Low-frequency oscillations of atrial fibrillation cycle length in goats: characterization and potentiation by class III antiarrhythmic almokalant

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Journal of Electrocardiology 41 (2008) 711 – 723 www.jecgonline.com

Low-frequency oscillations of atrial fibrillation cycle length in goats: characterization and potentiation by class III antiarrhythmic almokalant Paulo Eustáquio de Brito Santos, MD, PhD, a,⁎ Mattias Duytschaever, MD, b Maurits A. Allessie, MD, PhD b a Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, RJ, Brazil Physiology Department, Cardiovascular Research Institute, Maastricht University, Maastricht, Limburg, The Netherlands Received 31 May 2007; accepted 20 March 2008

b

Abstract

Background: In chronically fibrillating goats, low-frequency oscillations (LFOs) of atrial fibrillation cycle length (AFCL) with a deceleration-acceleration sequence have been observed. The present investigation characterized such oscillations in control conditions and during the infusion of class III antiarrhythmic almokalant, trying to understand their mechanism and possible relevance. Methods and results: The study was performed on fibrillating goats instrumented with multiple electrodes. LFOs were characterized in 64-s recording samples (1 electrode/atrium) before and during almokalant infusion. Filtering was applied to the raw sequence of AFCL. LFOs were completely random, non-flutterlike and potentiated by almokalant, as evinced by increases in oscillation frequency, duration and amplitude. As compared with nonoscillation periods, the upper part of LFOs displayed an increase in single (84.0 ± 11.4% vs 72.5 ± 12.9%) and a reduction in double spikes (12.1 ± 8.3% vs 20.2 ± 8.6%), suggesting an improvement of propagation. This was supported by the features of activation maps during LFOs: fast conduction, few wave fronts and many linking beats. Conclusions: Chronically fibrillating goats exhibit random LFOs, which are enhanced by almokalant. The improvement of propagation during oscillations suggests an increase in the excitable period/ excitable gap. These findings raise the question of LFOs involvement in atrial fibrillation termination. © 2008 Elsevier Inc. All rights reserved.

Keywords:

Atrial fibrillation; Class III Antiarrythmics; Almokalant; Cardioversion; Excitable gap

Introduction Class III antiarrhythmic drugs have been shown to acutely stop atrial fibrillation (AF) in human patients1 as well as in experimental models. 2-5 However, despite the intense research on the field, the mechanism underlying cardioversion by these drugs remains obscure.6 Classically, cardioversion has been explained by an increase in the excitation wavelength (WLAF, given by conduction velocity × refractory period) leading to a reduction in the number of reentering wavelets that fit the atria. This would raise the chance of AF termination by wavelet collision and resulting fusion/extinction.2,7,8 Thus, by inducing a prolongation of the atrial effective refractory period (AERP) due to action ⁎ Corresponding author. Departamento de Farmacologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte / MG, Brazil. Tel.: +55 31 3409 2723; fax: +55 31 3499 2695. E-mail address: [email protected] 0022-0736/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jelectrocard.2008.03.006

potential lengthening, class III drugs would promote an increase in WLAF and AF termination. Although there is fine experimental evidence supporting this hypothesis,2,5,8 in chronically fibrillating goats, Wijffels et al4 showed that cardioversion by d-sotalol, a class III drug that blocks the rapid delayed rectifier potassium channel (IKr), was not associated with changes in AERP, conduction velocity, or WLAF, but with a clear increase in excitable period and spatial excitable gap. Alternatively, if spiral-type reentry is considered as the basis of AF maintenance, prolongation of the action potential by class III drugs is expected to increase wave front/wave tail interactions, leading to frequent local conduction blocks and meandering of the circuit, thus raising the chance for rotation breakup.9 In our laboratory, we observed, in chronically fibrillating goats, the spontaneous occurrence of low-frequency, non– beat-to-beat, random oscillations of AF cycle length (AFCL), showing mostly a deceleration-acceleration pattern.

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Fig. 1. Electrode positioning, surface electrograms, and time evolution of the cycle length (AFCL). The drawing in the superior part of the figure depicts the position of epicardial electrodes in the goat atria. The middle panel shows 8-second simultaneous electrograms recorded by 2 unipolar electrodes, one placed in LA and the other in RA. The plots below the tracings represent the time series of the AFCL (upper) and the corresponding filtered data (lower). Smoothing in the lower plot was obtained by performing the running average of the 10 previous AFCL values. BB indicates Bachmann bundle; LAA, left atrial appendage; RAA, right atrial appendage; PV, pulmonary veins; SCV, superior (anterior) caval vein; ICV, inferior (posterior) caval vein.

Interestingly, such oscillations became more frequent and larger (showing a greater deceleration of AFCL) during the infusion of d-sotalol or almokalant, another class III antiarrhythmic and, like d-sotalol, a blocker of IKr.10-12 Because preliminary observations showed an enhancement of low-frequency oscillations (LFOs) by class III drugs, suggesting their possible involvement in AF cardioversion, the objective of the present study was to fully characterize these oscillations both in control conditions (awake goats with chronic AF) and under almokalant infusion. Specifically, information concerning the incidence, amplitude, and duration of oscillations were pursued, as well as the possibility of their being just transient shifts to atrial flutter. Furthermore, an initial investigation on the mechanisms underlying oscillations was carried out by indirect evaluation of refractoriness during oscillation and nonoscillation periods.

Methods Animal model and data acquisition Six female goats weighting between 50 and 65 kg (54 ± 6 kg) were used in this study. To induce chronic AF and

record atrial unipolar electrograms, the animals were submitted, under general anesthesia, to open-chest surgery, during which 45 (1 goat), 52 (1 goat), or 83 (4 goats) platinum electrodes were fixed to the atrial epicardial surface as previously described.13 Briefly, a left intercostal thoracotomy was executed, the pericardium was opened to expose the heart, and 3 silicon strips containing the electrodes (electrode diameter, 1.5 mm) were positioned as follows: 1 strip sutured to the lateral wall of the right atrium (RA), 1 to the lateral wall of the left atrium (LA), and 1 to the tips of both atrial appendages, lying on the Bachmann bundle. The interelectrode distance was 4 mm for electrodes on the atria free wall and 6 and 10 mm for those on the Bachmann bundle and atrial appendages. The most common arrangement (n = 4) of electrodes is shown in Fig. 1. Because of technical restrictions, goats 1 and 2 did not receive the complete array of electrodes. Two or 3 electrodes were sutured onto the left ventricle to register unipolar ventricular electrograms, and 3 silver plates were placed subcutaneously to be used as indifferent electrode and to record the electrocardiogram. The cables containing the electrodes wires were tunneled through the subcutaneous tissue and exteriorized in the back of the goat's neck.

