Name: optimization of transesophageal atrial pacing to assess atrial fibrillation susceptibility in mice
Uploaded: 2022-06-29T15:00:00.000+00:00
Duration: 8 min 5 s
Description: Watch this Scientific Journal Video about Optimization of Transesophageal Atrial Pacing to Assess Atrial Fibrillation Susceptibility in Mice at JoVE.com

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).

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
<ol>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>For acquired models, conduct pacing after mice achieve physical maturity (~12 weeks of age)10. As mentioned above, study both sexes.</li>
	<li>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.
	<ol>
		<li>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&#160;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.</li>
	</ol>
	</li>
	<li>Use the optimized model-specific parameters and definition of AF susceptibility for subsequent studies on additional mice.</li>
</ol>
2. Animal preparation
<ol>
	<li>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).</li>
	<li>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 &#176;C with the hindlimbs taped to the pad surface.</li>
	<li>Apply lubricating eye ointment to the eyes to prevent drying.</li>
	<li>Place an&#160;anesthetic mask securely over the mouse&#39;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.</li>
	<li>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.</li>
</ol>
3. Catheter placement
<ol>
	<li>Briefly remove the isoflurane mask from the mouse.</li>
	<li>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).
	<ol>
		<li>Insert to a depth that approximates the distance from the mouth (with neck extended) to just above the xiphoid cartilage.</li>
	</ol>
	</li>
	<li>Reposition the isoflurane mask over the mouse&#39;s nostrils.</li>
	<li>Begin data acquisition with continuous recording of ECG lead I using analysis software (see Table of Materials).</li>
	<li>Adjust the stimulus isolator mode to bipolar. Use the distal-most pair of electrodes during stimulation.</li>
	<li>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.</li>
</ol>
4. Threshold determination
<ol>
	<li>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 &#8804;0.75 mA.</li>
	<li>Adjust the stimulus amplitude to twice TH.</li>
</ol>
5. Determination of electrophysiologic properties
<ol>
	<li>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.</li>
</ol>
6. Atrial arrhythmia susceptibility
<ol>
	<li>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.).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
7. Post-procedure
<ol>
	<li>Stop data acquisition.</li>
	<li>Gently remove catheter and ECG electrodes.</li>
	<li>Stop anesthesia.</li>
	<li>Place the anesthetized mouse into a cage and observe for 10 min to ensure recovery.</li>
	<li>Save the data file. In the case of serial testing, wait for a minimum of 24 h before repeating the pacing procedure.</li>
</ol>

<ol>
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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+atrial+fibrillation+in+mice+by+rapid+transesophageal+atrial+pacing.">Induction of atrial fibrillation in mice by rapid transesophageal atrial pacing.</a> Basic Research in Cardiology. 97 (6), 452-460 (2002).</li><li>Verheule, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+vulnerability+to+atrial+fibrillation+in+transgenic+mice+with+selective+atrial+fibrosis+caused+by+overexpression+of+TGF-beta1.">Increased vulnerability to atrial fibrillation in transgenic mice with selective atrial fibrosis caused by overexpression of TGF-beta1.</a> Circulation Research. 94 (11), 1458-1465 (2004).</li><li>Faggioni, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+spontaneous+ca+elevations+prevents+atrial+fibrillation+in+calsequestrin+2-null+hearts.">Suppression of spontaneous ca elevations prevents atrial fibrillation in calsequestrin 2-null hearts.</a> Circulation: Arrhythmia and Electrophysiology. 7 (2), 313-320 (2014).</li><li>Murphy, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+transesophageal+atrial+pacing+in+mice+to+detect+atrial+fibrillation.">Optimizing transesophageal atrial pacing in mice to detect atrial fibrillation.</a> American Journal of Physiology - Heart and Circulatory Physiology. 332 (1), 36-43 (2022).</li><li>Prinsen, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+reactive+isolevuglandins+promote+atrial+fibrillation+caused+by+hypertension.">Highly reactive isolevuglandins promote atrial fibrillation caused by hypertension.</a> JACC: Basic to Translational Science. 5 (6), 602-615 (2020).</li><li>Aschar-Sobbi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+atrial+arrhythmia+susceptibility+induced+by+intense+endurance+exercise+in+mice+requires+TNF&#945;.">Increased atrial arrhythmia susceptibility induced by intense endurance exercise in mice requires TNF&#945;.</a> Nature Communications. 6, 6018 (2015).</li><li>Bruegmann, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+termination+of+atrial+fibrillation+in+mice.">Optogenetic termination of atrial fibrillation in mice.</a> Cardiovascular Research. 114 (5), 713-723 (2017).</li><li>Matsushita, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-1&#946;+plays+an+important+role+in+pressure+overload-induced+atrial+fibrillation+in+mice.">IL-1&#946; plays an important role in pressure overload-induced atrial fibrillation in mice.</a> Biological and Pharmaceutical Bulletin. 42 (4), 543-546 (2019).</li><li>Sato, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+overexpression+of+perilipin+2+induces+atrial+steatosis,+connexin+43+remodeling,+and+atrial+fibrillation+in+aged+mice.">Cardiac overexpression of perilipin 2 induces atrial steatosis, connexin 43 remodeling, and atrial fibrillation in aged mice.</a> American Journal of Physiology - Endocrinology and Metabolism. 317 (6), 1193-1204 (2019).</li><li>Li, N., Wehrens, X. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrial+fibrillation+in+aging+and+frail+mice.">Atrial fibrillation in aging and frail mice.</a> Circulation: Arrhythmia and Electrophysiology. 14 (9), 01077 (2021).</li><li>Luo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+atrial+histopathological+and+electrophysiological+changes+in+a+mouse+model+of+aging.">Characterization of atrial histopathological and electrophysiological changes in a mouse model of aging.</a> International Journal of Molecular Medicine. 31 (1), 138-146 (2013).</li><li>McCauley, M. 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The authors have nothing to disclose.

