Marc Wozniak for the use of Department of Cell and Developmental Biology videography equipment.
Vanderbilt University Cell Imaging Shared Resource.
This work is supported by AHA grant 12PRE10950005 to ERP, AHA grant 11GRNT7690040 to DMB, NIH grant R01 HL037675 to DMB. DMB is the Gladys P. Stahlman Chair in Cardiovascular Research.

Ensure all procedures involving animals are approved by the appropriate animal use and care body.
1.&#160;Prepare Stock Buffers in Advance
<ol>
	<li>Prepare 2 L stock Ca2+ free Tyrode&#8217;s (Table 1). Adjust the pH to 7.4 with NaOH.</li>
	<li>Prepare 2 L stock 1.2 mM Ca2+ Tyrode&#8217;s (Table 2). Adjust the pH to 7.4 with NaOH.</li>
</ol>
2.&#160;On the Day of the Experiment, Prepare Perfusion, Transfer, and Digestion Buffers
<ol>
	<li>Prepare 150 ml perfusion buffer in Ca2+&#160;free Tyrode&#8217;s (Table 3) and filter through a 0.2 &#956;m filter.</li>
	<li>Prepare 35 ml Ca2+ free transfer &#8220;Buffer A&#8221; (Table 4) and 25 ml Ca2+ containing &#8220;Buffer B&#8221; (Table 5). Filter each through a 0.2 &#956;m filter.</li>
	<li>Prepare solutions of 0.06, 0.24, 0.6, and 1.2 mM Ca2+ by mixing Buffer A and B per Table 6.</li>
	<li>Prepare 25 ml digestion buffer by dissolving digestion enzymes in 25 ml perfusion buffer (Table 7) based on the body weight (BW) of the mouse. Filter through a 0.2 &#956;m filter before use.</li>
</ol>
3.&#160;Experimental Setup
<ol>
	<li>Assemble a constant flow Langendorff apparatus, available commercially or using a peristaltic pump, tubing, and a heat exchanger coil to allow a flow rate of 3ml/min of perfusate heated to 37 &#176;C. A simple schematic is provided in the 2011 review by Bell, et al18.</li>
	<li>Set circulating water bath temperature (~47 &#176;C) of the Langendorff apparatus so that outflow from the cannula is at 37 &#176;C at a flow rate of 3 ml/min.</li>
	<li>Run 70% ethanol through the perfusion system for 15 min, followed by 100 ml of ultrapure type I water. Run perfusion buffer through the system for 5 min while eliminating air bubbles.</li>
	<li>Sterilize all surgical tools with a desired method.</li>
	<li>Create cannula by attaching a short piece (about 3 mm) of PE-50 tubing to the tip of a 23 G Luer-stub adaptor. Heat tip of tubing to create a lip over which the aorta will be positioned.</li>
</ol>
4.&#160;Cardiomyocyte Isolation
<ol>
	<li>Inject mouse with 0.2 ml Heparin solution (1,000 IU/ml), via intraperitoneal injection.</li>
	<li>After 5 min, anesthetize mouse with isofluorane. When fully anesthetized (no response to strong foot pinch), spray chest with 70% EtOH. Open the chest, quickly excise the heart, being careful not to damage the aorta, and place in 4 &#176;C perfusion buffer. Use curved forceps to grasp and lift under the heart. This creates space to trim attachments behind the heart with fine scissors.</li>
	<li>Cannulate the heart by gently grasping the edge of the aorta with two pairs of fine forceps and carefully pulling the opening of the aorta over the lip of the cannula. Ensure the tip of the cannula is not past the aortic valve and into the ventricle, as this will inhibit perfusion of the heart via the coronary arteries.</li>
	<li>Secure the aorta to the cannula by tying a loop of 5-0 silk suture around the aorta immediately above the lip of the cannula.</li>
	<li>Note: Steps 4.2 through 4.4 should be completed as quickly as possible for best quality digestion.</li>
	<li>Note: The heart can be hung directly onto the cannula already attached to the Langendorff Apparatus with flowing perfusion buffer. However, hanging the heart onto a cannula attached to a small syringe of perfusion buffer under a dissection microscope has been found to be most successful. This allows a small amount of perfusion buffer to be pushed through, watching for clearing of coronary arteries and ensuring heart is securely attached to cannula. The cannula is then hung on the Langendorff with running perfusion buffer.</li>
	<li>Perfuse the heart with Ca2+ free perfusion buffer at a flow of 3 ml/min for 2-3 min.</li>
