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>

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D., 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=Ion+channel+and+structural+remodeling+in+obesity-mediated+atrial+fibrillation.">Ion channel and structural remodeling in obesity-mediated atrial fibrillation.</a> Circulation: Arrhythmia and Electrophysiology. 13 (8), 00896 (2020).</li><li>Kato, 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=Spectral+analysis+of+heart+rate+variability+during+isoflurane+anesthesia.">Spectral analysis of heart rate variability during isoflurane anesthesia.</a> Anesthesiology. 77 (4), 669-674 (1992).</li><li>Schmeckpeper, 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=Abstract+11402:+Targeting+RyR2+to+suppress+ventricular+arrhythmias+and+improve+left+ventricular+function+in+chronic+ischemic+heart+disease.">Abstract 11402: Targeting RyR2 to suppress ventricular arrhythmias and improve left ventricular function in chronic ischemic heart disease.</a> Circulation. 144, 11402 (2021).</li><li>Kim, 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=Abstract+B-PO01-017:+RyR2+hyperactivity+promotes+susceptibility+to+ventricular+tachycardia+in+structural+heart+disease.">Abstract B-PO01-017: RyR2 hyperactivity promotes susceptibility to ventricular tachycardia in structural heart disease.</a> Heart Rhythm. 18, 57 (2021).</li></ol>

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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 averag...

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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. ...

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2.5K Views.  Vanderbilt University School of Medicine. The present protocol describes the optimization of experimental parameters when using transesophageal atrial pacing to assess atrial fibrillation susceptibility in mice.

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

Growth Assays to Assess Polyglutamine Toxicity in Yeast

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's disease, and Huntington's disease 1. Huntington's disease (HD) is caused by the abnormal expansion of a polyglutamine (polyQ) region within the protein huntingtin. The polyQ-expanded huntingtin protein attains an aberrant conformation (i.e. it misfolds) and causes cellular toxicity 2. At least eight further neurodegenerative diseases are caused by polyQ-expansions, including the Spinocerebellar Ataxias and Kennedy&#x2019;s disease 3.
The model organism yeast has facilitated significant insights into the cellular and molecular basis of polyQ-toxicity, including the impact of intra- and inter-molecular factors of polyQ-toxicity, and the identification of cellular pathways that are impaired in cells expressing polyQ-expansion proteins 3-8. Importantly, many aspects of polyQ-toxicity that were found in yeast were reproduced in other experimental systems and to some extent in samples from HD patients, thus demonstrating the significance of the yeast model for the discovery of basic mechanisms underpinning polyQ-toxicity. 
A direct and relatively simple way to determine polyQ-toxicity in yeast is to measure growth defects of yeast cells expressing polyQ-expansion proteins. This manuscript describes three complementary experimental approaches to determine polyQ-toxicity in yeast by measuring the growth of yeast cells expressing polyQ-expansion proteins. The first two experimental approaches monitor yeast growth on plates, the third approach monitors the growth of liquid yeast cultures using the BioscreenC instrument. 
Furthermore, this manuscript describes experimental difficulties that can occur when handling yeast polyQ models and outlines strategies that will help to avoid or minimize these difficulties. The protocols described here can be used to identify and to characterize genetic pathways and small molecules that modulate polyQ-toxicity. Moreover, the described assays may serve as templates for accurate analyses of the toxicity caused by other disease-associated misfolded proteins in yeast models.

This manuscript describes three complementary protocols for assessing the toxicity of polyglutamine (polyQ)-expansion proteins in the yeast Saccharomyces cerevisiae. These protocols can easily be modified to monitor the toxicity of other misfolded proteins in yeast.

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca2+ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).1 Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca2+ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca2+-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 &mu;M Ca2+. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca2+ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca2+ and Ca2+ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca2+ transient measurements, performed in these isolated myocytes.

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

High-resolution Optical Mapping of the Mouse Sino-atrial Node

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The development of novel pharmacological therapies to cure these patients relies on the thorough understanding of both normal physiology and pathophysiology of the SAN. Among the studies of cardiac pacemaking, the mouse SAN is widely used due to its feasibility for modifications in the expression of different genes that encode SAN ion channels or calcium handling proteins. Emerging evidence from electrophysiological and histological studies has also proved the representativeness and similarity of the mouse SAN structure and functions to larger mammals, including the presence of specialized conduction pathways from the SAN to the atrium and a complex pacemakers' hierarchy within the SAN. Recently, the technique of optical mapping has greatly facilitated the exploration and investigation of the origin of excitation and conduction within and from the mouse SAN, which in turn has extended the understanding of the SAN and benefited clinical treatments of SAN dysfunction associated diseases. In this manuscript, we have described in detail how to perform the optical mapping of the mouse SAN from the intact, Langendorff-perfused heart and from the isolated atrial preparation. This protocol is a useful tool to enhance the understanding of mouse SAN physiology and pathophysiology.

Here, we present a protocol for optical mapping of electrical activity from the mouse right atrium and especially the sino-atrial node, at a high spatial and temporal resolution.

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...