马克·沃兹尼亚克在细胞与发育生物学录像设备的使用部门。 范德堡大学细胞成像共享资源。 这项工作是由美国心脏协会授予12PRE10950005到ERP系统，美国心脏协会授予11GRNT7690040到DMB，美国国立卫生研究院资助R01 HL037675到DMB的支持。 DMB是格拉迪斯体育斯塔尔曼主席在心血管研究。

确保涉及动物所有的程序都通过适当的动物使用认可和照顾身体。 1，准备股票缓冲器提前<ol><li>准备2升股票的 Ca 2 +免费台氏（ 表1）。将pH调节至7.4，用NaOH。 </li><li>准备2升股票1.2mM的钙台氏（ 表2）。将pH调节至7.4，用NaOH。 </li></ol> 2，在实验当天，准备灌注，转移和消化缓冲器<ol><li>准备150毫升灌注缓冲液中的Ca 2 +免费台氏（ 表3），并通过0.2微米的过滤器过滤。 </li><li>准备35毫升的 Ca 2 +免费接送“缓冲液A”（ 表4）和25ml 钙含“缓冲液B”（ 表5）。每个过滤通过0.2微米的过滤器。 </li><li>准备为0的解决方案0.06，0.24，0.6，和1.2毫米的Ca 2 +混合缓冲液A和B按照表6。 </li><li>制备25毫升消化缓冲液通过在基于鼠标的体重（BW）25毫升灌注缓冲液（ 表7）中溶解消化酶。通过使用前用0.2微米的过滤器过滤。 </li></ol> 3，实验装置<ol><li>组装恒定的流量的Langendorff装置，市售或使用蠕动泵，管道和换热器盘管，使为3ml /分的流量的灌流液加热到37°C以​​上。一个简单的原理设在2011年的审查贝尔等[18]。 </li><li>设置循环水浴中的Langendorff装置的温度（〜47℃），以便从所述套管的外流是在37℃以3毫升/分钟的流速。 </li><li>运行70％的乙醇通过灌注系统15分钟，接着用100ml超纯I型水。通过SYSTE运行灌注缓冲米5分钟，同时消除气泡。 </li><li>消毒所有的手术工具有希望的方法。 </li><li>通过将PE-50管的一小段（约3mm）至23政鲁尔存根适配器的前端创建插管。管散热技巧来创建一个唇在其上主动脉将定位。 </li></ol> 4，心肌细胞分离<ol><li>注入小鼠用0.2ml肝素溶液（1,000 IU /毫升）中，通过腹膜内注射。 </li><li> 5分钟后，麻醉小鼠异氟烷。当完全麻醉（无反应强烈脚捏），胸部喷用70％乙醇。打开胸腔，迅速摘除心脏，小心不要损坏主动脉，并将其放置在4℃灌注缓冲区。用弯钳抓住并提起下的心脏。这将创建空间修剪后面用细剪刀心脏附件。 </li><li>通过轻轻地抓主动脉的边缘有两对细镊子，小心地拉动openi导管插入心脏纳克主动脉超过套管的唇。确保套管的前端不是过去的主动脉瓣和入脑室，因为这将通过冠状动脉抑制心脏的灌流。 </li><li>通过捆扎的5-0丝线缝合环围绕主动脉立即插管的嘴唇上面固定主动脉插管。 </li><li>注意：步骤4.2至4.4，应尽可能快地提供最好的质量消化完毕。 </li><li>注意：心脏可以直接挂到已附加到的Langendorff装置与流灌注缓冲套管。然而，悬在心脏上连接到一个小注射器的解剖显微镜下灌注缓冲套管已被发现是最成功的。这使得少量的血流灌注缓冲被推至，看的冠状动脉的结算和保证心脏牢固地连接套管。该套管，然后挂在Langendorff离与运行灌注缓冲区。 </li><li>灌注与钙离子游离灌注缓冲的心脏在3毫升/分钟2-3分钟的流量。 </li><li>切换到消化酶和缓冲液灌注7-10分钟，心脏会变得柔软，颜色较浅。 </li><li>注意：消解时间可以根据酶的活性和心脏的大小进行调整。 </li><li>一旦心脏是触觉松弛，在无菌P60菜用缓冲液A（ 钙免费接送缓冲区）移除插管和心脏的地方。 </li><li>卸下心房和大血管。如果需要除去右心室。用钳子，分离心室切成小块。吸管数次，用无菌5毫升移液管，以进一步分散细胞。 </li><li>通过250微米的尼龙网过滤器过​​滤细胞悬液到50毫升锥形管。 </li><li>使悬浮液沉淀约10分钟。 </li><li>转移球团入0.06毫米的Ca 2 +的解决方案。