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After recovering from surgery, the animals were connected to an external pacemaker controlled by a computer, which performed (through any of the atrium electrodes) a stimulation protocol designed to induce chronic AF.13 All procedures were conducted according to the guiding principles of the European Directive for Animal Research and approved by the Animal Investigation Committee of the Maastricht University. Electrograms were recorded simultaneously (gain, 200400; bandwidth, 1-500 Hz; sampling rate, 1 kHz) during the whole experiment by means of a high-impedance, multichannel amplifier (Maastricht University) and were stored in videotape. Data samples were then transferred to an IBMcompatible personal computer (Pentium III) in which analysis was carried out by using MAP, a homemade software developed by Dr MA Allessie. Basically, the study was divided in 2 parts. Initially, as the objective was to detect and characterize the LFOs, long (64-second) samples were obtained. Because of technical constraints, only 8 electrograms at a time were digitized from tape. Among these, 1 electrogram per atrium was selected for analysis. In the second part, attention was driven toward the mechanism underlying the oscillations. This was accomplished by studying electrogram morphology in the previously mentioned data (64-second electrograms) and mapping impulse propagation in 4-second time windows containing all electrograms (selected from tape and transferred to computer) during either oscillation or nonoscillation (baseline) periods. Experimental protocol Experiments were only conducted during chronic AF (range of 2-14 weeks' persistence). The animals were awake and not sedated. After 10 to 20 minutes of intravenous infusion of saline (control period), almokalant was infused (20 nmol kg−1 min−1) during 60 minutes. The electrocardiogram was continuously monitored to confirm the activity of the drug, indicated by a ∼10% increase in the QT interval, as well as to avoid the risk of ventricular toxicity, characterized by aberrant QRS complexes and/or a marked QT prolongation (beyond 20%) and/or the occurrence of ventricular arrhythmias. In no case was the drug infusion toxic or dangerous to the animals. Identification and characterization of oscillations Electrogram analysis was completely performed by MAP software. Among many functions, it contains algorithms for automatic detection of activation times (the maximal dV/dt of a negative deflection within the atrial complex), generation of color-coded activation maps, and interactive editing of local activation times. The value of AFCL was given by the difference between 2 consecutive activation times. As mentioned before, identification and characterization of LFO (1 electrogram per atrium) were carried out on 64-second data samples. For analysis, among the 8 digitized electrograms, 2 (1 per atrium) were selected randomly from those with nice signal quality (spikes easily distinguished from baseline) and little fragmentation, allowing more precision in determining activation times. Six samples were

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Fig. 2. Recognition and analysis of LFOs of AFCL. A, An atrial electrogram in which an oscillation is taking place. Observe the sustained increase in AFCL and the good quality of deflections during the oscillation (not to consider the low-voltage ventricular signals also recorded, as pointed by the 2 arrows). B, The sequence of AFCL obtained from the displayed electrogram is plotted as a function of time. The straight dotted line represents the baseline, defined as the modal value of AFCL within a 64-second recording. C, The time window of panel B is presented after filtering of AFCL values by applying the running average of the 10 previous cycles. The rule for filtered data validates it as an oscillation because (1) it begins with a sequence of at least 4 consecutive increasing cycles and (2) the deceleration amplitude is ≥20 milliseconds. The beginning of the oscillation and its highest value are indicated, respectively, by the start and peak arrows. The peak value divides the oscillation into deceleration and acceleration phases. The acceleration phase finishes when interrupted by at least 2 decelerating cycles (end, also indicated by arrow).

selected per experiment: 2 from the control period and 4 during almokalant infusion (10, 15, 25, and 30 minutes after drug start). Because AFCL showed a great beat-to-beat variation, thus precluding an easy detection of LFO, the time series of AFCL values were subjected, by trial and error, to low-pass filtering by applying a moving average to the original data. Preliminary screenings with running averages of 3 up to 20 previous cycles were tried, and the running average 10 (10 previous beats, RA10) was chosen because it was the best to delimit individual oscillations according to the researchers' conformity. Fig. 1 displays 8-second electrograms recorded simultaneously in both atria during control, as well as the time evolution of the AFCL in these tracings and the corresponding RA10-filtered plot. Oscillations were identified and characterized on RA10filtered data. Essentially, LFOs showed a deceleration phase followed by an acceleration phase back to baseline. The reverse pattern (acceleration-deceleration) was rarely seen and then was discarded. Decelerations were acknowledged

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whenever they started with a sequence of at least 4 progressively increasing cycles. The choice of 4 was not arbitrary: sequences of 3 frequently led to short-lived oscillations with low amplitudes (within the range of baseline beats); if sequences of 5 or more were taken, many obvious LFOs remained undetected. Again by trial and error, we observed that most of the accelerations were finished when accelerating beats were interrupted by 2 consecutive decelerating beats. The point of maximal AFCL was called the oscillation peak. Because it was clear that, during the oscillation-free periods, AFCL varied around a basal value, we decided to add a baseline to the tracings, which was defined as the mode (the value appearing most frequently) of AFCLs in the corresponding 64-second raw data. An illustrative example of the “rules” used to identify and characterize LFO is presented on Fig. 2. Considering that oscillations were a new phenomenon of unknown mechanism and that they might be related to cardioversion, we focused on the higher oscillations. A threshold of 20-millisecond amplitude in the deceleration phase (the difference between peak value and the first decelerating beat ≥20 milliseconds) was then set as the requirement for an oscillation to be considered and analyzed. After identifying LFOs, the next step was characterizing them in terms of incidence, amplitude, and duration, as well as the slope of deceleration and acceleration phases. Incidence was simply taken as the number of oscillations per minute. Oscillation amplitude was defined in terms of deceleration and acceleration amplitudes, respectively, calculated as the difference between peak and starting AFCL values and between peak and ending AFCL values. The duration of LFO was given by the difference between the activation times of the last acceleration beat and the first deceleration beat. Accordingly, the deceleration and acceleration rates were given by the amplitude-duration ratio of each phase.

