Name: isolation of atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction
Uploaded: 2018-07-26T14:00:00
Description: Watch this Scientific Journal Video about Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction at JoVE.com

This research was supported by the DZHK (German Centre for Cardiovascular Research, D.B.), the EKFS (Else-Kr&#246;ner-Fresenius Stiftung, F.H.), and by the BMBF (German Ministry of Education and Research), as well as the BIH-Charit&#233; clinical scientist program funded by the Charit&#233; - Universit&#228;tsmedizin Berlin and the Berlin Institute of Health (F.H.).

All experiments were approved by the local Ethics Committee (TVA T0060/15 and T0003-15) and performed in agreement with the Guidelines for the Care and Use of Laboratory Animals (National Institute of Health, U.S.A.).
NOTE: A simplified flowchart of the procedure is shown in Figure 1.
1. Prearrangements
<ol>
	<li>Prepare the buffers according to Table 1.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo...

<ol>
	<li>Alberti, K. 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=Harmonizing+the+metabolic+syndrome:+a+joint+interim+statement+of+the+International+Diabetes+Federation+Task+Force+on+Epidemiology+and+Prevention;+National+Heart,+Lung,+and+Blood+Institute;+American+Heart+Association;+World+Heart+Federation;+International+Atherosclerosis+Society;+and+International+Association+for+the+Study+of+Obesity.">Harmonizing the metabolic syndrome: a joint interim statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity.</a> Circulation. 120 (16), 1640-1645 (2009).</li><li>. IDF Consensus Worldwide Definition of the Metabolic Syndrome Available from: <a target="_blank" href="https://www.idf.org/e-library/consensus-statements/60-idfconsensus-worldwide-definitionof-the-metabolic-syndrome.html">https://www.idf.org/e-library/consensus-statements/60-idfconsensus-worldwide-definitionof-the-metabolic-syndrome.html</a> (2006)</li><li>Melenovsky, V., 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=Left+atrial+remodeling+and+function+in+advanced+heart+failure+with+preserved+or+reduced+ejection+fraction.">Left atrial remodeling and function in advanced heart failure with preserved or reduced ejection fraction.</a> Circulation: Heart Failure. 8 (2), 295-303 (2015).</li><li>Goette, A., 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=EHRA/HRS/APHRS/SOLAECE+expert+consensus+on+atrial+cardiomyopathies:+definition,+characterization,+and+clinical+implication.">EHRA/HRS/APHRS/SOLAECE expert consensus on atrial cardiomyopathies: definition, characterization, and clinical implication.</a> EP Europace. 18 (10), 1455-1490 (2016).</li><li>Schotten, U., Verheule, S., Kirchhof, P., Goette, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiological+mechanisms+of+atrial+fibrillation:+a+translational+appraisal.">Pathophysiological mechanisms of atrial fibrillation: a translational appraisal.</a> Physiological Reviews. 91 (1), 265-325 (2011).</li><li>Hohendanner, F., DeSantiago, J., Heinzel, F. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dyssynchronous+calcium+removal+in+heart+failure-induced+atrial+remodeling.">Dyssynchronous calcium removal in heart failure-induced atrial remodeling.</a> American Journal of Physiology-Heart and Circulatory Physiology. 311 (6), H1352-H1359 (2016).</li><li>Hohendanner, F., 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=Inositol-1,4,5-trisphosphate+induced+Ca2++release+and+excitation-contraction+coupling+in+atrial+myocytes+from+normal+and+failing+hearts.">Inositol-1,4,5-trisphosphate induced Ca2+ release and excitation-contraction coupling in atrial myocytes from normal and failing hearts.