Name: assessment of myofilament ca2+ sensitivity underlying cardiac excitation-contraction coupling
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这项研究是由基础科学研究计划通过教育，科学与技术部（2013068067）资助的韩国国家研究基金会（NRF）的支持;由教育部，科技部，首尔国立大学医院，高血压的韩国社会（2013年），SK电信研究基金的韩国部脑韩国21毕业生计划（编号3420130290）和中国国家自然科学基金（NSFC 31460265;国家自然科学基金81260035）。

该协议是按照指南卫生的联合国国家机构发布的实验室动物的护理和使用（NIH出版号85-23，1996年修订）。它是经首尔国立大学的机构动物护理和使用委员会（IACUC）（IACUC批准号:SNU-101213-1）。 1.缓冲液配制（表材料和设备） <ol><li>制备300毫升新鲜分离溶液对实验当天（以mM:氯化钠，135;氯化钾，5.4; MgCl 2的，3.5;葡萄糖，5; HEPES，5; 的 Na 2 HPO 4，0.4;牛磺酸，20; pH值7.4用NaOH）。 </li><li>制备100毫升新鲜的存储溶液在实验当天（以mM:NaCl的120;氯化钾5.4; MgSO 4上5; 氯化钙 0.2;钠丙酮酸5;葡萄糖5.5;牛磺酸20; HEPES 10;甘露醇29; pH 7.4的用NaOH）。 </li><li>制备灌注溶液1L（以mM:氯化钠，141.4;氯化钾，4;和NaH 2 PO 4，0.33; MGCL 2，1; HEPES，10;葡萄糖，5.5; 氯化钙 ，1.8;甘露醇，14.5; pH为7.4，用NaOH）。 </li><li>通过加入胶原酶（1毫克/毫升），蛋白酶（0.1毫克/毫升），牛血清白蛋白（BSA，1.67mg / ml）和钙离子 （0.05毫摩尔）制备加入30ml胶原酶溶液1至25隔离毫升解。 </li><li>通过加入胶原酶（1毫克/毫升），牛血清白蛋白（1.67毫克/毫升），和钙离子 （0.05毫摩尔）至16.7毫升隔离溶液制备20毫升胶原酶溶液2。 </li><li>通过加入0.4克的BSA至40ml的隔离溶液制备的40ml BSA溶液。单独10毫升和30ml成两个烧杯。添加的 Ca 2+至30毫升BSA溶液，使最终的Ca 2+浓度的BSA溶液是1毫米。 </li></ol> 2.准备左心室（LV）心肌细胞的隔离<ol><li>从动物设施的准备和隔离室转让清洁运输笼8-12周龄，雄性SD（SD）大鼠。 </li><li> H吃两水浴至37℃。 注意:使用一种水浴水 - 夹套容器和的Langendorff灌注系统的灌注管。使用其他水浴以分离单个细胞搅动心肌组织的树干。 </li><li>加入5 ml和3.3的BSA溶液（不含Ca 2+）至25毫升胶原酶溶液1和16.7 ml胶原酶溶液2 ml至补足体积至30ml和20ml，分别。 </li><li>在的Langendorff灌注系统，分别为（ 图1A）添加隔离溶液（100毫升）和胶原酶溶液1（30毫升）中，以第1列（第1栏）和第2列（第2列）。 </li><li>充氧在的Langendorff灌注系统（ 图1A） 通过氧连接管分离溶液和胶原酶溶液1中第1栏和Col.2。同样地，含氧化合物胶原酶溶液2，并在摇动的水槽用100％O 2的经由所述BSA溶液氧气连接管。 </li></ol> 3. LV心肌细胞的分离<ol><li>麻醉通过戊巴比妥钠（30毫克/千克）的腹膜内注射大鼠和脚趾捏和缺乏撤回反射的确认麻醉状态。 </li><li>移动老鼠解剖盘。在仰卧位，固定四条腿到主体用胶带的侧面。 </li><li>应用胸中期轴向切口手术剪刀打开胸腔，确保不损害心脏。使用另一一双干净的剪刀从血管连接（ 例如，上，下腔静脉，肺血管，主动脉）和心包膜13解剖心脏。 </li><li>留足够长的主动脉的一部分（5 - 8 mm产品），夹紧用细镊子主动脉，和1分钟（ 图1A）内快速安装的Langendorff灌注系统的插管。