Name: isolation of atrial myocytes from adult mice
Uploaded: 2019-07-25T14:00:00.000+00:00
Duration: 8 min 34 s
Description: Watch this Scientific Journal Video about Isolation of Atrial Myocytes from Adult Mice at JoVE.com

This work is supported by operating grants from the Canadian Institutes of Health Research (MOP 93718, 142486) and the Heart and Stroke Foundation of Canada to R.A. Rose.&#160; H.J. Jansen is the recipient of a Killam Postdoctoral Fellowship.

All animal procedures were approved by The University of Calgary Animal Care and Use Committee and were conducted in accordance with the guidelines of the Canadian Council on Animal Care. The atrial myocyte isolation, images, and representative results described below were obtained from a 15 week-old male wildtype C57Bl/6 mouse. We routinely use this protocol to isolate atrial myocytes from wildtype mice17,18, mice carrying genetic mutations19,20 and mouse models of disease such as chronic hypertension6,14. The protocol can be used similarly for male or female mice. We also utilized a similar version of this isolation procedure to isolate sinoatrial node myocytes from the mouse heart17,21,22,23. A flowchart of this experimental protocol is located in Figure 1.
1. Preparation of Stock Solutions and Equipment
<ol>
	<li>Prepare 1 dissecting dish by adding silicone elastomer according to the manufacturer&#8217;s instructions. Add enough silicone elastomer compound to cover the bottom of a 10 cm Petri dish to a depth of 1 cm. Allow to cure and then insert 6 insect pins into the dish. 
	NOTE: This silicone dissecting dish can be reused for months and stored at room temperature.</li>
	<li>Prepare 3 fire-polished Pasteur pipettes with an opening of 1 mm (small bore), 3 mm (medium bore), or 5 mm (large bore) in diameter as shown in Figure 2A. To make these pipettes, score a Pasteur pipette and then snap along the score mark to produce an opening that is slightly larger than the desired bore size. Use a metal file to smooth the surface and then fire-polish this opening using an open flame. 
	NOTE: This will produce a smooth, fire-polished edge with an opening of the desired diameter. It is important that the opening is free of cracks and rough surfaces. These fire-polished pipettes can be stored at room temperature and reused for months.</li>
	<li>Prepare stock solutions for Tyrode&#8217;s pH 6.9 solution and Tyrode&#8217;s pH 7.4 solution as listed in Table 1. Also prepare 10 mL each of 1 M MgCl2, 1 M CaCl2, and 100 mM CaCl2. Use ultrapure water for all solutions and store at 4 &#176;C for up to 2 months.</li>
	<li>Prepare 1 L of modified Kraft-Bru&#776;he (KB) solution as indicated in Table 2. Use ultrapure water. Divide the solution into 20 mL aliquots and store at -20 &#176;C for up to 2 months.</li>
</ol>
2. Preparation of Solutions and Isolation Setup for Atrial Myocyte Isolation
<ol>
	<li>Prepare 50 mL of a modified Tyrode&#8217;s pH 7.4 solution as described in Table 3 in a 125 mL Erlenmeyer flask, using 1 M CaCl2 and 1 M MgCl2 stock solutions. Place the Erlenmeyer flask in a 35 &#176;C water bath until use, as shown in Figure 2B.</li>
	<li>Prepare the modified Tyrode&#8217;s pH 6.9 solution as described in Table 4 in a 50 mL tube, using the 100 mM CaCl2 stock solution. Aliquot 2.5 mL of this solution into each of three 5 mL round bottom tubes. Place these tubes in a wire rack placed in a 35 &#176;C water bath until use, as shown in Figure 2B.</li>
	<li>Prepare the enzyme solution as described in Table 5 in a 14 mL round bottom tube. To make the protease solution, add 1 mg of protease per 100 &#956;L of ultrapure water. Place the tube containing this enzyme solution in the wire rack and incubate in a 35 &#176;C water bath until use.</li>
	<li>Thaw one aliquot of modified KB solution in a 35 &#176;C water bath. Aliquot 2.5 mL of KB solution into each of three 5 mL round bottom tubes and 2.5 mL into a 14 mL round bottom tube. Place these tubes in the wire rack and incubate in a 35 &#176;C water bath until use, as shown in Figure 2B.</li>
	<li>Lay out the dissecting plate, dissecting tools, a Pasteur pipette, and the fire-polished pipettes as shown in Figure 2B.</li>
</ol>
3. Dissection of Mouse Atrial Appendage(s)
<ol>
	<li>Inject the mouse with 0.2 mL of heparin (10 000 USP U/10 mL) via intraperitoneal injection and wait 5 min for absorption.</li>
