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Cardiovascular Research 2001 50(3):463-473; doi:10.1016/S0008-6363(01)00264-4
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Copyright © 2001, European Society of Cardiology

Induction of atrial tachycardia and fibrillation in the mouse heart

Hiroko Wakimotoa, Colin T Maguirea, Pramesh Kovoora, Peter E Hammera, Josef Gehrmanna, John K Triedmana,b and Charles I Berula,b,*

aDepartment of Cardiology, Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA
bDepartment of Pediatrics, Harvard Medical School, Boston, MA 02115, USA

* Corresponding author. Tel.: +1-617-355-6432; fax: +1-617-739-9058 berul{at}cardio.tch.harvard.edu

Received 7 August 2000; accepted 31 January 2001


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusion
 References
 
Background: Atrial tachycardia and fibrillation in humans may be partly consequent to vagal stimulation. Induction of fibrillation in the small heart is considered to be impossible due to lack of a critical mass of >100–200 mm2. Even with the recent progression of the technology of in vivo and in vitro mouse electrophysiological studies, few reports describe atrial tachycardia or fibrillation in mice. The purpose of this study was to attempt provocation of atrial tachyarrhythmia in mice using transvenous pacing following cholinergic stimulation. Methods and results: In vivo electrophysiology studies were performed in 14 normal mice. A six-lead ECG was recorded from surface limb leads, and an octapolar electrode catheter was inserted via jugular vein cutdown approach for simultaneous atrial and ventricular endocardial recording and pacing. Atrial tachycardia and fibrillation were inducible in one mouse at baseline electrophysiology study and eleven of fourteen mice after carbamyl choline injection. The mean duration of atrial tachycardia was 126±384 s. The longest episode lasted 35 min and only terminated after atropine injection. Reinduction of atrial tachycardia after administration of atropine was not possible. Conclusion: Despite the small mass of the normal mouse atria, sustained atrial tachycardia and fibrillation can be easily and reproducibly inducible with endocardial pacing after cholinergic agonist administration. This finding may contribute to our understanding of the classical theories of arrhythmogenesis and critical substrates necessary for sustaining microreentrant circuits. The techniques of transcatheter parasympathetic agonist-mediated atrial tachycardia induction may be valuable in further murine electrophysiological studies, especially mutant models with potential atrial arrhythmia phenotypes.

KEYWORDS Arrhythmia (mechanisms); Autonomic nervous system; Supraventr. arrhythmia


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 5 Conclusion
 References
 
Atrial tachycardia, especially atrial fibrillation is one of the most common sustained arrhythmias in humans, leading to significant morbidity, disability, and often results in limited quality of life. Numerous experimental and clinical studies have been devised with a goal of revealing the underlying mechanisms of atrial tachycardia and fibrillation, improving diagnostic testing and risk-stratification, and evaluating potential novel therapies. The experimental induction of atrial fibrillation with continuous infusion of acetylcholine in dog heart–lung preparations was well illustrated by Burn et al. [1]. Since then, many in vivo and in vitro large animal preparations demonstrating the inducibility, maintenance, and termination of atrial tachycardia have been established [2–8]. Atrial tachyarrhythmias are considered to be at least partially consequential to vagal stimulation. The cholinergic discharge increases the vulnerability to atrial arrhythmia through muscarinic receptor-mediated shortening of the atrial action potential duration and refractory period [9–11].

In small animals such as the mouse, it has been widely accepted that induction of fibrillatory tachycardias is impossible due to lack of a critical mass of the heart [12]. The murine atrial surface area is <35 mm2, and therefore it may be more difficult to sustain a micro-reentrant atrial tachyarrhythmia in an intact normal mouse. Studies of arrhythmia inducibility and electrophysiological characterization in mouse models are particularly useful to evaluate arrhythmia mechanisms using molecular and genetic approaches. We hypothesize that, despite the small mass of the normal mouse atria, it provides sufficient substrate at least as the minimal area for initiating and sustaining a reentrant circuit. Utilizing cholinergic stimulation to decrease atrial tissue refractoriness, and thereby shorten wavelength, should conceivably allow for the induction and maintenance of atrial tachycardia circuits. The aim of this report is to show the possibility of sustained atrial tachycardia and fibrillation in the normal mouse heart through an in vivo electrophysiological study, and to demonstrate standardized techniques of atrial arrhythmia induction by parasympathetic pharmacological stimulation.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 5 Conclusion
 References
 
