这项工作得到了德国研究基金会（DFG;血管医学临床科学家计划（PRIME），MA 2186/14-1至P. Tomsits和D. Schüttler;VO1568/3-1、IRTG1816 和 SFB1002 项目 A13 至 N. Voigt）、德国卓越战略下的德国研究基金会（EXC 2067/1- 390729940 至 N. Voigt）、德国心血管研究中心（DZHK;81X2600255 至 S. Clauss 和 N. Voigt;81Z0600206 至 S. Kääb）、科罗纳基金会（S199/10079/2019 至 S. Clauss）、ERA-NET 心血管疾病（ERA-CVD; 01KL1910 至 S. Clauss）， Heinrich-and-Lotte-Mühlfenzl Stiftung（致S. Clauss）和Else-Kröner-Fresenius基金会（EKFS 2016_A20致N. Voigt）。资助者在手稿准备中没有任何作用。

所有动物程序均由下萨克森州动物审查委员会（LAVES，AZ-18/2900）批准，并按照所有机构，国家和国际动物福利指南进行。
1. 预先安排
<ol><li>准备 1 L 10x 灌注缓冲液（表 1）、500 mL 1x 灌注缓冲液（表 2）、50 mL 消化缓冲液（表 3）、10 mL 终止缓冲液（表 4）、1 L Tyrode 溶液（表 5）、10 mL 每个钙阶梯溶液（含葡萄糖和相应钙量的 Tyrode 溶液，如所示）， 根据提供的配方，1 L 4-AP 溶液（表 5）、100 mL 移液器溶液（表 6）和 5 mL 普鲁龙酸。 注意：可以提前制备浴溶液（Tyrode和4-AP溶液）（不含葡萄糖）并储存在+ 4°C，在实验日加入葡萄糖。移液器溶液可储存在-20°C，在实验当天加入钙指示剂，然后将溶液储存在冰上直至进一步使用。10x灌注缓冲液可在室温下储存，1x灌注缓冲液，消解缓冲液和停止缓冲液应在实验当天新鲜制备。</li>
	<li>打开水浴和滚筒泵。</li>
	<li>用灌注缓冲液预填充朗根多夫装置;确保它是无空气的。</li>
	<li>通过将主动脉套管固定在解剖显微镜下来制备主动脉套管，用装有灌注缓冲液的 1 mL 注射器连接，并通过冲洗套管来清洁空气。 注意：避免灌注系统内有任何空气至关重要，因为这将直接影响冠状动脉灌注，从而影响消化效果。如有必要，可以在设置中添加气泡捕集器，以安全地避免任何空气滞留。</li>
	<li>用足够的灌注缓冲液准备培养皿，用于器官收集和显微镜检查（缓冲液应牢固地覆盖整个器官，几毫升 - 取决于使用的培养皿大小 - 应该足够）。</li>
	<li>用消化缓冲液准备3个培养皿，用于组织解离和显微镜检查，缓冲液应覆盖器官，以便在相应的培养皿内的显微镜下解剖，数量取决于使用的培养皿大小。对于解离，心室组织使用 3 mL，心房组织使用 1.5 mL。</li>
</ol>2. 摘取器官
<ol><li>使用带有27G套管的1mL注射器将小鼠与0.1mL肝素（1，000U / mL）腹腔注射，并等待5-10分钟。</li>
	<li>将小鼠与浸泡在约500μL异氟醚中的小组织一起放入诱导室中。动物不应与组织接触。为了避免这种情况，可以使用塑料活检包埋盒来覆盖组织。一旦动物完全麻醉，检查脚趾捏反射，一旦它不再存在，就通过颈椎脱位迅速对小鼠实施安乐死。</li>
	<li>将鼠标放在其背部的平台上（例如，在用纸巾覆盖的聚苯乙烯泡沫塑料上），并用套管固定爪子以将其固定到位。</li>
	<li>用剪刀从颈静脉向联合的清晰切口去除覆盖胸部和部分腹部的皮毛和皮肤，并在剑突下方打开腹部，而不会伤害任何器官结构。用手术钳抬起胸骨，用剪刀沿着肋骨边缘剪断横膈膜，然后在腋窝内侧线切割肋骨并取出肋骨以露出心脏。</li>
	<li>使用钝钳小心地取出心包，并通过用钝钳从下方提起心脏并用剪刀一次切割大血管来快速取出心脏。</li>
	<li>将心脏放入室温灌注缓冲液中，并在显微镜下尽快用钝端针插管主动脉。 注意：去除附着在器官上的任何肺组织和脂肪组织，而不会在其上浪费太多时间。插管时，请确保针头末端不会穿过主动脉瓣，因为这会阻止缓冲液进入冠状动脉，从而损害结果。</li>
	<li>用一块缝合丝将心脏牢固地绑在针头上，然后断开注射器。 注意：从获得心脏（大血管被切断的那一刻）到将主动脉缝合到针头上的整个过程应花费尽可能少的时间。建议从取出心脏到开始灌注的时间不要超过90-180秒。</li>
</ol>3. 酶消化
<ol><li>主动脉插管后，立即将空心连接到朗根道夫装置，避免任何空气进入系统。 注意：在Langendorff装置的底部悬挂一滴灌注缓冲液，并在针头顶部放置一滴灌注缓冲液，以避免任何空气进入系统，这很有帮助。</li>
	<li>在正好37°C的温度和恰好4mL / min的灌注速率下用灌注缓冲液灌注心脏1分钟。 注意：为了使灌注针尖的温度为37°C，水浴温度必须设置为略高于约40°C。 应通过测量灌注尖端的温度来定期测试。</li>
