通过在小鼠心脏中应用光遗传学多位点光刺激进行高级心律管理
1Research Group Biomedical Physics,Max Planck Institute for Dynamics and Self-Organization, 2Research Electronics Department,Max Planck Institute for Dynamics and Self-Organization, 3Department of Pharmacology and Toxicology,University Medical Center Goettingen, 4Institute for Nonlinear Dynamics,Georg-August-University Goettingen, 5Department of Cardiology and Pneumology,University Medical Center Goettingen, 6German Center for Cardiovascular Research,DZHK e.V., partner site Goettingen, 7Laboratory Animal Science Unit,German Primate Center Leibniz Institute for Primate Research, 8Department of Microsystems Engineering (IMTEK),University of Freiburg, 9Cluster of Excellence BrainLinks-BrainTools,University of Freiburg
Summary
这项工作报告了一种使用micro-LED阵列的局部光刺激和同时光学映射心外膜电位来控制转基因通道视紫红质-2（ChR2）小鼠完整鼠心脏心律的方法。
Abstract
室性快速性心律失常是全世界死亡和发病的主要原因。使用高能电击的电除颤是目前危及生命的心室颤动的唯一治疗方法。然而，除颤可能有副作用，包括无法忍受的疼痛、组织损伤和预后恶化，这表明开发更温和的心律管理策略的医学需求很大。除了降低能量的电方法外，心脏光遗传学还被引入，作为使用光敏膜离子通道和光脉冲影响心脏活动的有力工具。在本研究中，将描述一种基于应用3 x 3微发光二极管（micro-LED）阵列的多位点起搏成功光刺激Langendorff灌注的完整鼠心脏的稳健有效的方法。心外膜电压波的同时光学映射允许研究区域特异性刺激的影响，并直接在现场评估新诱导的心脏活动。获得的结果表明，除颤的功效在很大程度上取决于心律失常期间为光刺激选择的参数。将证明心脏的照明区域对于终止成功以及如何在照明期间实现心脏活动的靶向控制以改变心律失常模式起着至关重要的作用。总之，该技术提供了一种优化现场机制操作的可能性，以实现心律的实时反馈控制，并且与使用非特异性电击应用相比，在区域特异性方面，减少了对心脏系统的潜在危害的新方法。
Introduction
对心律失常期间时空动力学的早期研究表明，心脏颤动期间的复杂电模式是由涡状旋转激励波驱动的1。这一发现为心律失常的潜在机制提供了新的见解，从而开发了基于心肌多位点激发的新型电终止疗法2，3，4。然而，使用电场刺激的治疗是非局部的，可能会支配周围所有可兴奋的细胞，包括肌肉组织，导致细胞和组织损伤，以及无法忍受的疼痛。与电疗法相比，光遗传学方法提供了一种特异性和组织保护技术，以高空间和时间精度唤起心肌细胞动作电位。因此，光遗传学刺激具有对心脏颤动期间混沌激活模式进行微创控制的潜力。
通过遗传操作将光敏离子通道通道视紫红质-2（ChR2）引入可兴奋细胞5，6，7，使得使用光刺激可兴奋细胞的膜电位去极化成为可能。已经开发了几种医学应用，包括激活神经元网络，控制心脏活动，恢复视力和听力，治疗脊髓损伤等8，9，10，11，12，13，14。由于其毫秒响应时间15，ChR2在心脏病学中的应用具有巨大的潜力，使其非常适合心律失常的心脏动力学的靶向控制。
在这项研究中，显示了转基因小鼠模型完整心脏的多位点光刺激。总之，在欧洲共同体第七框架计划FP7/2007-2013（HEALTH-F2-2009-241526）的范围内建立了转基因α-MHC-ChR2小鼠系，并由S. E. Lehnart教授慷慨提供。一般情况下，在α-MHC控制下表达Cre-重组酶的转基因成年雄性C57/B6/J与雌性 B6.Cg-Gt（ROSA）26Sortm27.1（CAG-COP4*H134R/tdTomato）Hye/J配对交配。由于心脏STOP盒在第二代中被删除，后代表现出稳定的MHC-ChR2表达，并用于维持心脏光敏集落。所有实验均在36-48周龄的两性成年小鼠中进行。照明是使用3 x 3微型LED阵列实现的，该阵列如16，17 中所述制造，只是未实现硅基外壳和短光学玻璃光纤。它在心脏应用中的首次使用是在18年。基于类似制造技术的线性微型LED阵列已被用作心脏起搏的穿透探针19。微型 LED 排列在 3 x 3 阵列中，间距为 550 μm，可在非常小的区域内提供高空间分辨率和高辐射功率。作者在这项工作中展示了一种多功能的局部多位点光刺激，这可能为开发新的抗心律失常治疗方法开辟道路。
以下实验方案涉及离体逆行朗根道夫灌注，其空心主动脉用作灌注入口。由于施加的灌注压和心脏收缩，灌注液流经冠状动脉，冠状动脉从主动脉分支出来。在所展示的工作中，使用恒压设置灌注心脏，通过将灌注储液罐提升到 1 m 高度（相当于 73.2 mmHg）来实现，从而产生 2.633 ± 0.583 mL/min 的流速。在实验过程中使用两种Tyrode溶液作为灌注剂。常规Tyrode溶液支持稳定的窦性心律，而Low-K + Tyrode溶液与Pinacidil混合，以诱导小鼠心脏的心律失常。使用六角形水浴允许通过六个不同的平面窗口观察心脏，允许耦合多个光学元件，通过折射引起的失真较小。