2.1 Mouse preparation
Fourteen normal adult 12-week-old mice (C57BL6 strain, ten males and four females) were fully examined. The average weight was 24.9±3 g. All animal care protocol conformed to the American Association for the Accreditation of Laboratory Animal Care and the Harvard Medical School and Children's Hospital Animal Care and Use Committees. Mice were anesthetized by intraperitoneal administration of pentobarbital (initial dose of 0.033 mg/g, with a supplementary 0.016 mg/g dose as necessary for adequate sedation). Bupivacaine (0.25%) was infiltrated subcutaneously in the right neck for local anesthesia. A tracheotomy was performed as previously described [13,14], and intubation was achieved with a 3/4'' PTFE outer sheath from a 24-gauge intravenous catheter for continuous mechanical ventilation (FiO2=0.21, VT=0.3 cc, respiratory rate 130/min).

The in vivo electrophysiological studies were performed similarly to the protocol as previously reported [13–15]. The surface frontal-plane six-lead ECG was obtained from 25 gauge needle-electrodes placed subcutaneously in each limb. A 1.7 French octapolar catheter with an interelectrode spacing of 0.5 mm (CIBer mouse EP, NuMed, Hopkinton, NY, USA) was inserted via a jugular vein cutdown approach. Using this catheter, simultaneous atrial and ventricular pacing and recording were performed. ECG channels were filtered between 0.5 and 250 Hz. Intracardiac electrograms were filtered between 40 and 400 Hz. Surface ECG and the intracardiac recordings were displayed on an oscilloscope and simultaneously recorded to computer through an analog to digital converter (MacLab Systems, Milford, MA, USA) for detailed analysis and measurement.

2.2 Electrophysiological study protocols
Standard pacing protocols were used to determine the electrophysiologic parameters, including sinus node recovery, atrial, A–V nodal, and ventricular refractory periods and A–V nodal conduction properties [13–15]. Sinus node recovery time (SNRT) was measured between the last paced atrial depolarization and the first sinus return cycle after 15 s of atrial pacing at several pacing cycle lengths (CL). Rate corrected SNRT was defined as the sinus CL subtracted from SNRT. A–V nodal conduction properties were obtained by atrial and ventricular incremental pacing methods until Wenckebach and 2:1 A–V or retrograde ventriculoatrial block. Atrial, A–V, and ventricular effective refractory period (ERP) were analyzed by the extrastimulus method. Each mouse underwent an identical pacing and programmed stimulation protocol. Following completion of all pacing protocols, responses to muscarinic-cholinergic stimulation were studied using an intraperitoneal injection of carbamyl choline (Carbachol, CCH, 50 ng/g). In a pilot study, the pharmacokinetics and dose–response relationships of intravenous and intraperitoneal CCH administration were ascertained [16]. Although intravenous CCH had a low therapeutic index with an unfavorable effective/lethal dose ratio, intraperitoneal CCH injection was safe and clearly able to induce a demonstrable heart rate change with stable hemodynamic conditions. The CCH dosage utilized in the present study, determined by a dose–response curve (data not shown) leads to a 25% decrease of heart rate, considered to be effective muscarinic-cholinergic stimulation. A stable heart rate was observed within 5 min of intraperitoneal CCH injection and was maintained for a minimum of 30 min after CCH injection. The standard pacing and programmed stimulation protocol was then repeated similarly to the baseline status.