	<li>Place the mouse in an induction chamber and anesthetize by isoflurane inhalation (3-4%). Isoflurane and oxygen are delivered using an anesthetic machine and waste anesthetic gas is scavenged. Once the mouse is anesthetized, and does not exhibit a toe pinch reflex, euthanize the mouse by rapid cervical dislocation. Place the mouse on a paper towel or a cork board and tape the paws down to hold the mouse in place.</li>
	<li>Wet the chest of the mouse with 70% ethanol. Remove the fur and skin covering the chest using curved scissors. Next, use rat tooth forceps to lift the sternum and then cut the diaphragm along the edge of the ribs. Remove the entire rib cage using curved scissors to expose the heart.</li>
	<li>To remove the atrial appendage (right or left), gently lift the appendage using fine dissecting forceps and cut it out with spring scissors. Immediately transfer the atrial appendage to a silicone coated dissecting dish containing 20 mL of the warmed modified Tyrode&#8217;s pH 7.4 solution described in step 2.1.</li>
	<li>Place one dissecting pin at the top and one pin at the bottom of the opening of the atrial appendage. Using a pasture pipette, flush the atria with the warmed modified Tyrode&#8217;s pH 7.4 solution to remove blood. Open the atrial appendage by cutting along the top and bottom edge of the atrial appendage. Next, pin the corners of the atrial appendage down to create a flat, rectangular piece of tissue, as shown in Figure 3A.</li>
</ol>
4. Isolation of Atrial Myocytes
NOTE: Steps in this section are all performed at 35 &#176;C, with tubes submerged in a 35 &#176;C water bath. Be careful when transferring tissue strips between round bottom tubes to ensure that only the tissue (and not the solution) is transferred between tubes.
<ol>
	<li>Cut the atrial appendage into approximately 8-10 equal sized strips (approximately 0.7 mm in width) using spring scissors and fine forceps. An example of the strips of atrial tissue is shown in Figure 3B. Note that the strips contract once they are cut free from the main piece of tissue. Using the small bore fire-polished pipette, transfer the tissue strips into the first tube containing warmed modified Tyrode&#8217;s pH 6.9 solution described in step 2.2. Wait 5 min.</li>
	<li>Wash the tissue strips by transferring them to the second and then the third round bottom tube containing modified Tyrode&#8217;s pH 6.9 solution prepared in step 2.2 using the medium bore fire-polished pipette.
	<ol>
		<li>To wash the tissue strips, cap the 5 mL round bottom tube and gently invert the tube 3 times. Let the tissue strips settle to the bottom of the tube before transferring the tissue strips to the next tube using the medium bore fire-polished pipette.</li>
	</ol>
	</li>
	<li>Transfer the tissue strips into the enzyme solution described in step 2.3 using a medium bore fire-polished pipette and incubate for 30 min. Swirl the tube every 3-5 min to prevent the tissue strips from adhering together. 
	NOTE: At the beginning of the enzymatic digestion, tissue strips settle quickly following swirling. At approximately 20 min of digestion, the tissue strips begin to float in the enzymatic solution following swirling. During this time, the atrial tissue strips also change in appearance from pale pink to white as they are digested.</li>
	<li>After enzymatic digestion, perform three washes using 2.5 mL of KB solution in the 5 mL round bottom tubes prepared in step 2.4. For each wash, gently invert the tube 3 times before moving the tissue to the next tube using the medium bore fire-polished pipette. Following the final wash, transfer the strips into the 14 mL round bottom tube containing 2.5 mL of KB solution. Wait 5 min.</li>
	<li>Gently triturate the tissue for 7.5 min using the wide bore fire-polished pipette. This will mechanically dissociate the tissue strips and yield a cloudy solution filled with individual atrial myocytes. 
	NOTE: During trituration, the tissue becomes white and the solution becomes cloudy. The force of trituration, achieved by altering both the frequency and velocity of expelling the tissue strips from the wide bore fire-polished pipette, should be tailored to the individual isolation. If trituration is too gentle, the cell yield will be low, while trituration that is too harsh will yield many dead cells. Avoid bubbles while triturating.</li>
	<li>Fill the 14 mL round bottom tube containing the triturated tissue strips with KB solution to a final volume of 7-10 mL depending on the desired density of cells for experimental use. Place this tube at room temperature for 1 h. Following this incubation period, cells can be used for a variety of experiments for up to 7 h. Cells can also be stored at 4 &#176;C for up to 7 h.</li>
</ol>

