Bepridil (1-N-benzylanilino-2-pyrolidino-3-isobutoxy-propane) is an antianginal and antiarrhythmic drug with multiple pharmacologic actions (1). Recent electrophysiologic studies showed that bepridil inhibits both the fast sodium channels and the L-type calcium channels in single isolated myocytes of rats and guinea pigs (2) and in isolated guinea-pig atrial and ventricular muscles (3) and that it inhibits outward potassium currents including the inwardly rectifying K current (IKI), the delayed rectifier K current (IK), and the transient outward current (Ito) in sheep cardiac Purkinje fibers (4). The reduction of the outward K+ currents may account for class III antiarrhythmic action of this drug (4-7). Although bepridil is reported to have an inhibitor of the L-type calcium current (ICa; 2), the negative inotropic effect of this drug is significantly less than that of other Ca2+ antagonists, such as verapamil (6). For example, bepridil, at a concentration of 1 μM, which inhibits ICa of guinea pig ventricular myocytes by >50% (2), exerted only negligible effects on the tension developed in isolated ventricular muscles of rabbit hearts (6), suggesting the presence of some additional effects on contractile proteins.
Solaro et al. (8) reported that bepridil increased developed force and enhanced myofibrillar adenosine triphosphatase (ATPase) activity in myofibrils of skinned porcine ventricular muscles but not in myofibrils devoid of their regulatory proteins, such as troponin and tropomyosin (desensitized myofibrils). Also, because bepridil induced a leftward shift in the relation between the Ca2+ concentration and the amount of Ca2+ bound to troponin C in skinned porcine ventricular muscles, it may increase the Ca2+ sensitivity of troponin C (8). However, the extent to which this mechanism contributes to the overall positive inotropic response in intact cardiac muscles is still unknown. Therefore we investigated the effect of bepridil on intracellular Ca2+ and contraction by using intact (nonskinned) cell aggregates of cultured neonatal rat ventricular cells.
MATERIALS AND METHODS
Culture of ventricular myocytes
Primary cultured cardiomyocytes were prepared from ventricles of 1- to 5-day-old neonatal Wistar rats of either sex, according to Bollon et al. (9) with some modification by Yonemochi et al. (10). The hearts were excised aseptically from the rats anesthetized with ether. The ventricles were removed, cut into small pieces, and blood was washed out with phosphate-buffered solution (PBS, vide infra for the composition) containing 0.02% EDTA. The tissue was then transferred to Hanks' balanced salt solution (HBSS, vide infra for the composition) containing 10 mg/ml collagenase (Type IV; Worthington, Freehold, NJ, U.S.A.) held in a shaker in a water bath at 37°C. After 10 min of incubation, the supernatant was discarded, and the precipitant was further digested in Ca2+- and Mg2+-free balanced salt solution (CMF, vide infra for the composition) containing 1,000 U/ml dispase (Godo Shusei, Tokyo, Japan) for 10 min. The cell suspension was centrifuged at 2,000 rev/min at 4°C for 5 min, and the supernatant was discarded. This procedure was repeated 2 or 3 times. The myocytes were resuspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (Bioproducts, Walkersville, MD, U.S.A.), 10 mM HEPES, and 100 IU/ml kanamycin. The cells were seeded onto 100-mm plastic culture dishes and incubated for 90 min at 37°C in an incubator with air containing 5% CO2. The myocytes were separated from other components such as fibroblasts, endothelial cells, and smooth-muscle cells by decantation (11). The myocytes were suspended in DMEM at a density of ∼106 cells/ml DMEM, and 2 ml of the cell suspension was inoculated onto a sterile cover glass, 15 mm in diameter, and placed on the bottom of a 16-mm diameter culture dish. The myocyte cultures were replenished with fresh DMEM daily, after 2 days of culture, until the day of fura-2 experiments. This technique yielded myocyte aggregates, 100-300 μm in diameter, that beat synchronously at a rate of 0.45 ± 0.05 Hz (n = 68). Aggregates from 3- to 5-day cultures were used for experiments.
