代谢综合征心力衰竭模型大鼠心房心肌细胞的分离与保留射血分数的关系

Summary

在这里, 我们描述了一个优化的, Langendorff 的程序, 从一个大鼠模型的代谢综合征心力衰竭与保存的弹射分数分离的单细胞心房心肌细胞。对心脏腔腔内压进行人工调节, 以产生功能完整的心肌细胞, 适合于励磁收缩耦合研究。

Abstract

在本文中, 我们描述了一个优化的, Langendorff 的程序, 以隔离的大鼠代谢综合征 (飞机状态) 的模型, 与保存的射血分数 (HFpEF) 的一个人心肌细胞。随着心房重塑是死亡率的独立预测因子, 与心房重塑和心房颤动相关的心房心肌与 HFpEF 的发病率呈上升趋势。孤立单细胞心肌细胞的研究经常被用来证实和补充体内的发现。循环血管 rarefication 和间质组织纤维化是一个潜在的限制因素, 成功的单细胞分离飞机状态从动物模型的这种疾病。

我们通过使用一种能够在隔离过程中手动调节心脏腔腔内压力的装置来解决这个问题, 大大提高形态学和功能完整的飞机状态的产量。所获得的细胞可用于各种不同的实验, 如细胞培养和功能性钙成像 (即励磁收缩耦合)。

我们为研究员提供一步一步的协议, 一个优化的解决方案列表, 详细的说明准备必要的设备, 和全面的故障排除指南。虽然最初的实施程序可能是相当困难的, 一个成功的适应将使读者能够执行最先进的隔离在大鼠模型中与 HFpEF 有关的广泛的实验。

Introduction

大都会描述了糖尿病 type-2 和心血管疾病的一系列危险因素, 包括增加的动脉血压, 血脂异常 (升高甘油三酯和降低高密度脂蛋白胆固醇), 增加禁食葡萄糖和中央肥胖症¹。据估计, 世界大都会的流行率为 25–30%, 持续上升²。HFpEF 是一种异质临床综合征, 常与大都会相关。HFpEF 和前阶段 (即高血压性心脏病) 的心脏重塑也伴随着心房³的重塑。左心房的收缩功能和结构改变与死亡率增加、心房颤动和新发心力衰竭⁴有关。心房重塑的特点是离子通道功能的变化, 钙^{2 +}稳态, 心房结构, 成纤维细胞活化, 组织纤维化⁵。与 HFpEF 相关的左心房重塑及其潜在的病理机制尚不清楚, 需要进一步深入调查。动物模型已被证明是一个宝贵的工具, 并导致在心房心肌^6,7,8,9领域的许多进展。

孤立单细胞心肌细胞的研究经常被用来证实和补充体内的发现。一个孤立和潜在的后续细胞培养, 允许调查信号通路, 离子通道电流, 和励磁收缩耦合。在生理条件下, 心肌细胞不会增殖。在转基因小鼠中, 心房钠素转录调控序列与猿猴病毒40大 T 抗原的融合导致了第一个永生化的飞机状态, 命名为 AT-1¹⁰。AT-1 细胞的进一步发育引起了 HL-1 细胞的形成, 它不仅可以连续传代, 而且能自发收缩¹¹。然而, 与新近分离的细胞相比, 它们表现出结构和功能上的差异, 如组织超微结构、发育肌原纤维¹¹的高发生率和极化活化的内电流¹²。大鼠和小鼠心室心肌细胞 (VCM) 的分离是建立在^{13、14、15、16、17、18,19}. 一般情况下, 切除心脏被安装到一个 Langendorff 设备和 retrogradely 灌注与一个 Ca^{2 +}自由缓冲含有消化酶, 如胶原酶和蛋白酶。然后以逐步的方式将钙重新引入到生理条件中。然而, 即使专门用于隔离飞机状态的协议^20、21, 由于纤维化和压力相关的差异, 它们在心房重塑的疾病模型中的效用是有限的。

