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 ...

The overall goal of this experiment is to isolate and cultivate adult rat cardiomyocytes, which can be used in various experiments to understand their biological and pathophysiological behavior. This method can help answer key questions in the field of biology and pathophysiology of the heart, such as cardiac dedifferentiation, cardiac hypertension, or heart failure. The main advantage of this technique is that adult cardiomyocytes can be investigated individually without the influence of adjacent cells, such as fibroblasts or endothelial cells.The implications of this technique extend toward pathophysiological understanding of heart diseases because adult cardiac myocytes can be investigated individually. Though this method can provide insight into the behavior of adult rat cardiomyocytes, it can also be applied to other models, such as ischemia reperfusion models, spontaneously hypertensive rats, or other species. The Langendorff perfusion system consists of a long, double-walled glass tube with a glass funnel at the top and the bottom of the tube.The apparatus can be heated by water. The top and lower glass funnels are connected with a pump that transports the perfusion medium, which is fumigated with 95%oxygen. To prepare the perfusion system, heat the plating medium and washing medium to 37 degrees Celsius.Then, defreeze a tube of 500 microliters calcium chloride, and weigh 25 milligrams of collagenase. Next, flush the Langendorff perfusion system with aqua sterile. Afterwards, let Powell medium circulate the system for five minutes.Next, prepare a tube with 40 milliliters of Powell medium, and cover it with Parafilm. Heat the tube to 37 degrees Celsius, and gas it with 95%oxygen. Remove the circulating Powell medium, and fill the Langendorff perfusion system with 80 milliliters of fresh Powell medium without any air bubbles, and gas the medium with 95%oxygen.Then, prepare a thread of about 25 centimeters in length for attaching the removed heart to the cannula. Degrease a razor blade with alcohol, and fasten it to the chopper. Finally, clamp a plastic disk into the chopper.After sacrificing male, Wistar rats, as described in the text protocol, 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. 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.Remove the lung and thymus from the heart with a large dissecting scissor. Then, by fixating the material with capsule forceps, transfer the heart to a new saline solution. The following steps must be performed precisely but also quickly to minimize the time between extraction of the heart and fixation to the Langendorff perfusion system.This is essential for a good result in quality and number of the adult cardiomyocytes. Remove excess tissue, like residues of thymus, trachea, fat, and connective tissue from the heart, using capsule forceps and a dissecting scissor. Uncover the aorta, and sever it with a dissecting scissor between the first and second branchial arch.Next, 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.Dissolve 25 milligrams of collagenase in five to six milliliters of warm Powell medium, and add 12.5 microliters of calcium chloride. When the residue volume is 45 milliliters, close circulation by moving a glass funnel, which is connected with the Langendorff perfusion system over the dripping heart, and cover this funnel with Parafilm for protection of the heart. Add the solved collagenase to the perfusion system.Adjust the perfusion system for 25 minutes with a drop velocity of one drop per second. After 25 minutes of perfusion, the heart appears swollen and waxy. Stop the perfusion after 25 minutes, 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. Chop the heart two times at an angle of 90 degrees, repeating this process manually with two scalpels for 10 seconds each side. Next, transfer 12 milliliters of the perfusion medium into a new tube.Pour the cell slurry into this medium, and digest cells for another five minutes at 37 degrees Celsius. Mix the solution every minute. Filter the solution with the digested heart through a 200 micron nylon mesh into a new tube.Recalcification is a sensitive process. If other rat models or different species are used, the demonstrated protocol can be too aggressive, which would lead to a high mortality rate of the cells. Centrifuge the filtered solution at 29 g for three minutes.Discard the supernatant, and add six milliliters of warm Powell medium, including 12.5 microliters of calcium chloride to the cell pellet. Resuspend the pellet through smooth shaking movements. Then, centrifuge again at 29 g for two minutes.Discard the supernatant, and add six milliliters of warm Powell medium, substituted with 25 microliters of calcium chloride. Dissolve the cell pellet through gentle shaking movements, and add 12 milliliters of warm Powell medium, including 120 microliters of calcium chloride. After centrifuging for a third time at 16 g for one minute, remove the supernatant.Next, mix the cell pellet with the pre-warmed plating medium. Remove the pre-plating medium from the culture plates. Then, transfer one milliliter of plating medium, including the isolated cardiomyocytes, to each culture plate.Incubate the fresh isolated cardiomyocytes at 37 degrees Celsius for one hour. Remove the plating medium from the culture plates. Finally, add one milliliter of washing medium to each culture plate.Store the plates at 37 degrees Celsius up to six days without changing the medium. Freshly isolated ARVC show a typical rod-shaped morphology. ARVC with an irregular surface and no detectable intact cell membrane can be assumed not to be viable.This morphology changes over the period of cultivation in a medium containing FCS or oncostatin M.ARVCs round down and form pseudopodia-like structures, a process also called spreading. Through fluorescent staining with Phalloidin TRITC, the alteration of the contractile apparatus of ARVC can be investigated during cultivation. Freshly isolated cardiomyocytes display a clearly visible cross striation.During the first days of cultivation, ARVC lost all their contractile elements. This was followed by a reformation, implicating de novo sarcomerogenesis with actin stress fibers and actin bundles in the perinuclear region. This reformation was preceded by the formation of pseudopodia-like structures.At the end of the cultivation, a typical cross striation from newly assembled sarcomeres in the spread of ARVC was observed. At the end of the cultivation period, 14.7%plus or minus 1.39%of all counted ARVC showed pseudopodia-like structures. Once mastered, this technique can be performed in one to one and a half hour if it is performed properly.While attempting the procedure, please keep in mind to work as fast and as precise as possible. After watching this video, you should have a good understanding of how to isolate and cultivate adult cardiomyocytes. Don't forget that working with biological material and chemicals can be hazardous.After its development, this method paved the way for researchers in the field of cardiology to explore basic physiology of adult cardiac myocytes. Following this procedure, other methods like separation of cardiomyocytes of the left and right ventricle or other model species can be performed in order to answer additional questions, like anatomical or species-related differences.

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