Atrial pacing at rates exceeding the spontaneous sinus rate depresses automaticity of the sinoatrial (SA) node, and the first returning cycle length (1st RCL), second (2nd), and third (3rd) RCLs after the cessation of pacing are longer than the control cycle length. This is called "overdrive suppression," which is one of the functional characteristics of the SA-nodal pacemaker activity (1-3). The 1st RCL is determined by intrinsic SA-nodal pacemaker activity, pacemaker site, and SA conduction. It also has been suggested that the prolongation of the second and subsequent cycles is only the result of overdrive suppression or shifting of the pacemaker or both (3). The mechanism of this overdrive suppression is not completely understood. Three mechanisms of overdrive suppression are proposed as follows: (a) release of acetylcholine (ACh) induced by stimulation of the parasympathetic nerves during atrial pacing (4,5), (b) an intracellular accumulation of Ca2+ or Na+ or both by highfrequency pacing (6), and (c) activation of the Na+-K+ ATPase pump as reported in the Purkinje fibers (7).
Accumulation of ions as a mechanism underlying overdrive suppression is most convincing and deserves an intensive study. However, little is known concerning the role of K+ in overdrive suppression. The activation and deactivation of the delayed-rectifier K+ current, Ik, play an important role in pacemaking activity (8). The class III antiarrhythmic agent E-4031 is known to block a rapid type of Ik (Ikr) selectively (9,10). E-4031 prolongs the action-potential duration and the effective refractory period without affecting other electrophysiologic parameters (11). It was recently demonstrated that E-4031 prolongs cycle length, reduces both the upstroke velocity of the action potential and the diastolic depolarization rate, and even blocks the generation of action potential in single SA-nodal cells of the rabbit (12). E-4031 seems to depress automaticity of the SA node, from these observations on electrophysiologic parameters. Thus it was the first objective of our study to determine whether E-4031 enhances the prolongation of the 1st and subsequent (2nd and 3rd) RCLs after overdrive.
Although ACh release may not be a primary cause of overdrive suppression, the possibility still remains that parasympathetic nerves are simultaneously stimulated by the pacing stimuli. Stimulation of the discrete intracardiac parasympathetic nerves to the SA nodal region (SAP stimulation) increases the sinus cycle length (SCL) without affecting atrioventricular (AV) conductivity in the dog heart, whereas cervical vagus stimulation prolongs both SCL and AV conduction time (13). When the overdrive-suppression test is performed clinically, the pacing catheter electrodes are usually positioned at the high right atrium. This site for the electrophysiologic study is very close to the site of the SAP stimulation in dogs (13,14) and in humans (15). Accordingly, it is likely that in clinical practice, the parasympathetic neural elements are simultaneously stimulated by the pacing stimuli, and ACh release by the electrical stimulation may partially contribute to the prolongation of the 1st RCL and subsequent RCLs after overdrive. Thus it was the second objective of our study to determine whether the intracardiac parasympathetic stimulation enhances the prolongation of the 1st, 2nd, and 3rd RCLs after overdrive.
Recently we observed that E-4031 inhibits selectively the prolongation of SCL induced by vagus stimulation (16). An interaction between ACh-sensitive K+ current and the blockade of Ikr by E-4031 has been suggested. Therefore we also investigated the effects of E-4031 on the responses to SAP stimulation. To achieve these aims, we determined SA-node recovery time (SNRT) and corrected SNRT (CSNRT) after atrial pacing at rates of 120, 150, and 200% of the control rate for 1 min and also determined SA conduction time (SACT) by the constantatrial-pacing method in autonomically decentralized hearts of open-chest anesthetized dogs. Then we studied the effects of changing the number of pacing stimuli on the 1st, 2nd, and 3rd RCLs.
The experimental protocol was approved by the Animal Experimentation Ethics Committee, Shinshu University School of Medicine. Mongrel dogs, weighing 8-20 kg, were anesthetized with pentobarbital sodium (30 mg/kg, i.v.). A tracheal tube was inserted, and intermittent positive pressure ventilation was started. The chest was opened transversely at the fifth intercostal space. Each cervical vagosympathetic complex 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 virtually all tonic neural activity to the heart (17).