Electrogram morphology and mapping of oscillations With the objective of indirectly evaluating refractoriness and conduction velocity during the oscillations, studies on the morphology of atrial complexes and the characteristics of activation maps were conducted. In the former, complexes were categorized as single, double, fragmented, or block, whether they presented 1, 2, or more negative-going spikes or were blocked (very low voltage as compared with neighboring complexes). In the case of double potentials, spikes should be more than 10 milliseconds apart and the smaller component should bear at least 25% of the amplitude of the larger one.14 The percentage of each category in the baseline tracing was compared with that within the so-called top of oscillations (Top Osc), defined as the segment of the oscillations (in RA10filtered time series) showing AFCL ≥ baseline value + 1 SD of raw data. Again, the analysis was performed on 64-second recordings from 1 electrode per atrium. Activation maps were constructed from all electrograms using 4-second data samples containing nonoscillation (baseline) and oscillation periods. Maps were made automatically by the software and edited manually if necessary. Color-coded isochrones were drawn at 10-millisecond intervals for a better discrimination of the velocity and direction of propagation. Analysis was accomplished in terms of activation speed, propagation homogeneity, and presence of linking beats15 (consecutive waves propagating in the same direction). In the case of linking, whenever maps showed strong areas of block or orthogonal wave fronts (coming from inner layers), beats were considered as nonlinked. Conversely, bifurcation of the propagating wavelet or the presence of a secondary wavelet confined to the very border of the map (thus not disturbing the pathway of the main one) did not preclude classifying beats as linked, provided these events were repeated exactly in consecutive activations.

Table 1 Atrial fibrillation cycle length and ventricular parameters in chronically fibrillating goats before (control) and during almokalant infusion (almokalant) Goat

1 2 3 4 5 6 Mean ± SD

Site of atrial recording

AFCL (median, mean [ms])

RR interval (ms)

QRS interval (ms)

QT interval (ms)

Control

Almokalant

Control

Almokalant

Control

Almokalant

Control

Almokalant

LA RA LAA RA LA RA LA RA LAA RA LA

92, 93 82, 84 82, 82 106, 105 90, 90 127, 127 124, 126 119, 118 98, 98 111, 110 102, 102 103 ± 16, 103 ± 16

100, 100 91, 95 98, 100 114, 114 91, 90 134, 134 144, 147 139, 137 114, 116 124, 123 116, 116 115 ± 19 ⁎⁎, 116 ± 18 ⁎⁎

469 504

420 538

78 52

82 64

302 a 231

332 a 291

509

562

72

85

225

269

318

313

48

48

166

172

430

354

63

63

189

205

467

486

39

38

187

224

450 ± 71

445 ± 100

59 ± 15

63 ± 18

200 ± 28

232 ± 48 ⁎

The AFCL values were obtained from 64-second stored data samples (2 during control and 2 after 25-30 minutes of almokalant). Except for goat 1 QT interval, ventricular parameters are means of median (P50) values obtained online from samples containing 100 ventricular complexes (3-5 samples for control, 2 for almokalant). All data were collected by 1 electrode per atrium or ventricle. a Values are means of 20 QT intervals measured off-line from control and 25- to 30-minute drug infusion data. These values were not included in the calculation of final mean. ⁎ P b .05. ⁎⁎ P b .001.

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Fig. 3. Frequency of oscillations. The figure shows the incidence of oscillations in all atria during control and under almokalant infusion (10-15 and 25-30 minutes after drug start). Data were obtained from 64-second electrograms (2 for control and 4 for almokalant period) recorded by 1 electrode per atrium. *P b.05 (almokalant vs control).

Statistics All results were analyzed using the Student t test because only 2 groups were compared at a time. Whenever data were obtained from the same animal before and during drug infusion, the paired t test was applied. For the other cases, the simple t test was used as usual. Results were expressed in terms of mean ± SD or mean ± SEM as far as the emphasis was on the dispersion of values or on the variability around the mean. Statistical significance was set at P b .05.

Results General aspects Atrial fibrillation was persistent in all goats during the whole period of control. Within 60 minutes of drug infusion,

only 1 animal was cardioverted to sinus rhythm (goat 2, after 35 minutes). With the objective of collecting more information about precardioversion beats, AF was reinduced 3 times in this goat by stimulus bursts, so that 4 episodes of cardioversion were actually recorded. In 2 other animals, AF stopped in the following 24 hours; but no data from such period were recorded. The average AFCL during control (1 electrode per atrium, 11 atria, 2 × 64-second data samples, mean ± SD) was 103 ± 16 milliseconds (range of means, 82-127 milliseconds); and after 25 to 30 minutes of almokalant infusion (2 × 64-second data samples), it showed a small (13%) but significant increase to 116 ± 18 milliseconds. If, for the sake of evaluating the contribution of LFOs to the average AFCL, the cycles belonging to the oscillations are discarded, then these values shift to 102 ± 16 and 112 ± 19 milliseconds for control and almokalant periods, respectively (10% increase,

Table 2 Characteristics of LFOs (filtered data) during control Goat

AF duration (wk)

Electrode site

No. LFOs

1 2

4 2

3

2.5

4

5

5

14

6

2-4 a

All

2-14

LA RA LAA RA LA RA LA RA LAA RA LA RA LA RA + LA

1 9 0 5 2 3 7 5 0 0 5 22 15 37

Slope (ms s−1)

Amplitude (ms)

Duration (s)

