目的:通道视紫红质(ChRs)是一类具有独特特性的光门控离子通道,这对于选择给定应用的ChR变体非常重要。然而,缺乏指导心脏光遗传学应用选择的数据。因此,我们研究了接受各种光照方案的正常和病理性肥大心肌细胞中不同ChR变体的功能。方法和结果:用慢病毒载体转导分离的新生大鼠心室心肌细胞(NRVMs)以表达以下ChR变体之一:H134R,CatCh,ReaChR,或GtACR1。用去氧肾上腺素(PE)处理NRVM以诱导病理性肥大(PE组)或不进行处理[对照(CTL)组]。在这些群体中,ChR电流在通过单个1-s光脉冲(1mW/mm2:470、565或617nm)激活时,每个ChR变体都显示出独特且显着不同的特性。伴随的膜电位(Vm)反应也显示出ChR变体特异性谱,与其他ChR变体的V平台>-20mV相比,GtACR1在照明期间引起平均Vm的轻微增加(V平台:-38mV)。在不断增加的频率下重复激活(10毫秒脉冲,1-10Hz,持续30s),峰值电流,这对心脏起搏很重要,随着激活频率的增加而降低17-78%(p<0.05),而高原洋流,这对心律失常终止至关重要,下降了10-75%(p<0.05),两者都以变体特定的方式。相比之下,相应的V高原基本保持稳定。重要的是,PE组和CTL组之间的电流特性和Vm响应没有统计学差异,与使用的变体无关(p>0.05)。结论:我们的数据表明,ChR变异体在健康和病理肥厚心肌的细胞培养模型中同样功能良好,特定于变体的使用依赖性。在心脏光遗传学研究的设计及其实验发现的解释过程中,应考虑ChR功能的这种使用依赖性性质。
Aim: Channelrhodopsins (ChRs) are a large family of light-gated ion channels with distinct properties, which is of great importance in the selection of a ChR variant for a given application. However, data to guide such selection for cardiac optogenetic applications are lacking. Therefore, we investigated the functioning of different ChR variants in normal and pathological hypertrophic cardiomyocytes subjected to various illumination protocols. Methods and Results: Isolated neonatal rat ventricular cardiomyocytes (NRVMs) were transduced with lentiviral vectors to express one of the following ChR variants: H134R, CatCh, ReaChR, or GtACR1. NRVMs were treated with phenylephrine (PE) to induce pathological hypertrophy (PE group) or left untreated [control (CTL) group]. In these groups, ChR currents displayed unique and significantly different properties for each ChR variant on activation by a single 1-s light pulse (1 mW/mm2: 470, 565, or 617 nm). The concomitant membrane potential (V m) responses also showed a ChR variant-specific profile, with GtACR1 causing a slight increase in average V m during illumination (V plateau: -38 mV) as compared with a V plateau > -20 mV for the other ChR variants. On repetitive activation at increasing frequencies (10-ms pulses at 1-10 Hz for 30 s), peak currents, which are important for cardiac pacing, decreased with increasing activation frequencies by 17-78% (p < 0.05), while plateau currents, which are critical for arrhythmia termination, decreased by 10-75% (p < 0.05), both in a variant-specific manner. In contrast, the corresponding V plateau remained largely stable. Importantly, current properties and V m responses were not statistically different between the PE and CTL groups, irrespective of the variant used (p > 0.05). Conclusion: Our data show that ChR variants function equally well in cell culture models of healthy and pathologically hypertrophic myocardium but show strong, variant-specific use-dependence. This use-dependent nature of ChR function should be taken into account during the design of cardiac optogenetic studies and the interpretation of the experimental findings thereof.