This work was supported by grants of National Natural Science Foundation of China (No. 81770322, 81870244, 81500254, 81870243, 81470465) and the Beijing Natural Science Foundation (No. 7192051). Author contributions: Bai and Liu designed and conceived the project. Wen and Ruan provided valuable advices for the experiments. Wu and Linling Li carried out the experimental work and played key roles in data acquisition, analysis and interpretation. Li participated in the Langendorff apparatus assembling. Peng, Zhang, Wang and Yang participated in preparation of the reagents and solutions before experiment. Wu wrote the article.

Adult male C57BL/6 mice weighing 20-30 g, 8-10 weeks of age were purchased from the Animal Centre of Capital Medical University, Beijing, China. All experimental procedures were approved by the Animal Care and Use Committee of Capital Medical University and were performed in compliance with the Guide for the Care and Use of Laboratory Animals proposed by the Institute of Animal Resources and published by the National Institutes of Health. Excel and Origin 8.5 were used for data acquisition and analysis. Data are presented as means &#177; SD, for statistical evaluation, Student&#39;s t test was used and P &#60; 0.05 was considered statistically significant.
1. Solution and perfusion apparatus preparation
<ol>
	<li>Assemble a constant flow Langendorff apparatus (commercially available or self-made). Figure 1 provides a simple schematic.</li>
	<li>Prepare the solutions according to the reagents given in Table 1 and Table 2.</li>
	<li>Turn on the circulating water bath and adjust a suitable input temperature (42.7 &#176;C in our laboratory) to ensure that the perfusate circulation system&#39;s outflow from the cannula reaches 37 &#176;C.</li>
	<li>Clean the perfusion system by circulating deionized water. If a sterile condition is required, run 75% ethanol through the perfusion system for 15 min, followed by deionized water (at least 10 cycles to avoid alcohol toxicity effects on the heart).</li>
	<li>Measure the time and volume for one cycle of the perfusate circulation system to determine how many milliliters of oxygenated Tyrode&#39;s solution to be loaded in the following perfusion step.</li>
	<li>Sterilize all the surgical tools in the desired method.</li>
	<li>Set the flow rate of the perfusate perfusion system at 4 mL/min.</li>
	<li>Prepare two clean 35 mm Petri dishes-one containing Tyrode&#39;s solution (Petri dish 1), the other containing solution 1 (Petri dish 2).</li>
	<li>Prepare a 1 mL syringe filled with solution 1 and a 20 G blunt steel cannula needle with a notch from where the distance is 1 mm to the tip. Prepare a loose knot with 3-0 suture to the cannula shaft. Regulate the viewing field of the stereomicroscope and ensure that the cannula tip is below the liquid surface of Petri dish 2.</li>
</ol>
2. Animal preparation
<ol>
	<li>Administer heparin (concentration, 1,000 IU/mL; 0.2 mL/mouse) intraperitoneally to the mice to avoid blood clots. After 10 min, anesthetize the mice with sodium pentobarbital (50 mg/kg, I.P. injection) and ensure that the mice stop responding to tail/toe pinches. Perform cervical dislocation 20 min after heparin administration.</li>
	<li>Transfer the mice onto the surgical platform, fix the mice in a supine position, sterilize the chest with 75% ethanol, and dry it with gauze or napkin.</li>
</ol>
3. Heart excision and aorta cannulation
<ol>
	<li>Lift the skin of the xiphoid with tissue forceps and make a minor lateral incision through the skin with tissue scissors. Perform a blunt dissection between the skin and fascia and extend the incision of the skin in a V-shape toward the axillae on both sides.
	<ol>
		<li>Continue the same incision trace through the rib cage, and then, deflect the rib cage upward by clamping the sternum with tissue forceps to fully expose the heart and lungs.</li>
	</ol>
	</li>
	<li>Peel off the pericardium using a curved forceps. Tear the thymus gland toward both sides by two curved forceps if it covers the great vessels. Gently pull the base of the heart toward the tail with curved forceps until a &#34;Y&#34;-shaped blood vessel (the aorta and its branch arteries) can be seen.</li>
	<li>To reserve different lengths of the aorta for cannulation and comparison, transect the aorta at the left common carotid artery (green line in Figure 2) and cut the brachiocephalic artery at the same time in some mice. Transect at the ascending aorta in other mice (black line in Figure 2). 
	NOTE: For those who are new to this procedure, this step can also be done under a stereoscopic microscope.</li>
	<li>Excise the heart, and immediately immerse in Petri dish 1 to wash and pump out the residual blood.</li>
	<li>Transfer the heart to Petri dish 2, and trim any surplus tissue using fine iris scissors if necessary. For those who are new to this procedure, do this step under the stereomicroscope to avoid erroneously cutting the aorta or other heart structures.</li>
	<li>Push the syringe to expel air bubbles before aorta cannulation. Perform retrograde aorta cannulation with the assistance of two straight tying forceps (smooth jaws) under the stereomicroscope. Ensure that the whole cannulation process is under the liquid surface.</li>
	<li>Avoid penetrating the aortic valves and adjust the depths to the ascending aorta (the mouse that the aorta was transected at the left common carotid artery) and the aortic root (the mouse that the aorta was transected at the ascending aorta), respectively. 
	NOTE: The depth of aorta cannulation (referred to as simply as depth) is defined here as the position where the cannula tip is in the aorta or where the aorta is ligated.</li>
	<li>Ligate the aorta with the pre-knot 3-0 suture to the cannula notch. Gently press the syringe content to rinse the residual blood. 
	NOTE: The blood will leave the coronary arteries and effuse from the dorsal veins If the depth is properly positioned. The heart and the atrial appendages will expand and become pale.</li>
	<li>Remove and connect the cannula to the Langendorff apparatus. Once again, take care to avoid any air bubbles entering the heart. 
	&#8203;NOTE: Be aware that the time from thoracotomy to initial perfusion should not exceed 5 min.</li>
</ol>
4. Heart perfusion
<ol>
	<li>First, prime and perfuse oxygenated Tyrode&#39;s solution for approximately 10 s (the volume of liquid running 10 s in the system used in this protocol is approximately 0.7 mL) if there is residual blood in the atria, and then switch to solution 1. However, just perfuse with solution 1 If there is no residual blood in the atria. Perfuse the heart for approximately 2 min.</li>
	<li>Switch to solution 3. Suck about 2.5 mL of solution 3 with a single-use sterile polyethylene pipet and prewarm it in the water bath for later use, with the remainder used for perfusion for approximately 11-12 min. Discard solution 3 perfused in the first 2 min. Recycle the rest to the perfusate reservoirs by the peristaltic pump for reuse until digestion is completed.</li>
	<li>Terminate the digestion when the heart becomes swollen and turns slightly pale and flaccid (spongy texture) or an imprint exists if the myocardium is gently pinched using a toothed forceps.</li>
</ol>
5. Cell isolation and calcium reintroduction
<ol>
	<li>Remove the ventricles and the atria with forceps, and place them in different Petri dishes. Add the prewarmed solution 3 to the Petri dishes.</li>
	<li>Triturate the tissue into a turbid texture with blunt forceps and gently pipette the tissue for even digestion. Avoid introducing air bubbles.</li>
	<li>Transfer the turbid digested tissue with the pipette into solution 4 to arrest the remaining enzyme activity, and then centrifuge for 20 s at 192&#215;g. Remove the supernatant and add solution 5 into the cell sediment and pipette to evenly disperse the cells.</li>
	<li>Reintroduce calcium in a stepwise manner in order to avoid calcium paradox and calcium overload. Gradually add in a total of 50 &#181;L 100 mM/L CaCl2 (at every 5 min interval of 5 &#181;L, 10 &#181;L, 15 &#181;L, and 20 &#181;L, respectively) to the cell suspension.</li>
</ol>
6. Cell storage
<ol>
	<li>For the patch clamp study, store the cells in Tyrode&#39;s solution. For other cellular studies, store the cells in solution 6.</li>
	<li>To ensure the accuracy of acute functional studies&#39; (e.g., Ca2+ sparks, Ca2+ waves, Ca2+ release, Ca2+ leak, and patch clamp recordings) results, finish these kinds of functional experiments within the next 6 h.</li>
</ol>

