Delayed rectifier potassium current (IK) is one of the major currents constituting the sinoatrial (SA) nodal pacemaker current (1-3). IK contributes to repolarization and diastolic depolarization in mammalian nodal cells. IK is the composite of two components, i.e., a rapidly activating current that exhibits strong inward rectification (IKr) and a slowly activating current with only modest rectification (IKs) in mammalian cardiac tissues, including SA nodal cells (4-7). IKr is selectively blocked by E-4031, a class III antiarrhythmic agent (4,5), and IKs is activated by isoproterenol (ISO) (6). On the other hand, the sympathetic nervous system controls various cardiac functions. Sympathetic activation results in positive chronotropic, dromotropic, and inotropic responses and shortening of myocardial refractory period and frequently relates to an induction of arrhythmias. Shortening of the refractory period induced by sympathetic activation results from activation of IKs, and ISO indirectly antagonizes a prolongation of the refractory period of E-4031 (6).
Although IK has been investigated by electrophysiological studies, little is known about the role of IKr or IKs in the heart in situ when sympathetic nerves are activated. Therefore, to determine the actions of IKr of the heart of anesthetized dogs when sympathetic nerve fibers are activated, we investigated the effects of E-4031 on the chronotropic, dromotropic, and inotropic responses to stimulation of the ansae subclaviae in the autonomically decentralized heart of the anesthetized dog. After E-4031 treatment, sympathetic nerve stimulation changed sinus rhythm to atrioventricular (AV) junctional rhythm in half of the experiments, and we suggest that the role of IKr is different among the pacemaker cells, including SA and AV nodal cells.
MATERIALS AND METHODS
Eleven mongrel dogs of either sex weighing 10-17 kg were anesthetized with pentobarbital sodium (30 mg/kg intravenously i.v.). A tracheal cannula was inserted, and intermittent positive pressure ventilation was started. The chest was opened transversely at the fifth intercostal space. Each cervical vagus nerve was crushed with a tight ligature, and each stellate ganglion was ligated tightly at its junction with the ansa subclavia. These maneuvers have been shown to remove almost all tonic neural activity to the heart (8).
Two bipolar hook electrodes were placed on the cardiac side of each stellate ganglion to stimulate bilateral efferent sympathetic nerves. These electrodes were connected to an electrical stimulator (model SEN7103, Nihon Kohden, Tokyo, Japan). We stimulated the sympathetic nerves with 10 V and 1-ms pulse duration at a frequency of 1-2 Hz for 4 min.
Two bipolar electrodes were placed on the epicardial surface of the right atrial appendage and of the right ventricle, respectively, to record the electrical activity. The atrial and ventricular electrograms were filtered with a bandpass at 30-300 Hz. Two catheter-tip pressure transducers (Nihon Kohden TCP 2) were inserted in the middle of the right atrial cavity directly and in the basal portion of the right ventricle through the right external jugular vein, respectively, to measure the right atrial and ventricular pressures. Systemic arterial pressure was also measured through the left femoral artery. The right atrial and ventricular electrograms, right atrial pressure and its first derivative, right ventricular pressure and its first derivative, systemic arterial pressure, heart rate (HR), and AV conduction time were displayed on a thermo-writing rectigraph (Nihon Kohden RTA 1200). The HR was derived from the atrial electrogram, and AV conduction time was determined by measuring the time between the onset of the atrial and ventricular electrograms. The maximum rate of the development of the “a” wave component of the right atrial pressure (RAdP/dt) was determined as an indicator of the right atrial myocardial contractility (9). The maximum rates of the right ventricular pressure development (RV+dP/dt) and decrease (RV-dP/dt) were also determined. In some animals in which AV junctional rhythm was induced, His bundle activity was recorded from a bipolar electrode catheter inserted through the right femoral artery and positioned in the noncoronary cusp of the aortic valve. The His bundle electrogram was filtered with a bandpass at 30-300 Hz. We considered the rhythm to be AV junctional when the His bundle deflection was the initial deflection or when the ventricular deflection preceded the atrial deflection.
E-4031 in doses of 0.01-3 μmol/kg was injected cumulatively from a left femoral vein at 20-min intervals. Bilateral ansae subclaviae were stimulated 16 min after the injection of each dose. Direct cardiac effects of E-4031 were determined before sympathetic stimulation; the effects of E-4031 on the cardiac responses to sympathetic stimulation were determined 30 s and 2 min after initiation of the stimulation.
The drug used in the present experiments was N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl]-4-piperidinyl]carbonyl]phenyl] methanesulfonamide dihydrochloride dihydrate (E-4031), donated by Eisai, Tokyo, Japan.
All data are mean ± SEM. The data were analyzed by an analysis of variance (ANOVA) and Scheffé's method for multiple comparison data. Student's t test for paired data was used for comparison between the two groups. Chi-square test was used for analysis of frequency with which AV junctional rhythm was induced. p- Values <0.05 were considered statistically significant.
