Marc Wozniak per l&#39;utilizzo del Dipartimento di Cell e Developmental Biology attrezzature videografia. Vanderbilt University cellulare Imaging risorsa condivisa. Questo lavoro è supportato da AHA concessione 12PRE10950005 di ERP, AHA concessione 11GRNT7690040 a DMB, NIH concedere R01 HL037675 a DMB. DMB è il presidente Gladys P. Stahlman di Ricerca Cardiovascolare.

Assicurarsi che tutte le procedure che coinvolgono gli animali sono approvati con l&#39;uso appropriato degli animali e la cura del corpo. 1 Preparare buffer di archivio in anticipo <ol><li> Preparare 2 L stock Ca 2 + libero di Tyrode (Tabella 1). Regolare il pH a 7,4 con NaOH. </li><li> Preparare 2 L stock di 1.2 mM Ca 2 + Tyrode (Tabella 2). Regolare il pH a 7,4 con NaOH. </li></ol> 2 Sul giorno dell&#39;esperimento, preparare perfusione, trasferimento, e digestione Buffer <ol><li> Preparare il tampone di perfusione 150 ml di Ca 2 + libero di Tyrode (Tabella 3) e filtrare attraverso un filtro 0,2 micron. </li><li> Preparare 35 ml di Ca 2 + trasferimento gratuito &quot;Buffer A&quot; (Tabella 4) e 25 ml di Ca 2 + contenente &quot;Buffer B&quot; (Tabella 5). Filtrare ciascuno attraverso un filtro da 0,2 micron. </li><li> Preparare soluzioni di 0.06, 0.24, 0.6, e 1.2 mM Ca 2 + mescolando tampone A e B per ogni tabella 6. </li><li> Preparare tampone di digestione 25 ml sciogliendo enzimi digestivi in 25 ml di tampone di perfusione (Tabella 7) sulla base del peso corporeo (BW) del mouse. Filtrare attraverso un filtro da 0,2 micron prima dell&#39;uso. </li></ol> 3 setup sperimentale <ol><li> Assemblare un apparato Langendorff flusso costante, disponibile in commercio o utilizzando una pompa peristaltica, tubi, e una batteria di scambio termico per consentire un flusso di 3 ml / min di perfusato riscaldato a 37 ° C. Un semplice schema è previsto nel 2011 recensione di Bell, et al 18. </li><li> Impostare circolante temperatura del bagno d&#39;acqua (~ 47 ° C) dell&#39;apparato Langendorff cosicché deflusso dalla cannula è a 37 ° C ad una velocità di flusso di 3 ml / min. </li><li> Eseguire il 70% di etanolo attraverso il sistema di perfusione per 15 minuti, seguita da 100 ml di acqua ultrapura di tipo I. Buffer di perfusione Corri attraverso il system per 5 min, eliminando le bolle d&#39;aria. </li><li> Sterilizzare tutti gli strumenti chirurgici con un metodo desiderato. </li><li> Crea cannula allegando un breve pezzo (circa 3 mm) di PE-50 tubo alla punta di un adattatore Luer-stub 23 G. Punta di calore dei tubi per creare un labbro sul quale l&#39;aorta sarà posizionato. </li></ol> 4. Cardiomyocyte Isolamento <ol><li> Iniettare mouse con 0,2 ml di soluzione di eparina (1.000 UI / ml), mediante iniezione intraperitoneale. </li><li> Dopo 5 minuti, anestetizzare mouse con isofluorane. Quando è completamente anestetizzato (nessuna risposta al forte pizzico piedi), spruzzare il petto con il 70% EtOH. Aprite la cassa, asportare rapidamente il cuore, facendo attenzione a non danneggiare l&#39;aorta, e posto a 4 ° C buffer di perfusione. Utilizzare pinze curve di afferrare e sollevare sotto il cuore. Questo crea lo spazio per tagliare gli allegati dietro il cuore con le forbici sottili. </li><li> Cannulate cuore afferrando delicatamente il bordo della aorta con due coppie di pinze sottili e tirando con attenzione la opening dell&#39;aorta sopra il labbro della cannula. Assicurarsi che la punta della cannula non è oltre la valvola aortica e nel ventricolo, perché impedirebbe perfusione del cuore attraverso le arterie coronarie. </li><li> Fissare l&#39;aorta alla cannula legando un cappio di sutura di seta 5-0 intorno all&#39;aorta immediatamente sopra il labbro della cannula. </li><li> Nota: I passaggi 4.2 con 4.4 dovrebbe essere completata il più rapidamente possibile per la digestione migliore qualità. </li><li> Nota: Il cuore può essere appeso direttamente sulla cannula già attaccato al Apparato Langendorff con fluente buffer di perfusione. Tuttavia, appeso il cuore su una cannula collegata ad una piccola siringa di buffer di perfusione al microscopio dissezione è stato trovato per essere di maggior successo. Questo permette una piccola quantità di buffer di perfusione deve essere spinto attraverso, guardando per la compensazione delle arterie coronarie e garantendo il cuore è attaccato saldamente alla cannula. La cannula viene poi appeso alla Langendorff con tampone di corsa perfusione. </li><li> Perfuseil cuore con Ca 2 + buffer di perfusione libero a un flusso di 3 ml / min per 2-3 minuti. </li><li> Passare al buffer di digestione enzimatica e profumato per 7-10 minuti, il cuore diventerà più morbida e di colore più chiaro. </li><li> Nota: tempo di digestione può essere regolata in base alle attività enzimatica e la dimensione del cuore. </li><li> Una volta che il cuore è palpabile flaccido, rimuovere il cuore dalla cannula e mettere in un piatto p60 sterile con tampone A (Ca 2 + buffer di trasferimento gratuito). </li><li> Rimuovere atri e dei grossi vasi. Rimuovere ventricolo destro se lo si desidera. Con pinze, separare il ventricolo in piccoli pezzi. Pipettare più volte con una sterile 5 ml trasferimento pipetta per disperdere ulteriormente le cellule. </li><li> Filtrare la sospensione cellulare in una