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 &#8805;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 &#8804;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.

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.

<table><tbody><tr><td>27 G ECG electrodes</td><td>ADInstruments</td><td>MLA1204</td><td></td></tr><tr><td>2-F octapolar electrode catheter</td><td>NuMED</td><td>CIBercath</td><td></td></tr><tr><td>Activated carbon canister</td><td>VetEquip</td><td>931401</td><td></td></tr><tr><td>Analysis software</td><td>ADInstruments</td><td>LabChart v8.1.13</td><td></td></tr><tr><td>Biological amplifier</td><td>ADInstruments</td><td>FE231</td><td></td></tr><tr><td>Data acquisition hardware</td><td>ADInstruments</td><td>PowerLab 26T</td><td></td></tr><tr><td>Eye ointment</td><td>MWI Veterinary</td><td>NC1886507</td><td></td></tr><tr><td>Heating pad</td><td>Braintree Scientific</td><td>DPIP</td><td></td></tr><tr><td>Isoflurane</td><td>Piramal</td><td>66794-017-25</td><td></td></tr><tr><td>Stimulator</td><td>Bloom Associates</td><td>DTU-210</td><td></td></tr><tr><td>Stimulus Isolator</td><td>World Precision Instruments</td><td>Model A365</td><td></td></tr></tbody></table>

optimization of 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.

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.
<img alt="Electrocardiogram comparison: sinus rhythm vs atrial fibrillation; graph depicts heart electrical activity." class="xfigimg" src="/files/ftp_upload/64168/64168fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64168/64168fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Rat brain study; ganglionic plexi detection setup, electrode close-up, neuroscience diagram." class="xfigimg" src="/files/ftp_upload/64168/64168fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64168/64168fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Atrial and AV block rhythm analysis; electrocardiogram charts; cardiovascular study research." class="xfigimg" src="/files/ftp_upload/64168/64168fig03.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64168/64168fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Optimization of Transesophageal Atrial Pacing to Assess Atrial Fibrillation Susceptibility in Mice at JoVE.com

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

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. ...

optimization-transesophageal-atrial-pacing-to-assess-atrial

Nanyang Technological University

Research

JoVE Journal

Biology

2.6K Views.  Vanderbilt University School of Medicine. Geprogrammeerde elektrische stimulatie met behulp van transesofageale atriale pacing kan elektrofysiologische eigenschappen en gevoeligheid voor atriumfibrilleren bij muizen bepalen. Hier hebben we een gestandaardiseerde procedure beschreven voor protocolontwikkeling die de reproduceerbaarheid kan optimaliseren. De transesofageale benadering voor atriale pacing is typisch een overlevingsprocedure, waardoor seriële tests bij hetzelfde dier mogelijk zijn.Belangrijke componenten voor pro...