</a> The Journal of Physiology. 593 (6), 1459-1477 (2015).</li><li>Tada, 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=Role+of+mineralocorticoid+receptor+on+experimental+cerebral+aneurysms+in+rats.">Role of mineralocorticoid receptor on experimental cerebral aneurysms in rats.</a> Hypertension. 54 (3), 552-557 (2009).</li><li>Iwasaki, Y. 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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. (91), e51109 (2014).</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=Isolation+and+cultivation+of+Ca2++tolerant+cardiomyocytes+from+the+adult+rat:+improvements+and+applications.">Isolation and cultivation of Ca2+ tolerant cardiomyocytes from the adult rat: improvements and applications.</a> Xenobiotica. 30 (11), 1063-1077 (2000).</li><li>Egorova, M. V., Afanas'ev, S. A., Popov, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+isolation+of+cardiomyocytes+from+adult+rat+heart.">A simple method for isolation of cardiomyocytes from adult rat heart.</a> Bulletin of Experimental Biology and Medicine. 140 (3), 370-373 (2005).</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. (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. (92), e51823 (2014).</li><li>Hohendanner, F., 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=Cellular+mechanisms+of+metabolic+syndrome-related+atrial+decompensation+in+a+rat+model+of+HFpEF.">Cellular mechanisms of metabolic syndrome-related atrial decompensation in a rat model of HFpEF.</a> Journal of Molecular and Cellular Cardiology. 115, 10-19 (2017).</li><li>Seluanov, A., Vaidya, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+primary+adult+fibroblast+cultures+from+rodents.">Establishing primary adult fibroblast cultures from rodents.</a> Journal of Visualized Experiments. (44), e2033 (2010).</li><li>Bootman, M. D., Higazi, D. R., Coombes, S., Roderick, H. 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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=Diastolic+dysfunction+and+left+atrial+volume:+a+population-based+study.">Diastolic dysfunction and left atrial volume: a population-based study.</a> Journal of the American College of Cardiology. 45 (1), 87-92 (2005).</li><li>Linz, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cathepsin+A+mediates+susceptibility+to+atrial+tachyarrhythmia+and+impairment+of+atrial+emptying+function+in+Zucker+diabetic+fatty+rats.">Cathepsin A mediates susceptibility to atrial tachyarrhythmia and impairment of atrial emptying function in Zucker diabetic fatty rats.</a> Cardiovascular Research. 110 (3), 371-380 (2016).</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>Ramanathan, T., Skinner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+blood+flow.">Coronary blood flow.</a> Continuing Education in Anaesthesia Critical Care &amp; Pain. 5 (2), 61-64 (2005).</li><li>Bond, M. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[On+the+Quantitative+Determination+of+Collagenase].">[On the Quantitative Determination of Collagenase].</a> Hoppe-Seyler's Zeitschrift f&#252;r physiologische Chemie. 333, 149-151 (1963).</li><li>Conceicao, G., Heinonen, I., Lourenco, A. P., Duncker, D. 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Here, we first described a protocol for the isolation of single-cell ACMs from a rat model of MetS-related HFpEF that shows marked atrial remodeling22. The procedure is uniquely challenging as excessive fatty tissue can make the surgical preparation, as well as the cannulation of the aorta, increasingly difficult. The troubleshooting guide provided in Table 2 addresses the most common issues of the isolation procedure.
<table border="1" fo:keep-together.within-page="1" fo:keep...