2.3 Induction of atrial tachycardia and fibrillation
To induce atrial tachycardia and fibrillation, programmed extrastimulation techniques and burst pacing were utilized. Programmed right atrial and right ventricular double and triple extrastimulation techniques were performed at 150-ms drive cycle length, down to a minimum coupling interval of 10 ms [13]. Right atrial and right ventricular burst pacing was performed as eight 50-ms and four 30-ms cycle length trains episodes repeated several times, up to a maximum 1-min time limit of total stimulation. For comparison of the inducibility in each mouse, programmed extrastimulation techniques and stimulation duration of atrial and ventricular burst pacing were alike in all mice. Reproducibility was defined as greater than one episode of induced atrial tachycardia. The identical burst pacing and stimulation protocols were repeated after carbamyl choline administration (50 ng/g) to attempt atrial tachyarrhythmia induction. This protocol consistently induced atrial tachycardia and fibrillation without adjusting any major technical factors, such as catheter position, medication dosage, or level of anesthesia. To determine the effect of a cholinergic antagonist on the induced atrial tachyarrhythmia, reinduction was attempted by the burst pacing and programmed extrastimulation protocols after intraperitoneal atropine administration (40 µg). Reinduction following atropine was attempted multiple times in mice with consistent inducibility of sustained atrial tachycardia (more than three episodes induced after CCH administration), or atrial tachycardia greater than 10 min in duration.

2.4 Atrial rate analysis of the tachycardia
To characterize the regularity of atrial tachycardia in the mouse heart, computerized atrial rate calculation was performed using the customized software written in MATLAB (The Math Works, Natick, MA, USA) programming language. Low-pass filtering of digitized right atrial intracardiac electrogram signals obtained from the proximal pair of recording electrodes was performed to 150 Hz. Ventricular beats were subtracted, and atrial beats were detected using a threshold and lockout algorithm. Rate analysis of the atrial tachycardia segments was performed to compare tachycardia episodes within each mouse and episodes between mice with induced atrial tachycardia.

2.5 Statistical analysis
All statistical analyses were performed with EXCEL and STATA software. Values are presented as the mean±1 standard deviation. Surface ECG and intracardiac conduction parameters were measured by two independent observers and compiled for statistical interpretation. Differences between groups were tested using a two-tailed Student t-test, analysis of variance (ANOVA) or {chi}2 distribution test, where appropriate. A two-tailed t-test was used in comparisons of electrophysiological data before and after carbamyl choline. A P value of <0.05 was considered significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 5 Conclusion
 References
 
3.1 Influence of carbamyl choline on electrophysiological parameters
Carbamyl choline slowed the mean heart rate, sinus nodal recovery time and corrected SNRT, the A–V Wenckebach and 2:1 A–V block cycle length. Following CCH administration, ventricular conduction, refractoriness, and retrograde ventriculoatrial conduction were not affected (Table 1). The exact point of atrial refractoriness could not be precisely determined in every animal due to pseudopseudofusion of the preceding conducted ventricular electrogram with potential atrial electrogram responses at coupling intervals between 20 and 50 ms. In a subgroup of mice (n=6) in which the atrial effective refractory period (AERP) could be accurately determined, baseline AERP (paced cycle length=200 ms) was 38±7 ms, while AERP (paced CL=200 ms) shortened after CCH injection to 20±5 ms (P=0.012) (Fig. 1).


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  		Table 1 Electrophysiological data summarya

 

Figure 1
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  		Fig. 1 Atrial refractoriness at baseline electrophysiology study (A and B) and after carbamyl choline injection (C and D). In each panel, the upper figure depicts lead I of a surface ECG and the lower one depicts an atrial intracardiac electrogram. The cycle length of the eight basic pacing beats (S1–S1 interval) is 150 ms. (A) The coupling interval of the extrastimulus (S1–S2 interval) is 55 ms and captures the atrium (A2). (B) Extrastimulus coupling interval is shortened to 50 ms and no A2 is induced by S2. After carbamyl choline, in (C) shows that S2 induces A2 at coupling interval of 25 ms, whereas in (D) no A2 is induced by S2 at 20 ms coupling. P, P wave of surface ECG; QRS, QRS complex of surface ECG; A, atrial electrogram; V, ventricular electrogram; S, stimulation artifact; ms, milliseconds; CCH, carbamyl choline.