在这篇文章中, 我们已经实施了一个协议, 以隔离的动物, 显示心房重塑 (特别是为 ZFS1大鼠模型 HFpEF)²²的心房单细胞心肌细胞。现有的隔离协议得到了优化, 并辅以一个简单的, 定制的装置来控制和修改心脏腔腔内压力, 导致更高的形态和功能完整的心肌细胞的产量。下面的协议为研究员提供了一步一步的指南, 详细描述了定制的设备, 一个解决方案列表, 以及一个全面的故障排除指南。

Protocol

所有实验均经当地伦理委员会 (TVA T0060/15 和 T0003-15) 批准, 并与《关于护理和使用实验动物指南》 (美国国立卫生研究院) 达成一致。 注意: 过程的简化流程图如图 1所示。 1. Prearrangements 根据表1准备缓冲区。 解…

Representative Results

在21周的年龄, 60–90% 的可行飞机状态 (估计如步骤6.1 所述), 在钙重新适应 (步骤 5.4–5.7), 可以从 ZSF-1 肥胖大鼠的这种方法 (图 4A)。在大鼠, 飞机状态的特点是一个不同的和更异质表型相比, VCMs24,25。图 4B显示了一个人与保存的膜和小节结构, 两个功能完整的细胞的强指标。 <p class="…

Discussion

在这里, 我们首先描述了一个协议, 从大鼠模型中分离出的单细胞飞机状态, 显示显着心房重塑²²。这一过程具有独特的挑战性, 因为过量的脂肪组织可以使手术准备, 以及主动脉插管, 越来越困难。表 2中提供的故障排除指南解决了隔离过程中最常见的问题。

