The authors thank Nadine Woitasky and Peter Volk for technical assistance. Additionally, the authors thank Mrs. Claudia Lorenz (medical writer, ACCEDIS) for her help in preparing the manuscript.&#160;This manuscript was financially supported by DFG (Schlu 324/7-1).

The investigation is conducted according to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996). In general, male wistar rats aged 3 to 4 months and with an average weight of 250 - 350 g are used for this protocol. One rat heart is sufficient for 20 culture dishes (1 mL per dish; inner diameter: 35 mm) with an approximate cell density of 1.5 x 104 cells/1000 mm2.
1. Preparation of Media and Reagents
<ol>
	<li>Creatine-carnitine-taurine medium (CCT medium) 
	NOTE: CCT medium is a complex medium based on medium 199 with the addition of creatine, carnitine, and taurine.
	<ol>
		<li>Prepare 1 L of medium 199: add 3.6 g Hepes and mix for 1 h. Then add 655.5 mg creatine (5 mM), 395.4 mg carnitine (2 mM), and 625.5 mg taurine (5 mM). Carnitine and taurine change pH to &#60;7. In order to inhibit the growth of any contaminating cells, e.g. endothelial cells or fibroblasts, add 10 &#181;M cytosine &#946;-D-arabinofuranoside to the medium. Adjust the pH with NaOH (2 mM) to 7.4 and sterile filter the medium. Store the CCT medium at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Powell medium
	<ol>
		<li>For 1 L Powell medium, dissolve 6.43 g NaCl (110 mM) with 0.19 g KCl (2.5 mM), 0.16 g KH2PO4 (1.2 mM), 0.3 g MgSO4 7H2O (1.2 mM), 5.96 g Hepes (25 mM), and 1.98 g D(+)-Glucose monohydrate (10 mM) in Aqua sterile. Adjust pH with NaOH (2 M) to 7.4 and sterile filter the medium. Store Powell medium at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Calcium chloride (CaCl2)
	<ol>
		<li>Prepare a 100 mM CaCl2 solution (50 mL) and prepare aliquots containing 500 &#181;L CaCl2. Freeze aliquots at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Preparation of culture medium
	<ol>
		<li>Prepare three cell culture mediums: pre-plating medium, plating medium, and washing medium. Use CCT medium as the basis for all three mediums (Table 1). Calculate CCT medium with 1 mL per culture dish. Therefore, prepare 20 mL CCT medium for 20 culture dishes (inner diameter: 35 mm).</li>
		<li>Cell culture plates: Coat each cell culture plate (inner diameter: 35 mm) with 1 mL pre-plating medium. Store the coated plates at 37 &#176;C overnight or for at least 2 h before using.</li>
	</ol>
	</li>
</ol>
2. Isolation of Adult Cardiomyocytes
<ol>
	<li>Preparation of Langendorff perfusion system
	<ol>
		<li>Heat plating medium and washing medium to 37 &#176;C. Defreeze a tube of 500 &#181;L CaCl2 and weigh in 25 mg of collagenase.</li>
		<li>Flush the Langendorff perfusion system with aqua sterile, afterwards let Powell medium circulate the system for 5 min.</li>
		<li>Fill the Langendorff perfusion system with 80 mL Powell medium without any air bubbles and gas the medium with 95% oxygen.</li>
		<li>Prepare a tube (50 mL) with 40 mL Powell medium, heat it to 37 &#176;C, and gas it with 95% oxygen.</li>
		<li>Prepare a thread of about 25 cm in length for attaching the removed heart to the cannula.</li>
		<li>Degrease a razor blade with alcohol (70% by volume) and fasten it to the chopper. Clamp a plastic disc into the chopper.</li>
	</ol>
	</li>
	<li>Extraction of heart
	<ol>
		<li>Anesthetize a male wistar rat with 4% to 5% isoflurane and sacrifice it with cervical dislocation. Open the abdomen behind the costal arch with an abdominal shear and, with the same pair of scissors, cut through the diaphragm to open the thoracic cavity.</li>