 
3.2 Induction of atrial tachycardia and fibrillation
Fig. 2 illustrates a typical example of a disorganized appearing atrial tachycardia induced by atrial burst pacing after carbamyl choline injection. This atrial tachyarrhythmia was easily and reproducibly inducible in eleven out of fourteen mice (seven out of ten male mice and all four female mice). Reinducibility varied between two and ten episodes per mouse. There were no differences in tachycardia induction protocols between mice that were reinducible more frequently. Mean duration of all induced atrial tachycardias was 126±384 s (Table 2). Without carbamyl choline, short bursts of atrial tachycardia lasting up to 2 s could be induced by either atrial programmed triple extrastimulation (Fig. 3), or burst pacing methods. However, the majority of mice had atrial tachyarrhythmias inducible only after carbamyl choline injection, and the tachycardia duration was significantly longer following cholinergic stimulation. Atrial tachycardia or fibrillation was provoked with atrial burst pacing in seven mice, programmed atrial stimulation in two mice, ventricular burst pacing in five mice and with programmed ventricular stimulation in one mouse (Table 2). The termination of all induced atrial tachyarrhythmias occurred abruptly as shown in Fig. 4, and spontaneously except one episode, which was abruptly terminated by atropine. All mice that had inducible atrial tachycardia or fibrillation could be reinduced at least one (range 1–9) additional episode using the same pacing protocol, as described above. There were no differences in the electrophysiological characteristics measured, including atrial, A–V nodal, and ventricular conduction and refractoriness, in the animals that had tachycardia reinduced 4–10 times compared with those with tachycardia induced 1–3 times. Using custom rate-analysis software, the episodes of recurrent induced atrial tachycardia showed both similar and different tachycardia cycle lengths within the same animal as well as between mice (Table 3). Only atrial tachycardia episodes greater than 1 s in duration are included in tachycardia cycle length analysis. The longest atrial tachycardia recording was for 35 min, induced by atrial burst pacing and could be terminated only by intraperitoneal atropine (40 µg) injection. Atrial tachycardia was never successfully terminated with burst atrial pacing at attempted coupling intervals of 75–93% of the tachycardia cycle length. Among mice with reliable inducibility of sustained atrial tachycardia or fibrillation, reinduction was attempted in four mice multiple times after intraperitoneal atropine administration (40 µg). None of these four mice that were consistently inducible prior to atropine could be induced following atropine injection using the identical pacing and programmed stimulation protocols.


Figure 2
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  		Fig. 2 Example of sustained atrial tachycardia induced by atrial pacing at 50 ms following carbamyl choline injection. (A) Lead I of a surface ECG. (B) Distal low right atrial bipolar intracardiac electrogram with disorganized fibrillatory activity. (C) High right atrial bipolar intracardiac electrogram, 2–3 mm proximal to distal electrodes, revealing a somewhat more organized atrial electrical activity.

 

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  		Table 2 Inducibility of atrial tachycardia and fibrillationa

 

Figure 3
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  		Fig. 3 Atrial tachycardia induced without carbamyl choline. Atrial tachycardia was initiated by atrial triple extrastimulation (three extrastimulations following eight paced beats at cycle length 100 ms). The ventricular cycle length (R–R) intervals are measured on the lower panel. (A) Lead I of a surface electrocardiogram (ECG). (B) Right ventricular intracardiac electrogram. (C) Right atrial intracardiac electrogram.

 

Figure 4
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  		Fig. 4 Conversion of an unorganized atrial tachycardia to a more organized appearing atrial tachycardia, and redegeneration into an unorganized atrial tachycardia. Atrial tachycardia was induced by 8 s of right ventricular burst pacing. After 3 s from the initiation of arrhythmia, it converted to an intra-atrial reentrant tachycardia (IART) with 4-to-1 atrioventricular (A–V) conduction. One minute later, A–V conduction became irregular, and 91 s after initiation of arrhythmia, atrial organization degenerated. After this episode, there were several conversions between IART and unorganized atrial tachycardia, and the termination of this arrhythmia occurred spontaneously. In each panel, the upper graph is lead I surface ECG, and the lower panel is the intracardiac electrogram. (A) Initiation of atrial tachycardia during atrial pacing. (B) More organized atrial tachycardia with 4-to-1 A–V conduction. (C) Variable AV conduction. (D) Spontaneous termination of atrial tachycardia after 599 s from the initiation of arrhythmia. SCL, sinus cycle length.