领带缝合线4/0紧紧过主动脉。 </li><l我&gt;阀门打开上校1和10分钟预热和含氧隔离解决方案（灌注率:12-14毫升/分钟）灌注离体心脏。关闭阀上的第1栏，打开阀门上西2和8胶原酶溶液灌注1的心脏 - 10分钟。 </li><li>通过削减主动脉卸除消化心脏，并通过召开用钳子将主动脉含有新鲜的隔离解决方案（ 图1Bi）烧瓶中传送。用细剪刀和镊子大部分LV游离壁（包括隔膜），以切成小块（〜22毫米， 图1Bii）。 </li><li>转移片放入含预热和含氧的新鲜胶原酶溶液2（ 图1Biii）的烧瓶中。振摇10分钟。请提供氧气的含肌细胞胶原酶溶液2。 </li><li>移动心肌细胞-悬浮液10 ml离心管中，使用滴管带吸球，并添加钙 -含BSA溶液（1:1的体积比）。离心在30克2分钟，弃上清。重悬在5ml BSA溶液的肌细胞沉淀。离心并弃去上清液。 </li><li>驱散心肌细胞颗粒，并在RT（ 图1Biv-VI）保持心肌细胞在10毫升预氧化的存储解决方案。 </li><li>在烧瓶内的剩余的LV组织 - 重复程序（3.9步骤3.7）。 注意:不断重复的过程（步骤3.7 - 3.9）再次直到大部分LV组织消失，细胞获得了良好的收益。 </li><li>保持在存储溶液中的细胞在室温6-8小时，直至实验结束。 </li></ol> 4.细胞内钙瞬变和心肌细胞收缩的同时测量<ol><li>负载的LV心肌细胞与乙酰氧甲基酯的Fura-2 AM（2μM）。 注:在黑暗的管（ 图1Bvii）装载的细胞执行所有加载的程序和实验。 <ol><li> CENTRifuge在2000 xg离心肌细胞悬浮液（1ml）中，持续10秒。弃去上清液并重新悬浮于1ml台罗德氏溶液具有低Ca 2+浓度（250μM，表材料和设备）的肌细胞沉淀。 </li><li>添加的Fura-2AM和泊洛沙姆407（2微升），轻轻分散肌细胞悬浮液，并保持该混合物在RT（20 - 24℃）15分钟（ 图1Bvii）。 </li><li>离心混合物，持续5秒，弃去上清液并分散肌细胞沉淀在1ml含有500μM 的 Ca 2+灌注溶液。 10分钟后，离心该混合物，持续5秒，并丢弃上清液。 </li><li>加入新鲜的灌注溶液（500μM 钙 1毫升），并保持的Fura-2AM -加载 ​​心肌细胞颗粒在此解决方案的记录。 </li></ol></li><li> LV的细胞收缩和细胞内Ca 2+测定<ol><li>录像之前，填补通过运行输液管与台氏溶液，这是水套预热到36℃。 </li><li>放置的Fura凌晨2点几滴 - 加载的LV心肌悬浮于5倒置荧光显微镜的腔室 - 8分钟。慢慢灌注台罗德氏溶液（2.2毫升/分钟）。 </li><li>按数字刺激开始字段刺激（2赫兹）的前面板上的"启动"按钮。 注意:输出电压（10V，5毫秒的持续时间）通过设置在腔室的任一侧铂导线施加到心肌细胞中的腔室。 <ol><li>选择细胞的合同稳定（而不是那些显示高血糖或低收缩）记录。 </li></ol></li><li>调整选择的心肌细胞中的基于视频的肌节检测系统的水平位置和调整显微镜的焦点，以获得肌节的最佳图像。在其中肌节清晰明确，直到平均观察到的区域紫色矩形框的位置肌节长度（红色峰）的显示单一尖峰（ 图2A，下图）。 </li><li>记录响应于场刺激（ 图2A和B）小节的长度的变化。 注意事项:1范围内使用加载的细胞 - 2小时。 </li><li>调节摄像机的光圈，使得在基于视频的肌节的检测系统中的场是心肌细胞（ 图2A）的尺寸。刺激与360纳米/ 380纳米激发和发射510纳米（收购频率1000赫兹）的心肌细胞。记录肌节缩短和与场刺激（ 图2B）的Fura2调幅比。 </li></ol></li></ol> 5.肌丝钙离子敏感性评价<ol><li>平均肌节长度和Ca在稳态2+瞬变（10 - 20痕量）和绘制的Fura-2比对相同的心肌细胞的肌节长度的相平面环（都与测量值S和增量变化; 图2C）。 </li><li>在每个小区，定义的Fura -2在50％松弛（EC 50， 图2C）的比例。比较这两个循环，每个干预的EC 50。 </li></ol>

