A Canine Model of Sustained Atrial Fibrillation Induced by Rapid Atrial Pacing and Phenylephrine

Abstract

Atrial fibrillation is a common arrhythmia with considerable morbidity and mortality. Limitations in studying both the mechanisms and therapy of atrial fibrillation arise due to the paucity of models that yield sufficiently high-quality data, are not costly, and in which atrial fibrillation is sustained long enough to make the necessary observations. The canine model we present is based on the hypothesis that atrial fibrillation requires heterogeneity of repolarization, that distribution of vagal fibers is heterogeneous in the atria, and that atrial fibrillation will persist after reflex stimulation of vagal efferents by increased systemic arterial pressure. Dogs were anesthetized with morphine–chloralose because this combination maintains nearly intact autonomic control. Systemic arterial pressure was elevated approximately 75 mm Hg during infusion of phenylephrine (2 μg/kg · min⁻¹). The right atrium was paced for 20 min at 40 Hz. Atrial fibrillation was sustained after cessation of atrial pacing in dogs receiving phenylephrine, but terminated within seconds in normotensive animals. In conclusion, atrial fibrillation can be maintained for at least 40 min after cessation of rapid atrial pacing in dogs with phenylephrine-induced hypertension.

Atrial fibrillation is a common arrhythmia that affects more than 2 million persons in the United States.¹ This condition is characterized by chaotic asynchronous activation and contraction of hundreds of regions of the atria, resulting in both absence of active atrial transport of blood and a rapid ventricular response. With chronic atrial fibrillation, patients can develop thromboembolism and stroke;²² and 15% of strokes in the United States occur in patients with atrial fibrillation.¹ Despite prodigious efforts to understand the mechanism of this condition and to prevent and remediate it, atrial fibrillation leads to enormous morbidity and mortality.³ One factor hindering studies of atrial fibrillation is the absence of a model in which fibrillation can be sustained for more than several seconds, although the arrhythmia can be sustained nearly permanently after weeks of rapid atrial pacing in animals with either heart failure or physical injury to the left atrium.⁸

Rapid atrial pacing decreases the atrial effective refractory period, slows atrial conduction, and increases electrophysiologic heterogeneity.^10,11,20 Recently, phenylephrine was shown to increase the difference between left and right atrial and intraatrial refractory periods, thus creating heterogeneity of atrial refractoriness.¹⁶ We therefore postulated that rapid atrial pacing together with phenylephrine infusion would induce relatively sustained atrial fibrillation for at least 40 min in dogs—a duration likely to be sufficient for testing of agents with potential to convert atrial fibrillation. This report describes a simple canine model using rapid atrial pacing in which atrial fibrillation was sustained for at least 40 min.

Materials and Methods

Approval.

These studies were conducted after approval of the Institutional Animal Care and Use Committee of QTest Labs, LLC. The facility is in compliance with USDA regulations.²⁶

Animals.

Beagle dogs were purchased from Marshall BioResources (North Rose, NY). The animals were housed individually in stainless steel cages from the time of arrival until euthanasia. The cages conformed to standards set forth in the Guide for the Care and Use of Laboratory Animals.²¹ All animals participated in an environmental enrichment program consisting of Kong toys and frequent positive interactions with humans. The room conditions were: temperature, 65 °F to 80 °F; relative humidity, 30% to 70%; and 12:12-h light:dark cycle. All animals received commercial chow (Certified Dog Diet 2021C, Harlan Teklad Diets, Madison, WI) once daily, and water was provided ad libidum in stainless steel containers.

Fourteen healthy, male Beagles, weighing between 8 and 12 kg, were anesthetized intravenously with morphine (1.5 mg/kg; Baxter Health Corporation, Deerfield, IL) followed by α-chloralose (100 mg/kg; Sigma–Aldrich, St Louis, MO).²⁵ Anesthetized dogs were ventilated mechanically with room air at a rate of 8 to 12 breaths per minute and a tidal volume of approximately 20 ml/kg, thus sustaining the arterial partial pressure of CO₂ between 35 and 45 mm Hg, that of O₂ greater than 80 mm Hg, and pH at 7.38 to 7.45. Body temperature was maintained between 37.0 and 37.5 °C. A surgical plane of anesthesia was maintained by infusion of α-chloralose at a rate of 50 mg/kg hourly.

A bipolar pacing catheter (CR Bard, Lowell, MA) was placed in the right atrium by way of the left jugular vein, and the atrium was paced at a rate of 40 Hz with square waves of 20 V and 2 ms duration. A catheter for measuring systemic arterial blood pressure (model SPC-464D, Millar Instruments, Houston, TX) was inserted through the left femoral artery. Lead II electrocardiographic (ECG) data were acquired (EMKA-IOX system, EMKA Technologies, Falls Church, VA) for analysis of rhythm and rate (ECG Auto software, EMKA Technologies).

