Name: analysis of tubular membrane networks in cardiac myocytes from atria and ventricles
Uploaded: 2014-10-15T13:00:00.000+00:00
Duration: 10 min 30 s
Description: Watch this Scientific Journal Video about Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles at JoVE.com

This work received support through Deutsche Forschungsgemeinschaft SFB 1002 (subprojects A05 and B05 to S.E.L.) and KFO 155 (subproject 4 to S.E.L.), a Halbach Foundation award to S.E.L. supporting E.W.; a grant from the German Cardiac Society to S.B.; and a DAAD exchange program supporting T.K. as visitor at the University of Maryland. The research leading to these results has received funding from the European Community&#8217;s Seventh Framework Program FP7/2007-2013 under grant agreement No. HEALTH-F2-2009-241526, EUTrigTreat (to S.E.L.). S.E.L. is a principal investigator of the German Center of Cardiovascular Research (DZHK).

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The authors declare that no conflict of interest exists.

虽然心肌细胞已被分离，并研究了几十年的32，最近一次检讨的结论是一致的高质量的心肌细胞的隔离仍然充满挑战27。这反映了主要的心肌细胞VIS-à-VIS缺乏共同的标准方法，共享的元数据，而透明细胞质量文件的隔离相对复杂的协议。细胞分离的协议通常是由单独的组定制，产生可变结果的细胞株，取决于个体模式设置（ 例如 ，物种，年龄，并存心脏疾病），通常调整为特定的实验条件。在定量TATS膜的研究和协议这里提出，质量评估和文档的基本水平的情况下涉及单个细胞的膜结构容易出现代谢和隔离协议相关变动或共聚焦显微镜超高分辨率，无论是在上午或VM。重要的是，即使细胞株高产建议健康完好的细胞，研究人员需要记录和批判仔细判断每个细胞对抗与非特异性损害表面和TATS细胞膜的完整性，由于分离过程对由于不同类型的具体变化形态标准干预措施相比，控制的条件。在心肌细胞的分离的一个重要变量是一个给定的胶原酶大量的特定活性。以选择一组新的胶原酶，胶原酶几个样品的酶活性，应互相通过评估心肌细胞产率和质量，并根据制造商的说明书进行试验。理想情况下，一个新的大量的胶原酶是确定与类似于以前成功地使用大量的胶原酶活性（对可能的酶活性进一步评估是指在材料和方法表中的"胶原酶很多选择工具"）。牛逼阿肯一起的TATS膜的可视化定量方法严重依赖于细胞的分离和质量，反之亦然，心肌细胞隔离导致非特异性膜破坏记录在案的TATS显微镜应触发的隔离程序，严格审查和纠正。由于细胞分离质量和TATS膜的可视化和量化有着内在的联系，本文所讨论的协议，涵盖所有主要方面作为一个连续的策略。 一个额外的挑战和心脏研究中，细胞损伤和/或细胞损失的常见问题的发生是由于代谢损害的干预，例如 ，心肌梗死后如图9所示 ，还需要针对潜在的意外损坏，例如判断，下列过程中细胞分离忽视空气栓塞。从患病心脏的心肌细胞的分离可能导致额外的，显著细胞损失和降低细胞的产量。因此，对比例如果细胞的分离和计数是通过标准化的协议一致地施加控制和病态心脏之间分离的完整细胞的总数的ISON可以是有意义的。因此，重要的是通过适当的对照组，这反映了最佳的心肌细胞的分离的质量来判断细胞的完整性。重要的是，单个细胞的质量和健康与患病细胞与活细胞显微镜的分离过程中不慎损坏，可能显著影响TATS膜的网络分析。这里介绍的协议，在细胞的分离和胎膜完整的活细胞显微因此强调生理膜元件的完整性和稳定性。整个工作流被设计为一个连续的策略来实现并保持完好TATS膜元件同时排除损伤的细胞，因为这些将表现出隔离依赖性膜器物等破坏膜小管，卷材胶粘ê泡，并改变TATS网络错误地控制条件和损害进一步定量分析。反之，同样的策略是至关重要的与潜在的干预研究扰乱TATS膜，它主要取决于真正健康与同TATS膜变化真患病细胞之间进行有意义的比较对照。 此外，我们要解决的程序，实现了技术上更有挑战性的隔离上午细胞。尽管进步和改善的协议，它强调的是，这是不平凡重现虚拟机的高品质小区隔离，甚至不太可靠的肺泡是很重要的。这是由于整体产量较低AM的细胞，其中在细胞的分离，即使小的误差或变化可能导致完全的调幅细胞分离失败，而VM的细胞损伤轻微程度可能是在细胞悬浮液中不那么明显，由于相对高的细胞数字相比，PM。由于上午细胞可能成为ç分离后urved，通过几个感兴趣区分析可能是有利的根据步骤4.3所概述。继细胞分离步骤的详细过程，我们提供了一个协议，直接整合膜染色和共聚焦或TATS都为虚拟机和AMS网络的STED超分辨成像。这些协议使通过先前建立的参数定量分析和TATS膜的选择组件的分化。相比于虚拟机，三维组织和肺泡心房TATS网络功能的行为是目前还不了解。 该程序形象TATS膜在活细胞中（步骤3.1至3.7）的开发与商业焦（表材料/设备）和定制的受激发射损耗荧光显微镜9。以优化荧光图像生成和定量分析TATS显微镜设置，以下几点是具有普遍意义: <ul><li> 目的 </str翁&gt; 为了解决TATS膜结构的小细节，实证检验的目标提供最高的图像质量，同时注重几个微米深的细胞。某些共焦显微镜可以与水或甘油的目标，以此处使用的63X 1.4 NA油浸物镜有更好的表现，与此相反。目标与100X的放大倍率用于STED超分辨显微术，权衡的视场较小的纳米级分辨率的34。 </li><li> 激发和增益 励磁功率和检测器增益的最佳设置取决于显微镜光路，激光表演和样本性质上。理想情况下，激光功率和增益调节以利用全范围的检测器的，但避免图像的饱和度。商业显微镜软件包通常提供了可视化的动态范围的下限和上限的查找表。此外，为了最小化染料漂白采用尽可能低的升ASER电源仍然提供足够的结构TATS膜的详细信息。此外，激励功率应足够低，以避免导致心肌细胞挛缩和死亡的累积光损伤。 </li><li> 像素大小 使用像素大小与奈奎斯特采样兼容，大约有一半实现了与给定设置的分辨率。对于共焦成像的100纳米×100nm的像素大小是兼容的，这也将限制漂白。对于超分辨率显微镜显著更小的像素尺寸，例如使用20纳米×20纳米的STED显微镜9。 </li><li> 停留时间 共焦显微镜提供了一个平均功能。在一般情况下，用最短的像素停留时间，以避免漂白结合信号平均化，例如 ，线路均≥8，以改善信号噪声比。 </li><li> 文档的应用显微设置通过元数据 一旦设置如何TATS膜结构的图像细节进行了优化，在一个特定的激光共聚焦显微镜，安全和/或文档的设置（协议的元数据）。掌握细胞内的（或间）组（S）的所有图像具有相同的目标，激励功率，增益，像素尺寸，像素停留时间和平均功能。平等的成像条件允许的范围内细胞（或间）组（S）直接比较和定量。 </li><li>对于一般的指导和有关原则和共聚焦显微镜的应用程序的详细信息请参阅生物共轭焦显微镜（帕利JB，第3版，2006年，施普林格科学+商业媒体有限责任公司）的手册。 </li></ul>相反，这里介绍的直接分析的策略，描述TATS膜与疾病相关的变化以前的出版物中，使用了T型小管密度区域总读数定量策略16,17，或间接的区域战略基于傅立叶互感器纹状的膜的信号，以评估T-细管部件规律性7本署可能分析。相反，此处所描述的定量方法是直接关系到个人TATS组件，并提供了一​​些额外的参数，包括膜的网络性能和像的A-肾小管的百分数特定组件。此外，TATS网络密度可被量化为每ROI区域的整个提取骨架的归一化长度。三个单独的三联点数目，连续地连接小管组件可以用来作为TATS膜网络的分支的复杂性的度量。我们注意到，最小TATS组件的任何分析依赖于染色程序。根据我们的经验，800微升的50微米二8 ANEPPS解决方案，足以完成染色TATS网络，包含50000 VM 9细胞的细胞沉淀。但是，如果将细胞沉淀中含有心肌细胞的下数如果强大的荧光检测器是可用的，并且如果根据经验测试可以使用整体TATS网络分布的共焦成像，而不是最小的膜细节和量的变化是感兴趣的，低级的染料浓度。最后，所描述的分析编写的软件的宏可用于自动化图像处理步骤，以促进分析的更大的数据集，这对于不同的治疗组（ 例如 ，药物），细胞类型之间的比较是特别有用的（ 例如 ，AM与VM ），和病理生理干预（ 例如 ，假与心肌梗死）。 为TATS网络的图像分析，主要步骤如下顺序应用:1）滚动的球背景减法（4.5.1），以除去在背景强度的空间变化; 2）局部对比度增强（4.5.2）; 3）图像平滑（4.5.3）; 4）统计区域合并（4.5.4）; 5）定义图像二值化的阈值（4.5.6）;和6）计算该骨架数据的（4.5.8）。的荧光TATS图像的骨架化过程中的一个关键步骤是在图6所示的图像的二值化的阈值相关联的步骤，最终确定被检测到表示底层TATS部件与从背景噪声确定错误可能的错误结构，其真正的膜结构。正确的阈值进行二值化图像分析，识别要与真实TATS膜结构，它依赖于足够高的信号 - 噪声（SNR）的各比值为共焦和超高分辨率显微镜方法对应。因此，足够的图像质量，应首先建立，随后结合单个细胞的质量包括亮场图像文件的关键判断，概括。替代方案，以适应图像分割协议对于给定的显微镜数据输出和/或物理iological问题包括图像去卷积和像"大津"或"ISO数据"作为ImageJ的插件，其他的阈值程序。不管最终的分割过程中，我们考虑了图像叠加的强制性质量控制步骤提取原始数据之间的比较。总之，个人的离体心肌细胞，足以染色细胞TATS膜，用于荧光成像参数优化，并提取骨​​架数据的叠加控制形态和膜的完整性都将有助于荧光TATS图像和定量结果的质量。 如果较大的物种比小鼠用于细胞的分离，该协议可以很容易地修改为适当的。为下一个较大的物种，大鼠心脏可插管的钝地下14套管（外2.1mm直径），并灌注以8毫升/分钟。显著年长或患病的心脏，可能需要更大的套管尺寸。在基因拉尔，心脏灌注可以或者由恒压例如进行，使用储层和主动脉之间或通过使用蠕动泵恒定流动的高1米水柱。用于从小型啮齿动物的心像小鼠和大鼠细胞分离恒定流可能是有利的，因为胶原酶消化最终将破坏冠状动脉阻力血管，导致过度的灌注速度漏出血管床将由恒定流的协议来控制到某种程度。与此相反，恒压灌注是有利的，如果监视的流量和正确的插管是一个优先，这是有利的干预模式具有改变血管阻力的行为，以及用于在细胞分离程序的培训。 如以上概述的，足够的细胞质量为内源性膜系统的定量研究很重要。然而，在心脏灌注和胶原酶消化众多因素CRitically影响细胞的分离，这不应该在协议优化和故障排除27被低估的质量。特别是，在给定的胶原酶大量的活性应为兴趣如心房或在执行前的实验真正研究建立隔离条件心室的特定组织被确定为保持在整个研究的剩余部分。此外，水的质量，pH值，温度，灌注设置的优化和清洗会无意中破坏污染物和栓塞的风险最小化，并有可能附加因素必须被监视，以在细胞分离建立最佳稳态条件。 BDM（2,3 - 丁二酮-肟）肌球蛋白ATP横桥可逆抑制剂的组织解剖和消化过程中是常用的，以维持心脏肌肉松弛，从而增加细胞分离物的产率。不过，调查人员需要吨ö意识到BDM可能施加导致脱靶效应，例如 ，在某些条件下33抑制钠 / 钙交换电流的非特异性磷酸酶的活性。对于一些实验中，可能有利的由II型肌球蛋白的心脏停搏液，抑制剂与肌球蛋白在微摩尔浓度的高亲和性然而这是，中毒性且相对昂贵的更换BDM，并且可以具有其他的脱靶效应。搁健康心肌不应显示在没有电刺激与这些细胞的任何收缩应被排除在进一步分析。另一方面，响应于在生理细胞外的 Ca 2 +浓度的电刺激心肌细胞的收缩和舒张，可用于建立正常的收缩行为作为一种附加措施来评估心脏疾病与健康控制功能单元的质量和/或反常的行为细胞。 9和AM单元21以及用于微管网络在定量分析定心肌细胞（数据未示出）。的协议，这些和未来的应用可能会打开渠道，多种实验问题，如TATS膜在不同发育阶段的特征或相关膜蛋白或细胞器结构的联系TATS网络的分析，以发挥高度本地化的，特定的域信号功能在上午和VM细胞。

