隔离与人类心室肌细胞的功能研究从新鲜手术样品
Summary
心脏疾病的细胞基础上的现有知识大多依赖于对动物模型的研究。在这里，我们描述和验证了一种新的方法从人心肌小型手术样品得到单一可行的心肌细胞。人心肌细胞可用于电生理学研究和药物测试。
Abstract
从患病心脏的心肌细胞受到涉及改变细胞结构，激发收缩偶联和膜离子流复杂重塑过程。这些变化很可能是负责增加心律失常的风险和收缩变化导致的收缩和舒张功能不全的心脏病患者。然而，在心脏疾病心肌细胞功能的改变大部分信息都来自动物模型。
在这里，我们描述和验证协议从心室肌从接受心脏外科手术的病人手术小样本分离出可行的心肌细胞。的协议进行详细说明。电生理学和细胞内钙的测量报告来演示了一些在用这种方法获得的人左心室心肌细胞的单细胞测量的可行性。
该协议的报道，他再可用于功能性改变人的心脏在不同心脏疾病的存在的细胞和分子基础的未来的调查非常有用。另外，该方法可用于鉴定在细胞水平上新的治疗靶点，并测试新化合物的效力对人心肌细胞，用直接平移值。
Introduction
的心肌的电生理特性解剖技术的发展为单个心肌细胞分离后已取得进展显着。在心肌兴奋收缩偶联（EC-耦合）的认识的最新发展，也已成为可能，可行的隔离单个心肌细胞可在保留完整的组织的所有生理特性的能力。膜片钳方法被常规使用，研究心肌细胞膜离子电流的功能和药理学调制。细胞内钙动力学与钙离子敏感染料的录音也定期进行单心肌细胞从不同的健康和疾病模型，提供对EC-耦合的生理以及对病理改变细胞内Ca 2 +的重要数据动态平衡，导致机械损伤和心脏疾病的增加心律失常的负担。信息tion从这些研究对于了解药物在临床上的电和机械作用的关键。但是，也有物种的跨膜电流和在该占心脏动作电位和心脏力学的具体特征对EC-偶联蛋白特异性差异。因此，当细胞从非人类哺乳动物中分离的研究已阐明的生物物理属性和特定的跨膜离子通道和EC-偶联蛋白的生理功能，它们不一定提供人类心肌细胞的相关模型。因此，从人类心肌存活心肌细胞的隔离是必要的，充分了解心脏疾病的病理生理学和验证新的治疗方法。
人类心房组织是现成的作为心耳在手术过程中通常被丢弃。成人人心肌动作电位和离子立方米初步定量研究rrents采用酶解分离心房细胞1-4。动作电位或从孤立的成人心室肌细胞电流记录已随后报告3,5-10。大部分这些研究使用了从外植心脏获得和利用胶原酶或冠状动脉段或数量较大的切除的组织暴露于胶原酶，得到分离的细胞的灌注细胞。这些研究使一些来自健康人的心脏心室肌细胞，并从患者的终端心脏衰竭跨膜离子电流的详细特征。 L型的录音的Ca 2 +电流（I CA-L）5-7，瞬时外向钾电流（I 至 ）8，内向整流钾电流（Iκ1）8，延迟整流钾电流的不同组成部分（我κ ）9已有报道。进步和精炼分离过程10，允许在终端心脏衰竭，包括动作电位延长11增加的潜在致心律失常的离子基础的明确表征，去极化12后延迟并增加有趣电流13到舒张期去极化和早搏。
成年心肌细胞通常是从小型动物的整个心脏与各种酶的混合物，一种能够产生的Ca 2 +的容错电池14的高产量技术的逆行灌注隔离。从组织碎片心肌细胞的分离是可能是因为酶的有限访问单个肌细胞与由冠状动脉灌注取得了比较本来就不太成功。由于未使用捐助者的心非常有限的，唯一可行的方法，以获得正常的人心肌细胞定期是通过酶digestio不适用不择期外科手术的切除往往非常小的组织碎片。已经彻底的特点在细胞水平上的唯一人类疾病模型是终端心脏衰竭，因无障碍移植后的心脏。然而，终端心脏衰竭发生在少数患者并且经常涉及心肌细胞的严重重塑，这是相对独立的根本原因15的共同通路。以评估患者的单个心肌细胞的功能在疾病的早期非故障段的能力是至关重要的理解不同遗传或后天条件的特定病理生理学。肥厚型心肌病（HCM）是一个生动的例子。 HCM是一种常见的（1/500的个体）的特征在于心脏肥大可继承的心脏状况，由于流出道梗阻和舒张功能障碍16增加心律失常风险和收缩变化。从HCM心心肌ündergo参与细胞结构的变化（肥大，肌原纤维混乱）和EC-17耦合复杂的重塑过程。然而，在胡志明市心肌功能障碍多数信息都来自转基因动物模型。由于HCM患者，只有少数演变对终端心脏衰竭，需要心脏移植，HCM的心中都很少适用于细胞的分离与标准方法。然而，HCM患者中至少有30％的收缩（HCM）18发展过程中由于大量室间隔肥厚改变流出道血流梗阻症状。最有效的可用的治疗选择梗阻的HCM的救济是手术室间隔心肌切除术：这种手术过程中，上部隔一个大小可变的部分是由反式主动脉瓣的方法去除。肥大隔膜的这一部分，因此可用于从新鲜组织细胞的分离。
一种用于人类ventricula的分离方法r是单一的，小静脉心内膜心肌活检标本细胞先前已经制定和公布19。我们实现了一个方法，从接受心脏手术，包括HCM患者接受室间隔心肌切除术和心脏瓣膜置换手术的患者患者分离单个心肌细胞室间隔从心室心肌标本。除了 隔离协议，有代表性的电生理学和Ca 2 +的荧光的详细描述测量呈现，显示了分离的人心肌细胞的生存能力和膜片钳和细胞内Ca 2 +的研究的可行性。
Protocol
Representative Results
Discussion
我们已经描述和验证的方法从人心肌的手术标本分离出可行的心肌细胞。从前面描述的协议已被成功地用于分离的细胞从心房手术样品，该技术允许从患病心室肌单个活细胞分离的开发和微调开始。早期的报道表明，从心房和心室组织块选择性受损复极钾电流，导致改变的电生理特性和响应于生理刺激8,24，单个心肌细胞的分离，而输送通过冠状动脉灌注含有缓冲液的酶没有损害延迟整流?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项工作是由欧盟（STREP项目241577“大心脏”，第七届欧洲框架计划，CP），梅纳里尼国际业务卢森堡（AM），马拉松式节目GGP07133（CP）和Gilead Sciences公司（AM）的支持。
Materials
| Potassium phosphate monobasic (KH2PO4) | Sigma-Aldrich | P9791 | |