A quadripolar electrode was placed on the epicardial surface of the base of the right atrial appendage; one pair was used for electrical pacing and the other for recording atrial electrograms. Atrial pacing was performed by an electrical stimulator (SEN 7103; Nihon Kohden, Tokyo, Japan), which delivered a 1-ms rectangular pulse at twice the diastolic voltage threshold for activation. The atrial electrograms were passed through a bandpass filter at 30-300 Hz (AP621G; Nihon Kohden) and displayed on a thermowriting rectigraph (RTA 1200; Nihon Kohden).
A bipolar silver electrode with 2-mm interelectrode distance was placed on the fatty tissue overlying the right atrial juncture of the right pulmonary veins and was used to stimulate the intracardiac parasympathetic nerve fibers to the SA-nodal region (13,14). We refer to this stimulation as "SAP stimulation." This electrode was connected to an electrical stimulator (SEN7103; Nihon Kohden). The stimulation was subthreshold for activation of pacemaker cells and cardiac muscle cells, when a quite narrow stimulation-pulse duration (0.01-0.05 ms) was used for parasympathetic nerve stimulation (13). The left femoral artery and vein were cannulated for monitoring the systemic arterial blood pressure and for drug administration, respectively.
Measurement and definition of parameters
SA-node recovery time (SNRT). Pacing was performed for 1 min at three different pacing rates: 120, 150, and 200% of the control rate. SNRT was further corrected for the basic cycle length (BCL: spontaneous cycle length immediately before the pacing); that is, the CSNRT was determined by subtracting BCL from SNRT. Two-minute intervals for recovery were given between each pacing run. It has been suggested that the prolongation of the second and subsequent cycles is only the result of overdrive suppression or shifting of the pacemaker or both (3). Therefore we also determined SNRT and CSNRT for the 2nd and 3rd RCLs.
SA conduction time (SACT). The constant-atrial-pacing method was adopted (18). The atrium was paced for eight beats at 10 beats/min faster than the control rate. SACT was calculated as the difference between the 1st RCL and BCL. The mean of four determinations was taken as SACT.
To block the responses mediated by β-adrenergic receptors during experiments, we administered propranolol (1 mg/kg, i.v.) and an additional dose of 0.5 mg/kg hourly. Propranolol completely blocked the positive cardiac responses to stimulation of the cardiac sympathetic nerves.
We conducted three series of experiments. In the first series, we investigated the effects of E-4031 (0.1-3 μmol/kg, i.v.) in eight dogs. E-4031 was administered cumulatively to determine the direct effects of the drug on SCL, SNRT, CSNRT, and SACT.
In the second series, we investigated the effects of SAP stimulation (10 Hz, 30 Hz) on SCL, SNRT, CSNRT, and SACT in eight dogs. After control recordings had been taken, SAP stimulation was used. The measurement was started 2 min after the onset of the stimulation to ensure the stabilization of the cycle length. At least 10-min intervals were set to allow the heart to return to the control state after the cessation of the electrical stimulation. We also examined the effects of E-4031 on the responses to SAP stimulation. The increases in SCL, SNRT, CSNRT, and SACT in response to SAP stimulation at 30 Hz were compared between the control condition and after E-4031 treatment at a dose of 3 μmol/kg, i.v.
In the third series, we examined the effects of E-4031 on the 1st, 2nd, and 3rd RCLs when the number of pacing stimuli was changed in six dogs. Pacing rate was kept constant at 200% of the spontaneous sinus rate, but the number of pacing stimuli was changed from eight to 50, 100, 200, and the number of stimuli at 200% of the control rate for 1 min. We determined the prolongation of the 1st, 2nd, and 3rd RCLs after pacing stimuli was stopped. We repeated the same protocol after the administration of E-4031 at a dose of 1 μmol/kg, i.v.
Drugs used in the present experiments were N-[4[[1-[2-(6-methyl-2-pyridinyl) ethyl]-4-piperidinyl] carbonyl] phenyl] methanesulfonamide dihydrochloride dihydrate (E-4031), generously donated by Eisai, Tokyo, Japan, and propranolol hydrochloride (Sumitomo Chemicals, Tokyo, Japan).
All data were expressed as mean ± SEM. The data were analyzed with an analysis of variance and Bonferroni's method for multiple comparisons. Pacing rate, E-4031 treatment, SAP stimulation, the number of pacing stimuli, and the order of the RCLs were considered to be fixed factors. Student's t test was applied for paired observations. p Values < 0.05 were considered statistically significant.