<ol>
	<li>Bartos, D. C., Grandi, E., Ripplinger, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+Channels+in+the+Heart.">Ion Channels in the Heart.</a> Comprehensive Physiology. 5 (3), 1423-1464 (2015).</li><li>Heijman, J., Voigt, N., Nattel, S., Dobrev, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+electrophysiology+of+atrial+fibrillation+initiation,+maintenance,+and+progression.">Cellular and molecular electrophysiology of atrial fibrillation initiation, maintenance, and progression.</a> Circulation Research. 114 (9), 1483-1499 (2014).</li><li>Jalife, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+persistent+atrial+fibrillation.">Mechanisms of persistent atrial fibrillation.</a> Current Opinion in Cardiology. 29 (1), 20-27 (2014).</li><li>Nattel, S., Maguy, A., Le Bouter, S., Yeh, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arrhythmogenic+ion-channel+remodeling+in+the+heart:+heart+failure,+myocardial+infarction,+and+atrial+fibrillation.">Arrhythmogenic ion-channel remodeling in the heart: heart failure, myocardial infarction, and atrial fibrillation.</a> Physiological Reviews. 87 (2), 425-456 (2007).</li><li>Jansen, H. 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+structure,+function+and+arrhythmogenesis+in+aged+and+frail+mice.">Atrial structure, function and arrhythmogenesis in aged and frail mice.</a> Scientific Reports. 7, 44336 (2017).</li><li>Jansen, H. 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=Distinct+patterns+of+atrial+electrical+and+structural+remodeling+in+angiotensin+II+mediated+atrial+fibrillation.">Distinct patterns of atrial electrical and structural remodeling in angiotensin II mediated atrial fibrillation.</a> Journal of Molecular and Cellular Cardiology. 124, 12-25 (2018).</li><li>Nerbonne, J. M., Kass, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+physiology+of+cardiac+repolarization.">Molecular physiology of cardiac repolarization.</a> Physiological Reviews. 85 (4), 1205-1253 (2005).</li><li>Grant, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+ion+channels.">Cardiac ion channels.</a> Circulalation: Arrhythmia and Electrophysiology. 2 (2), 185-194 (2009).</li><li>Schmitt, N., Grunnet, M., Olesen, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+potassium+channel+subtypes:+new+roles+in+repolarization+and+arrhythmia.">Cardiac potassium channel subtypes: new roles in repolarization and arrhythmia.</a> Physiological Reviews. 94 (2), 609-653 (2014).</li><li>Lomax, A. E., Kondo, C. S., Giles, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+time-+and+voltage-dependent+K++currents+in+myocytes+from+left+and+right+atria+of+adult+mice.">Comparison of time- and voltage-dependent K+ currents in myocytes from left and right atria of adult mice.</a> American Journal of Physiology Heart and Circulatory Physiology. 285 (5), H1837-H1848 (2003).</li><li>Li, D., Zhang, L., Kneller, J., Nattel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+ionic+mechanism+for+repolarization+differences+between+canine+right+and+left+atrium.">Potential ionic mechanism for repolarization differences between canine right and left atrium.</a> Circ Res. 88 (11), 1168-1175 (2001).</li><li>Wirth, K. J., Knobloch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+dofetilide,+amiodarone,+and+class+lc+drugs+on+left+and+right+atrial+refractoriness+and+left+atrial+vulnerability+in+pigs.">Differential effects of dofetilide, amiodarone, and class lc drugs on left and right atrial refractoriness and left atrial vulnerability in pigs.</a> Naunyn Schmiedebergs Archives of Pharmacology. 363 (2), 166-174 (2001).</li><li>Qi, A., Yeung-Lai-Wah, J. A., Xiao, J., Kerr, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+differences+in+rabbit+atrial+repolarization:+importance+of+transient+outward+current.">Regional differences in rabbit atrial repolarization: importance of transient outward current.</a> American Journal of Physiology. 266 (2 Pt 2), H643-H649 (1994).</li><li>Jansen, H. 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=NPR-C+(Natriuretic+Peptide+Receptor-C)+Modulates+the+Progression+of+Angiotensin+II-Mediated+Atrial+Fibrillation+and+Atrial+Remodeling+in+Mice.">NPR-C (Natriuretic Peptide Receptor-C) Modulates the Progression of Angiotensin II-Mediated Atrial Fibrillation and Atrial Remodeling in Mice.</a> Circulation: Arrhythmia and Electrophysiology. 12 (1), e006863 (2019).</li><li>Mangoni, M. E., Nargeot, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genesis+and+regulation+of+the+heart+automaticity.">Genesis and regulation of the heart automaticity.</a> Physiological Reviews. 88 (3), 919-982 (2008).</li><li>Voigt, N., Pearman, C. M., Dobrev, D., Dibb, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+isolating+atrial+cells+from+large+mammals+and+humans.">Methods for isolating atrial cells from large mammals and humans.</a> Journal of Molecular and Cellular Cardiology. 86, 187-198 (2015).</li><li>Springer, 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=The+natriuretic+peptides+BNP+and+CNP+increase+heart+rate+and+electrical+conduction+by+stimulating+ionic+currents+in+the+sinoatrial+node+and+atrial+myocardium+following+activation+of+guanylyl+cyclase-linked+natriuretic+peptide+receptors.">The natriuretic peptides BNP and CNP increase heart rate and electrical conduction by stimulating ionic currents in the sinoatrial node and atrial myocardium following activation of guanylyl cyclase-linked natriuretic peptide receptors.</a> Journal of Molecular and Cellular Cardiology. 52 (5), 1122-1134 (2012).</li><li>Hua, R., Adamczyk, A., Robbins, C., Ray, G., Rose, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+patterns+of+constitutive+phosphodiesterase+activity+in+mouse+sinoatrial+node+and+atrial+myocardium.">Distinct patterns of constitutive phosphodiesterase activity in mouse sinoatrial node and atrial myocardium.</a> PLoS One. 7 (10), e47652 (2012).</li><li>Egom, E. E., 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=Impaired+sinoatrial+node+function+and+increased+susceptibility+to+atrial+fibrillation+in+mice+lacking+natriuretic+peptide+receptor+C.">Impaired sinoatrial node function and increased susceptibility to atrial fibrillation in mice lacking natriuretic peptide receptor C.</a> Journal of Physiology. 593 (5), 1127-1146 (2015).</li><li>Hua, 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=Effects+of+Wild-Type+and+Mutant+Forms+of+Atrial+Natriuretic+Peptide+on+Atrial+Electrophysiology+and+Arrhythmogenesis.">Effects of Wild-Type and Mutant Forms of Atrial Natriuretic Peptide on Atrial Electrophysiology and Arrhythmogenesis.</a> Circulation: Arrhythmia and Electrophysiology. 8 (5), 1240-1254 (2015).</li><li>Krishnaswamy, P. 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=Altered+parasympathetic+nervous+system+regulation+of+the+sinoatrial+node+in+Akita+diabetic+mice.">Altered parasympathetic nervous system regulation of the sinoatrial node in Akita diabetic mice.</a> Journal of Molecular and Cellular Cardiology. 82, 125-135 (2015).</li><li>Mackasey, 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=Natriuretic+peptide+receptor+C+(NPR-C)+protects+against+angiotensin+II+mediated+sinoatrial+disease+in+mice.">Natriuretic peptide receptor C (NPR-C) protects against angiotensin II mediated sinoatrial disease in mice.</a> JACC Basic to Translational Science. , (2018).</li><li>Azer, J., Hua, R., Vella, K., Rose, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natriuretic+peptides+regulate+heart+rate+and+sinoatrial+node+function+by+activating+multiple+natriuretic+peptide+receptors.">Natriuretic peptides regulate heart rate and sinoatrial node function by activating multiple natriuretic peptide receptors.</a> Journal of Molecular and Cellular Cardiology. 53 (5), 715-724 (2012).</li></ol>