	<li>将灌注切换到消化缓冲液并在正好 37 °C 的温度和正好 4 mL/min 的灌注速率下灌注正好 9 分钟。</li>
	<li>将消化的心脏转移到具有足够消化缓冲液的培养皿中，以使其完全覆盖。然后在显微镜下仔细解剖心房和心室。</li>
	<li>将心房转移到装有 1.5 mL 消化缓冲液的培养皿中，将心室转移到另一个装有 3 mL 消化缓冲液的培养皿中。</li>
	<li>心房夹层<ol><li>小心地，但不浪费时间，使用钝钳将心房拉开成小块。</li>
		<li>使用 1，000 μL 移液器吸头小心地上下移液来溶解组织，该吸头之前已被切割以扩大吸头开口。</li>
		<li>将溶液转移到 15 mL 离心管中，并通过小心地从管侧面移液以结束反应，加入等量的终止缓冲液 （1.5 mL）。</li>
		<li>小心地将所有 3 mL 细胞/组织溶液通过 200 μm 尼龙网，以去除尚未完全消化的剩余较大组织碎片。 注意：成功的消化几乎不会留下任何固体块。</li>
	</ol></li>
	<li>心室夹层<ol><li>使用解剖剪刀快速将心室组织切成小块，上下移液以溶解。使用另一个 1，000 μL 移液器吸头上下移液，可以缩短以扩大开口。</li>
		<li>将细胞/组织溶液转移到 15 mL 离心管中，并通过小心地从管侧面移液以结束反应，加入等量的终止缓冲液 （3 mL）。</li>
		<li>小心地将所有 6 mL 细胞/组织溶液通过 200 μm 尼龙网，以去除尚未完全消化的较大组织碎片。 注意：成功的消化几乎不会留下任何固体块。</li>
	</ol></li>
	<li>将两个管（心房和心室细胞悬液）在室温下在工作台上放置6分钟以沉淀。</li>
	<li>将两个 15 mL 管以 5 x g 离心 2 分钟。</li>
</ol>4. 钙再引入
注意：以下步骤对于心房和心室细胞（除非另有说明）是相同的，并且在室温下进行。
<ol><li>使用塑料巴斯德移液管弃去上清液，并小心地将沉淀重悬于10mL无钙Tyrode溶液中。</li>
	<li>让细胞沉降8分钟。</li>
	<li>以5× g 离心1分钟（仅心房细胞，沉降足以用于心室细胞）。</li>
	<li>弃去上清液，小心地将沉淀重悬于100μM钙浓度的10mL Tyrode溶液中。</li>
	<li>让细胞沉降8分钟。</li>
	<li>以5× g 离心1分钟（仅心房细胞，沉降足以用于心室细胞）。</li>
	<li>弃去上清液，小心地将沉淀重悬于10mL钙浓度为400μM的Tyrode溶液中。</li>
	<li>让细胞沉降8分钟。</li>
	<li>以5× g 离心1分钟（仅心房细胞，沉降足以用于心室细胞）。</li>
	<li>弃去上清液，小心地将沉淀重悬于1mL（心房）/ 5mL（心室）的Tyrode溶液中，钙浓度为1mM。</li>
</ol>5.用荧光钙指示剂Fluo-3 AM加载肌细胞
注意：由于荧光钙指示剂的光敏性，应执行以下步骤，避光（例如，用铝箔覆盖管）。
<ol><li>通过将 44 μL 20% 多朗 F-127 无水 DMSO 添加到 50 μg Fluo-3AM 中（可在避光下储存在 -20 °C 中）来制备 Fluo-3 AM 储备溶液。</li>
	<li>将 10 μL Fluo-3 AM 储备溶液加入 1 mL 细胞悬液中，并在室温避光下孵育 10 分钟。</li>
	<li>以5× g 离心1分钟。</li>
	<li>使用塑料巴斯德移液管弃去上清液，并将沉淀重悬于合理量的浴液中，以获得良好的工作浓度（1-5mL浴液，取决于细胞密度）。</li>
	<li>在开始实验之前，静置30分钟进行脱酯。</li>
</ol>6. 如前所述同时膜片钳和落射荧光 Ca2+ 瞬态测量16
注意：膜片钳测量不是本文的主题，感兴趣的读者可以参考主要出版物，其中提供了该方法的深入描述17，18，19，20，21，22。然而，为了更好地理解，提供了测量L型钙电流以及电流诱导的钙瞬变的协议摘要。
<ol><li>将肌细胞转移到细胞室中，并在37°C下用浴液过度融合。</li>
	<li>如 表5所示，通过在浴液中加入4-氨基吡啶和氯化钡来阻断钾电流。</li>
	<li>确保硼硅酸盐微电极的尖端电阻为2-5 MΩ，填充有移液器溶液（表6）。</li>
	<li>设置测量以允许同时记录电信号和落射荧光。电压钳位模式用于测量 L 型Ca 2+ 电流，协议将电池保持在 -80 mV，600 ms 斜坡脉冲保持在 -40 mV 以停用快速 Na+ 电流，然后在 0.5 Hz 时将 100 ms 测试脉冲保持在 +10 mV（图 2）。</li>
	<li>使用488 nm处的激发，在<520 nm处发射的光进行检测并转换为[Ca2+]I 假设 <img alt="Equation 1" src="/files/ftp_upload/61964/61964equ01.jpg"> 其中 kd = Fluo-3的解离常数（864 nM）， F = Fluo-3荧光; Fmax = 在每个实验结束时获得的Ca 2+ 饱和荧光19.</li>
</ol>