		<li>Remove the heart, together with the lung and thymus, by cutting above the thymus highly cranial in the thoracic cavity. Transfer the material to ice-cold saline solution immediately.</li>
		<li>Remove the lung and thymus from the heart with a dissecting scissor (large) and by fixating the material with capsule forceps, transfer the latter to a new saline solution.</li>
	</ol>
	</li>
	<li>Isolation
	<ol>
		<li>Remove excess tissue, like residues of thymus, trachea, fat, and connective tissue from the heart using capsule forceps and a dissecting scissor (large or small). Uncover the aorta and sever it with a dissecting scissor (large or small) between the first and second branchial arch.</li>
		<li>Start the dripping of the Langendorff perfusion system. Place the heart on the cannula of the Langendorff perfusion system and fixate it first with a crocodile clamp and later with the prepared thread. Rinse the heart until it is free of blood.</li>
		<li>Dissolve 25 mg Collagenase in 5 - 6 mL warm Powell medium and add 12.5 &#181;L CaCl2 (30 &#181;M).</li>
		<li>Close circulation by moving a glass funnel, which is connected with the Langendorff perfusion system, over the dripping heart and add the solved collagenase to the perfusion system. Start the perfusion for 25 min with a drop velocity of 1 drop per second. 
		NOTE: During perfusion, the heart will swell and get a waxy appearance.</li>
		<li>Stop the perfusion after 25 min and remove the heart from the Langendorff perfusion system. Remove the aorta, atria, and connective tissue from the heart and open the right and left ventricles.</li>
		<li>Chop the heart two times at an angle of 90&#176; (cutting width: 0.7 mm; velocity: 0.15 cm/s). Repeat this process manually with two scalpels for 10 s each side.</li>
		<li>Transfer 12 mL of the perfusion medium into a new tube (50 mL). Pour the cell slurry into this medium and digest cells for another five minutes at 37 &#176;C. Mix the solution every minute.</li>
		<li>Filter the solution with the digested heart through a nylon mesh (200 &#181;m) into a new tube (50 mL).</li>
		<li>Centrifuge the filtered solution at 29 x g for 3 min. Discard the supernatant and add 6 mL warm Powell medium including 12.5 &#181;L CaCl2 (250 &#181;M) to the cell pellet. Resuspend the pellet through smooth shaking movements. Centrifuge again at 29 x g for 2 min. Discard the supernatant and add 6 mL warm Powell medium substituted with 25 &#181;L CaCl2 (500 &#181;M). Dissolve the cell pellet through gentle shaking movements and add 12 mL warm Powell medium including 120 &#181;L CaCl2 (1 mM). Centrifuge for a third time at 16 x g for 1 min. Again, remove the supernatant.</li>
		<li>Mix the cell pellet with the pre-warmed plating medium.</li>
		<li>Remove pre-plating medium from culture plates. Transfer 1 mL plating medium, including the isolated cardiomyocytes, to each culture plate. Incubate fresh isolated cardiomyocytes at 37 &#176;C for 1 h.</li>
		<li>Remove the plating medium from culture plates. Add 1 mL washing medium to each culture plate and store the plates at 37 &#176;C up to six days without changing the medium.</li>
		<li>For investigating the influence of different chemicals and treatments on ARVC, first refresh plating medium by washing medium and thereafter adding different chemicals. 
		NOTE: Evaluation with light microscopy: With each cell preparation, 150 to 300 cardiomyocytes should be monitored per day by light microscopy. Subdivide all counted cardiomyocytes into groups according to their appearance (e.g., &#34;rod-shaped&#34;, &#34;round down&#34;, &#34;spreading&#34;, and &#34;unusual appearance&#34;). The category &#34;spreading&#34; includes all cardiomyocytes with pseudopodia-like structures. &#34;Unusual appearance&#34; includes all ARVC with an irregular surface and no detectable intact cell membrane.</li>