Effects of E-4031
Figure 1 shows the effects of E-4031 on SCL, SACT, SNRT, and CSNRT in eight anesthetized dogs, and Table 1 presents control values before E-4031 administration. E-4031 (0.1-3 μmol/kg, i.v.) increased SCL dose dependently (p < 0.001; Fig. 1A). E-4031 increased SNRT, and the increases in SNRT were dependent on pacing rate (p < 0.01; Fig. 1C). On the other hand, E-4031 dose-dependently decreased CSNRT at pacing rates of 120 and 150% of the control rate, although CSNRT did not change significantly when the pacing rate was 200% of the control rate (Fig. 1D): effects of E-4031 on CSNRT were changed by pacing rate (p < 0.01), and the changes in CSNRT by pacing rate were affected by a dose of E-4031 (p < 0.001). In Fig. 1C and D, data for the 3rd RCL are also shown (closed symbols). SNRT and CSNRT for the second and the third returning cycles were shorter than those for the first returning cycle, values for the second returning cycle being always between those for the first and the third returning cycles. E-4031 dose-dependently (p < 0.001) decreased CSNRT for the third returning cycle at pacing rates of 120 and 150% of the control rate. E-4031 slightly increased CSNRT for the third returning cycle at a pacing rate of 200%, but the change was not significant. E-4031 also decreased SACT in a dose-dependent manner (p < 0.01; Fig. 1B). After E-4031 treatment, initial configurations of the atrial electrograms were changed in some cases, suggesting the possibility of pacemaker shift. Changes in configuration of the atrial electrograms were also observed after overdrive.
Effects of the intracardiac parasympathetic stimulation
When SAP stimulation at a frequency of 10 or 30 Hz increased SCL (Fig. 2A), it prolonged SNRT (Fig. 2C) and CSNRT (Fig. 2D) in eight animals. The higher the pacing rate was, the more pronounced the effect of SAP stimulation on CSNRT became (p < 0.05). SAP stimulation similarly increased the SNRT and CSNRT for the third returning cycle, but the changes in these responses were smaller than those for the first returning cycle. SAP stimulation increased SACT as the frequency of the stimulation was increased (Fig. 2B). SACT in response to SAP stimulation was shortened, with changes in configuration of the atrial electrograms, in two of eight experiments. Control values before applying SAP stimulation are shown in Table 1.
Effects of E-4031 on the responses to intracardiac parasympathetic stimulation
After we observed the increases in SCL, SNRT, CSNRT, and SACT in response to SAP stimulation, we investigated the effects of E-4031 on the responses to SAP stimulation. As shown in Table 2, E-4031 at a dose of 3 μmol/kg, i.v., attenuated the prolongation of SCL induced by SAP stimulation (p < 0.05) but did not affect the prolongations of SNRT, CSNRT, and SACT. Similarly, E-4031 had no effect on the prolongations of SNRT and CSNRT for the second and the third returning cycles (data not shown). In one experiment, SACT in response to SAP stimulation after E-4031 treatment was 244 ms, which was extraordinarily large compared with the other cases, suggesting a possibility of the exit block. Thus we excluded the SACT datum of this dog.
Effects of E-4031 on the RCLs after changing the number of pacing stimuli
Because E-4031 decreased CSNRT when the atrium was paced at 120 and 150% of the control rate (Figs. 1 and 2), we next designed the experiment to determine whether E-4031 inhibits the prolongation of the 1st, 2nd, and 3rd RCLs when the number of pacing stimuli is small. We fixed the pacing rate at 200% of the control rate but changed the number of pacing stimuli from eight to 200 beats because the control rate was ∼100 beats/min after 1 μmol/kg of E-4031 treatment.
