Name: Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy (Video) | JoVE Journal
Uploaded: 2014-05-21T14:00:00.000+00:00
Duration: 6 min 51 s
Description: Learn to isolate and culture adult mouse cardiomyocytes for cardiac signaling studies using collagenase digestion and calcium reintroduction. This reliable protocol enables functional assays across genetically modified mouse models.

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5 minutes

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
<ol>
	<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 hours&#160;or overnight.</li>
	<li>The next morning, adjust the thermocycler temperature to 37 &#176;C and allow the solution to warm up. Remove the detergent solution and flush the system thoroughly with sterilized distilled water. Do not forget all the side branches.</li>
	<li>Fill the system with perfusion buffer via a constant peristaltic pump and draw any air bubbles out from the aorta chamber (bubble trap) with a side-mounted syringe to protect the heart from coronary occlusion. The flow rate of the peristaltic pump should be checked routinely.</li>
</ol>
2. Preparation of Buffers, Culture Media, and Dishes
<ol>
	<li>Perfusion Buffer: Make 500 ml of 1x&#160;perfusion buffer prior to use. This calcium-free buffer contains 113 mM NaCl, 4.7 mM KCl, 0.6 mM KH2PO4, 0.6 mM Na2HPO4, 1.2 mM MgSO4, 10 mM Na-HEPES, 12 mM NaHCO3, 10 mM KHCO3, 0.032 mM phenol red, 30 mM taurine, 10 mM BDM, and 5.5 mM glucose. Adjust the pH to 7.0 with 2 M HCl and filter through a 0.2 &#956;m&#160;filter, store at 4&#160;&#176;C. 
	Note: a)&#160;The perfusion solution should be prepared on the same day when it is used. In some cases, it is acceptable to store the solution for less than 3 days at 4 &#176;C. b)&#160;This buffer has a high potassium concentration up to 15.3 mM, which can keep the cardiomyocytes quiescent, reduce ATP consumption and increase Ca2+-tolerant ability.</li>
	<li>Digestion Buffer (300 U/ml, 25-50 ml/heart): prepare just prior to perfusion. Weigh 15,000 U of total activity of type II collagenase, dissolve in 50 ml perfusion buffer in culture hood. Add 25 &#956;l of 100 mM CaCl2 (stock solution, filter sterilized) to make the final calcium concentration 50 &#956;M. Warm the buffer to 37 &#176;C when ready for isolation. Unlike trypsin, collagenase works best in the presence of 50-100 &#956;M Ca2+.</li>
	<li>Stop Buffer (50 ml/heart): Operate in culture hood. Mix 45 ml of perfusion buffer with 5 ml sterile FBS.
	<ol>
		<li>Ca2+ solution I (12.5 &#956;M Ca2+): Add 2.5 &#956;l of 100 mM CaCl2 to 20 ml of stop buffer and mix.</li>
		<li>Ca2+ solution II (100 &#956;M Ca2+): Add 10 &#956;l of 100 mM CaCl2 to 10 ml of stop buffer and mix.</li>
		<li>Ca2+ solution III (400 &#956;M Ca2+): Add 40 &#956;l of 100 mM CaCl2 to 10 ml of stop buffer and mix.</li>
		<li>Ca2+ solution IV (900 &#956;M Ca2+): Add 90 &#956;l of 100 mM CaCl2 to 10 ml of stop buffer and mix.</li>
	</ol>
	</li>
	<li>Plating medium (50 ml/heart): Transfer 43 ml of MEM containing 2 mM L-glutamine and 1.26 mM CaCl2 into a sterile 50-ml conical tube in culture hood and add 5 ml of FBS, 1 ml of BDM stock solution (500 mM, filter sterilized), and 0.5 ml penicillin/streptomycin (10,000 U/ml). Mix and equilibrate for 2-3 hr&#160;prior to myocyte isolation in 2% CO2, 37 &#176;C incubator with the caps loose. Right before myocyte plating, add 0.5 ml of 200 mM ATP (pH 7.2, stock solution, filter sterilized) into the medium.</li>
	<li>Culture medium (50 ml/heart): Transfer 48 ml of MEM containing 2 mM L-glutamine and 1.26 mM CaCl2 into a sterile 50-ml conical tube in culture hood and add 1 ml of BDM stock solution (500 mM, filter sterilized), and 0.5 ml penicillin/streptomycin (10,000 U/ml). Mix and equilibrate in 2% CO2, 37 &#176;C incubator prior to myocyte isolation. Prior to use, add 0.5 ml of 100 mg/ml BSA (filter sterilized) into the medium. 
