成年小鼠和大鼠心肌细胞的隔离、转染和长期培养
Summary
在这里，我们提出了成人小鼠和大鼠心肌细胞的分离、转染和长期培养的协议。
Abstract
成年哺乳动物心肌细胞（CMs）的外体培养是心脏生物学体外研究最相关的实验系统。成年哺乳动物CMs是端端分化的细胞，繁殖能力最小。成人CMs的后位位状态不仅限制了心肌细胞周期的进展，也限制了CMs的高效培养。此外，成人CM的长期培养对于许多研究是必要的，如CM增殖和基因表达分析。
老鼠和老鼠是两个最喜欢用于心肌细胞分离的实验室动物。虽然大鼠CMs的长期培养是可能的，但成年小鼠CMs很容易死亡，在正常情况下不能培养超过五天。因此，迫切需要优化成人 murine CMs 的细胞隔离和长期培养协议。通过此修改后的协议，可以成功地分离和培养成年小鼠和大鼠的 VM 超过 20 天。此外，与之前的报告相比，分离CM的siRNA转染效率显著提高。对于成年小鼠CM分离，Langendorf灌注方法采用最佳酶溶液和足够的时间进行完整的细胞外基质分离。为了获得纯心室的CMs，在进行分离和电镀之前，对两个腹膜进行解剖和丢弃。细胞分散在层压涂层板上，从而能够高效、快速地连接。在SIRNA转染之前，CMs被允许在4-6小时内安顿下来。培养基每24小时刷新20天，随后，CMs被固定和染色的心脏特异性标记，如肌龙宁和细胞周期标记，如KI67。
Introduction
心脏病是全世界主要死亡原因之一。几乎所有类型的心脏损伤都会导致成人心肌细胞 （CMs） 的显著损失。由于成人CM1的衰老性质，成年哺乳动物的心脏无法修复其心脏损伤。因此，任何对成年哺乳动物心脏的侮辱都会导致CMs的永久丧失，导致心脏功能下降和心力衰竭。与成年哺乳动物不同，斑马鱼和纽特心脏等小动物可以通过现有的CM增殖2、3、43再生心脏损伤。2正在全球范围内努力通过增殖和非增殖方法寻找一种新的治疗性治疗性治疗性治疗性治疗手段。在过去的几十年里，已经开发出各种基因小鼠模型来研究心脏损伤和修复。然而，在体内使用动物模型仍然是一种昂贵的方法，从二次效应中破译细胞自主机制的复杂性更高。此外，体内系统很难分析从CM诱导心脏保护信号的药理干预的CM特异性效果。
此外，成人CM的长期文化对于进行CM增殖分析是必要的。CM增殖检测需要至少4-5天细胞被诱导到细胞周期，并在此之后获得准确的数据。此外，利用分离的CMs进行电生理学研究、药物筛选、毒性研究和Ca+平衡研究的研究都需要改进培养系统5、6、7。,6,7此外，最近的研究表明，从CMs（心因子）8，9中分泌的细胞因子具有心脏保护意义。为了研究这些心因子在心脏修复和再生过程中的治疗作用和分子机制，需要长期培养。
成年大鼠CMs在体外系统10、11、12中，足够坚固，可以进行单细胞分离和长期培养。然而，成人小鼠CM是极大的兴趣的体外测定，由于各种转基因小鼠模型的可用性，这允许设计和执行各种创新分析，这是不可能与大鼠CM13。与成年大鼠CM分离相比，从成年小鼠心脏获得单细胞悬浮是相当具有挑战性的，成人小鼠CM在培养中的长期培养更具挑战性。
成人CM隔离从老鼠和老鼠的心脏使用兰根多夫系统已经建立，以研究CM功能5,5，14，15。,15在这里，我们详细介绍了成人CM从大鼠和小鼠分离的协议，以及经过修改的长期培养，转染和CM增殖的分离细胞。
Protocol
Representative Results
Discussion
迫切需要建立成人心肌细胞分离和长期培养的规程，以进行细胞特定的机械研究。只有少数报告讨论成人CM隔离协议，甚至更少用于成人小鼠CM15，16，17,16,的长期培养。研究表明，成年大鼠CM对体外培养的耐受性比成年大鼠CM10、11、12,11,高。在这份报告中，?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了辛辛那提大学医学院病理学和实验室医学系对奥努尔·卡尼西卡克博士的资助;国家卫生研究院（R01HL148598）向奥努尔·卡尼西卡克博士提供赠款。奥努尔·卡尼西卡克博士获得美国心脏协会职业发展奖（18CDA34110117）的支持。Perwez Alam博士得到美国心脏协会博士后资助（AHA_20POST35200267）。Malina J. Ivey 博士得到 NIH T32 赠款 （HL 125204-06A1） 的支持。
Materials
| 2,3-Butane Dione monoxime | Sigma-Aldrich | B-0753 | |
| Blebbistatin | APExBIO | B1387 | |
| Bovine serum albumin | Sigma-Aldrich | A3059 | |
| CaCl2 | Sigma-Aldrich | 449709 | |
| Cell culture plate | Corning Costar | 3526 | |
| Cell strainer | BD Biosciences | 352360 | |
