Light-Induced Depolarization to Stimulate Cardiomyocytes with High Spatio-Temporal Resolution and to Modulate their Differentiation in Vitro

Abstract

Electrical pacing of cells stimulates only excitable cells by evoking free-running action potentials and has limitations in long term application because of electrolysis. Here we report the use of the light-gated cation channel channelrhodopsin 2 (ChR2) to depolarize non-excitable embryonic stem (ES) cells, to modulate action potentials, and to stimulate long-term cell cultures.ES cells were transfected with the ChR2-EYFP fusion protein under control of the chicken β-actin promoter. Both, ES cells and differentiated cardiomyocytes showed membrane bound EYFP fluorescence. Application of light (450nm) evoked a non-selective current and depolarization. Constant application of low-intensity light enhanced spontaneous beating frequencies of cardiomyocytes, whereas brief (5ms) high-intensity light stimulation triggered free-running action potentials. Longer illumination led to prolonged action potentials and to an increase of absolute refractory periods.Monolayers of ChR2-expressing cardiomyocytes plated on multi-electrode-arrays could be locally stimulated by application of spatially confined (∼400μm diameter) brief (5ms) illumination whereas constant stimulation induced electrical uncoupling of illuminated areas. Further experiments with different spatio-temporal stimulation patterns can be used to investigate the development of arrhythmias in vitro.To study the impact of depolarization on cardiac development, ES cells were differentiated within embryoid bodies (EBs) on transparent fluorocarbon film on top of light-emitting-diodes. Pulsed light (450nm, 0.6mW/mm2) was applied starting at day 4 of the in vitro differentiation with different stimulation patterns. At day 9 all EBs contracted upon light stimulation. Initial experiments showed higher EYFP fluorescence within beating, cardiac-α-actinin positive areas in illuminated EBs compared to non-stimulated control EBs, suggesting enhanced differentiation and/or hypertrophy of cardiomyocytes. Further long-term experiments with ChrR2 expressing ES cells and different stimulation patterns will be used to investigate the effect of excitation-transcription coupling during ES cell differentiation.