Marc Wozniak pour l&#39;utilisation du Département de biologie cellulaire et de l&#39;équipement de la vidéographie de développement. Université Vanderbilt imagerie cellulaire de ressources partagées. Ce travail est soutenu par AHA subvention 12PRE10950005 à l&#39;ERP, AHA subvention 11GRNT7690040 de DMB, NIH R01 HL037675 à DMB. DMB est le président Gladys P. Stahlman en recherche cardiovasculaire.

S&#39;assurer que toutes les procédures impliquant des animaux sont approuvés par l&#39;utilisation des animaux appropriés et soins du corps. 1 Préparer Stock tampons à l&#39;avance <ol><li> Préparer 2 L stocks de Ca2 + libre de Tyrode (Table 1). Ajuster le pH à 7,4 avec NaOH. </li><li> Préparer 2 L stocks 1.2 (la Table 2) de mM Ca2 + Tyrode. Ajuster le pH à 7,4 avec NaOH. </li></ol> 2 Le jour de l&#39;expérience, Préparer la perfusion, transfert et Digestion tampons <ol><li> Préparer tampon de perfusion de 150 ml en Ca 2 + libre de Tyrode (tableau 3) et filtrer à travers un filtre de 0,2 um. </li><li> Préparer 35 ml de Ca 2 + libre transfert &quot;tampon A&quot; (Tableau 4) et de 25 ml contenant du Ca2 + &quot;tampon B» (Tableau 5). Filtre chacun à travers un filtre de 0,2 um. </li><li> Préparer des solutions de 00,06, 0,24, 0,6, et 1,2 mM de Ca 2 + par le mélange tampon A et B au tableau 6. </li><li> Préparer du tampon de digestion de 25 ml en dissolvant les enzymes de digestion dans 25 ml de tampon de perfusion (tableau 7) sur la base du poids corporel (BW) de la souris. Filtrer à travers un filtre de 0,2 um avant utilisation. </li></ol> 3. installation expérimentale <ol><li> Assemblage d&#39;un appareil de Langendorff à débit constant, disponible dans le commerce ou à l&#39;aide d&#39;une pompe, un tube, et une bobine d&#39;échangeur de chaleur péristaltique pour permettre un débit de 3 ml d&#39;écoulement / min de solution de perfusion chauffé à 37 ° C. Un schéma simple est fourni dans l&#39;examen de 2011 par Bell et al 18. </li><li> Régler la température de circulation du bain d&#39;eau (~ 47 ° C) de l&#39;appareil de Langendorff, afin que la canule de sortie est à 37 ° C à un débit de 3 ml / min. </li><li> Exécuter éthanol à 70% à travers le système de perfusion pendant 15 min, puis 100 ml d&#39;eau de type I ultrapure. Tampon de perfusion courir à travers la systématisationm pendant 5 min, tout en éliminant les bulles d&#39;air. </li><li> Stériliser tous les instruments chirurgicaux avec une méthode souhaitée. </li><li> Créer une canule en fixant un morceau court (environ 3 mm) de tube PE-50 à l&#39;extrémité 23 d&#39;un embout Luer-G adaptateur. pointe de chaleur de tube pour créer une lèvre sur laquelle l&#39;aorte sera positionné. </li></ol> 4. cardiomyocytes isolement <ol><li> Injecter souris avec 0,2 ml de solution d&#39;héparine (1000 IU / ml), par injection intrapéritonéale. </li><li> Après 5 min, anesthésier la souris avec isofluorane. Une fois complètement anesthésiés (pas de réponse à une forte pincée de pied), pulvériser poitrine avec EtOH à 70%. Ouvrez le coffre, exciser rapidement le coeur, en faisant attention à ne pas endommager l&#39;aorte, et dans 4 ° C tampon de perfusion. Utilisez une pince courbes de saisir et de soulever dans le cœur. Cela crée un espace pour couper les pièces jointes derrière le cœur avec des ciseaux fins. </li><li> Cathétériser coeur en saisissant délicatement le bord de l&#39;aorte avec deux paires de pinces fines et en tirant soigneusement la mise en service:ng de l&#39;aorte au-dessus de la lèvre de la canule. S&#39;assurer que la pointe de la canule n&#39;est pas passé la valve aortique et dans le ventricule, comme cela va inhiber la perfusion du coeur par les artères coronaires. </li><li> Fixer l&#39;aorte à la canule en attachant une boucle de fil de suture en soie 5-0 autour de l&#39;aorte juste au-dessus de la lèvre de la canule. </li><li> Remarque: Les étapes 4.2 à 4.4 devraient être achevés aussi rapidement que possible pour une meilleure digestion de la qualité. </li><li> Remarque: Le cœur peut être accroché directement sur la canule déjà fixé à l&#39;appareil de Langendorff avec coule tampon de perfusion. Cependant, le coeur accroché sur une canule fixée à une seringue de petit tampon de perfusion sous un microscope de dissection a été trouvé pour être plus efficace. Cela permet à une petite quantité de tampon de perfusion pour être poussée à travers, en regardant à la compensation des artères coronaires et d&#39;assurer le coeur est solidement fixé à la canule. La canule est ensuite accroché sur le Langendorff avec la gestion du tampon de perfusion. </li><li> Perfusele coeur de Ca2 + libre tampon de perfusion à un débit de 3 ml / min pendant 2-3 min. </li><li> Mettre à l&#39;enzyme tampon de digestion et perfuser pour 7-10 min, le cœur devient plus doux et plus légers en