问题

… Disclosures The authors have nothing to disclose. Acknowledgements 这项研究得到了 DZHK (德国心血管研究中心)、EKFS (Kröner 费森尤斯基金会、卡尔亨利) 和 BMBF (德国教育部和研究部) 的支持, 以及波黑-Charité临床科学家项目资助由 Charité-Universitätsmedizin 柏林和柏林健康学院 (卡尔亨利)。 Materials ZSF-1 Obese rat Charles River Laboratories, Inc. 21 weeks old Fine Iris Scissors Fine Science Tools GmbH 14094-11 Surgical Scissors Fine Science Tools GmbH 14001-18 Micro Dressing Forceps (curved, serrated) Aesculap, Inc. BD312R Tissue Forceps (straight, 1 x 2 teeth) Aesculap, Inc. BD537R Tying Forceps (angled) Aesculap, Inc. MA624R Rodent and Small Animal Guillotine Kent Scientific Corp. DCAP Low Cost Induction Chamber 3.0 L Kent Scientific Corp. SOMNO-0730  Butterfly Winged Infusion Set 21 G Hospira, Inc. 181106101 Abbocath 16 G Hospira, Inc. 0G7149702 Microlance Hypodermic Needle Becton Dickinson GmbH 301300 modify needle to make cannula Braun Original Perfusor Syringe 50 ml B. Braun Melsungen AG 8728810F Braun Inject Solo Syringe 10 ml B. Braun Melsungen AG 2057926 Beaker 50ml Duran Group (DWK Life Sciences GmbH) 21 106 17 Duroplan petri dish (100 x 20 mm) Duran Group (DWK Life Sciences GmbH) 21 755 48 Seraflex Suture USP 3/0 SERAG-WIESSNER GmbH & Co. KG IC208000 VWR disposable Square Weighin Boats 100ml VWR, Inc. 10803-148 Styrofoam surface Sodium chloride Sigma-Aldrich, Inc. 71380 Potassium chloride Sigma-Aldrich, Inc. P4504 Potassium phosphate monobasic Sigma-Aldrich, Inc. P5379 Sodium phosphate dibasic Sigma-Aldrich, Inc. S0876 Magensium sulfate heptahydrate Sigma-Aldrich, Inc. 230391 Magensium chloride Sigma-Aldrich, Inc. M8266 HEPES Sigma-Aldrich, Inc. H3375 Taurine Sigma-Aldrich, Inc. T0625 Glucose Sigma-Aldrich, Inc. G7528 2,3-Butanedione monoxime Sigma-Aldrich, Inc. B0753 Calcium chloride solution (1 M) Sigma-Aldrich, Inc. 21115 Bovine Serum Albumin Sigma-Aldrich, Inc. A9647 Liberase Roche (Sigma-Aldrich, Inc.) LIBTM-RO Heparin Rotexmedica GmbH 3862357 Forene (Isoflurane) Abbvie Deutschland GmbH & Co. KG 10182054 Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane Sigma-Aldrich, Inc. L2020 WillCo glass-bottom dish 500µl 0.005mm WillCo Wells B.V. HBST-3522 Fluo4 AM Invitrogen (Thermo Fisher Scientific, Inc.) F14201 5µM for 20min at RT Di-8-ANNEPS Invitrogen (Thermo Fisher Scientific, Inc.) D3167 10µM for 45 min at 37° C  Mitotracker RED FM Invitrogen (Thermo Fisher Scientific, Inc.) M22425 20nM for 30 min at 37° C Jacketed reaction vessel 500 ml Gebr. Rettberg GmbH 107024414 Jacketed reaction vessel 1000 ml Gebr. Rettberg GmbH 107025414 Jacketed bubble trap Gebr. Rettberg GmbH 134720001 ED heating immersion circulator Julabo GmbH 9116000 Reglo Digital MS-2/6 peristaltic pump Ismatec (Cole-Parmer Gmbh) ISM 831 Voltcraft Thermometer 302 K/J Conrad Electronic SE 030300546 Tubing LSM 700 microscope Carl Zeiss, Inc. ZEN 2.3 imaging software Carl Zeiss, Inc. 410135-1011-240  Single channel heater controller TC-324B Warner Instruments, LLC 64-2400 8 channel perfusion system Warner Instruments, LLC 64-0185 8 channel Multi-Line In-Line Solution Heaters Warner Instruments, LLC 64-0105 References Alberti, K. G., et al. Harmonizing the metabolic syndrome: a joint interim statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity. Circulation. 120 (16), 1640-1645 (2009). . IDF Consensus Worldwide Definition of the Metabolic Syndrome Available from: https://www.idf.org/e-library/consensus-statements/60-idfconsensus-worldwide-definitionof-the-metabolic-syndrome.html (2006) Melenovsky, V., et al. 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A., Wolska, B. M. Methods in cardiomyocyte isolation, culture, and gene transfer. Journal of Molecular and Cellular Cardiology. 51 (3), 288-298 (2011). Gunduz, D., Hamm, C. W., Aslam, M. Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart. Journal of Visualized Experiments. (123), e55601 (2017). 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. (87), e51357 (2014). Graham, E. L., et al. Isolation, culture, and functional characterization of adult mouse cardiomyoctyes. Journal of Visualized Experiments. (79), e50289 (2013). Roth, G. M., Bader, D. M., Pfaltzgraff, E. R. Isolation and physiological analysis of mouse cardiomyocytes. Journal of Visualized Experiments. (91), e51109 (2014). Thum, T., Borlak, J. 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Netherlands Heart Journal. 24 (4), 275-286 (2016). Horgan, S., Watson, C., Glezeva, N., Baugh, J. Murine models of diastolic dysfunction and heart failure with preserved ejection fraction. Journal of Cardiac Failure. 20 (12), 984-995 (2014). Tags Atrial CardiomyocytesRat ModelMetabolic SyndromeHeart FailurePreserved Ejection FractionLangendorff ApparatusCannulationCardiomyocyte IsolationExcitation-contraction-coupling Play Video PDF DOI Cite This Article Bode, D., Guthof, T., Pieske, B. M., Heinzel, F. R., Hohendanner, F. Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction. J. Vis. Exp. (137), e57953, doi:10.3791/57953 (2018). 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Materials