<ol>
	<li>Mahmood, S. S., Levy, D., Vasan, R. S., Wang, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+framingham+heart+study+and+the+epidemiology+of+cardiovascular+diseases:+a+historical+perspective.">The framingham heart study and the epidemiology of cardiovascular diseases: a historical perspective.</a> Lancet. 383 (9921), 999-1008 (2014).</li><li>Olejnickova, V., Novakova, M., Provaznik, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+heart+models:+cardiovascular+system+studies+and+technological+advances.">Isolated heart models: cardiovascular system studies and technological advances.</a> Medical &amp; Biological Engineering &amp; Computing. 53 (7), 669-678 (2015).</li><li>Guinamard, R., Hof, T., Sall&#233;, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+recordings+at+the+single+channel+level+in+adult+mammalian+isolated+cardiomyocyte.">Current recordings at the single channel level in adult mammalian isolated cardiomyocyte.</a> Methods in Molecular Biology. 1183, 291-307 (2014).</li><li>Santana, L. F., Kranias, E. G., Lederer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+sparks+and+excitation-contraction+coupling+in+phospholamban-deficient+mouse+ventricular+myocyte.">Calcium sparks and excitation-contraction coupling in phospholamban-deficient mouse ventricular myocyte.</a> The Journal of Physiology. 503, 21-29 (1997).</li><li>Chou, C. C., 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=Intracellular+calcium+dynamics+and+anisotropic+reentry+in+isolated+canine+pulmonary+veins+and+left+atrium.">Intracellular calcium dynamics and anisotropic reentry in isolated canine pulmonary veins and left atrium.</a> Circulation. 111 (22), 2889-2897 (2005).</li><li>Brittsan, A. G., 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=Chronic+SR+Ca2+-ATPase+inhibition+causes+adaptive+changes+in+cellular+Ca2++transport.">Chronic SR Ca2+-ATPase inhibition causes adaptive changes in cellular Ca2+ transport.</a> Circulation Research. 92 (7), 769-776 (2003).</li><li>Mohler, P. 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=Ankyrin-B+mutation+causes+type+4+long-QT+cardiac+arrhythmia+and+sudden+cardiac+death.">Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death.</a> Nature. 421 (6923), 634-639 (2003).</li><li>Paigen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+miracle+enough:+the+power+of+mice.">A miracle enough: the power of mice.</a> Nature medicine. 1 (3), 215-220 (1995).</li><li>Ni, L., 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-specific+gene+delivery+using+an+adeno-associated+viral+vector.">Atrial-specific gene delivery using an adeno-associated viral vector.</a> Circulation Research. 124 (2), 256-262 (2019).</li><li>Dobrev, D., Wehrens, X. H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+cardiac+arrhythmias.">Mouse models of cardiac arrhythmias.</a> Circulation Research. 123 (3), 332-334 (2018).</li><li>Jian, Z., 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=In+vivo+cannulation+methods+for+cardiomyocytes+isolation+from+heart+disease+models.">In vivo cannulation methods for cardiomyocytes isolation from heart disease models.</a> PLoS One. 11 (8), 0160605 (2016).</li><li>Li, D., Wu, J., Bai, Y., Zhao, X., Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+adult+mouse+cardiomyocytes+for+cell+signaling+and+in+vitro+cardiac+hypertrophy.">Isolation and culture of adult mouse cardiomyocytes for cell signaling and in vitro cardiac hypertrophy.</a> Journal of Visualized Experiments: JoVE. (87), e51357 (2014).</li><li>Graham, E. L., 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=Isolation,+culture,+and+functional+characterization+of+adult+mouse+cardiomyoctyes.">Isolation, culture, and functional characterization of adult mouse cardiomyoctyes.</a> Journal of Visualized Experiments JoVE. (79), e50289 (2013).</li><li>Roth, G. M., Bader, D. M., Pfaltzgraff, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+physiological+analysis+of+mouse+cardiomyocytes.">Isolation and physiological analysis of mouse cardiomyocytes.</a> Journal of Visualized Experiments: JoVE. (91), e51109 (2014).</li><li>Motayagheni, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+Langendorff+technique+for+mouse+heart+cannulation:+Improved+heart+quality+and+decreased+risk+of+ischemia.">Modified Langendorff technique for mouse heart cannulation: Improved heart quality and decreased risk of ischemia.</a> MethodsX. 4, 508-512 (2017).</li><li>Kohncke, C., 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=Isolation+and+Kv+channel+recordings+in+murine+atrial+and+ventricular+cardiomyocytes.">Isolation and Kv channel recordings in murine atrial and ventricular cardiomyocytes.</a> Journal of Visualized Experiments: JoVE. (73), e50145 (2013).</li><li>Wagner, E., Brandenburg, S., Kohl, T., Lehnart, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+tubular+membrane+networks+in+cardiac+myocytes+from+atria+and+ventricles.">Analysis of tubular membrane networks in cardiac myocytes from atria and ventricles.</a> Journal of Visualized Experiments: JoVE. (92), e51823 (2014).</li><li>Pla&#269;ki&#263;, J., Kocksk&#228;mper, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+atrial+and+ventricular+cardiomyocytes+for+in+vitro+studies.">Isolation of atrial and ventricular cardiomyocytes for in vitro studies.</a> Methods in Molecular Biology. 1816, 39-54 (2018).