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Cardiovascular Research 1998 37(1):130-140; doi:10.1016/S0008-6363(97)00216-2
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Combined potassium and calcium channel antagonistic activities as a basis for neutral frequency dependent increase in action potential duration: comparison between BRL-32872 and azimilide

Antoine Bril*, Marie-Claire Forest, Brigitte Cheval and Jean-François Faivre

SmithKline Beecham Laboratoires Pharmaceutiques, 4 rue Chesnay Beauregard, BP 58, 35762 Saint-Grégoire Cedex, France

* Corresponding author. Tel. +33-299-28-04-56; Fax +33-299-28-04-44; E-mail: antoine_bril@sbphrd.com

Received 6 January 1997; accepted 5 August 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Material and methods
 3 Results
 4 Discussion
 References
 
Objective: The effects of BRL-32872, azimilide and a selective blocker of the delayed rectifier potassium current, E-4031, were measured at two different basic cycle lengths (BCL), 300 and 1000 ms. Calcium channel antagonists of sarcolemmal (verapamil and nitrendipine) and sarcoplasmic reticulum (ryanodine) membranes were used to investigate whether the inhibition of the calcium current or the calcium release from the sarcoplasmic reticulum could alter the reverse-rate dependence of E-4031 on action potential duration (APD). Methods: Guinea pig isolated papillary muscles were superfused with a Tyrode solution maintained at 37°C and stimulated at a BCL of 300 or 1000 ms. The standard microelectrode technique was used to record action potential parameters and to study the effects of azimilide, BRL-32872 and E-4031. E-4031 was superfused at increasing concentrations (0.01, 0.03, 0.1 and 0.3 µM) in the absence or in the presence of verapamil (0.3 µM), nitrendipine (0.03 µM) or ryanodine (0.1 µM). Results: BRL-32872 and azimilide induced a self-limited concentration-dependent increase in APD. The effect of BRL-32872 was not dependent on the stimulation frequency whereas the effect of azimilide was significantly reduced at the shorter BCL. E-4031 induced a concentration-dependent increase in APD at both stimulation BCL. The increase in APD was significantly more pronounced in fibres stimulated at a BCL of 1000 ms than in fibres stimulated at a BCL of 300 ms, characterising the reverse-frequency dependent effect of class III antiarrhythmic agents. The reverse-frequency dependence in action potential prolongation induced by E-4031 was significantly reduced in the presence of a low concentration of verapamil (0.3 µM), nitrendipine (0.03 µM), or ryanodine (0.1 µM). Conclusion: The results show that BRL-32872, in contrast to azimilide, does not induce the reverse-rate dependency of action potential prolongation typically produced by class III antiarrhythmic agents such as E-4031. Our results also show that reverse-rate dependency induced by E-4031 can be reduced by the simultaneous administration of a low concentration of a calcium channel antagonist or an inhibitor of the release of calcium from the sarcoplasmic reticulum. It is thus suggested that compounds with a suitable balance of potassium and calcium antagonistic activities may have less adverse effects than purely selective potassium channel blockers.

KEYWORDS Class III antiarrhythmic compounds; Calcium channel antagonists; Verapamil; Nitrendipine; Ryanodine; E-4031; BRL-32872; Azimilide; Reverse-frequency dependent increase in action potential duration


   1 Introduction
Top
 Abstract
 1 Introduction
2 Material and methods
 3 Results
 4 Discussion
 References
 
In species where the delayed rectifier potassium current is the main repolarising current, the cardiac action potential duration is shorter at faster stimulation rates in the physiological range of frequencies. This adaptation to stimulation frequency has been related to the cumulative effects of activation and inactivation of potassium and calcium currents and also to the effects of the electrogenic sodium potassium pump together with the accumulation of extracellular potassium [1]. Numerous studies have shown that the concentration-dependent increase in action potential duration observed with selective blockers of the rapid component of the delayed rectifier potassium current is more pronounced at slow than at fast stimulation frequency. This effect, termed "reverse-frequency dependence", has been observed with most class III antiarrhythmic compounds, e.g. sematilide, dofetilide, E-4031, MK499 and d-sotalol [2–7], and represents an important issue for the clinical use of these agents. The reduced action potential prolonging effect of class III antiarrhythmic compounds at short basic cycle length may decrease drug efficacy during ventricular tachyarrhythmias [8]and, perhaps of greater importance, the excessive prolongation of ventricular action potential at slow heart rates may increase the propensity of these agents for inducing early afterdepolarisations and torsades de pointes [9].