Protocol
所有实验都严格遵守动物福利法规，符合德国立法、当地规定，并符合欧洲实验动物科学协会联合会 （FELASA） 的建议。动物实验的批准申请已获得负责动物福利当局的批准，所有实验均已报告给我们的动物福利代表。 1. 实验准备和材料 光学映射设置注：光学设置以及电气设置如图 1所示。材料表中详细列出了光学和电气设置中使用的所有组?…
Representative Results
该协议允许使用LED 1和LED 2（图1）产生的光刺激脉冲诱导完整鼠心脏中的室性心律失常，频率f ind在25 Hz和35 Hz之间，脉冲持续时间Wind在2 ms和10 ms之间。请注意，这种快速光脉冲的目的不是捕捉心律，而是使心脏活动不平衡，从而产生不稳定的电波，从而促进心律失常。用光诱导心律失常比用电刺激诱导的优点是心电图中不会引起伪影，从而可以不受限制地对…
Discussion
成功治疗快速性心律失常是心脏治疗的关键。然而，心律失常发生、延续和终止的生物物理机制尚不完全清楚。因此，心脏研究旨在优化电击疗法，以更温和地终止心律失常，从而提高患者的生活质量28，29，30，31。低能量电方法有望显着减少严重的副作用，但仍可能诱发不必要的肌肉兴奋。心脏光?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢Marion Kunze和Tina Althaus在实验过程中的出色技术支持。导致结果的研究已获得欧洲共同体第七框架方案FP7/2007-2013的资助，资助协议号为HEALTH-F2-2009-241526。德国心血管研究中心、DZHK e.V.（MD28项目）、合作伙伴网站哥廷根、德国研究基金会CRC 1002（项目C03）和马克斯·普朗克学会也提供了支持。这项工作得到了德国研究基金会（DFG，批准号EXC 1086）资助的卓越集群BrainLinks-BrainTools的部分支持。
Materials
| Chemical Components | |||
| Blebbistatin | TargetMol | T6038 | 10 mM stock solution |
| BSA/Albumin | Sigma-Aldrich | A4919 | |
| Calcium Chloride | Sigma-Aldrich | C1016 | CaCl2 |
| Carbogen | Westfalen | 50 l bottle | |
| DI-4-ANBDQPQ | AAT Bioquest | 21499 | Dye for Optical Mapping |
| Glucose | Sigma-Aldrich | D9434 | C6H12O6 |
| Heparin | LEO Pharma | Heparin-Natrium Leo 25.000 I.E./5 ml, available only on prescription | |
| Hydrochlorid Acid | Merck | 1.09057.1000 | HCl, 1 M stock solution |
| Isoflurane | CP Pharma | 1 ml/ml, available only on prescription | |
| Magnesium Chloride | Merck | 8.14733.0500 | MgCl2 |
| Monopotassium Phosphate | Sigma-Aldrich | 30407 | KH2PO4 |
| Pinacidil monohydrate | Sigma-Aldrich | P154-500mg | 10 mM stock solution |
| Potassium Chloride | Sigma-Aldrich | P5405 | KCl |
| Sodium Bicarbonate | Sigma-Aldrich | S5761 | NaHCO3 |
| Sodium Chloride | Sigma-Aldrich | S5886 | NaCl |
| Sodium Hydroxide | Merck | 1.09137.1000 | NaOH, 1 M stock solution |
| Electrical Setup | |||
| Biopac MP150 | Biopac Systems | MP150WSW | data acquisition and analysis system |
| Custom-built ECG, alternative ECG100C | Biopac Systems | ECG100C | Electrocardiogram Amplifier |
| Custom-built water bath heater using heating cable | RMS Heating System | HK-5,0-12 | Heating cable 120W |
| Hexagonal water bath | |||
| LED Driver Power supply | Thorlabs | KPS101 | 15 V, 2.4 A Power Supply Unit with 3.5 mm Jack Connector for One K- or T-Cube. |
| LEDD1B LED Driver | Thorlabs | LEDD1B | T-Cube LED Driver, 1200 mA Max Drive Current |