在健康横纹肌细胞，管状膜结构与"横向"方向（T管）垂直于主细胞轴是丰富的。因此，T管已被定性为肌肉细胞主要的"横向"的表膜（肌膜）持续扩展，深入渗透朝小区中心的细胞质。的T管连续的外表面的膜的生理作用是通过SER细胞器接触结构域在整个较大的心肌细胞体积的电压活化的L-型Ca纳米邻近耦合形成远程胞内区室的快速电耦合离子通道（Cav1.2）内向电流（I Ca）的激活相邻兰尼碱受体（RyR2的）SER的钙离子释放。在交界SER域和T之间的心室肌细胞（虚拟机）中，非连续的膜的接触（"结"）-tubules被认为控制数千个体细胞内的 Ca 2 +释放纳米畴的每个单元1中。 对于任何给定的接触领域，在并置的膜的每个T形小管和外周（连接）的SER的部分是大约彼此接近15纳米，因此定义为nanodomain。因此，极个别小细胞质子空间隔离这使准小区自治仓行为。当传入的动作电位活化的T管虚拟机，一个相对较小的Ca Cav1.2 离子通道内向电流会迅速增加的子空间中钙离子浓度的钙离子浓度S IN的attoliter大小nanodomain 1。接下来，[ 钙 ] S增加激活钙离子门控的兰尼碱受体（RyR2的）中的并列SER膜纳米路口附近，并且这种耦合过程发生在所有电连接ð心肌细胞纳米畴。 RyR2s发生致密多通道簇的1 Cav1.2通道的估计的化学计量为5-10的RyR2通道2。由于SER到胞浆钙离子浓度梯度很陡（10比4:1）和RyR2s作为高电导钙离子的功能结合型集群的释放渠道，在数量上大RyR2的钙激活导致2 +释放的T小管加上交界的SER域增加局部子空间的[Ca 2 +] S以100微米或更高在1-2毫秒3,4电流。这种心脏信号放大行为也被称为钙离子诱导的Ca 2 +释放（CICR）。总之，T管是必要的膜结构，可以快速激活兴奋收缩（EC）的耦合过程中通过交界nanodomain SER的接触和细胞性CICR ​​钙释放的信号。 除了T管，轴向细管S（A-小管）平行于主（纵向）细胞轴的显著不同的方向已经证实了通过电子显微镜（EM），共聚焦和双光子显微镜的研究。举例来说，肌原纤维相互连接的带T管附近肌Z-线之间的A-肾小管细胞的宽连续晶格所表现出细胞外示踪剂和固定豚鼠虚拟机5的EM图像。使用外葡聚糖联荧光素染色和生活大鼠虚拟机的双光子成像，复杂的三维网状小管网络中包含可视〜60％T管和〜40％，小管6。这项研究不仅导致了大量的A-小管三维可视化，同时也实现了切片的EM可视化本质上是有限的复杂和动态膜网络，如横向轴小管系统（TATS）的分析。因此，TATS膜的共聚焦活细胞成像直接沾上二8 ANNEPS开发。如果活细胞TATS网络由傅里叶变换分析，经常出现在靠近肌Z-线空间的T-细管部件是通过从条纹信号7的区域中的集合功率谱反射。这种间接的分析策略已经被用于检测在疾病模型7中TATS成分规律性小区宽的区域的变化。例如，shRNA的介导junctophilin-2击倒导致心脏衰竭和亚型特异性蛋白质不足造成的T小管重组与nanodomain 钙释放功能障碍8。我们通过直接定量方法，并进一步利用受激发射损耗（STED）纳米显微镜9在小鼠单独的虚拟机TATS组件的活细胞超高分辨率显微镜最近延长TATS膜网的分析。允许直接分析较小的单个TATS组件，其中接近50:50的横向分布的纳米图像分辨率与小管的轴向方向，定量确定2丰富的差异还面向个人TATS在健康小鼠心脏9成分。这些策略将在下面的协议部分中进一步概述的。 而丰富的A-小管组成部分，在成人心脏的生理作用仍然神秘莫测，新兴市场的研究已经证明了A-小管提示内源性钙豚鼠和大鼠的VM 5,10释放纳米畴相关的SER膜结构。 Cav1.2和RyR2的共聚焦分析发现，高度共存的A-小管路口10。由于自发钙火花在大鼠虚拟机起源比较远的地方，从T管，通常会出现Z线条纹〜20％，有一个参数一直是A-小管相关的纳米畴可能确实存在，并作为钙释放位点11,12。有趣的是，T-细管形成和成熟OC只有出生后小人和P5平行心脏细胞， 例如生长，通过对前体肌膜内陷发芽和未成熟的支TATS网络组件在P10小鼠13。看来，Junctophilin-2是产后TATS网络成熟特别重要，因为的shRNA敲除抑制T型小管膜锚定到SER路口，导致延迟钙释放及病理TATS组织，在不成熟的A-小管为主的架构一致虚拟机13。这些意见最终可能导致验证性的概念，T管通过膜内陷的过程，而A-小管形成可变形通过额外的，甚至替代细胞内的机制14。 在心脏疾病TATS膜特性的变化已成为一个重要的研究领域病理生理问题。在起搏诱发心脏failu狗模型初步报告再次表明了T管和Cav1.2电流（I Ca）的 15亏损。缺血性心肌病的动物模型表现出减少的T小管密度和细胞内钙的减少同步离子释放16。采用自发性高血压大鼠（SHR）模型心脏衰竭中，T管的亏损与Cav1.2和RyR2的的减少nanodomain耦合关联由"RyR2的孤儿"所提出的机制7。的T管的亏损也已缺血，扩张型，肥厚型心肌病样17表明在人类的虚拟机。此外，增加了在A-肾小管报道，在人的扩张型心肌病18的组织切片。心肌梗死后，我们发现TATS重组小鼠虚拟机与T管对比显著降低差分机制中的A-小管组件9的增加。重要的是，改善了当地的膜对比，通过活细胞superre实现解决方案STED显微镜启用了详细的定量成分分析通过直接测量，这表明了A-小管与TATS网络长度的整体增加，复杂的分支9显著增殖。此外，还表明，运动训练可逆转大鼠的T-细管重塑后心肌梗死19和心脏再同步治疗可能导致反向的T管重构犬心房快速起搏诱导的心脏衰竭20。合在一起，无论是在患病的人和动物的VM以及潜在的治疗性干预的研究将可以说从高质量细胞分离程序和详细的定量分析策略受益在协议中所概述和下面的效果的部分。 此外，最近由侧面证明了对ATP敏感性钾通道的TATS膜贩运亚型21，考虑心房m是重要的yocytes（AMS）作为生物鲜明以及比较心肌细胞模型与虚拟机。 T管最近被记录在羊和人的肺泡22。目前的证据表明，存在于细胞上午几个T管，通常像羊和人类，而不是较大的哺乳动物在小型啮齿动物23。相反，虚拟机，在肺泡巨细胞内的 Ca 2 +释放出现从细胞表面传播通过向小区中心其导致标记的空间和时间的 Ca 2 +梯度23扩散发生。在这个框架内就阐明细胞内Ca2 +信号不稳的常见病，多发病形式的机制，如房颤24显得重要。总之，无论是上午和VM细胞的分离和分别用于健康和患病的心是常用的协议。只有当细胞分离做正确作为判断的足够的细胞质量的微观文件，AM和虚拟机的样品应该是CArried向前定量TATS分析。因此，下面的协议部分是极其依赖于高质量细胞从小鼠或其它物种接着活细胞显微术来分析完整TATS膜分离。正如前面所指出的，TATS膜的特性是有倾向的固定和编制文物6时，由于渗透压的改变细胞膜的变化，传统的光学显微镜9的分辨率限制，一个具有挑战性的研究领域。我们注意到，国家的最先进的最新协议，对人类肺泡大鼠虚拟机用于细胞培养的钙成像和膜片钳和隔离此前已发表 ​​在该刊25,26。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Chemicals and Enzymes&lt;/strong&gt;</td></tr><tr><td>2,3-Butanedione monoxime</td><td>Sigma-Aldrich, Munich, Germany</td><td>B0753</td><td></td></tr><tr><td>Bovine calf serum</td><td>Thermo Scientific, Schwerte, Germany</td><td>SH30073</td><td>Triple 0.1 &amp;micro;m sterile filtered.</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich, Munich, Germany</td><td>21115</td><td>Diluted 1:10 in MQ water to obtain 100 mM CaCl&lt;sub&gt;2 &lt;/sub&gt;stock concentration.</td></tr><tr><td>Collagenase type II</td><td>Worthington via Cell Systems, Troisdorf, Germany</td><td>on request</td><td>Enzymatic activity depends on individual collagenase batches. Collagenase II and other enzyme activities (Caseinase, Clostripain, Tryptic) can be assessed in the &quot;collagenase lot selection tool&quot;. Determine cell yield and quality individually for each new lot of collagenase.</td></tr><tr><td>Glucose</td><td>Carl Roth, Karlsruhe, Germany</td><td>HN06.1</td><td></td></tr><tr><td>Heparin</td><td>Rotexmedica, Trittau, Germany</td><td>PZN-03862340</td><td>Diluted in 0.9% NaCl and injected subcutaneuosly in abdominal skin.</td></tr><tr><td>HEPES</td><td>Carl Roth, Karlsruhe, Germany</td><td>9105.4</td><td></td></tr><tr><td>Forene 100% (V/V)</td><td>Abbott, Libertyville, IL, USA</td><td>B506</td><td>Active agent: isoflurane, 250 ml. Use approximately 2 Vol% in air/oxygen dispenser instrument.</td></tr><tr><td>KCl</td><td>Carl Roth, Karlsruhe, Germany</td><td>6781.3</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Carl Roth, Karlsruhe, Germany</td><td>3904.2</td><td></td></tr><tr><td>Laminin (2 mg/ml)</td><td>BD Biosciences, Heidelberg, Germany</td><td>354232</td><td>Lamination is described under step 2.1.