 

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  		Table 3 Rate analysis of atrial tachycardia episodesa

 
Although most mice showed a fibrillation-like unorganized tachycardia on the surface ECG, several mice exhibited a more organized atrial tachyarrhythmia, appearing analogous to an intra-atrial macro-reentrant tachycardia (i.e. ‘atrial flutter’). Fig. 4 shows an example of an atrial fibrillation converting to a more organized atrial tachycardia. It started as an atrial fibrillation at the beginning of the arrhythmia, but in 3 s, it converted to a more organized-appearing atrial tachycardia. After 90 s of a stable atrial cycle length of 32 ms, the atrial rhythm again degenerated, periodically converting back to a more organized atrial tachycardia and repeated several times, before finally terminating spontaneously. To characterize the regularity of atrial tachycardia induced in the mouse heart, rate analysis of the atrial intracardiac electrogram was performed. All episodes of induced atrial tachycardia lasting at least 1 s were analyzed (Table 3). Some of the induced atrial tachycardia episodes had visually appearing disorganization, while others had relatively regular cycle length intervals, more consistent with a single reentrant circuit. The A–A interval range during atrial tachycardia confirmed significant variability in cycle length frequency between mice (Fig. 5). The majority of episodes had low cycle length rate variation, but some episodes appeared distinctly more fibrillatory with large A–A interval standard deviation.


Figure 5
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  		Fig. 5 Rate analysis of atrial tachycardia: the left four panels show a representative example of an organized atrial tachycardia and the right side illustrates a typical disorganized fibrillatory atrial tachycardia. In both examples, from the top to bottom are: surface ECG (lead I), intra-atrial electrogram, beat-to-beat rate analysis of 3 s of atrial tachycardia and a histogram of rate bins. On the left, a regular A–A cycle length is illustrated with all intervals falling within a narrow rate range, and on the right, there is a wide range of A–A cycle length intervals.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
5 Conclusion
 References
 
4.1 Mechanisms of atrial tachycardia and fibrillation
This study demonstrates consistent induction of atrial tachycardia and fibrillation in normal mice using an in vivo electrophysiological pacing protocol, facilitated by pharmacologic parasympathetic stimulation. The induction of sustained atrial tachyarrhythmias was attainable and reliably reproducible with endocardial pacing after cholinergic agonist administration. The atrial tachycardia and fibrillation induced in mice was characterized by parasympathetic-agonist mediated induction and abrupt termination, which implies a mechanism favoring reentrant activity rather than enhanced automaticity of atrial myocytes or triggered activity. Cholinergic stimulation may be necessary to promote a shortening of atrial tissue refractoriness, providing a substrate for reentrant atrial tachycardia and fibrillation vulnerability. The complicated atrial structure and surface area may allow for inhomogeneous repolarization and varied conduction velocities, which are both important factors in the initiation and the maintenance of atrial arrhythmias.

The critical mass theory of fibrillation mechanics was initially presented by Garrey in 1914, and this hypothesis went nearly unchallenged for over 60 years [12]. Although some more recent investigations have more precisely defined the minimal area of critical mass [17–19], the impossibility of fibrillation-induction in the small heart has been widely accepted. Garrey proposed that multiple, changing reentrant circuits, modified by refractory tissue areas, caused the irregular activity seen during atrial fibrillation [12]. Moe expanded upon this premise with his initial proposal of the ‘multiple wavelet hypothesis’ [20], predicting that the number of wavelets would be dependent on the atrial mass, the mean duration of the refractory period, and the average conduction velocity. Allessie and colleagues satisfactorily demonstrated multiple wavelets using dog atria, in which they visualized the circus movements of atrial flutter and reported that the localization of the reentrant circuits was highly variable [21,22]. Schuessler et al. examined this critical concept by using a study designed to detect the number and location of reentrant circuit and to relate them to the activation patterns, cycle length, number of wavelets, and duration of atrial fibrillation [11]. They concluded that multiple circuits were present during an induced arrhythmia, but to sustain the fibrillation was the result of a ‘relatively stable single’ reentrant circuit. Gray et al. clearly illustrated examples of incomplete reentry circuits and frequent breakthrough patterns of activity using high-resolution optical mapping in the sheep heart [23]. They suggested that three-dimensional structure of the atria, as well as the complex atrial anatomy and transmural activation, play an important role in the atrial excitation patterns during atrial fibrillation.