Amiodarone is an antiarrhythmic agent which has been reported to show a large number of different effects [10]. Focusing on direct cardiac activity, amiodarone, in addition to blocking several potassium currents, inhibits the sodium and calcium currents and also has an antiadrenergic action [11]. When administered chronically, amiodarone prolongs action potential duration and this effect is not dependent on the stimulation rate [12]. However, because of its complex mechanism of action, the absence of reverse rate-dependent increase in action potential duration of amiodarone remains unexplained. Recently, we described the electrophysiological effects of a novel antiarrhythmic agent, BRL-32872, and showed that its effects on action potential duration were related to a dual inhibition of the delayed rectifier potassium and of the L-type calcium currents [13]. Similarly, Fermini et al. suggested that azimilide, which inhibits the rapid and the slow component of the delayed rectifier potassium current, may also reduce the calcium current in cardiac preparations [14]. Because the plateau of the action potential is controlled by a precise equilibrium between many different ionic currents and notably the outward potassium and inward calcium currents, compounds affecting these two conductances may exhibit a frequency dependence different from that observed with pure class III antiarrhythmic agents.

The aim of the present study was to compare the effect of stimulation frequency on the increase in action potential duration induced by BRL-32872, azimilide and a selective blocker of the delayed rectifier potassium current, E-4031 [15]. Furthermore, we evaluated whether the reverse-rate dependence of action potential prolongation induced by potassium blockade may be reduced or suppressed by concomitant calcium current antagonism and by the alteration of intracellular calcium handling. To this aim the effects of E-4031 were measured at two different basic cycle lengths, 300 and 1000 ms, in the absence and in the presence of calcium channel antagonists or ryanodine. E-4031 was used in the present study because this compound was shown to block selectively the rapid component of the delayed rectifier, also called the E-4031 sensitive component [15], and to exhibit a clear reverse-frequency dependent increase in action potential duration and effective refractory period [6]. Since calcium current antagonists may differ in their ability to decrease action potential duration in relation to the stimulation frequency [16], both verapamil and nitrendipine were used to investigate the role of sarcolemmal calcium current inhibition.


   2 Material and methods
Top
 Abstract
 1 Introduction
 2 Material and methods
3 Results
 4 Discussion
 References
 
2.1 Tissue preparation and recordings
Male Hartley guinea pigs (250–300 g, Elevage St. Antoine, Pleudaniel, France) were maintained in accordance with National Institute of Health (NIH) guidelines (publication no. 85-23) for animal care. Guinea pigs were anesthetized with sodium pentobarbital (60 mg/kg). The hearts were rapidly removed and placed in a modified Tyrode solution gassed with 95%O2/5%CO2. The composition of this solution was as follows (mM): NaCl 125, KCl 4.0, CaCl2 1.8, MgCl2 1.0, NaHCO3 24, NaH2PO4 0.9, glucose 11 (pH 7.35). Thin papillary muscles were excised from either right or left ventricle, fixed to the silastic bottom of an organ bath and superfused with Tyrode solution maintained at 37°C. After a stabilisation period (at least 2 hours), transmembrane potentials were recorded by conventional glass microelectrodes filled with 3 M KCl (10–30 M{Omega}) and connected to a high input impedance amplifier (Biologic VF180, Claix, France). External stimuli (2 ms; twice threshold) were delivered by a Pulsar 6I stimulator (Frederick Haer and Co, Brunswick, ME) upon bipolar platinum electrodes applied at the base of the preparation. Depending on the protocol, the basic cycle length of stimulation was 300 or 1000 ms. Signals were monitored on a storage 20 MHz oscilloscope (Gould 1604, Ballainvilliers, France) and simultaneously recorded on a digital tape recorder (Biologic DTR 1200). Action potentials were acquired and analysed with a microcomputer. Measurements were made of the following: resting membrane potential, action potential amplitude, maximum upstroke velocity (Vmax) and action potential duration measured at 30%, 50%, and 90% of repolarisation when estimated from the peak of the action potential (APD30, APD50 and APD90, respectively).