When we determined the effects of increasing the number of pacing stimuli from eight to 50, 100, 200, and 275 ± 13 beats (200% of the control rate) or 216 ± 19 beats (200% of the control rate after 1 μmol/kg of E-4031) on the 1st RCL, the 1st RCL was prolonged with increasing pacing stimuli significantly (p < 0.05), and the prolongation of the 1st RCL after E-4031 treatment was less (p < 0.05) than that before E-4031 (Fig. 3A, open symbols). On the other hand, the prolongation of the third returning cycle after E-4031 treatment was larger (p < 0.05) than that before E-4031 (Fig. 3A, closed symbols). Fig. 3B shows the effects of E-4031 at 1 μmol/kg on the prolongation of the 1st and 3rd RCLs when the atrial muscle was paced at eight, 50, and 200 beats with the pacing rate at 200% of the control rate. RCLs decreased with the order of RCL (p < 0.001), and the reduction of the RCLs was attenuated by E-4031 [i.e., the interaction between E-4031 treatment and the RCLs was significant (p < 0.005)]. In two of six experiments, the prolongation of the 1st RCLs after eight pacing stimuli was 28 and 33 ms, which were extremely short compared with the mean SACT value obtained in our study (Table 1). It was suggested that the pacemaker site was located outside the SA-nodal region, possibly in the region close to the recording electrodes. Thus we excluded these two data from the statistical analysis.
In this study, we demonstrated the following:
- An Ikr blocker, E-4031, decreased the prolongation of the 1st RCL after overdrive when the atrial pacing rate was low or the number of pacing stimuli was small but did not affect the prolongation of the 1st RCL when pacing stimuli were sufficient in autonomically decentralized hearts of the open-chest anesthetized dogs;
- E-4031 delayed the recovery of the second and third RCLs after overdrive;
- Stimulation of the intracardiac parasympathetic neural elements to the SA-nodal region (SAP stimulation) prolonged SNRT, CSNRT, and SACT, as well as SCL; and
- E-4031 attenuated the prolongation of SCL induced by SAP stimulation but did not affect other cardiac responses, SACT, SNRT, and CSNRT.
These results suggest that IKr inhibition by E-4031 attenuates the prolongation of the RCLs when the number of the preceding ectopic beats is small in the dog heart in situ, and E-4031 does not affect the responses except SCL to parasympathetic nerve activation.
The 1st RCL is determined by the intrinsic SA-nodal pacemaker activity, pacemaker site, and SA conduction. The prolongation of the subsequent RCLs, 2nd RCL and 3rd RCL, are the result of overdrive suppression or shifting of the pacemaker or both, but not because of the conduction (3). Thus in this study, we observed the atrial electrogram at a site of the base of the right auricle, which is close to the crista terminalis, determined the 1st, 2nd, and 3rd RCLs after overdrive by using the overdrive-suppression test and assessed SACT by Narula's method (i.e., a constant atrial pacing method; 18). However, our observations were limited to investigate the precise mechanism of overdrive suppression, because we could not determine the SA-nodal pacemaker activity directly by electrophysiologic methods and the pacemaker site by the mapping sequence by using the multiple electrodes.
In the autonomically denervated, open-chest anesthetized dog, E-4031 attenuated the prolongation of the 1st, 2nd, and 3rd RCLs when atrial pacing rates were ≤ 150% of the basal rate but not 200% (Fig. 1). E-4031 prolonged the 2nd and 3rd RCLs when the atrial-pacing rate was 200% of the control basal rate with enough of stimuli, although E-4031 attenuated the 1st RCL when the number of pacing stimuli was small (Fig. 3). These results suggest that IKr inhibition by E-4031 attenuates the developing and recovery of the overdrive suppression in the dog heart in situ.
E-4031 is a blocker of the IKr(9,10) and does not affect Na+ and Ca2+ currents (11,12). Verheijck et al. (12) showed that E-4031 prolonged cycle length and actionp-otential duration, depolarized maximal diastolic potential, and reduced both the upstroke velocity of the action potential and the diastolic depolarization rate. In their experiment in single SA-nodal myocytes of the rabbit, half of the cells were arrested completely after E-4031 treatment. Therefore we first expected that E-4031 might enhance the prolongation of the 1st RCL after overdrive. Our data, on the contrary, demonstrated that E-4031 attenuated the prolongation of the 1st as well as 2nd and 3rd RCLs after overdrive when the pacing rate was low. Because we did not measure changes in the membrane potential before and after E-4031 treatment and before and after overdrive pacing, we could not provide direct evidence to account for the underlying mechanism. The definitive mechanism would be best clarified after performing single-cell electrophysiologic studies.