</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth, Karlsruhe, Germany</td><td>2189.2</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth, Karlsruhe, Germany</td><td>8283.2</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth, Karlsruhe, Germany</td><td>4984.2</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Carl Roth, Karlsruhe, Germany</td><td>HN01.1</td><td></td></tr><tr><td>Taurin</td><td>Carl Roth, Karlsruhe, Germany</td><td>4721.2</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Dyes&lt;/strong&gt;</td></tr><tr><td>Di-8-ANEPPS</td><td>Molecular Probes, Life Technologies, Darmstadt, Germany</td><td>D-3167</td><td>Stock solution 2 mM in DMSO</td></tr><tr><td>Trypan blue</td><td>Sigma-Aldrich, Munich, Germany</td><td>T8154</td><td>Trypan blue is gently mixed 1:1 via tip-cut 1 ml plastic pipette with cell suspension prior to cell counting in Neubauer cytometer.</td></tr><tr><td colspan="4">&lt;strong&gt;Langendorff Perfusion Setup&lt;/strong&gt;</td></tr><tr><td>Circulation thermostat</td><td>Lauda, Lauda-K&amp;ouml;nigshofen, Germany</td><td></td><td>Please refer to Louch &lt;em&gt;et al.&lt;/em&gt; (JMCC 2011). Heat up thermostat und buffers in perfusion tubing to 37 &amp;deg;C 15 min prior to use.</td></tr><tr><td>Flexible silicone tubing Tygon for peristaltic pump</td><td>VWR, Darmstadt, Germany</td><td>224-2252</td><td>Tubing needs to be changed regularly.</td></tr><tr><td>Flexible silicone tubing Tygon for thermostat</td><td>VWR, Darmstadt, Germany</td><td>228-4340</td><td></td></tr><tr><td>Heating coil surroundung perfusion tubing</td><td>Rettberg, G&amp;ouml;ttingen, Germany</td><td>custom-made</td><td>Heating coil and tubing needs to be cleaned thoroughly via MQ water after using. Do not use detergents. Glass components should be bathed regularly&amp;nbsp; in 10 mM NaOH overnight.</td></tr><tr><td>Peristaltic pump</td><td>Ismatec, Wertheim, Germany</td><td>ISM830</td><td></td></tr><tr><td>Three way stop cock Discofix C Luer Lock 10 cm</td><td>Braun, Melsungen, Switzerland</td><td>16500C</td><td></td></tr><tr><td>Three way stop cock Discofix 3SC</td><td>Braun, Melsungen, Switzerland</td><td>4095146</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Instruments&lt;/strong&gt;</td></tr><tr><td>42 mm glass coverslips</td><td>Menzel Gl&amp;auml;ser via Thermo Scientific, Schwerte, Germany</td><td>on request</td><td>0.13-0.16 mm thickness</td></tr><tr><td>Cannula 21 G</td><td>Becton, Dickinson and Company, Franklin Lake, NY, USA</td><td>304432</td><td>Cut to a length of ~5 mm, roughened with sandpaper.</td></tr><tr><td>Coverslips for Neubauer cytometer 24 x 24 mm</td><td>Menzel Gl&amp;auml;ser via Thermo Scientific, Schwerte, Germany</td><td>on request</td><td>0.38-0.42 mm thickness</td></tr><tr><td>Graefe forceps, 0.5 mm tips, slight curve</td><td>Fine Science Tools, Heidelberg, Germany</td><td>11151-10</td><td></td></tr><tr><td>LSM 710 NLO</td><td>Carl Zeiss, Jena, Germany</td><td></td><td>63X 1.4 NA oil objective</td></tr><tr><td>Neubauer improved cytometer</td><td>Labor Optik, Friedrichsdorf, Germany</td><td>1100000</td><td>Counting procedure: Wipe cytometer and coverslip provided with the counting chamber with 70 % ethanol. Press coverslip gently on the counting chamber so that the two glass surfaces are in contact and Newton&#039;s rings can be observed. Subsequently, 10-20 &amp;micro;l cell suspension can be applied to the edge of the coverslip to be sucked into the void by capillary action. Count the intact vs. defect myocytes using the squares of the cytometer grid which reflects 100 nl. Repeat counting procedure on the second grid provided on the cytometer. Calculate the density of cells in your original cell suspension by taking account of any dilutions and counting shortcuts.</td></tr><tr><td>POC-R2 Imaging Chamber</td><td>Pecon, Erbach, Germany</td><td></td><td>Cell suspension volume: 800 &amp;micro;l; desired plating density: ~1,000 AM and ~10,000 VM</td></tr><tr><td>Spring scissors, 8 mm blades straight, blunt</td><td>Fine Science Tools, Heidelberg, Germany</td><td>15025-10</td><td></td></tr><tr><td>Student dumont #7 forceps, inox</td><td>Fine Science Tools, Heidelberg, Germany</td><td>91197-00</td><td></td></tr><tr><td>Student iris scissors, curved, 11.5 cm</td><td>Fine Science Tools, Heidelberg, Germany</td><td>91461-11</td><td></td></tr><tr><td>Student iris scissors, straight, 11.5 cm</td><td>Fine Science Tools, Heidelberg, Germany</td><td>91460-11</td><td></td></tr><tr><td>Student surcigal scissors, straight, sharp, 12 cm</td><td>Fine Science Tools, Heidelberg, Germany</td><td>91402-12</td><td></td></tr><tr><td>Tissue forceps, 1 x 2 teeth, slim, 10 cm</td><td>Fine Science Tools, Heidelberg, Germany</td><td>11023-10</td><td></td></tr></tbody></table>

