Optimization of Transesophageal Atrial Pacing to Assess Atrial Fibrillation Susceptibility in Mice

Podsumowanie

The present protocol describes the optimization of experimental parameters when using transesophageal atrial pacing to assess atrial fibrillation susceptibility in mice.

Streszczenie

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.

Wprowadzenie

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 phenotypes^1,2,3. 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 protocols^7,8 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.

Protokół

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

1. 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.
   1. 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.
2. For acquired models, conduct pacing after mice achieve physical maturity (~12 weeks of age)¹⁰. As mentioned above, study both sexes.
3. 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.
   1. 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 min^13,14) 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 studies^4,7. 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.
4. Use the optimized model-specific parameters and definition of AF susceptibility for subsequent studies on additional mice.

2. Animal preparation

1. 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).
2. 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.
3. Apply lubricating eye ointment to the eyes to prevent drying.
4. 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.
5. 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

1. Briefly remove the isoflurane mask from the mouse.
2. 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).
   1. Insert to a depth that approximates the distance from the mouth (with neck extended) to just above the xiphoid cartilage.
3. Reposition the isoflurane mask over the mouse's nostrils.
4. Begin data acquisition with continuous recording of ECG lead I using analysis software (see Table of Materials).
5. Adjust the stimulus isolator mode to bipolar. Use the distal-most pair of electrodes during stimulation.
6. 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

1. 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.
2. Adjust the stimulus amplitude to twice TH.

5. Determination of electrophysiologic properties

1. 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

1. 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.).
2. 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 ms^8,10. 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.
3. 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.
4. 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

1. Stop data acquisition.
2. Gently remove catheter and ECG electrodes.
3. Stop anesthesia.
4. Place the anesthetized mouse into a cage and observe for 10 min to ensure recovery.
5. Save the data file. In the case of serial testing, wait for a minimum of 24 h before repeating the pacing procedure.

Wyniki

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 averag...

Dyskusje

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 mice^18,19, and individual genetic models may demonstrate AF inducibility over...

Materiały

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