	<li>Switch to enzyme digestion buffer and perfuse for 7-10 min, heart will become softer and lighter in color.</li>
	<li>Note: Digestion time can be adjusted based on enzyme activity and heart size.</li>
	<li>Once heart is palpably flaccid, remove heart from cannula and place in a sterile p60 dish with Buffer A (Ca2+ free transfer buffer).</li>
	<li>Remove atria and great vessels. Remove right ventricle if desired. With forceps, separate the ventricle into small pieces. Pipette several times with a sterile 5 ml transfer pipette to further disperse cells.</li>
	<li>Filter the cell suspension into a 50 ml conical tube through a 250 &#956;m nylon mesh filter.</li>
	<li>Allow the suspension to pellet for about 10 min.</li>
	<li>Transfer the pellet into the 0.06 mM Ca2+ solution. Allow cells to pellet for 10 min. Healthy cells will pellet, while most dead cells will float.</li>
	<li>Aspirate the supernatant, and transfer the pellet to the 0.24 mM Ca2+ solution.</li>
	<li>Repeat 10 min incubation, aspiration, and transfer through the remaining two Ca2+ solutions until cells are in the 1.2 mM solution. At this point, a majority of the cells should be rod shaped myocytes.</li>
</ol>
5.&#160;Measuring Contractility and Calcium Transients
<ol>
	<li>Note: Both contractility and calcium transients can be measured simultaneously.</li>
	<li>Prepare Fura-2 AM solution by resuspending in anhydrous DMSO to a concentration of 1 &#956;g/&#956;l. Fura-2 AM solution can be stored in a desiccator at -20 &#176;C.</li>
	<li>Load 1 ml of suspended cells with 1 &#956;l Fura-2 AM. Allow cells to sit in dark 10 min then aspirate supernatant. Wash twice with Ca2+ Tyrode&#8217;s solution, allowing cells to pellet in the dark for 10 min between washes.</li>
	<li>Place a glass coverslip in a stage insert that allows heated perfusion/aspiration and electrode pacing with a myocyte field stimulator. Place stage insert on inverted microscope set up for fluorescence measurement, adding a drop of oil to lens if using an oil immersion objective.</li>
	<li>Set up heated gravity perfusion with 1.2 mM Ca2+ Tyrode&#8217;s solution so solution on stage reaches 37 &#176;C. Attach aspiration to vacuum. Attach electrodes.</li>
	<li>Turn on the microscope system, and open the ratiometric fluorescence and cell dimensioning data acquisition software.</li>
	<li>Add a few drops of Fura-2 AM loaded cells to the stage insert, blocking perfusion momentarily to allow healthy cells to settle.</li>
	<li>Use 40X&#160;objective to locate desired cell. A healthy cell should be rod shaped and not spontaneously contracting. It should visibly and uniformly contract when cell is paced at 5-20 V.</li>
	<li>Note: Physiological analysis requires a high quality digestion. It is possible to have cells that look healthy but do not pace well for physiological analysis. Digestion protocol may require refinement to get high quality, healthy myocytes. Additionally, mutant or diseased hearts may require modifications to the protocol.</li>
	<li>Adjust lens rotation and change aperture so desired cell is lined up horizontally on screen and background is not visible.</li>
	<li>Align edge detection bars to each end of myocyte and adjust threshold. Align sarcomere detection on a portion of cell with uniform sarcomeres. The longer you can make this bar, the more accurate the mathematical model of sarcomere length will be, so long as it is not on two populations of sarcomeres.</li>
	<li>Pace cells at 2 Hz and 5-20 V for 10-15 sec before recording.</li>
	<li>Record tracing. Ambient light should be minimized for best calcium transient recording.</li>
</ol>
6.&#160;Analysis
<ol>
	<li>Analyze traces using ratiometric fluorescence and cell dimensioning data acquisition software.</li>
</ol>
7.&#160;Further Studies
<ol>
	<li>This protocol provides excess cells which can be used for a variety of other experiments including: fixation and immunostaining, assessing direct drug effects, short term culture, and atomic force microscopy.</li>
</ol>