	Note: Using a 2% CO2 incubator facilitates maintaining the culture medium pH at 6.9-7.0, which helps the myoctyes maintain their rod-shaped morphology up to 24 hr.</li>
	<li>Laminin (1-2 &#956;g/cm2)-coated dishes or plates: Transfer 200 &#956;l of 1 mg/ml natural mouse laminin to 20 ml of sterilized PBS (w/o calcium and magnesium) and mix. Add 1 ml to 35-mm dishes, 2.5 ml to 60-mm dishes, or 5 ml to 100-mm dishes, shake to evenly cover the surfaces, and place in 2% CO2, 37 &#176;C incubator until myocyte plating. 
	Note: If the dishes or plates are not used on the day coated, they can be sealed with Parafilm and stored at 4&#176;C overnight&#160;(Slow Coating). Sealing is required to prevent evaporation and contamination. Coated plates can be kept at 4 &#176;C for up to 1 week.</li>
</ol>
3. Mouse Heart Removal and Cannulation
<ol>
	<li>Soak the scissors and forceps in 70% ethanol for 30 min and let them dry.</li>
	<li>Weigh the mouse to the nearest 0.1 gram. Record its body weight, strain, sex, and date of birth.</li>
	<li>Inject 200 &#956;l heparin (100 IU/mouse) i.p. 10 min prior to anesthesia to prevent coagulation of blood in the coronary arteries. Anesthetize the mouse with 200 mg/kg body weight of ketamine and 10 mg/kg body weight of xylazine by i.p. injection and wait for 5-10 min&#160;until the mouse stops responding to tail/toe pinches.</li>
	<li>Secure the mouse in the supine position by gently fixing the forepaws and hindpaws to a pinnable work surface (i.e. styrofoam) on an animal surgery tray or table bench near the perfusion system.</li>
	<li>Wipe the chest and abdomen with 70% ethanol. Make a midline skin incision from mid abdomen to the diaphragm with a surgical scissor.</li>
	<li>Cut the diaphragm with another surgical scissor and hold the sternum with curved serrated forceps. Cut bilaterally and retroflect thoracic cage to expose the heart.</li>
	<li>Lift the heart slightly using fine curved serrated and atraumatic forceps and dissect the heart out of the thoracic cavity as close as possible to the dorsal thoracic wall.</li>
	<li>Transfer the heart to a 100-mm dish containing cold perfusion buffer. Carefully trim away the connective tissues such as lungs, thymus, and bronchi.</li>
	<li>Identify the aorta and its cranial branches, which are usually hidden by thymus and fat pad. Cut the aorta below its first branch13, grasp the aortic wall, lift the heart, and slightly slip it on to the perfusion fluid-filled aortic cannula (1.0 mm OD stainless steel cannula with blunt/flat tip and notches 1 and 2 mm above the tip; or a 22 G reusable feeding needle) using two ultra fine-tip forceps. 
	Note: a)&#160;The perfusion fluid should be dripping during heart cannulation to prevent gas bubbles from entering the coronary arteries; b)&#160;Keep the tip of the cannula just above the aortic valve.</li>
	<li>Clamp the aorta to the cannula and ligate the aorta to the cannula with 6-0 surgical silk. The whole cannulation should take less than 120 sec.</li>
</ol>
4. Heart Perfusion and Digestion
<ol>
	<li>Perfuse the heart with calcium-free perfusion buffer at flow rate of 4 ml/min about 4-5 min until the effluents become clear, then switch to digestion buffer containing 50 &#956;M CaCl2 and perfuse for 3.5-20 min depending on mouse strain, perfusion pressure and collagenase activity. If applicable, increase the aorta pressure to 70-80 mmHg when digesting. When necessary, enzyme perfusate can be recirculated for digestion. Typically, 300 U/ml enzyme buffer will digest a heart in 10-12 min at a flow rate of 4 ml/min; If applying afterload pressure at 70 mmHg, 300 U/ml will take only 3.5-5.5 min at a flow rate of 4 ml/min.</li>
	<li>Stop digestion when the heart becomes slightly pale and flaccid. It should be spongy when gently pinched.</li>
</ol>
5. Cells dissociation and Calcium reintroduction
<ol>