</li><li>Andrade, J., Khairy, P., Dobrev, D., 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=The+clinical+profile+and+pathophysiology+of+atrial+fibrillation:+relationships+among+clinical+features,+epidemiology,+and+mechanisms.">The clinical profile and pathophysiology of atrial fibrillation: relationships among clinical features, epidemiology, and mechanisms.</a> Circulation Research. 114 (9), 1453-1468 (2014).</li><li>Louch, W. E., Sheehan, K. A., Wolska, B. 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+in+cardiomyocyte+isolation,+culture,+and+gene+transfer.">Methods in cardiomyocyte isolation, culture, and gene transfer.</a> Journal of Molecular and Cellular Cardiology. 51 (3), 288-298 (2011).</li><li>Bers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+Na/Ca+exchange+function+in+rabbit,+mouse+and+man:+what's+the+difference.">Cardiac Na/Ca exchange function in rabbit, mouse and man: what's the difference.</a> Journal of Molecular and Cellular Cardiology. 34 (4), 369-373 (2002).</li><li>Chen, X., O'Connell, T. D., Xiang, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=With+or+without+Langendorff:+a+new+method+for+adult+myocyte+isolation+to+be+tested+with+time.">With or without Langendorff: a new method for adult myocyte isolation to be tested with time.</a> Circulation Research. 119 (8), 888-890 (2016).</li><li>Fern&#225;ndez, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+coronary+arteries+of+the+C57BL/6+mouse+strains:+implications+for+comparison+with+mutant+models.">The coronary arteries of the C57BL/6 mouse strains: implications for comparison with mutant models.</a> Journal of Anatomy. 212 (1), 12-18 (2008).</li><li>Zimmerman, A. N., Hulsmann, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paradoxical+influence+of+calcium+ions+on+the+permeability+of+the+cell+membranes+of+the+isolated+rat+heart.">Paradoxical influence of calcium ions on the permeability of the cell membranes of the isolated rat heart.</a> Nature. 211 (5049), 646-647 (1966).</li><li>Schl&#252;ter, K. D., Schreiber, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+ventricular+cardiomyocytes:+isolation+and+culture.">Adult ventricular cardiomyocytes: isolation and culture.</a> Methods in Molecular Biology. 290, 305-314 (2005).</li><li>Thum, T., Borlak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Butanedione+monoxime+increases+the+viability+and+yield+of+adult+cardiomyocytes+in+primary+cultures.">Butanedione monoxime increases the viability and yield of adult cardiomyocytes in primary cultures.</a> Cardiovascular Toxicology. 1 (1), 61-72 (2001).</li><li>Hall, A. R., Hausenloy, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+respiratory+inhibition+by+2,3-butanedione+monoxime+(BDM):+implications+for+culturing+isolated+mouse+ventricular+cardiomyocytes.">Mitochondrial respiratory inhibition by 2,3-butanedione monoxime (BDM): implications for culturing isolated mouse ventricular cardiomyocytes.</a> Physiological Reports. 4 (1), 12606 (2016).</li><li>Scaduto, R. C., Grotyohann, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2,3-butanedione+monoxime+unmasks+Ca+(2+)-induced+NADH+formation+and+inhibits+electron+transport+in+rat+hearts.">2,3-butanedione monoxime unmasks Ca (2+)-induced NADH formation and inhibits electron transport in rat hearts.</a> American Journal of Physiology. Heart and Circulatory Physiology. 279 (4), 1839-1848 (2000).</li><li>Watanabe, Y., 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=Inhibitory+effect+of+2,3-butanedione+monoxime+(BDM)+on+Na+(+)/Ca+(2+)+exchange+current+in+guinea-pig+cardiac+ventricular+myocytes.">Inhibitory effect of 2,3-butanedione monoxime (BDM) on Na (+)/Ca (2+) exchange current in guinea-pig cardiac ventricular myocytes.</a> British Journal of Pharmacology. 132 (6), 1317-1325 (2001).</li><li>Benndorf, K., Boldt, W., Nilius, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+current+in+single+myocardial+mouse+cells.">Sodium current in single myocardial mouse cells.</a> European Journal of Physiology. 404 (2), 190-196 (1985).</li><li>Fiset, C., Clark, R. B., Larsen, T. 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=A+rapidly+activating+sustained+K++current+modulates+repolarization+and+excitation-contraction+coupling+in+adult+mouse+ventricle.">A rapidly activating sustained K+ current modulates repolarization and excitation-contraction coupling in adult mouse ventricle.</a> The Journal of Physiology. 504, 557-563 (1997).</li><li>Isenberg, G., Klockner, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+tolerant+ventricular+myocytes+prepared+by+preincubation+in+a+"KB+medium".">Calcium tolerant ventricular myocytes prepared by preincubation in a "KB medium".</a> European Journal of Physiology. 395 (1), 6-18 (1982).</li><li>Ackers-Johnson, 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=A+simplified,+Langendorff-free+method+for+concomitant+isolation+of+viable+cardiac+myocytes+and+nonmyocytes+from+the+adult+mouse+heart.">A simplified, Langendorff-free method for concomitant isolation of viable cardiac myocytes and nonmyocytes from the adult mouse heart.</a> Circulation Research. 119 (8), 909-920 (2016).</li><li>Jiang, L., 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=Ibrutinib+promotes+atrial+fibrillation+by+inducing+structural+remodeling+and+calcium+dysregulation+in+the+atrium.">Ibrutinib promotes atrial fibrillation by inducing structural remodeling and calcium dysregulation in the atrium.</a> Heart Rhythm. 16 (9), 1374-1382 (2019).</li></ol>