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	<li>Babick, A. P., Dhalla, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Subcellular+Remodeling+in+Cardiac+Dysfunction+due+to+Congestive+Heart+Failure.">Role of Subcellular Remodeling in Cardiac Dysfunction due to Congestive Heart Failure.</a> Medical Principles and Practice. 16, 81-89 (2007).</li><li>Nogami, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purkinje-Related+Arrhythmias+Part+I:+Monomorphic+Ventricular+Tachycardias.">Purkinje-Related Arrhythmias Part I: Monomorphic Ventricular Tachycardias.</a> Pacing and Clinical Electrophysiology. 34, 624-650 (2011).</li><li>Clancy, R. M., Kapur, R. P., Molad, Y., Askanase, A. D., Buyon, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohitologic+Evidence+Supports+Apoptosis,+IgG+Deposition,+and+Novel+Macrophage/Fibroblast+Crosstalk+in+the+Pathologic+Cascade+Leading+to+Congenital+Heart+Block.">Immunohitologic Evidence Supports Apoptosis, IgG Deposition, and Novel Macrophage/Fibroblast Crosstalk in the Pathologic Cascade Leading to Congenital Heart Block.</a> Arthritis and Rheumatism. 50, 173-182 (2004).</li><li>Splawski, I., 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=CaV1.2+Calcium+Channel+Dysfunction+Causes+a+Multisystem+Disorder+Including+Arrhythmia+and+Autism.">CaV1.2 Calcium Channel Dysfunction Causes a Multisystem Disorder Including Arrhythmia and Autism.</a> Cell. 119, 19-31 (2004).</li><li>Berry, M. N., Friend, D. S., Scheuer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+and+Metabolism+of+Intact+Muscle+Cells+Isolated+from+Adult+Rat+Heart.">Morphology and Metabolism of Intact Muscle Cells Isolated from Adult Rat Heart.</a> Circulation Research. 26, 679-687 (1970).</li><li>Fang, F., 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=Luteolin+Inhibits+Apoptosis+and+Improves+Cardiomyocyte+Contractile+Function+through+the+PI3K/Akt+pathway+in+Simulated+Ischemia/Reperfusion.">Luteolin Inhibits Apoptosis and Improves Cardiomyocyte Contractile Function through the PI3K/Akt pathway in Simulated Ischemia/Reperfusion.</a> Pharmacology. 88, 149-158 (2011).</li><li>Feng, W., 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=Coordinated+Regulation+of+Murin+Cardiomyocyte+Contractility+by+Nanomolar+(-)-Epigallocatechin-3-Gallate+the+Major+Green+Tea+Catechin.">Coordinated Regulation of Murin Cardiomyocyte Contractility by Nanomolar (-)-Epigallocatechin-3-Gallate the Major Green Tea Catechin.</a> Molecular Pharmacology. 82, 993-1000 (2012).</li><li>Claycomb, W. C., 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=HL-1+cells:+A+cardiac+muscle+cell+line+that+contracts+and+retains+phenotypic+characteristics+of+the+adult+cardiomyocyte.">HL-1 cells: A cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte.</a> Proceedings of the National Academy of Science. 95, 2979-2984 (1998).</li><li>Kaestner, L., 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=Isolation+and+Genetic+Manipulation+of+Adult+Cardiac+Myocytes+for+Confocal+Imaging.">Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging.</a> J Vis Exp. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longstanding+Hyperthyroidism+is+Associated+with+Normal+or+Enhanced+Intrinsic+Cardiomyocyte+Function+despite+Decline+in+Global+Cardiac+Function.">Longstanding Hyperthyroidism is Associated with Normal or Enhanced Intrinsic Cardiomyocyte Function despite Decline in Global Cardiac Function.</a> PLoS One. 7, e46655 (2012).</li><li>Papanicolaou, K. N., 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=Preserved+heart+function+and+maintained+response+to+cardiac+stresses+in+a+genetic+model+of+cardiomyocyte-targeted+deficiency+of+cyclooxygenase-2.">Preserved heart function and maintained response to cardiac stresses in a genetic model of cardiomyocyte-targeted deficiency of cyclooxygenase-2.</a> Journal of Molecular and Cellular Cardiology. 49, 196-209 (2010).</li><li>Despa, S., Lingrel, J. B., Bers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na+/K+-ATPase+a2-isoform+preferntially+modulates+Ca2++transients+and+sarcoplasmic+reticulum+Ca2++release+in+cardiac+myocytes.">Na+/K+-ATPase a2-isoform preferntially modulates Ca2+ transients and sarcoplasmic reticulum Ca2+ release in cardiac myocytes.</a> Cardiovascular Research. 95, 480-486 (2012).</li><li>Touchberry, C. D., 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=FGF23+is+a+novel+regulator+of+intracellular+calcium+and+cardiac+contractility+in+addition+to+cardiac+hypertophy.">FGF23 is a novel regulator of intracellular calcium and cardiac contractility in addition to cardiac hypertophy.</a> American Journal of Physiology - Endocrinology and Metabolism. , (2013).</li><li>Helmes, M., 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=Titin+Determines+the+Frank-Starling+Relation+in+Early+Diastole.">Titin Determines the Frank-Starling Relation in Early Diastole.</a> The Journal of General Physiology. 121, 97-110 (2003).</li><li>Bell, R. M., Mocanu, M. M., Yellon, D. 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We have nothing to disclose.