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The authors declare that they have no competing financial interests.

这里，我们描述了协议，以评估肌丝的 Ca 2+在单个孤立心肌细胞的敏感性的变化，并强调测定该参数一起电生理特性，细胞内Ca 2+瞬变，和肌丝动力学的重要性。这是因为一个或两个参数的记录可能没有阐明心脏收缩和舒张的基本机制。不同于测量肌细胞收缩和细胞内Ca 2+信息单独1的常规方法，本方法检查这两个参数在相同的心肌细胞同时。 <...

心肌兴奋收缩（EC）耦合是分析心肌， 即心脏1,2的收缩功能的机械性能的根本方案。 EC耦合 ​​通过膜去极化继发于细胞膜离子通道的活动发起（ 例如，电压门控钠通道，它可以通过膜片钳技术测量）。电压门控的L型的随后活化的Ca 2+通道（LTCCS）和Ca 经由 LTCCS 2+内流触发散装的Ca 2+通过兰尼碱受体（RyRs的）的释放，从纳摩尔提高胞质Ca 2+浓度（纳米）到微摩尔（μM）的水平。这种增加在细胞内钙离子促进钙离子结合在薄丝肌钙蛋白C（TNC）和抒发灯丝复杂的构象变化，促进肌动球蛋白相互作用和达到myocardi人收缩3。相反，细胞内钙重新摄取回SR中的肌质网（SR）通过的 Ca 2+ -ATP酶（SERCA2a的），或者是通过 钠 / 钙交换和质膜挤出肌纤维钙离子 ATP酶1,2。因此，在细胞内的Ca的下降2+指使细长丝的构象变化返回到原来的状态，从而导致肌动蛋白-肌球蛋白和肌细胞松弛1-3的解离。在这个方案中，SERCA2a的活性通常被认为是，以确定心肌松弛的速度，因为它占70 -在大多数哺乳动物的心脏细胞1细胞内的Ca 2+去除的90％。正如LTCC，碱受体和SERCA2a的等这样的，不正常的钙处理一直被认为是受损的收缩和放松患病心脏1-4的主要机制。 离子 ，其功能如同在EC耦合 ​​账户信使约1％的总细胞内Ca 2+和广大钙势必细胞内钙离子缓冲5,6。这是由于这样的事实，即各个的 Ca 2+缓冲液是在心肌细胞丰富， 例如，膜磷脂，ATP，磷酸，钙调蛋白，小清蛋白，肌原纤维TNC，肌球蛋白，SERCA2a的，并且在对SR肌集钙蛋白。5,6,7。其中，SERCA2a的和跨国公司是主要的钙缓冲5,6,7。此外，钙离子结合到其缓冲器是抽搐期间一个动态的过程（ 例如，钙2+ 30-50％结合于肌钙蛋白和Ca期间将它解离2+瞬变7）和中的 Ca 2+结合的原因的其他变化在细胞内钙的改变游离钙离子的"释放"到细胞质，结果2+浓度entration。因此，细胞内Ca 2+水平的扰动引起异常肌丝的运动，这是收缩功能障碍和心律失常8,9的前体。许多因素（包括生理和病理）可以是肌丝蛋白质的转录后修饰，影响肌丝的 Ca 2+缓冲和肌丝的 Ca 2+敏感性8-10的来源。最近，据报道，在肌丝蛋白突变增加的 Ca 2+结合亲和力和细胞内Ca 2+的处理，触发的 Ca 2+瞬变，异常的 Ca 2+释放，和心律失常8的暂停依赖性增强。根据这一理念，我们也表现出高血压大鼠心脏继发于神经元型一氧化氮合酶上调的肌丝钙脱敏与高架舒张和收缩的 Ca 2+水平有关11，这又增加了LTCC的对于Ca 2+依赖性失活12的脆弱性。因此，肌丝的 Ca 2+敏感性是细胞内Ca 2+稳态和肌细胞收缩功能的"有效的"调节器。它已成为必要分析肌丝和Ca之间的相互作用2+处理的蛋白质为心肌兴奋收缩耦联及心功能的彻底调查。 在这里，我们描述了评估肌丝钙离子敏感性在孤立的心肌细胞变化的协议。细胞内钙离子分布的综合分析，肌丝钙离子敏感性和收缩会发掘新机制基本心肌力学。