The authors have nothing to disclose.

Single CM is a valuable and indispensable tool in cellular level studies of cardiac function and diseases20. Hence, the isolation of viable CMs from the hearts is the initial and most crucial step. Cell quality is one of the significant determinants of conducting successful experiments, especially in optical and electrophysiological experiments. Compared with the CMs of other animals, rodent CMs are more vulnerable to ischemia and hypoxia because of a higher concentration of intracellular sodium ions, which favors calcium influx through the Na+/Ca2+ exchanger21. Moreover, the number of AMs is far lesser than that of VMs; thus, successful isolation is extremely difficult to achieve. The Langendorff method is excellent for isolating mice VMs22, but the success rate in isolating AMs is low, and few reports are available. The proper depth of aorta cannulation is also an essential factor in yielding ideal VMs aside from temperature, enzyme activity, PH, and water quality used for buffer preparation. The principle of the Langendorff method relies on retrograde perfusion of the heart. Upon perfusion, the aortic valve is closed; thereby, the perfusate is forced into the coronary arteries, delivering enzyme solution through the vessel branches, and the myocardial tissue is evenly digested. To achieve this kind of circulation pattern, the aorta must reserve enough length for cannulation and ligation, also the cannula tip must not penetrate the aortic valves or block the CA ostium. Thus, it is reasonable to speculate that the depth of aorta cannulation is also associated with atria perfusion, which affects the digestion efficacy of the atria and the yields of AM in a similar way. The protocol presented here confirmed the hypothesis, and crucial steps for optimizing cell yield along with the suggestions are noted below.
In step 1.9, to better secure the aorta, a blunt 20 G cannula with a notch (or a circumferential groove) from where the distance is 1 mm to the tip is recommended. Based on our experiences, the cannula size that is a little bit larger than the diameter of the aorta was found to prevent the cannula tip from puncturing the aortic valves during cannulation because the aorta, relying on its intrinsic elasticity, can snugly fit into the cannula and produce friction, which acts as a protective factor when moving forward or backward adjusting the cannulation depth. In terms of the notch&#39;s position, the heart will easily slip off during cannulation because of gravity if it is too near the cannula tip. Conversely, the space for adjusting the cannulation depth and ligation position will be very confined if too far. Given these circumstances, transecting the aorta between the left common carotid artery (as the green line in Figure 2) to ensure the aorta is long enough and can be cannulated and ligated at the ascending aorta is better in step 3.3. Whereas transecting at the ascending aorta, the reserved length for cannulation at least could secure the cannula tip close to the aortic root and will not penetrate the aortic valves after ligation. The anatomy of the aorta and the measurement of the distance from the cannula tip to the CA ostium indicate that transecting the aorta between the left common carotid artery is an ideal position to achieve a proper aorta cannulation depth.
Cannulation depth was found to be associated with the atria&#39;s perfusion, which in turn acts as a depth indicator. Figure 4 shows that atria perfusion is good when the depth is at the ascending aorta, and both atrial appendages are inflated. However, atria perfusion is insufficient when the depth is at (or approaching) the aortic root and both atrial appendages are wizened. The total and viable count of AM yielded from the inflated atrial appendages were higher (Figure 8). These findings indicate that the aorta cannulation depth can affect the perfusion and digestion of the atria and atrial appendages in a certain way and finally affect AM yield and quality. The yield and quality of AM were inferred to be associated with the distribution of the vessels that supply the atria. The study of Fern&#225;ndez et al.23 has demonstrated various anomalies in the origin and course of mouse CA. They found that the CAs ostia were highly variable and were not all located in the aortic sinus. Some CAs may originate anomalously from above the aortic sinuses, named the high take-off ostium. Some CAs may originate from the same aortic sinus, and the ostium of the atrium vessel is just nearby. The anatomy of the aorta in the present study (Figure 10) is also consistent with the finding of Fern&#225;ndez. This may be the cause why attempts to isolate AM by the Langendorff method have been largely unsuccessful if the cannulation depth is not appropriate. Thus, the cannula tip will have a greater chance of blocking the atrium vessel ostium that is adjacent to the CA ostium if not enough space is available between the cannula tip and the CA ostium. By contrast, the perfusion and cell yield of the ventricle were hardly affected as long as the aortic valves were not penetrated by the cannula tip. This is probably because the CAs that supply blood to the ventricle have bigger ostia and more origins. If one ostium was occluded by the cannula, ventricle perfusion can be compensated by another CA or the collateral circulation, whereas the blood vessel that supplies the atrium is rather small and has no substitute. Thus, the influence of the depth in aorta cannulation is important.
Other noteworthy factors and trouble shootings in the digestion and cell storage process are listed as follows. First, consider perfusing oxygenated Tyrode&#39;s solution in step 4.1 to make the muscle contract and pump out residual blood if the blood in the atria has not been rushed out after aorta ligation. This can help avoid the adverse effect of ca2+ and other materials released from the damaged erythrocytes. Second, perfusing ca2+-free solution ahead of time to dissociate the connection and expand the space between cells can improve the efficacy of enzyme digestion because intercalated disks between CMs are calcium-dependent intercellular junctions. However, time should be limited to 3-5 min to avoid the calcium paradox phenomenon24. A mixed enzyme solution is recommended. Collagenase type II disrupts the extracellular matrix network, and trypsin helps clear the granular material that remains on the cell surface if collagenase II digestion is incomplete. This ensures a smooth cell surface, which is critical to forming a G&#937; seal in the patch clamp recording. Nevertheless, trypsin concentration should be controlled in the proper range to avoid over digestion and cell injury because it can degrade the membrane protein. Using collagenase type II alone to improve cell yield may often lead to tissue over digestion, and the isolated CMs will be calcium intolerant after prolonged collagenase exposure25. The use of 2,3-butanedione monoxime (BDM), a substance that prevents spontaneous contraction by inhibiting the myosin ATPase and preventing cross-bridge formation, is still controversial26,27,28,29. According to previous experience, adding BDM is necessary for this protocol. Enzyme solution is prepared with solution 1 although solution 1 does not contain calcium, and calcium is added to activate the enzyme. The benefit of adding BDM in the perfusion solution includes (1) inhibiting myocytes contraction and reducing oxygen consumption during enzyme solution perfusion and (2) preventing myocytes from hypoxia and improving the quality of the isolated myocytes. Some studies reported that BDM may have a potential adverse influence on cellular electrical properties. However, the results of the whole-cell patch clamp recording of the sodium current did not indicate an undesirable effect. In the cell storage step (step 6), numerous studies have chosen the KB buffer, a calcium-free but high potassium concentration solution, in which cells can maintain a better state because they are in polarized and low metabolic conditions. However, the glycocalyx of the cell membrane will separate from the lipid bilayer in the absence of exogenous calcium for a certain length of time and the membrane permeability will increase, affecting the subsequent functional analysis30,31,32.
All myocytes isolation techniques can be essentially categorized currently into either chunk (a small piece of tissue) digestion in an enzymatic solution or CA perfusion with enzymatic solution (the Langendorff perfusion)22. Compared with the Langendorff method, the chunk digestion method is easier to perform and is also routinely used for isolating CMs in many laboratories. However, this method normally produces a low yield of CMs of poor quality from adult tissues22. Moreover, cells isolated by this method may not be suitable for conducting comparative experiments. For example, when testing the cell type-specific drug effects between AM and VM, the impact of different isolation conditions cannot be neglected or excluded. This is because the myocardium and the tissue density of the ventricle are much thicker and denser than that of the atrium, resulting in differing digestion times and enzyme concentrations. Furthermore, excessive agitation and pipetting of the tissue during digestion will damage the cells and significantly impact functional studies. Moreover, many previous studies have shown that AMs are more vulnerable to calcium. However, AMs isolated by the current protocol can be tolerant of gradient calcium reintroduction probably because the tissue is easy to rupture at the end of the digestion process. Thus, mechanical damage is less, whereas cells would suffer more mechanical damages as steps of the chunk method need repeated rupture and centrifugal. More recently, Ackers et al.33 reported a simplified, Langendorff-free method for the isolation of viable cardiac myocytes and nonmyocytes. The VMs and the fibroblasts can be effectively isolated, but the amount of the AMs was not mentioned. However, this protocol has several limitations. First, the distribution of the heart blood vessels may have variations for individual differences and mice strain, and the recommended cannulation depth cannot guarantee a successful AMs isolation for each time. Second, for those who are new to this procedure may take a certain amount of time to practice aorta transection and retrograde aorta cannulation. Finally, this method was not tested in other heart disease models except in healthy and elderly mice. Hence, it will require adjustments in enzyme concentrations and time of digestion because of the heart&#39;s fibrosis extent. The disadvantage of different digestion conditions encountered in the chunk method will not be a problem in the Langendorff method in which the enzyme solution is evenly distributed to the tissue by the vessel beds.
In summary, the protocols for the simultaneous isolation of single AM and VM described here have demonstrated that the proper aorta cannulation depth can effectively improve atrium perfusion and AM yield. The CMs isolated by this method are of high quality, possess good calcium tolerance, and have been successfully applied to patch clamp recording and calcium handling (Ca2+ release and Ca2+ wave measurement by the IonOptix system) in the team34. It is anticipated that the isolation protocol can be used for cell preparation in a series of cellular and subcellular investigations, which will help deepen the understanding of cardiac physiology and pathology. Importantly, it will enable the discovery of more clinically relevant heart disease mechanisms and intervention methods.