| MAP, ECG Electrode | Hugo Sachs Elektronik | BS4 73-0200 | Mini-ECG Electrode for isoalted hearts |
| micro-LED Driver e.g. AFG | Agilent Instruments | A-2230 | Arbitrary function generator (AFG) |
| Signal Generator | Agilent Instruments | A-2230 | AFG |
| micro-LED Array Components | |||
| Epoxid glue | Epoxy Technology | EPO-TEK 353ND | Two component epoxy |
| Fluoropolymer | Asahi Glass Co. Ltd. | Cytop 809M | Fluoropolymer with high transparency |
| Image reversal photoresist | Merck KGaA | AZ 5214E | Image Reversal Resist for High Resolution |
| LED chip | Cree Inc. | C460TR2227-S2100 | Blue micro-LED |
| Photoresist | Merck KGaA | AZ 9260 | Thick Positive Photoresists |
| Polyimide | UBE Industries Ltd. | U-Varnish S | Polyimide Solution |
| Silicone | NuSil Technology LLC | MED-6215 | Low viscosity silicone elastomer |
| Solvent free adhesive | John P. Kummer GmbH | Epo-Tek 301-2 | Epoxy resin with low viscosity |
| Optical Mapping | |||
| Blue Filter | Chroma Technology Corporation | ET470/40x | Blue excitation filter |
| Camera | Photometrics | Cascade 128+ | High performance EMCCD Camera |
| Camera Objective | Navitar | DO-5095 | Navitar high speed fixed focal length lenses work with CCD and CMOS cameras |
| Dichroic Mirror | Semrock | FF685-Di02-25×36 | 685 nm edge BrightLine® single-edge standard epi-fluorescence dichroic beamsplitter |
| Emmision Filter | Semrock | FF01-775/140-25 | 775/140 nm BrightLine® single-band bandpass filter |
| Heatsink | Advanced Thermal Solutions | ATSEU-077A-C3-R0 | Heat Sinks – LED STAR LED Heatsink, 45mm dia., 68mm, Black/Silver, Unthreaded Baseplate Hardware |
| LED 1 and LED 2 | LED Engin Osram | LZ4-00B208 | High Power LEDs – Single Colour Blue, 460 nm 130 lm, 700mA |
| LED 3 | Thorlabs | M625L3 | 625 nm, 700 mW (Min) Mounted LED, 1000 mA |
| Lenses | LED Engin Osram | LLNF-2T06-H | LED Lighting Lenses Assemblies LZ4 LENS NARROW FLOOD BEAM |
| Photodiode for power meter | Thorlabs | S120VC | Standard Photodiode Power Sensor |
| Power Meter | Thorlabs | PM100D | Compact Power and Energy Meter |
| Red Filter | Semrock | FF02-628/40-25 | BrightLine® single-band bandpass filter |
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Cite This Article
Diaz-Maue, L., Steinebach, J., Schwaerzle, M., Luther, S., Ruther, P., Richter, C. Advanced Cardiac Rhythm Management by Applying Optogenetic Multi-Site Photostimulation in Murine Hearts. J. Vis. Exp. (174), e62335, doi:10.3791/62335 (2021).