Isolation of quality cardiomyocytes requires some level of practice and optimization. This protocol contains key steps that can greatly affect the outcome of the digestion. These should be considered carefully when performing and troubleshooting the protocol. The time from removal of the heart to cannulation and perfusion should be minimized, ideally less than five minutes. Rinsing the heart immediately in ice-cold perfusion buffer after removing from the mouse and dissecting and cannulating the heart in ice-cold buffer ...

Cardiomyocytes provide the contractile force for the heart. Each myocyte contains organized cytoskeletal and contractile elements essential to contraction. The heart not only contains cardiomyocytes but also fibroblasts, connective tissue, and modified myocytes such as Purkinje fibers. Changes in any or all of these cell types can be seen in heart failure, making determination of the ultimate cause of heart dysfunction difficult. Decreased ventricular contractility of a failing heart is preceded by varying degrees of fibrous tissue build-up and hypertrophy and dysfunction of the cardiomyocytes1. Arrhythmias can be caused by dysfunction in the conducting cells2, fibrosis interrupting cardiac conduction3, or changes in the expression of ion channels in cardiomyocytes1,4. Indeed, these problems often co-exist in a complicated fashion.
Isolating and studying cardiomyocytes allows one to examine contractile and electrical dysfunction of individual myocytes in the context of overall heart function. Techniques to isolate individual cardiac myocytes were developed over 40 years ago in an effort to better study physiology of this &#8216;workhorse&#8217; cell of the heart. Isolation techniques have been improved to increase yield and quality of cells, with the vital development of coronary perfusion via cannulation of the aorta, first performed in 19705. This article describes cardiomyocyte isolation followed by measurement of contractility and calcium transients using ratiometric fluorescence and cell dimensioning data acquisition software. In both genetic and environmental models of heart disease, utilizing this protocol provides key information about cell contractility, sarcomeric contractility and relaxation, calcium transients, and cytoskeletal disruption. This system is often used to test the effect of drugs on isolated cardiomyocytes6,7. We employ the system to compare contractility and calcium transients of cardiomyocytes between wild type mice and mice with a mutation leading to dilated cardiomyopathy.
Studying individual cardiac myocytes has numerous advantages over other commonly used methods to study cardiac anatomy and physiology. Echocardiography and electrocardiography provide information about the contractile function and electrical conduction of the heart overall. Neither of these methods explains the cause or nature of the dysfunction below the level of the whole organ. Measuring contractility of individual cardiomyocytes using edge-detection and sarcomere length algorithms can demonstrate that changes within the contractile cell of the heart are present within global heart dysfunction. Measuring calcium transients demonstrates the influence of changes in ion flux at a cellular level to contractile dysfunction or arrhythmias.
In addition to complementing whole organ studies, this protocol provides cells easily stained for sub-cellular components. It is possible to see cytoskeletal changes by staining tissue sections of the whole heart; however, one is only able to visualize a cross section of cells. A whole cardiomyocyte is thicker than the typical tissue section, and given the arrangement and length of the cardiomyocytes, it is difficult to see whole cells in a single section. Isolated cardiomyocytes can be fixed immediately and stained for a variety of cytoskeletal elements and ion channels. Confocal images can be compiled into full thickness z-stacks of the cell. Additionally, staining isolated cardiac myocytes allows for staining of components that are impossible to visualize in sections, such as the membrane invaginations known as t-tubules.
Cultured cell lines of cardiac myocytes do exist, some of which have similar transcriptional profiles and phenotypic features to cardiac myocytes. Some, such as the HL-1 cell line, even retain some sarcomeric organization and rudimentary contractile ability8. Despite these qualities, cultured cells lack the &#8220;box car&#8221; shape and t-tubule structure of cardiomyocytes and have less sarcomeric organization. These components are essential to the function of cardiomyocytes in vivo. In addition, using cultured cells allows genetic manipulation but not in vivo physiologic manipulation. Isolated cardiomyocytes retain their cellular organization long enough to study but are still easily imaged and even transduced9. 
Isolated cardiomyocytes are extremely versatile, providing a substrate to identically analyze cells from varied experimental conditions. Isolation and physiological analysis of myocytes have been used by countless labs for a variety of experiments including studying the effect of drugs6,7 or small molecules10, environmental stressors11, infection12, illness13, or genetic mutation14,15 on contractility and/or calcium transients and studying the regulation of contractility and calcium release within a cardiomyocyte15-17. With the application of any of these stressors, changes in heart function could be due to any combination of changes in the cardiomyocytes themselves and changes in the surrounding environment of the heart, such as scarring, changes in conductivity, or changes to the extracellular matrix. This is an ideal protocol for answering research questions regarding individual myocyte structure and function in the context of heart failure from any cause.
