Name: voltage and calcium dual channel optical mapping of cultured hl-1 atrial myocyte monolayer
Uploaded: 2015-03-23T15:00:00.000+00:00
Duration: 8 min 25 s
Description: Watch this Scientific Journal Video about Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer at JoVE.com

我们要感谢Claycomb博士提供HL-1细胞和详细的维护协议。我们还要感谢埃琳娜·卡里略女士和赛斯Robia博士为他们与产生细胞电影和皮特卡隆先生的协助做一些配件为双通道光学标测系统的援助。 这项工作是由美国心脏协会（10GRNT3770030＆12GRNT12050478为XA），国立卫生研究院（HL113640到XA），和Loyola大学的研究发展基金（以XA）的支持。

1.溶液制备<ol><li>去甲肾上腺素（NP）溶液<ol><li>溶解40毫克的NP的成将25ml 30mM的抗坏血酸（0.1475克抗坏血酸在25ml纯化的Milli-Q（MQ）水。该溶液在制备过程中避免直接光照，因为去甲肾上腺素（NP）是光敏感的。 </li><li>过滤用0.22μm注射器滤器对NP溶液。分装，在-20℃下将溶液在1ml无菌微量管和存储。一旦冻结，NP为稳定一个月。 </li></ol></li><li>辅以洗净，最后Claycomb媒体<ol><li>先从43.5毫升Claycomb培养基和补充用5毫升胎牛血清（FBS），0.5 ml的青霉素/链霉素，0.5毫升的NP和1:100稀释的L-谷氨酰胺，使50最后补充Claycomb培养基中。这种溶液的制备过程中应避免直射光照射，因为Claycomb介质对光敏感。 </li><li>使用相同的配方为SUP准备洗Claycomb媒体除了执行完成Claycomb中添加5％FBS和省略NP和L-谷氨酰胺的组件。 注:存放于4℃无直射光照射时，最后的补充和冲洗Claycomb媒体是很好的两周。 </li></ol></li><li>大豆胰蛋白酶抑制解决方案<ol><li>溶解的大豆胰蛋白酶抑制剂25毫克到100ml无钙和镁的Dulbecco磷酸盐缓冲盐水（PBS）中。 </li><li>过滤用0.22微米注射器过滤该溶液。 注:储存在4℃下时，灭菌溶液是稳定一个月。 </li></ol></li><li>明胶/盘纤连蛋白涂液<ol><li>将0.1g明胶到500ml MQ水中，使0.02％的明胶。 </li><li>反应釜，然后稀释1毫升纤维连接蛋白进入199毫升0.02％的明胶，轻轻混匀。 </li><li>等分6毫升的明胶溶液成单独的管中并储存在-20℃。 </li></ol></li><li>汉克斯&#39;盐溶液<ol><li>使用搅拌板，溶解在MQ水中（毫摩尔/升）以下的盐类，氯化钠（136.9），氯化钾（5.366），KH 2 PO 4（0.4409），将NaH 2 PO 4（0.4000），硫酸镁 （0.8142），D-葡萄糖（5.051）， 碳酸氢钠 （4.162），HEPES（5.042）， 氯化钙 （1.261）。 </li></ol></li><li>钙敏感染料（Rhod2）解决方案<ol><li>稀释的1mg Rhod2干粉在400微升20％的Pluronic F-127的DMSO作Rhod2染料原液并储存在-20℃。 </li><li>这种稀释原液1:1000用Hanks&#39;盐溶液在实验开始前，作最后的工作方案。 </li></ol></li><li>电压敏感染料（Rh237）解决方案<ol><li>稀释5毫克Rh237的用2毫升DMSO中，使储液并储存在-20℃。