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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+atrial+cytosolic+calcium+handling+contributes+to+the+development+of+postoperative+atrial+fibrillation.">Altered atrial cytosolic calcium handling contributes to the development of postoperative atrial fibrillation.</a> Cardiovascular Research. , (2020).</li><li>Chen, W., Frangogiannis, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+inflammatory+and+fibrogenic+pathways+in+heart+failure+associated+with+aging.">The role of inflammatory and fibrogenic pathways in heart failure associated with aging.</a> Heart Failure Reviews. 15 (5), 415-422 (2010).</li><li>Pla&#269;ki&#263;, J., Kocksk&#228;mper, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+atrial+and+ventricular+cardiomyocytes+for+in+vitro+studies.">Isolation of atrial and ventricular cardiomyocytes for in vitro studies.</a> Methods in Molecular Biology. 1816, 39-54 (2018).</li><li>D&#237;az, M. E., Trafford, A. W., Eisner, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+exogenous+calcium+buffers+on+the+systolic+calcium+transient+in+rat+ventricular+myocytes.">The effects of exogenous calcium buffers on the systolic calcium transient in rat ventricular myocytes.</a> Biophysical Journal. 80 (4), 1915-1925 (2001).</li><li>Zimmerman, A. N., H&#252;lsmann, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paradoxical+influence+of+calcium+ions+on+the+permeability+of+the+cell+membranes+of+the+isolated+rat+heart.">Paradoxical influence of calcium ions on the permeability of the cell membranes of the isolated rat heart.</a> Nature. 211 (5049), 646-647 (1966).</li><li>Chen, X., O'Connell, T. D., Xiang, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=With+or+without+Langendorff:+A+new+method+for+adult+myocyte+isolation+to+be+tested+with+time.">With or without Langendorff: A new method for adult myocyte isolation to be tested with time.</a> Circulation Research. 119 (8), 888-890 (2016).</li><li>Kappadan, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+optical+measurement+of+cardiac+restitution,+contraction,+and+fibrillation+dynamics+in+beating+vs.+blebbistatin-uncoupled+isolated+rabbit+hearts.">High-resolution optical measurement of cardiac restitution, contraction, and fibrillation dynamics in beating vs. blebbistatin-uncoupled isolated rabbit hearts.</a> Frontiers in Physiology. 11, 464 (2020).</li><li>Brack, K. E., Narang, R., Winter, J., Ng, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanical+uncoupler+blebbistatin+is+associated+with+significant+electrophysiological+effects+in+the+isolated+rabbit+heart.">The mechanical uncoupler blebbistatin is associated with significant electrophysiological effects in the isolated rabbit heart.</a> Experiment in Physiology. 98 (5), 1009-1027 (2013).</li><li>Seibertz, F., Reynolds, M., Voigt, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+optical+action+potential+measurement+in+human+induced+pluripotent+stem+cell-derived+cardiomyocytes.">Single-cell optical action potential measurement in human induced pluripotent stem cell-derived cardiomyocytes.</a> Journal of Visual Experiment. , e61890 (2020).</li></ol>