| Cel-miR-67 | Dharmacon | CN-001000-01-50 | |
| Collagenase type2 | Worthington | LS004177 | |
| Disposable Graduated Transfer Pipettes | Fisherbrand | 13-711-20 | |
| Disposable polystyrene weighing dishes | Sigma-Aldrich | Z154881-500EA | |
| Dulbecco's Modified Eagle's medium | Thermo Scientific | SH30022.01 | |
| EdU | Life Technologies | C10337 | |
| Fetal bovine serum | Corning | 35-015-CV | |
| Fine Point High Precision Forceps | Fisherbrand | 22-327379 | |
| Glucose | Sigma-Aldrich | G-5400 | |
| Hemocytometer | Hausser Scientific | 1483 | |
| Heparin | Sagent Pharmaceuticals | PSLAB-018285-02 | |
| HEPES | Sigma-Aldrich | H3375 | |
| High Precision Straight Broad Strong Point Tweezers/Forceps | Fisherbrand | 12-000-128 | |
| Hyaluronidase | Sigma | H3506 | |
| Insulin | Sigma-Aldrich | I0516-5ML | |
| K2HPO4 | Sigma-Aldrich | P-8281 | |
| KCl | Sigma-Aldrich | 746436 | |
| Light Microscope | Nikon | ||
| Lipofectamine RNAiMAX | Life Technologies | 13778-150 | |
| MgSO4 | Sigma-Aldrich | M-2643 | |
| NaCl | Sigma-Aldrich | S9888 | |
| NaOH | Fisher Scientific | S318-500 | |
| Natural Mouse Laminin | Invitrogen | 23017-015 | |
| Penicillin/Streptomycin | Corning | 30-002-CI | |
| Pentobarbital | Henry Schein | 24352 | |
| Phosphate buffered saline | Life Technologies | 20012-027 | |
| Protease XIV | Sigma-Aldrich | P5147-1G | |
| Selenium | Sigma-Aldrich | 229865+5G | |
| siMeis2 | Dharmacon | s161030 | |
| siRb1 | Dharmacon | s128325 | |
| Straight Blunt/SharpDissecting Scissors | Fisher Scientific | 28252 | |
| Straight Very Fine Precision Tip Forceps | Fisherbrand | 16-100-120 | |
| Taurine | Sigma-Aldrich | T0625 | |
| Transferrin | Sigma-Aldrich | T8158-100MG | |
| Ultra-smooth, beveled-edge finish scissor | Fisherbrand | 22-079-747 | |
| Water Bath | Fisher Scientific | 3006S |
References
- van Amerongen, M. J., Engel, F. B. Features of cardiomyocyte proliferation and its potential for cardiac regeneration. Journal of Cellular and Molecular Medicine. 12, 2233-2244 (2008).
- Parente, V., et al. Hypoxia/reoxygenation cardiac injury and regeneration in zebrafish adult heart. PLoS One. 8, 53748 (2013).