	<li>Pull the aorta from the cannula with 70% ethanol-soaked forceps and put the heart into a sterile 60-mm dish containing 2.5 ml digestion buffer. Move into the culture hood using sterile supplies, staying mindful of sterile techniques for this and follow-up steps.</li>
	<li>Remove aorta, atria, and great vessels with fine surgical scissors. Gently tease the ventricle into 10-12 small pieces with two fine-tip forceps.</li>
	<li>Gently pipette the heart pieces and cells up and down ~10x&#160;with a 10-ml transfer pipette and then transfer to a 15-ml polypropylene conical tube. Rinse the dish with 7.5 ml calcium solution I containing 12.5 &#956;M CaCl2 and transfer this into the tube, resulting in a final volume of 10 ml.</li>
	<li>Gently pipette the cell suspension up and down using a fine-tip transfer pipette to disperse the large pieces of heart tissue.</li>
	<li>Then centrifuge for 3 min at 20 x g to separate out small non-myocyte cells such as endothelial cells and fibroblasts.</li>
	<li>Aspirate off the supernatant and resuspend the myocyte pellet in 10 ml calcium solution I with a supplement of 100 &#956;l of 200 mM ATP (pH 7.2, stock solution, filter sterilized). Leave the tube in the hood for 3-5 min.</li>
	<li>Transfer duplicate 10 &#956;l aliquots to a hemacytometer to count rod-shaped and round myocytes. Calculate the total number of myocytes and calculate the percentage of rod-shaped myocytes.</li>
	<li>Centrifuge the myocytes for 3 min at 20 x g. Remove the supernatant and resuspend the pellets in 10 ml calcium solution II containing 100 &#956;M CaCl2. Disperse the myocytes with the fine-tip transfer pipette by pipetting up and down.</li>
	<li>Repeat the above step (5.8) using calcium solution III (400 &#956;M CaCl2) and IV (900 &#956;M CaCl2), respectively.</li>
	<li>For mouse myocyte primary culture, resuspend the final myocyte pellet in 5 ml of plating medium. Count the total number of myocytes and calculate the percentage of rod-shaped myocytes.</li>
</ol>
6. Cell Culture
<ol>
	<li>Adjust the concentration of rod-shaped myocytes to 25,000 rod-shaped myocytes/ml in a 50-ml tube. Gently pipette the myocytes using a 10-ml pipette to suspend them well. Avoid cell sedimentation in the pipette and tube to ensure equal cell density for each dish or plate.</li>
	<li>After aspirating off the laminin coating solution, plate the myocytes into those dishes or plates. Gently slide the dishes or plates forward and backward and side-to-side in a cross-like pattern three to four times on the surface of the culture hood.</li>
	<li>Place the dishes or plates in a 2% CO2 incubator at 37 &#176;C. Incubate for 1-3 hr to allow myocyte attachment at a rate of about 80%.</li>
	<li>After attachment, gently aspirate off the medium containing unattached myocytes and cell debris. Gently add culture medium to the sides of the dishes or plates. Perform this medium change one by one. Immediately return the dishes or plates to the incubator.</li>
	<li>After O/N culture, the myocytes can be changed to the same medium without BDM before studies of short term cell signaling. Keep the BDM in long term culture for hypertrophic or apoptosis assays. 
	Note: BDM (2,3-butanedione monoxime) can inhibit the myosin-ATPase and prevent contraction. Its inhibition of myosin-based contraction is readily reversible. It is reported that BDM may have some potential side effects, such as acting as a non-specific phosphatase, increasing calcium release from SR, and alteration of free calcium concentration and cellular electrical properties, so before the treatment, the BDM should be removed. Moreover, in some cases, a specific myosin II inhibitor Blebbistatin could be an alternative drug to inhibit cardiomyocyte contraction8,9.&#160; However, in our cases, BDM is better than Blebbistatin for the sake of the quality and quantity of the cardiomyocytes.</li>
</ol>