MetS describes a cluster of risk factors for diabetes mellitus type-2 and cardiovascular disease and includes an increased arterial blood pressure, dyslipidemia (raised triglycerides and lowered high-density lipoprotein cholesterol), increased fasting glucose, and central obesity1. The worldwide prevalence of MetS is estimated to be 25&#8211;30% and constantly rising2. HFpEF is a heterogenous clinical syndrome often associated with MetS. The cardiac remodeling during HFpEF and its preceding phases (i.e., hypertensive heart disease) is also accompanied by a remodeling of the atria3. Reduced contractile function and structural changes of the left atrium have been associated with increased mortality, atrial fibrillation, and new-onset heart failure4. Atrial remodeling is characterized by changes in the ion channel function, Ca2+ homeostasis, atrial structure, fibroblast activation, and tissue fibrosis5. Left atrial remodeling in MetS-related HFpEF and its underlying pathological mechanisms are still poorly understood and require a further in-depth investigation. Animal models have proven to be a valuable tool and lead to many advances in the field of atrial cardiomyopathies6,7,8,9.
Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. An isolation, and the potential subsequent cell culture, allow for the investigation of signaling pathways, ionic channel currents, and excitation-contraction-coupling. Under physiologic conditions, cardiomyocytes do not proliferate. The fusion between the transcriptional regulatory sequences of an atrial natriuretic factor and a simian virus 40 large T antigen in transgenic mice led to the creation of the first immortalized ACMs, named AT-110. The further development of AT-1 cells gave rise to HL-1 cells, which cannot only be serially passaged but also contract spontaneously11. They do, however, show structural and functional differences compared to freshly isolated cells, such as a less organized ultrastructure, a high occurrence of developing myofibrils11, and a hyperpolarization-activated inward current12. The isolation of ventricular cardiomyocytes (VCM) in rats and mice from a variety of models is well established13,14,15,16,17,18,19. Generally, the excised heart is mounted to a Langendorff apparatus and retrogradely perfused with a Ca2+-free buffer containing digestive enzymes, such as collagenases and proteases. Calcium is then reintroduced in a stepwise manner to the physiological conditions. However, even though protocols dedicated to the isolation of ACMs are available20,21, due to increased fibrosis and pressure-related differences, their usefulness in disease models with atrial remodeling is limited.
In this article, we have implemented a protocol for the isolation of atrial single-cell cardiomyocytes from animals that show atrial remodeling (i.e., in particular for the ZFS1 rat model for MetS-related HFpEF)22. Existing isolation protocols were optimized and complemented by a simple, custom-made device to control and modify the intraluminal pressure of the cardiac cavities, leading to higher yields of morphologically and functionally intact cardiomyocytes. The following protocol provides the researcher with a step-by-step guide, a detailed description of the custom-made equipment, a list of solutions, as well as a comprehensive troubleshooting guide.