<table><tbody><tr><td>Sprague Dawley rat</td><td>Koatech</td><td></td><td>8-12 weeks</td></tr><tr><td>Pentobarbital Sodium</td><td>Hanlim Pharmaceutical (Korea)</td><td>AHN901</td><td>Insurance code:645301220</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9625</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P4504</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>S8282</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>G8270</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Biosesang</td><td>C2002</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Biosesang</td><td>M2001</td><td></td></tr><tr><td>Mannitol</td><td>Sigma</td><td>M4125</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>M5921</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Sigma</td><td>P2256</td><td></td></tr><tr><td>Taurine</td><td>Merck</td><td>8.08616.1000</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&amp;nbsp;&lt;/sub&gt;</td><td>Sigma</td><td>71649</td><td></td></tr><tr><td>Bovine Fetal Albumin</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>Collagenase Type 2</td><td>Worthington</td><td>LS004177</td><td></td></tr><tr><td>Protease</td><td>Sigma</td><td>P6911</td><td></td></tr><tr><td>Fura-2 (AM)</td><td>Molecular Probes</td><td>F1221</td><td></td></tr><tr><td>Pluronic F127 20% solution in DMSO</td><td>Invitrogen</td><td>P3000MP</td><td></td></tr><tr><td>Shaking Water Bath</td><td>Chang Shin Scientific</td><td>Model: C-108</td><td></td></tr><tr><td>IonWizard Softwae Suite</td><td>IonOptix Ltd</td><td>Experimental Builder</td><td>Acquisition and Analysis of EC Coupling Data in Myocytes</td></tr><tr><td>Myocyte Calcium &amp;amp; Contractility Recording System</td><td>IonOptix Ltd</td><td></td><td></td></tr><tr><td>Circulating Water Bath</td><td>BS-Tech</td><td>BW2-8</td><td></td></tr><tr><td>Myocyte Fluorescence Microscope</td><td>Nikon</td><td>DIATPHOTO 200</td><td></td></tr><tr><td>MyoCam-S Power</td><td>IonOptix</td><td></td><td></td></tr><tr><td>Fluorescence &amp;amp; Video Detection</td><td>IonOptix</td><td>MyoCam-S</td><td></td></tr><tr><td>CFA300</td><td></td><td></td><td></td></tr><tr><td>PMT400</td><td></td><td></td><td></td></tr><tr><td>Fluorescence &amp;amp; System Interface</td><td>IonOptix</td><td>FSI700</td><td></td></tr><tr><td>Excitation Light Source</td><td>IonOptix</td><td>mSTEP</td><td></td></tr><tr><td>High intensity ARC Lamp Power supply</td><td>Cairn Reseach</td><td></td><td></td></tr><tr><td>Filter wheel controller</td><td>IonOptix</td><td>GB/MUS200</td><td></td></tr><tr><td>Digital Stimulator</td><td>Medical Systems Corportion</td><td>S-98 Mutimode</td><td></td></tr><tr><td>&lt;strong&gt;Compositions of Experimental Solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>Isolation Solution (pH: 7.4, NaOH)</td><td></td><td></td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9625</td><td>Concentration (mmol) 135</td></tr><tr><td>KCl</td><td>Sigma</td><td>P4504</td><td>Concentration (mmol) 5.4</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td>Concentration (mmol) 5</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G8270</td><td>Concentration (mmol) 5</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Biosesang</td><td>M2001</td><td>Concentration (mmol) 3.5</td></tr><tr><td>Taurine</td><td>Sigma</td><td>CB2742654</td><td>Concentration (mmol) 