稀释原液1:1000用Hanks&#39;盐溶液在实验开始前，作最后的工作方案。 </li></ol></li></ol> ＆＃160;注:1-4的解决方案是基于Claycomb HL-1细胞培养协议轻微修改。 2.传代培养和HL-1心房肌细胞到盖玻片注:HL-1细胞可以从Claycomb博士（路易斯安娜州立大学）获得。 <ol><li>生长的HL-1细胞在T25烧瓶和饲料用5ml补充Claycomb中等每日。通道中的细胞在T25根据Claycomb HL-1细胞培养物的协议（以下描述）10烧瓶每五天。 </li><li>地方圆形盖玻片（20毫米直径）到12孔培养板中之前24小时，将细胞在T25烧瓶中完全汇合，并准备进行传代，然后盖上明胶/纤连蛋白过夜，在37℃的盖玻片。 </li><li>第二天，吸出并丢弃从培养板中的明胶/纤连蛋白和替换用1.5ml在每个补充最终Claycomb介质的井。 </li><li>从T25文化性拆分单元格Ë烧瓶中，在5天达到完全汇合后，弃从含有汇合培养的T25烧瓶中的培养基和冲洗培养简要地用PBS在37℃。 </li><li>抽吸PBS并且加入3毫升0.05％胰蛋白酶/ EDTA，在37℃吹打的胰蛋白酶/ EDTA到T25烧瓶的底部，避免与细胞直接接触。孵育在37℃下1分钟。 </li><li>通过抽吸除去胰蛋白酶/ EDTA，并添加每个T25烧瓶另一个1.3毫升胰蛋白酶/ EDTA中。在室温下孵育1分钟。检查在显微镜下，如果细胞仍然附着在烧瓶中，点击要充分打跑剩余的细胞。 </li><li>添加1.3毫升大豆胰蛋白酶抑制剂，从烧瓶内的细胞和细胞转移到15ml离心管中。冲洗用5毫升洗净Claycomb介质的空烧瓶中。漂洗添加到细胞中，在15ml离心管中。离心，在500×g离心5分钟。 </li><li>离心，吸出上清液，并轻轻后重新悬浮细胞沉淀6毫升补充决赛Claycomb介质。 </li><li>种子在12孔平板的每个孔用0.5ml从细胞传代细胞悬浮液。每天喂细胞新鲜辅以最终Claycomb媒介。 注:密切监测细胞的生长，通常细胞单层达到汇合状态在第4天，并自发活动保持可见。然后，将培养的单层将准备在4-5天的光学测绘的。 </li></ol> 3.装载钙和电压敏感染料的细胞<ol><li>用于光学作图研究，轻轻地从12孔细胞培养板除去盖玻片和冲洗用Hanks&#39;盐溶液。 </li><li>染色的细胞用钙敏感染料Rhod2，孵育在盖玻片的细胞单层用3ml的充氧，在37℃的水浴中30分钟Rhod2工作溶液（用5％的CO 2/95％O 2气鼓泡） 。 </li><li>染色的细胞在V 米敏感的染料Rh237，浸入Rhod2加载细胞单层分为3毫升含氧Rh237工作溶液，在37℃的水浴中另外15分钟。 </li></ol> 4.双通道光映射<ol><li>将单层细胞，这是双沾满V M和钙染料，插入一个倒置荧光显微镜循环室。使用加热的循环体系，以保持细胞室的温度在35±1℃。小心通过调节循环流的速度通过​​腔室，以避免超过0.3℃，在整个腔室的梯度控制所述灌流液的温度。 <ol><li>记录V M /钙信号之前，用新鲜的Hanks液洗细胞单层约5分钟，以从先前的染色步骤除去剩余的染料。 </li></ol></li><li>使用双极电极的玻璃吸移管填充用Hanks&#39;盐溶液和SILV组成步伐健康细胞单层呃丝枪头周围卷绕如前所述12,17。 </li><li>吴佩慈健康的细胞单层在200-500毫秒，脉冲2毫秒宽度的周期长，期待起搏阈值约为1毫伏。 注:对于每一批培养单层的，至少有两个非处理单层应当用于测试细胞批次的状态。健康对照单层应当能够被电起搏在200-500毫秒周期长度捕获。如果控制单层不能被电节奏，这表明，整个细胞批次可能不适合用于光学测绘实验。 </li><li>激励两个V M和钙敏感性染料，可使用的LED光源（520纳米波长）加上一个激励滤光器（510纳米/ 40）和一个二向色镜（561纳米）。 <ol><li>收集通过40X物镜和分割用分色镜（594纳米）成V 米组分和Ca成分由细胞发出的荧光信号。过滤在V 米通道的发射光具有长通滤波器（700纳米），发射的光中的钙通道有一个带通滤波器（四十分之五百八十五纳米）。 </li><li>收集用两台CCD照相机（80×80像素，1000 fps的;红衫成像）将过滤的荧光。 </li><li>为了避免染料光漂白，使录音为小于2秒上的每个视场限制光曝光的时间。这里，典型的图像的每个单层四个不同的区域。 </li></ol></li></ol>的V M或Ca录音5.数据处理<ol><li>为V M和Ca信号（80×80像素），平均的25（5×5）的强度值的相邻像素成在不同的记录时间点的一个强度值以减少背景噪声。 注:该数据平均后，个人动作电位和钙瞬变每次录制时间点产生含有16×16的平均亮度值的数据矩阵（平均数据信道）为50微米的空间分辨率。 </li><li>定义的记录，V M或Ca信号激活时间（AT）。 <ol><li>以限定的单个动作电位或钙瞬变的激活时间（AT），确定的在V m或者使用一个基于MATLAB定做算法钙信号的峰值幅度的50％的时间，如前所述。12 </li><li>手动校对所分析的AT来消除程序分析期间可能发生的任何错误。 </li><li>存储所述所定义的AT矩阵（16×16平均数据信道）分别记录动作电位和钙瞬变，在所使用的软件。 </li></ol></li><li>计算上使用矢量场分析方法如先前稍作修改12,18中描述的AT矩阵动作电位或钙瞬变传导速度（CV）的载体。 <ol><li>计算数据信道相对于其周围的AT的数据点的CV载体（5×; 5相邻平均数据信道），使用中的MATLAB程序12,18最小二乘算法，模拟的激活时间（AT表面光滑多项式表面）。 </li><li>使用以下公式计算传导矢量 式1: <img alt="式（1）" fo:content-width="1.5in" src="/files/ftp_upload/52542/52542eq1.jpg" /> 配方2: θ=反正切[（DY / DT）/（DX / DT）] 注:Ve为在x轴和y轴投射在简历矢量; DX / DT是在X轴上的CV矢量的投影; DY / DT是在y轴的CV向量的投影。 T是所述AT表面上;的x，y指的是2-D坐标为的T x =∂T/狓是T的关于x的偏导数; ŤY =∂T/狔是T的相对于y的偏导数; θ是一个简历矢量的最终方向。 </li><li>所述AT矩阵的组中的所有矢量成36通路，其每一个覆盖指南针上的10度扇区。&lt;/ li&gt; <li>选择包含​​36传导通路之间的CV向量的人口最多的途径。计算这个途径的平均CV代表单层的CV。 </li></ol></li></ol> 6.可视化的波前传播<ol><li>正常化的在相对于利用MATLAB软件其峰值不同的记录时间点的单个动作电位或Ca瞬态信号的强度。 </li><li>每记录的时间点的归一化强度数据暂时存储到在所使用的MATLAB软件一个16×16的强度矩阵。 </li><li>构造从使用surf.m函数的每个记录时间点的存储的强度矩阵的彩色编码强度地图。 注:在强度图，红色代表的V 米或钙信号强度高，蓝色体现的荧光信号的强度低。 </li><li>创建使用avifile.m功能的.avi文件。 </li><li>保存所有的I在动作电位或Ca瞬态不同的记录时间点使用addframe.m函数ntensity地图。 注意:在这一点上，将产生的V M和Ca传播两个独立的AVI文件。 </li></ol>

