings were in good agreement with the EC50 values for mibefradil (10 μM) and for diltiazem (40 μM) reported for Ca2+-channel currents (28,29). The higher potency of mibefradil in comparison to diltiazem in guinea pig cardiomyocytes may be a result of the more selective blockade of T-type Ca2+ channels at lower concentrations and the additional blockade of L-type Ca2+ channels at higher concentrations. Mibefradil showed a 3 times higher preference for T- in comparison with L-type Ca2+ channels (13). As mibefradil exerts higher potency to inhibit T-type Ca2+ influx and to reduce contractility, our study could be an indication that both T- and L-type Ca2+-channel inhibition may be important for the regulation of contraction, at least in guinea pig cardiomyocytes. However, whether this negative inotropic action of mibefradil results from L-type Ca2+-channel blockade cannot be ruled out. The differences in potency of the negative inotropic effects of mibefradil and diltiazem may result from their different potency to inhibit L-type Ca2+-channel influx in guinea pig cardiomyocytes as well.
T-type Ca2+ channels are found in a variety of tissues including the heart, vascular smooth muscle, and adrenal cortex. The density of T-type Ca2+ channels in myocytes specialized for contraction of most species including humans is low (10,12). T-type Ca2+ channels are not present in human ventricular myocardium (30). Because of the T-type Ca2+ channels' comparatively low threshold in the sinoatrial node, T-type Ca2+ channels are able to participate in pacemaker activity (31,32). Thus, mibefradil has been reported to reduce heart rate (33).
The physiologic function of T-type Ca2+ channels is not yet clear. They probably play a role in pacemaking and spontaneous excitations (34), but their significance in relation to contractility is not well defined. However, in this study with ventricular cardiomyocytes from guinea pig, it cannot be ruled out whether opening of T-type Ca2+ channels can indeed admit enough Ca2+ ions to trigger the contraction process. Ca2+ influx via T-type Ca2+ channels reduces the vascular tone, and probably in some species, it may also influence cardiomyocyte shortening.
Various studies showed the existence of T-type Ca2+ channels in embryonic tissues and their density in parallel to development (35). In contrast, the density of L-type Ca2+ channels is constant during development. These results led to the assumption that T-type Ca2+ channels are involved in cell growth and development. At this time, it cannot be ruled out that T-type Ca2+ channels may be expressed under certain pathophysiologic conditions (i.e., hypertrophic myocardium, ischemic or dilated cardiomyopathy). Studies in cardiomyopathic hamsters showed that the density of T-type Ca2+ currents was increased, and abnormal channel activation and inactivation kinetics were observed (36). Thus, there may be the possibility of expression of T-type Ca2+ channels during hypertrophy and remodeling of cardiac tissue during human heart failure.
Because of their vasodilatory effects, Ca2+ antagonists are used in the treatment of stroke, hypertension (37), and coronary heart disease (4). However, the use of Ca2+ antagonists may be limited by their direct negative inotropic actions. These are different in the classes of Ca2+-channel blockers (38,39). In human cardiovascular tissue, mibefradil shares potent vasodilatory potency and efficacy with only minor cardiodepressant effects (20). In this study, mibefradil up to a concentration of 10 μM reduced neither the intracellular Ca2+ transient measured by Fura 2 nor the force of contraction. Thus, T-type Ca2+ channels may not be present in human ventricular myocardium (30). In consequence, T-type Ca2+-channel blockade can reduce vascular force with only minor negative inotropic action. At very high concentrations, mibefradil can inhibit L-type Ca2+ influx as well.
As T-type Ca2+-channel blockade may affect force development only in some species-as shown for guinea pig cardiomyocytes-additional studies on the expression and function of T-type Ca2+ channels, especially in diseased myocardium, are needed. It must be stressed that changes in the disease states in isolated cardiomyocytes, as well as in multicellular papillary muscle preparations, were observed (22).
Acknowledgment: This study was supported by a grant of the Deutsche Forschungsgeselischaft (to Dr. Schwinger) and Köln Fortune and the Graduiertenkolleg "Molekularbiologische Grundlagen pathophysiologischer Vorgänge" of the University of Cologne to Dr. Schwinger.
This work contains part of doctoral thesis of S. Hoischen, University of Cologne (in preparation). Our special thanks are due to Prof. Dr. C.H. Orchard (University of Leeds, Great Britain) for help with methods. We also thank ASTA MEDICA AWD GmbH for support.
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