<ol><li>Louch, W. E., Sheehan, K. A., Wolska, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+in+cardiomyocyte+isolation%2C+culture%2C+and+gene+transfer%5BTitle%5D)+AND+%22J.+Mol.+Cell%22%5BJournal%5D)">Methods in cardiomyocyte isolation, culture, and gene transfer.</a> J. Mol. Cell. 51, 288-298 (2011).</li>
<li>Gross, D. R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGross%2C+DR+%22Isolated+heart+preparations%2C+problems%2C+and+pitfalls.+In%3A+Animal+Models+in+Cardiovascular+Research%22">Isolated heart preparations, problems, and pitfalls. In: Animal Models in Cardiovascular Research</a>. Gross, D. R. , 3rd edition, Springer. 109-130 (2009).</li>
<li>Schlüter, K. D., Piper, H. M. Isolation and culture of adult ventricular cardiomyocytes. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSchl%C3%BCter%2C+KD+%22Practical+Methods+in+Cardiovascular+Research%22">Practical Methods in Cardiovascular Research</a>. Dheim, S., Mohr, F. W., Delmar, M. , Springer. 557-567 (2005).</li>
<li>O'Connell, T. D., Ni, Y. G., Lin, K. M., Han, H. P., Yan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+culture+of+adult+cardiac+myocytes+for+signaling+studies%5BTitle%5D)+AND+%22AfCS+research+reports%22%5BJournal%5D)">Isolation and culture of adult cardiac myocytes for signaling studies.</a> AfCS research reports. 1 (5), 1-9 (2003).</li>
<li>O'Connell, T. D., Rodrigo, M. C., Simpon, P. C. Isolation and culture of adult mouse cardiac myocytes. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aO%27Connell%2C+TD+%22Methods+in+Molecular+Biology%22">Methods in Molecular Biology</a>. Vivanco, F. 35, Humana Press. 271-296 (2007).</li>
<li>Nilolskaya, A., Sharma, V. Cell culture models and methods. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aNilolskaya%2C+A+%22Cardiac+Electrophysiology+Methods+and+Models%22">Cardiac Electrophysiology Methods and Models</a>. Sigg, D. C., Laizzo, P. S., Xiao, Y. F. , Springer. 213-235 (2010).</li>
<li>Merx, M. W., Schrader, M. The working heart. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMerx%2C+MW+%22Practical+Methods+in+Cardiovascular+Research%22">Practical Methods in Cardiovascular Research</a>. Dhein, S., Mohr, F. W., Delmar, M. , Springer-Verlag. Berlin Heidelberg, Germany. 173-189 (2005).</li>
<li>Kabaeva, Z., Zhao, M., Michele, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blebbistatin+extends+culture+life+of+adult+mouse+cardiac+myocytes+and+allows+efficient+and+stable+transgene+expression%5BTitle%5D)+AND+%22Am.+J.+Physiol.+Heart+Circ.+Physiol%22%5BJournal%5D)">Blebbistatin extends culture life of adult mouse cardiac myocytes and allows efficient and stable transgene expression.</a> Am. J. Physiol. Heart Circ. Physiol. 294, 1667-1674 (2008).</li>
<li>Lou, Q., Li, W., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+dynamic+instability+and+wavelength+in+arrhythmia+maintenance+as+revealed+by+panoramic+imaging+with+blebbistatin+vs.+2%2C3-butanedione+monoxime%5BTitle%5D)+AND+%22Am.+J.+Physiol.+Heart+Circ.+Physiol%22%5BJournal%5D)">The role of dynamic instability and wavelength in arrhythmia maintenance as revealed by panoramic imaging with blebbistatin vs. 2,3-butanedione monoxime.</a> Am. J. Physiol. Heart Circ. Physiol. 302, 262-269 (2012).</li>
<li>Liu, C. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reduction+of+Na%2FK-ATPase+potentiates+Marinobufagenin-induced+cardiac+dysfunction+and+myocyte+apoptosis%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">Reduction of Na/K-ATPase potentiates Marinobufagenin-induced cardiac dysfunction and myocyte apoptosis.</a> J. Biol. Chem. 287 (20), 16390-16398 (2012).</li>
<li>Bai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Different+roles+of+the+cardiac+Na%2B%2FCa2%2B-exchanger+in+ouabain-induced+inotropy%2C+cell+signaling%2C+and+hypertrophy%5BTitle%5D)+AND+%22Am.+J.+Physiol.+Heart+Circ.+Physiol%22%5BJournal%5D)">Different roles of the cardiac Na+/Ca2+-exchanger in ouabain-induced inotropy, cell signaling, and hypertrophy.</a> Am. J. Physiol. Heart Circ. Physiol. 304, 427-435 (2013).</li>
<li>Liu, L. J., Zhao, X. C., Pierre, S. V., Askari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Association+of+PI3K-AKT+signaling+pathway+with+digitalis-induced+hypertrophy+of+cardiac+myocytes%5BTitle%5D)+AND+%22Am.+J.+Physiol.+Cell+Physiol%22%5BJournal%5D)">Association of PI3K-AKT signaling pathway with digitalis-induced hypertrophy of cardiac myocytes.</a> Am. J. Physiol. Cell Physiol. 293, 1489-1497 (2007).</li>
<li>Flynn, J. M., Santana, L. F., Melov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+Cell+Transcriptional+Profiling+of+Adult+Mouse+Cardiomyocytes%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes.</a> J. Vis. Exp. (58), (2011).</li>
</ol>