At the end of the experiment, all animals were euthanized while they were under general anesthesia with sodium pentobarbital (200 mg/kg; Somnasol, Butler Animal Health Supply, Dublin, OH) in accordance with accepted American Veterinary Medical Association guidelines.²

Experimental procedures.

In 2 dogs, the right atrium was paced from 20 to 90 min at various frequencies, but within seconds after cessation of pacing, dogs returned to sinus rhythms (Figure 1). One published report¹² stated that atrial fibrillation can be sustained during acetylcholine infusion; therefore the procedure was repeated after dosing with carbachol, a long-acting parasympathomimetic agent, but again all dogs returned to sinus rhythms seconds after termination of rapid atrial pacing. Based on our assumptions that (1) all atrial fibers were affected by the intravenous exposure to carbachol and therefore an increase in spatial dispersion of refractoriness would not occur, (2) spatial heterogeneity is required for sustenance of atrial fibrillation, and (3) the vagus nerve is distributed variably to regions of the atria and when stimulated should result in spatial inhomogeneities in electrophysiology, the following protocol was used.

Figure 1.

Lead II electrocardiogram (top panel) and arterial blood pressure curve (bottom panel) in the anesthetized dog receiving rapid atrial pacing (40 Hz, 20 V, and 2 ms) only. Atrial fibrillation converted to normal sinus rhythm within seconds after rapid atrial pacing was stopped.

In 12 dogs, infusion of phenylephrine (2 μg/kg per minute) was initiated simultaneously with rapid atrial pacing as described earlier. After 20 min of pacing, the pacemaker was inactivated, and the dogs were monitored for heart rate and rhythm. The surface lead II electrocardiogram and blood pressure were continuously monitored during the experiment. Atrial fibrillation was identified when (1) the ventricular response was rapid and irregularly irregular, (2) P waves were absent, (3) low-frequency, irregular oscillations (f waves) were present, and (4) systemic arterial pressure pulses occurred irregularly, were variable in amplitude, and accompanied a pulse deficit (that is, fewer pulsations than QRS complexes occurred).⁹ The time between cessation of rapid atrial pacing and return to sinus rhythm was measured.

Data analysis.

Electrocardiographic data were analyzed (ECG Auto software, EMKA Technologies) for rhythm and rate. Systemic arterial pressure was obtained for calculation of mean arterial pressure. Statistical analysis was performed by using SigmaStat software (version 2.03, SigmaStat, Point Richmond, CA). Comparison of mean heart rate and arterial blood pressure between before and after phenylephrine infusion were performed by using a 1-tailed t test. Values were considered statistically different when the P value was less than 0.05.

Results

Heart rate and mean systemic arterial pressure before onset of phenylephrine infusion (mean ± 1 SD) were 70 ± 12.1 beats per minute and 88.5 ± 15.9 mm Hg, respectively (Figure 2). After infusion of phenylephrine and immediately after cessation of pacing, heart rate and mean systemic arterial pressure were 61 ± 11.7 beats per minute and 164.3 ± 17.9 mm Hg, respectively (Figure 3). The decrease in heart rate was significant (P < 0.05); the increase in arterial pressure was significant (P < 0.001). Therefore, infusion of phenylephrine increased mean systemic arterial pressure by approximately 75 mm Hg and decreased heart rate by approximately 9 beats per minute.

Figure 2.

Lead II electrocardiogram (top panel) and arterial blood pressure curve (bottom panel) of the anesthetized dog (morphine and α-chloralose) baseline just before the start of rapid atrial pacing.

Figure 3.

Lead II electrocardiogram (top panel) and arterial blood pressure curve (bottom panel) in the anesthetized dog receiving rapid atrial pacing (40 Hz, 20 V, and 2 ms) and phenylephrine infusion (2 μg/kg per minute). Atrial fibrillation characterized by fibrillatory waves that varied in amplitude, shape, and timing occurred after cessation of rapid atrial pacing. The systemic arterial pressure was higher than the baseline level because of the phenylephrine infusion.

In 6 of the 12 dogs, atrial fibrillation persisted for more than 40 min after cessation of pacing. In the other 6 dogs, atrial fibrillation persisted for 18.8, 8.3, 6.5, 5.4, 5.0, and 2.7 s (mean ± 1 SD, 7.78 ± 5.6 s). Therefore 50% of the dogs maintained atrial fibrillation for more than 2400 s, whereas the other 50% showed a rapid (less than 19 s) return to sinus rhythm.