analysis of tubular membrane networks in cardiac myocytes from atria and ventricles

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.

此外，向TATS膜网络分析若干像细胞内的 Ca 2 +成像，膜片钳电生理学或药理学剂量反应研究中常用的细胞生物学技术从心房或心室严重地依赖于高品质的初级细胞分离或选择心脏组织的部分，以使成熟的分化，结构和生理完好心肌细胞的表征。因此，隔离和质量评估第1节上午和VM细胞是许多不同的问题，包括这里所描述TATS网络分析，这主要取决于完整的膜和细胞的完整性，最终很有用。 图1提供了图像的逐步手册如何进行心脏组织解剖开始，在小鼠心脏的心房室。随后，心室腔和隔膜都准备了ð需要解剖。可靠地建立调幅和VM隔离以足够的细胞纯度精确选择和制备正确的组织部位是很重要的。以下胶原酶消化它可以是相对难以确定心房和心室组织之间夹层的正确路线，但一旦AM和VM细胞混合不受控制的细胞悬浮液中，这是不可能的扭转混合细胞群。因此，解剖定位，三维组织可视化，足够的经验与消化的组织处理，正确识别的特定组织部位及其解剖线将所有向细胞分离的成功。 <img alt="图1" fo:content-width="5.75in" src="/files/ftp_upload/51823/51823fig1highres.jpg" width="600" /> 心房组织的图1.Dissection。 （一）面对心脏的前部，两种手术Šutures修复近端主动脉到21克钢套管的残肢末端（B）对剩余肺组织解剖阻碍在心房腔心切的心脏基础影音浏览。洛杉矶，左心房;类风湿性关节炎，右心房（C）剩余lungtissue及大血管被拆除访问心房室。填充黑色三角形，肺动脉;三角纹，肺静脉;填充白色三角形，上腔静脉;盒装三角形，下部腔静脉（D）首先，在右心房壁被切开，而钳握住心耳（E）查看进入右心房室腔。黑色三角形标记完好房间隔（F）的解剖继续进入左心房室腔（G）以下的左，右心房的完全剥离，房室瓣变得可见。纤维气门传动装置被解剖并丢弃到SUBSEQuently收获仅心室肌肉组织。分离的左和右心房（H）的后视图。洛杉矶，左心房;类风湿性关节炎，右atrium.Scale条:700微米。 以确定细胞隔离的质量， 图2提供了典型的细胞的例子的产率和典型的杆或砖状横纹肌完整细胞的存活力的评估过程中，无论是用于AMS和VM。小损坏，在视觉上不明显，以及与异常过度弯曲的形态，或不规则球面状细胞严重受损的细胞，可以容易地确定包括台盼蓝排除在1.11节中描述。虽然完整的细胞保持明亮，均匀条纹，当暴露于外台盼蓝，受损细胞通常显示多个小泡膜和/或迅速积累台盼蓝细胞内表明膜的损伤。不过，台盼蓝本身会伤害过unnecessari细胞LY长潜伏期和直接的细胞质量评估，因此强制性的。对像肌丝挛缩或总表面损伤影响细胞的完整性细胞损伤较为明显的实例是如图4C和4D， <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51823/51823fig1highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图2" fo:content-width="7in" src="/files/ftp_upload/51823/51823fig2highres.jpg" width="700" /> 如图2台盼蓝排除细胞染色。隔离（A） 的 AM和（B）中的VM的细胞混合在悬浮液用锥虫蓝和通过在40Xmagnification所示的倒置光学显微镜下观察。注意thatabnormal球形细胞（A）和（B）占台盼蓝，这indicatesmembrane leakaGE和结构损坏。与此相反，centralAM和VM细胞与完整的膜排斥台盼蓝，如图所示。此外，请注意，完整的AM和VM细胞显示肌条纹整个细胞体积，nomembrane泡，并在两侧边尖锐的边缘和两闰盘。比例尺为20μm。 下面的VM的成功分离，细胞产量从5×5月 10日至6月 10日可从单个小鼠心脏消化可以预期的。 AMS产率是在3×10 3〜3×10 4个杆形，台盼蓝排除单元的顺序显著更低。相反，虚拟机，甚至有经验的人员隔离偶尔失败。步骤1.11总结程序如何估计分离的健康细胞在悬浮液中的产率。另外，判断为如图3所示的单个AM或VM细胞株通过步骤1.13的平均泡孔尺寸<html>或者比较调幅与VM的细胞群并排侧（如需要）。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51823/51823fig2highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图3" fo:content-width="7in" src="/files/ftp_upload/51823/51823fig3highres.jpg" width="700" /> 图心肌细胞3.Bright场形态分析，虚拟机的轮廓由下步1.13中描述的图像分析工具检测到。使用多边形选择工具以可视记号和定义由外表面的膜用于分析所定义的细胞的细胞外边界。广告的感兴趣区域（ROI）的投资回报率管理器所选择的区域接着1D距离测量。宽度比调幅与VM尺寸的比较研究，它以记录单元的长度，宽度和面积，并计算出的长度是有用的。 NT"&gt;要荧光染色TATS膜无论是在AM或VM的细胞中，整合膜染料二- 8-ANEPPS是如在第2随后通过共聚焦显微镜，导致TATS网络在图4A中所示的代表图像和描述用于4B，此外， 图4A示出一个完整的调幅边并排的共焦TATS图像的相应的透射光的图像（图像采集见第3节）。正如所描述的步骤1.12，活的形态和表面细胞膜的完整性健康VM的细胞是通过所发射的光的图像进行判断。二 - 8-ANEPPS信号的放大区域突出在肺泡的TATS网络的基本形态，其特征在于丰富的轴向（纵向）膜小管。相反，TATS形态如图4B所示 ，其特征在于由大约相同的数字轴向的中ð横向部件。与此相反， 图中应排除在进一步分析受损心肌细胞4C和4D示出的例子。特别地，在图4C中的调幅细胞收缩，就证明了1.2微米的异常短肌节长度和不规则的，扭曲的TATS网络，而在图4D中的VM的细胞表现出的多个膜泡（部分强调红色三角形）表示膜破坏，这可能发生在外表面上的膜，而且在TATS膜。