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 
			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 &#956;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 ...

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15.4K 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 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 ...

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 ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

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 ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

Ultrasonic-augmented Primary Adult Fibroblast Isolation

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary fibroblasts cannot divide indefinitely due to myofibroblast differentiation or senescence induction, new cultures must be established regularly. However, there are several obstacles to overcome during the processes of developing a reliable isolation protocol and primary fibroblast isolation itself: the method&#8217;s degree of difficulty (especially for beginners), the risk of bacterial contamination, the required time until primary fibroblasts can be used for experiments, and subsequent cell quality and viability. In this study, a fast, reliable and easy-to-learn protocol to isolate and culture primary adult fibroblasts from mouse heart, lung, liver and kidney combining enzymatic digestion and ultrasonic agitation is provided.

We present a protocol to isolate primary adult fibroblasts in an easy, fast and reliable way, performable by beginners (e.g., students). The procedure combines enzymatic tissue digestion and mechanical agitation with ultrasonic waves to obtain primary fibroblasts. The protocol can easily be adapted to specific experimental requirements (e.g., human tissue).

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...

Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous high-quality cells for experimental requirements. mBMSCs could be widely used in therapeutic applications as tissue engineering cells in case of craniofacial diseases and cranio-maxillofacial regeneration in the future due to the excellent self-renewal ability and multi-lineage differentiation potential. Therefore, it is important to obtain mBMSCs in large numbers.
In this study, bone marrow was flushed from the mandible and primary mBMSCs were isolated through whole bone marrow adherent cultivation. Furthermore, CD29+CD90+CD45&#8722; mBMSCs were purified through fluorescent cell sorting. The second generation of purified mBMSCs were used for further study and displayed potential in differentiating into osteoblasts, adipocytes, and chondrocytes. Utilizing this in vitro model, one can obtain a high number of proliferative mBMSCs, which may facilitate the study of the biological characteristics, the subsequent reaction to the microenvironment, and other applications of mBMSCs.

This article presents a method that combines whole bone marrow adherence and flow cytometry sorting for isolating, cultivating, sorting, and identifying bone marrow mesenchymal stem cells from rat mandibles.

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous ...

Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells provide promising sources for cardiac disease modeling. For cardiomyopathies, sarcomere shortening is one of the standard physiological assessments that are used with adult cardiomyocytes to examine their disease phenotypes. However, the available methods are not appropriate to assess the contractility of PSC-CMs, as these cells have underdeveloped sarcomeres that are invisible under phase-contrast microscopy. To address this issue and to perform sarcomere shortening with PSC-CMs, fluorescent-tagged sarcomere proteins and fluorescent live-imaging were used. Thin Z-lines and an M-line reside at both ends and the center of a sarcomere, respectively. Z-line proteins &#8212;&#160;&#945;-Actinin (ACTN2), Telethonin (TCAP), and actin-associated LIM protein (PDLIM3) &#8212; and one M-line protein &#8212; Myomesin-2 (Myom2) &#8212;&#160;were tagged with fluorescent proteins. These tagged proteins can be expressed from endogenous alleles as knock-ins or from adeno-associated viruses (AAVs). Here, we introduce the methods to differentiate mouse and human pluripotent stem cells to cardiomyocytes, to produce AAVs, and to perform and analyze live-imaging. We also describe the methods for producing polydimethylsiloxane (PDMS) stamps for a patterned culture of PSC-CMs, which facilitates the analysis of sarcomere shortening with fluorescent-tagged proteins. To assess sarcomere shortening, time-lapse images of the beating cells were recorded at a high framerate (50-100 frames per second) under electrical stimulation (0.5-1 Hz). To analyze sarcomere length over the course of cell contraction, the recorded time-lapse images were subjected to SarcOptiM, a plug-in for ImageJ/Fiji. Our strategy provides a simple platform for investigating cardiac disease phenotypes in PSC-CMs.

This method can be used to examine sarcomere shortening using pluripotent stem cell-derived cardiomyocytes with fluorescent-tagged sarcomere proteins.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells ...

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

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

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

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