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No conflicts of interest declared.

本文介绍了培养HL-1心房肌细胞单层染色钙和电压敏感的荧光染料的光映射的关键方面。它包括培养的最佳的HL-1细胞单层，设置的映射设备，映射培养单层，和数据分析。 要成功映射培养细胞，关键是要准备一个均匀分布的单层细胞。当播种到盖玻片细胞，一定要均匀地分散细胞。始终映射完全长大，完​​全融合细胞单层，因为这些将最好的电起搏回应。同样重要的是，保持在加载染料氧化的细胞，但该氧气泡不应该接触到的细胞，因为这可能会杀死或从盖玻片撞出它们直接接触。降低染料浓度，减少光强度将降低信号幅度和降低信噪比，同时增加染料concentrati并提高光的强度会增加信号幅度也增加噪音。因此，荧光色素的浓度和激发光的强度的最佳平衡，必须确定以增强信号，以记录信号的信噪比。此外，光映射录制过程中，一定要避免暴露单层细胞，以集中光长时间曝光，因为这可能会导致光暴露区域内的光动力细胞损伤。最后，光学测绘系统，提供的记录信号的高的时间和空间分辨率，需要用于获得高质量的数据。 而在完整心脏双通道光学测绘具有用于研究心脏电生理由于它能够同时收集电压和钙信号的潜力很大，一个限制是该技术收集作为二维投影图，从一个三维表面信息。从完整心脏获得的2-D数据与三维curvat忽略信息心脏的URE可能会导致错误的数据分析，尤其是传导速度的分析。利用培养的2-D HL-1细胞模型的优点，我们能够形象化定期和不定期的节奏，评估钙瞬变和动作电位动力学，确定传导速度和表征Ca和动作电位传播的模式（均匀或非均匀传播），而不从完整心脏的3-D结构的干扰。因此，在培养的HL-1心房肌细胞单层作为一个免费的方式成功地映射两种钙瞬变和动作电位可以大大提高心房肌细胞的电生理特性和心房心律失常的基本机制的理解。

独特的钙（Ca）和电压（V M）双通道光映射技术1-5正在成为一种有效的工具，同时记录V M和钙信号在两个完整的心和培养细胞单层。该技术使得有可能获得关于钙瞬变和动作电位之间的关系，以便更好地理解心律失常的底层电机制有力的信息。 培养的细胞单层已被证明是研究心脏电生理和心律失常的基本机制的有用的细胞模型。4,6-8的HL-1细胞是充分表征的心房肌细胞培养线。 HL-1细胞也保持着独特的差异化基因型和表现型，包括形态，电生理和成人心房肌细胞的药理特性。这些细胞表达心肌基因和蛋白质，包括性重要nt的心脏离子通道（ 即 ，L和T型钙通道）9和肌节收缩蛋白的成熟异构体通常在成人心房肌细胞作为其他人发现，我们以前曾报道10-13此外，HL-1细胞可以是培养，以形成一个2维（2-D）的肌细胞单层。因此，使用培养的HL-1单层的优点包括:1）较低的成本，更容易保持一个心肌细胞培养系不是分离和培养原代新生心肌细胞; 2）细胞的汇合单层减少而导致的心脏的3-D结构的结构的复杂性; 3）细胞单层可以消除间质纤维化的发生在完整心脏的干扰。这可以用来解剖一组肌细胞的特定电功能，而不成纤维细胞和间质基质的干扰; 4）药物或基因操纵的小路评估功能的后果捕获的原始图像的细胞单层，可以有效地实现。因此，HL-1细胞已经成为研究肌细胞生理学的各个方面以及起搏诱导的异常电活动的一个广泛使用的细胞模型13-16然而，特殊的处理和护理需要培养健康细胞单层，为了响应外部电起搏光学定位研究。此外，双荧光染料染色过程可能会很容易损坏的培养融合细胞单层的完整性。因此，在执行V 米/ CA双通道培养HL-1单层光学标测受到了极大的挑战。 该方法的目标是提供的关键步骤为在培养的HL-1单层成功执行双通道光学测绘的。在这里，我们提供了大量具体的细节上的优化协议HL-1细胞单层的准备，培养的细胞单层双通道光学测绘，测绘数据处理。

