Antiarrhythmic drugs terminate reentry either by prolonging the refractory period or by suppressing conduction. In various models of canine atrial flutter caused by macro reentry, some drugs (e.g., class III drugs) prolong the effective refractory period (ERP) sufficiently to abolish the excitable gap, leading to termination of atrial flutter (1,2). In contrast, class Ia and Ic drugs suppress conduction to a crucial point beyond which propagation of conduction is impossible (2,3).
For termination of fibrillation caused by random reentry with a short excitable gap, drugs that prolong ERP seem to be superior to drugs that suppress conduction (4). This could be reasonable, because shortening the wavelength for reentry is necessary for fibrillation to be sustained. In a canine model of atrial fibrillation (AF) induced by vagal stimulation, Wang et al. (5) showed that class III drugs terminated AF by increasing the refractory period and the wavelength for reentry. The class Ic drug, propafenone, also could prolong the refractory period at rapid atrial rates, leading to termination of AF through an increase in the wavelength (6).
In this study, we determined the efficacy of a new class Ic drug, pilsicainide, and a new investigational class III drug, MS-551, in the canine model of AF induced during vagal stimulation.
MATERIALS AND METHODS
All experiments were performed in accordance with the "Guideline for Animal Experiment" at our University.
Fourteen mongrel dogs of either sex, weighing 8-15 kg, were anesthetized with intravenous sodium pentobarbital at a dose of 30 mg/kg. Additional amounts of pentobarbital were injected as needed to maintain anesthesia. No measurements were obtained for <15 min after pentobarbital administration. The dog was intubated with a tracheal tube and artificially ventilated with room air by using a volume-cycled respirator (Harvard Model 607, Chicago, IL, U.S.A.). A femoral venous cannula was used to infuse normal saline at a rate of 100-200 ml/h to replace spontaneous fluid losses. The chest was opened through a median sternotomy, and the heart was suspended in a pericardial cradle. The thoracotomy was covered by a plastic sheet, and an operating lamp was used to maintain temperature of the chest.
The cervical vagi were isolated, doubly ligated, and cut in all dogs. Two Teflon-coated wire electrodes were embedded in the cardiac end of each vagal nerve to stimulate efferent vagal nerves. The ansae subclaviae were isolated as they exited from the stellate ganglia, doubly ligated, and cut. Three bipolar hook electrodes with an interhook distance of ∼1 mm were placed in the high, middle, and low right atrium, and another two, in the high and low left atrium. These electrodes were used to determine atrial activation. The electrodes that served as a cathode for unipolar stimulation were placed in the high right and high left atrium (Fig. 1). The anode was placed in the abdominal wall. Atrial electrograms filtered at 50-300 Hz were recorded simultaneously with surface electrocardiographic lead II on a thermal recorder (RTA-1200M; Nihon Kohden, Tokyo, Japan) at a paper speed of 100 mm/s and stored by using a digital data recorder (RD-130TE, TEAC, Tokyo, Japan) for later analyses. A storage oscilloscope also was used to monitor the electrocardiogram and atrial electrogram.
Measurement of effective refractory period
ERP was determined at each test site by the extrastimulus technique by using a digital programmable stimulator (SEC-202; Nihon Kohden). Each test site was driven with a 2-ms rectangular stimulus at twice diastolic threshold. Late diastolic thresholds of test sites were measured during each intervention. A train of eight stimuli (S1) at constant cycle length of 200 ms was followed by a late extrastimulus (S2) that produced a propagated atrial response. The atrial response to S2 was obtained from a bipolar electrode in the right atrium and displayed on a storage oscilloscope. The S1-S2 interval was shortened in steps of 2 ms until S2 failed to produce a propagated response. The ERP was defined as the longest S1-S2 interval at which S2 failed to produce a propagated response. Measurements were repeated to ascertain the reproducibility of ERP.
Measurement of conduction time in the atrium
Intraatrial conduction time (CT) was measured during atrial pacing at constant pacing-cycle lengths of 300, 200, and 150 ms. The CT in each atrium was determined as the time from the pacing stimulus to the onset of activation in the lower part of the ipsilateral atrium.
Wavelength index (WLI) was calculated as the ratio of ERP to CT at a pacing-cycle length of 200 ms, as in the previous study (7). This index was used in our study as an alternative variable for the wavelength (ERP × conduction velocity) for reentry.