Cardiac disease is one of the leading global causes of mortality1. To address this burden on the healthcare system, an in-depth understanding of the heart&#39;s physiology and pathology is essential. Besides whole animal and intact heart preparation, cellular preparation is another indispensable tool for functional and disease study2. By applying the patch clamp, calcium imaging, molecular biology, and other advanced technologies, researchers can obtain more information on the electrophysiological properties, calcium homeostasis, signaling pathways, metabolic states, and gene transcriptions in a single cardiomyocyte (CM). This is extremely helpful in revealing the physiological and pathological mechanisms of the cardiac disease process3,4,5,6,7. For animal research, species ranging from small (e.g., mice, rats, and guinea pigs) to large (e.g., rabbits and dogs) animals can be used. Small animals are usually preferred, particularly mice, because they are amenable to genetic and disease model manipulation8,9,10.
Techniques of acutely isolated CMs have undergone a long development period and are still evolving11. The Langendorff retrograde perfusion is the most successful and reproducible CMs isolation method applied in rats and mice, especially for isolating ventricular myocytes (VMs)12,13,14,15. However, reports of successfully isolated atrial myocytes (AMs) are scarce16,17,18. Further investigations on both levels of the whole organ/system and the cellular/subcellular to reveal the mechanisms and explore new therapeutic approaches are warranted because atrial fibrillation (AF), the most common type of arrhythmia, is increasingly becoming prevalent globally, and current treatment modalities in both pharmacological therapies and cardiac ablation remain ineffective in approximately 40%-50% of AF patients19. Successful adult mouse CMs isolation is the first step for cellular study. Two primary isolation methods can be used: the chunk and Langendorff methods. In the Langendorff perfusion method, tissue digestion depends on the enzyme solution delivered by the coronary arteries and their branches to the capillary beds. A proper aorta cannulation depth that can avoid penetrating the aortic valves and blocking the coronary artery ostia is the prerequisite of achieving such a perfusion pattern, which is also the essential step for efficient digestion and ideal VM yield. Hence, it is reasonable to assume that the aorta cannulation&#39;s depth may similarly affect the atria vessel&#39;s perfusion and finally affect the AM yield. To test this hypothesis, aorta cannulation at different depths was performed and the corresponding AM yields were compared. The data showed that the aorta cannulation depth is directly relevant to the AM yield. Herein, a protocol to isolate AMs and VMs simultaneously is introduced.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2,3-butanedione monoxime (BDM)</td>
			<td>Sigma-Aldrich</td>
			<td>31550</td>
			<td></td>
		</tr>
		<tr>
			<td>Bull Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type II</td>
			<td>Worthington</td>
			<td>43D14160</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td></td>
			<td></td>
			<td>data acquisition and analysis</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Zhejiang Tianhang Biotechnology</td>
			<td>150207</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>KHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>237205</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>S7907</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Origin 8.5</td>
			<td>OriginLab, Northampton, MA,US</td>
			<td></td>
			<td>data acquisition and analysis</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Longerpump</td>
			<td>BT100-2J</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pentobarbital</td>
			<td>Shanghai Reagent Factory</td>
			<td>810923</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich</td>
			<td>T0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Solarbio</td>
			<td>C0040</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Invitrogen</td>
			<td>15090046</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>JULABO</td>
			<td>ED (v.2)</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 paper, where the cannula tip is positioned in the aorta, which is defined as the depth of aorta cannulation (referred to simply as depth), also represents where the aorta is ligated. The AM and VM isolated from a heart that was cannulated and ligated at the ascending aorta are represented as AMAA and VMAA, respectively. Moreover, AM and VM isolated from a heart that was cannulated and ligated at the aortic root are represented as AMAR and VMAR, respectively. Figure 2 shows the overview of the aorta and the transection positions. Also, Figure&#160;3 shows the depths and the ligation places that correspond to the aorta transection positions. Depth was associated with the atria and atrial appendages&#39; perfusion. Both atrial appendages are inflated when the cannula tip is at the ascending aorta, indicating sufficient atria perfusion. However, when at the aortic root, atria perfusion is insufficient and both atrial appendages are wizened (Figure 4). The CM morphology and viability that are isolated from the cannulated hearts at different depths after calcium reintroduction (Figure 5). Cell morphologies of AMAA, AMAR, VMAA, and VMAR are under the confocal microscope before and after calcium reintroduction. AMs are spindle-shaped, whereas VMs are rod-shaped with rectangular ends. Moreover, AMs and VMs have intact membranes, clear contours, clear striated sarcomeres, and smooth-surfaced (Figure 6). Cell viability was assessed via trypan blue staining. Normal cells with intact membranes can exclude trypan blue and will not be stained, whereas cells loss activity will intracellularly quickly accumulate trypan blue (Figure 7). AMs and VMs were placed in different object slides to perform cell counting. The total yields of AM and the percentage of viable spindle-shaped CMs (survival rate) were determined by transferring 10 &#181;L of the cell suspension to an object slide and counted under an inverted phase-contrast microscope at 4 &#215; 10 fields of view. The same method was used to determine the total yields of VMs (rod-shaped) and the percentage of viable VMs. This method is preferred over using a hemocytometer, because the CMs did not easily distribute into the counting area of the hemocytometer because of their size and shape.