The authors declare that they have no competing financial interests.

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.

<table><tbody><tr><td>Isolated heart system for small rodent</td><td>Harvard Apparatus</td><td>IHSR-mouse 73-4019</td><td></td></tr><tr><td>Aortic Cannula, OD 1.0 mm</td><td>Harvard Apparatus</td><td>73-2816</td><td></td></tr><tr><td>Dumont #4 Forceps</td><td>Fine science tools</td><td>11294-00</td><td></td></tr><tr><td>Straight sharp serrated scissors</td><td>Fine science tools</td><td>14070-12</td><td></td></tr><tr><td>Serrated Graefe forceps</td><td>Fine science tools</td><td>11050-10</td><td></td></tr><tr><td>Curved, serrated Graefe forceps</td><td>Fine science tools</td><td>11052-10</td><td></td></tr><tr><td>Straight Mini Serrefines clamps</td><td>Fine science tools</td><td>18054-28</td><td></td></tr><tr><td>MEM</td><td>Gibco</td><td>11575-032</td><td></td></tr><tr><td>Collagenase II</td><td>Worthington</td><td>4176</td><td></td></tr><tr><td>Mucosal universal detergent</td><td>Sigma</td><td>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

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 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. Typicall...

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 ...

[Narrator] The overall goal of this procedure is to yield a consistent preparation for the culture of functional adult mouse cardiomyocytes. First remove the mouse heart under anesthesia and cannulation. Perfuse the heart and digest with collagenase. Then dissociate the cells and reintroduce the calcium. Next seed the cells and culture the primary cardiomyocytes. Results from western blots and tritiated leucine incorporation assays can demonstrate function such as the activation of PI3 kinase-dependent AKT phosphorylation and the increase of protein synthesis rate induced by Ouabain.- After isolation of the heart, quickly fan the aorta from many other vessels, then handle the aorta very carefully and hook it up quickly on the first try. With practice you will be able to determine when to stop the collagenase digestion of the heart by accurately monitoring the color and the softness change.- Demonstrating the procedure will be Dr Daxiang Li, a postdoctoral fellow in my laboratory.- [Narrator] Just prior to the perfusion, prepare fresh digestion buffer containing type II collagenase and warm the buffer to 37 degrees Celsius when ready for isolation. Soak scissors and forceps in 70% ethanol and set them to dry. Now weigh the mouse to the nearest 10th of a gram. Record its body weight, strain, sex and date of birth. Inject 200 microliters of Heparin intraperitoneally to prevent coagulation of blood in the coronary arteries. After 10 minutes, anesthetize the mouse. After five to 10 minutes, check that the mouse is non-responsive to a tail or toe pinch, then secure the mouse in a supine position by gently fixing the fore paws and hind paws to a work surface. Wipe the chest and abdomen with 70% ethanol, then make a midline skin incision from mid abdomen to the diaphragm. Hold the sternum and cut bilaterally. Next, cut the diaphragm and retroflect the thoracic cage to expose the heart. Now lift the heart slightly and dissect the heart out of the thoracic cavity as close as possible to the dorsal thoracic wall. Transfer the heart to cold profusion buffer in a 100 millimeter dish. Carefully trim away the connective tissues such as lungs, thymus, bronchi and esophagus. Now identify the aorta and it's cranial branches which are hidden by the thymus and fat pad. Cut the aorta below its first branch, grasp the aortic wall and lift the heart. Check that the profusion fluid is dripping, then slightly slip the heart onto the perfusion fluid-filled aortic cannula keeping the tip of the cannula just above the aortic valve. Clamp the aorta to the cannula and ligate the aorta to the cannula with 6-0 surgical silk. Perfuse the heart with calcium-free profusion buffer at a flow rate of four milliliters per minute for about four to five minutes until the effluents become clear, then switch to digestion buffer containing 50 micromolar calcium chloride. If applicable, increase the after load pressure to 70 to 80 millimeters of mercury when digesting. When the heart becomes slightly pale and flaccid, check that it is spongy when gently pinched, then stop the digestion. With 