2.2 Protocol
After equilibration, the fibres were randomly assigned to stimulation rate at basic cycle length of either 300 or 1000 ms. Then, stable control action potential parameters (maintained for 30 minutes) were determined. Papillary muscles were superfused with increasing concentrations of BRL-32872 (0.3, 1.0, 3.0 and 10.0 µM), azimilide (1.0, 3.0, 10.0, 30.0 and 100.0 µM) or E-4031 (0.01, 0.03, 0.1 and 0.3 µM). Each concentration was applied for 30 minutes, a duration which was considered as the time necessary to reach steady state effect. Only fibres in which the same impalement was maintained during the whole experiments were used for analysis. In a second set of experiments, increasing concentrations of E-4031 (0.01, 0.03, 0.1 and 0.3 µM) were tested in the absence or in the presence of verapamil (0.3 µM), nitrendipine (0.03 µM) or ryanodine (0.1 µM). These concentrations of verapamil and nitrendipine slightly decreased action potential duration whereas ryanodine slightly prolonged action potential, but these effects were not significant (data not shown). The concentration of ryanodine was however expected to fully inhibit calcium release from the sarcoplasmic reticulum [17]. In these experiments, verapamil, nitrendipine or ryanodine was added to the superfusion medium 30 minutes prior to the administration of the first concentration of E-4031 and remained present during the whole experiment.

2.3 Drugs
BRL-32872 (N-(3,4- dimethoxyphenyl)- N- [3 [[2- (3,4- dimethoxyphenyl) ethyl] propyl] -4-nitrobenzamide hydrochloride, SmithKline Beecham, Saint-Grégoire, France), azimilide (NE10064, Procter and Gamble, synthesised at SmithKline Beecham, Saint-Grégoire, France), E-4031 (N- (4- (1- [2- (6- methyl- 2- pyridyl) ethyl] -4-piperidyl) -carbonyl] phenyl) methanesulfonamide dihydrochloride dihydrate, (Eisai) synthesised at SmithKline Beecham, Saint-Grégoire, France), Verapamil (Sigma, St Louis, MO), Nitrendipine (Sigma, St. Louis, MO), and Ryanodine (Calbiochem, La Jolla, CA) were prepared daily as a stock solution in water. The concentration of compound for all these stock solutions was 2.5 mM. Further dilutions were made in Tyrode solution. Because nitrendipine may be sensitive to light, all tubing and reservoirs containing this compound were protected with aluminium foil. All other chemicals were of analytical grade.

2.4 Statistics
Results are presented as mean±SEM. The comparison of the action potential parameters measured at the two cycle lengths was made using the two-tailed unpaired Student's t test. The other statistical comparisons were performed using a two factor repeated measures analysis of variance followed by simple main effects to assess the effect of a drug concentration at a given stimulation frequency and to compare the effect of the two stimulation rates for a given concentration. The ANOVA design consisted of a between group comparison for the effects of stimulation rate on action potential parameters and a within group comparison for the effects of different concentrations of drugs. For parameters in which a simple main effect for drug concentration was found significant, further comparisons of the effects of drugs at a given stimulation rate were made using a within group analysis of variance followed by multiple comparisons using the Sidak procedure [18]. For comparisons with equal group sizes, the Greenhouse–Geisser adjustment was used to calculate p value, and for comparisons with unequal group sizes, the Geisser–Greenhouse conservative adjustment was taken into consideration [18]. All statistical analyses were performed by means of the microcomputer statistical program CRUNCH 4.0 (Crunch Software Corporation, Oakland, CA). A p value of less than 0.05 was considered as statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Material and methods
 3 Results
4 Discussion
 References
 
3.1 Effect of stimulation frequency on action potential parameters
Stimulation frequency is well known to influence action potential parameters, principally action potential duration. Table 1 shows the effect of the stimulation frequency on action potential parameters in guinea pig isolated papillary muscle superfused with a normal Tyrode solution. Stimulation frequency had no effect on resting membrane potential and on maximum upstroke velocity. However when the stimulation rate was increased from basic cycle length of 1000 ms (1 Hz) to 300 ms (3.3 Hz), action potential amplitude was slightly but significantly reduced. The principal effect induced by the increase in stimulation frequency, when basic cycle length was reduced from 1000 to 300 ms, was a significant reduction in action potential duration measured at 30, 50 and 90% of the repolarisation.