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

Programmed Electrical Stimulation in Mice

Genetically-modified mice have emerged as a preferable animal model to study the molecular mechanisms underlying conduction abnormalities, atrial and ventricular arrhythmias, and sudden cardiac death.1 Intracardiac pacing studies can be performed in mice using a 1.1F octapolar catheter inserted into the jugular vein, and advanced into the right atrium and ventricle. Here, we illustrate the steps involved in performing programmed electrical stimulation in mice. Surface ECG and intracardiac electrograms are recorded simultaneously in the atria, atrioventricular junction, and ventricular myocardium, whereas intracardiac pacing of the atrium is performed using an external stimulator. Thus, programmed electrical stimulation in mice provides unique opportunities to explore molecular mechanisms underlying conduction defects and cardiac arrhythmias.

Programmed electrical stimulation provides the ability to determine conduction properties of the heart, and the possibility to induce and terminate cardiac arrhythmias using various pacing protocols. Using a transvenous catheter, intracardiac electrogram recordings can be obtained in mice following programmed electrical stimulation protocols to identify arrhythmogenic substrates.

Genetically-modified mice have emerged as a preferable animal model to study the molecular mechanisms underlying conduction abnormalities, atrial and ...

Medicine

Ambulatory ECG Recording in Mice

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although the surface ECG is utilized for short-term measurements of waveform intervals, it is not practical for long-term studies of heart rate variability or the capture of rare episodes of arrhythmias. Implantable ECG telemeters offer the advantages of simple surgical implantation, long-term recording of electrograms in ambulatory mice, and scalability with simultaneous recordings of multiple animals. Here, we present a step-by-step guide to the implantation of telemeters for ambulatory ECG recording in mice. Careful adherence to aseptic technique is required for favorable survival results with the possibility of implantation and recording from weeks to months. Thus, implantable ECG telemetry is a valuable tool for detection of critical information on cardiac electrophysiology in ambulatory animal models such as the mouse. 

Telemetric ECG has emerged as an essential tool in evaluating animal models for cardiac arrhythmias and sudden cardiac death. Here, we present a stepwise guide to telemetric ECG recordings for application in long-term ambulatory ECG monitoring in mice.

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although ...

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

Since its invention in the late 19th century, the Langendorff ex vivo heart perfusion system continues to be a relevant tool for studying a broad spectrum of physiological, biochemical, morphological, and pharmacological parameters in centrally denervated hearts. Here, we describe a setup for the modulation of the intracardiac autonomic nervous system and the assessment of its influence on basic electrophysiology, arrhythmogenesis, and cyclic adenosine monophosphate (cAMP) dynamics. The intracardiac autonomic nervous system is modulated by the mechanical dissection of atrial fat pads-in which murine ganglia are located mainly&#8212;or by the usage of global as well as targeted pharmacological interventions. An octapolar electrophysiological catheter is introduced into the right atrium and the right ventricle, and epicardial-placed multi-electrode arrays (MEA) for high-resolution mapping are used to determine cardiac electrophysiology and arrhythmogenesis. F&#246;rster resonance energy transfer (FRET) imaging is performed for the real-time monitoring of cAMP levels in different cardiac regions. Neuromorphology is studied by means of antibody-based staining of whole hearts using neuronal markers to guide the identification and modulation of specific targets of the intracardiac autonomic nervous system in the performed studies. The ex vivo Langendorff setup allows for a high number of reproducible experiments in a short time. Nevertheless, the partly open nature of the setup (e.g., during MEA measurements) makes constant temperature control difficult and should be kept to a minimum. This described method makes it possible to analyze and modulate the intracardiac autonomic nervous system in decentralized hearts.

Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

Here, we present a protocol for the modulation of the intracardiac autonomic nervous system and the assessment of its influence on basic electrophysiology, arrhythmogenesis, and cAMP dynamics using an ex vivo Langendorff setup.

Since its invention in the late 19th century, the Langendorff ex vivo heart perfusion system continues to be a relevant tool for studying a broad spectrum ...

Analyzing Long-Term Electrocardiography Recordings to Detect Arrhythmias in Mice

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ineffective in many patients. To improve patient care, novel and innovative therapeutic concepts causally targeting arrhythmia mechanisms are needed. To study the complex pathophysiology of arrhythmias, suitable animal models are necessary, and mice have been proven to be ideal model species to evaluate the genetic impact on arrhythmias, to investigate fundamental molecular and cellular mechanisms, and to identify potential therapeutic targets.
Implantable telemetry devices are among the most powerful tools available to study electrophysiology in mice, allowing continuous ECG recording over a period of several months in freely moving, awake mice. However, due to the huge number of data points (&gt;1 million QRS complexes per day), analysis of telemetry data remains challenging. This article describes a step-by-step approach to analyze ECGs and to detect arrhythmias in long-term telemetry recordings using the software, Ponemah, with its analysis modules, ECG Pro and Data Insights, developed by Data Sciences International (DSI). To analyze basic ECG parameters, such as heart rate, P wave duration, PR interval, QRS interval, or QT duration, an automated attribute analysis was performed using Ponemah to identify P, Q, and T waves within individually adjusted windows around detected R waves. 
Results were then manually reviewed, allowing adjustment of individual annotations. The output from the attribute-based analysis and the pattern recognition analysis was then used by the Data Insights module to detect arrhythmias. This module allows an automatic screening for individually defined arrhythmias within the recording, followed by a manual review of suspected arrhythmia episodes. The article briefly discusses challenges in recording and detecting ECG signals, suggests strategies to improve data quality, and provides representative recordings of arrhythmias detected in mice using the approach described above.