ZSF-1 Obese rat	Charles River Laboratories, Inc.		21 weeks old
Fine Iris Scissors	Fine Science Tools GmbH	14094-11
Surgical Scissors	Fine Science Tools GmbH	14001-18
Micro Dressing Forceps (curved, serrated)	Aesculap, Inc.	BD312R
Tissue Forceps (straight, 1 x 2 teeth)	Aesculap, Inc.	BD537R
Tying Forceps (angled)	Aesculap, Inc.	MA624R
Rodent and Small Animal Guillotine	Kent Scientific Corp.	DCAP
Low Cost Induction Chamber 3.0 L	Kent Scientific Corp.	SOMNO-0730
Butterfly Winged Infusion Set 21 G	Hospira, Inc.	181106101
Abbocath 16 G	Hospira, Inc.	0G7149702
Microlance Hypodermic Needle	Becton Dickinson GmbH	301300	modify needle to make cannula
Braun Original Perfusor Syringe 50 ml	B. Braun Melsungen AG	8728810F
Braun Inject Solo Syringe 10 ml	B. Braun Melsungen AG	2057926
Beaker 50ml	Duran Group (DWK Life Sciences GmbH)	21 106 17
Duroplan petri dish (100 x 20 mm)	Duran Group (DWK Life Sciences GmbH)	21 755 48
Seraflex Suture USP 3/0	SERAG-WIESSNER GmbH & Co. KG	IC208000
VWR disposable Square Weighin Boats 100ml	VWR, Inc.	10803-148
Styrofoam surface
Sodium chloride	Sigma-Aldrich, Inc.	71380
Potassium chloride	Sigma-Aldrich, Inc.	P4504
Potassium phosphate monobasic	Sigma-Aldrich, Inc.	P5379
Sodium phosphate dibasic	Sigma-Aldrich, Inc.	S0876
Magensium sulfate heptahydrate	Sigma-Aldrich, Inc.	230391
Magensium chloride	Sigma-Aldrich, Inc.	M8266
HEPES	Sigma-Aldrich, Inc.	H3375
Taurine	Sigma-Aldrich, Inc.	T0625
Glucose	Sigma-Aldrich, Inc.	G7528
2,3-Butanedione monoxime	Sigma-Aldrich, Inc.	B0753
Calcium chloride solution (1 M)	Sigma-Aldrich, Inc.	21115
Bovine Serum Albumin	Sigma-Aldrich, Inc.	A9647
Liberase	Roche (Sigma-Aldrich, Inc.)	LIBTM-RO
Heparin	Rotexmedica GmbH	3862357
Forene (Isoflurane)	Abbvie Deutschland GmbH & Co. KG	10182054
Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane	Sigma-Aldrich, Inc.	L2020
WillCo glass-bottom dish 500µl 0.005mm	WillCo Wells B.V.	HBST-3522
Fluo4 AM	Invitrogen (Thermo Fisher Scientific, Inc.)	F14201	5µM for 20min at RT
Di-8-ANNEPS	Invitrogen (Thermo Fisher Scientific, Inc.)	D3167	10µM for 45 min at 37° C
Mitotracker RED FM	Invitrogen (Thermo Fisher Scientific, Inc.)	M22425	20nM for 30 min at 37° C
Jacketed reaction vessel 500 ml	Gebr. Rettberg GmbH	107024414
Jacketed reaction vessel 1000 ml	Gebr. Rettberg GmbH	107025414
Jacketed bubble trap	Gebr. Rettberg GmbH	134720001
ED heating immersion circulator	Julabo GmbH	9116000
Reglo Digital MS-2/6 peristaltic pump	Ismatec (Cole-Parmer Gmbh)	ISM 831
Voltcraft Thermometer 302 K/J	Conrad Electronic SE	030300546
Tubing
LSM 700 microscope	Carl Zeiss, Inc.
ZEN 2.3 imaging software	Carl Zeiss, Inc.	410135-1011-240
Single channel heater controller TC-324B	Warner Instruments, LLC	64-2400
8 channel perfusion system	Warner Instruments, LLC	64-0185
8 channel Multi-Line In-Line Solution Heaters	Warner Instruments, LLC	64-0105