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  		Table 1 Effect of stimulation frequency on action potential parameters of guinea pig isolated papillary muscles

 
3.2 Comparative effects of BRL-32872 and azimilide on action potential duration in guinea pig isolated papillary muscle
BRL-32872 and azimilide have been shown to inhibit the delayed rectifier potassium current and at higher concentrations the L-type calcium current. The results summarised in Table 2 show that in papillary muscles stimulated at basic cycle length of either 1000 ms or 300 ms, both compounds did not significantly change action potential amplitude, resting membrane potential and Vmax except at the highest concentrations tested. The main effect induced by BRL-32872 and azimilide is a prolongation of the action potential duration at low concentrations with a limitation of this effect when the concentration is increased further (Fig. 1). Larger concentrations of azimilide than BRL-32872 are required for this bell-shaped response, with the potency difference in fibres stimulated at a basic cycle length of 1000 ms (as shown in Fig. 1) being approximately ten fold. However, the results summarised in Fig. 2 clearly show that azimilide prolonged action potential duration more at slow (basic cycle length 1000 ms) than at fast stimulation frequency (300 ms). In contrast, the increase in action potential duration induced by BRL-32872 was similar at both stimulation frequencies (Fig. 2).


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  		Table 2 Effects of BRL-32872 and azimilide on action potential parameters of guinea pig isolated papillary muscle

 

Figure 1
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  		Fig. 1 A: Representative traces showing the effect of BRL-32872 (0.3–10.0 µM, left panel) and azimilide (1.0–100.0 µM, right panel) on action potentials recorded from guinea pig isolated papillary muscle stimulated at basic cycle length 1000 ms. B: Change in action potential duration measured at 90% of the repolarisation (APD90, ms) of guinea pig isolated papillary muscle stimulated at basic cycle length 1000 ms induced by increasing concentrations of BRL-32872 (closed symbols) or azimilide (open symbols). Each value represents the mean±SEM of 3–6 experiments.

 

Figure 2
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  		Fig. 2 Comparison between the frequency dependent effects of BRL-32872 (A) and azimilide (B). Values represent the mean±SEM of 3–6 experiments and are presented as a function of the drug concentration. Values represent the changes in action potential duration measured at 90% of the repolarisation (APD90, ms) of guinea pig isolated papillary muscle stimulated at basic cycle length 1000 ms (closed symbols) or 300 ms (open symbols) and were analysed using analysis of variance followed by multiple comparison with the Sidak procedure. *p<0.05, **p<0.01 between the two basic cycle lengths.

 
3.3 Effect of E-4031 in the absence and in the presence of verapamil or nitrendipine
Because a calcium blocking action has been involved to explain the electrophysiological effects of BRL-32872 and azimilide, the effects of a pure IKr blocker, E-4031, administered at concentrations between 0.01 and 0.3 µM, were investigated in guinea pig isolated papillary muscle in the absence and presence of verapamil or nitrendipine. The concentrations of verapamil (0.3 µM) and nitrendipine (0.03 µM) that were used in these experiments did not significantly change the action potential duration parameters when given alone (data not shown). Administered alone, E-4031 had no effect on action potential amplitude, resting membrane potential or maximum upstroke velocity (Table 3). The main electrophysiological effect of E-4031 was a concentration-dependent increase in action potential duration at both cycle lengths (300 and 1000 ms; Fig. 3). The absolute increase in action potential duration was significantly greater at the longer (1000 ms) than the shorter (300 ms) basic cycle length (Fig. 3). This effect, which was observed for all the concentrations of E-4031, illustrated the reverse-frequency dependence of E-4031 on action potential duration.


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  		Table 3 Effects of E-4031 in the absence or in the presence of nitrendipine or verapamil on action potential parameters of guinea pig isolated papillary muscle

 

Figure 3
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  		Fig. 3 Changes in action potential duration measured at 90% of the repolarisation (APD90, ms) of guinea pig isolated papillary muscle stimulated at basic cycle length 1000 ms or 300 ms induced by increasing concentration of E-4031. Columns represent the mean±SEM of 6 experiments. Values were analysed using analysis of variance followed by multiple comparison with the Sidak procedure. **p<0.01, ***p<0.001 between the two basic cycle lengths. E-4031-induced increase in APD90 was significant (p<0.001) vs. control value for all tested concentrations at both basic cycle lengths.