	</ol>
	</li>
</ol>
3. Example Experiments
<ol>
	<li>Fluorescence/Immunofluorescence staining of adult cardiomyocytes
	<ol>
		<li>Analyze morphological and structural conversions of ARVC during cultivation by confocal laser microscopy. Use Phalloidin-TRITC to investigate F-actin structures in &#34;rod-shaped&#34;, &#34;round down&#34;, and &#34;spreading&#34; ARVC. Perform staining according to the manufacturer&#39;s protocol. An example for Phalloidin-TRITC staining is given in reference Nippert et al.11. With fluorescence/immunofluorescence staining, differences in the de- and re-differentiation of the contractile apparatus in cultivated adult cardiomyocytes between experimental treatments (e.g., with Swiprosin-1, ionomycin) can be investigated.</li>
	</ol>
	</li>
	<li>Real-time quantitative RT-PCR (qRT-PCR)
	<ol>
		<li>Perform qRT-PCR to investigate changes in the mRNA expression of different genes (e.g., OSM, Swiprosin-1, &#946;-MHC) during cultivation of ARVC. For sufficient sample size, use larger culture dishes (inner diameter: 60 mm) with 2 mL volume. ARVC of five culture dishes yields one sample. Perform isolation of mRNA and transformation of cDNA according to the manufacturer&#39;s protocol.</li>
	</ol>
	</li>
	<li>Immunoblot techniques
	<ol>
		<li>Perform Western Blots to investigate changes in protein expression (e.g., for Swiprosin-1) during cultivation of ARVC. Use one culture dish (1 mL) per sample.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Schl&#252;ter, K. -. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cardiomyocytes - Active Players in Cardiac Disease. , (2016).</li><li>Piper, H. M., Probst, I., Schwartz, P., Hutter, F. J., Spieckermann, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+of+calcium+stable+adult+cardiac+myocytes.">Culturing of calcium stable adult cardiac myocytes.</a> J. Mol. Cell. Cardiol. 14 (7), 397-412 (1982).</li><li>Schl&#252;ter, K. -. D., Schreiber, D., Fabbro, D., McCormick, F. <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> Protein Tyrosine Kinases: From Inhibitors to Useful Drugs. 290, 305-314 (2005).</li><li>Eppenberger, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+analysis+of+the+regeneration+of+myofibrils+in+long-term+cultures+of+adult+cardiomyocytes+of+the+rat.">Immunocytochemical analysis of the regeneration of myofibrils in long-term cultures of adult cardiomyocytes of the rat.</a> Dev. Biol. 130 (1), 1-15 (1988).</li><li>Burrows, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cultivation+of+tissues+of+the+chick-embryo+outside+the+body.">The cultivation of tissues of the chick-embryo outside the body.</a> JAMA. 55 (24), 2057-2058 (1910).</li><li>Cavanaugh, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsation,+migration+and+division+in+dissociated+chick+embryo+heart+cells+in+vitro.">Pulsation, migration and division in dissociated chick embryo heart cells in vitro.</a> J Exp Zool A Eco Genet Physiol. 128 (3), 573-589 (1955).</li><li>Muir, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+observation+on+the+cellular+structure+of+cardiac+muscle.">Further observation on the cellular structure of cardiac muscle.</a> J. Anat. 99, 27-46 (1965).</li><li>Powell, T., Twist, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+technique+for+the+isolation+and+purification+of+adult+cardiac+muscle+cells+having+respiratory+control+and+a+tolerance+to+calcium.">A rapid technique for the isolation and purification of adult cardiac muscle cells having respiratory control and a tolerance to calcium.