&#160;A bar graph (Figure 8) shows the survival rates of AMAA, AMAR, VMAA, and VMAR before and after calcium reintroduction. Each value represents the mean &#177; SD from 10 mice. Before calcium reintroduction, the survival rates of AMAA are significantly higher than those of AMAR (70.9% &#177; 2.8% and 41.0% &#177; 5.2%, respectively; p &#60; 0.01). After calcium reintroduction, the survival rates of AMAA are significantly higher than those of AMAR (69.4% &#177; 3.0% and 37.7% &#177; 4.9%, respectively; p &#60; 0.01).
The VMAA survival rates did not differ from those of the VMAR (89.5% &#177; 2.7% vs 88.1% &#177; 2.6%, respectively; p &#62; 0.05) before calcium reintroduction. Similarly, the VMAA survival rates did not differ from those of the VMAR (82.2% &#177; 1.9% vs 82.9% &#177; 1.6%, respectively; p &#62; 0.05) after calcium reintroduction.
A heart cannulated at depth as this protocol recommended (at the ascending aorta), a viable VMs and AMs yield of approximately 4.1 million and approximately 180,000, respectively, after calcium reintroduction. The whole-cell patch clamp recording of the sodium current (Figure 9A, B) and current densities (Figure 9C) in the isolated AM and VM confirm that the cell quality has met the requirements for electrophysiological experiments. The anatomy of the aorta (Figure 10) shows that the ostium of the atrial vessel is near the aortic root and is adjacent to the ostium of the coronary artery (CA). Table 3 shows the distance from the cannula tip to the CA ostium of hearts cannulated and ligated at the ascending aorta and the aortic root, respectively.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62514/62514fig01.jpg" /> 
Figure 1. Schematic diagram of an assembled modified Langendorff system. This system is economical and portable for the users. It has two parts of plastic tubing-one for the water (inner diameter, 8 mm) and one for the perfusate (inner diameter, 1.5 mm)-of which the distal end is connected to a PE medical three-way stopcock with a Luer lock. A glass bottle containing water and with a rubber stopper form as a heat exchanger. The perfusate is pumped into the plastic tubing in the heat exchanger by the peristaltic pump and comes out from the three-way stopcock connected to the cannula. <a href="https://www.jove.com/files/ftp_upload/62514/62514fig01large.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/62514/62514fig02.jpg" /> 
Figure 2.&#160;Aorta and tissues around. A &#34;Y&#34;-shaped blood vessel structure is the aorta and its branches: the brachiocephalic artery (arrow 1) and the left common carotid artery (arrow 2). Transect the aorta between the left common carotid artery (green line) and simultaneously cut the brachiocephalic artery in some mice; transect at the ascending aorta (black line) in other mice (stereomicroscope 10 &#215; 2 magnification). <a href="https://www.jove.com/files/ftp_upload/62514/62514fig02large.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/62514/62514fig03.jpg" /> 
Figure 3. Frontal view of the cannulated heart. The black curved line represents the incisal edge of the aorta that was transected between the left common carotid artery. The red curved line represents the incisal edge of the aorta transected at the ascending aorta. The straight line represents where the aorta was ligated: black at the ascending aorta and red at the aortic root (stereomicroscope 10 &#215; 2 magnification). <a href="https://www.jove.com/files/ftp_upload/62514/62514fig03large.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/62514/62514fig04.jpg" /> 
Figure 4. Perfusion state of the cannulated hearts. The atria are sufficiently perfused, and both atrial appendages are inflated in the heart (A) cannulated and ligated at the ascending aorta. However, both atrial appendages are wizened in the heart (B) cannulated and ligated at the aortic root, indicating poor atria perfusion. <a href="https://www.jove.com/files/ftp_upload/62514/62514fig04large.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/62514/62514fig05.jpg" /> 
Figure 5. Cell morphology and viability evaluation after calcium reintroduction. AMs (A) and VMs (B) isolated from the heart cannulated and ligated at the ascending aorta are represented as AMAAs and VMAAs, respectively. AMs (C) and VMs (D) isolated from the heart cannulated and ligated at the aortic root are represented as AMARs and VMARs, respectively. High-quality CMs are quiescent and have intact membranes, clear contours, clear striated sarcomeres, and a smooth cell surface. AMs are spindle-shaped, whereas VMs are rod or brick-shaped with rectangular ends. CMs that spontaneously contract or contain blebs in the membrane are of poor quality, and those that shrink into a spherical shape are dead cells. A random field of view (fluorescence microscope, 10 &#215; 10 magnification; scale bars, 100 &#181;m). AM = atrial myocyte, VM = ventricular myocyte. <a href="https://www.jove.com/files/ftp_upload/62514/62514fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62514/62514fig06.jpg" /> 
