Name: isolation and cultivation of adult rat cardiomyocytes
Uploaded: 2017-10-19T15:00:00
Description: Watch this Scientific Journal Video about Isolation and Cultivation of Adult Rat Cardiomyocytes at JoVE.com

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 Me...

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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...

Watch this Scientific Journal Video about Isolation and Cultivation of Adult Rat Cardiomyocytes at JoVE.com

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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