Discussion

This report describes the use of rapid atrial pacing in the presence of increased systemic arterial pressure to produce atrial fibrillation that can be sustained for at least 40 min after cessation of rapid atrial pacing. This duration of atrial fibrillation is likely to be long enough to allow evaluation of both electrophysiologic mechanisms and potential therapeutic interventions.

An important aspect of this study was the anesthesia. For humane reasons and for restraint, anesthesia must be used with this canine model of atrial fibrillation. However most anesthetics profoundly affect autonomic balance, and many (for example, barbiturates, propofol) are potently parasympatholytic. Morphine–chloralose is an anesthetic regimen that maintains autonomic balance nearly identical with that of spontaneous sleep and is thought to result in absence of sensation.¹⁵ Morphine–chloralose anesthesia has been used for years as the agent of choice in cardiovascular studies requiring physiological autonomic control.¹⁵

This study shows that atrial fibrillation can be sustained sufficiently long after cessation of rapid atrial pacing to enable investigation of the antiarrhythmic potential of a drug. Phenylephrine-induced elevation of systemic arterial pressure likely stimulated the high-pressure baroreceptor reflex, through which increased parasympathetic efferent activity slowed the rate of sinoatrial nodal discharge and presumably increased spatial heterogeneity of atrial refractoriness.¹³ If the distribution of parasympathetic fibers were homogeneous to the atrium, then all fibers would have experienced the vagal effect, and all would have responded with similar electrophysiologic changes (that is, no increased heterogeneity), and phenylephrine (like carbachol) would not have increased the likelihood of atrial fibrillation. However atrial fibrillation persisted for at least 40 min after cessation of pacing in 6 dogs that received phenylephrine. Therefore we presume that these 6 dogs had heterogeneous enough distribution of vagal efferents; in contrast, the 6 dogs that converted quickly to sinus rhythm may have lacked the heterogeneity required to sustain the arrhythmia. The amount of heterogeneity in vagal distribution that is necessary to support the electrophysiological heterogeneity that will sustain atrial fibrillation is unknown, but apparently dogs fall into one of 2 subclasses: those with and those without sufficient heterogeneity. This notion is consistent with previous observations⁴ of 2 classes of dogs—sensitive and resistant—with regard to the development of ventricular fibrillation after coronary occlusion during exercise.

As with methoxamine, α₁-adrenergic stimulation is torsadogenic, α₁-adrenergic stimulation precipitates some forms of ventricular arrhythmias after coronary artery occlusion,⁶ and α₁-adrenergic stimulation mediates arrhythmias with thiobarbiturate anesthesia.¹⁷ The relative contributions of α₁-adrenergic stimulation in the absence and presence of systemic arterial pressure are unknown. The predominant arrhythmogenic property of phenylephrine is attributed to its increasing of systemic arterial pressure, which reflexively increased parasympathetic tone, rather than to a direct α₁-adrenergic effect on cellular electrophysiology.^16,23,24

Some models of atrial fibrillation produce durations of fibrillation that are too brief to permit evaluation of either mechanisms or antiarrhythmic potential.¹⁴ In contrast, some models in which fibrillation persists for relatively long periods require either longterm pacing or concomitant atrial injury (as in heart failure or modifications of the Maze procedure).^7,10 Increased susceptibility to atrial fibrillation similar to that of our model can be produced by electrical stimulation of the vagus.¹⁴ However that method is painful to the animal, results in stimulus artifacts that might obfuscate electrocardiographic findings, and does not result in the same physiologic elevation of parasympathetic tone due to increased blood pressure, because delivery of current to the nerve(s) can be variable.¹⁸ Advantages of the present model are that it can be produced quickly, inexpensively, and reliably and that fibrillation is sustained.

Our model has some disadvantages. Elevating systemic arterial pressure is an artificial condition that may itself alter electrophysiology. Because only half of the dogs developed longterm fibrillation—perhaps due to the lack of heterogeneity of vagal distribution—a greater number of animals must be used to achieve a number that provides adequate statistical power for associated studies. In addition, even when fibrillation occurred, it lasted for only about 40 min. Whether the yield would have been higher had greater elevations in the systemic arterial pressure been used is unknown. Conversely, the same arrhythmogenicity may have been attainable with less elevation of pressure but longer periods of rapid atrial pacing. We also do not know whether the mechanism for sustenance of fibrillation mimicked more closely so-called ‘lone’ atrial fibrillation or that associated with left atrial enlargement due to mitral regurgitation and heart failure.^5,19 Despite the many questions that remain to be addressed during refinement of our model, atrial fibrillation was maintained for at least 40 min after cessation of rapid atrial pacing in anesthetized dogs rendered hypertensive due to phenylephrine infusion.