The solutions used had the following compositions (in mM). PBS: NaCl, 137; KCl, 2.7; Na2HPO4, 8.1; KH2PO4, 1.5; HBSS: NaCl, 137; KCl, 5.4; MgSO4, 0.8; Na2HPO4, 0.34; KH2PO4, 0.44; Ca2+/Mg2+-free balanced salt solution (CMF): NaCl, 137; KCl, 4.0; Na2HPO4, 0.32; and KH2PO4, 0.18. HEPES-buffered Tyrode's solution used in the Ca2+ microfluorometry experiments contained (in mM): NaCl, 135; KCl, 5.4; MgSO4, 1; NaH2PO4, 1; NaHCO3, 3; glucose, 10; and HEPES, 10 (pH adjusted to 7.4 by adding NaOH). The final Ca2+ concentration was adjusted by adding an appropriate amount of a 1 M CaCl2 stock solution to make various concentrations as indicated in Results. This solution was introduced to the test chamber (volume, 2 ml) at a rate of 2 ml/min. All experiments were performed at room temperature (24-26°C).
Intracellular loading of fura-2
The myocytes were loaded with pentaacetoxymethyl ester fura-2 (fura-2/AM; Wako Pure Chemical Industries, Osaka, Japan) at a concentration of 10 μM in culture medium that contained 0.2% vol/wt cremophor EL (Sigma, St. Louis, MO, U.S.A.). Fura-2/AM was dissolved, in advance, in dimethylsulfoxide (Wako Pure Chemical Industries, Osaka, Japan) at a concentration of 1 mM. Fura-2/AM (10 μM) was loaded to the cells in a carbon dioxide incubator at 37°C for 8-10 min (12). The excess fura-2/AM was washed out with HEPES-buffered Tyrode's solution (1.8 mM Ca2+), and the cells were maintained at room temperature (24-26°C) for 30-60 min before measurements of [Ca2+]i to attain deesterification of fura-2/AM in the cytosol.
Measurement of fura-2 fluorescence
The aggregates loaded with fura-2 were field-stimulated at 0.5, 1.0, and 2.0 Hz with rectangular pulses of 10-ms duration and of twice the threshold intensity by a stimulator (SEN-3201; Nihon Kohden, Tokyo, Japan) through a pair of platinum plate electrodes coupled to the isolation unit. Fluorescent light from fura-2 was recorded by using a fluorometer with a dual-wavelength excitation device (CAM-200; Nihonbunkou, Tokyo, Japan) equipped with an inverted microscope in the epifluorescence mode (TMD; Nikon, Tokyo, Japan). At the same time, images of the cell aggregates, made by white light from a halogen lamp source, was focused on a CCD video camera (CS3320; Tokyo Electronic Industry, Tokyo, Japan). A light-path exchanger (Nikon, Tokyo, Japan) was used to select the fluorescent light signals for the measurement of [Ca2+]i or the white light for recording of cell image to measure the contraction (shortening of cell aggregates). The excitation lights with a wavelength of either 340 or 380 nm (alternated at 1,000 Hz) were introduced onto the aggregate, and the emission from a particular small area (80 × 80 μm) located close to the central part of the aggregate was detected through a ×40 objective lens (Nikon CF UV, Fluor; Nikon, Tokyo, Japan). The emission light (fluorescent light from fura-2) at a wavelength of 500 nm was detected by a photomultiplier and recorded. To minimize the photobleaching of fura-2, the excitation light was applied intermittently and attenuated by 90% with the use of a neutral density filter. The light signals were stored on magnetic tapes by using a DAT recorder (RD-101T PCM/DATE recorder; TEAC, Tokyo, Japan), replayed and processed by a computer (Power Macintosh 8500/120; Apple Japan, Tokyo, Japan). To increase signal-to-noise ratio, five to 15 signals of the Ca2+ transients were averaged in some experiments (Figs. 1, 3, and 4). After subtracting background fluorescence signals obtained in the absence of the specimen, the ratio (R340/380) was calculated by dividing the signal intensity obtained at excitation wavelength of 340 nm with that obtained at 380 nm (Fig. 1). Because the fura-2 ratio is related to [Ca2+]i in a nonlinear fashion (13), and as we did not perform in vitro calibration of the fura-2 signal, we used the ratio (R340/380) to indicate the intracellular level of free Ca2+ concentration (i.e., [Ca2+]i, throughout this study).