本文提供了一种简单实用的方法来从同一只小鼠获得高质量的心房和心室肌细胞，用于膜片钳研究，同时进行钙瞬时记录。所获得数据的质量在很大程度上取决于细胞分离的质量。如上所述，许多分离小鼠心肌细胞的方法已在前面描述9，10，11，12。分离的细胞用于各种分析，从膜片钳实验到收缩力研...

心律失常很常见，也是当前主要的医疗保健挑战之一，因为它们影响着全球数百万人。心律失常与高发病率和死亡率有关1，2，是大多数心脏性猝死的根本原因3。最新的治疗方案提高了患者的生存率，但仍然主要是对症治疗，而不是针对潜在的机制。因此，这些治疗的疗效有限，并且可能经常引起严重的副作用4，5，6。当前治疗方案的改进需要深入了解潜在的病理生理学，从而需要合适的模型进行研究。小动物模型 - 特别是小鼠模型 - 在心律失常研究中起着至关重要的作用，因为它们可以研究心律失常发生的关键机制，例如对细胞电生理学，离子通道功能或钙处理的遗传影响7，8。
为此，需要足够数量和活力的孤立心房和心室心肌细胞。先前已经描述了获得心房和心室肌细胞的广谱不同分离方法9，10，11，12，13，并且一些小组提供了同时测量来自心房14或心室15的L型电流和钙电流诱导的钙瞬变的数据鼠心肌细胞。然而，据我们所知，没有一种动物的心房和心室测量数据。研究人员专注于各种各样的主题，从电生理学到蛋白质组学，细胞收缩力或蛋白质相互作用的功能研究，线粒体功能或遗传学 - 所有这些都需要分离的心肌细胞。因此，许多已发表的方案尚未专门针对膜片钳研究开发，导致膜片钳研究的产量有限且细胞质量不足。因此，不能使用既定的方案对从一只动物分离的心房和心室细胞同时进行膜片钳和钙瞬时测量。
分离鼠（尤其是心房）肌细胞进行膜片钳实验仍然具有挑战性。本文提供了一种简单快速的方法，通过基于逆行酶的Langendorff灌注分离高质量的鼠心房和心室肌细胞，随后可以同时测量一只动物的净膜电流和电流诱导的钙瞬变。本文详细阐述了分离来自野生型小鼠和携带基因突变的小鼠的心房和心室肌细胞的方案。该协议可用于雄性和雌性小鼠。下面描述的肌细胞分离，图像和代表性结果是从6（±1）个月大的野生型C57Bl / 6小鼠中获得的。然而，该方案已成功用于具有不同基因型的2至24个月不同年龄的小鼠。 图1 显示了酶灌注过程中分离设置和空心的特写。