The authors have to nothing to disclose.

Our lab routinely uses this protocol to isolate mouse atrial myocytes for use in patch-clamp experiments in order to investigate the effects of different forms of cardiovascular disease, genetic mutations, or pharmacological compounds on atrial myocyte electrophysiology. Although highly reproducible, the quality of the data obtained from the isolated atrial myocytes depends on the quality of the isolation. In addition, reintroduction of calcium following the atrial myocyte isolation will result in cell death for a population of isolated myocytes due to the calcium paradox16. Accordingly, isolating viable, high-quality atrial myocytes using this approach requires practice and optimization at multiple points throughout the isolation. Once optimized, it is estimated that between 70-90% of the total atrial myocytes isolated using this approach will be both calcium tolerant and rod shaped. The steps requiring the most practice and optimization are discussed below.
The speed and efficiency of the dissection will have downstream effects on the quality of the isolated cells. It is important to take time to ensure all blood is removed from the atrial tissue and that tissue strips are cut to a similar size. It should take approximately 5 min to remove the atrial appendage, cut the tissue into strips, and transfer the tissue strips into the first tube of modified Tyrode&#8217;s pH 6.9 solution. However, if this step takes too long, the quality of the tissue may be compromised.
It is also important that tissue strips are cut to a uniform size within an isolation and between hearts. If tissue strips are too large or too small, or if they are not uniform within an isolation, this can cause problems during both enzymatic digestion and trituration. This is because small strips will be more thoroughly digested and large strips will be under digested. It is equally important to consider the genotype and disease setting being studied as the size of the atrial appendage can vary between animals. For example, hypertrophic hearts have larger atrial appendages compared to healthy hearts, and therefore the experimenter can cut more strips in hypertrophic hearts compared to normal sized hearts. Accordingly, optimizing the size of the cut tissue strips and applying these dimensions to each individual atrial appendage will greatly improve the reproducibility of myocyte isolations between experimental conditions.
The delicate balance between the enzymatic digestion and mechanical dissociation is key to a successful atrial myocyte isolation using this protocol. If tissue is not adequately swirled during the enzymatic&#160;digestion the individual tissue strips will tend to clump and stick together, which will limit the effectiveness of the enzymatic digestion. If agitated too frequently or vigorously, this can damage the atrial tissue, which will result in the isolation of nonviable cells. Mechanical dissociation of isolated atrial myocytes from tissue strips during trituration is the most critical step to practice and optimize using this approach to isolate atrial myocytes. If trituration is too gentle, cell yield will be low. On the other hand, if trituration is too harsh, then an abundance of non-viable myocytes will be isolated, and the quality of data obtained during patch-clamp experiments will be jeopardized. In addition, the composition of the atria can affect the isolation. For example, if tissue is fibrotic, the enzymatic digestion and the trituration steps may need to be modified. It is therefore important to take the time to develop the skills required to obtain high quality cells during trituration that can be used for patch-clamp experiments.
As with all experimental techniques there are limitations. This technique requires practice in order to reproducibly isolate viable, high quality myocytes, which in turn will impact the feasibility of any experiments to be performed using these myocytes. This approach is also terminal and atrial myocytes isolated using this approach can be used on the day of the isolation only. Our lab uses the cells within 6-7 h of isolation.
This approach of isolating atrial cardiomyocytes has several applications. For example, this approach can be modified to isolate atrial myocytes (as well as cardiac fibroblasts) from other species including human atrial tissue biopsies. In addition, a benefit to using this chunk method for atrial myocyte isolation (in contrast to retrograde perfusion of the heart) is that it can be modified to isolate cardiomyocytes from other regions of the heart, such as the sinoatrial node or other specific regions of the atrial myocardium, or encompass the entire supraventricular region of the heart. Our lab uses atrial cardiomyocytes for patch-clamp experiments to measure action potentials and ionic currents, although this approach need not be limited to this technique. For example, isolated myocytes can be used to investigate alterations in calcium transients and contractility in a variety of experimental settings. Atrial cardiomyocytes can also be used in immunofluorescence studies to study the location of proteins or structures of interest. Accordingly, this approach is highly versatile with many possible applications.

The atria, which are the thin walled, low pressure chambers of the heart that receive blood from the superior and inferior vena cavae as well as the pulmonary veins, are integral in normal cardiac physiology. Like other regions of the heart, the atria contain a number of cell types, including cardiomyocytes, fibroblasts, endothelial cells, vascular smooth muscle cells, and others. Atrial myocytes are electrically excitable cells that play an essential role in the conduction of electrical signals through the heart, thereby ensuring proper atrial contraction during each heart beat1. Electrical dysfunction in the atria can lead to a number of atrial specific arrhythmias such as atrial flutter and atrial fibrillation2,3. These are highly prevalent, yet poorly understood, atrial arrhythmias that lead to significant morbidity and mortality. Atrial fibrillation can occur in association with genetic mutations, in association with aging or in the setting of acquired forms of heart disease, including hypertension, heart failure and diabetes2,4,5,6. These conditions can alter the electrical properties of atrial myocytes which can create a substrate that increases the prevalence of arrhythmogenesis1,2.
Normal electrical function in the atria, as well as atrial arrhythmogenesis, are importantly affected by the morphology of the action potential (AP) produced in atrial myocytes. The atrial AP is generated from the activity of a number of ionic currents, including the sodium current (INa, carried by NaV&#173;1.5 channels), the L-type calcium current (ICa,L, carried by Cav1.2 and CaV1.3 channels), and several potassium currents including the ultra-rapid delayed rectifier potassium current (IKur, carried by KV1.5 channels), the transient outward potassium current (Ito, carried by KV&#173;4.2 and KV4.3 channels), a steady state potassium current (IKss, carried by KV2.1 channels), and the inward rectifier potassium current (IK1, carried by Kir2.1 channels)1,7,8. Although they do not play a major role in the mouse atria, the rapid and slow components of the delayed rectifier K+ current (IKr and IKs) also contribute to AP repolarization in some species7. Alterations in one or more of these ionic currents can significantly alter the electrical properties of atrial myocytes, which can lead to atrial arrhythmias. For example, a reduction in INa can slow conduction velocity across the atria by reducing the AP upstroke velocity. On the other hand, a reduction in repolarizing potassium currents or an increase in either ICa,L or the late INa can result in the development of afterdepolarizations that can trigger spontaneous activity in the atria1,2,9.
It is important to recognize that there are differences in AP morphology in different parts of the atrial myocardium that are likely due to differences in the expression or regulation of these underlying ion channels. For example, differences in AP duration between the right and left atria in association with differences in Ito current density have been well described10,11,12,13. Also, we have recently demonstrated that there are distinct patterns of electrical remodeling in the right and left atria of mice with chronic hypertension6,14. The right atrial posterior wall also contains the sinoatrial node, which has its own distinct patterns of AP morphology and firing patterns15. The distinct properties of myocytes in each of these different parts of the atria can be investigated in detail using isolated myocytes from each of these regions.
There are different approaches that can be used to isolate atrial myocytes for patch-clamp electrophysiology studies16. One possibility is to use a retrograde perfusion approach where the heart is cannulated via the aorta for the delivery of enzymes. While this is a viable approach, it can produce variability in atrial myocyte quality due to inconsistencies in perfusion of the atria. We have adopted a &#8216;chunk&#8217; digestion approach for the isolation of atrial myocytes that eliminates the need for retrograde perfusion of the heart. Our approach uses a combination of enzymatic digestion and mechanical dissociation of atrial tissue that consistently and reliably yields large numbers of isolated atrial myocytes that are suitable for patch-clamp studies. While we describe our approach here using atrial appendage tissue, the approach can be used on any region of the atrial myocardium (i.e., right or left atrial appendages, free walls, posterior walls) that the investigator chooses. This approach is ideal for studies of atrial myocyte electrophysiology in genetically modified mice, in mouse models of cardiovascular disease, or for studying the effects of pharmacological compounds5,6,17,18,19.