</a> Biochem. Biophys. Res. Com. 72 (1), 327-333 (1976).</li><li>Schwarzfeld, T. <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+development+in+cell+culture+of+myocardial+cells+of+the+adult+rat.">Isolation and development in cell culture of myocardial cells of the adult rat.</a> J. Mol. Cell. Cardiol. 13 (6), 563-575 (1981).</li><li>Kubin, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncostatin+M+is+a+major+mediator+of+cardiomyocyte+dedifferentiation+and+remodeling.">Oncostatin M is a major mediator of cardiomyocyte dedifferentiation and remodeling.</a> Cell Stem Cell. 9 (5), 420-432 (2011).</li><li>Nippert, F., Schreckenberg, R., Hess, A., Weber, M., Schl&#252;ter, K. -. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+Swiprosin-1+on+the+formation+of+pseudopodia-like+structures+and+&#946;-adrenoceptor+coupling+in+cultured+adult+rat+ventricular+cardiomyocytes.">The effects of Swiprosin-1 on the formation of pseudopodia-like structures and &#946;-adrenoceptor coupling in cultured adult rat ventricular cardiomyocytes.</a> PLoS ONE. 11 (12), e0167655 (2016).</li><li>Moses, R. L., Claycomb, 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=Disorganization+and+reestablishment+of+cardiac+muscle+cell+ultrastructure+in+cultured+adult+rat+ventricular+muscle+cells.">Disorganization and reestablishment of cardiac muscle cell ultrastructure in cultured adult rat ventricular muscle cells.</a> J. Ultrastruct. Res. 81 (3), 358-374 (1982).</li><li>Eppenberger-Eberhardt, M., Flamme, I., Kurer, V., Eppenberger, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reexpression+of+&#945;-smooth+muscle+actin+isoform+in+cultured+adult+rat+cardiomyocytes.">Reexpression of &#945;-smooth muscle actin isoform in cultured adult rat cardiomyocytes.</a> Dev. Biol. 139 (2), 269-278 (1990).</li><li>Fedak, P. W., Verma, S., Weisel, R. D., Skrtic, M., Li, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+remodeling+and+failure:+from+molecules+to+man+(Part+III).">Cardiac remodeling and failure: from molecules to man (Part III).</a> Cardiovasc. Pathol. 14 (3), 109-119 (2005).</li><li>Leri, A., Kajstura, J., Anversa, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+myocardial+regeneration.">Mechanisms of myocardial regeneration.</a> Trends Cardiovasc. Med. 21 (2), 52-58 (2011).</li><li>Cohn, J. N., Ferrari, R., Sharpe, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+remodeling-concepts+and+clinical+implications:+A+consensus+paper+from+an+international+forum+on+cardiac+remodeling.">Cardiac remodeling-concepts and clinical implications: A consensus paper from an international forum on cardiac remodeling.</a> J. Am. Coll. Cardiol. 35 (3), 569-582 (2000).</li><li>Alberts, 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=.">.</a> Molecular biology of the cell. , (2015).</li><li>P&#246;ling, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Janus+face+of+OSM-mediated+cardiomyocyte+dedifferentiation+during+cardiac+repair+and+disease.">The Janus face of OSM-mediated cardiomyocyte dedifferentiation during cardiac repair and disease.</a> Cell Cycle. 11 (3), 439-445 (2014).</li><li>Decker, M. L., Behnke-Barclay, M., Cook, M. G., Lesch, M., Decker, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphometric+evaluation+of+the+contractile+apparatus+in+primary+cultures+of+rabbit+cardiac+myocytes.">Morphometric evaluation of the contractile apparatus in primary cultures of rabbit cardiac myocytes.</a> Circ. Res. 69 (1), 86-94 (1991).</li></ol>

