d-Sotalol (d-sot) and dofetilide (dof) are class III compounds, the potential antiarrhythmic usefulness of which has been investigated in large clinical trials. Dof is the prototype of a highly selective Ikr antagonist (1-2). The pharmacologic profile of d-sot, on the other hand, is not as well defined. Available data are mostly consistent with nonspecific potassium channel-blocking properties of d-sot (3-5). The results of the SWORD clinical trial have indicated severe proarrhythmic effects of d-sot in postinfarction patients with impaired left ventricular function (6). In contrast, dof seems to carry a more favorable therapeutic potential in a comparable patient group (7). The beneficial clinical role of the combined β blocker and class III agent d,l-sotalol (d,l-sot) is widely accepted (8,9). Results of large clinical trials directly comparing the effects of d,l-sot, d-sot, and dof in a risk-stratified patient population at risk for rhythmogenic events are not available. In a clinical setting, a complex interplay of modulating factors interferes with class III drug action. Increased sympathetic tone known to be associated with different states of chronic organic heart disease additionally has been identified as a crucial determinant of class III activity (10-12). In a conscious canine model of sudden cardiac death, d,l-sot, unlike d-sot, was effective in preventing ventricular fibrillation in the presence of ischemia and high catecholaminergic activity (13). In contrast, experimental models in which adrenergic activity was not primarily involved in the generation of tachyarrhythmias provided evidence of significant antifibrillatory potency of d-sot (14).
The clinical significance of reverse rate-dependent effects of class III agents is still controversial (15). It has been speculated that reduced efficacy of class III agents at high frequencies might limit their clinical usefulness, whereas excessive lengthening of repolarization at physiologic rates might be proarrhythmic. Based on guinea pig experimental models, reverse rate dependence of selective Ikr blockers has been attributed to the accumulation of Iks at faster pacing rates secondary to its slow deactivation kinetics (16). The specific interaction of adrenergic stimulation with the rate-response pattern of class III agents is not well established. Respective mechanisms are of particular interest in that the Ik component Iks is likely to play a major role both in mediating adrenergic activity (10) and in regulating the rate-dependent action of class III agents (17). Our experimental study was designed to evaluate the modulating effects of isoproterenol (iso) on the class III action of the selective class III agents dof and d-sot, and the combined β blocker and class III compound d,l-sot.
MATERIALS AND METHODS
Solutions and cell-isolation procedure
Ten healthy beagle dogs (12-15 kg, 1-2 years old) were used in these studies. All procedures performed were in accordance with institutional guidelines and with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1985). Under pentobarbital and N2O anesthesia, the chest was opened through a left thoracotomy, and the heart was excised. Small strips of left ventricular endocardium were dissected from transmural left anterior wall specimen. The tissue material was immediately placed in a zero calcium Tyrode's solution (NaCl, 115 mM; KCl, 5 mM; glucose, 45 mM; HEPES buffer, 10 mM; taurine, 4 mM; MgSO4, 2.7 mM; at pH 7.2) at 36.5°C bubbled with 100% O2. In a flat glass container, the endocardial tissue strips were cut in small chunks of 1 mm3 and washed extensively during continuous gasing with 100% O2. The chunks were then incubated with 20 ml collagenase solution (Sigma collagenase type 5, 317 U/mg) in a water bath at 36.5°C. Oxygen flow was maintained through glass cannulae agitating the chunks throughout the isolation procedure. After an isolation cycle of 20 min, samples of supernatant were screened for cell yield under the microscope. Intact myocytes in the supernatant were collected by gentle hand pipetting 30-40 times. Three to four isolation cycles were performed with new collagenase solution. Finally, the myocyte suspension was centrifuged at 700 rev/min for 10 min. The supernatant was discarded, and the sediment resuspended in 10 ml standard Hank's solution containing no collagenase but 10 μM Ca2+. After completion of the dissociation procedure, isolated cells were routinely obtained. On average, maximum length and width was 134 ± 15 by 33 ± 6 μm.
Data acquisition and processing
A small drop of cell suspension was placed in a microelectrode bath and mounted on a Nikon microscope stage. Cells were allowed to settle and were equilibrated for half an hour by constant superfusion with normal Tyrode's solution at a flow rate of 5-10 ml/min before the experiments were started. By means of flow-rate changes, the temperature in the bath solution was kept at 36.5 ± 0.5°C. Action potentials were recorded through glass capillary microelectrodes filled with 3 M KCl solution. The tip resistances ranged from 20 to 50 MΩ. The bath was grounded through a KCl Ag/AgCl salt bridge. The cells were stimulated individually by injection of brief (2 ms) depolarizing current pulses generated by a Grass stimulator (model S44). Recorded signals were transferred to a high-input resistance amplifier (Axoclamp 2A; Axon Instruments), displayed on an oscilloscope (Tektronix; type A 564 B), amplified with a programmable amplifier and digitized with a model 1401 AD/DA system. Data acquisition was controlled by an IBM personal computer with installed action-potential recording and processing program. Baseline action-potential recordings were obtained at stimulation rates of 0.1-3 Hz. At each frequency, action-potential duration (APD) values stabilized within 1 min of stimulation before the AP registrations were performed.
