Name: isolation and culture of adult mouse cardiomyocytes for cell signaling and in vitro cardiac hypertrophy
Uploaded: 2014-05-21T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy at JoVE.com

This work was supported by National Heart, Lung, and Blood Institute Grant HL-36573. We thank Mr. David Sowa for editing this manuscript.

All research on mice was done according to procedures and guidelines of the National Institutes of Health, and the protocols were approved by the Institutional Animal Care and Use Committee of the University of Toledo, College of Medicine and Life Sciences.
1. Perfusion System Preparation
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	<li>The day before isolation, fill the entire perfusion system (including reservoirs) with a solution of fast alkaline residue-free detergent. Allow the solution to soak into the system for a few hour...

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	<li>Louch, W. E., Sheehan, K. A., Wolska, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+cardiomyocyte+isolation,+culture,+and+gene+transfer.">Methods in cardiomyocyte isolation, culture, and gene transfer.</a> J. Mol. Cell. 51, 288-298 (2011).</li><li>Gross, D. R., Gross, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Isolated heart preparations, problems, and pitfalls. In: Animal Models in Cardiovascular Research. , 109-130 (2009).</li><li>Schl&#252;ter, K. D., Piper, H. M., Dheim, S., Mohr, F. 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For the best preparation, the most critical steps are: 1) promptly hooking up the mouse heart to the cannula after excision; 2) appropriate heart perfusion. Also, note that water quality, perfusion temperature, pH of buffer, sterilization, chemical purity, and clean, non-contaminated tubing and chambers are also important factors. 18.2 m&#937; molecular biology-grade H2O is highly recommended for buffer preparation. The pH of 200 mM ATP stock solution should be adjusted to 7.2, a step which is easily missed.

It is e...

Cardiomyocytes are not proliferative. There are some atrial cardiomyocyte cell lines, like HL-1 and AT-1 cells derived from mouse atrial tumors; however, there are no adult ventricular cardiomyocyte cell lines available for research. Primary cell cultures of adult mouse cardiomyocytes provide a powerful model for heart research at the cellular and molecular levels. To date, they have been used extensively for biochemical, physiological, and pharmacological research1. Additionally, the frequent use of genetically modified mice has necessitated effective methods of cardiomyocyte isolation. Pure culture allows for conditions free from interaction with other organs and the systemic circulation, such as through endogenous neurohormonal and hormone-like factors2,3. However, successful isolation of cardiomyocytes can be challenging.
The protocol we introduce herein is based on our experiences with adult rat cardiomyocytes and the method described by O&#39;Connell et al4,5. Especially in 20075, they described detailed techniques from buffer preparations to cell culture and functional assay. Since mouse myocytes are highly susceptible to contracture compared to the rat cardiomyocytes, the buffers or media used for the perfusion, digestion, Ca2+ toleration, plating and culture in the mouse protocol are supplemented with a nonspecific excitation-contraction coupling inhibitor, 2,3-Butanedione monoxime (BDM) to inhibit their spontaneous contraction, hence the viability and yield of rod-shaped myocytes improve significantly. In the protocol introduced here, the myocytes are separated in a high potassium buffer from the isolated heart by modified Langendorff perfusion with type II collagenase. Collagenase II effectively breaks down the intercellular matrix and releases cells. The perfusion solution also keeps cellular metabolism at a low level6. In addition, the isolated heart system from Harvard Apparatus we used here is well-designed for precise temperature and constant pressure control7. This approach provides highly reproducible preparations and uniform populations of single cell type, which can be used in overnight&#160;culture for measuring signaling proteins or 2-3 day culture for cardiac hypertrophic assays.

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			<td height="41" rowspan="2" style="height:41px;width:257px;">Isolated heart system for small rodent</td>
			<td style="width:131px;">Harvard Apparatus</td>
			<td style="width:99px;">IHSR-mouse 73-4019</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:257px;">Aortic Cannula, OD 1.0 mm</td>
			<td style="width:131px;">Harvard Apparatus</td>
			<td style="width:99px;">73-2816</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:257px;">Dumont #4 Forceps</td>
			<td style="width:131px;">Fine science tools</td>
			<td style="width:99px;">11294-00</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:257px;">Straight sharp serrated scissors</td>
			<td style="width:131px;">Fine science tools</td>
			<td style="width:99px;">14070-12</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:257px;">Serrated Graefe forceps</td>
			<td style="width:131px;">Fine science tools</td>
			<td style="width:99px;">11050-10</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:257px;">Curved, serrated Graefe forceps</td>
			<td style="width:131px;">Fine science tools</td>
			<td style="width:99px;">11052-10</td>
			<td style="width:288px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:257px;">Straight Mini Serrefines clamps</td>
			<td style="width:131px;">Fine science tools</td>
			<td style="width:99px;">18054-28</td>
			<td style="width:288px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:257px;">MEM</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:99px;">11575-032</td>
			<td style="width:288px;"></td>
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			<td height="20" style="height:20px;width:257px;">Collagenase II</td>
			<td style="width:131px;">Worthington</td>
			<td style="width:99px;">4176</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mucosal universal detergent</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:99px;">Z637181</td>
			<td>Mucasol is a fast alkaline residue-free detergent.</td>
		</tr>
	</tbody>
</table>

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.

1. Successful Isolation Quantification
Two criteria are used to quantify the success of the isolation: first, the total number of cardiomyocytes isolated, and second, the ratio of rod-shaped calcium-tolerant to round non-tolerant myocytes. Generally this protocol takes around 75-90 min&#160;from heart removal to myocyte plating and yields around 1 million rod-shaped cardiomyocytes (70-90% of total myocytes harvested) from one adult mouse heart. This may vary with mouse body weight and strain. Typ...

Watch this Scientific Journal Video about Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy at JoVE.com

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.

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 ...

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