Summary
The present protocol describes the optimization of experimental parameters when using transesophageal atrial pacing to assess atrial fibrillation susceptibility in mice.
Abstract
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.
Introduction
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.
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Protocol
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
- For genetic models, subject mice to biweekly (i.e., every other week) atrial pacing as described below (see step 6.) to determine the optimal period of AF susceptibility.
- Begin biweekly pacing at 8 weeks of age. Use wild-type littermates as controls to reduce variability. Study both sexes, as one may not develop an AF phenotype9.
- For acquired models, conduct pacing after mice achieve physical maturity (~12 weeks of age)10. As mentioned above, study both sexes.
- During these preliminary studies, perform both burst pacing8 (using a fixed pacing cycle length [CL]) and decremental pacing7 (with a progressively shorter pacing CL) to determine the optimal pacing mode. Separate each procedure by a minimum of 24 h.
NOTE: As an increasing number of mice are studied, review the accumulated data to determine the optimal age, sex, and pacing mode that promotes AF in AF susceptible mice but not controls.- Analyze the data using multiple definitions of AF susceptibility (e.g., number of AF episodes8, total AF duration9, AF incidence4, and sustained AF incidence, commonly defined as 10 s11 or 15 s12, and even up to 5 min13,14) as some models may display an AF phenotype for one but not all definitions9.
NOTE:The definition of an AF episode and AF susceptibility differ between published studies4,7. AF episodes8 are commonly defined as rapid atrial activity with an irregularly irregular ventricular response occurring for at least 1s (Figure 1). In addition to AF, atrial pacing may also induce atrial flutter with either a regular or irregular ventricular response.
- Analyze the data using multiple definitions of AF susceptibility (e.g., number of AF episodes8, total AF duration9, AF incidence4, and sustained AF incidence, commonly defined as 10 s11 or 15 s12, and even up to 5 min13,14) as some models may display an AF phenotype for one but not all definitions9.
- Use the optimized model-specific parameters and definition of AF susceptibility for subsequent studies on additional mice.
2. Animal preparation
- Anesthetize the mouse in an induction chamber using 3% isoflurane (see Table of Materials) in 1 L/min of 100% oxygen.
NOTE: Isoflurane is harmful. It may irritate the skin or eye and can cause dizziness, fatigue, and headache, among other central nervous system toxicities. Use in a well-ventilated area with an appropriate scavenging method (e.g., activated carbon canister). - After the loss of the pedal reflex, place the mouse in a supine position on a heating pad designed to maintain body temperature at approximately 37 °C with the hindlimbs taped to the pad surface.
- Apply lubricating eye ointment to the eyes to prevent drying.
- Place an anesthetic mask securely over the mouse's nose. Begin maintenance of anesthesia using 1% isoflurane in 1 L/min of 100% oxygen. Ensure the nostrils are free from obstruction as mice are obligate nose breathers.
- Obtain a surface electrocardiogram (ECG, lead I) by the subcutaneous placement of 27 G ECG needle electrodes (see Table of Materials) connected to a biological amplifier and data acquisition hardware into the forelimbs. Ground the signal by placing a needle electrode into the left hind limb.
3. Catheter placement
- Briefly remove the isoflurane mask from the mouse.
- Insert a 2-F octapolar electrode catheter (electrode width and spacing = 0.5 mm) connected to a stimulator and stimulus isolator (see Table of Materials) into the esophagus (Figure 2).
- Insert to a depth that approximates the distance from the mouth (with neck extended) to just above the xiphoid cartilage.
- Reposition the isoflurane mask over the mouse's nostrils.
- Begin data acquisition with continuous recording of ECG lead I using analysis software (see Table of Materials).
- Adjust the stimulus isolator mode to bipolar. Use the distal-most pair of electrodes during stimulation.
- Properly position the catheter within the esophagus to enable capture. To do so, apply a 1.5 mA stimulus with a pulse width of 2 ms at a CL slightly shorter than the sinus CL (e.g., use a CL of 100 ms if the sinus CL is 120 ms). Carefully position the catheter until consistent atrial capture is obtained.
4. Threshold determination
- To determine the atrial diastolic capture threshold (TH), initiate pacing at 1.5 mA with a pulse width of 2 ms at the CL used for atrial capture. Decrease the stimulus amplitude by 0.05 mA increments until the loss of atrial capture, with subsequent increase until capture.
NOTE: The lowest amplitude at which consistent atrial capture is obtained is the atrial TH. Due to concern for parasympathetic stimulation at high stimulus amplitudes, which is reflected by excessive AV block during pacing with artifactual AF induction9, the maximum acceptable TH is 0.75 mA. If necessary, reposition the catheter to achieve a TH ≤0.75 mA. - Adjust the stimulus amplitude to twice TH.
5. Determination of electrophysiologic properties
- Measure electrophysiologic parameters, including the sinus node recovery time (SNRT), Wenckebach cycle length (WCL), and atrioventricular effective refractory period (AVERP) prior to rapid atrial pacing for AF induction15.
6. Atrial arrhythmia susceptibility
- Perform pacing at twice TH with a pulse width of 2 ms using either burst pacing at different CLs or decremental pacing as determined from initial studies (steps 1.1.-1.4.).
- For burst pacing, pace at an initial CL of 50 ms for 15 s with subsequent trains occurring at CLs of 40 ms, 30 ms, 25 ms, 20 ms, and 15 ms8,10. Pause pacing for 30 s after each pacing train to allow for recovery before proceeding. If AF occurs after a pacing train, wait for 30 s after termination before proceeding with subsequent pacing.
- For decremental pacing, pace at a CL of 40 ms and decrease the CL by 2 ms every 2 s until termination at 20 ms7. Perform pacing trains in triplicate16 or quintuplicate17, with a 30 s pause for recovery following each train. As above, if AF develops, wait for 30 s after termination before proceeding.
NOTE: When optimizing protocol parameters during preliminary experiments (i.e., steps 1.1.-1.5.), perform decremental pacing with five trains. Conduct a post hoc analysis to determine if three or five trains provide the greatest sensitivity. - Terminate the procedure upon 30 s of sinus rhythm following the last pacing train or after a 10 min episode of AF, whichever comes first.
7. Post-procedure
- Stop data acquisition.
- Gently remove catheter and ECG electrodes.
- Stop anesthesia.
- Place the anesthetized mouse into a cage and observe for 10 min to ensure recovery.
- Save the data file. In the case of serial testing, wait for a minimum of 24 h before repeating the pacing procedure.
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Representative Results
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.
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Discussion
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.
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Disclosures
The authors have nothing to disclose.
Acknowledgments
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).
Materials
| Name | Company | Catalog Number | Comments |
| 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 |
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