Vagal stimulation. Stimulation of the right and left vagal nerves was performed with separate isolated constant-current sources (SS-202J; Nihon Kohden), driven by a programmable stimulator (SEN-7103; Nihon Kohden). The current strength was set to prolong sinus-cycle length by 100% with right vagal stimulation and to produce 2:1 atrioventricular (AV) block with left vagal stimulation by using 2-ms rectangular pulses at 10 Hz. In some dogs, left vagal stimulation produced complete AV block without producing 2:1 block. In these dogs, the current strength that produced complete AV block was used. Between interventions, vagal nerves were stimulated to be certain that the sinus and AV nodal responses remained constant.
After determining the baseline values, the vagi were stimulated bilaterally, and electrophysiologic variables were determined. Then AF was induced by bursts of atrial pacing at cycle lengths of 90-120 ms under continuous bilateral vagal stimulation. Diagnosis of AF was based on the surface ECG with no discrete P waves, irregular RR intervals, and irregular atrial activation without isoelectric line. After initiation of AF, a period of 5 min was allowed in each dog to ensure that AF was sustained. Antiarrhythmic drugs, pilsicainide and MS-551, were administered intravenously in the following doses: 1.0 mg/kg pilsicainide in six dogs and 0.5 mg/kg MS-551 in eight dogs. Pilsicainide, a class Ic drug, was used to determine the effect of suppression of conduction, and MS-551, a class III drug, that of prolongation of ERP. Antiarrhythmic drugs were injected over 3-min periods, and injection was discontinued if AF was terminated during injection of antiarrhythmic drugs. An additional dose of 0.5 mg/kg of MS-551 was necessary in five dogs in which AF had not been terminated by the initial injection of MS-551. When AF was not terminated within 4 min after completion of second injection of MS-551, vagal stimulation was interrupted to terminate AF. Changes in electrophysiologic variables induced by test drugs were determined during bilateral vagal stimulation after termination of AF. A blood sample was obtained to determine the plasma concentration of antiarrhythmic drugs after the experiment.
At each recording site, mean of atrial-activation intervals (FF intervals), standard deviation (SD) of FF intervals, and coefficient of variance (CV), calculated as the ratio of SD/mean, were determined with 10 consecutive FF intervals at four different times (i.e., just before the administration of test drugs, and 10 s, 5 s, and just before termination of AF by test drugs). These variables also were determined during the baseline AF and just before termination of AF by cessation of vagal stimulation but without test drugs.
The data are expressed as mean ± SD. The difference among mean values of FF intervals was determined by using an analysis of variance (ANOVA) for repeated measures. Multiple comparisons were made by Fisher's method, if indicated. A paired t test was performed when only two measures were compared. Statistical significance level was set at p value < 0.05.
Effects of bilateral vagal stimulation
Current strength for right vagal stimulation was 0.27 ± 0.15 mA and did not differ from that for left vagal stimulation (0.25 ± 0.15 mA). Changes in ERP and CT are summarized in Table 1. In one dog of the MS-551 group, ERP was not determined, because AF was repeatedly induced with extrastimulation during vagal stimulation. Therefore data are summarized by using the remaining 13 dogs. The ERP was shortened during bilateral vagal stimulation in both atria. The CT in each atrium did not change significantly during bilateral vagal stimulation at each pacing-cycle length; therefore the WLI determined at a pacing-cycle length of 200 ms in the right and left atrium decreased significantly during bilateral vagal stimulation. AF was induced repeatedly with burst atrial pacing during bilateral vagal stimulation in each dog.
Effects of antiarrhythmic drugs
AF was terminated by pilsicainide in all six dogs tested within 114 ± 31 s after starting the injection (Fig. 2, Table 2). After termination of AF, rapid atrial pacing was repeated to reinitiate AF in five dogs. Nonsustained AF lasting only ≤10 s was inducible in four of five dogs but not in the remaining dog. Pilsicainide increased ERP and also CT during vagal stimulation significantly in both atria (Table 3). The increase in CT did not show an apparent use dependence at the basic pacing-cycle lengths from 300 to 150 ms. After administration of pilsicainide, WLI was decreased only slightly but significantly in the right atrium but tended to decrease in the left atrium (Table 3). This indicates that pilsicainide certainly did not increase WLI but could terminate AF in our canine model.