<table><tbody><tr><td>1, 2-Bis(2-Aminophenoxy)ethane-N, N, N&#039;, N&#039;-tetraacetic acid 98%</td><td>Sigma</td><td>A4926-1G</td><td></td></tr><tr><td>Adenosine 5&#039;-triphosphate disodium salt hydrate BioXtra, &lt;u&gt;&gt;&lt;/u&gt; 99%, from microbial</td><td>Sigma</td><td>A7699-1G</td><td></td></tr><tr><td>Adenosine 5&#039;-triphosphate magnesium salt &lt;u&gt;&gt;&lt;/u&gt; 95%, bacterial</td><td>Sigma</td><td>A9187-1G</td><td></td></tr><tr><td>Amphocetericin B from Streptomyces sp. ~80% (HPLC), powder</td><td>Sigma</td><td>A4888-500 MG</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A3059-50G</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma</td><td>223506-500G</td><td></td></tr><tr><td>Cesium chloride ReagentPlus, 99.9%</td><td>Sigma</td><td>289329-100G</td><td></td></tr><tr><td>Cesium hydroxide monohydrate &lt;u&gt;&gt;&lt;/u&gt; 99.5% trace metals basis</td><td>Sigma</td><td>562505-1KG</td><td></td></tr><tr><td>Collagenase Type 2</td><td>Worthington Biochemical Corporation</td><td>LS004176</td><td></td></tr><tr><td>Creatine anhydrous</td><td>Sigma</td><td>C0780</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma</td><td>G7021-1KG</td><td></td></tr><tr><td>DL-Aspartic acid potassium salt</td><td>Sigma</td><td>A2025-100G</td><td></td></tr><tr><td>Elastase suspension</td><td>Worthington Biochemical Corporation</td><td>LS002279</td><td></td></tr><tr><td>Ethylene glycol-bis(2-amino-ethylether)-N,N,N&#039;,N&#039;-tetraacetic acid &lt;u&gt;&gt;&lt;/u&gt;97.0%</td><td>Sigma</td><td>E4378-25G</td><td></td></tr><tr><td>Guanosine 5&#039;-triphosphate sodium salt hydrate &lt;u&gt;&gt;&lt;/u&gt; 95% (HPLC), powder</td><td>Sigma</td><td>G8877-250MG</td><td></td></tr><tr><td>Heparin 10 000 USP U/10 mL</td><td>SANDOZ</td><td>10750</td><td></td></tr><tr><td>HEPES &lt;u&gt;&gt;&lt;/u&gt; 99.5% (titration)</td><td>Sigma</td><td>H3375-500G</td><td></td></tr><tr><td>L-Glutamic acid potassium salt monohydrate &lt;u&gt;&gt;&lt;/u&gt; 99% (HPLC), powder</td><td>Sigma</td><td>G1501-500G</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Sigma</td><td>M2643-500G</td><td></td></tr><tr><td>Nifedipine &lt;u&gt;&gt;&lt;/u&gt; 98% (HPLC), powder</td><td>Sigma</td><td>N7634-1G</td><td></td></tr><tr><td>Phosphocreatine disodium salt hydrate enzymatic, approx 98%</td><td>Sigma</td><td>P7936-5G</td><td></td></tr><tr><td>Potassium chloride ACS reagent, 99.0-100.5%</td><td>Sigma</td><td>P3911-500G</td><td></td></tr><tr><td>Potassium hydroxide</td><td>EM Science</td><td>PX1480-1</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>EMD</td><td>PX1565-1</td><td></td></tr><tr><td>Protease from Streptomyces griseus, type XIV, &lt;u&gt;&gt;&lt;/u&gt;3.5 units/mg solid, powder</td><td>Sigma</td><td>P5147-1G</td><td></td></tr><tr><td>Sodium chloride ACS reagent, &lt;u&gt;&gt;&lt;/u&gt; 99.0%</td><td>Sigma</td><td>S9888-2.5KG</td><td></td></tr><tr><td>Sodium hydroxide, pellets, 97+%, A.C.S. reagent</td><td>Sigma</td><td>221465-500G</td><td></td></tr><tr><td>Sylgard 184 silicone elastomer kit</td><td>World Precision Instruments Inc</td><td>SYLG184</td><td></td></tr><tr><td>Taurine</td><td>Sigma</td><td>T0625-100G</td><td></td></tr><tr><td>Tetraethylammonium chloride &lt;u&gt;&gt;&lt;/u&gt; 98% (titration)</td><td>Sigma</td><td>T2265-100G</td><td></td></tr></tbody></table>

isolation of atrial myocytes from adult mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