One possibility is the mechanism related to a decrease in K+ conductance. Kline and Morad (19) demonstrated that rapid pacing caused an increase in the extracellular K+ concentration and concomitant depolarization of the membrane in the frog myocardial tissue. It is therefore anticipated that an accumulation of K+ in the extracellular space may contribute to the decrease in the maximal diastolic potential during overdrive, which is observed by many investigators (3,6,20,21). A decrease in K+ conductance induced by E-4031 is likely to attenuate the accumulation of K+, to lessen the decrease in the maximal diastolic potential during overdrive, and thereby to lessen the prolongation of the 1st RCL after overdrive when the pacing rate is low or the pacing number is small. That is, E-4031 attenuates the developing of the overdrive suppression. However, when the pacing rate is high and the pacing number is big, the decrease in the maximal diastolic potential might be developed even after E-4031 treatment. Then the inhibition by E-4031 of the hyperpolarization might prolong the 2nd and 3rd RCLs more than those in the control (Fig. 3) and delay the recovery from the overdrive suppression.
The attenuation of overdrive suppression by E-4031 might be the result of an attenuation of the effects of transmural vagal stimulation caused by the pacing stimuli. Intracardiac parasympathetic nerve stimulation (SAP stimulation) prolonged SCL, SNRT, CSNRT, and SACT, but these parasympathetic effects, except the effects on SCL, were not affected by E-4031. We paced the atrial muscle electrically at the base of the right atrial appendage. However, stimulation of this site hardly decreases sinus rate (22,23), although right atrial muscles are innervated from the parasympathetic nerve fibers through ganglionic cells in the fatty tissue overlying the right atrial juncture of the right pulmonary veins (i.e., SAP stimulation site), as well as those in the other sites (24). Additionally, E-4031 attenuated the negative chronotropic response to vagus stimulation but not other cardiac responses, myocardial contractile force, and AV conduction in anesthetized dogs (16). Thus it is less likely that the attenuation of overdrive suppression by E-4031 is caused by an attenuation of the effects of parasympathetic stimulation by pacing stimuli, although we could not neglect its possibility completely.
E-4031 dose-dependently decreased SACT in anesthetized dogs (Fig. 1). Changes in SACT result either from a change in conduction velocity itself or from a shift of the pacemaker site (2,25). However, E-4031 was shown not to affect Na+ and Ca2+ currents (11,12). Therefore it is suggested that the shortening of SACT induced by E-4031 is related not to a change in conduction velocity itself but to a shift of the pacemaker site in the anesthetized dog. E-4031 treatment changed the initial configurations of the atrial electrograms. This observation supports the possibility of pacemaker shift. Dose-dependent shortening of SACT induced by E-4031 may relate to the series changes in characteristics of the membrane potential of the pacemaker cells in the SA-nodal region (21).
In this study, we demonstrated that direct intracardiac parasympathetic nerve stimulation prolonged SNRT, CSNRT, SACT, and SCL in the anesthetized dog heart. Therefore when the overdrive-suppression test is performed clinically, it is possible that the parasympathetic neural elements are simultaneously stimulated by the pacing stimuli, and ACh release by the electrical stimulation may partially contribute to the prolongation of the 1st RCL after overdrive. Thus we must pay attention to the role of the release of ACh during the overdrive-suppression test in clinical practice. ACh activates a muscarinic receptor followed by opening ACh-sensitive K+ channels (8,26). It has been also reported that ACh inhibits other ionic currents, Ca2+ current (27) and hyperpolarization-activated current (28,29). Therefore the prolongation of CSNRT and SACT induced by SAP stimulation may involve these ionic changes in addition to changes in the pacemaker site.
Prolongation of SACT by SAP stimulation was in agreement with the previous studies in which cervical vagal nerve stimulation was applied (30,31). Vagal stimulation changes a pacemaker site in the SA-nodal pacemaker complex (31-33). Therefore the prolongation of SACT induced by SAP stimulation is probably the result of the reduction in conduction velocity or pacemaker shift or both.
E-4031 selectively attenuated the prolongation of SCL but not the prolongation of atrioventricular interval and decreases in myocardial contractility, induced by SAP stimulation, as previously reported (16). However, E-4031 did not affect the prolongation of CSNRT and SACT induced by SAP stimulation (Table 2). These results may suggest that effects of E-4031 on the pacemaker activity induced by SAP stimulation are different from the effects of E-4031 on the pacemaker activity after overdrive; that is, activated pacemaker cells before and after overdrive are different in the dog heart in situ. The underlying mechanism of the fact that E-4031 selectively inhibited the prolongation of SCL is unclear at the moment.
Acknowledgment: We thank Eisai (Tokyo, Japan) for the generous supply of E-4031.
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