Dec

Acc

Dec

Acc

Osc

Dec

Acc

34 29 – 25 22 31 25 25 – – 26 28 ± 7 26 ± 5 27 ± 7

18 28 – 22 32 26 28 26 – – 23 26 ± 9 26 ± 10 26 ± 9

0.96 1.76 – 1.71 1.85 1.78 1.54 1.13 – – 1.26 1.61 ± 0.57 1.45 ± 0.29 1.54 ± 0.48

3.03 2.02 – 1.69 2.05 1.86 2.01 0.95 – – 1.62 1.68 ± 0.82 1.95 ± 0.83 ⁎ 1.79 ± 0.82

3.99 3.78 – 3.40 3.89 3.64 3.54 2.04 – – 2.88 3.26 ± 1.12 3.40 ± 0.86 3.33 ± 1.02

35 18 – 16 12 17 17 24 – – 21 19 ± 6 19 ± 6 19 ± 6

6 15 – 14 18 17 15 29 – – 16 18 ± 9 15 ± 6 17 ± 8

Data obtained from two 64-second samples recorded by 1 electrode per atrium. Results are expressed either as mean alone (for the sake of clarity) or mean ± SD. Dec indicates deceleration phase; Acc, acceleration phase. a Atrial fibrillation duration not accurate. ⁎ Dec vs Acc: P b .05.

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P b .001). The results related to AFCL are presented in Table 1. As expected from a pure class III drug, the atrialventricular conduction and ventricular propagation were not affected by almokalant. The values of RR interval and QRS duration (Table 1) obtained during control were not statistically different from those found at 25/30 minutes of drug infusion: 450 ± 71 vs 445 ± 100 milliseconds for RR interval and 59 ± 15 vs 63 ± 18 milliseconds for QRS duration (control vs drug). On the other hand, after 25 to 30 minutes of almokalant infusion, the QT interval showed a 16% prolongation (P b .05) as compared with control (232 ± 48 vs 200 ± 28 milliseconds, n = 5), thus confirming the typical class III action of the drug. Characteristics of spontaneous oscillations A typical LFO of AFCL is presented in Fig. 2A. As we can see, during the oscillation, there is a sustained increase in cycle length; and the quality of the deflections markedly improves (single, high-amplitude spikes) as compared with the baseline (double, fragmented, low-voltage spikes).

The analysis of two 64-second data samples during control (1 electrode per atrium) clearly confirmed the presence of spontaneous LFOs in chronic AF for all goats (Fig. 3). Nevertheless, the frequency of oscillations showed a great variation from atrium to atrium (Fig. 3), even within the same goat. For instance, whereas goat 2 RA electrode and goat 4 LA electrode recorded, respectively, 4.2 and 3.3 LFOs per minute, no oscillations were detected in goat 2 left atrial appendage (LAA), goat 5 LAA, and goat 6 RA. The number of oscillations recorded in the RA was almost twice that obtained in the LA/LAA (mean ± SD, 2.3 ± 1.5 vs 1.2 ± 1.4 min−1), but the difference did not reach significance. There was no correlation between the median AFCL and the frequency of oscillations (r = 0.22). No matter their location or their frequency, LFOs seemed to be a completely random phenomenon, without any sign of periodicity. The analysis of LFO parameters was performed on RA10filtered data. Table 2 shows the results of amplitude, duration, and slope of the deceleration and acceleration phases for all spontaneous LFOs (n = 37) recorded before the

Fig. 4. Regional nature of LFOs. The upper figure shows simultaneous electrograms recorded from LAA (ele 19), BB (ele 30), RAA (ele 40), low RA (ele 93), and high RA (ele 88) in goat 2, under almokalant infusion. In the lower figure are depicted plots of unfiltered fibrillation cycle lengths (AFCL), as a function of time, obtained from the respective electrograms, together with goat 2 electrode map. Notice that the oscillation taking place in the beginning of the tracings (1.23.6 seconds) in low RA, high RA, and RAA was not observed in BB and LAA. Similarly, the oscillation occurring between 11.6 and 13.8 seconds in the RA and RAA did not reach BB. On the other hand, the huge oscillation seen in the second half of ele 19 was not present in BB and in the RA. LRA indicates low RA; HRA, high RA; ele, electrogram/electrode; *, RAA.

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Fig. 5. Characteristics of spontaneous and almokalant-related LFOs. The figure shows the pooled results for the duration of LFOs (deceleration and acceleration components as well as whole oscillation) and for the amplitude and slope of deceleration and acceleration phases. Results were obtained from 64-second electrograms (2 for control and 4 for almokalant [10, 15, 25, and 30 minutes of infusion] period) recorded by 1 electrode per atrium and are expressed as mean ± SEM. ⁎P b .05, ⁎⁎P b .01, and ⁎⁎⁎P b .001 (control vs almokalant).

infusion of almokalant. As we can see, no differences were found in terms of amplitude and slope between the 2 processes. With respect to duration, acceleration lasted significantly (P b .05) longer than deceleration in LA (1.95 ± 0.83 vs 1.45 ± 0.29 seconds), but not in RA. Oscillations in the presence of almokalant Oscillations were markedly enhanced during the infusion of almokalant. After only 10 to 15 minutes of drug start, the overall frequency of LFOs (Fig. 3) had more than

doubled as compared with control (3.8 ± 2.3 vs 1.7 ± 1.5 min−1, n = 11, P b .05). A similar figure was obtained at 25/30 minutes of infusion (4.1 ± 2.4 min−1, n = 11, P b .05 vs control; P = .739 vs 10-15 minutes), probably suggesting the achievement of the maximal incidence. Curiously, the areas showing no oscillations during control displayed quite variable responses to almokalant. Therefore, whereas goat 5 LAA electrode recorded only 1 oscillation in 2 minutes (0.5 min−1) at 25 to 30 minutes of drug, the number of LFOs increased considerably in goat 2 LAA and goat 6 RA, reaching 2.8 and 3.5 min −1,

Table 3 Characteristics of LFOs (filtered data) during almokalant infusion Goat

1 2 3 4 5 6 All

Electrode site

No. LFOs

LA RA LAA RA LA RA LA RA LAA RA LA RA LA RA + LA

11 27 8 22 9 10 19 17 2 8 18 84 67 151

Slope (ms s−1)