At first the baseline activity of iso on AP parameters was assessed by exposing six individual cells to iso at a concentration of 10−7 and 10−6M subsequently. The combined action of two different concentrations of d,l-sot (Bristol Myers Squibb Pharmaceuticals), d-sot (Bristol Myers Squibb Pharmaceuticals), and dof (Pfizer Pharmaceuticals) + iso at a fixed concentration was studied with a two-step protocol; In a first step, individual cells were exposed to concentration A (10−5M d,l-sot; 10−5M d-sot; 10−7M dof) of one of the three drugs and subsequently to 10−6M iso. After a complete drug washout was obtained, the same cells were subjected to the protocol with concentration B (10−4M d,l-sot; 10−4M d-sot; 10−6M dof) + 10−6M iso. After obtaining the final measurements, again a complete drug washout was performed. The complete study protocol is summarized in Fig. 1. Under all conditions studied, the initial impalement was maintained throughout the whole experiment. Six cardiomyocytes were used to evaluate the baseline activity of iso on AP parameters. Six cardiomyocytes were subjected to the protocol with d,l-sot A + iso/d,l-sot B + iso; six cardiomyocytes were subjected to the protocol with d-sot A + iso/d-sot B + iso; and six cardiomyocytes were subjected to the protocol with dof A + iso/dof B + iso. In addition, stimulation protocols A1 and B1 (see Fig. 1) was applied on another four individual cardiomyocytes without drug to exclude a spontaneous variability of the APD over time. Thus a total of 32 cells was studied. The following parameters were registered: resting membrane potential (RMP), AP amplitude (APA), APD at 90% repolarization (APD 90), and effective refractory period (ERP). The ERP was determined by a two stimulus technique as the shortest coupling interval eliciting two APs of normal morphology. The ΔAPD 90 (i.e., the difference in APD 90 on maximal drug dose and at baseline) was used to quantify absolute class III activity.
Results are expressed as mean ± SD. Changes in AP parameters induced by d,l-sot, d-sot, or dof at different concentrations in combination with iso were analyzed with the nonparametric Wilcoxon test for repeated measurements. The same test was used to assess prolongation of APD and ERP by different concentrations of iso alone. Differences in ΔAPD and ΔERP after exposure to d,l-sot/d-sot/dof or to d,l-sot+iso/d-sot+iso/dof+iso were compared using the Mann-Whitney U test. A confidence level of 95% was considered statistically significant.
Baseline AP configuration and parameters were in congruity with previous reports on isolated canine cardiomyocytes (18,19). Original registrations of APs under the various conditions studied are illustrated in Fig. 2. As shown in Table 1, RMP and APA values recorded under control conditions were not significantly affected by the drugs.
The baseline effects of iso at two different concentrations on APD and ERP are illustrated in Fig 3. Iso significantly decreased APD 90 and ERP in a dose-dependent manner. However, the interrate variability of the effects of iso on APD and ERP was not statistically significant. As previously stated, the combined action of d,l-sot+iso, d-sot+iso, and dof+iso was investigated in a second experimental step. For each drug, the protocol was applied at two different concentrations (A and B) in combination with a fixed concentration of iso. APD 90 and ERP values recorded in these experiments are listed in Table 2. After exposing the cardiomyocytes to d,l-sot, d-sot, or dof, and additionally to iso, a complete washout was obtained with restoration of the baseline AP parameters. The experiments conducted by merely applying the stimulation protocol without any drug revealed no significant spontaneous variability in APD or ERP over time. With concentrations A and B, d,l-sot, d-sot, and dof exhibited statistically significant class III action throughout the range of frequencies studied. As shown in Fig. 4, significant reverse use dependence was equally prominent with high and low concentrations of all three drugs. With d,l-sot, d-sot, and dof, the absolute use-dependent AP prolongation with concentration B was significantly more pronounced than that with concentration A. However, at the same concentration level, the class III effects were not significantly different among the three drugs.