Although it has countless advantages over other techniques, physiological analysis of isolated cardiomyocytes is not ideal for every research question. Isolation is a terminal procedure, thus cardiomyocytes can only be assessed at one time in the mouse&#8217;s life. This can be partially overcome using cohorts and sacrificing individual mice along the course of illness development or by monitoring the mouse to determine whether it has clinically significant heart failure before isolation. Although this protocol can determine whether cardiomyocyte dysfunction has occurred, one cannot draw the conclusion that this dysfunction is the root cause of heart failure without additional experimental information. It is possible that cardiomyocyte dysfunction itself is secondary to other changes in the heart. Despite these challenges, studying cardiomyocytes offers valuable information in determining the nature of dysfunction in the heart, especially when combined with other experiments. 
The following protocol is adapted from one provided by Dr. Chee Lim, Vanderbilt University. Although contractility and calcium flux data are highly reproducible and useful in comparing hearts with genetic or environmental manipulation, isolation itself remains a technique dependent protocol, requiring optimization. Minimizing the time between removing the heart from the mouse and perfusing the heart via aortic cannulation is essential for a quality digestion. In addition, digestion time and enzyme concentration can be optimized for the highest quality myocytes. Steps likely requiring optimization, along with suggestions for optimization, are noted below.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">NaCl</td>
			<td style="width:221px;">RPI</td>
			<td style="width:159px;">S23020</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">KCl</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">P-3911</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:253px;">MgCl2</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">M-8266</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">HEPES</td>
			<td style="width:221px;">EM Science</td>
			<td style="width:159px;">S320</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:253px;">NaH2PO4</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">S5011</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:253px;">CaCl2 &#376; 2H2O</td>
			<td style="width:221px;">Fisher</td>
			<td style="width:159px;">C79</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">D-(+)-Glucose</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">G7528</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">2,3-Butanedione-monoxime (BDM)</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">B-0753</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Taurine</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">T-0625</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">A-7030</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Collagenase B</td>
			<td style="width:221px;">Roche Diagnostics</td>
			<td style="width:159px;">1-088-823</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Collagenase D</td>
			<td style="width:221px;">Roche Diagnostics</td>
			<td style="width:159px;">1-088-882</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Protease XIV</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">P-5147</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Heparin</td>
			<td style="width:221px;">APP Pharmaceuticals, LLC</td>
			<td style="width:159px;">401586D</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Isofluorane</td>
			<td style="width:221px;">Butler Schein</td>
			<td style="width:159px;">11695-6776-2</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Fura-2 AM</td>
			<td style="width:221px;">Teflabs</td>
			<td style="width:159px;">103</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">DMSO &#8211; anhydrous</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">276855</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:253px;">IonOptix cell pacing and fluorescent analysis system</td>
			<td style="width:221px;">IonOptix</td>
			<td style="width:159px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:253px;">250 um nylon mesh filter</td>
			<td style="width:221px;">Sefar America</td>
			<td style="width:159px;">Lab Pak 03-250/50</td>
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			<td height="22" style="height:22px;width:253px;">23G leur-stub adaptor</td>
			<td style="width:221px;">Becton Dickinson</td>
			<td style="width:159px;">1482619E</td>
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			<td height="22" style="height:22px;width:253px;">PE-50 tubing</td>
			<td style="width:221px;">Becton Dickinson</td>
			<td style="width:159px;">427410</td>
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</table>

isolation and physiological analysis of mouse cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Once the contractility and transient tracings are collected (Figure 2), data are easily analyzed with the appropriate software. The whole tracing, or portions thereof can be averaged (Figure 3). Contractility can be analyzed in a variety of ways. For systolic function, one can assess the magnitude of contraction with fractional shortening, or speed of contraction with time to peak shortening, and contraction velocity. Diastolic function can be analyzed similarly with time to 50% shorteni...

Watch this Scientific Journal Video about Isolation and Physiological Analysis of Mouse Cardiomyocytes at JoVE.com

Isolation and Physiological Analysis of Mouse Cardiomyocytes

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.

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

isolation-and-physiological-analysis-of-mouse-cardiomyocytes

Research

JoVE Journal

Biology

22.8K Views.  Vanderbilt University.  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. 

Video: Isolation and Physiological Analysis of Mouse Cardiomyocytes

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

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.

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

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.

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

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.

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.

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

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.

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...