Parameters of Ca2+ transient
As shown in Fig. 1, the following parameters of the Ca2+ transient were defined: dCa (the minimum R340/380 at diastole), sCa (the maximal R340/380 at systole), and the half decay time of Ca2+ transient [the time to 50% decay of Ca2+ transient or the level of (sCa + dCa)/2].
Measurements of contraction of cell aggregates
The image of cell aggregates was recorded by using a CCD video camera and stored on a VTR (NV-G40; Panasonic, Tokyo, Japan). Contractions of the cells were measured as a shortening of the peripheral margins of a particular cell in the aggregate, by using the public domain NIH image program written by Wayne Rasband (National Institutes of Health, NIHM, Bethesda, MD, U.S.A.). In brief, images of a cell in the aggregate stimulated at the rate of 1.0 Hz were stored on a computer every 33 ms for the entire cycle length of the contraction (consisting of 30 frames for 1 s), and each cell length was measured along the longitudinal axis. We then determined the maximal length (diastolic length) and the minimal length (systolic length) from 30 frames of cell images and calculated the shortening [(diastolic length) - (systolic length)]. This calculation was repeated on five consecutive contractions and averaged. The averaged shortening was normalized by dividing the averaged shortening by the averaged diastolic length and was expressed as the percent shortening, which is deemed to be a measure of contractile intensity in the cell aggregate.
Bepridil and D600 were kind gifts from Nippon Organon Co., Ltd., and Knoll/Taisho Pharmaceutical Co., Ltd., respectively. A stock solution of bepridil (1 mM) was prepared by dissolving the compound in dimethyl sulfoxide (DMSO); D600 (10 mM) was prepared by dissolving the compound in ethanol. The vehicles (DMSO and ethanol) alone did not affect either Ca2+ transients or contractions, even at the maximal concentrations used in our present experiments.
All values were expressed as mean ± standard error of the mean (SEM). The statistical significance of the results was evaluated with analysis of variance (ANOVA), and probability values were determined by Student's paired t test. A p value of <0.05 was considered significant.
Effects of bepridil and D600 on sCa, dCa, and contractions
To test the validity of the measurement of contractions of cell aggregates with the use of the system consisting of a CCD video camera and the NIH image program (see Methods), we examined the effect of isoproterenol, a well-known β-adrenoceptor agonist, on the shortening of aggregates of cultured rat ventricular cells stimulated at 1.0 Hz. As expected, isoproterenol at 10−8, 10−7, and 10−6M increased the percent shortening of cell aggregates from 11.2 ± 0.27% (control, n = 5) to 15.4 ± 0.96% (n = 4), 16.9 ± 0.75% (n = 4) and 23.4 ± 0.38% (n = 4), respectively, thereby suggesting that our system can reliably measure contraction in the cell aggregates.
With our measurement system and microfluorometry, we examined the effects of bepridil and D600 on sCa (peak systolic level of Ca2+ transient), dCa (end-diastolic level of Ca2+ transient), and the shortening of cell aggregates stimulated at three different frequencies of 0.5, 1.0, and 2.0 Hz. The results are summarized in Fig. 2. The drug effect attained the steady state ∼2 min after application of the drugs (at 0.5 Hz) and after each step-wise increase of the stimulation frequencies to 1.0 and 2.0 Hz. In the absence of the drugs (open circles), both sCa and dCa increased with increasing stimulation frequency (0.5, 1.0, and 2.0 Hz; Fig. 2A and B). However, application of bepridil (10 μM, solid circles) or D600 (10 μM, solid triangles) decreased sCa, which was more pronounced when the rate of stimulation was increased (frequency-dependent decrease of the Ca2+ transient; Fig. 2A). Figure 2C shows the effects of bepridil and D600 on the contraction. In the absence of the drugs, the percent shortening was significantly larger at 2.0 Hz than at 0.5 Hz (frequency-dependent increase). In the presence of 10 μM D600, the shortening was markedly decreased at all stimulation frequencies tested, albeit the decrease was more prominent at higher frequencies. In contrast, bepridil had no significant effect on the percent shortening despite its significant depressant effect on sCa (cf. Fig. 2A).
