The present protocol describes the optimization of experimental parameters when using transesophageal atrial pacing to assess atrial fibrillation susceptibility in mice.
Mouse models of genetic and acquired risk factors for atrial fibrillation (AF) have proven valuable in investigating the molecular determinants of AF. Programmed electrical stimulation can be performed using transesophageal atrial pacing as a survival procedure, thus enabling serial testing in the same animal. However, numerous pacing protocols exist, which complicates the reproducibility. The present protocol aims to provide a standardized strategy to develop model-specific experimental parameters to improve reproducibility between studies. Preliminary studies are performed to optimize the experimental methods for the specific model under investigation, including age at the time of the study, sex, and parameters of the pacing protocol (e.g., mode of pacing and definition of AF susceptibility). Importantly, care is taken to avoid high stimulus energies, as this can cause stimulation of the ganglionic plexi with inadvertent parasympathetic activation, manifested by exaggerated atrioventricular (AV) block during pacing and often associated with artifactual AF induction. Animals demonstrating this complication must be excluded from the analysis.
Atrial fibrillation (AF) represents a final common pathway for multiple acquired and genetic risk factors. For studies investigating the pathophysiologic mechanisms of the AF substrate, mouse models are advantageous given the ease of genetic manipulation and the fact that, in general, they reproduce the AF susceptibility observed in humans for different clinical phenotypes1,2,3. However, mice rarely develop spontaneous AF4, necessitating the use of provocative atrial pacing studies.
Programmed electrical stimulation (PES) can be performed to assess murine atrial electrophysiology and AF susceptibility using either intracardiac5 or transesophageal6 pacing. While the transesophageal approach is particularly advantageous as a survival procedure, its use is complicated by the numerous published experimental protocols7,8 and sources of variability that can hinder reproducibility9. Moreover, limited reported protocol comparisons make selecting an appropriate pacing protocol challenging.
The current protocol aims to utilize a systematic strategy to develop model-specific transesophageal PES methods for assessing murine AF susceptibility in order to increase reproducibility. Importantly, initial pilot studies are performed to optimize the pacing protocol by accounting for age, sex, and pacing mode variability, with pacing designed to minimize inadvertent parasympathetic stimulation that can confound results9.
This procedure was approved by the Vanderbilt Institutional Animal Care and Use Committee and is consistent with the Guide for the Care and Use of Laboratory Animals. The protocol was developed using both genetic9 and acquired10 (e.g., hypertension) mouse models of AF susceptibility. The operator was blinded to the phenotype of the mouse under study.
1. Animal selection
2. Animal preparation
3. Catheter placement
4. Threshold determination
5. Determination of electrophysiologic properties
6. Atrial arrhythmia susceptibility
7. Post-procedure
Transesophageal atrial pacing studies assess the electrophysiologic properties of the SA and AV nodes by determining the SNRT and AVERP, as well as AF susceptibility6 (Figure 1). ECG recording enables measurements of P wave duration, PR interval, QRS duration, and QT/QTc intervals. Continuous recording of the ECG during rapid atrial pacing can provide the following measurements of AF vulnerability: the number of episodes induced during the study, cumulative and average duration of the episodes, and the number of sustained AF episodes. Episodes of excessive AV block during pacing can demonstrate periods of pacing-induced parasympathetic stimulation (Figure 3), signifying that the associated AF is an artifact of this phenomenon rather than the pathophysiology of the model itself9.
Figure 1: Representative results of atrial pacing. Surface ECG recordings depicting (A) sinus rhythm and (B) atrial fibrillation after rapid atrial pacing. The pacing rate exceeds Wenckebach CL, resulting in the loss of 1:1 AV nodal conduction during pacing. The baseline artifact is related to mouse respiration. Please click here to view a larger version of this figure.
Figure 2: Visual representation of the transesophageal catheter and its proximity to the ganglionic plexi. (A) A photograph depicting the 2-F octapolar catheter. (B) Depiction of the catheter's proximity to the posterior left atrial ganglionic plexi. Please click here to view a larger version of this figure.
Figure 3: Representative results of excessive AV block during rapid atrial pacing. Surface ECG recordings demonstrating atrial paced rhythm with (A) and without (B) excessive AV block that can occur during atrial pacing, especially during pacing with higher stimulus intensity and at short CLs. Red arrows denote QRS complexes. Please click here to view a larger version of this figure.
Transesophageal atrial pacing not only allows serial studies in the same animal, but its duration is typically shorter than intracardiac studies (~20 min), thus minimizing anesthetic use and its effects on electrophysiologic parameters.
It is critical to optimize the methods initially for each individual mouse model. Aging increases AF inducibility in normal mice18,19, and individual genetic models may demonstrate AF inducibility over a limited period of time. Performing pilot studies every other week can determine an age window during which the AF phenotype mouse is inducible but control mice are not. Sex can be a determining factor, as either one or both sexes can display inducible AF9. In addition, specific mice can show AF susceptibility in response to only one type of pacing mode, whereas others demonstrate AF susceptibility to a different mode or to multiple modes9.
During rapid atrial pacing, mice may experience excessive AV block that is often coincident with AF induction. This phenomenon is caused by inadvertent stimulation of the ganglionic plexi located on the posterior left atrium, resulting in parasympathetic activation9. Significant AV block is defined as ventricular bradycardia that lasts ≥10% of a single pacing train and is most frequently encountered when pacing with high stimulus intensities and at short pacing CLs. This type of arrhythmia induction increases the incidence of AF in control mice and causes greater arrhythmia variability within an experimental group. Given these contaminating features, animals that experience AF under these conditions must be excluded from the analysis.
Should profound AV block occur during pacing despite TH ≤0.75 mA, it is reasonable to reduce the pacing amplitude to 1.5x TH7. Furthermore, if an AF phenotype is not observed during preliminary experiments, it is conceivable to reattempt using 10 ms as the lowest pacing CL16. If an AF phenotype is not observed at 12 weeks of age for an acquired model, consider biweekly preliminary studies to explore the effects of increasing phenotype maturity20.
A limitation of this approach is the use of isoflurane anesthesia. Isoflurane is known to suppress autonomic function21, and this effect cannot be ruled out despite a relatively short exposure. This protocol represents the first detailed report of an optimized strategy to develop transesophageal PES methods in mice. While this study focuses on AF susceptibility, future applications of this protocol could be used to assess ventricular arrhythmias22,23.
The authors have nothing to disclose.
Figure 2 was created with BioRender.com. This work was supported by grants from the National Heart, Lung, and Blood Institute at the National Institutes of Health (HL096844 and HL133127); the American Heart Association (2160035, 18SFRN34230125 and 903918 [MBM]); and the National Center for Advancing Translational Sciences of the National Institute of Health (UL1 TR000445).
27 G ECG electrodes | ADInstruments | MLA1204 | |
2-F octapolar electrode catheter | NuMED | CIBercath | |
Activated carbon canister | VetEquip | 931401 | |
Analysis software | ADInstruments | LabChart v8.1.13 | |
Biological amplifier | ADInstruments | FE231 | |
Data acquisition hardware | ADInstruments | PowerLab 26T | |
Eye ointment | MWI Veterinary | NC1886507 | |
Heating pad | Braintree Scientific | DPIP | |
Isoflurane | Piramal | 66794-017-25 | |
Stimulator | Bloom Associates | DTU-210 | |
Stimulus Isolator | World Precision Instruments | Model A365 |