Introduction
Optogenetics is a valuable technique to investigate biological processes. It allows controlling of excitable cells with high spatial and temporal resolution. Here we aimed to generate human cardiomyocytes with a chemo- and optogenetic off-on switch by introducing the luminopsin iLMO4, consisting of the light-sensitive ion channel, improved chloride-conducting Channelrhodopsin (iChloC), the Gaussia luciferase M23 (GLucM23), and EYFP.

Methods

iLMO4 was knocked into the AAVS1 locus of human induced pluripotent stem cells (iPSC) using CRISPR/Cas9. The selection of EYFP-positive cells for clonal expansion was performed by FACS. iLMO4-hiPSCs were subjected to cardiac differentiation. Engineered heart tissue (EHT) was generated by casting cardiomyocytes into a fibrin-based matrix. iLMO4-EHTs were analyzed morphologically (immunostainings) and physiologically (video-optical force measurement and action potential (AP) measurements with a sharp microelectrode). 

Results

iLMO4-iPSCs could be differentiated to cardiomyocytes. iLMO4-EHTs developed regularly and morphologically resembled WT-EHTs. Force development was more variable and overall lower in iLMO4-EHTs compared to WT-EHTs (mean force 0.12±0.04 mN vs. 0.18±0.02 mN). Baseline frequency was also more variable but overall similar to WT-EHTs (mean frequency 67.7±6.84 bpm vs. 57.96±2.28 bpm). iLMO4-EHT contractility could be stopped reversibly by photostimulation (470 nm; light intensity: 0.66 mW/mm²). EHTs were not electrically paceable during photostimulation. Application of the GLucM23 substrate Coelenterazine (CTZ) resulted in light emission, but the bioluminescence induced off-switch relied on high CTZ concentrations and was inconsistent. Photostimulation during AP measurements showed an elevation of the maximum diastolic potential. 

Discussion

Integration of iLMO4 allowed to generate cardiomyocytes with an optogenetic off-on switch. The chemogenetic off-on switch was inconsistent. Nevertheless, this cell line can serve as a versatile tool for future studies, including disease modeling and regenerative medicine.