Table 1 summarized the frequency-dependent effects of various concentrations (1, 5, 10, and 30 μM) of bepridil and D600. Both drugs decreased sCa in a frequency- and concentration-dependent manner. In addition, bepridil at the highest concentration (30 μM) significantly decreased dCa. Moreover, moderate concentrations of bepridil and D600 (5 and 10 μM) significantly decreased the sCa as well. It must be noted here that D600, at concentrations (5-30 μM) that significantly decreased sCa, decreased the contraction (percent shortening) significantly, whereas bepridil at concentrations (5-30 μM) that significantly depressed the sCa did not decrease the contraction at all.
Concentration-dependent effects of bepridil and D600 on the Ca2+ transient
Figure 3 shows typical examples of the effects of altered concentrations of bepridil (A) and D600 (B) on the Ca2+ transient (evoked at 1.0 Hz) in the presence of 1.8 mM [Ca2+]o. It is apparent that both drugs markedly decreased the sCa, whereas bepridil, but not D600, slightly decreased the dCa in a concentration-dependent manner. Figure 4A illustrates tracing examples of the Ca2+ transient and the cell shortening seen in the absence (control) and presence of bepridil or D600, in the presence of 1.8 mM [Ca2+]o. Bepridil (10 μM) decreased sCa but did not decrease the percent shortening; in contrast, D600 (10 μM) deceased both sCa and percent shortening. We then compared the effects of bepridil and D600 on the half decay time of the Ca2+ transient and the time course of the percent shortening (contraction) in the same experimental settings. As shown in Fig. 4B, bepridil (10 μM), and not D600 (10 μM), shortened the half decay time of the Ca2+ transient and prolonged the time course of the contraction. In addition, the effect of bepridil on the half decay time of the Ca2+ transient depended on the concentrations used. On average, the drug shortened the half decay time of Ca2+ transients (at 1.0 Hz) from 208 ± 6 ms (control, n = 8) to 180 ± 6 ms (1 μM, n = 7), 152 ± 3 ms (5 μM, n = 7), 125 ± 3 ms (10 μM, n = 7), and 111 ± 3 ms (30 μM, n = 7). In contrast, the equivalent concentrations of D600 (1-30 μM) did not alter either the half-decay time of the Ca2+ transient or the time course of the contraction, an example of which is shown in Fig. 4B (bottom) with 10 μM D600.
Effects of bepridil and D600 on Ca2+ transient and contraction under varied [Ca2+]o
The results so far showed that both bepridil and D600 decreased the peak amplitude of Ca2+ transients (sCa) in a frequency- and concentration-dependent manner. This may be due to the drug-induced, frequency-dependent blockage of calcium influx through the calcium channels. To confirm the notion that the Ca2+ transient and thus the contraction of the cell aggregate may depend on the Ca2+ influx from the extracellular solution via the 1-type calcium channels, we studied the effects of altered external Ca2+ concentration on these two parameters. As expected, both sCa and percent shortening increased in a parallel manner with increases in extracellular Ca2+ concentration step-wise from 0.45 to 0.9, 1.8, and 3.6 (Fig. 5A and B, open circles). No significant changes were observed in the time course of Ca2+ transients for each [Ca2+]o tested (0.45, 0.9, 1.8, and 3.6 mM). In the presence of D600 (10 μM, solid triangles), the Ca2+ transient and contraction were suppressed over all [Ca2+]o tested, thereby shifting sCa or percent shortening versus [Ca2+]o relations downward to the right. In contrast, bepridil altered these relations differently. Bepridil (10 μM) decreased sCa to the same extent as did D600 (Fig. 5A, solid circles), whereas this drug did not alter the relation between the contraction and [Ca2+]o at all (Fig. 5B, solid circles).