<table border="0" cellpadding="0" cellspacing="0" width="935">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">ZSF-1 Obese rat</td>
			<td style="width:292px;">Charles River Laboratories, Inc.</td>
			<td style="width:136px;"></td>
			<td style="width:227px;">21 weeks old</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Fine Iris Scissors</td>
			<td style="width:292px;">Fine Science Tools GmbH</td>
			<td style="width:136px;">14094-11</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Surgical Scissors</td>
			<td style="width:292px;">Fine Science Tools GmbH</td>
			<td style="width:136px;">14001-18</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Micro Dressing Forceps (curved, serrated)</td>
			<td style="width:292px;">Aesculap, Inc.</td>
			<td style="width:136px;">BD312R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Tissue Forceps (straight, 1 x 2 teeth)</td>
			<td style="width:292px;">Aesculap, Inc.</td>
			<td style="width:136px;">BD537R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Tying Forceps (angled)</td>
			<td style="width:292px;">Aesculap, Inc.</td>
			<td style="width:136px;">MA624R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Rodent and Small Animal Guillotine</td>
			<td style="width:292px;">Kent Scientific Corp.</td>
			<td style="width:136px;">DCAP</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Low Cost Induction Chamber 3.0 L</td>
			<td style="width:292px;">Kent Scientific Corp.</td>
			<td style="width:136px;">SOMNO-0730&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Butterfly Winged Infusion Set 21 G</td>
			<td style="width:292px;">Hospira, Inc.</td>
			<td style="width:136px;">181106101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Abbocath 16 G</td>
			<td style="width:292px;">Hospira, Inc.</td>
			<td>0G7149702</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Microlance Hypodermic Needle</td>
			<td style="width:292px;">Becton Dickinson GmbH</td>
			<td style="width:136px;">301300</td>
			<td>modify needle to make cannula</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Braun Original Perfusor Syringe 50 ml</td>
			<td style="width:292px;">B. Braun Melsungen AG</td>
			<td style="width:136px;">8728810F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Braun Inject Solo Syringe 10 ml</td>
			<td style="width:292px;">B. Braun Melsungen AG</td>
			<td style="width:136px;">2057926</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Beaker 50ml</td>
			<td style="width:292px;">Duran Group (DWK Life Sciences GmbH)</td>
			<td>21 106 17</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Duroplan petri dish (100 x 20 mm)</td>
			<td style="width:292px;">Duran Group (DWK Life Sciences GmbH)</td>
			<td style="width:136px;">21 755 48</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Seraflex Suture USP 3/0</td>
			<td style="width:292px;">SERAG-WIESSNER GmbH &#38; Co. KG</td>
			<td style="width:136px;">IC208000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">VWR disposable Square Weighin Boats 100ml</td>
			<td style="width:292px;">VWR, Inc.</td>
			<td style="width:136px;">10803-148</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Styrofoam surface</td>
			<td style="width:292px;"></td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Sodium chloride</td>
			<td style="width:292px;">Sigma-Aldrich, Inc.</td>
			<td style="width:136px;">71380</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Potassium chloride</td>
			<td style="width:292px;">Sigma-Aldrich, Inc.</td>
			<td style="width:136px;">P4504</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Potassium phosphate monobasic</td>
			<td style="width:292px;">Sigma-Aldrich, Inc.</td>
			<td style="width:136px;">P5379</td>
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			<td style="width:292px;">Invitrogen (Thermo Fisher Scientific, Inc.)</td>
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			<td height="20" style="height:20px;width:281px;">Di-8-ANNEPS</td>
			<td style="width:292px;">Invitrogen (Thermo Fisher Scientific, Inc.)</td>
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			<td height="20" style="height:20px;width:281px;">Mitotracker RED FM</td>
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			<td style="width:292px;">Gebr. Rettberg GmbH</td>
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			<td style="width:292px;">Gebr. Rettberg GmbH</td>
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			<td style="width:292px;">Ismatec (Cole-Parmer Gmbh)</td>
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			<td style="width:292px;">Conrad Electronic SE</td>
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			<td height="20" style="height:20px;width:281px;">Tubing</td>
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			<td style="width:292px;">Warner Instruments, LLC</td>
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			<td height="20" style="height:20px;width:281px;">8 channel perfusion system</td>
			<td style="width:292px;">Warner Instruments, LLC</td>
			<td style="width:136px;">64-0185</td>
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			<td height="20" style="height:20px;width:281px;">8 channel Multi-Line In-Line Solution Heaters</td>
			<td style="width:292px;">Warner Instruments, LLC</td>
			<td style="width:136px;">64-0105</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