 
In the presence of a low concentration of either verapamil (0.3 µM) or nitrendipine (0.03 µM), the main electrophysiological effect induced by E-4031 was a concentration-dependent increase in action potential duration (Table 3; Fig. 4A and 4B). The only change observed in the other action potential parameters, was a reduction of the action potential amplitude with the larger concentrations of E-4031 and verapamil (Table 3). In the presence of nitrendipine, E-4031 had no effect on action potential amplitude, resting membrane potential and maximum upstroke velocity. Fig. 4A and 4B clearly illustrate that both calcium channel antagonists reduce the dependence of the increase in action potential duration produced by E-4031 on the stimulation basic cycle length. In the presence of verapamil, the effect of E-4031 was similar at both basic cycle lengths. Although action potential prolongation remained greater in fibres stimulated at 1000 ms than at 300 ms in the presence of nitrendipine, the difference in effect was less than that observed with E-4031 alone (compare Fig. 4B with Fig. 3). The results obtained with both verapamil and nitrendipine reveal that a low concentration of a calcium blocking agent may blunt the reverse-rate dependence on action potential duration of E-4031. However, verapamil was more effective than nitrendipine in this respect.


Figure 4
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  		Fig. 4 Changes in action potential duration measured at 90% of the repolarisation (APD90, ms) of guinea pig isolated papillary muscle stimulated at basic cycle length 1000 ms or 300 ms induced by increasing concentration of E-4031 in fibres superfused by a low concentration of verapamil (0.3 µM, A) or nitrendipine (0.03 µM, B). Columns represent the mean±SEM of 6 experiments. Values were analysed using analysis of variance followed by multiple comparison with the Sidak procedure. *p<0.05 between the two basic cycle lengths. E-4031-induced increase in APD90 was significant (p<0.01 for 0.01 µM E-4031 in the presence of nitrendipine; p<0.001 for all other conditions) for all tested concentrations at both basic cycle lengths vs. control value (i.e. in the presence of verapamil or nitrendipine given alone).

 
3.4 Effect of ryanodine on the reverse-rate dependence of E-4031 on action potential duration
As evidenced by the results obtained with BRL-32872 on the first hand and with E-4031 in the presence of verapamil and nitrendipine on the other hand, a slight inhibition of the calcium current can reduce the reverse-frequency dependence in the increase in action potential duration induced by a class III antiarrhythmic action. However, other mechanisms may also have similar effect. To test this hypothesis further, an additional group of experiments was performed to measure the effect of E-4031 in the presence of a blocker (namely ryanodine) of the sarcoplasmic reticulum calcium channel. The results summarised in Table 4 show that the principal effect induced by E-4031 in the presence of ryanodine was a concentration-related prolongation in action potential duration (Fig. 5). Furthermore, the increase in action potential duration was similar at both the basic cycle lengths of 300 and 1000 ms (Fig. 5) suggesting that the blockade of the ryanodine receptor can abolish the reverse-frequency dependent effect of E-4031. It is interesting to note that ryanodine suppresses the reverse-rate dependency of E-4031 by elevating the response to 300 ms to that of 1000 ms whereas verapamil and nitrendipine reduce the response to 1000 ms to that of 300 ms.


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  		Table 4 Effects of E-4031 in the absence or in the presence of ryanodine on action potential parameters of guinea pig isolated papillary muscle

 

Figure 5
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  		Fig. 5 Changes in action potential duration measured at 90% of the repolarisation (APD90, ms) of guinea pig isolated papillary muscle stimulated at basic cycle length 1000 ms or 300 ms induced by increasing concentration of E-4031 in fibres superfused by a low concentration of ryanodine (0.1 µM). Columns represent the mean±SEM of 6–13 experiments. Values were analysed using analysis of variance followed by multiple comparison with the Sidak procedure. E-4031-induced increase in APD90 was not significantly modified by stimulation rate increase. E-4031-induced increase in APD90 was significant (p<0.001) vs. control value (i.e. in the presence of ryanodine given alone) for all tested concentrations at both basic cycle lengths.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Material and methods
 3 Results
 4 Discussion
References
 