To demonstrate the differential effects of bepridil (10 μM) and D600 (10 μM) on contraction, we plotted the relation between the percent shortening and sCa as shown in Fig. 5C. In this figure, the magnitude of Ca2+ transient was changed by altering [Ca2+]o in the absence of the drugs (open circles) and in the presence of bepridil (10 μM, solid circles) or D600 (10 μM, solid triangles). The curve showing the relation between sCa and percent shortening in the absence of the drugs is sigmoidal and overlaps that obtained in the presence of D600. In contrast, in the presence of bepridil, the curve was markedly shifted leftward to the top, suggesting that bepridil might have increased Ca2+ sensitivity of the contractile proteins.
In Fig. 6, we replotted the relation between the percent shortening and sCa, in that both parameters were changed by altering [Ca2+]o in the absence of the drugs or by altering the concentrations of bepridil or D600 in the presence of a standard [Ca2+]o of 1.8 mM. The curve in the absence of the drugs (open circles) is identical to that shown in Fig. 5C (open circles) in which the percent shortening-sCa relation was obtained by changing [Ca2+]o. The relation between sCa and percent shortening obtained by varying the concentration of D600 (solid triangles) was superposable on the one obtained by changing [Ca2+]o in the absence of the drug (open circles). In contrast, the curve obtained by changing the concentration of bepridil was located markedly leftward to the upward with no practical changes in % shortening despite fairly large changes in sCa, thereby suggesting that suppression of Ca2+ influx by bepridil was not accompanied by the depression of contraction, at least under our experimental conditions.
Reversibility from the drug effects
To determine the reversibility of sCa, dCa, and the percent shortening of cell aggregates, we washed out the cell aggregates with HEPES-buffered Tyrode's solution after the drug effects reached steady state (usually ∼2 min of application) and measured the recovery of these parameters; the recovery was excellent. For example, changes of sCa, dCa, and percent shortening caused by 30 μM bepridil (at 2.0 Hz) restored to as much as 94.3 ± 0.48% (n = 4), 95.2 ± 1.12% (n = 4), and 92.9 ± 2.09% (n = 4) of predrug values, respectively. The same levels of reversibility were also seen in case of 30 μM D600 (at 2.0 Hz). Therefore we regarded the effects seen in the presence of bepridil and D600 to be real drug effects and not caused by spontaneous deterioration of cell function.
By using dual-wavelength microfluorometry with fura-2 and video image-analyzing techniques, we studied the effects of bepridil on the intracellular Ca2+ concentration ([Ca2+]i) and contraction (shortening of cell aggregates) in cultured rat ventricular myocytes and compared the results with those of D600 (methoxyverapamil). Major findings are as follows.
- Bepridil (1-30 μM) and D600 (1-30 μM) decreased the peak amplitude of Ca2+ transients (sCa) in a concentration- and frequency-dependent manner.
- Bepridil did not produce any significant decrease in the contraction (measured as the percent shortening), whereas D600 suppressed contraction in a concentration- and frequency-dependent manner.
- Bepridil, but not D600, abbreviated the Ca2+ transients with a significant shortening of the half decay time (p < 0.05) and prolonged the time course of the contractions (percent shortening).
- Bepridil, but not D600, shifted the curve relating the shortening of cell aggregates to sCa toward the direction of lower [Ca2+]i.
We monitored the shortening of the cell aggregates as an index of contraction amplitude (14,15), although there may be some differences between the contractions in cell aggregates and muscle fibers in vivo. The movement of cell aggregates is restricted by the internal compliance of the tissue rather than by external loading, as is the case for muscles in vivo. In addition, as cross-bridges are randomly oriented in the cell aggregates, the resistance for the conformation change is enhanced during contraction (14). The random orientation of cross-bridges might suppress the cellular shortening, particularly when the aggregates are in positive inotropic state. To circumvent this problem, we examined the effect of a β-adrenergic agonist isoproterenol on the shortening of the cell aggregates.
The magnitude of the cell shortening increased with increasing concentrations of isoproterenol, indicating that the shortening could be a measure of contractile force exerted by the cell aggregates, even under positive inotropic states.