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51823/51823fig3highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图4" fo:content-width="6in" src="/files/ftp_upload/51823/51823fig4highres.jpg" width="600" /> 完整的心房和心室肌的图4.Live膜染色核细胞。对应的透射光及生活的共焦图象二- 8-ANEPPS染色的完整的（A）的 AM和（B）VM细胞。相反，部分收缩，并可能损坏调幅为1.2微米的肌节长度显示在（C）。承包细胞通常显示异常缩短，因而扭曲TATS结构，排除进一步分析。对于细胞膜缺陷另一个重要指标是膜泡（红色三角形）所示在虚拟机中（D）。膜泡代表破坏表面的膜结构和细胞泡应排除进一步TATS分析。此外，虚拟机显示了毛损坏显然缺少其左下部分的整个部分（标以星号）。总之，通过比较透射光及共焦图象，细胞形态和表面完整性是记录和结合荧光信号的信息。 "N"标记NUCLEI从TATS膜染色的分析被删去。黄色柱线表示从上面介绍的相同的共聚焦图像放大投资回报。比例尺:10微米<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51823/51823fig4highres.jpg" target="_blank">，请点击这里查看该图的放大版本。</a> 如示于图4A和4B TATS膜具有足够的信号-噪声比的共焦图象接受进一步的定量分析。该TATS膜的分析是基于从荧光直线的信号分量导出镂空的内容图5示出了这是由以下步骤进行详细说明4.3至4.5的个别图像的处理步骤的流程图。这些步骤产生代表直线TATS膜网镂空图像，示出了每个独立的虚拟机（ 图5A）和AM的细胞（ 连接gure 5B）。 <img alt="图5" fo:content-width="7in" src="/files/ftp_upload/51823/51823fig5highres.jpg" width="700" /> 有图5.Workflow的荧光TATS图像骨架。图像处理步骤，这导致TATS网络的骷髅图像由单独的一步一步的图像例子都为二-8-ANEPPS代表的虚拟机染色（A）和AM （B）。对于单个图像处理步骤，请参见第4。注意比例尺的不同:20微米（A）和10微米（ 二） <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51823/51823fig5highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> 图像处理过程中的关键步骤，确定合适的阈值二值化数据如第4.5节所述。6，得到的二进制图像应包括TATS网络唯一真正的膜信号，但不是从背景信号中的噪声阈值的错误虚假的衍生结构。然而，重要的是，该阈值是足够的低，以检测所有真TATS结构使得真TATS组件不会错误地在图像分析丢失。 图6示出的过程中如何选择数据进行二值化时的阈值。而40的阈值， 如图6B和6C似 ​​乎是恰当的，以检测所有真TATS结构单独地，选择较高的阈值，例如 ，60， 如图6A中没有检测到微弱的轴向膜结构（AT）的由黄色三角形所指示。与此相反，选择一个较低的阈值，例如 ，20， 如图6D导致误检测的本底噪声视为假阳性TATS结构由所指示的黄色三角形。 <img alt="图6" fo:content-width="7in" src="/files/ftp_upload/51823/51823fig6highres.jpg" width="700" /> 图6.How确定skeletonizing TATS图像数据的过程中的信号的阈值的例子显示每个所产生的数据进行二值化过程中不同的阈值（在步骤4.5中所述）TATS骨架。上图片:显示阈值调整使用斐济。下图片:骷髅图像与相应的输入荧光图像和放大区域的覆盖所示。高门槛，例如 ，60英寸采用（A）是显然不适合检测所有真正的TATS结构，由黄色三角形的放大部分所示。 40中的（B）和（C）应用阈值检测所有TATS结构和正确不检测背景噪音，而低门槛，例如，在20（D）的误识别的背景噪声如TATS结构及由此产生的不存在的膜结构的假阳性信号。假阳性信号由黄色三角形的放大的插图所示。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51823/51823fig6highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> "分析骷髅（2D / 3D）"插件支持镂空TATS结构的详细分析。一旦执行，该插件生成一个数据表具有以下骨架参数:#branches，#junctions，＃终点体素，平均枝长，#triple点，#quadruple点，最大分支长度。对于所有可能的输出参数的详细说明，请参考tohttp://fiji.sc/wiki/index.php/AnalyzeSkeleton及相关物品29-31。一个典型的数据表的输出被示于图7A。 <p类="jove_content"&gt;参数可以进一步用于导出每个区域的总骨架长度或单位面积结的数目。分支乘以平均分支长度的数目的示例性的计算给出了连续的骨架中的2D的总长度: 总长度为骨架每投资回报率: Σ（#branches点¯x平均枝长）= 5155像素= 515.5微米骨架的总长度可以是归一化的图像区域。对于图7中的归一化的骨架长度0.64μm的中所示的示例个/μm2和所有路口的总和来计算，如下所示: 归骨架长度: 515.5微米/ 803.6微米2 = 0.64微米/微米2 归号路口: 155路口/ 803.6微米2 </SUP&gt; = 0.19路口/微米2 <img alt="图7" fo:content-width="7in" src="/files/ftp_upload/51823/51823fig7highres.jpg" width="700" /> 图从镂空的图像输出7.Automated数据。 （一）由"分析骨架（2D / 3D）"插件从（B）所示的镂空图像生成一个典型的数据表格。对于可能的输出参数的详细说明请参考<a href="http://fiji.sc/wiki/index.php/AnalyzeSkeleton">http://fiji.sc/wiki/index.php/AnalyzeSkeleton</a> 。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51823/51823fig7highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> 在图5中在4.5中描述和图示的图像处理步骤，可以使用作为补充代码文件斐济宏实现自动化</str翁&gt;。该命令通过反复的步骤定义图像处理的重复。宏可以应用来完成堆叠通过以下步骤4.3和4.4产生的输入图像。宏可以通过制备单独的输入图像栈用于使用宏命令每个独立治疗组用于自动分析是有利的完整的数据集组的分析，例如。 概述的软件战略进一步使TATS网络导向分析的所有组件。为此，使用"方向性"插件（http://fiji.sc/Directionality）29,31产生直方图数据显示所有TATS组件取向的取向分布。如果输入图像的x轴对应于一个给定的AM或​​虚拟小区的主轴线，轴向（纵向）TATS部件将由0°仓表示，而横向的部件用90来表示＃176;宾图8显示了镂空TATS图像的虚拟机（8A）与调幅（8B）电池示范方向性直方图。而典型的VM的方向性的直方图示出了在0°和90°的双峰值分布，则AM直方图显示在0℃显性单峰。这些实施例中与先前观察该虚拟机的单个TATS分量几乎相等的T-管和A-肾小管间分布，而在肺泡的TATS组分可以主要成分的A-肾小管协议。 <img alt="图8" fo:content-width="6in" src="/files/ftp_upload/51823/51823fig8highres.jpg" width="600" /> 图8.Representative方向性直方图从单个细胞TATS网络从单个VM（一 ）与AM的骷髅图像产生方向性直方图（B </STRONG&gt;）TATS网络。因为所分析的图像的x轴对应于如图所示的心肌细胞的主要（纵）轴，A小管部件由0°仓表示，而T-细管部件用90°的槽来表示。高斯只适合于大型直方图peakasshown作为背景图。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51823/51823fig8highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 灌注缓冲 </td><td> 毫 </td></tr><tr><td>氯化钠</td><td> 120.4 </td></tr><tr><td>氯化钾</td><td> 14.7 </td></tr><tr><td> KH 2 PO 4的</td><td> 0.6 </td></tr><tr><td>的Na 2 HPO 4 </td><td> 0.6 </td></tr><tr><td> 硫酸镁 </td><td> 1.2 </td></tr><tr><td> HEPES </td><td> 10 </td></tr><tr><td> 碳酸氢钠 </td><td> 4.6 </td></tr><tr><td>牛磺酸</td><td> 30 </td></tr><tr><td> 2,3 - 丁二酮，肟</td><td> 10 </td></tr><tr><td>血糖</td><td> 5.5 </td></tr><tr><td colspan="2"> pH值7.4 </td></tr><tr><td> 消化缓冲液 </td><td> 毫（如果未指定） </td></tr><tr><td>氯化钠</td><td> 120.4 </td></tr><tr><td>氯化钾</td><td> 14.7 </td></tr><tr><td> KH 2 PO 4的</td><td> 0.6 </td></tr><tr><td>的Na 2 HPO 4 </td><td> 0.6 </td></tr><tr><td> 硫酸镁 </td><td> 1.2 </td></tr><tr><td> HEPES </td><td> 10 </td></tr><tr><td> 碳酸氢钠 </td><td> 4.6 </td></tr><tr><td>牛磺酸</td><td> 30 </td></tr><tr><td> 2,3 - 丁二酮，肟</td><td> 10 </td></tr><tr><td>血糖</td><td> 5.5 </td></tr><tr><td>胶原酶II型</td><td> 600 U / ml的</td></tr><tr><td colspan="2"> pH值7.4 </td></tr><tr><td> 挡车器 </td><td> 毫（如果未指定） </td></tr><tr><td>氯化钠</td><td> 120.4 </td></tr><tr><td>氯化钾</td><td> 14.7 </td></tr><tr><td> KH 2 PO 4的</td><td> 0.6 </td></tr><tr><td>的Na 2 HPO 4 </td><td> 0.6 </td></tr><tr><td> 硫酸镁 </td><td> 1.2 </td></tr><tr><td> HEPES </td><td> 10 </td></tr><tr><td> 碳酸氢钠 </td><td> 4.6 </td></tr><tr><td>牛磺酸</td><td> 30 </td></tr><tr><td> 2,3 - 丁二酮，肟</td><td> 10 </td></tr><tr><td>血糖</td><td> 5.5 </td></tr><tr><td> 氯化钙 </td><td> 0.0125 </td></tr><tr><td>小牛血清</td><td> 10％ </td></tr><tr><td colspan="2"> pH值7.4 </td></tr></tbody></table> 表1.Buffer SOLUT离子。三种不同的生理缓冲液的细胞分离和成像的内容进行了总结。