MS-551 terminated AF in six of eight dogs within 224 ± 129 s after starting the initial injection. After termination of AF, nonsustained AF was inducible by burst atrial pacing in two dogs but not in the remaining four dogs. ERP was prolonged significantly in both atria after administration of MS-551 (Table 4). The increase in ERP was greater in the right than in the left atrium (50 ± 35% vs. 37 ± 17%; p < 0.05). CT was not affected by MS-551 in both atria at any pacing-cycle lengths. Accordingly, WLI increased significantly in both atria after administration of MS-551. The increase in WLI induced by MS-551 was similar to that induced by simple cessation of bilateral vagal stimulation (e.g., from 3.3 ± 1.0 to 4.1 ± 1.0 in the right atrium; Table 1).
Changes in FF intervals
Mean FF intervals during the baseline AF were shorter in the right atrium (85 ± 22 ms; mean of the three recording sites) than in the left atrium (107 ± 29 ms; mean of the two recording sites; p < 0.01). The SD of FF intervals did not differ between the right and left atrium. After injection of pilsicainide, AF intervals increased at each recording site (Fig. 3) before termination of AF. The increase in FF intervals did not differ among test sites. SD did not show any significant changes in each recording site after injection of pilsicainide. CV therefore decreased in each recording site (e.g., electrode 3, 26 ± 8% → 15 ± 5%; p < 0.05; and electrode 4, 16 ± 7% → 11 ± 6%; p < 0.05).
After injection of MS-551, indices of FF intervals changed as after injection of pilsicainide except for SD (Fig. 3). SD increased transiently in the right atrium after injection of MS-551. CV decreased in each recording site before termination of AF (e.g., electrode 3, 28 ± 5% → 21 ± 4%; p < 0.05; and electrode 4, 22 ± 12% → 16 ± 9%; p < 0.05). These results indicate that FF intervals increased significantly after injection of test drugs irrespective of changes in WLI induced by the drug [i.e., a decrease in WLI by pilsicainide and an increase in WLI by MS-551 (Figs. 4 and 5)].
The major findings of this study are as follows. AF induced under vagal stimulation was terminated by pilsicainide and MS-551. FF interval increased before termination of AF with test drugs, whereas changes in WLI were different between the two drugs. Therefore vagotonic AF could be terminated by either prolongation of ERP or suppression of conduction.
Effect of vagal stimulation on atrial refractory period
As expected from previous studies (8,9), vagal stimulation shortened atrial ERP. Because this effect of vagal stimulation on atrial refractoriness is not uniformly distributed, reentry is prone to occur without anatomic obstacle (8). Lammers et al. (10) showed that inhomogeneities in refractoriness are important in the initiation of atrial reentry in isolated rabbit atria. Clinically, paroxysmal AF is documented in some patients during sleep when vagal tone increases (11). In our study, bilateral vagal stimulation decreased ERP but did not change CT in each atrium. Consequently, WLI, an estimate of the minimal length of the reentrant circuit, decreased under bilateral vagal stimulation, and AF was inducible with burst atrial pacing in all dogs (12).
The right vagal nerve predominantly innervates the right atrium and the sinus node, whereas the left vagal nerve predominantly innervates the left atrium and the AV node (8,13). In the previous study, the ERP in each atrium was shortened by ipsilateral vagal stimulation, but the degree of ERP shortening with right vagal stimulation was greater than that with left vagal stimulation in both atria (14). In this study, the shortening of the ERP during bilateral vagal stimulation was greater in the right than in the left atrium. These results could be explained, at least in part, by greater density of the muscarinic receptor in the right atrium than in the left atrium (15).
Mechanisms of antiarrhythmic drugs for termination of atrial fibrillation
Pilsicainide (16), a new class Ic antiarrhythmic drug, preferentially inhibits Na+ conductance; the maximal rate of increase of action potentials (Vmax) is therefore reduced. This reduction of Vmax is use dependent, and the recovery of use-dependent block is markedly slow (16). In a clinical study, AF could be successfully terminated by the oral administration of pilsicainide (17). Plasma concentration of this drug was slightly higher in this clinical trial (1.03 ± 0.65 μg/ml) compared with the experimental study. In this study, atrial ERP and intraatrial conduction time in each atrium were prolonged with pilsicainide. Because the prolongation of the conduction time was slightly greater than that of ERP, the WLI decreased in the right atrium. This decrease in WLI is expected to sustain the reentry (12); however, pilsicainide successfully terminated AF induced during vagal stimulation. It is likely that pilsicainide suppressed the intraatrial conduction at faster atrial rates to a crucial point, beyond which propagation of activation would be impossible. Wang et al. (6) showed that the class Ic drug, propafenone, prolonged atrial ERP in a use-dependent manner and increased the wavelength, leading to termination of AF in the canine model similar to this study. They determined atrial ERP at basic pacing-cycle lengths of ≥200 ms, and their atrial ERP was much shorter because of stronger vagal stimulation compared with our study. We determined ERP at a cycle length of 200 ms; therefore the different results could be explained, at least in part, by the different protocol for vagal stimulation and different properties of test drugs used.