Here we present a step-by-step protocol for a semiautomated approach to analyze murine long-term electrocardiography (ECG) data for basic ECG parameters and common arrhythmias. Data are obtained by implantable telemetry transmitters in living and awake mice and analyzed using Ponemah and its analysis modules.

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ...

Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or bradycardia. Reliable identification of cardiac pacemaking defects requires the measurement of intrinsic heart rates by largely preventing the influence of the autonomic nervous system, which can mask rate deficits. Traditional methods to analyze intrinsic cardiac pacemaker function include drug-induced autonomic blockade to measure in vivo heart rates, isolated heart recordings to measure intrinsic heart rates, and sinoatrial strip or single-cell patch-clamp recordings of sinoatrial pacemaker cells to measure spontaneous action potential firing rates. However, these more traditional techniques can be technically challenging and difficult to perform. Here, we present a new methodology to measure intrinsic cardiac firing rate by performing microelectrode array (MEA) recordings of whole-mount sinoatrial node preparations from mice. MEAs are composed of multiple microelectrodes arranged in a grid-like pattern for recording in vitro extracellular field potentials. The method described herein has the combined advantage of being relatively faster, simpler, and more precise than previous approaches for recording intrinsic heart rates, while also allowing easy pharmacological interrogation.

This protocol aims to describe a new methodology to measure intrinsic cardiac firing rate using microelectrode array recording of the whole sinoatrial node tissue to identify pacemaking defects in mice. Pharmacological agents can also be introduced in this method to study their effects on intrinsic pacemaking.

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or ...

Sterile Pericarditis in Aachener Minipigs As a Model for Atrial Myopathy and Atrial Fibrillation

Atrial fibrillation (AF) is the most common arrhythmia caused by structural remodeling of the atria, also called atrial myopathy. Current therapies only target the electrical abnormalities and not the underlying atrial myopathy. For the development of novel therapies, a reproducible large animal model of atrial myopathy is necessary. This paper presents a model of sterile pericarditis-induced atrial myopathy in Aachener minipigs. Sterile pericarditis was induced by spraying sterile talcum and leaving a layer of sterile gauze over the atrial epicardial surface. This led to inflammation and fibrosis, two crucial components of the pathophysiology of atrial myopathy, making the atria susceptible to the induction of AF. Two pacemaker electrodes were positioned epicardially on each atrium and connected to two pacemakers from different manufacturers. This strategy allowed for repeated non-invasive atrial programmed stimulation to determine the inducibility of AF at specified time points after surgery. Different protocols to test AF inducibility were used. The advantages of this model are its clinical relevance, with AF inducibility and the rapid induction of inflammation and fibrosis-both present in atrial myopathy-and its reproducibility. The model will be useful in the development of novel therapies targeting atrial myopathy and AF.

We describe a sterile pericarditis model in minipigs to study atrial myopathy and atrial fibrillation (AF). We present surgical and anesthetic techniques, strategies for vascular access, and a protocol to study the inducibility of AF.

Atrial fibrillation (AF) is the most common arrhythmia caused by structural remodeling of the atria, also called atrial myopathy. Current therapies only ...

Transesophageal Atrial Burst Pacing for Atrial Fibrillation Induction in Rats

Animal studies have brought important insights into our understanding regarding atrial fibrillation (AF) pathophysiology and therapeutic management. Reentry, one of the main mechanisms involved in AF pathogenesis, requires a certain mass of myocardial tissue in order to occur. Due to the small size of the atria, rodents have long been considered 'resistant' to AF. Although spontaneous AF has been shown to occur in rats, long-term follow-up (up to 50 weeks) is required for the arrhythmia to occur in those models. The present work describes an experimental protocol of transesophageal atrial burst pacing for rapid and efficient induction of AF in rats. The protocol can be successfully used in rats with healthy or remodeled hearts, in the presence of a wide variety of risk factors, allowing the study of AF pathophysiology, identification of novel therapeutic targets, and evaluation of novel prophylactic and/or therapeutic strategies.

