In cardiomyocytes, the ryanodine receptor-2 (RyR2) gates Ca2+-induced Ca2+ release from the intracellular stores of the sarcoplasmic reticulum (SR). Pathological Ca2+ leak from the SR caused by gain-of-function mutations or altered phosphorylation of RyR2 plays an important role in the development of arrhythmias and heart failure. It remains challenging to investigate the importance of location and duration of pathological SR Ca2+ leak because of the diffusion-limited pharmacological RyR2 opening. We aim to develop an optogenetic tool for precise spatial and temporal control of SR Ca2+ release by targeting a light-gated Ca2+-permeable cation channel Channelrhodopsin-2 (ChR2) with L132C, H134R, T159C mutations (CatChUP) to the SR close to the RyR2 in order to obtain localized light-induced Ca2+ release that is subsequently triggering RyR2 opening. Therefore, CatChUP-EYFP was c-terminally fused to the SR membrane-spanning and RyR-interacting protein Junctin (CatChUP-EYFP-Junctin). After expression in HL-1 cells, CatChUP-EYFP-Junctin was detected in intracellular compartments but not on the plasma membrane (PM). Ca2+ imaging with X-Rhod-1 was performed to analyze effects of the RyR2 opener caffeine (10 mM) and blue-light (470 nm). Interestingly, blue-light illumination led to Ca2+ release in CatChUP-EYFP-Junctin HL-1 cells (ΔF/F0: 10.6±1.3% for 1.1 mW/mm2, n=6 and 18.3±2.0% for 2.4 mW/mm2, n=6), which was significantly higher (p<0.01, students t-test) than in wild-type HL-1 cells but Ca2+ release by caffeine was not different (ΔF/F0: ~45%, p>0.4, students t-test). Next, we generated a transgenic mouse embryonic stem cell (ESC) line with stable expression of CatChUP-EYFP-Junctin. ESCs were differentiated into cardiomyocytes in spontaneously beating embryoid bodies (EBs). Using beating EBs from ChR2-EYFP ESCs with PM targeted ChR2 as the controls; spontaneous beating rate of EBs was examined by infrared video microscopy. Application of low-intensity blue-light (20 s, 4-20 µW/mm2) led to an instantaneous and sustained increment of spontaneous beating rate in CatChUP-EYFP-Junctin EBs but not in ChR2-EYFP EBs, which required higher light intensities (>20 µW/mm2) to accelerate beating. Importantly, at light intensities between 30-300 µW/mm2 rate acceleration was always higher in CatChUP-EYFP-Junctin EBs (e.g. increase by 159.2±27.8%, n=7 at 102 µW/mm2) compare to ChR2-EYFP EBs (increase by 67.4±12.9%, n=4 at 102 µW/mm2) (p=0.04 students t-test). This indicates the light-induced Ca2+ release in ESC-derived CatChUP-EYFP-Junctin expressing cardiomyocytes that speeds up the Ca2+ clock machinery, which is the main pacemaking mechanism in these cells. Further patch-clamp and Ca2+ imaging experiments are required to discriminate between PM-bound ChR2 photocurrent and SR Ca2+ release-induced Na+/Ca2+ exchanger current and to proof light-induced Ca2+ release by CatChUP-EYFP-Junctin.