Amplitude (ms)

Duration (s)

Dec

Acc

Dec

Acc

Osc

Dec

Acc

30 35 38 43 29 32 39 35 29 25 30 36 ± 12 33 ± 11 35 ± 12

31 42 48 45 26 38 40 34 23 29 31 39 ± 17 35 ± 15 37 ± 16

1.81 1.94 4.55 2.94 1.68 1.83 3.54 1.64 1.39 2.01 2.13 2.13 ± 1.31 2.68 ± 1.97 2.38 ± 1.65

1.96 1.58 2.63 ⁎ 2.11 1.65 2.06 2.70 1.15 ⁎ 0.92 2.32 1.66 1.76 ± 0.71 ⁎ 2.10 ± 1.08 ⁎ 1.91 ± 0.91 ⁎⁎

3.79 3.52 7.17 5.05 3.33 3.89 6.24 2.79 2.49 4.33 3.79 3.90 ± 1.63 4.78 ± 2.63 4.29 ± 2.17

17 21 11 17 17 18 14 25 20 13 16 20 ± 7 15 ± 6 18 ± 7

18 28 ⁎⁎ 19 ⁎ 23 ⁎⁎ 17 21 18 30 24 14 21 25 ± 11 ⁎⁎⁎ 19 ± 9 ⁎⁎ 22 ± 10 ⁎⁎⁎

Data were obtained from four 64-second samples recorded by 1 electrode per atrium at 10, 15, 25, and 30 minutes of drug infusion. Results are expressed either as mean alone (for the sake of clarity) or mean ± SD. ⁎ Dec vs Acc: P b .05. ⁎⁎ Dec vs Acc: P b .01. ⁎⁎⁎ Dec vs Acc: P b .001.

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Table 4 Morphology of electrograms recorded at baseline and during the Top Osc (mean cycle length + 1 SD) as referred to the percentage of single, double, and fragmented spikes or block Goat

1 2 3 4 5 6 Mean ± SD Total

Electrode site

LA RA LAA RA LA RA LA RA LAA RA LA –

Single spikes (%)

Double spikes (%)

Fragmented spikes and block (%)

Total no. of spikes (atrial complexes)

Baseline

Top Osc

Baseline

Top Osc

Baseline

Top Osc

Baseline

Top Osc

60.3 85.6 81.0 74.8 80.0 79.2 92.8 61.9 72.2 53.2 56.1 72.5 ± 12.9 –

72.4 92.5 96.3 83.7 89.9 88.8 99.1 60.6 83.3 74.1 83.7 84.0 ± 11.4 ⁎ –

31.3 11.6 13.3 20.6 15.4 17.3 5.7 26.6 20.6 31.2 28.4 20.2 ± 8.6 –

23.5 6.3 3.5 13.2 10.1 9.3 1.1 26.0 8.3 22.2 9.8 12.1 ± 8.3 ⁎ –

8.4 3.5 5.6 6.0 4.6 3.7 1.3 11.5 7.3 15.6 15.6 7.6 ± 4.8 –

5.3 1.3 0.2 3.1 0 1.8 0 13.5 8.3 3.7 7.7 4.1 ± 4.3 ⁎⁎ –

3587 2830 3644 2315 3925 2539 1593 2534 3544 2705 2362 – 31 578

170 544 429 326 89 107 346 104 12 27 245 – 2399

Results comprise data obtained either during control or under almokalant infusion. Data obtained from 1 electrode per atrium. Pooled results expressed as mean ± SD. ⁎ Baseline vs Top Osc: P b .05. ⁎⁎ Baseline vs Top Osc: P = .089.

respectively. Again, no significant difference was found in the analyzed time windows between the right (4.1 ± 2.6 and 5.0 ± 2.1 min−1 for 10-15 and 25-30 minutes, respectively)

and left atria (3.6 ± 2.2 and 3.3 ± 2.4 min−1 for 10-15 and 25-30 minutes, respectively), despite the tendency toward a greater frequency in the former.

Fig. 6. Mapping of activation times in AF: comparison between an LFO and baseline. The figure shows a series of AF cycles (goat 3 RA) starting in the baseline, then decelerating and accelerating to make up an oscillation, and finally going back to baseline. Together are shown representative samples of sequential activation maps with respect to baseline (before [AB interval] and after the oscillation [CD interval]) and during the top beats of the oscillation. Activation maps are color-coded in 10-millisecond steps according to the convention of the vertical bar (bottom right), with intervals increasing from top to bottom: red (0-9 milliseconds), yellow (10-19 milliseconds), light green (20-29 millisecond), and so on, as far as violet (80-89 millisecond) and block (#). Observe that, whereas the baseline is characterized by slow activation times, multiple lines of block, and beat-to-beat shift in the direction of propagation, oscillation top beats present a rapid conduction velocity, only one direction of propagation, and a large number (11) of linking beats.

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719

Fig. 7. Atrial fibrillation cycle length before cardioversion. The figure shows the evolution of AFCL during the last 36 to 42 seconds before conversion to sinus rhythm in 4 episodes (A-D) of AF termination in goat 2. Data were obtained from 2 electrodes, ele 88 and ele 19, positioned, respectively, in high RA and LAA. All episodes happened under almokalant infusion. As we can see, AF termination was an abrupt process, not preceded by a progressive change in the baseline.

Differently from flutter waves, LFOs did not spread to both atria, as illustrated in Fig. 4. In this example, taken from the almokalant period, between 1.2 and 3.6 seconds, an obvious oscillation occurs simultaneously in low RA, high RA, and right atrial appendage (RAA), but is absent in the Bachmann bundle and LAA. Similarly, another clear oscillation takes pace between 11.6 and 13.8 seconds in low RA, high RA, and RAA; but again, it is missing in the Bachmann bundle. Conversely, a long-lasting (N6 seconds) oscillation starts by the middle and continues until the end of the tracing in LAA, which seems to be completely unrelated to the second oscillation observed in the other sites. This feature of LFOs was not restricted to the almokalant period, being observed also during control (data not shown).