The atrial myocytes isolated using this protocol can be used to characterize the electrophysiological properties of these cells using the patch-clamp technique. Aliquots of atrial myocytes in KB solution can be added to the recording chamber of a standard patch-clamp apparatus and superfused with solutions appropriate for the kind of recording the experimentalist wishes to perform. Atrial myocytes isolated using this protocol are best used for electrophysiological studies within 6-7 h of isolation. Representative patch-clamp data from our laboratory is presented below.
Figure 4 illustrates examples of isolated atrial myocytes from normal mice prepared using the protocol above. Isolated atrial myocytes are typically on the order of 100 &#181;m in length and 10 &#181;m in width with clear striations. The capacitance of isolated atrial myocytes is typically 40-70 pF.
Figure 5A illustrates an example of an atrial myocyte AP recorded using the perforated patch-clamp technique in current clamp mode, as we have described previously6,19,20. Summary data illustrating typical atrial myocyte AP parameters are provided in Table 6. Specifically, we present summary data for measurements of resting membrane potential (RMP), maximum upstroke velocity (Vmax), overshoot (OS) and AP duration at 50% (APD50), 70% (APD70) and 90% (APD90) repolarization time (Table 6). APs can also be recorded in the whole cell configuration14. The superfusion and pipette solutions for recording APs are available in Table 7 and Table 8.
Figure 5B illustrates a representative family of Na+ currents (INa) recorded in the whole cell configuration of the patch-clamp technique. These currents were recorded using 50 ms voltage clamp steps between -100 and +10 mV from a holding potential of -120 mV. We have described approaches and protocols for recording INa previously6,14,20. A summary INa IV relationship is also presented in Figure 5B. Solutions used to record INa are presented in Table 7 and Table 8.
Figure 5C illustrates a representative family of Ca2+ currents (ICa,L) recorded in the whole cell configuration of the patch-clamp technique. These currents were recorded using 250 ms voltage clamp steps between -60 and +80 mV from a holding potential of -70 mV. The experimental conditions that can be used to measure ICa,L have been previously described17,18,20. A summary ICa,L IV relationship is also presented in Figure 5C. The solutions used to record ICa,L are available in Table 7 and Table 8.
Figure 5D illustrates a representative family of K+ currents (IK) recorded in the whole cell configuration of the patch-clamp technique. These currents were recorded from a holding potential of -80 mV using 500 ms voltage clamp steps between -120 mV and +80 mV, as we have described previously6,14. The summary IV relationship for total IK is also presented in Figure 5D. The solutions used to record IK are available in Table 7 and Table 8.
Using these approaches to record APs and major families of ionic currents, including Na+, Ca2+ and K+ currents (as illustrated above), permits the investigator to rigorously interrogate atrial myocyte electrophysiology in a plethora of experimental conditions. Our laboratory has routinely employed these techniques to study atrial myocyte electrophysiology in normal mice, in mouse models of heart disease, and in genetically modified mice6,14,17,18,19,20.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59588/59588fig1.jpg" /> 
Figure 1: Flowchart for the atrial myocyte isolation protocol. Summary of the steps used to isolate atrial myocytes using this protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59588/59588fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59588/59588fig2.jpg" /> 
Figure 2: Experimental setup and dissection tools for atrial myocyte isolation. (A). A small bore fire-polished pipette with an opening 1 mm in diameter (left) is used for tissue transfer following dissection, a medium bore fire-polished pipette with an opening 3 mm in diameter (middle) is used to transfer tissue strips during the isolation, and a large bore fire-polished pipette with an opening 5 mm in diameter (right) is used for trituration of digested atrial tissue. (B). Experimental setup for atrial myocyte isolation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59588/59588fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59588/59588fig3.jpg" /> 
Figure 3: Image of the atrial appendage dissection. (A). Representative bright field image of an excised atrial appendage cut open and pinned out. (B). Representative bright field image of the atrial appendage cut into tissue strips of approximately 0.7 mm in width. Scale bar = 1 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59588/59588fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59588/59588fig4.jpg" /> 
Figure 4: Images of isolated atrial myocytes. (A). Brightfield image of isolated atrial myocytes immediately after isolation. Scale bar = 50 &#956;m. (B). Brightfield image of a single isolated atrial myocyte. Scale bar = 100 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59588/59588fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59588/59588fig5.jpg" /> 
Figure 5: Representative patch-clamp data obtained from isolated atrial myocytes. (A). Representative stimulated AP recording from an isolated atrial myocyte. Summary of AP parameters is presented in Table 6. Amphotericin B (200 &#956;g/mL) was added to the pipette solution to permeabilize the cellular membrane. (B). Representative INa recordings (left) and summary INa IV curve (right) from an isolated atrial myocyte. Nifedipine (10 &#956;M) was added to the modified Tyrode&#8217;s solution to block ICa,L&#173; when recording INa&#173;. C. Representative ICa,L recordings (left) and summary ICa,L IV curve (right) from an isolated atrial myocyte. (D). Representative IK recordings (left) and summary IK IV curve (right) from an isolated atrial myocyte. The solutions used to record each of these currents are listed in Table 7 and Table 8. Summary IV curves are averaged measurements from 10 atrial myocytes isolated from a 15 week-old male wildtype C57Bl/6 mouse. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59588/59588fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Stock Tyrode&#39;s pH 6.9</td>
			<td>Stock Tyrode&#39;s pH 7.4</td>
		</tr>
		<tr>
			<td>Chemical</td>
			<td>in mM</td>
			<td>in mM</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>140</td>