4.2 Autonomic modulation of atrial rhythm
Atrial tachycardia and fibrillation is commonly facilitated by cholinergic agonist administration, both experimentally and possibly contributing to some clinical episodes [2,24–26]. The cholinergic discharge following parasympathetic pharmacological stimulation causes shortening of the refractory period of atrial myocytes, mediated by muscarinic receptors, and thereby increases the vulnerability to atrial tachycardia and fibrillation [24,27]. Our present data using the intact mouse model support the finding that atrial refractoriness is shortened by cholinergic stimulation, allowing for provocation of atrial tachyarrhythmias. The mechanism of cholinergic induced atrial tachycardia and fibrillation was well described in several papers using large animal heart models [20–22,28–31]. Suppression of the sinus node and supra-atrioventricular nodal junctional pacemaker induced by cholinergic stimulation may cause the initiation of the tachyarrhythmia [28]. Other studies have shown the participation of multiple pacemakers distributed in the atria [29,30]. Asynchronous recovery of multiple right atrial pacemakers can generate premature depolarizations that initiate a reentrant tachyarrhythmia [9]. Acetylcholine, which shortens wavelength, decreases the tachycardia cycle length and the line of block [21,32].

4.3 Animal models of atrial arrhythmia
Animal models of chronic atrial fibrillation were developed using long term rapid atrial pacing in large animals [7,31,33]. Morillo et al. demonstrated a new model of atrial fibrillation to investigate the pathophysiology of sustained atrial fibrillation using dog atria [7]. Furthermore, Yue et al. investigated the molecular mechanisms of ionic remodeling using the dog chronic atrial fibrillation models [31]. They found that chronic rapid atrial pacing reduced mRNA concentrations of Kv4.3, the {alpha}1c subunit of L-type Ca2+ channels genes, and the {alpha}-subunit of cardiac Na+ channels genes [31]. A chronic atrial fibrillation model in goats was utilized to demonstrate distinct structural changes in the persistently fibrillating atrial myocytes [33]. These observations suggest several potential molecular mechanisms for the electrical remodeling caused by and resulting in long-term atrial fibrillation. These mechanisms include alterations in ion channel receptor density, isoform regulation, and spatial distribution, as well as analogous mechanisms for potential modulation of gap junction protein isoforms.

Large animal models are useful to observe the macro study of atrial arrhythmia, and these studies have greatly contributed to our understanding of the underlying mechanisms of atrial tachycardias. However, to investigate molecular and genetic mechanisms, small animal models of atrial tachyarrhythmias are advantageous and may be of unique value. Previously, only a few papers to our knowledge have reported atrial tachycardia and fibrillation in rats [34] or mice [35,36]. However, no study has clearly described the detailed method of atrial arrhythmia induction or illustrated the actual ECG and atrial electrograms during atrial arrhythmia. Our study aimed to demonstrate the consistent and reproducible provocation of sustained atrial tachycardia and fibrillation in mice, utilizing standardized electrophysiologic techniques.

The mechanism of the induction of atrial tachyarrhythmias by atrial premature depolarization is considered to be due to provoking an abnormally delayed and inhomogeneously conducted atrial activation [25]. The mechanism of how an atrial arrhythmia could be induced by programmed ventricular stimulation is not clear. It is most conceivably related to the collision of atrioventricular node-conducted retrograde atrial activation with sinus node-generated antegrade atrial activation. The role of atrial stretch associated with ventricular pacing may also be important in the genesis of atrial tachyarrhythmias.