- Wang, J., et al. The regenerative capacity of zebrafish reverses cardiac failure caused by genetic cardiomyocyte depletion. Development. 138, 3421-3430 (2011).
- Gonzalez-Rosa, J. M., Martin, V., Peralta, M., Torres, M., Mercader, N. Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish. Development. 138, 1663-1674 (2011).
- Graham, E. L., et al. Isolation, culture, and functional characterization of adult mouse cardiomyoctyes. Journal of Visualized Experiments. , e50289 (2013).
- Brette, F., Orchard, C. T-tubule function in mammalian cardiac myocytes. Circulation Research. 92, 1182-1192 (2003).
- Müller, J. G., et al. Differential regulation of the cardiac sodium calcium exchanger promoter in adult and neonatal cardiomyocytes by Nkx2.5 and serum response factor. Journal of Molecular and Cellular Cardiology. 34, 807-821 (2002).
- Zhou, H., et al. Exosomes in ischemic heart disease: novel carriers for bioinformation. Biomedicine & Pharmacotherapy. 120, 109451 (2019).
- Wu, Y. S., Zhu, B., Luo, A. L., Yang, L., Yang, C. The Role of Cardiokines in Heart Diseases: Beneficial or Detrimental. BioMed Research International. 2018, 8207058 (2018).
- Eppenberger, H. M., Hertig, C., Eppenberger-Eberhardt, M. Adult rat cardiomyocytes in culture A model system to study the plasticity of the differentiated cardiac phenotype at the molecular and cellular levels. Trends in Cardiovascular Medicine. 4, 187-193 (1994).
- Alam, P., et al. Inhibition of Senescence-Associated Genes Rb1 and Meis2 in Adult Cardiomyocytes Results in Cell Cycle Reentry and Cardiac Repair Post-Myocardial Infarction. Journal of the American Heart Association. 8, 012089 (2019).
- Arif, M., et al. MicroRNA-210-mediated proliferation, survival, and angiogenesis promote cardiac repair post myocardial infarction in rodents. Journal of Molecular Medicine. 95, 1369-1385 (2017).
- Rosenthal, N., Brown, S. The mouse ascending: perspectives for human-disease models. Nature Cell Biology. 9, 993-999 (2007).
- Nippert, F., Schreckenberg, R., Schlüter, K. D. Isolation and Cultivation of Adult Rat Cardiomyocytes. Journal of Visualized Experiments. , e56634 (2017).
- Judd, J., Lovas, J., Huang, G. N. Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes. Journal of Visualized Experiments. , e54012 (2016).
- Ackers-Johnson, M., et al. A Simplified, Langendorff-Free Method for Concomitant Isolation of Viable Cardiac Myocytes and Nonmyocytes From the Adult Mouse Heart. Circulation Research. 119, 909-920 (2016).
- Li, D., Wu, J., Bai, Y., Zhao, X., Liu, L. Isolation and culture of adult mouse cardiomyocytes for cell signaling and in vitro cardiac hypertrophy. Journal of Visualized Experiments. , e51357 (2014).
- Pinz, I., Zhu, M., Mende, U., Ingwall, J. S. An improved isolation procedure for adult mouse cardiomyocytes. Cell Biochemistry and Biophysics. 61, 93-101 (2011).
- O’Connell, T. D., Rodrigo, M. C., Simpson, P. C. Isolation and culture of adult mouse cardiac myocytes. Methods in Molecular Biology. 357, 271-296 (2007).
- Dou, Y., Arlock, P., Arner, A. Blebbistatin specifically inhibits actin-myosin interaction in mouse cardiac muscle. American Journal of Physiology: Cell Physiology. 293, 1148-1153 (2007).
- Kabaeva, Z., Zhao, M., Michele, D. E. Blebbistatin extends culture life of adult mouse cardiac myocytes and allows efficient and stable transgene expression. American Journal of Physiology: Heart and Circulatory Physiology. 294, 1667-1674 (2008).
- Sellin, L. C., McArdle, J. J. Multiple effects of 2,3-butanedione monoxime. Pharmacology & Toxicology. 74, 305-313 (1994).