			<td>140</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>5.4</td>
			<td>5.4</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>1.2</td>
			<td>1.2</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>5</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Final volume</td>
			<td>500 mL</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>Final pH with NaOH</td>
			<td>6.9</td>
			<td>7.4</td>
		</tr>
	</tbody>
</table>
Table 1: Stock Tyrode&#8217;s pH 7.4 and stock Tyrode&#8217;s pH 6.9 solutions. Composition of stock Tyrode&#8217;s solutions (pH 7.4 and pH 6.9) that can be made in advance and stored at 4 &#176;C for up to 2 months.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemical</td>
			<td>in mM</td>
		</tr>
		<tr>
			<td>K-glutamate</td>
			<td>100</td>
		</tr>
		<tr>
			<td>K-aspartate</td>
			<td>10</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>25</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>10</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Creatine</td>
			<td>5</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>20</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>5</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>Final volume&#160;</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>Final pH with KOH</td>
			<td>7.2</td>
		</tr>
	</tbody>
</table>
Table 2: Modified KB solution. Recipe for modified KB solution that can be made in advance, aliquoted, and stored at -20 &#176;C for up to 2 months.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemical</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>5.55 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>1.8 mM</td>
		</tr>
		<tr>
			<td>Stock Tyrode&#39;s pH 7.4</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>250 &#956;L</td>
		</tr>
	</tbody>
</table>
Table 3: Modified Tyrode&#8217;s pH 7.4 solution with glucose, magnesium, calcium, and heparin. Composition of modified Tyrode&#8217;s pH 7.4 solution used for the atrial tissue dissection. This solution should be made fresh and kept in a 35 &#176;C water bath until use.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemical</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>18.5 mM</td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>49.96 mM</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>15 mg</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>0.066 mM</td>
		</tr>
		<tr>
			<td>Stock Tyrode&#39;s pH 6.9</td>
			<td>15 mL</td>
		</tr>
	</tbody>
</table>
Table 4: Modified Tyrode&#39;s pH 6.9 solution containing glucose, taurine, BSA, and low calcium. Composition of the modified Tyrode&#8217;s pH 6.9 solution used for the atrial myocyte isolation. This solution should be made fresh and kept in a 35 &#176;C water bath until use.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemical</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>1,064 U</td>
		</tr>
		<tr>
			<td>Elastase</td>
			<td>9 U</td>
		</tr>
		<tr>
			<td>Protease solution</td>
			<td>65.2 &#956;L&#160;</td>
		</tr>
		<tr>
			<td>Modified Tyrode&#39;s pH 6.9</td>
			<td>5 mL</td>
		</tr>
	</tbody>
</table>
Table 5: Enzymatic solution. Composition of the enzymatic solution used to enzymatically digest atrial tissue strips. This solution should be made fresh and kept in a 35 &#176;C water bath until use.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parameter</td>
			<td>Average</td>
		</tr>
		<tr>
			<td>RMP (mV)</td>
			<td>-74.2 &#177; 0.7</td>
		</tr>
		<tr>
			<td>Vmax (V/s)</td>
			<td>144.6 &#177; 5.8</td>
		</tr>
		<tr>
			<td>OS (mV)</td>
			<td>71.9 &#177; 3.0</td>
		</tr>
		<tr>
			<td>APD50 (ms)</td>
			<td>11.1 &#177; 1.7</td>
		</tr>
		<tr>
			<td>APD70 (ms)</td>
			<td>23.0 &#177; 4.6</td>
		</tr>
		<tr>
			<td>APD90 (ms)</td>
			<td>54.7 &#177; 7.8</td>
		</tr>
	</tbody>
</table>
Table 6: Summary of AP parameters from isolated atrial myocytes. Data are presented as mean &#177; SEM, n = 10 atrial myocytes isolated from a 15 week male wildtype C57Bl/6 mouse.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Potassium currents and APs</td>
			<td>Sodium currents</td>
			<td>Calcium currents</td>
		</tr>
		<tr>
			<td>Chemical</td>
			<td>in mM</td>
			<td>in mM</td>
			<td>in mM</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>140</td>
			<td>5</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>5.4</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>1</td>
			<td>1</td>
			<td>2</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>10</td>
			<td>10</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>5.5</td>
			<td>5.5</td>
			<td>5.5</td>
		</tr>
		<tr>
			<td>CsCl</td>
			<td></td>
			<td>130</td>
			<td></td>
		</tr>
		<tr>
			<td>TEA-Cl</td>
			<td></td>
			<td>5.4</td>
			<td>145.5</td>
		</tr>
		<tr>
			<td>pH</td>
			<td>7.4 with NaOH</td>
			<td>7.4 with CsOH</td>
			<td>7.4 with CsOH</td>
		</tr>
	</tbody>
</table>
Table 7: Composition of Tyrode&#8217;s solutions used during patch-clamp experiments. Composition of the Tyrode&#8217;s solutions used to record APs, INa&#173;, ICa,L, and IK from isolated atrial myocytes.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Potassium currents and APs</td>
			<td>Sodium currents</td>
			<td>Calcium currents</td>
		</tr>
		<tr>
			<td>Chemical</td>
			<td>in mM</td>
			<td>in mM</td>
			<td>in mM</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>5</td>
			<td>5</td>
			<td>5</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>140</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>0.2</td>
			<td>0.2</td>
			<td>0.2</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>10</td>
			<td>10</td>
			<td>10</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>5</td>
			<td></td>
			<td>5</td>
		</tr>
		<tr>
			<td>Mg-ATP</td>
			<td>4</td>
			<td>5</td>
			<td>4</td>
		</tr>
		<tr>
			<td>Na-GTP</td>
			<td>0.3</td>
			<td>0.3</td>
			<td>0.3</td>
		</tr>
		<tr>
			<td>Na-phosphocreatine</td>
			<td>6.6</td>
			<td></td>
			<td>6.6</td>
		</tr>
		<tr>
			<td>CsCl</td>
			<td></td>
			<td>130</td>
			<td>135</td>
		</tr>
		<tr>
			<td>BAPTA</td>
			<td></td>
			<td>5</td>
			<td></td>
		</tr>
		<tr>
			<td>pH</td>
			<td>7.2 with KOH</td>
			<td>7.2 with CsOH</td>
			<td>7.2 with CsOH</td>
		</tr>
	</tbody>
</table>
Table 8: Composition of internal pipette solution used during patch-clamp experiments. Composition of the pipette filling solutions used to record APs, INa&#173;, ICa,L, and IK from isolated atrial myocytes.