使细胞沉淀10分钟。健康的细胞会沉淀，而大部分死亡的细胞会漂浮。 </li&gt; <li>吸出上清液，将沉淀转移到0.24毫米的Ca 2 +溶液。 </li><li>重复温育10分钟，吸出，并通过剩余的两个钙离子溶液转移至细胞中的1.2mM的溶液。在这一点上，大多数的细胞应该是杆状细胞。 </li></ol> 5，测量收缩和钙瞬变<ol><li>注意：两个收缩力和钙瞬变可以同时测量。 </li><li>由在无水DMSO中重悬浮至1微克/微升的浓度制备的Fura-2 AM溶液。的Fura-2 AM溶液能在-20℃下储存在干燥器中。 </li><li>负载1毫升悬浮细胞用1微升的Fura-2 AM。让细胞坐在黑暗中10分钟，然后吸出上清液。与钙台氏液洗两次，使细胞在黑暗中以沉淀的洗涤之间进行10分钟。 </li><li>放置盖玻片的阶段插入，使加热PErfusion /愿望和电极与心肌细胞领域的刺激起搏。在倒置显微镜地方台刀片设置了荧光测量，如果使用油浸物镜加一滴油镜头。 </li><li>设置加热重力灌注1.2毫米的Ca 2 +台氏液等阶段溶液达到37℃。附吸真空。连接电极。 </li><li>打开显微镜系统上，并打开成比例的荧光和细胞尺寸的数据采集软件。 </li><li>加入几滴的Fura-2 AM负载细胞的阶段插入，阻断血流灌注瞬间，让健康的细胞来解决。 </li><li>使用40倍物镜找到所需的细胞。一个健康的细胞应该是杆形，而不是自发收缩。它应该明显地，均匀地合同时，电池节奏5-20五。 </li><li>注意：某些生理分析需要高品质的消化。它能够具有细胞看起来健康但不啪<html> CE以及生理分析。消化协议可能需要细化，以获得高品质，健康的细胞。此外，突变体或患病的心脏，可能需要修改的协议。 </li><li>调整镜头旋转和改变光圈，使所需的细胞被一字排开横在屏幕上，背景是不可见的。 </li><li>对齐边缘检测杆到心肌细胞的每一端，并调整阈值。对齐肌检测细胞的均匀肌节的一部分。可以使这条越长，肌节长度的更精确的数学模型将是，只要它不是对肌节的两个群体。 </li><li>吴佩慈细胞2 Hz和5-20 V对于拍摄前10-15秒。 </li><li>跟踪记录。环境光线应尽量减少最佳钙瞬变记录。 </li></ol> 6，分析<ol><li>分析使用比率荧光和细胞尺寸数据采集软件的痕迹。 </li></ol>标题“&gt; 7。升学<ol><li>这个协议提供了过量的细胞，可用于各种其他实验，包括：固定和染色，评估直接药物作用，短期培养，和原子力显微镜。 </li></ol>

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我们什么都没有透露。

的质量的心肌细胞的分离需要实践和优化的一些水平。该协议包含可大大影响消化的结果的关键步骤。执行和解决方案时，这些应慎重考虑。从拆除的心脏插管灌注的时间应尽量减少，最好不超过五分钟。从鼠标在冰冷的缓冲液去除，并解剖和cannulating的心脏后，在冰冷的缓冲液灌注冲洗，立即的心脏也将增加优质消化的机会。插管本身是关键的一步。插管的尖端必须放在下面主动脉的第一分支?...

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.

一旦收缩和瞬态描收集（ 图2），数据很容易与适当的软件进行分析。整个跟踪，或它们的部分可以被平均化（ 图3）。收缩可以以各种不同的方式进行分析。对于收缩功能，人们可以评估收缩与缩短分数，或收缩随时间的速度的幅度峰值缩短和收缩速度。舒张功能同样可以用50％的时间缩短，减少速度进行分析。对于钙瞬变，类似的数据可以比包括基线和峰的Fura-2的比率...

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.

鼠心肌细胞的分离及生理分析

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 次观看.  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. 