Name: isolation and physiological analysis of mouse cardiomyocytes
Uploaded: 2014-09-07T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Physiological Analysis of Mouse Cardiomyocytes at JoVE.com

Marc Wozniak para el uso de Departamento de la célula y el equipo videografía Biología del Desarrollo. Universidad Vanderbilt de imágenes de células de recursos compartidos. Este trabajo es apoyado por AHA subvención 12PRE10950005 a ERP, AHA subvención 11GRNT7690040 a DMB, NIH subvención R01 HL037675 de DMB. DMB es el Presidente Gladys P. Stahlman en Cardiovascular Research.

Asegurar que todos los procedimientos con animales son aprobados por el uso adecuado con animales y el cuidado del cuerpo. 1. Preparar Imagenes Buffers in Advance <ol><li> Prepare 2 L de stock Ca 2 + de Tyrode libre (Tabla 1). Ajustar el pH a 7,4 con NaOH. </li><li> Prepare 2 L de stock 1,2 mM de Ca2 + Tyrode (Tabla 2). Ajustar el pH a 7,4 con NaOH. </li></ol> 2. En el día del experimento, Preparar perfusión, T...

<ol>
	<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. 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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. (31), e1433 (2009).</li><li>Vainio, 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=Neronostatin,+a+Novel+Peptide+Encoded+by+Somatostatin+Gene,+Regulates+Cardiac+Contractile+Function+and+Cardiomyocytes+Survival.">Neronostatin, a Novel Peptide Encoded by Somatostatin Gene, Regulates Cardiac Contractile Function and Cardiomyocytes Survival.</a> The Journal of Biological Chemistry. 287, 4572-4580 (2012).</li><li>Park, 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=Novel+mechanisms+for+caspase+inhibition+protecting+cardiac+function+wth+chronic+pressure+overload.">Novel mechanisms for caspase inhibition protecting cardiac function wth chronic pressure overload.</a> Basic Research in Cardiology. , 108 (2013).</li><li>Novaes, R. 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=Effects+of+Trypanosoma+cruzi+infection+on+myocardial+morphology,+single+cardiomyocyte+contractile+function+and+exercise+tolerance+in+rats.">Effects of Trypanosoma cruzi infection on myocardial morphology, single cardiomyocyte contractile function and exercise tolerance in rats.</a> International Journal of Experimental Pathology. 92, 299-307 (2011).</li><li>Weltman, N. Y., Wang, D., Redetzke, R. A., Gerdes, A. 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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Aislamiento de cardiomiocitos de calidad requiere un cierto nivel de la práctica y la optimización. Este protocolo contiene pasos clave que pueden afectar en gran medida el resultado de la digestión. Estos deben ser cuidadosamente considerados al realizar la solución de problemas y el protocolo. El tiempo desde la extracción del corazón de la canulación y la perfusión debe reducirse al mínimo, idealmente menos de cinco minutos. Enjuagar el corazón inmediatamente en tampón de perfusión enfriado con hielo desp...

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>
			<td></td>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr height="22">
			<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>
			<td></td>
		</tr>
		<tr height="22">
			<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>
			<td></td>
		</tr>
	</tbody>
</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.

Una vez que la contractilidad y trazados transitorios se recogen (figura 2), los datos se analizan con facilidad con el software adecuado. Todo el rastreo, o porciones de los mismos se pueden promediar (Figura 3). La contractilidad se puede analizar en una variedad de maneras. Para la función sistólica, se puede evaluar la magnitud de la contracción con la fracción de acortamiento, o la velocidad de la contracción con el tiempo hasta el pico acortamiento, y la velocidad de contracc...

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

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Aislamiento y fisiológica Análisis de Ratón cardiomiocitos

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 ...

Research

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

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Aislamiento simultáneo de cardiomiocitos de alta calidad, células endoteliales y fibroblastos de un corazón de rata adulto

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 ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

Aislamiento de las glándulas mamarias del ratón intacto, todo para el análisis de expresión de la matriz extracelular y la morfología de la glándula

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...