<table><tbody><tr><td>Rhod2 dry powder</td><td>AAT Bioquest</td><td>21062</td><td></td></tr><tr><td>pluronic</td><td>AAT Bioquest</td><td>20050</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>276855</td><td></td></tr><tr><td>Rh237</td><td>Invitrogen</td><td>S-1109</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P3911</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P0662</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S9638</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>M7506</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma</td><td>G8270</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S6014</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>C3881</td><td></td></tr><tr><td>HEPEs</td><td>Sigma</td><td>H3375</td><td></td></tr></tbody></table>

voltage and calcium dual channel optical mapping of cultured hl-1 atrial myocyte monolayer

光学映射已被证明是有价值的技术来检测心电活动上都完好体外心和在培养的心肌细胞单层。的HL-1细胞已被广泛地用作用于研究心脏生理学的各个方面的2维细胞模型。但是，它已经受到了极大的挑战光学地图钙（Ca）瞬态和动作电位同时从相同的视野中培养HL-1心房细胞单层。这是因为特殊的处理和护理，需要准备一个可以电捕获并光学映射健康细胞。因此，我们已开发了一种最佳的工作协议进行双通道光学测绘的。在这个手稿中，我们已经详细介绍了如何进行双通道光的映射实验。这个协议是一个有用的工具，以提高动作电位传播和Ca动力学心律失常发展的理解。

有文化的融合单层呈现有规律的内在节奏表现在电影1。然后，我们执行的V 米/钙双通道光映射在一个完全融合的HL-1的单层。 图1A显示了V M和钙信号的例子痕迹从记录单击败。均匀地传播V M和使用该双通道光学测绘系统的Ca信号代表等时的地图显示在图1B和1C。均匀地传播动作电位和钙瞬变代表时间顺序的图像是从同一的HL-1培养单层获得。最后， 影2和3提供的均匀传播动作电位（ 电影2）和在所述的HL-1融合单层钙瞬变（ 电影3）动画的例子。 -page ="总是"&gt; <img alt="图1" src="/files/ftp_upload/52542/52542fig1highres.jpg" /> 图1: （A），从电的节奏跳动V M（蓝色）和钙（红色）在500毫秒的周期长度（CL）示例痕迹。 （B）我们的光学标测系统的HL-1单层的图像。 （C）动作电位传播从整个视场相同的节奏跳动的等时的地图。 （D）钙瞬变传播从整个视场相同的节奏跳动的等时的地图。在这两个等时的地图，时间的推移表示颜色，从深蓝色的启动和暗红色，在50毫秒。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52542/52542fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> /52542/52542fig2highres.jpg"/&gt; 图2: 在60（A）中的一系列的V M分布图，80，140，和记录开始后200毫秒。钙分布的（B）的地图，在60，80，140，和记录开始后200毫秒。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52542/looped HL-1 7-20-14.avi">电影1:</a>记录从融合HL-1的单层文化日5，光学显微镜下用40X的目标代表的电影。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52542/vm 07_21_14 jy.avi">电影2</a> :从一个节奏跳动传播的动作电位在500毫秒的CL代表电影。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52542/ca 07_21_14 jy.avi">电影3</a> :传播的钙瞬变从PAC代表电影ED击败在500毫秒的CL。

Watch this Scientific Journal Video about Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer at JoVE.com

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

培养HL-1心房肌细胞单层的电压和钙双通道光映射

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

voltage-calcium-dual-channel-optical-mapping-cultured-hl-1-atrial

Universidad San Sebastian

Research

JoVE Journal

Biology

10.2K 次观看.  Loyola University Chicago. This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

视频: Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

多参数的Langendorff灌注兔心脏的光学测绘

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

科研

生物工程

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

在小鼠心脏的动作电位和钙瞬变的光学测绘

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

分离的月毛心室肌细胞的光学成像

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

人体器官性心脏切片双电压和钙光学映射的临床前心脏电生理评估

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

测量心肌细胞的快速钙助焊剂

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

生物学

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

高分辨率光学制图对小鼠心房电生理的评价

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

医学

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+ ...

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

的内肌浆网钙光学标测<sup&gt; 2+</sup&gt;和的Langendorff灌注兔心脏跨膜电位

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

分离高质量鼠心房和心室肌细胞，同时测量 Ca2+ 瞬变和 L 型钙电流

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

培养HL-1心房肌细胞单层的电压和钙双通道光映射

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