The present work describes an experimental protocol of transesophageal atrial burst pacing for efficient induction of atrial fibrillation (AF) in rats. The protocol can be used in rats with healthy or remodeled hearts, allowing the study of AF pathophysiology, identification of novel therapeutic targets, and evaluation of new therapeutic strategies.

Animal studies have brought important insights into our understanding regarding atrial fibrillation (AF) pathophysiology and therapeutic management. ...

Translational Rabbit Model of Chronic Cardiac Pacing

Animal models of cardiac pacing are beneficial for testing novel devices, studying the pathophysiology of artificially paced heart rhythms, and studying arrhythmia-induced cardiomyopathies and subsequent heart failure. Currently, only a few such models are available, and they mostly require extensive resources. We report a new experimental cardiac pacing model in small mammals with the potential to study arrhythmia-induced heart failure.
In six New Zealand white rabbits (mean weight: 3.5 kg) under general inhalational anesthesia the jugular region was dissected and a single pacing lead was inserted via the right external jugular vein. Using fluoroscopic guidance, the lead was further advanced to the right ventricular apex, where it was stabilized using passive fixation. A cardiac pacemaker was then connected and buried in a subcutaneous pocket.
The pacemaker implantation was successful with good healing; the rabbit anatomy is favorable for the lead placement. During 6 months of follow-up with intermittent pacing, the mean sensed myocardial potential was 6.3 mV (min: 2.8 mV, max: 12 mV), and the mean lead impedance measured was 744 &#937; (min: 370 &#937;, max: 1014 &#937;). The pacing threshold was initially 0.8 V &#177; 0.2 V and stayed stable during the follow-up.
This present study is the first to present successful transvenous cardiac pacing in a small-mammal model. Despite the size and tissue fragility, human-size instrumentation with adjustments can safely be used for chronic cardiac pacing, and thus, this innovative model is suitable for studying the development of arrhythmia-induced cardiomyopathy and consequent heart failure pathophysiology.

We present a minimally invasive leporine model of long-term cardiac pacing that can be utilized for artificial pacing and heart failure development in preclinical studies.

Animal models of cardiac pacing are beneficial for testing novel devices, studying the pathophysiology of artificially paced heart rhythms, and studying ...

Optimizing Pulmonary Artery Banding for Consistent Right Heart Failure Induction

Rat Model of Right-Sided Cardiac Remodeling and Arrhythmia Using Pulmonary Artery Banding

Clinical conditions, including chronic obstructive pulmonary disease or pulmonary arterial hypertension (PAH), can lead to chronic right ventricle pressure overload and progressive right heart failure (RHF). RHF can be identified by right-sided cardiac hypertrophy and dilation associated with abnormal myocardial function affecting the RV and the right atrium (RA). We recently demonstrated that severe RHF is accompanied by an increased risk of atrial inflammation, atrial fibrosis, and atrial fibrillation (AF), the most common type of cardiac arrhythmia (CA). Recent studies have shown that RV and RA inflammation plays an important role in the arrhythmogenesis of CA, including AF. However, the impact of inflammation in the development of CA and AF in RHF is poorly described.
Experimental models of RHF are required to better understand the association between right-sided myocardial inflammation and CA. The rat model of monocrotaline (MCT)-induced pulmonary hypertension (PH) is well-established to provoke RHF. However, MCT triggers severe pneumo-toxicity and pulmonary inflammation. Hence, MCT-induced RHF does not help to distinguish whether the subsequent myocardial inflammation originates from the RHF per se or circulating inflammatory signals secreted by the injured lung.
In this article, a mechanical method involving pulmonary artery trunk banding (PAB) was used to provoke right-sided cardiac arrhythmogenesis. The PAB consists of performing a permanent suture of the pulmonary artery trunk for 3 weeks. Such an approach generates increased right-sided pressure overload. At D21 post-PAB, the suture results in hypertrophied, dilated, and inflamed RV and RA. The PAB-induced RHF is also accompanied by vulnerability to ventricular and atrial arrhythmias, including AF.

Right heart failure (RHF) is characterized by right-sided cardiac dilation and hypertrophy, leading to ventricular and atrial malfunction. Cardiopulmonary conditions associated with RHF are accompanied by an increased risk for cardiac arrhythmias. This article describes a standardized model of pulmonary artery banding-induced RHF associated with enhanced ventricular and atrial arrhythmogenesis.

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

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