In the presence of almokalant, LFOs became markedly higher and slower; and there was a speeding up of the acceleration phase. As we can see in Fig. 5 (RA10-filtered results), the mean amplitude of all decelerations recorded at 10/15 and 25/30 minutes of drug (35 ± 1 milliseconds) was 29% higher (P b .001) than the control value (27 ± 1 milliseconds). The increase in amplitude of the almokalantrelated accelerations was still more pronounced, reaching 43% (37 ± 1 vs 26 ± 2 milliseconds, P b .001). With respect to duration, the drug caused a marked lengthening (55%) of the deceleration phase (2.38 ± 0.13 vs 1.54 ± 0.08 seconds, P b .001) without changes in the acceleration time (1.91 ± 0.07 vs 1.79 ± 0.13 seconds, P N .05), thus leading to a 29% increase in the duration of the whole oscillation (4.29 ± 0.18

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vs 3.33 ± 0.17 seconds, P b .05). The slope of acceleration was quite enhanced by the drug (22.2 ± 0.9 vs 17.0 ± 1.3 ms s−1, P b .01), whereas the rate of deceleration remained unchanged (17.7 ± 0.6 vs 18.6 ± 1.0 ms s−1, P N .05). Differently from what we observed in control LFOs (Table 2), in almokalant-related oscillations (Table 3), the duration and the rate of acceleration were, respectively, shorter (17%-22%) and faster (25%-27%) than those of deceleration in both atria. Alternatively, no differences were found in amplitude between deceleration and acceleration. Searching for a mechanism underlying LFOs Although valuable to introduce the phenomenon, the descriptive characterization does not provide a mechanistic explanation for the oscillations. Searching for clues about the mechanisms underlying LFOs, we tried to evaluate conduction and refractoriness in the fibrillating atria by studying electrogram morphology and activation maps during baseline and LFO recordings. In the first case (Table 4), except for goat 5 RA, in all other atria (n = 10), there was an evident increase in the percentage of single spikes and a reduction of double spikes within the Top Osc as compared with baseline. That was confirmed statistically (P b .05) for pooled data: single spikes—84.0% ± 11.4% vs 72.5% ± 12.9% for Top Osc and baseline, respectively; double spikes—12.1% ± 8.3% vs 20.2% ± 8.6% for Top Osc and baseline, respectively. Similarly, the percentage of fragmented/ blocked potentials was markedly reduced in 9 of 11 recordings during Top Osc. However, when taken together, the difference was not significant (4.1% ± 4.3% for Top Osc and 7.6% ± 8.3% for baseline, P = .089). On the other hand, during baseline alone (LFOs not considered), the quality of propagation worsened by the infusion of almokalant, as the percentage of single, double, and fragmented/blocked potentials shifted from (mean ± SEM) 74.8% ± 3.5%, 18.7% ± 2.4%, and 6.5% ± 1.2%, respectively, to 70.7% ± 4.2% (P b .01), 21.1% ± 2.7% (P b .01), and 8.2% ± 1.7% (P b .05). Consistently, activation maps of beats taking place around the oscillation peak showed much better patterns of propagation than those obtained from baseline. A typical example from goat 3 RA is presented in Fig. 6. As clearly displayed there, AF was completely chaotic before the oscillation start (AB interval), showing multiple wave fronts, beat-to-beat variation of the propagation route, and slow conduction, as evinced by delayed activation times and several areas of block. Remarkably, in the beats around oscillation peak, activation times became shorter; propagation followed a single, well-defined direction; and a sequence of 11 linking beats was detected. Back to baseline (CD interval), AF resumed the previous chaotic pattern. The sequences of linking beats were compared in baseline and during oscillations. This was accomplished on maps from 3 goats, one atrium per animal: goat 1 LA, goat 3 RA, and goat 4 LA. These were chosen because of the more adequate number of functioning electrodes and the nice quality of electrograms. Results were not separated into control and almokalant periods. As expected from Fig. 6, in

goat 3 RA, the percentage of linked beats increased unequivocally from 17.3% in baseline to 35.3% during LFO. A result in the same direction, although less remarkable, was obtained in goat 1 LA: 27.4% vs 37.4% for baseline and oscillations, respectively. Yet, to our surprise, in goat 4 LA, linked beats were fairly more frequent in baseline (30.6% vs 28.6%). Cardioversion in goat 2 Unfortunately, almokalant-induced cardioversion was only recorded in one goat (goat 2). Nevertheless, 4 episodes of cardioversion were actually registered because AF was reinduced 3 times by burst stimulation. Fig. 7 shows the evolution of the AFCL recorded simultaneously at 2 sites (LAA, electrode 19; high RA, electrode 88; same map as in Fig. 4) during the last 36 to 42 seconds before cardioversion episodes. Interestingly, AF termination was not preceded by remarkable increases in AFCL: during the last precardioversion minute, the mean AFCL (average of 4 episodes) increased 16.9% and 12.5%, respectively, for electrodes 19 and 88, as compared with the control period (without drug). In addition, as observed most of the time (Fig. 4, for example), LFOs did not occur simultaneously at the 2 sites (Fig. 7). Furthermore, the evolution of the final fibrillation cycles varied among the 4 episodes. In the first episode (Fig. 7A), cardioversion happened when a large oscillation took place in electrode 19, whereas in 88, only a very mild deceleration was seen, which was followed by 2 long cycles just before sinus rhythm. In the second (Fig. 7B), an obvious deceleration was recorded by electrode 88, whereas in electrode 19, AFCL was fairly irregular. The third episode (Fig. 7C) was more or less the opposite of the second, with a sustained deceleration occurring in electrode 19 and a short-lived oscillation just being finished in 88. In the fourth (Fig. 7D), cardioversion was preceded by few decelerating beats in electrode 19 and was totally abrupt in 88.