20</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&amp;nbsp;&lt;/sub&gt;</td><td>Sigma</td><td>71649</td><td>Concentration (mmol) 0.4</td></tr><tr><td>Storage Solution (pH: 7.4, NaOH)</td><td></td><td></td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9625</td><td>Concentration (mmol) 120</td></tr><tr><td>KCl</td><td>Sigma</td><td>P4504</td><td>Concentration (mmol) 5.4</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td>Concentration (mmol) 10</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G8270</td><td>Concentration (mmol) 5.5</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Biosesang</td><td>C2002</td><td>Concentration (mmol) 0.2</td></tr><tr><td>Mannitol</td><td>Sigma</td><td>M4125</td><td>Concentration (mmol) 29</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>M5921</td><td>Concentration (mmol) 5</td></tr><tr><td>Sodium Pyruvate</td><td>Sigma</td><td>P2256</td><td>Concentration (mmol) 5</td></tr><tr><td>Taurine</td><td>Sigma</td><td>CB2742654</td><td>Concentration (mmol) 20</td></tr><tr><td>Perfusion Solution (Tyrode solution, pH: 7.4, NaOH)</td><td></td><td></td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9625</td><td>Concentration (mmol) 141.4</td></tr><tr><td>KCl</td><td>Sigma</td><td>P4504</td><td>Concentration (mmol) 4</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>S8282</td><td>Concentration (mmol) 0.33</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td>Concentration (mmol) 10</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G8270</td><td>Concentration (mmol) 5.5</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Biosesang</td><td>C2002</td><td>Concentration (mmol) 1.8&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; For Fura 2AM loading, CaCl2 concentrations are 0.25 mM and 0.5 mM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Biosesang</td><td>M2001</td><td>Concentration (mmol) 1</td></tr><tr><td>Mannitol</td><td>Sigma</td><td>M4125</td><td>Concentration (mmol) 14.5</td></tr><tr><td>Collangenase Solution 1</td><td></td><td></td><td></td></tr><tr><td>Isolation Solution (30mL)</td><td></td><td></td><td></td></tr><tr><td>Bovine Fetal Albumin (BSA solution 5 ml)</td><td></td><td></td><td>Concentration (mmol) 1.67 mg/mL</td></tr><tr><td>Collagenase Type 2</td><td>Worthington</td><td>LS004177</td><td>Concentration (mmol) 1 mg/mL</td></tr><tr><td>Protease</td><td>Sigma</td><td>P6911</td><td>Concentration (mmol) 0.1 mg/mL</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Biosesang</td><td>C2002</td><td>Concentration (mmol) 0.05 mM</td></tr><tr><td>Collangenase Solution 2</td><td></td><td></td><td></td></tr><tr><td>Isolation Solution (20mL)</td><td></td><td></td><td></td></tr><tr><td>Bovine Fetal Albumin (BSA solution 3.3 mL)</td><td></td><td></td><td>Concentration (mmol) 1.67 mg/mL</td></tr><tr><td>Collagenase Type 2</td><td>Worthington</td><td>LS004177</td><td>Concentration (mmol) 1 mg/mL</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Biosesang</td><td>C2002</td><td>Concentration (mmol) 0.05 mM</td></tr><tr><td>BSA solution</td><td></td><td></td><td></td></tr><tr><td>Isolation Solution (40mL)</td><td></td><td></td><td></td></tr><tr><td>Bovine Fetal Albumin</td><td>Sigma</td><td>A7906</td><td>Concentration (mmol) 400 mg</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Biosesang</td><td>C2002</td><td>Concentration (mmol) 1mM</td></tr></tbody></table>