MS-551, a new investigational class III antiarrhythmic drug, inhibits K+ conductance but does not affect Na and Ca channels (18). Consequently this drug prolongs both action-potential duration and ERP but does not reduce Vmax of action potential in experimental animals. In this study, the ERP in each atrium was prolonged, but the CT was not changed with MS-551 as expected. The dosage of MS-551 used in our study was very similar to that (0.3 mg/kg + 0.05 mg/kg/min) found to be effective in suppressing sustained atrial flutter in a canine aseptic pericarditis model (19). However, plasma concentration of this drug was not determined in this previous experimental study (19). The FF interval was prolonged before termination of AF with MS-551. This prolongation of the FF interval could therefore be explained by the prolongation of wavelength, leading to prolongation of the functional reentrant circuit.
Both drugs affect acetylcholine-induced K+ currents (20,21). Pilsicainide exerts an anticholinergic effect through blocking the muscarinic acetylcholine receptors (20). In contrast, MS-551 blocks the muscarinic receptors, as well as depresses the function of the K channel itself (21). Therefore antiarrhythmic effects of these drugs are attributed to their direct electrophysiologic effects and also, at least in part, to anticholinergic effects in this model of vagotonic AF.
Our study was limited for several reasons. First, the properties of AF induced by vagal stimulation in dogs are different from those in the diseased, dilated atria as seen in chronic AF of humans. However, this dog model may resemble paroxysmal AF seen in subjects with relatively normal atria. Patients with lone AF have a shorter atrial ERP and a greater dispersion of atrial refractoriness than normal subjects do (22,23). The difference of right atrial ERP between patients with chronic lone AF and control subjects is ∼10% (23) and slightly smaller compared with vagal-induced shortening of ERP in our study. Moreover, occurrence of paroxysmal AF appears to depend on increases in vagal tone in some patients (11,24).
Second, we stimulated bilateral vagal nerves with nonphysiologic strength, as mentioned in Methods, to sustain AF. This stimulation decreased ERP by 19% in the right atrium and by 10% in the left atrium. These decreases in ERP were similar to those of AF model induced by rapid atrial pacing in a previous study (25) but were smaller compared with the results of Wang et al. (5,6). Criteria for selection of current strength for vagal stimulation differed between the right and the left vagus in our study. However, the current strength used did not differ significantly between the right and the left vagus. Therefore the difference in ERP changes between the atria could not be attributed to the current strength used.
Third, ERP was determined only at a basic cycle length of 200 ms, and use-dependent effects of test drugs on ERP were not determined in this study. Other investigators (6,26) stressed use-dependent effects of class Ic drugs on atrial ERP as the mechanism for termination of AF.
Last, we used WLI calculated by ERP/CT instead of wavelength itself, because conduction velocity was not determined in this study. However, this WLI has been widely used in clinical studies (7,27,28). In the baseline AF of our study, WLI was smaller, and FF intervals were shorter in the right than in the left atrium. Therefore this index could be used to determine relative changes in the wavelength. After pilsicainide injection, FF intervals increased, and WLI decreased. This paradox could be explained by the greater suppression of conduction induced with this class Ic drug. This suggests that changes in wavelength induced by drugs may not predict changes in local activation intervals during AF.
Although caution is required when extrapolating the experimental data to clinical patients, our study might provide some insight into the mechanisms by which antiarrhythmic drugs terminate AF. Class Ic antiarrhythmic drugs could terminate AF by suppression of conduction, whereas class III antiarrhythmic drugs could do so by prolongation of the ERP, leading to abolition of the excitable gap.
Acknowledgment: We are grateful for Mitsui Pharmaceutical Co. and Suntory Limited for providing test drugs and measuring plasma concentrations of test drugs.
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