<table>
	<tbody>
		<tr>
			<td>2,3-Butanedione monoxime</td>
			<td>Sigma-Aldrich</td>
			<td>31550</td>
			<td></td>
		</tr>
		<tr>
			<td>27G cannula</td>
			<td>Servoprax</td>
			<td>L10220</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Aminopyridine</td>
			<td>Sigma-Aldrich</td>
			<td>A78403</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D12345</td>
			<td></td>
		</tr>
		<tr>
			<td>Aortic cannula</td>
			<td>Radnoti</td>
			<td>130163-20</td>
			<td></td>
		</tr>
		<tr>
			<td>BaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>342920</td>
			<td></td>
		</tr>
		<tr>
			<td>blunt surgical forceps</td>
			<td>Kent Scientific</td>
			<td>INS650915-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Calf Serum</td>
			<td>Sigma-Aldrich</td>
			<td>12133C</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C5080</td>
			<td></td>
		</tr>
		<tr>
			<td>Caffeine</td>
			<td>Sigma-Aldrich</td>
			<td>C0750</td>
			<td></td>
		</tr>
		<tr>
			<td>Circulating heated water bath</td>
			<td>Julabo</td>
			<td>ME</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type II</td>
			<td>Worthington</td>
			<td>LS994177</td>
			<td></td>
		</tr>
		<tr>
			<td>disscetion scissors</td>
			<td>Kent Scientific</td>
			<td>INS600124</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-aspartat K+-salt</td>
			<td>Sigma-Aldrich</td>
			<td>A2025</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-3</td>
			<td>Invitrogen</td>
			<td>F3715</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-3 AM</td>
			<td>Invitrogen</td>
			<td>F1242</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanosine 5&#8242;-triphosphate tris salt</td>
			<td>Sigma-Aldrich</td>
			<td>G9002</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating coil</td>
			<td>Radnoti</td>
			<td>158821</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Ratiopharm</td>
			<td>25.000 IE/5ml</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H9136</td>
			<td></td>
		</tr>
		<tr>
			<td>induction chamber</td>
			<td>CWE incorporated</td>
			<td>13-40020</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Cp-pharma</td>
			<td>1214</td>
			<td></td>
		</tr>
		<tr>
			<td>Jacketed heart chamber</td>
			<td>Radnoti</td>
			<td>130160</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Merck</td>
			<td>1049360250</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl x 6H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M0250</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4 x 7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M9397</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2ATP</td>
			<td>Sigma-Aldrich</td>
			<td>A2383</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4 x 2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>S5136</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon mesh (200 &#181;m)</td>
			<td>VWR-Germany</td>
			<td>510-9527</td>
			<td></td>
		</tr>
		<tr>
			<td>pasteur pipette</td>
			<td>Sigma Aldrich</td>
			<td>Z331759</td>
			<td></td>
		</tr>
		<tr>
			<td>petri-dishes</td>
			<td>Thermo Fisher</td>
			<td>150318</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic Acid F-127</td>
			<td>Sigma-Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>Probenecid</td>
			<td>Sigma-Aldrich</td>
			<td>P8761</td>
			<td></td>
		</tr>
		<tr>
			<td>Roller Pump</td>
			<td>Ismatec</td>
			<td>ISM597D</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical forceps</td>
			<td>Kent Scientific</td>
			<td>INS650908-4</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical scissors</td>
			<td>Kent Scientific</td>
			<td>INS700540</td>
			<td></td>
		</tr>
		<tr>
			<td>suturing silk</td>
			<td>Fine Science Tools</td>
			<td>NC9416241</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe</td>
			<td>Merck</td>
			<td>Z683531-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurin</td>
			<td>Sigma-Aldrich</td>
			<td>86330</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of high quality murine atrial and ventricular myocytes for simultaneous measurements of ca2+ transients and l-type calcium current

小鼠模型在心律失常研究中起着至关重要的作用，并允许研究心律失常发生的关键机制，包括离子通道功能的改变和钙处理。为此，需要高质量的心房或心室心肌细胞来进行膜片钳测量或探索钙处理异常。然而，通过当前分离方案获得的高质量心肌细胞的有限产量不允许在同一只小鼠中进行两次测量。本文描述了一种通过逆行基于酶的Langendorff灌注分离高质量鼠心房和心室肌细胞的方法，以便随后同时测量一只动物的钙瞬变和L型钙电流。获得小鼠心脏，并迅速插管主动脉以去除血液。然后首先用无钙溶液（37°C）灌注心脏以在插层盘水平上解离组织，然后用含有少量钙的酶溶液破坏细胞外基质（37°C）。消化的心脏随后被解剖成心房和心室。将组织样品切成小块，并通过小心地上下移液溶解。停止酶消化，并逐步将细胞重新引入生理钙浓度。用荧光Ca2+指示剂加载后，制备分离的心肌细胞，用于同时测量钙电流和瞬变。此外，还讨论了隔离陷阱，并提供了膜片钳方案和L型钙电流的代表性迹线，同时在如上所述分离的心房和心室鼠肌细胞中进行钙瞬时测量。