provetta conica da 50 ml attraverso un filtro a rete di nylon da 250 micron. </li><li> Lasciare che la sospensione a pellet per circa 10 min. </li><li> Trasferire il pellet nella soluzione 0,06 mM Ca 2 +. Consentono alle cellule di pellet per 10 min. Le cellule sane saranno pellet, mentre la maggior parte delle cellule morte galleggia. </li&gt; <li> Aspirare il surnatante e trasferire il pellet al Ca 2 + 0,24 mM soluzione. </li><li> Ripetere 10 min di incubazione, aspirazione, e trasferire attraverso i restanti due Ca 2 + soluzioni fino a quando le cellule sono nella soluzione 1.2 mM. A questo punto, la maggioranza delle cellule dovrebbe essere miociti forma di bastoncello. </li></ol> 5. misura contrattilità e calcio Transients <ol><li> Nota: Sia la contrattilità e transienti di calcio possono essere misurate simultaneamente. </li><li> Preparare Fura-2 AM soluzione risospendendo in DMSO anidro ad una concentrazione di 1 mg / mL. Soluzione Fura-2 AM può essere immagazzinato in un essiccatore a -20 ° C. </li><li> Carico 1 ml di cellule in sospensione con 1 ml Fura-2 del mattino. Consentono alle cellule di sedersi nel buio 10 min poi aspirare il surnatante. Lavare due volte con la soluzione di Ca 2 + Tyrode, permettendo alle cellule di pellet al buio per 10 minuti tra un lavaggio. </li><li> Posizionare un coprioggetto di vetro in un inserto fase che permette pe riscaldatarfusion / aspirazione ed elettrodo di stimolazione con uno stimolatore campo miociti. Luogo inserto sul palco microscopio invertito istituito per la misurazione della fluorescenza, aggiungendo una goccia di olio di lente se si utilizza un obiettivo ad immersione in olio. </li><li> Impostare perfusione gravità riscaldata con soluzione 1,2 mM Ca 2 + Tyrode così soluzione sul palco raggiunge i 37 ° C. Fissare l&#39;aspirazione a vuoto. Attaccare gli elettrodi. </li><li> Accendere il sistema microscopio, e aprire la fluorescenza e di acquisizione dati dimensionamento delle cellule software raziometrico. </li><li> Aggiungere qualche goccia di cellule caricate Fura-02:00 all&#39;inserto fase, bloccando perfusione momentaneamente per consentire le cellule sane a stabilirsi. </li><li> Utilizzare obiettivo 40X per individuare cella desiderata. Una cellula sana dovrebbe essere cilindriche e non spontaneamente contraente. Dovrebbe visibilmente e contratto uniforme quando la cella viene stimolato a 5-20 V. </li><li> Nota: Analisi fisiologica richiede una digestione di alta qualità. E &#39;possibile avere le cellule che sembrano in buona salute, ma non lo fanno pa<html> Ce bene per l&#39;analisi fisiologica. Protocollo di digestione può richiedere raffinatezza per ottenere alta qualità, miociti sani. Inoltre, cuori mutanti o malati possono richiedere modifiche al protocollo. </li><li> Regolare la rotazione della lente e il cambiamento di apertura così cella desiderata è allineata orizzontalmente sullo schermo e lo sfondo non è visibile. </li><li> Allineare barre di rilevamento bordo a ciascuna estremità di miociti e regolare la soglia. Allineate rilevamento sarcomere su una porzione di cella con sarcomeri uniformi. Più a lungo si può fare questo bar, il più accurato il modello matematico di lunghezza sarcomero sarà, fintanto che non è su due popolazioni di sarcomeri. </li><li> Cellule Pace a 2 Hz e 5-20 V per 10-15 secondi prima della registrazione. </li><li> Record tracciato. La luce ambiente deve essere ridotta al minimo per la migliore registrazione di calcio transitori. </li></ol> 6 Analisi <ol><li> Analizzare le tracce utilizzando la fluorescenza raziometrico e software di acquisizione dati dimensionamento delle cellule. </li></ol>titolo &quot;&gt; 7. Ulteriori studi <ol><li> Questo protocollo fornisce le cellule in eccesso che possono essere utilizzati per una varietà di altri esperimenti, tra cui: fissazione e immunostaining, valutando gli effetti diretti della droga, la cultura a breve termine, e la microscopia a forza atomica. </li></ol>

<ol>
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Isolamento di cardiomiociti di qualità richiede un certo livello di pratica e di ottimizzazione. Questo protocollo contiene passaggi chiave che possono influenzare notevolmente il risultato della digestione. Questi dovrebbero essere considerate attentamente quando si esegue la risoluzione dei problemi e il protocollo. Il tempo dalla rimozione del cuore di cannulazione e perfusione deve essere ridotto al minimo, idealmente meno di cinque minuti. Sciacquare immediatamente il cuore in tampone di perfusione ghiacciato dopo...

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 volta che la contrattilità e tracciati transitori vengono raccolti (Figura 2), i dati possono essere facilmente analizzati con il software appropriato. L&#39;intero tracciato, o parti di essi possono essere mediate (Figura 3). Contrattilità può essere analizzato in una varietà di modi. Per la funzione sistolica, si può valutare l&#39;ampiezza della contrazione con accorciamento frazionale, o la velocità di contrazione con il tempo al picco accorciando, e la velocità di contra...

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.

Isolamento e fisiologica Analisi di mouse Cardiomyocytes

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. 