Direct cardiac effects of E-4031
E-4031 (0.01-3 μmol/kg i.v.) decreased the HR dose dependently (p < 0.01) but did not change the AV conduction time, RAdP/dt, RV+dP/dt, RV-dP/dt, or mean arterial blood pressure (MAP) significantly (Fig. 1). E-4031 (3 μmol/kg i.v.) decreased the HR by 42 ± 6 beats/min (37 ± 4%) from the control basal HR (113 ± 5 beats/min). Cardiac control values before treatment with E-4031 are shown in Table 1.
Effects of E-4031 on the cardiac responses to bilateral ansae subclaviae stimulation
Bilateral ansae subclaviae stimulation increased the HR, RAdP/dt, RV+dP/dt, and MAP and decreased the AV conduction time (Table 1). Figure 2 shows a typical experiment in which AV junctional rhythm was induced by bilateral ansae subclaviae stimulation after treatment with E-4031. E-4031 at a dose of 1 μmol/kg i.v. decreased the HR from 114 to 53 beats/min. Thirty seconds after initiation of the stimulation, the HR increased from 53 to 77 beats/min and the His bundle deflection preceded the atrial deflection by 5 ms, showing that the AV junctional rhythm was induced. Two minutes after the stimulation, the HR increased further and the atrial deflection preceded the His bundle deflection, showing that sinus rhythm was restored.
Table 2 shows the number of animals in which AV junctional rhythm was induced by sympathetic nerve stimulation. Before E-4031 administration, all 11 hearts beat with sinus rhythm before and during the stimulation. After treatment with E-4031, sinus rhythm was maintained before sympathetic stimulation. However, sympathetic stimulation changed the sinus rhythm to the AV junctional rhythm in 6 of 11 animals 30 s after the beginning of the stimulation when E-4031 at doses ≥1 μmol/kg intravenously. Frequency of induction of the AV junctional rhythm increased in a dose-dependent manner (p < 0.01). In 3 of 6 animals in which the AV junctional rhythm was induced, the rhythm continued until 120 s after the beginning of the stimulation. In the other 3 animals, sinus rhythm was restored during stimulation. HR before and 30 and 120 s after the beginning of the stimulation was 66 ± 4, 97 ± 2, and 106 ± 7 beats/min, respectively, in 3 dogs in which the AV junctional rhythm continued and 66 ± 6, 91 ± 4 and 105 ± 11 beats/min, respectively, in 3 dogs in which the AV junctional rhythm was induced transiently. On the other hand, in 5 dogs in which sinus rhythm was maintained, HR was 84 ± 10, 125 ± 17, and 141 ± 17 beats/min, respectively. HR before the stimulation in sinus rhythm dogs tended to be faster (p = 0.09) than that of dogs with induced AV junctional rhythm.
In animals in which sinus rhythm was maintained during the stimulation, E-4031 had no effect on the positive chronotropic and dromotropic responses to sympathetic nerve stimulation (Fig. 3A). Sympathetic stimulation increased the HR by 49 ± 7 and 50 ± 8 beats/min before and after treatment with E-4031 at a dose of 1 μmol/kg, respectively. E-4031 had no significant effect on the positive inotropic responses of the right atrium and right ventricle to the stimulation (Fig. 3B).
Effects of E-4031 on the cardiac responses to sympathetic stimulation
E-4031 is a selective blocker of a rapidly activating current component of the delayed rectifier potassium current IKr(4,5). E-4031 has no effect on inward sodium or calcium current (10), although high concentration of E-4031 (10-5M) has been shown to block the calcium current slightly in rabbit single SA nodal myocytes (11). In the present study, E-4031 decreased the HR without affecting the AV conduction time, RAdP/dt, RV+dP/dt, RV-dP/dt, or MAP in autonomically decentralized hearts of the anesthetized dogs (Fig. 1). These results are in accord with those obtained in in vitro (10-12) and in vivo (13) experiments. Furthermore, we provide evidence that E-4031 does not affect positive chronotropic, dromotropic, atrial, or ventricular inotropic responses to sympathetic nerve stimulation in anesthetized dogs (Fig. 3). Therefore, E-4031 probably has no β-adrenoceptor blocking property and an IKr blocker such as E-4031 probably does not interact directly with the positive chronotropic, dromotropic, or inotropic responses to sympathetic nerve activation in anesthetized dog hearts. Similarly, Yang and colleagues (14) showed that dofetilide, another IKr inhibitor, did not competitively antagonize the positive chronotropic response to ISO in guinea pig atrium.