couleur. </li><li> Remarque: la durée de minéralisation peut être ajustée en fonction de l&#39;activité enzymatique et la taille du cœur. </li><li> Une fois que le cœur est manifestement flasque, retirez le cœur de canule et le placer dans un plat de p60 stérile avec le tampon A (Ca2 + tampon de transfert gratuit). </li><li> Retirer les oreillettes et les gros vaisseaux. Retirer le ventricule droit, si désiré. Avec une pince, séparer le ventricule en petits morceaux. Introduire à la pipette plusieurs fois avec 5 ml d&#39;une pipette de transfert stérile à disperser en outre des cellules. </li><li> On filtre la suspension cellulaire dans un tube conique de 50 ml à travers un filtre à mailles de nylon de 250 um. </li><li> Laisser la suspension de culot pour environ 10 min. </li><li> Transférer le culot dans la solution 2 + Ca 0,06 mM. Permettent aux cellules de granulés pour 10 min. Les cellules saines vont sédimenter, tandis que la plupart des cellules mortes flottent. </li&gt; <li> Aspirer le surnageant et transférer le culot à l&#39;mM Ca2 + solution de 0,24. </li><li> Répétez 10 minutes d&#39;incubation, l&#39;aspiration, et de les transférer dans les deux solutions 2 + Ca reste jusqu&#39;à ce que les cellules sont dans la solution de 1,2 mM. A ce moment, la majorité des cellules doit être myocytes en forme de tige. </li></ol> 5. mesure contractilité et calcium transitoires <ol><li> Remarque: Les contractilité et transitoires de calcium peuvent être mesurées simultanément. </li><li> Préparer Fura-2 AM solution par remise en suspension dans du DMSO anhydre à une concentration de 1 pg / pl. Solution Fura-2 AM peut être stocké dans un dessiccateur à -20 ° C. </li><li> Charge 1 ml de cellules en suspension avec 1 pi Fura-2 heures. Permettent aux cellules de s&#39;asseoir dans l&#39;obscurité 10 minutes, puis aspirer le surnageant. Laver deux fois avec la solution de Ca2 + Tyrode, permettant aux cellules de granulés dans l&#39;obscurité pendant 10 min entre les lavages. </li><li> Déposer une lamelle de verre dans un insert de phase qui permet chauffé perfusion / aspiration et l&#39;électrode de stimulation d&#39;un stimulateur domaine des myocytes. insert de scène de la Place de microscope inversé mis en place pour la mesure de la fluorescence, en ajoutant une goutte d&#39;huile à l&#39;aide d&#39;un objectif si l&#39;objectif à immersion d&#39;huile. </li><li> Mettre en place une perfusion par gravité chauffée avec une solution de 1,2 mM Ca2 + Tyrode donc solution sur scène atteint 37 ° C. Fixez aspiration à vide. Appliquez les électrodes. </li><li> Allumez le système de microscope, et ouvrir le logiciel de fluorescence et d&#39;acquisition de données de dimensionnement des cellules quotientométrique. </li><li> Ajouter quelques gouttes de cellules chargées de Fura-2 heures à l&#39;insert de la scène, bloquant momentanément la perfusion afin de permettre aux cellules saines de se déposer. </li><li> Utilisez objectif 40X pour localiser la cellule désirée. Une cellule saine doit être en forme de bâton et pas se contractant spontanément. Il faut visiblement et uniformément contrat lorsque la cellule est stimulée à 5-20 V. </li><li> Remarque: l&#39;analyse physiologique nécessite une digestion de haute qualité. Il est possible d&#39;avoir des cellules qui semblent saines, mais ne le font pas pa<html> CE et à l&#39;analyse physiologique. protocole de digestion peut être affinée pour obtenir de haute qualité, les myocytes sains. En outre, coeurs mutantes ou malades peuvent nécessiter des modifications au protocole. </li><li> Régler la rotation de la lentille et le changement ouverture de manière souhaitée cellule est aligné horizontalement sur l&#39;écran et le fond n&#39;est pas visible. </li><li> Aligner les barres de détection de bord à chaque extrémité de myocytes et ajuster seuil. Aligner la détection des sarcomères sur une partie de cellule de sarcomères uniformes. Le plus vous pouvez faire ce bar, le plus précis du modèle mathématique de la longueur de sarcomère sera, tant que ce n&#39;est pas sur deux populations de sarcomères. </li><li> cellules de Pace à 2 Hz et 5-20 V pour 10-15 secondes avant l&#39;enregistrement. </li><li> traçage d&#39;enregistrement. La lumière ambiante doit être minimisée pour le meilleur enregistrement calcium transitoire. </li></ol> Analyse 6. <ol><li> Analyser des traces par fluorescence ratiométrique et logiciel d&#39;acquisition de données de dimensionnement de cellule. </li></ol>titre &quot;&gt; 7. D&#39;autres études <ol><li> Ce protocole fournit les cellules excédentaires qui peuvent être utilisés pour une variété d&#39;autres expériences, notamment: la fixation et coloration immunologique, l&#39;évaluation des effets directs de la drogue, de la culture à court terme, et la microscopie à force atomique. </li></ol>