The results shown are part of the doctoral thesis of Franziska Nippert.

The behavior of adult cardiomyocytes in vivo is influenced by many interactions with other cells (e.g., neurons, endothelial cells, fibroblasts, inflammatory cells) and the electrical syncytium which they form1. Therefore, studying stress adaptation of adult cardiomyocytes exclusively requires the isolation and cultivation of ARVC. The main effects of isolating and cultivating ARVC are: 1) disconnecting them from extracellular matrix and cell-cell contacts; 2) disconnecting them ...

Adult cardiomyocytes in vivo work as an electrical syncytium based on cell-cell contacts between myocytes. In addition, they are influenced by adjacent cells like cardiac fibroblasts, endothelial cells, neurons, and inflammatory cells1. In order to study the ability of cardiomyocytes to adapt their intracellular organization to altered load conditions, as seen during cardiac hypertrophy, which is an initial step leading to heart failure, the isolation and cultivation of adult ventricular rat cardiomyocytes (ARVC) is necessary2,3,4. Historically, cardiomyocytes were first isolated from embryonic chick hearts5,6. A few years later, the first isolation of terminally differentiated cardiomyocytes was described by using calcium depletion7. However, these adult cardiomyocytes were not calcium tolerant and could therefore not be used for functional assays. Finally, in 1976 a new protocol enabled Powell and Twist to investigate adult ventricular cardiomyocytes under physiological conditions8. As a first step, they isolated adult cardiomyocytes under low calcium concentrations and thereafter increased calcium to physiological concentrations in a stepwise procedure. Today, most protocols for the isolation and cultivation of adult cardiomyocytes work with this calcium protocol and use collagenase for the enzymatic digestion of the dense cell-cell contacts1.
For a successful cultivation, fetal calf serum (FCS) or oncostatin M (OSM) is required. ARVC perform a de- and re-differentiation with extensive structural changes including sarcomere disassembly and reformation9,10,11,12. This process is accompanied by a re-expression of fetal-type genes, like &#946;-myosin heavy chain (&#946;-MHC), as known from hypertrophy, and a formation of pseudopodia-like structures, also called spreading4,11,13. Furthermore, swiprosin-1 (EFHD2), a newly identified protein, plays a major role in the process of re-differentiation of cultivated ARVC11. As a result, ARVC in culture transform into widespread, polymorphic cells, which spontaneously show contractions after two to three weeks in culture2,4,14.
Recent discoveries have revealed that cardiac re- and de-differentiation as it occurs under culture conditions mimics features seen in vivo during cardiac remodeling10,15. Cardiac remodeling is a key process during cardiac diseases16. As cardiac diseases are still the main cause of death in industrialized societies, a better understanding of the biology of adult cardiomyocytes is important (WHO; 2015). Isolation and cultivation of ARVC can help to develop new strategies and medicines for the treatment of cardiac diseases. With this manuscript, a protocol for the isolation and cultivation of ARVC is provided. Furthermore, some critical parts of this method are highlighted in the discussion section.

<table><tbody><tr><td>Langendorff perfusion system</td><td>inhouse construction</td><td></td><td>double-walled with a water&lt;br/&gt; based heating system</td></tr><tr><td>Tissue chopper Mc Ilwain</td><td>Cavey Laboratory Engeneering Co. Ltd.</td><td></td><td></td></tr><tr><td>Aortic Cannula, OD 1,8 mm</td><td>inhouse construction</td><td></td><td></td></tr><tr><td>Abdominal shears</td><td>Aeskulap</td><td>BC772R</td><td></td></tr><tr><td>Capsule forceps</td><td>Eickemeyer</td><td>171307</td><td></td></tr><tr><td>Dissecting scissor large</td><td>Aeskulap</td><td>BC562R</td><td></td></tr><tr><td>Dissecting scissor small</td><td>Aeskulap</td><td>BC163R</td><td></td></tr><tr><td>Mash with Polyamid</td><td>Neolab</td><td>4-1413</td><td>mash size 200 &amp;mu;m</td></tr><tr><td>plastic disc</td><td>Cavey Laboratory Engeneering Co. Ltd.</td><td></td><td></td></tr><tr><td>Collagenase Typ II</td><td>Worthington</td><td>LS004177</td><td></td></tr></tbody></table>

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.

Adult cardiomyocytes in culture: Figure 1 shows an overview of freshly isolated adult cardiomyocytes 2 h after the last washing. Approximately 75% of all cardiomyocytes had a rod-shaped morphology. The remaining 25% showed an unusual appearance with a round morphology and no detectable intact cell membrane (Figure 1). At the end of cultivation (day 6), up to 15% of all cardiomyocytes showed spreading, about 10% r...

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Isolation and Cultivation of Adult Rat 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-and-cultivation-of-adult-rat-cardiomyocytes

Research

JoVE Journal

Medicine

15.5K Views.  Justus Liebig University Giessen. 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.

Video: Isolation and Cultivation of Adult Rat Cardiomyocytes

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

Developmental Biology

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Biology

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

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

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

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

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

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

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

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

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

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

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

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

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

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

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

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

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

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Isolation and Cultivation of Adult Rat Cardiomyocytes

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