Discussion Characterization of LFOs In the present article, we describe, for the first time, spontaneous (non–beat-to-beat) LFOs of the AFCL in chronically fibrillating goats. These oscillations are composed of a deceleration phase, during which AFCL increases, followed by an acceleration phase, when cycles become shorter and return to baseline values, the whole process lasting for a few seconds. Interestingly, LFOs were markedly enhanced by almokalant, a class III antiarrhythmic, suggesting their possible role in cardioversion by these drugs. To avoid beat-to-beat variation of the AFCL, low-pass filtering was applied to raw data. By trial and error, a series of rules was developed to identify and characterize the oscillations. Because these rules were developed empirically and applied on filtered data, absolute figures are likely to be meaningless. However, the results are quite useful for comparing oscillation characteristics before and during drug infusion.

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Spontaneous LFOs showed a marked variation of incidence among the animals and even between both atria in the same goat (Fig. 3). Although not significant, there was a tendency toward a greater LFO incidence in the RA as compared with the LA. Interestingly, the basic cycle length had no influence on the occurrence of LFO. Furthermore, no matter how frequent they were, LFOs did not show any sign of periodicity. With respect to mechanisms, this might suggest that oscillations are not associated to respirationrelated fluctuations of autonomic discharge to the heart. Thus, taking all results into consideration, not a single predictive factor concerning the incidence of spontaneous LFO was identified. The analysis of 64-second recordings revealed that LFOs do not take place simultaneously in all parts of the atria (Fig. 4) as in flutter,16 thus suggesting that decelerations are not just transient shifts from AF to atrial flutter. Low-frequency oscillations were clearly enhanced by the class III antiarrhythmic almokalant, as evinced by a marked increase in oscillation frequency, amplitude, and duration as compared with the control period (Figs. 3 and 5, Tables 2 and 3). The increase in duration was due to a prolongation of deceleration because acceleration time was unchanged. Under almokalant infusion, the rate of deceleration was similar to that of control since amplitude and duration increased proportionally (Fig. 5). In contrast, because acceleration amplitude increased and duration was unaltered, acceleration rate became noticeably faster. On the other hand, the lack of periodicity and the non–flutter-like nature of oscillations were maintained in the presence of the drug (Fig. 4). The nature of LFO The mechanism that generates LFO is unknown. In the context of the multiple wavelet hypothesis,17 the deceleration phase of an oscillation seems to reflect a reduction in the number of reentrant wavelets taking place simultaneously in the atria. This reduction would occur by chance (because of block or collision-induced fusion/extinction of wavelets), as predicted in the hypothesis and suggested by the lack of periodicity, and could explain sudden, short-lived increases in AFCL, as well as the initial jump of abrupt decelerations. Gradual decelerations, however, are not so easily explained. They might involve, for instance, a progressive lengthening of the revolution time of a functional reentrant circuit. According to the leading circle reentry concept,18 such a lengthening would be caused by an increase in refractory period and/or a decrease in stimulation efficacy. Yet, that seems not to be the case. Although we did not measure the refractory period directly, during LFO, indirect evidence strongly points toward a reduction instead of an increase in refractoriness. This evidence comes from the analysis of electrogram morphology and activation maps, which revealed an improvement of propagation, thus indicating less refractoriness. In a scenario of improved propagation, the second hypothesis, a decrease in stimulation efficacy, would be rather unlikely. The reduction in refractoriness comes out from the following reasoning. It is well known that unipolar

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electrograms showing single, high-amplitude, biphasic potentials are associated with a uniform, high-quality propagation of fibrillating waves, whereas double and fragmented potentials reflect conduction disturbances such as collision, asynchronous activation, block, pivot points, and slow conduction.14,19 As shown in Table 4, during the oscillation top beats, the percentage of single spikes increased and that of double spikes decreased significantly as compared with baseline values. Regarding fragmented/ blocked potentials (Table 4), a tendency toward reduction was evident, occurring in 9 of 11 atria and almost reaching significance. Favoring the hypothesis of improved propagation is the increase in electrogram voltage,14,19 which was frequently found during LFO, as illustrated in Figs. 2 and 4. The results above strongly suggest that, when oscillations take place, propagation is improved, probably reflecting a decrease in refractoriness. In most cases, activation maps readily confirmed the improvement of propagation as revealed by the shorter activation times, less wave fronts, less areas of block,20 and enhanced linking15 shown by LFO in relation to baseline. Sometimes, the transition from very poor to optimal propagation was outstanding as in the example of Fig. 6. With respect to the percentage of linked beats, quantitative analysis showed an obvious increase in goat 3 RA (LFO 35.3% vs baseline 17.3%) and goat 1 LA (LFO 37.4% vs baseline 27.4%) and a small reduction in goat 4 LA (LFO 28.6% vs baseline 30.6%). The reason for such a discrepancy is unknown but might be related to the poor pattern of propagation during baseline in goat 3 RA and goat 1 LA (60.3% and 74.8% of single spikes, respectively), contrasting with the nice propagation in goat 4 LA (92.8% of single spikes). If the refractory period is not lengthened and the stimulation efficacy is not lowered, the increase in AFCL seems to reflect an enlargement of circuit sizes and a reduction in the number of wave fronts. The increase of functional reentrant circuits decreases the curvature of the turning wavelets, leading to improved propagation.21 In addition, when pathways are enlarged, anatomical obstacles tend to be incorporated into functional circuits,4 with the concomitant creation of excitable gaps (and increase in preexisting ones), which facilitates conduction. The observation of simultaneous LFOs in extensive areas of RA and RAA in Fig. 4 (electrodes 40, 88, and 93) corroborates the hypothesis of large circuits underlying oscillations. Although the increase in pathway is a reasonable explanation for the prolongation of AFCL, the mechanism involved in such an increase during gradual decelerations is obscure. Conversely, acceleration back to baseline could express an increase in the amount of wave fronts. If our hypothesis is correct, as deceleration renders circuits larger and bearing a wider excitable gap, they also become more vulnerable to short-circuiting and invasion by neighboring wavelets. Afterward, circuits are broken and acceleration ensues. Back to baseline, the number of wavelets returns to preoscillation values, the excitable gap is reduced, and circuits are stabilized.