70% ethanol-soaked forceps, pull the aorta from the cannula and put the heart into a sterile 60 millimeter dish containing 2.5 milliliters of digestion buffer. Now move into the cell culture hood and use sterile supplies and sterile techniques. Remove the aorta, atria and great vessels with fine surgical scissors. Then gently tease the ventricle into 10 to 12 small pieces. With a 10 milliliter transfer pipette, gently pipette the heart pieces and cells and transfer the sample to a 15 milliliter polypropylene conical tube. Rinse the dish with 7.5 milliliters of calcium solution I and transfer the wash to the conical tube, resulting in a final volume of 10 milliliters. Next, disperse the large pieces of heart tissue by gently pipetting with a fine tip transfer pipette. Then centrifuge for three minutes at 20 G's to separate out small non-myocyte cells such as endothelial cells and fibroblasts. Aspirate off the supernatant. Resuspend the myocyte pellet in 10 milliliters of calcium solution I supplemented with ATP and equilibrate for three to five minutes. Next transfer duplicate 10 microliter aliquots to a hemocytometer. Count the rod-shaped and the round myocytes, then calculate the total number of myocytes and determine the percentage of rod-shaped myocytes. Pellet the myocytes by centrifugation. Remove the supernatants and wash the pellet successively in 10 milliliters of increasing concentrations of calcium. For mouse myocyte primary culture, resuspend the final myocyte pellet in five milliliters of plating medium to get calcium-tolerant cardiomyocytes. Count the total number of myocytes and calculate the percentage of rod-shaped cyocytes. Adjust the concentration of rod-shaped myocytes to 25,000 rod-shaped myocytes per milliliter. Gently pipette the myocytes to ensure equal cell density for each dish or plate. After aspirating off the laminen coating solution from the culture plates, seed the cardiomyocytes and evenly disperse the cells by gently sliding the dishes forward and backward and side to side in a cross-like pattern on the surface of the culture hood. Place the cultures in a 2% carbon dioxide incubator at 37 degrees Celsius to allow myocyte attachment at a rate of about 80%. Now gently aspirate off the medium containing unattached myocytes and cell debris. Replenish the culture medium by aiming at the sides of the dishes one plate at a time. Immediately return the cells to the culture incubator. In a successful preparation, the isolated myocytes normally have a distinct rode shape with rectangular ends and clear cross striations. As a functional assay, adult C57 black six mouse cardiomyocytes were cultured and treated with Ouabain and ET1. These data show that Ouabain and ET1 can increase AKT phosphorylation in a dose-dependent manner and also induce tritiated leucine incorporation during protein synthesis. This technique paved the way for researchers in the field of heart disease to explore the mechanism of cardiac regulation in genetic-modified mice.

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JoVE Journal

Biology

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.

Isolation and Culture of Neonatal Mouse Cardiomyocytes

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.

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

Isolation and Physiological Analysis of Mouse Cardiomyocytes

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.

Isolation, Culture and Transduction of Adult Mouse 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.

Isolation and Cultivation of Adult Rat Cardiomyocytes

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

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.

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

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.

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Single cell expression profiling allows the detailed gene expression analysis of individual cells.  We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

Learn to isolate and culture adult mouse cardiomyocytes for cardiac signaling studies using collagenase digestion and calcium reintroduction. This reliable protocol enables functional assays across genetically modified mouse models.

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

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