Action potential duration is known to be physiologically shorter at fast than at slow stimulation rates in species where the delayed rectifier potassium current is the main repolarising current, and this effect is classically attributed to an increased activation in the potassium current [19]. Through a blockade of the cardiac potassium currents, class III antiarrhythmic agents are well known to prolong action potential duration and effective refractory period, an effect which occurs for most agents in a reverse-frequency dependent manner. The reverse-frequency dependence of class III antiarrhythmic agents may limit their therapeutic potential because of reduced effectiveness at rapid frequencies and proarrhythmic potential at slow rates as a consequence of excessive action potential prolongation [8]. The results obtained in the present study with E-4031 provide confirmation of the reverse-frequency dependent effect of a typical class III antiarrhythmic agent. A similar range of concentration of E-4031 as the one used in the present study (0.03 to 1.0 µM) has been shown to prolong effective refractory period in ferret isolated papillary muscle [6]and action potential duration in guinea pig isolated papillary muscle [20]in a reverse-frequency dependent manner. A similar effect in both animal and human studies, has been reported for other class III antiarrhythmic agents such as dofetilide, MK-499, sematilide and d-sotalol [2–7]. Similarly, azimilide has been shown to cause a reverse-frequency dependent increase in effective refractory period in ferret isolated papillary muscle [14]and we observed a similar effect in the present study on action potential duration in guinea pig papillary muscle. In contrast, as shown in Fig. 2, BRL-32872 did not exhibit any reverse-frequency dependence in its ability to increase action potential duration.

The reverse-frequency dependent effect of class III antiarrhythmic agents, which has been shown to occur mainly during steady-state conditions [7], can be attributed to the blockade of the rapid component (IKr) of the delayed rectifier potassium current [15, 21, 22]. This allows the slowly activating component (IKs) to make a greater contribution to repolarisation at shorter basic cycle lengths due to its slow deactivation [21]. However, blockade of IKs may not be sufficient to eliminate completely the reverse-frequency dependent increase in action potential duration since azimilide, which still shows a reverse-frequency effect on action potential duration, has been shown to block IKs in several species [23, 24].

It has been previously shown that calcium blocking agents may inhibit drug-induced afterdepolarisations and excessive action potential prolongation at slow stimulation rates [25, 26]. The present study shows that low concentrations of calcium channel antagonists reduced the reverse-rate dependent effect of E-4031 on action potential duration but did not induce any bell shape concentration-dependent effect on action potential duration as obtained with BRL-32872 and azimilide. Indeed, we have shown that increasing concentrations of calcium channel antagonist are required to induce such a bell shape effect [27]. In contrast, the present experiments were carried out with a single low concentration of calcium channel antagonist. In addition to an effect on membrane calcium current, calcium channel antagonists have been shown to have other properties; for example, verapamil has been found to inhibit the delayed rectifier potassium current, though at high concentration (>10 µM) [28, 29], and dihydropyridines have been shown to alter the deactivation gating of IK but not its amplitude [30]. However, it is unlikely that these properties are involved in the reduction of the reverse-rate dependent effect of E-4031 on action potential duration and it seems reasonable to propose that blockade of membrane calcium current may be responsible for this action.

It is interesting to speculate why a reduction of calcium entry into the cell or inhibition of calcium release from the sarcoplasmic reticulum should reduce the reverse-frequency dependence of class III antiarrhythmic agents on action potential duration. Even if verapamil- and nitrendipine-induced decrease in action potential duration were not significant, it is possible that these small changes have led to a decreased IKr/IKs ratio at slow stimulation rate (BCL 1000 ms) and thus to a decreased efficacy of E-4031. At high stimulation rate (BCL 300 ms), lack of complete deactivation of IKs might largely explain action potential shortening and reduction of E-4031 activity which occurs independently of the presence of a calcium current antagonist. Another possibility is that an increase in intracellular calcium concentration within the physiological range may enhance the delayed rectifier potassium current in guinea pig cardiac myocytes [31, 32]. As the calcium-sensitive component of the delayed rectifier is believed to be IKs [32], calcium transients, which result mainly from calcium release from the sarcoplasmic reticulum, may be involved in action potential termination particularly at higher frequencies. In the presence of ryanodine, calcium release from the sarcoplasmic reticulum is inhibited and may explain, at least in part, the ryanodine-induced increase in E-4031 efficacy at high stimulation frequency (BCL 300 ms). These hypotheses could account for the observation that calcium current antagonists decrease efficacy of E-4031 at BCL1000 ms and that ryanodine increases efficacy of E-4031 at BCL 300 ms. It is difficult from the present data to speculate whether lack of reverse-rate dependency of BRL-32872 results from the former or the latter mechanism. Whereas it has been found that BRL-32872 inhibits the L-type calcium current of heart cells [13], its effect on calcium release from the sarcoplasmic reticulum remains to be investigated.