With concentrations A and B, the effects of d,l-sot were well preserved after application of 10−6M iso, independent of the stimulation rate applied. The AP prolongation at either concentration of d-sot or dof, on the other hand, was partially antagonized by iso. For details see Fig. 5A-C. Compared with the lower concentration A, with the higher concentration B, the class III action of both d-sot and dof was significantly better preserved in the presence of iso. At faster pacing rates, the effects of d-sot were inhibited by iso to a significantly greater extent than were the effects of dof. The difference in ΔAPD 90 after combined exposure to d-sot and iso, on the one hand, and after exposure to dof and iso, on the other hand, was statistically significant at stimulation rates >1 Hz. Under all conditions studied, ERP values paralleled APD 90 values, indicating absence of significant postrepolarization refractoriness.
This study was designed to determine the modulating effects of iso on the electropharmacologic properties of the class III agents d,l-sot, d-sot, and dof. Microelectrode recordings were chosen because APD and ERP represent the clinically most relevant electrophysiologic target parameters of antiarrhythmic drug action.
Our results provide evidence of a differential rate-response pattern of d,l-sot, d-sot, and dof in response to adrenergic stimulation. In contrast to d-sot and dof, the β blocking component of d,l-sot seems to preserve its class III action after exposure to iso. These observations are in congruity with results obtained in guinea pig in vitro models demonstrating preserved class III action of d,l-sot, unlike d-sot, after exposure to iso (11). In our study, particularly at a concentration of 10−6M, dof, in combination with iso, still induced a significant AP prolongation throughout the range of frequencies studied. The class III action of d-sot, on the other hand, was even overcompensated by iso at faster pacing rates, leading to APD values shorter than those recorded at baseline.
The underlying mechanism of this differential rate-response pattern of d-sot and dof in response to iso is unclear. The drug concentrations of d,l-sot, d-sot, and dof used in combination with iso were pharmacologically equipotent in terms of absolute prolongation of APD and ERP. Differential rate-response effects on adrenergic stimulation could equally be demonstrated at low and high concentrations of d-sot and dof. However, it cannot be ruled out completely that differences in pharmacologic potency or activation and deactivation kinetics between the two drugs might be involved. At baseline, iso exhibited dose-dependent but rate-independent effects to decrease APD and ERP. It thus seems likely that specific interactions between iso, on the one hand, and the two class III compounds, on the other hand, rather than differences in use-dependent baseline activity of iso are responsible for the differential rate-response pattern of d-sot+iso and dof+iso, as demonstrated in our study.
The differential contribution of Iks to total Ik amplitude at different stimulation rates is probably of major importance. Based on assumptions derived from single-cell guinea pig models (16), a highly selective Ikr blocking agent like dof would be expected to be particularly susceptible to the antagonizing effects of Iks-stimulating compounds like iso. In this case, marked reverse use dependence should be prominent on adrenergic stimulation because of the accumulation of Iks at high frequencies. Conversely, a presumably less selective Ikr and Iks blocking agent like d-sot would be more likely to preserve its class III action after exposure to iso. However, as this hypothesis is incompatible with our findings, we have to assume either additional, differential mechanisms of action of d-sot and dof on the two Ik components or marked electrophysiologic dissimilarities between canine and guinea pig cardiomyocytes. A previous study performed on isolated canine cardiomyocytes revealed Ikr activation and deactivation kinetics inconsistent with findings in guinea pig cardiomyocytes (17). The fact that the antagonism of d-sot and iso, unlike findings in guinea pig papillary muscle (11), was significantly more pronounced at faster pacing rates additionally argues in favor of species-specific regulation mechanisms in guinea pigs and canines. Further patch-clamp studies are warranted to identify the underlying ionic channel substrate of this differential rate-response pattern. In conclusion, the existing concepts of reverse rate-dependent class III action must be refined to explain fully our findings of differential rate- and iso-dependent effects of d-sot and dof.
Severe proarrhythmic effects have put into great doubt the clinical usefulness of antiarrhythmia agents (6,20). Based on experimental findings, a more individualized therapeutic approach with regard to the underlying arrhythmogenic mechanism-substrate dependent versus catecholamine induced-might prove advantageous. In adrenergic-triggered tachyarrhythmias, combined β blocking and class III agents like d,l-sot are likely to be superior. Selective class III compounds like dof or d-sot, on the other hand, would be preferable in the treatment of substrate-related reentry tachyarrhythmias. Further studies on human cardiomyocytes are required to substantiate possible clinical implications of our findings. An unfavorable rate-response pattern of d-sot on adrenergic stimulation compared with dof and particularly with d,l-sot, as documented in our study, might provide a potential electrophysiologic basis for reduced efficacy of d-sot in high-catecholamine states.
Acknowledgment: This study was supported by a grant of the Deutsche Forschungsgemeinschft within the SFB 320 "Herzfunktion und ihre Regulation," University of Heidelberg.
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