Abstract
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 (Vm) responses also showed a ChR variant-specific profile, with GtACR1 causing a slight increase in average Vm during illumination (Vplateau: −38 mV) as compared with a Vplateau > −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 Vplateau remained largely stable. Importantly, current properties and Vm 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.
Highlights
Optogenetics uses the light-sensitive proteins as actuators to take control over cellular function (Fenno et al, 2011; Ferenczi et al, 2019)
Since Ca2+-translocating ChR (CatCh) possesses increased activation kinetics and light sensitivity compared with ChR2, it has been the microbial rhodopsin of choice in several previous studies in cardiac optogenetics (Bingen et al, 2014; Feola et al, 2017; Watanabe et al, 2017; Majumder et al, 2018)
Neonatal rat ventricular cardiomyocytes (NRVMs) expressing ChR variants were kept in the dark for 3 min before the application of each light pulse
Summary
Optogenetics uses the light-sensitive proteins as actuators to take control over cellular function (Fenno et al, 2011; Ferenczi et al, 2019). Optogenetics has become an important experimental approach during the past decade in the context of both basic and translational research, mostly owing to its unique capacity to precisely and reversibly modulate the membrane potential of cardiomyocytes by the use of microbial rhodopsins This method has been successfully applied to achieve a wide range of research goals, including cardiac pacing (Arrenberg et al, 2010; Bruegmann et al, 2010), shaping of action potential (AP) waveforms (Park et al, 2014; Govorunova et al, 2016), sympathetic (Yu et al, 2017) and parasympathetic (Moreno et al, 2019; Machhada et al, 2020) neuromodulations of the heart, termination (Bingen et al, 2014), and spatiotemporal control (Majumder et al, 2018) of spiral waves in vitro, as well as the termination of ventricular (Bruegmann et al, 2016; Nyns et al, 2017; Li et al, 2021) and atrial fibrillation (Bruegmann et al, 2018; Nyns et al, 2019) in vivo. Considering their prevalence in the literature on cardiac optogenetics and their unique features, these four ChR variants have been included in this study
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