assessment of myofilament ca2+ sensitivity underlying cardiac excitation-contraction coupling

心脏衰竭和心律失常是全世界发病率和死亡率的主要原因。然而，在患病心脏发病和心肌故障的机制仍有待完全澄清。最近令人信服的证据表明，在肌丝的 Ca 2+敏感性的变化会影响细胞内Ca 2+稳态和离子通道活动在心肌细胞，负责在健康和患病心脏心脏动作电位和收缩的主要机制。事实上，潜在的心脏动作电位（如钠离子 ，钙离子和钾离子通道，钠+ -Ca 2+交换）和细胞内钙离子处理的蛋白质（ 例如 ，兰尼碱受体和钙离子通道的活动和运输2+ -ATP酶肌浆网（SERCA2a的）或磷蛋白和磷酸化其）的常规测量evalu吃了心脏兴奋收缩（EC）耦合的基本机制。在细胞膜和细胞内钙离子的变化包括电活动有兴奋收缩耦联的触发信号，而肌丝的收缩和舒张的功能单位，肌丝钙离子敏感性是在肌原纤维性能的实施势在必行。然而，很少有研究纳入肌丝钙离子敏感性到心肌的功能分析，除非它是研究的重点。在这里，我们描述了测量肌缩短/再延长使用的Fura-2 AM（比率检测），并评估肌丝的 Ca 2+从大鼠心脏心肌细胞敏感性的变化，细胞内钙离子水平的协议。其主要目的是强调肌丝钙离子敏感性，应考虑到EC联轴器机械分析。我的全面排查对背后的健康和患病心脏肌细胞收缩将提供有价值的信息设计平移和治疗价值的更为有效的战略通道，离子转运，细胞内钙离子处理和肌丝钙离子敏感性。

LV肌细胞正常和高血压大鼠心脏分离。有明确的条纹（代表肌节），并稳定收缩响应于场刺激棒状细胞被认为是最佳的细胞和被选择用于记录（ 图2A）。在F中igure 2A所示的例子中，一个的Fura上午02点-loaded左心室心肌细胞水平定位并调节摄像机的光圈，使心肌细胞占据了大部分的记录区和最小的背景区域被包括在内。在录制现场，通过点击和拖动?...

Watch this Scientific Journal Video about Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling at JoVE.com

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

This paper describes a protocol that assesses the changes of myofilament Ca2+ sensitivity during contraction in isolated cardiac myocytes from rat heart. Together with cardiac electrophysiology, systolic/diastolic cytosol Ca2+ levels and contraction/relaxation, this measurement is imperative in underpinning the mechanisms mediating cardiac excitation-contraction coupling in healthy and diseased hearts.

肌丝的Ca评估<sup&gt; 2+</sup&gt;灵敏度潜在心脏兴奋收缩耦联

Heart failure and cardiac arrhythmias are the leading causes of mortality and morbidity worldwide. However, the mechanism of pathogenesis and myocardial ...

assessment-myofilament-ca2-sensitivity-underlying-cardiac-excitation

Research

JoVE Journal

Biology

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

9.4K 次观看.  Seoul National University College of Medicine. This paper describes a protocol that assesses the changes of myofilament Ca2+ sensitivity during contraction in isolated cardiac myocytes from rat heart. Together with cardiac electrophysiology, systolic/diastolic cytosol Ca2+ levels and contraction/relaxation, this measurement is imperative in underpinning the mechanisms mediating cardiac excitation-contraction coupling in healthy and diseased hearts. 

视频: Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric&#160;deformation. Our understanding of the internal relationships between these events has been limited because we can neither &#39;see&#39; inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue&#39;s geometric&#160;changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 &#181;m by 540 &#181;m) and the length of a typical trabecula (&#62;2000 &#181;m). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric&#160;data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

同时布莱特菲尔德， 荧光， 和光学相干断层成像收缩心脏特拉贝库莱 Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and ...

科研

生物工程

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

体外评估对离体骨骼肌的收缩，疲劳性和交替“

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

生物学

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

使用皮肤心肌细胞进行心脏功能的体外评估

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...

医学

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

对离体乳头肌的心脏收缩力对小鼠的研究收缩尺寸

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

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 ...