The present results in guinea pig isolated ventricular preparations might have clinical relevance since a reverse-frequency dependent increase in action potential duration has been observed in humans treated with selective class III antiarrhythmic agents such as sematilide [5]. It has already been reported that calcium antagonism may decrease proarrhythmic profile of selective class III antiarrhythmic compounds [33–35]. On the other hand, compounds with an intrinsic diversity of electrophysiological actions such as amiodarone [12]showed no reverse-frequency dependent effect and, in view of our present results, it is tempting to suggest that its ability to inhibit both potassium and calcium currents may at least in part be responsible for this lack of reverse-rate dependency. Therefore, the development of antiarrhythmic agents with a certain diversity of electrophysiological activity, such as potassium and calcium channel antagonism, may lead to compounds with reduced proarrhythmic potential and indeed the results of the present study suggest this might be the case. Thus, the novel antiarrhythmic agent, BRL-32872, like amiodarone [12], prolongs action potential duration to a similar extent at short and long stimulation basic cycle lengths. Whereas amiodarone exhibits very complex electrophysiological and biochemical activities [11, 36, 37], BRL-32872 induces a potent blockade of the rapid component of the delayed rectifier potassium current together with a moderate blockade of the L-type calcium current [13]. This dual electrophysiological activity has been suggested to be responsible for the low proarrhythmic profile of BRL-32872 in vivo [38]. It was indeed shown that the proarrhythmic profile of E-4031 was markedly reduced by co-administration of verapamil [38]. Whereas azimilide has a diversity of electrophysiological activity (blockade of the rapid and the slow components of the delayed rectifier potassium current and the calcium and the sodium inward currents [14, 39], it prolonged action potential duration and effective refractory period more at slow than at fast stimulation frequency both in ferret isolated papillary muscle [14]and in guinea pig isolated papillary muscle (present study). This suggests that, in contrast to BRL-32872, which blocks IKr and ICa with EC50s=30 nM and 3 µM, respectively [13], the balance between the potassium and calcium antagonistic effects of azimilide (EC50s=0.3 µM and 17.8 µM for IKr and ICa, respectively [40]is not adequate to prevent a reverse frequency dependent increase in action potential duration. Moreover, other mechanisms, yet to be identified, might also explain the absence of a reverse-frequency dependent effect of amiodarone and BRL-32872.

A further development of the present pharmacological study would consist in a mechanistic analysis that could be performed using several approaches. Indeed a computerised simulation could demonstrate whether rate-dependency of IKr inhibitors on action potential duration is altered by calcium current inhibition. An alternative approach would be to evaluate whether the IKr/IKs ratio changes when the membrane is clamped by action potentials recorded either in the presence or in the absence of calcium channel inhibitors.

In conclusion, the results of the present study confirm that stimulation rate has a significant influence on the effect of class III antiarrhythmic agents, the increase in action potential duration produced by E-4031 being inversely related to cycle length. Our results also demonstrate that in guinea pig isolated papillary muscles, BRL-32872, in contrast to azimilide, induces a similar increase in the action potential duration at fast and slow stimulation rates. This lack of reverse-rate dependency has been recently shown to be relevant in human patients where action potential duration was increased similarly by BRL-32872 whatever the cycle length (from 300 to 600 ms) [41]. The fact that BRL-32872 shows no reverse rate dependency, whereas azimilide does, suggests that the balance between potassium and calcium antagonistic activities may be of key value in rate-dependence. However, because the rate dependence of action potential duration is a complex mechanism involving the interaction of many currents, a reduction of the intracellular calcium movement, as evidenced by the effect of ryanodine, or modulation of other currents such as the inward rectifier current, the sodium-calcium exchange current, the slowly inactivating sodium current, the sodium «window» current or the sodium-potassium pump current may also be considered. Nevertheless, our results reinforce the hypothesis [27, 42]that compounds with a diversity of electrophysiological actions, including potassium and calcium blocking activity, might exhibit less adverse effects than selective class III antiarrhythmic agents.

Time for primary review 21 days.


   Acknowledgements
 
The authors would like to acknowledge Sonia Metayer-Saïdi for the preparation of the manuscript, Dr. Robert H. Poyser for useful criticisms and Caroline Dusseau for technical help in some of these experiments.


   References
Top
 Abstract
 1 Introduction
 2 Material and methods
 3 Results
 4 Discussion
 References
 

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