Introduction

Optogenetics offers multifold opportunities to unravel cell-type specific behaviour in complex biological tissues, such as the heart [1]. More specifically, light-activated ion channels, commonly referred to as channelrhodopsins (ChR), can be selectively targeted to the cell type of interest to assess effects of optically induced transmembrane currents. We have previously shown that cation non-selective ChR can effectively depolarize different cardiac cell populations [2]. Moreover, we demonstrated that anion-selective channelrhodopsins depolarize cardiomyocytes, thereby enabling both optical pacing and inhibition, depending on the duration of the applied light stimulus used for channel activation [3]. Here, we present the novel class of K⁺-selective ChR, so-called Kalium Channelrhodopsins (KCR) [4,5]. These channels are promising candidates for targeted optical silencing of action potentials (AP) without change in diastolic membrane potentials.

Methods

We expressed three recently identified KCR (HcKCR1, HcKCR2 and WiChR) by either transfection or adenoviral delivery of target cells. Photocurrent properties were determined by patch-clamp recordings of human embryonic kidney (HEK) cells. Atrial cardiomyocytes (aCM) derived from human induced pluripotent stem cells served as model system to assess effects of K⁺ currents on spontaneous AP and contractions. Sharp electrode recordings were used to measure light-induced changes in membrane potential and AP properties. Contractions were visualized by camera-based imaging of cultured aCM. Primary ventricular cardiomyocytes (vCM) were isolated from tissue slices of hearts obtained from New Zealand white rabbits and were cultured on laminin-coated coverslips. vCM were characterized 48-72 h post-adenoviral transduction using patch-clamp recordings and sarcomere length tracking experiments, as previously described [3].

Results and Conclusion

KCR mediate large outward currents upon blue/green light stimulation, with different photocurrent kinetics of channel variants. Spontaneous AP generation in aCM cultures expressing WichR was blocked during blue light, by keeping syncytia close to end-diastolic potentials. Accordingly, contractions of WiChR expressing aCM were effectively blocked by blue light application [5]. In contrast, aCM expressing HcKCR1 and HcKCR2 showed reversible AP shortening and an increase in spontaneous AP rate during illumination. In pilot experiments on rabbit vCM, K⁺ channels mediated a reversible inhibition of AP and contractions. Based on these preliminary data, we conclude that KCR are potent optogenetic tools to silence and/or shape cardiac AP, depending on the chosen variant and light stimulation protocol.

References

[1]      Zgierski-Johnston CM, Schneider-Warme F. Observing and Manipulating Cell-Specific Cardiac Function with Light.  Adv Exp Med Biol 2021/1293:377-388.

[2]      Fernandez MC et al. Channelrhodopsins for Cell-Type Specific Illumination of Cardiac Electrophysiology. Methods Mol Biol 2021/2191:287-307.

[3]      Kopton RA et al. Cardiac Electrophysiological Effects of Light-Activated Chloride Channels. Front Physiol 2018/17:1806.

[4]      Govorunova EG et al. Kalium rhodopsins: Natural light-gated potassium channels. Nat Neurosci 2022/25:967-974.

[5]      Vierock J et al. WiChR, a highly potassium selective channelrhodopsin for low-light one- and two-photon inhibition of excitable cells. Sci Adv 2022/eadd7729.