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
1Department of Medicine I, University Hospital Munich, Campus Großhadern,Ludwig-Maximilians University Munich (LMU), 2Partner Site Munich, Munich Heart Alliance (MHA),DZHK (German Centre for Cardiovascular Research), 3Walter Brendel Center of Experimental Medicine,Ludwig-Maximilians University Munich (LMU), 4Institute of Pharmacology and Toxicology,University Medical Center Göttingen, 5Partner Site Göttingen,DZHK (German Centre for Cardiovascular Research), 6Cluster of Excellence “Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells” (MBExC),University of Göttingen
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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…
Representative Results
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?…
Discussion
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…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 2,3-Butanedione monoxime | Sigma-Aldrich | 31550 | |
| 27G cannula | Servoprax | L10220 | |
| 4-Aminopyridine | Sigma-Aldrich | A78403 | |
| Anhydrous DMSO | Sigma-Aldrich | D12345 | |
| Aortic cannula | Radnoti | 130163-20 | |
| BaCl2 | Sigma-Aldrich | 342920 | |
| blunt surgical forceps | Kent Scientific | INS650915-4 | |
| Bovine Calf Serum | Sigma-Aldrich | 12133C | |
| CaCl2 | Sigma-Aldrich | C5080 | |
| Caffeine | Sigma-Aldrich | C0750 | |
| Circulating heated water bath | Julabo | ME | |
| Collagenase Type II | Worthington | LS994177 | |
| disscetion scissors | Kent Scientific | INS600124 | |
| DL-aspartat K+-salt | Sigma-Aldrich | A2025 | |
| EGTA | Sigma-Aldrich | E4378 | |
| Fluo-3 | Invitrogen | F3715 | |
| Fluo-3 AM | Invitrogen | F1242 | |
| Glucose | Sigma-Aldrich | G8270 | |
| Guanosine 5′-triphosphate tris salt | Sigma-Aldrich | G9002 | |
| Heating coil | Radnoti | 158821 | |
| Heparin | Ratiopharm | 25.000 IE/5ml | |
| HEPES | Sigma-Aldrich | H9136 | |
| induction chamber | CWE incorporated | 13-40020 | |
| Isoflurane | Cp-pharma | 1214 | |
| Jacketed heart chamber | Radnoti | 130160 | |
| KCl | Merck | 1049360250 | |
| KH2PO4 | Sigma-Aldrich | P5655 | |
| MgCl x 6H2O | Sigma-Aldrich | M0250 | |
| MgSO4 x 7H2O | Sigma-Aldrich | M9397 | |
| Na2ATP | Sigma-Aldrich | A2383 | |
| Na2HPO4 x 2H2O | Sigma-Aldrich | S5136 | |
| NaCl | Sigma-Aldrich | S3014 | |
| NaHCO3 | Sigma-Aldrich | S5761 | |
| Nylon mesh (200 µm) | VWR-Germany | 510-9527 | |
| pasteur pipette | Sigma Aldrich | Z331759 | |
| petri-dishes | Thermo Fisher | 150318 | |
| Pluronic Acid F-127 | Sigma-Aldrich | P2443 | |
| Probenecid | Sigma-Aldrich | P8761 | |
| Roller Pump | Ismatec | ISM597D | |
| surgical forceps | Kent Scientific | INS650908-4 | |
| surgical scissors | Kent Scientific | INS700540 | |
| suturing silk | Fine Science Tools | NC9416241 | |
| syringe | Merck | Z683531-100EA | |
| Taurin | Sigma-Aldrich | 86330 |
Referenzen
- Camm, A. J., et al. Guidelines for the management of atrial fibrillation: the Task force for the management of atrial fibrillation of the European Society of Cardiology (ESC). Europace. 12 (10), 1360-1420 (2010).
- Chugh, S. S., et al. Worldwide epidemiology of atrial fibrillation: a global burden of disease 2010 study. Circulation. 129 (8), 837-847 (2014).
- Tonchev, I., Luria, D., Orenstein, D., Lotan, C., Biton, Y. For whom the bell tolls : Refining risk assessment for sudden cardiac death. Current Cardiology Reports. 21 (9), 106 (2019).
- Kirchhof, P., et al. 2016 ESC Guidelines for the management of atrial fibrillation developed in collaboration with EACTS. European Heart Journal. 37 (38), 2893-2962 (2016).
- Dobrev, D., Nattel, S. New antiarrhythmic drugs for treatment of atrial fibrillation. Lancet. 375 (9721), 1212-1223 (2010).
- Heijman, J., Voigt, N., Carlsson, L. G., Dobrev, D. Cardiac safety assays. Current opinion in pharmacology. 15, 16-21 (2014).
- Schüttler, D., et al. Animal models of atrial fibrillation. Circulation Research. 127 (1), 91-110 (2020).
- Clauss, S., et al. Animal models of arrhythmia: classic electrophysiology to genetically modified large animals. Nature Reviews. Cardiology. 16 (8), 457-475 (2019).