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The effect of almokalant Previously, the effects of d-sotalol, another class III antiarrhythmic, were studied in the same model.4 The authors observed a moderate increase in AFCL (24%) and marked increases in excitable period (102%) and spatial excitable gap (126%). The AERP and conduction velocity, both measured directly during AF, did not change, the same happening to WLAF. In our study, the analysis of electrogram morphology and activation maps showed improved propagation during LFO, suggesting an increase in excitable gap. On the other hand, during baseline (oscillations discarded), indirect evidence (reduction in the number of single spikes and elevation in that of double and fragmented spikes) pointed toward an almokalant-induced increase in refractoriness. As in the study of Wijffels et al4 oscillations remained unnoticed, it is possible that part of the measurements was performed while they took place. It is logical then to suppose that the overall increase in the excitable period (and AFCL) was due to oscillation enhancement caused by d-sotalol. In other words, the increase in excitable period and spatial excitable gap might have been just a consequence of the elevated number of LFOs (with higher amplitude and longer duration) taking place under d-sotalol infusion. The possibility that oscillations are not potentiated by d-sotalol has been rejected by preliminary experiments (data not shown). The mechanism underlying LFO potentiation by almokalant is unknown. One of the possibilities would consider the kinetic properties of the drug-channel interaction. Assuming that the drug binds mainly to the channel in the resting state, causing the so-called reverse use dependence characteristic of class III antiarrhythmics,6 we can predict the following: if AFCL is suddenly increased, the blocking action will be enhanced, leading to longer repolarization and refractoriness and, consequently, to slower propagation and further increase in AFCL (deceleration process). However, as discussed above, tissue refractoriness during LFO seems to be reduced instead of augmented. Moreover, in rabbit ventricular cells, Carmeliet11 demonstrated that almokalant has actually a direct, not reverse, use-dependent blocking action on the IKr channel. This was confirmed by studies on action potential rate-dependent effects performed in guinea pig papillary muscles and pig atrium and ventricle.12,22 Alternatively, the potentiating effect of almokalant on deceleration might result from a spatial change in reentrant circuits. In atrial flutter, d-sotalol was able to terminate reentry by initiating destabilizing ectopic beats outside the main stimulus pathway.23,24 Similarly, an almokalantinduced premature beat in front of the propagating wavelet would force it to make a contour around the region of refractoriness, causing an enlargement of the circuit and consequently an increase in AFCL. The enlargement of the circuit would reduce the available area for functional reentry, leading to a reduction in the number of wavelets.18 Cardioversion and LFO Although 4 cardioversion episodes were recorded, they were restricted to only one goat. This precludes any

conclusion about the participation of LFOs in AF termination. Differently from several studies with class III antiarrhythmics showing a marked prolongation of cycle length by drug infusion,2,3,5 in the present investigation, AFCL was just mildly increased in the last minute before cardioversion as compared with control, prealmokalant period. Interestingly, until the end of fibrillation, LFOs continued to occur in a random and unsynchronized fashion, at least in the 2 sites recorded (Fig. 7). Thus, in our case, AF termination was not a gradual process related to AFCL prolongation but rather a sudden, random, and probably oscillation-triggered event. Similarly, spontaneous termination of paroxysmal AF in humans has recently been shown to take place abruptly.25 In summary, the present study characterizes LFOs of the AFCL in chronically fibrillating goats. These oscillations occur spontaneously and are enhanced by the class III drug almokalant. The mechanism underlying them is unknown. During LFO, impulse propagation improves, suggesting reduction in tissue refractoriness and increase in excitable period and spatial excitable gap. This condition would favor cardioversion by wavelet collision and resulting fusion/extinction. During oscillation-free periods, AFCL and propagation characteristics are only mildly affected by almokalant. References 1. Agarwal A, York M, Kantharia BK, Ezekowitz M. Atrial fibrillation: modern concepts and management. Annu Rev Med 2005;65:475. 2. Wang J, Bourne GW, Wang Z, Villemaire C, Talajic M, Nattel S. Comparative mechanisms of antiarrhythmic drug action in experimental atrial fibrillation. Importance of use-dependent effects on refractoriness. Circulation 1993;88:1030. 3. Nattel S, Liu L, St-Georges D. Effects of the novel antiarrhythmic agent azimilide on experimental atrial fibrillation and atrial electrophysiologic properties. Cardiovasc Res 1998;37:627. 4. Wijffels MCEF, Dorland R, Mast F, Allessie MA. Widening of the excitable gap during pharmacological cardioversion of atrial fibrillation in the goat. Effects of cibenzoline, hydroquinidine, flecainide and d-sotalol. Circulation 2000;102:260. 5. Blaauwn Y, Gögelein H, Tieleman RG, Van Hunnik A, Schotten U, Allessie MA. “Early” class III drugs for the treatment of atrial fibrillation. Efficacy and atrial selectivity of AVE0118 in remodeled atria of the goat. Circulation 2004;110:1717. 6. Davy JM, Raczka F, Beck L, Piot C. Méchanismes d´action des antiarythmiques dans la cardioversion de la fibrillation et du flutter auriculaires. Arch Mal Coeur 2003;96(IV):20. 7. Tse HF, Lau CP. Electrophysiological properties of the fibrillating atrium: implications for therapy. Clin Exp Pharmacol Physiol 1998;25: 293. 8. Rensma PL, Allessie MA, Lammers WJEP, Bonke FIM, Schalij MJ. Length of excitation wave and susceptibility to reentrant atrial arrhythmias in normal conscious dogs. Circ Res 1988;62:395. 9. Amino M, Yamasaki M, Nakagawa H, et al. Combined effects of nifekalant and lidocaine on the spiral-type re-entry in a perfused 2dimensional layer of rabbit ventricular myocardium. Circ J 2005;69: 576. 10. Wettwer E, Grundke M, Ravens U. Differential effects of the new class III antiarrhythmic agents almokalant, E-4031 and D-sotalol, and of quinidine, on delayed rectifier currents in guinea pig ventricular myocytes. Cardiovasc Res 1992;26:1145. 11. Carmeliet E. Use-dependent block and use-dependent unblock of the delayed rectifier K current by almokalant in rabbit ventricular myocytes. Circ Res 1993;73:857.

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