Watch this Scientific Journal Video about Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles at JoVE.com

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

在心肌细胞，管式膜结构形成细胞内的网络。我们描述了优化方案从小鼠心脏肌细胞I）的隔离，包括质量控制，二）活细胞染色国家的最先进的荧光显微镜，及iii）直接图像分析来量化成分的复杂性和膜细胞的可塑性网络。

管式膜网络在心肌细胞的心房和心室分析

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. ...

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Universidad de Cartagena UDEC

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JoVE Journal

Bioengineering

20.5K 次观看.  Heart Research Center Goettingen. 在心肌细胞，管式膜结构形成细胞内的网络。我们描述了优化方案从小鼠心脏肌细胞I）的隔离，包括质量控制，二）活细胞染色国家的最先进的荧光显微镜，及iii）直接图像分析来量化成分的复杂性和膜细胞的可塑性网络。 

视频: Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

在胚胎小鼠心脏采用免疫荧光心肌发展分析

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

科研

发育生物学

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

隔离与人类心室肌细胞的功能研究从新鲜手术样品

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

医学

Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal microscopy, the scientific community has unprecedented access to large datasets at sub-micrometer resolution that characterize the architectural remodeling that accompanies changes in cardiomyocyte function in health and disease. However, these datasets have been under-utilized for&#160;investigating the role of cellular architecture remodeling in cardiomyocyte function. The purpose of this protocol is to outline how to create an accurate finite element model of a cardiomyocyte using high resolution electron microscopy and confocal microscopy images. A detailed and accurate model of cellular architecture has significant potential to provide new insights into cardiomyocyte biology, more than experiments alone can garner. The power of this method lies in its ability to computationally fuse information from two disparate imaging modalities of cardiomyocyte ultrastructure to develop one unified and detailed model of the cardiomyocyte. This protocol outlines steps to integrate electron tomography and confocal microscopy images of adult male Wistar (name for a specific breed of albino rat) rat cardiomyocytes to develop a half-sarcomere finite element model of the cardiomyocyte. The procedure generates a 3D finite element model that contains an accurate, high-resolution depiction (on the order of ~35 nm) of the distribution of mitochondria, myofibrils and ryanodine receptor clusters that release the necessary calcium for cardiomyocyte contraction from the sarcoplasmic reticular network (SR) into the myofibril and cytosolic compartment. The model generated here as an illustration does not incorporate details of the transverse-tubule architecture or the sarcoplasmic reticular network and is therefore a minimal model of the cardiomyocyte. Nevertheless, the model can already be applied in simulation-based investigations into the role of cell structure in calcium signaling and mitochondrial bioenergetics, which is illustrated and discussed using two case studies that are presented following the detailed protocol.

This protocol outlines a novel method to create a spatially detailed finite element model of the intracellular architecture of cardiomyocytes from electron microscopy and confocal microscopy images. The power of this spatially detailed model is demonstrated using case studies in calcium signaling and bioenergetics.

建立一种结构逼真的心肌细胞的有限元几何模型研究蜂窝结构在心肌系统生物学中的作用

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal ...

生物工程

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca2+ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).1 Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca2+ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca2+-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 &mu;M Ca2+. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca2+ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca2+ and Ca2+ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca2+ transient measurements, performed in these isolated myocytes.

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.

CA同时测量人的心房肌细胞分离<sup&gt; +</sup&gt;瞬变和膜电流

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

生物学

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.

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 of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

从成年小鼠分离心膜肌细胞

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction and electrical activity. Hence, isolating viable, high-quality cardiomyocytes from the heart is the initial and most crucial experimental step. Comparing the various protocols for isolating the cardiomyocytes of adult mice, the Langendorff retrograde perfusion is the most successful and reproducible method reported in the literature, especially for isolating ventricular myocytes. However, isolating quality atrial myocytes from the perfused heart remains challenging, and few successful isolation reports are available. Solving this complicated problem is extremely important because apart from ventricular disease, atrial disease accounts for a large part of heart diseases. Therefore, further investigations on the cellular level to reveal the mechanisms are warranted. In this paper, a protocol based on the Langendorff retrograde perfusion method is introduced and some modifications in the depth of aorta cannulation and the steps that may affect the digestion process to isolate atrial and ventricular myocytes were simultaneously made. Moreover, the isolated cardiomyocytes are confirmed to be amenable to patch clamp investigation.

This protocol describes modifications of the Langendorff method including the depth of aorta cannulation for simultaneous Isolation of atrial and ventricular myocytes from adult mice.

朗根多夫方法的修改，用于从成年小鼠中同时分离心房和心室肌细胞

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction ...

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury and/or remodeling. One approach for assessing these functional alterations utilizes unloaded shortening and Ca2+ transient analyses in primary adult cardiac myocytes. For this approach, adult myocytes are isolated by collagenase digestion, made Ca2+ tolerant, and then adhered to laminin-coated coverslips, followed by electrical pacing in serum-free media. The general protocol utilizes adult rat cardiac myocytes but can be readily adjusted for primary myocytes from other species. Functional alterations in myocytes from injured hearts can be compared to sham myocytes and/or to in vitro therapeutic treatments. The methodology includes the essential elements needed for myocyte pacing, along with the cell chamber and platform components. The detailed protocol for this approach incorporates the steps for measuring unloaded shortening by sarcomere length detection and cellular Ca2+ transients measured with the ratiometric indicator Fura-2 AM, as well as for raw data analysis.

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

A set of protocols are presented that describe the measurement of contractile function via sarcomere length detection along with calcium (Ca2+) transient measurement in isolated rat myocytes. The application of this approach for studies in animal models of heart failure is also included.

啮齿动物心肌细胞心脏收缩功能障碍和Ca2+ 瞬变分析

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

来自成人鼠心脏的心外肌细胞、心室肌细胞和非肌细胞的同步隔离和文化

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the &#945;-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

Analyzing the &#945;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

利用单分子定位显微镜分析人类 iPSC 衍生肌细胞中的α-Actinin 网络

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...

管式膜网络在心肌细胞的心房和心室分析

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