The presence of cholinergic stimulation by carbamyl choline suppressed potential focal activity, i.e. pacemaker or triggered activity. Thus, the most likely underlying mechanism of the atrial tachycardia and fibrillation described in this study is functional or anatomical reentry [11,37]. A major challenge in this study has been the precise classification of reentrant atrial arrhythmias in the small mouse heart. After close examination, surface ECG recordings exhibited differential variations in conduction between cycle length oscillations. As illustrated in Fig. 5, the intracardiac electrograms of atrial tachycardia were further characterized by rate analysis. This technique was quantitatively accurate and ensured a reproducible method of measurement. In some cases, even in the presence of cycle length variability, the atrial intracardiac electrogram morphology was relatively constant, despite the fact that the surface ECG often appeared visibly unorganized and irregular. In addition, some mice had regular cycle length intervals, which suggested having a quite stable single reentrant circuit. Finally, there was clearly a significant proportion of atrial tachycardias, including wide A–A cycle length variability without an obvious pattern, and simultaneous differing atrial rates and morphologies at different right atrial electrode sites within a few millimeters.

These studies add to our knowledge regarding the electrophysiological characterization of atrial tachycardia and fibrillation in mice. Despite these new techniques and findings, the electrophysiologic properties of atrial tachyarrhythmias in mice are not yet fully defined. Spatiotemporal analysis by optical mapping techniques may be useful in identifying mechanisms that underlie arrhythmogenic sequences of reentry. This may explain how some unorganized appearing rhythms on a surface ECG appear organized with atrial rate analysis.

As cholinergic stimulation was shown to shorten atrial refractoriness, and thereby wavelength, the difficulty of tachycardia induction without carbamyl choline administration may indicate that wavelength is prohibitively long for reliable induction and maintenance of tachycardia. Furthermore, the failure to terminate atrial tachycardia by atrial burst pacing suggests that the reentrant circuit must have a small excitable gap, or alternatively that multiple reentrant circuits or a non-reentrant mechanism are at work. The present data demonstrates a small heart size with induced atrial tachyarrhythmias, using the technique of widening the potential atrial electrical surface area with shortening of action potential duration and refractoriness. The variation of the atrial action potential duration and refractoriness may also play an important role in this model.

4.4 Limitations
Limitations of the mouse electrophysiology model in general include the fact that induced arrhythmias during electrophysiologic testing may be dissimilar from the mechanisms occurring during spontaneous cardiac arrhythmias. In addition, parasympathetic stimulation with carbamyl choline may affect non-cardiac physiologic functions and systemic muscarinic actions, which might indirectly influence conduction characteristics and arrhythmia susceptibility. Alternate methods of parasympathetic activation are feasible, including direct stimulation of the vagus nerve, intracardiac administration of cholinergic agonists, and ex vivo isolated heart approaches. We utilized each of these approaches, and concluded that intraperitoneal carbamyl choline led to a consistent and reproducible parasympathetic activation with a longer cholinergic effect than direct vagal stimulation and a higher therapeutic index (ED50/LD50) than the intracardiac route. These findings are similar to prior studies in rats in vivo [38] and ex vivo [39]. Future refinements in the acquisition of conduction velocity, atrial refractoriness, and action potential duration data in intact mice may supplement information regarding arrhythmia wavelength and other mechanistic features. Finally, mouse models of atrial tachyarrhythmia and miniaturization of catheter-based in vivo EP study methodologies may not be relevantly extrapolatable to clinical disease pathophysiology or electrophysiologic risk stratification in humans.


   5 Conclusion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusion
References
 
We have provided a consistent method of atrial tachyarrhythmia provocation in mice and further electrophysiological characterization of these induced murine atrial arrhythmias. Despite the small size of the non-pathologic mouse atria, muscarinic stimulation causes shortening of refractoriness, allowing for sustained atrial arrhythmia induction consequently affirming the critical mass hypothesis. In vivo parasympathetic agonist mediated pacing-induced atrial tachycardia susceptibility experiments may play a valuable role in future murine electrophysiology studies, particularly utilizing mutant mouse models that may have a phenotype of potential atrial arrhythmia vulnerability or arrhythmia resistance [40–44].

Time for primary review 31 days.


   Acknowledgements
 
CIB is supported in part by NIH grants K08-HL03607 and P50-HL61036.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusion
 References
 

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