通过将 10 μL 细胞悬液移液到显微镜载玻片上，在重新引入钙后确定分离产量。超过 100 个活的杆状非收缩细胞/10 μL 用于心房细胞分离和超过 1，000 个活的杆状、非收缩细胞/10 μL 用于心室细胞分离被认为是足够的产量，并且通常使用此协议获得。通过该方案获得的心房细胞显示出各种不同的细胞类型，其中包含心脏传导系统的细胞，特别是窦房结，以及来自左右心房以及心耳的不同肌细胞。由?...

Watch this Scientific Journal Video about Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current at JoVE.com

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

小鼠模型允许研究心律失常发生的关键机制。为此，需要高质量的心肌细胞来进行膜片钳测量。这里描述了一种通过逆行基于酶的Langendorff灌注分离鼠心房和心室肌细胞的方法，该方法允许同时测量钙瞬变和L型钙电流。

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 ...

isolation-high-quality-murine-atrial-ventricular-myocytes-for

Research

JoVE Journal

Biology

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

2.9K 次观看.  Ludwig-Maximilians University Munich (LMU). 小鼠模型允许研究心律失常发生的关键机制。为此，需要高质量的心肌细胞来进行膜片钳测量。这里描述了一种通过逆行基于酶的Langendorff灌注分离鼠心房和心室肌细胞的方法，该方法允许同时测量钙瞬变和L型钙电流。

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

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

成人的心肌细胞的分离和遗传操作共聚焦成像

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

科研

生物学

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

在小鼠的心房和心室肌细胞的分离及钾离子通道的录音

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

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+ ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

在肾小管上皮细胞胞浆钙的测量流式细胞仪

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

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

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

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 ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

从小鼠骨骼肌高通量微孔板呼吸测量最低数量的隔离线粒体

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

发展与表征<em&gt;体外</em&gt;微血管网络和内皮的定量测量的[Ca<sup&gt; 2+</sup&gt;]<sub&gt;我</sub&gt;和一氧化氮含量

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

线粒体钙和线粒体膜的同时测量通过荧光显微镜活细胞的潜在

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

从成年大鼠心脏同时分离高质量心肌细胞，内皮细胞和成纤维细胞

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of intracellular calcium release are the major intracellular calcium storage organelles, the endoplasmic reticulum (ER) and the more specialized sarcoplasmic reticulum (SR) in muscle cells. The dynamic release of calcium from these organelles is mediated by the ryanodine receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R) with refilling occurring through the sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. A genetically encoded calcium sensor (GECI) called CatchER was created to monitor the rapid calcium release from the ER/SR. Here, the detailed protocols for the transfection and expression of the improved, ER/SR-targeted GECI CatchER+ in HEK293 and C2C12 cells and its application in monitoring IP3R, RyR, and SERCA pump-mediated calcium transients in HEK293 cells using fluorescence microscopy is outlined. The receptor agonist or inhibitor of choice is dispersed in the chamber solution and the intensity changes are recorded in real time. With this method, a decrease in ER calcium is seen with RyR activation with 4-chloro-m-cresol (4-cmc), the indirect activation of IP3R with adenosine triphosphate (ATP), and inhibition of the SERCA pump with cyclopiazonic acid (CPA). We also discuss protocols for determining the in situ Kd and quantifying basal [Ca2+] in C2C12 cells. In summary, these protocols, used in conjunction with CatchER+, can elicit receptor mediated calcium release from the ER with future application in studying ER/SR calcium related pathologies.

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

We present protocols for the application of our targeted genetically-encoded calcium indicator (GECI) CatchER+ for monitoring rapid calcium transients in the endoplasmic/sarcoplasmic reticulum (ER/SR) of HEK293 and skeletal muscle C2C12 cells using real-time fluorescence microscopy. A protocol for the in situ Kd measurement and calibration is also discussed.

监测ER / SR钙释放与目标Ca<sup&gt; 2+</sup&gt;传感器捕捉器<sup&gt; +</sup

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of ...