At 21 weeks of age, 60&#8211;90% of viable ACMs (estimated as described in step 6.1), after the calcium re-adaptation (step 5.4&#8211;5.7), can be isolated from ZSF-1 obese rats by this method (Figure 4A). In rats, ACMs are characterized by a different and more heterogenous phenotype compared to VCMs24,25. Figure 4B shows an individual ACM with preserved membranes and sar...

Watch this Scientific Journal Video about Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction at JoVE.com

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

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 ...

This method can help answer key questions in the field of individual cardiomyocyte function and allows studying excitation-contraction-coupling. The main advantage of this technique is that a high yield of viable atrial cardiomyocytes from hearts with atrial cardiomyopathy can be isolated and used for subsequent experiments. Begin by placing a cannula with the long, sharp tip removed on top of the Langendorff apparatus and start the flow.Turn on the heating module and adjust so that the perfusion buffer at the tip of the needle maintains the desired temperature for perfusion. Once the temperature calibration is complete, transfer the cannula to a 10 ml syringe filled with ice-cold cannulation buffer. Place two ready-prepared double overhand knots over the cannula for rapid subsequent cannulation of the aorta.Remove the heart from the euthanized, heparinized rat by pinching the base of the heart with forceps and gently pulling down towards the tail of the animal. Cut across the aorta while maintaining a pull on the heart, leaving a 5-mm-long segment of the aorta attached to the heart. Quickly transfer the heart into 50 ml of ice-cold cannulation buffer in a 50 ml beaker.Wait approximately 10 s for the heart to cool and cease contractions. Then, transfer the heart into a Petri dish, containing 50 ml of fresh, ice-cold cannulation buffer and carefully remove the fatty tissues surrounding the aorta using forceps and scissors. Insert the modified cannula 3 mm into the aorta without damaging the aortic valve.Fix the aorta onto the cannula by tying one of the two cannulation knots at the indentation proximal to the tip of the cannula. Tie the second cannulation knot at the indentation distal to the tip of the cannula. Gently flush the aorta with 5 ml of ice-cold cannulation buffer using the syringe attached to the cannula until no blood is visible in the coronary arteries.Then, use forceps to gently massage the left atrium while injecting the remaining 5 ml of cannulation buffer to flush any remaining blood. Unmount the cannula with the attached heart from the syringe. Mount the cannula with the attached heart to the Langendorff, using an appropriate adapter.Start the peristaltic pump of the Langendorff apparatus to initiate the perfusion of the cardiac tissue. Quickly tie a double overhand knot around the base of the heart excluding the aorta. When the right and left atrium and coronary sinus inflate, puncture the atrium with the butterfly needle of the pressure control device and allow the atrium to deflate.Manipulate the intraluminal pressure of the atrium by adjusting the elevation of the hose attached to the butterfly needle. Keep the atrium slightly inflated throughout the rest of the procedure. It is critical that the step is performed swiftly, as a prolonged inflation of the atrium will result in cardiomyocyte death.Position a temperature probe between the left atrium and the left ventricle to measure the approximate temperature of the left atrium. Adjust the temperature of the Langendorff heating module accordingly, targeting an approximate temperature of 37 C of the left atrium. After 3 min of perfusion with perfusion buffer, switch to digestion buffer and perfuse for approximately 14-18 min.When the left atrial structure collapses and the tissue acquires a milky texture, pinch the atrium using forceps and an act of slight pull. Remove the left atrium using fine scissors and transfer the atrium into a large weighing boat containing 2 ml of stopping buffer, fully submerging the tissue. Use fine scissors to mince the atrial tissue into small pieces of roughly 2 sq mm.Disperse the tissue by gently triturating the tissue with a transfer pipette for approximately 5 min until a macroscopic dissociation of the tissue can be observed. Avoid generating air bubbles, as exposing the cells to air will result in cardiomyocyte death. Then, transfer the cell suspension to a 15 ml conical tube.After allowing the tissue chunks to settle for 30 s, remove the supernatant and transfer to another 15 ml conical tube. Then allow the cells to settle for 15 min. After removing and discarding the supernatant, add 2 ml of Step 1 Buffer and allow the cardiomyocytes to settle for 10 min.After 10 min, remove and discard the final supernatant. Add 250 microliters of normal Tyrode, containing 1 mM calcium. Transfer 50 microliters of the atrial cardiomyocyte suspension onto a laminin-coated glass bottom dish and allow the cardiomyocytes to settle at room temperature for 10 min.After 10 min, add 500 microliters of normal Tyrode containing 1 mM calcium chloride to the cells. Evaluate cell morphology and viability under a light microscope. Randomly select approximately 100 cells and classify them as either viable or unviable to estimate the viability of the cell isolation procedure.Viable cells are characterized by symmetric sarcomere structure, the absence of membrane blebs and a rod shape. Add 10 micromolar Fluo-4-AM to 500 microliters of normal Tyrode and use this to replace the normal Tyrode containing 1 mM calcium chloride in the glass bottom dish. After 20 min, remove the Fluo-4-AM solution and wash the cells twice with 500 microliters of normal Tyrode.Transfer the dish to a confocal microscope and visualize the calcium excitation, using 40 x magnification. Then, perfuse the cells with normal Tyrode heated to 37 C using an appropriate superfusion device. Stimulate the cardiomyocytes in an electrical field with commercially available, microscope-mounted stimulator electrodes at a frequency of 1 Hz and an electrical current of 24 Amps.After waiting for 1 min to allow the cells to reach a steady state of calcium ion handling, acquire line scan images by repetitive scanning. This image shows the transverse line scan of a single cardiomyocyte. The change of cytosolic calcium concentration is shown by excitation of the calcium-sensitive fluorescent dye, Fluo-4-AM.The arrows indicate the electrical stimulation conducted at 1 Hz.This image shows the transverse calcium transients derived from this single cell. Once mastered, this technique can be performed in 3 h. While attempting this protocol, it is important to remember the time sensitivity of certain steps in the isolation procedure.Following this procedure, other methods of sub-cellular imaging can be performed in order to answer additional questions regarding atrial cardiomyocyte remodeling.

Research

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