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 ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

使用纳米-牛顿分辨率力转换器从骨骼肌肉活检中隔离肌异物并确定收缩函数

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

天然骨骼肌细胞中的功能性定点荧光测定

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability

Functional site-directed fluorometry has been the technique of choice to investigate the structure-function relationship of numerous membrane proteins, including voltage-gated ion channels. This approach has been used primarily in heterologous expression systems to simultaneously measure membrane currents, the electrical manifestation of the channels&#39; activity, and fluorescence measurements, reporting local domain rearrangements. Functional site-directed fluorometry combines electrophysiology, molecular biology, chemistry, and fluorescence into a single wide-ranging technique that permits the study of real-time structural rearrangements and function through fluorescence and electrophysiology, respectively. Typically, this approach requires an engineered voltage-gated membrane channel that contains a cysteine that can be tested by a thiol-reactive fluorescent dye. Until recently, the thiol-reactive chemistry used for the site-directed fluorescent labeling of proteins was carried out exclusively in Xenopus oocytes and cell lines, restricting the scope of the approach to primary non-excitable cells. This report describes the applicability of functional site-directed fluorometry in adult skeletal muscle cells to study the early steps of excitation-contraction coupling, the process by which muscle fiber electrical depolarization is linked to the activation of muscle contraction. The present protocol describes the methodologies to design and transfect cysteine-engineered voltage-gated Ca2+ channels (CaV1.1) into muscle fibers of the flexor digitorum brevis of adult mice using in vivo electroporation and the subsequent steps required for functional site-directed fluorometry measurements. This approach can be adapted to study other ion channels and proteins. The use of functional site-directed fluorometry of mammalian muscle is particularly relevant to studying basic mechanisms of excitability.

作者聚焦：天然细胞中的功能性定点定向荧光测定法研究骨骼肌兴奋性  

Functional site-directed fluorometry is a method to study protein domain motions in real time. Modification of this technique for its application in native cells now allows the detection and tracking of single voltage-sensor motions from voltage-gated Ca2+ channels in murine isolated skeletal muscle fibers.

天然细胞中的功能性定点荧光测定法，用于研究骨骼肌兴奋性

Functional site-directed fluorometry has been the technique of choice to investigate the structure-function relationship of numerous membrane proteins, ...

肌丝的Ca评估

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同时布莱特菲尔德，荧光，和光学相干断层成像收缩心脏特拉贝库莱 Ex Vivo

体外评估对离体骨骼肌的收缩，疲劳性和交替“

使用皮肤心肌细胞进行心脏功能的体外评估

对离体乳头肌的心脏收缩力对小鼠的研究收缩尺寸

的内肌浆网钙光学标测

啮齿动物心肌细胞心脏收缩功能障碍和^Ca2+ 瞬变分析

使用纳米-牛顿分辨率力转换器从骨骼肌肉活检中隔离肌异物并确定收缩函数

天然细胞中的功能性定点荧光测定法，用于研究骨骼肌兴奋性

天然细胞中的功能性定点荧光测定法，用于研究骨骼肌兴奋性

天然细胞中的功能性定点荧光测定法，用于研究骨骼肌兴奋性

肌丝的Ca评估

摘要

探索更多视频

此视频中的章节

同时布莱特菲尔德， 荧光， 和光学相干断层成像收缩心脏特拉贝库莱 Ex Vivo

体外评估对离体骨骼肌的收缩，疲劳性和交替“

使用皮肤心肌细胞进行心脏功能的体外评估

对离体乳头肌的心脏收缩力对小鼠的研究收缩尺寸

的内肌浆网钙光学标测

啮齿动物心肌细胞心脏收缩功能障碍和Ca2+ 瞬变分析

使用纳米-牛顿分辨率力转换器从骨骼肌肉活检中隔离肌异物并确定收缩函数

天然细胞中的功能性定点荧光测定法，用于研究骨骼肌兴奋性

天然细胞中的功能性定点荧光测定法，用于研究骨骼肌兴奋性

天然细胞中的功能性定点荧光测定法，用于研究骨骼肌兴奋性

同时布莱特菲尔德，荧光，和光学相干断层成像收缩心脏特拉贝库莱 Ex Vivo

啮齿动物心肌细胞心脏收缩功能障碍和^Ca2+ 瞬变分析