As demonstrated in Fig. 2, when the stimulation frequency was increased from 0.5 to 2.0 Hz under control conditions (in the absence of drugs), both systolic (sCa) and diastolic levels (dCa) of [Ca2+]i and contraction (shortening) increased significantly. These findings suggest that the preparation has characteristics of a positive staircase (16). Application of D600 decreased sCa and contraction of the cell aggregates, which was especially marked with higher stimulation frequencies (1.0 and 2.0 Hz), whereas dCa remained unchanged. This observation could be explained by the frequency (use)-dependent inhibition of the L-type Ca2+ current by D600 (17). By contrast, although bepridil also decreased sCa in a frequency-dependent manner (Fig. 2A), it did not affect the contraction, even at the highest concentration tested (30 μM;Fig. 2C), which significantly decreased sCa at 1.0 and 2.0 Hz. Lack of contractile depression with significant decreases in sCa and dCa (Table 1) suggested that bepridil might have exerted such an effect via some direct effect(s) on the contractile elements other than well-documented inhibitory effects on the Ca2+ channels in the sarcolemma (2,5).
Bepridil is classified as a Ca2+ antagonist, as it suppressed the L-type Ca2+ current with high efficacy (apparent dissociation constant, 0.5 μM; 2). However, it was reported that the negative inotropic effect of bepridil is not strong compared with other authentic Ca2+ antagonists, such as verapamil (6,18). In the study of Anno et al. (6), bepridil decreased the maximal rate of slow-response action potentials and L-type Ca2+ currents but had no practical depressant effects on the contraction. Solaro et al. (8) first reported that bepridil increases Ca2+ sensitivity of troponin C and of ATPase activity in myofibrils of skinned porcine ventricular muscles. These findings were later confirmed by Herzig and Quast (19) by using the same preparation.
The finding that bepridil significantly shortened the half decay time of the Ca2+ transient and prolonged the time course of the contraction (percent shortening; Fig. 4) lends support to the notion that bepridil does increase the Ca2+ sensitivity of the contractile protein(s), supposing that the decaying phase of Ca2+ transient is determined by the combination of the rate of release of Ca2+ from contractile protein and the rate of Ca2+ uptake into the sarcoplasmic reticulum (SR; 20,21). When the Ca2+ sensitivity of the contractile protein(s) is increased, Ca2+ may bind rather tightly to the protein(s), which, in turn, shortens the half decay time of Ca2+ transients and prolongs the time course of the contraction (20,22). Indeed, EMD57033, a selective Ca2+ sensitizer, shortened the decay time course of aequorin signals and prolonged the time course of contraction in the isolated ferret ventricular muscles (22).
Among the drugs classified as Ca2+ antagonists, bepridil is unique in that it exerts extremely small negative inotropic effects in comparison with the coronary dilating action (18). A part of the strong vasodilating effect of this agent may be due to its inhibitory effect on calmodulin (23). In myocardium, the inhibition of calmodulin increased Ca2+ release from the SR (24). Thus the calmodulin antagonistic action of bepridil, together with its Ca2+-sensitizing effect, might have counteracted the negative inotropic effect otherwise known to be caused by this Ca2+-antagonistic agent.
In conclusion, this study showed that the negative inotropic effect of bepridil as a Ca2+ entry blocker could be offset by the increased Ca2+ sensitivity. Although the Ca2+-sensitizing effect of bepridil was seen by using skinned cardiac muscles (8), our study, by using an intact (nonskinned) cardiac preparation (cultured neonatal rat ventricular cell aggregates), first confirmed that the depressed contraction due to Ca2+ entry-blocking action of bepridil could be nullified by virtue of its simultaneous Ca2+-sensitizing effect on contractile protein(s). Ca2+ channel-blocking action with no significant depressant effects on the contractility of working cardiac muscles is useful for the treatment of reentrant arrhythmias mediated by Ca2+ channel-dependent slow conduction (25) or triggered activity originating from the delayed afterdepolarization (26), both of which are thought to occur in acute and chronic myocardial ischemia. The Ca2+-sensitizing effect may have another advantage in increasing the energetic efficiency of contraction with eventual production of more force with relatively small increases in O2 consumption (27). Final evaluation must await further clinical studies.
Acknowledgment: We thank Ms. K. Goto and Ms. K. Moriyama for their secretarial services.
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