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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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Nous n&#39;avons rien à divulguer.

Isolation des cardiomyocytes de qualité nécessite un certain niveau de pratique et d&#39;optimisation. Ce protocole contient des étapes clés qui peuvent grandement influer sur le résultat de la digestion. Ceux-ci devraient être examinés avec soin lors de l&#39;exécution et le dépannage du protocole. Le temps de retrait de la canule de coeur et la perfusion doit être réduit au minimum, de préférence moins de cinq minutes. Rinçage du coeur immédiatement dans un tampon de perfusion glacé après le retrait d...

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.

Une fois la contractilité et de tracés de passage sont recueillies (figure 2), les données sont facilement analysées avec le logiciel approprié. Le tout suivi, ou des parties de ceux-ci peuvent être en moyenne (Figure 3). Contractilité peut être analysé de différentes façons. Pour la fonction systolique, on peut évaluer l&#39;ampleur de la contraction de la fraction de raccourcissement, ou la vitesse de contraction de temps pour culminer raccourcir, et la vitesse de contract...

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.

L&#39;isolement et l&#39;analyse physiologique de cardiomyocytes de souris

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 vues.  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. 

Vidéo: 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.

Dissection de l&#39;hippocampe gyrus denté de souris adulte

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 ...

Recherche

Biologie

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.

L&#39;imagerie in vivo et des traitements thérapeutiques dans un modèle de souris orthotopique de cancer des ovaires

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.

Profilage Single Cell transcriptionnelle des cardiomyocytes de souris adultes

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.

Isolement et enregistrements Chaîne Kv en cardiomyocytes murins auriculaire et ventriculaire

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.

Isolement et culture de cardiomyocytes de souris néonatales

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.

Isolement et culture de cardiomyocytes adultes de souris pour Signalisation cellulaire et<em&gt; In vitro</em&gt; Hypertrophie cardiaque

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.

Isolement et analyse fonctionnelle des mitochondries de cellules cultivées et de tissus de souris

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.

L&#39;isolement, la culture et la transduction des adultes cardiomyocytes souris

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.

Isolation simultanée de cardiomyocytes de haute qualité, de cellules endothéliales et de fibroblastes d&#39;un coeur de rat adulte

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.

TRANS-intérieur cellulaire massive Injection de cellules souches embryonnaires conduit à des taux plus élevés de chimérisme

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