Figure 6. Cell morphology under a confocal microscope before and after calcium reintroduction. Before calcium reintroduction, AMAA (A) and AMAR (C) are spindle-shaped with clear contours, striated sarcomeres, and smooth membrane surfaces. After calcium reintroduction, AMAA (B) and AMAR (D) are also spindle-shaped with clear contours, striated sarcomeres, and smooth membrane surfaces. Before calcium reintroduction, VMAA (E) and VMAR (G) are rod- or brick-shaped with clear contours, striate sarcomeres, and smooth membrane surfaces with rectangular ends. After calcium reintroduction, VMAA (F) and VMAR (H) are also rod- or brick-shaped with clear contours, striated sarcomeres, and smooth membrane surfaces with rectangular ends (confocal microscope oil, 63 &#215; 10 magnification; scale bars, 50 &#181;m). <a href="https://www.jove.com/files/ftp_upload/62514/62514fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62514/62514fig07.jpg" /> 
Figure 7. Cell viability assessment. Damaged cells that lose activity are stained by trypan blue. In contrast, viable cells cannot be stained by trypan blue (fluorescence microscope, 4 &#215; 10 magnification; scale bars, 100 &#181;m). <a href="https://www.jove.com/files/ftp_upload/62514/62514fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/62514/62514fig08.jpg" /> 
Figure 8. Bar graph showing the survival rates of AMAA, AMAR, VMAA, and VMAR before and after calcium reintroduction. CR = calcium reintroduction. Each value represents the mean &#177; SD from 10 mice. ***p &#60; 0.01. <a href="https://www.jove.com/files/ftp_upload/62514/62514fig08large.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>Solution</td>
			<td>Contents (final concentration in mmol/L, if not specified differently)</td>
			<td>noted</td>
		</tr>
		<tr>
			<td>Perfusion solution (solution 1)</td>
			<td>113 NaCl, 4.7 KCl, 0.6 KH2PO4, 0.6 Na2HPO4, 1.2 MgSO4, 12 NaHCO3, 10 KHCO3, 10 HEPES, 15 Taurine, 5 Glucose, 10 2,3-butanedione monoxime(BDM)</td>
			<td>Which can be stored for 3 days at 4 &#176;C. Glucose, Taurine and BDM are added on the day of experiment. Prepare 200 mL of perfusion solution for each heart.</td>
		</tr>
		<tr>
			<td>Tyrode&#8217;s solution (solution 2)</td>
			<td>140 NaCl, 4 KCl, 1 MgCl2, 10 HEPES, 1 CaCl2, 5 Glucose</td>
			<td>Which can be stored for 3 days at 4 &#176;C. CaCl2 and Glucose are added on the day of experiment, adjust pH to 7.3-7.4 with saturated NaOH at room temperature (28-30&#176;C) with a PH meter.</td>
		</tr>
	</tbody>
</table>
Table 1. Solutions for adult mouse CM isolation.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution</td>
			<td>Contents&#160;</td>
			<td>noted</td>
		</tr>
		<tr>
			<td>Digestion solution(solution 3)</td>
			<td>25 mL solution 1, 6&#956;L 100 mM/L CaCl2, 25mg collagenase II, 50&#956;L (2.5 % 10&#215;) trypsin</td>
			<td>for each heart</td>
		</tr>
		<tr>
			<td>Stop solution 1(solution 4)</td>
			<td>9 mL solution 1, 1 mL FBS (Fetal Bovine Serum), 4&#956;L 100 mM/L CaCl2</td>
			<td>for each heart</td>
		</tr>
		<tr>
			<td>Stop solution 2(solution 5)</td>
			<td>9.5 mL solution 1, 0.5 mL FBS (Fetal Bovine Serum), 4&#956;L 100 mM/L CaCl2</td>
			<td>for each heart</td>
		</tr>
		<tr>
			<td>Cell resuspension solution(solution 6)</td>
			<td>13 mL solution 1, 7&#956;L 1M/L CaCl2, BSA (Bull Serum Albumin) at a dose that can form a thin layer covering the surface of the liquid</td>
			<td>for each heart</td>
		</tr>
	</tbody>
</table>
Table 2. Solutions for adult mouse CM isolation and storage.
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/62514/62514fig09.jpg" /> 
Figure 9. Current-voltage curves of the sodium channels.&#160;Whole-cell patch clamp techniques were used to record sodium currents of the isolated cardiomyocytes in the voltage clamp mode. The voltage clamp protocol is shown in the inset. Sodium currents recorded from the AM (A) and VM (B) are shown. The current densities at potentials of &#8722;45mV, &#8722;40mV, and &#8722;35 mV are significantly higher in AM (&#8722;32.71 &#177; 1.597 pA/pF, &#8722;31.49 &#177; 1.820 pA/pF, and &#8722;29.34 &#177; 1.939 pA/pF, respectively; n = 10) than in VM (&#8722;17.66 pA/pF &#177; 1.976 pA/pF, &#8722;21.09 &#177; 1.560 pA/pF, and &#8722;21.86 &#177; 1.381 pA/pF, respectively; n = 8; P &#60; 0.05). (C) The I-V curves show the sodium current densities that were normalized to cell capacitance.&#160;<a href="https://www.jove.com/files/ftp_upload/62514/62514fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/62514/62514fig10.jpg" /> 
Figure 10. Origin and distribution of the atrial vessel. The atrial vessel (arrow 1) and the CA (arrow 2) in panel A. Panel B is a closer look at the atrial vessel (arrow 3). (C) Two different-sized ostia exist; one (arrow 4) corresponds to the atrial vessel that irrigated the atrial appendages, and the other one (arrow 5) corresponds to the CA (stereomicroscope 10 &#215; 5 magnification). CA = coronary artery. <a href="https://www.jove.com/files/ftp_upload/62514/62514fig10large.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 colspan="3">The serial number of the mice</td>
			<td>1</td>
			<td>2</td>
			<td>3</td>
			<td>4</td>
			<td>5</td>
			<td>6</td>
			<td>7</td>
			<td>8</td>
			<td>9</td>
			<td>10</td>
		</tr>
		<tr>
			<td colspan="3">The depth of aorta cannulation is at the ascending aorta</td>
			<td>0.5</td>
			<td>0.6</td>
			<td>0.3</td>
			<td>0.4</td>
			<td>0.8</td>
			<td>0.3</td>
			<td>0.8</td>
			<td>0.7</td>
			<td>0.5</td>
			<td>0.7</td>
		</tr>
		<tr>
			<td colspan="3">The depth of aorta cannulation is at the aortic root</td>
			<td>0.2</td>
			<td>-0.1</td>
			<td>0.1</td>
			<td>0</td>
			<td>-0.1</td>
			<td>0.1</td>
			<td>-0.2</td>
			<td>0</td>
			<td>-0.1</td>
			<td>0</td>
		</tr>
	</tbody>
</table>
Table 3. Distance from the cannula tip to the coronary artery ostium. The hearts of C57BL/6 mice (n = 20) were used to cannulate and ligate the aorta at depths of the ascending aorta and the aortic root, respectively. The aorta is anatomized, and the distance from the cannula tip to the CA ostium was measured. The distance is measured in millimeters, and positive or negative signs represent the cannula tip above and below the CA ostium, respectively.