Watch this Scientific Journal Video about Isolation of Atrial Myocytes from Adult Mice at JoVE.com

Isolation of Atrial Myocytes from Adult Mice

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

isolation-of-atrial-myocytes-from-adult-mice

Nanyang Technological University

Research

JoVE Journal

Medicine

10.3K Views.  University of Calgary. Het isoleren van de atrium myocyten voor gebruik in patch-clamp experimenten heeft sterk geavanceerde onze kennis en het begrijpen van atrium elektrofysiologie op zowel de cellulaire en moleculaire niveaus. De aanpak die we gebruiken levert effectief grote aantallen geïsoleerde atrium myocyten op die kunnen worden gebruikt om cellulaire atriumelelektrofysiologie en aritmogenese te onderzoeken in een breed scala van experimentele modellen en aandoeningen. We hebben gekozen voor een stam spijs...

Video: Isolation of Atrial Myocytes from Adult Mice

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+ ...

Biology

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of metabolic syndrome (MetS)-related heart failure with preserved ejection fraction (HFpEF). The prevalence of MetS-related HFpEF is rising, and atrial cardiomyopathies associated with atrial remodeling and atrial fibrillation are clinically highly relevant as atrial remodeling is an independent predictor of mortality. Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. Circulatory vessel rarefication and interstitial tissue fibrosis pose a potentially limiting factor for the successful single-cell isolation of ACMs from animal models of this disease.
We have addressed this issue by employing a device capable of manually regulating the intraluminal pressure of cardiac cavities during the isolation procedure, substantially increasing the yield of morphologically and functionally intact ACMs. The acquired cells can be used in a variety of different experiments, such as cell culture and functional Calcium imaging (i.e., excitation-contraction-coupling).
We provide the researcher with a step-by-step protocol, a list of optimized solutions, thorough instructions to prepare the necessary equipment, and a comprehensive troubleshooting guide. While the initial implementation of the procedure might be rather difficult, a successful adaptation will allow the reader to perform state-of-the-art ACM isolations in a rat model of MetS-related HFpEF for a broad spectrum of experiments.

Here, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction. A manual regulation of intraluminal pressure of cardiac cavities is implemented to yield functionally intact myocytes suitable for excitation-contraction-coupling studies.

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction and electrical activity. Hence, isolating viable, high-quality cardiomyocytes from the heart is the initial and most crucial experimental step. Comparing the various protocols for isolating the cardiomyocytes of adult mice, the Langendorff retrograde perfusion is the most successful and reproducible method reported in the literature, especially for isolating ventricular myocytes. However, isolating quality atrial myocytes from the perfused heart remains challenging, and few successful isolation reports are available. Solving this complicated problem is extremely important because apart from ventricular disease, atrial disease accounts for a large part of heart diseases. Therefore, further investigations on the cellular level to reveal the mechanisms are warranted. In this paper, a protocol based on the Langendorff retrograde perfusion method is introduced and some modifications in the depth of aorta cannulation and the steps that may affect the digestion process to isolate atrial and ventricular myocytes were simultaneously made. Moreover, the isolated cardiomyocytes are confirmed to be amenable to patch clamp investigation.

This protocol describes modifications of the Langendorff method including the depth of aorta cannulation for simultaneous Isolation of atrial and ventricular myocytes from adult mice.

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.

The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and ...

Camera-based Measurements of Intracellular [Na+] in Murine Atrial Myocytes

Intracellular sodium concentration ([Na+]i) is an important regulator of intracellular Ca2+. Its study provides insight into the activation of the sarcolemmal Na+/Ca2+ exchanger, the behavior of voltage-gated Na+ channels and the Na+,K+-ATPase. Intracellular Ca2+ signaling is altered in atrial diseases such as atrial fibrillation. While many of the mechanisms underlying altered intracellular Ca2+ homeostasis are characterized, the role of [Na+]i and its dysregulation in atrial pathologies is poorly understood. [Na+]i in atrial myocytes increases in response to increasing stimulation rates. Responsiveness to external field stimulation is therefore crucial for [Na+]i measurements in these cells. In addition, the long preparation (dye-loading) and experiment duration (calibration) require an isolation protocol that yields atrial myocytes of exceptional quality. Due to the small size of mouse atria and the composition of the intercellular matrix, the isolation of high quality adult murine atrial myocytes is difficult. Here, we describe an optimized Langendorff-perfusion based isolation protocol that consistently delivers a high yield of high quality atrial murine myocytes.
Sodium-binding benzofuran isophthalate (SBFI) is the most commonly used fluorescent Na+ indicator. SBFI can be loaded into the cardiac myocyte either in its salt form through a glass pipette or as an acetoxymethyl (AM) ester that can penetrate the myocyte&#8217;s sarcolemmal membrane. Intracellularly, SBFI-AM is de-esterified by cytosolic esterases. Due to variabilities in membrane penetration and cytosolic de-esterification each cell has to be calibrated in situ. Typically, measurements of [Na+]i using SBFI whole-cell epifluorescence are performed using a photomultiplier tube (PMT). This experimental set-up allows for only one cell to be measured at one time. Due to the length of myocyte dye loading and the calibration following each experiment data yield is low. We therefore developed an EMCCD camera-based technique to measure [Na+]i. This approach permits simultaneous [Na+]i measurements in multiple myocytes thus significantly increasing experimental yield.

The intracellular Na+ concentration ([Na+]i) in cardiac myocytes is altered during cardiac diseases. [Na+]i is an important regulator of intracellular Ca2+. We introduce a novel approach to measure [Na+]i in freshly isolated murine atrial myocytes using an electron multiplying charged coupled device (EMCCD) camera and a rapid, controllable illuminator.

Intracellular sodium concentration ([Na+]i) is an important regulator of intracellular Ca2+. Its study provides insight into the activation of 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. ...

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.

Isolation of Atrial Myocytes from Adult Mice

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video