- Voigt, N., Pearman, C. M., Dobrev, D., Dibb, K. M. Methods for isolating atrial cells from large mammals and humans. Journal of Molecular and Cellular Cardiology. 86, 187-198 (2015).
- Jansen, H. J., Rose, R. A. Isolation of atrial myocytes from adult mice. Journal of Visualized Experiments. (149), e59588 (2019).
- Blackwood, E. A., Bilal, A. S., Azizi, K., Sarakki, A., Glembotski, C. C. Simultaneous isolation and culture of atrial myocytes, ventricular myocytes, and non-myocytes from an adult mouse heart. Journal of Visualized Experiments. (160), e61224 (2020).
- Omatsu-Kanbe, M., Yoshioka, K., Fukunaga, R., Sagawa, H., Matsuura, H. A simple antegrade perfusion method for isolating viable single cardiomyocytes from neonatal to aged mice. Physiological Report. 6 (9), 13688 (2018).
- Köhncke, C., et al. Isolation and Kv channel recordings in murine atrial and ventricular cardiomyocytes. Journal of Visualized Experiments. (73), e50145 (2013).
- Brandenburg, S., et al. Axial tubule junctions activate atrial Ca(2+) release across species. Frontiers in Physiology. 9, 1227 (2018).
- Hofhuis, J., et al. Dysferlin links excitation-contraction coupling to structure and maintenance of the cardiac transverse-axial tubule system. Europace. 22 (7), 1119-1131 (2020).
- Voigt, N., Zhou, X. B., Dobrev, D. Isolation of human atrial myocytes for simultaneous measurements of Ca2+ transients and membrane currents. Journal of Visualized Experiment. (77), e50235 (2013).
- Voigt, N., Makary, S., Nattel, S., Dobrev, D. Voltage-clamp-based methods for the detection of constitutively active acetylcholine-gated I(K,ACh) channels in the diseased heart. Methods in Enzymology. 484, 653-675 (2010).
- Voigt, N., Nattel, S., Dobrev, D. Proarrhythmic atrial calcium cycling in the diseased heart. Advances in Experimental Medicine and Biology. 740, 1175-1191 (2012).
- Trafford, A. W., Díaz, M. E., Eisner, D. A. A novel, rapid and reversible method to measure Ca buffering and time-course of total sarcoplasmic reticulum Ca content in cardiac ventricular myocytes. Pflugers Archiv: European Journal of Physiology. 437 (3), 501-503 (1999).
- Hamill, O. P., Marty, A., Neher, E., Sakmann, B., Sigworth, F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. European Journal of Physiology. 391 (2), 85-100 (1981).
- Voigt, N., et al. Enhanced sarcoplasmic reticulum Ca2+ leak and increased Na+-Ca2+ exchanger function underlie delayed afterdepolarizations in patients with chronic atrial fibrillation. Circulation. 125 (17), 2059-2070 (2012).
- Fakuade, F. E., et al. Altered atrial cytosolic calcium handling contributes to the development of postoperative atrial fibrillation. Cardiovascular Research. , (2020).
- Chen, W., Frangogiannis, N. G. The role of inflammatory and fibrogenic pathways in heart failure associated with aging. Heart Failure Reviews. 15 (5), 415-422 (2010).
- Plačkić, J., Kockskämper, J. Isolation of atrial and ventricular cardiomyocytes for in vitro studies. Methods in Molecular Biology. 1816, 39-54 (2018).
- Díaz, M. E., Trafford, A. W., Eisner, D. A. The effects of exogenous calcium buffers on the systolic calcium transient in rat ventricular myocytes. Biophysical Journal. 80 (4), 1915-1925 (2001).
- Zimmerman, A. N., Hülsmann, W. C. Paradoxical influence of calcium ions on the permeability of the cell membranes of the isolated rat heart. Nature. 211 (5049), 646-647 (1966).
- Chen, X., O’Connell, T. D., Xiang, Y. K. With or without Langendorff: A new method for adult myocyte isolation to be tested with time. Circulation Research. 119 (8), 888-890 (2016).
- Kappadan, V., et al. High-resolution optical measurement of cardiac restitution, contraction, and fibrillation dynamics in beating vs. blebbistatin-uncoupled isolated rabbit hearts. Frontiers in Physiology. 11, 464 (2020).
- Brack, K. E., Narang, R., Winter, J., Ng, G. A. The mechanical uncoupler blebbistatin is associated with significant electrophysiological effects in the isolated rabbit heart. Experiment in Physiology. 98 (5), 1009-1027 (2013).
- Seibertz, F., Reynolds, M., Voigt, N. Single-cell optical action potential measurement in human induced pluripotent stem cell-derived cardiomyocytes. Journal of Visual Experiment. , e61890 (2020).
Diesen Artikel zitieren
Tomsits, P., Schüttler, D., Kääb, S., Clauss, S., Voigt, N. Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current. J. Vis. Exp. (165), e61964, doi:10.3791/61964 (2020).