| Magnesium sulfate heptahydrate(MgSO4 * 7H2O) | Sigma-Aldrich | M1880 | |
| HEPES | Sigma-Aldrich | H3375 | |
| Adenosine | Sigma-Aldrich | A9251 | |
| D-(+)-Glucose | Sigma-Aldrich | G8270 | |
| Mannitol | Sigma-Aldrich | M4125 | |
| Taurine | Sigma-Aldrich | T0625 | |
| Potassium hydroxide (KOH) | Sigma-Aldrich | P5958 | |
| Sodium chloride (NaCl) | Sigma-Aldrich | S7653 | |
| Potassium chloride (KCl) | Sigma-Aldrich | P9333 | |
| Sodium phosphate dibasic (Na2HPO4) | Sigma-Aldrich | S7907 | |
| Sodium bicarbonate (NaHCO3) | Sigma-Aldrich | S6297 | |
| Potassium bicarbonate (KHCO3) | Sigma-Aldrich | 237205 | |
| Sodium pyruvate | Sigma-Aldrich | P2256 | |
| 2,3-Butanedione monoxime | Sigma-Aldrich | B0753 | |
| Sodium hydroxide(NaOH) | Sigma-Aldrich | S8045 | |
| L-Glutamic acid monopotassium salt monohydrate | Sigma-Aldrich | 49601 | |
| Pyruvic acid | Sigma-Aldrich | 107360 | |
| 3-Hydroxybutyric acid | Sigma-Aldrich | 166898 | |
| Adenosine 5′-triphosphate dipotassium salt dihydrate (K2-ATP) | Sigma-Aldrich | A8937 | |
| Creatine | Sigma-Aldrich | C0780 | |
| Succinic Acid | Sigma-Aldrich | S3674 | |
| Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) | Sigma-Aldrich | E0396 | |
| Albumin from bovine serum | Sigma-Aldrich | A0281 | |
| Magnesium chloride (MgCl2) | Sigma-Aldrich | M8266 | |
| Collagenase from Clostridium histolyticum, Type V | Sigma-Aldrich | C9263 | |
| Proteinase, Bacterial, Type XXIV | Sigma-Aldrich | P8038 | |
| Calcium chloride solution, ~1 M in H2O | Sigma-Aldrich | 21115 | |
| Calcium chloride 0.1 M solution | Sigma-Aldrich | 53704 | |
| Potassium methanesulfonate | Sigma-Aldrich | 83000 | |
| FluoForte Reagent | Enzo Life Sciences | ENZ-52015 | |
| Powerload concentrate, 100X | Life Technologies | P10020 | |
| Perfusion Fast-Step System | Warner Instruments | VC-77SP | |
| Amphotericin B solubilized | Sigma-Aldrich | A9528 | |
| Multiclamp 700B patch-clamp amplifier | Molecular Devices | ||
| Digidata 1440A | Molecular Devices | ||
| pClamp10.0 | Molecular Devices | ||
| Digestion Device | CUSTOM | CUSTOM | The device is custome made in our laboratory using plastic tubes, cast Sylgard and a motor; it is described in detail in Fig 1 C-D and in Fig.7. We can provide further details if requested |
| Silicone elastomer for the digestion device's brushes | Dow Corning | SYLGARD® 184 | |
| Variable speed rotating motor for the digestion device | Crouzet | Crouzet 178-4765 | |
| Mold for brushes casting | N.A. | N.A. | The mold is custom made from standard PTFE 2.5 cm diameter rods |
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