Electrophysiological Properties and Viability of Neonatal Rat Ventricular Myocyte Cultures with Inducible ChR2 Expression.
Electrophysiological Properties and Viability of Neonatal Rat Ventricular Myocyte Cultures with Inducible ChR2 Expression.
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DOI：
10.1038/s41598-017-01723-2
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发表时间：
2017-05-08
影响因子：
4.6
通讯作者：
Zhou L
中科院分区：
文献类型：
--
作者：
Li Q;Ni RR;Hong H;Goh KY;Rossi M;Fast VG;Zhou L
基于通道视紫红质-2(ChR2)的光遗传技术已越来越多地应用于心血管研究。然而，ChR2蛋白过表达对心肌细胞的潜在影响尚不完全清楚。本工作旨在研究多西环素诱导的慢病毒介导的ChR2表达如何影响培养的新生大鼠心室肌细胞(NRVM)的细胞活力和电生理特性。原代NVRM感染含ChR2或YFP基因的慢病毒，进行细胞毒性分析。然后，用蓝色发光二极管对表达ChR2的培养物进行电或光学起搏，同时使用光学映射记录激活扩散。结果表明，ChR2可以通过多西环素诱导的慢病毒系统很容易地转导到NRVMs；然而，高水平的ChR2(而不是YFP)的表达与大量的细胞毒性有关，这阻碍了光学起搏。应用溴脱氧尿嘧啶核苷显著减少细胞损伤，允许用光刺激。同时光学Vm标测显示，ChR2表达细胞和对照细胞的传导速度、动作电位时程和dvm/dtmax相似。最后，表达ChR2的培养物可以在多个位置进行光学控制，显著减少了总的激活时间。综上所述，我们证明了可诱导慢病毒介导的ChR2过表达可能会在NRVM培养中引起细胞毒性，这种毒性可以在不损害电生理功能的情况下得到缓解，允许同时进行光学起搏和VM标测。
Channelrhodopsin-2 (ChR2)-based optogenetic technique has been increasingly applied to cardiovascular research. However, the potential effects of ChR2 protein overexpression on cardiomyocytes are not completely understood. The present work aimed to examine how the doxycycline-inducible lentiviral-mediated ChR2 expression may affect cell viability and electrophysiological property of neonatal rat ventricular myocyte (NRVM) cultures. Primary NVRMs were infected with lentivirus containing ChR2 or YFP gene and subjected to cytotoxicity analysis. ChR2-expressing cultures were then paced electrically or optically with a blue light-emitting diode, with activation spread recorded simultaneously using optical mapping. Results showed that ChR2 could be readily transduced to NRVMs by the doxycycline-inducible lentiviral system; however, high-level ChR2 (but not YFP) expression was associated with substantial cytotoxicity, which hindered optical pacing. Application of bromodeoxyuridine significantly reduced cell damage, allowing stimulation with light. Simultaneous optical Vm mapping showed that conduction velocity, action potential duration, and dVm/dtmax were similar in ChR2-expressing and control cultures. Finally, the ChR2-expressing cultures could be optically paced at multiple sites, with significantly reduced overall activation time. In summary, we demonstrated that inducible lentiviral-mediated ChR2 overexpression might cause cytotoxicity in NRVM cultures, which could be alleviated without impairing electrophysiological function, allowing simultaneous optical pacing and Vm mapping.