Induction of AV junctional rhythm by sympathetic stimulation after treatment with E-4031
The AV junctional region contains pacemaker cells that are subsidiary to the atrial pacemaker cells (15-17). Pacemaker cells in the AV junctional region become manifest either when the SA node is selectively suppressed surgically (18,19) or pharmacologically (20,21) or when their own automaticity is sufficiently enhanced. Left stellate stimulation or stimulation of the ventrolateral cardiac nerves caused a pacemaker shift to the AV junctional region (22-24). Stimulation of the intracardiac sympathetic nerve fibers to the AV nodal region quite frequently induces the AV junctional rhythm (25,26). However, no report indicates that bilateral ansae subclaviae stimulation frequently induced the AV junctional rhythm in anesthetized dog heart; i.e., the SA node pacemaker activity was dominant during sympathetic stimulation and its activity suppressed AV junctional pacemaker cells by overdrive suppression (17,27). In the present study, we confirmed that bilateral ansae subclaviae stimulation did not induce the AV junctional rhythm before treatment with E-4031. However, we demonstrated that bilateral ansae subclaviae stimulation frequently induced AV junctional rhythm after systemic administration of E-4031 (Table 2). In a previous study, we demonstrated that sympathetic stimulation did not induce the AV junctional rhythm after treatment with other bradycardic agents such as verapamil, an L-type calcium channel blocker, and zatebradine, a hyperpolarization-activated current blocker (28). Therefore, the induction of the AV junctional rhythm was not merely a rate-dependent effect, although HR of the dogs with induced AV junctional rhythm tended to be lower than that of the dogs with sinus rhythm. Therefore, our present results suggest that E-4031 has different influences on sympathetically induced increase in automaticities between the SA node and the AV junctional region.
We observed previously that E-4031 decreased AV junctional rate in hearts of the anesthetized dogs with AV junctional rhythm, but the decrease in junctional rate induced by E-4031 was less than the decrease in sinus rate (29). Voltage-dependent inhibition of IK induced by quinidine has been reported in rabbit SA and AV nodal cells (30). E-4031 also inhibits IKr voltage dependently in guinea pig ventricular myocytes (31). Diastolic membrane potential of the SA nodal cell is less negative than that of the AV nodal cell. Therefore, E-4031 might be more effective on the SA nodal pacemaker activity than on the AV nodal pacemaker activity in anesthetized dog heart. Furthermore, the distribution of the two components of IK, IKr and IKs, is tissue and species dependent (7,32). In guinea pig heart, IKs is a dominant current in the SA node, and both IKr and IKs exist in atrial and ventricular myocardial cells (4,5,7), but IKr is dominant in rabbit SA and AV nodal cells (4,5,30). Although the distribution of two components of IK in dog heart is not known, the difference in density of IKr between SA and AV nodal cells might cause different effects of E-4031 on sinus and junctional rates. In addition, Wallick and co-workers (21) showed that the same frequency of sympathetic stimulation resulted in increases of 17 and 102% in sinus rate and AV junctional rate, respectively, in anesthetized dogs. They concluded that the AV junctional pacemaker cells were more responsive to sympathetic stimulation than were the SA nodal pacemaker cells in dog hearts. From the results of these previous reports, we suggest that E-4031 may unmask subsidiary pacemaker activity such as AV junctional rhythm due to the combination of the different participation of IKr, the different resting potentials, and the different sensitivity to sympathetic activation of cardiac pacemaker cells. However, the mechanisms by which AV junctional rhythm was induced were not precisely determined from our in vivo experiment.
In the present study, E-4031 affected neither positive chronotropic, dromotropic, or atrial or ventricular inotropic responses to sympathetic nerve stimulation in the anesthetized dog heart, suggesting that E-4031 interacts directly with neither slow inward calcium current nor hyperpolarization-activated current in addition to IKs in the heart. However, sympathetic nerve stimulation-induced AV junctional rhythm in E-4031-treated hearts and half of the AV junctional rhythm hearts changed to sinus rhythm during sympathetic stimulation (Table 2). These results may suggest that attenuation of IKr by E-4031 in SA nodal pacemaker cells indirectly delays the activation of other pacemaker currents in response to sympathetic nerve stimulation. When AV junctional rhythm changed to sinus rhythm during sympathetic stimulation in E-4031-treated anesthetized dogs, sinus rate became faster than the induced junctional rate.
E-4031 has been reported to be effective against electrically induced ventricular tachycardia in dogs with previous myocardial infarction (33,34). Prolongation of refractory period by E-4031 was indirectly antagonized by ISO (6). Therefore, effects of E-4031 on refractory period could be reduced in conditions of high sympathetic tone. Furthermore, our results suggest that, at least under our experimental conditions, activation of the sympathetic nerves causes pacemaker shift in E-4031-treated dogs. Extrapolation of these findings to clinical conditions is speculative because of species difference and the high dose of E-4031 at which AV junctional rhythm was induced in our study. However, sympathetic activation such as acute myocardial ischemia or strong exercise may induce pacemaker shift in humans treated with E-4031. This effect of E-4031 therefore may be involved in arrhythmogenic properties of class III antiarrhythmic agents.
Acknowledgment: We thank Eisai (Tokyo, Japan) for the generous supply of E-4031.
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