Watch this Scientific Journal Video about Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice at JoVE.com

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

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 ...

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5.2K Views.  Capital Medical University. 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. 

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

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

The WATCHMAN Left Atrial Appendage Closure Device for Atrial Fibrillation

Atrial fibrillation (AF) is the most common cardiac arrhythmia, affecting an estimated 6 million people in the United States 1. Since AF affects primarily elderly people, its prevalence increases parallel with age. As such, it is expected that 15.9 million Americans will be affected by the year 2050 2. Ischemic stroke occurs in 5% of non-anticoagulated AF patients each year. Current treatments for AF include rate control, rhythm control and prevention of stroke 3.
The American College of Cardiology, American Heart Association, and European Society of Cardiology currently recommended rate control as the first course of therapy for AF 3. Rate control is achieved by administration of pharmacological agents, such as &beta;-blockers, that lower the heart rate until it reaches a less symptomatic state 3. Rhythm control aims to return the heart to its normal sinus rhythm and is typically achieved through administration of antiarrhythmic drugs such as amiodarone, electrical cardioversion or ablation therapy. Rhythm control methods, however, have not been demonstrated to be superior to rate-control methods 4-6. In fact, certain antiarrhythmic drugs have been shown to be associated with higher hospitalization rates, serious adverse effects 3, or even increases in mortality in patients with structural heart defects 7. Thus, treatment with antiarrhythmics is more often used when rate-control drugs are ineffective or contraindicated. Rate-control and antiarrhythmic agents relieve the symptoms of AF, including palpitations, shortness of breath, and fatigue 8, but don't reliably prevent thromboembolic events 6.
Treatment with the anticoagulant drug warfarin significantly reduces the rate of stroke or embolism 9,10. However, because of problems associated with its use, fewer than 50% of patients are treated with it. The therapeutic dose is affected by drug, dietary, and metabolic interactions, and thus requires detailed monitoring. In addition, warfarin has the potential to cause severe, sometimes lethal, bleeding 2. As an alternative, aspirin is commonly prescribed. While aspirin is typically well tolerated, it is far less effective at preventing stroke 10. Other alternatives to warfarin, such as dabigatran 11 or rivaroxaban 12 demonstrate non-inferiority to warfarin with respect to thromboembolic events (in fact, dabigatran given as a high dose of 150 mg twice a day has shown superiority). While these drugs have the advantage of eliminating dietary concerns and eliminating the need for regular blood monitoring, major bleeding and associated complications, while somewhat less so than with warfarin, remain an issue 13-15.
Since 90% of AF-associated strokes result from emboli that arise from the left atrial appendage (LAA) 2, one alternative approach to warfarin therapy has been to exclude the LAA using an implanted device to trap blood clots before they exit. Here, we demonstrate a procedure for implanting the WATCHMAN Left Atrial Appendage Closure Device. A transseptal cannula is inserted through the femoral vein, and under fluoroscopic guidance, inter-atrial septum is crossed. Once access to the left atrium has been achieved, a guidewire is placed in the upper pulmonary vein and the WATCHMAN Access Sheath and dilator are advanced over the wire into the left atrium. The guidewire is removed, and the access sheath is carefully advanced into the distal portion of the LAA over a pigtail catheter. The WATCHMAN Delivery System is prepped, inserted into the access sheath, and slowly advanced. The WATCHMAN device is then deployed into the LAA. The device release criteria are confirmed via fluoroscopy and transesophageal echocardiography (TEE) and the device is released.

The accompanying video describes a procedure for percutaneous placement of the WATCHMAN Left Atrial Appendage (LAA) Device. The WATCHMAN is a nitinol device designed to be permanently implanted at, or slightly distal to, the opening of the left atrial appendage (LAA) to trap blood clots before they exit the LAA, preventing thromboembolic stroke.

Atrial fibrillation (AF) is the most common cardiac arrhythmia, affecting an estimated 6 million people in the United States 1. Since AF affects primarily ...

Catheter Ablation in Combination With Left Atrial Appendage Closure for Atrial Fibrillation

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, affecting millions of individuals worldwide 1-3. The rapid, irregular, and disordered electrical activity in the atria gives rise to palpitations, fatigue, dyspnea, chest pain and dizziness with or without syncope 4, 5. Patients with AF have a five-fold higher risk of stroke 6.
Oral anticoagulation (OAC) with warfarin is commonly used for stroke prevention in patients with AF and has been shown to reduce the risk of stroke by 64% 7. Warfarin therapy has several major disadvantages, however, including bleeding, non-tolerance, interactions with other medications and foods, non-compliance and a narrow therapeutic range 8-11. These issues, together with poor appreciation of the risk-benefit ratio, unawareness of guidelines, or absence of an OAC monitoring outpatient clinic may explain why only 30-60% of patients with AF are prescribed this drug 8.
The problems associated with warfarin, combined with the limited efficacy and/or serious side effects associated with other medications used for AF 12,13, highlight the need for effective non-pharmacological approaches to treatment. One such approach is catheter ablation (CA), a procedure in which a radiofrequency electrical current is applied to regions of the heart to create small ablation lesions that electrically isolate potential AF triggers 4. CA is a well-established treatment for AF symptoms 14, 15, that may also decrease the risk of stroke. Recent data showed a significant decrease in the relative risk of stroke and transient ischemic attack events among patients who underwent ablation compared with those undergoing antiarrhythmic drug therapy 16.
Since the left atrial appendage (LAA) is the source of thrombi in more than 90% of patients with non-valvular atrial fibrillation 17, another approach to stroke prevention is to physically block clots from exiting the LAA. One method for occluding the LAA is via percutaneous placement of the WATCHMAN LAA closure device. The WATCHMAN device resembles a small parachute. It consists of a nitinol frame covered by fabric polyethyl terephthalate that prevents emboli, but not blood, from exiting during the healing process. Fixation anchors around the perimeter secure the device in the LAA (Figure 1). To date, the WATCHMAN is the only implanted percutaneous device for which a randomized clinical trial has been reported. In this study, implantation of the WATCHMAN was found to be at least as effective as warfarin in preventing stroke (all-causes) and death (all-causes) 18. This device received the Conformit&eacute; Europ&eacute;enne (CE) mark for use in the European Union for warfarin eligible patients and in those who have a contraindication to anticoagulation therapy 19.
Given the proven effectiveness of CA to alleviate AF symptoms and the promising data with regard to reduction of thromboembolic events with both CA and WATCHMAN implantation, combining the two procedures is hoped to further reduce the incidence of stroke in high-risk patients while simultaneously relieving symptoms. The combined procedure may eventually enable patients to undergo implantation of the WATCHMAN device without subsequent warfarin treatment, since the CA procedure itself reduces thromboembolic events. This would present an avenue of treatment previously unavailable to patients ineligible for warfarin treatment because of recurrent bleeding 20 or other warfarin-associated problems.
The combined procedure is performed under general anesthesia with biplane fluoroscopy and TEE guidance. Catheter ablation is followed by implantation of the WATCHMAN LAA closure device. Data from a non-randomized trial with 10 patients demonstrates that this procedure can be safely performed in patients with a CHADS2 score of greater than 1 21. Further studies to examine the effectiveness of the combined procedure in reducing symptoms from AF and associated stroke are therefore warranted.

Catheter ablation is combined with placement of the WATCHMAN Left Atrial Appendage Closure Device to prevent ischemic stroke in a patient with non-valvular atrial fibrillation.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, affecting millions of individuals worldwide 1-3. The rapid, irregular, and ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

Isolation and Cultivation of Adult Rat Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).
The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.
Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are &#946;-myosin heavy chain (&#946;-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

Here, we present a protocol for the isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). Isolated ARVC can be used for short and long-term cultivation. The isolation and cultivation of ARVC can play a key role in developing new treatment regimens for cardiac diseases.

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise ...

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 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.

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 ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. However, the evaluation of right ventricular pressure (RVP) and pulmonary artery pressure (PAP) remains technically challenging in mice. RVP and PAP are more difficult to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems. In this paper, we describe a stable right heart hemodynamic measurement method and its validation using healthy and PAH mice. This method is based on open-chest surgery and mechanical ventilation support. It is a complicated procedure compared to closed chest procedures. While a well-trained surgeon is required for this surgery, the advantage of this procedure is that it can generate both RVP and PAP parameters at the same time, so it is a preferable procedure for the evaluation of PAH models.

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...