Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB033

Poster Communications

Optogenetic stimulation of Gq signaling in cardiomyocytes using Neuropsin (OPN5)

C. Jin1, D. Malan1, P. Sasse1, T. Bruegmann1

1. Institute of Physiology I, Medical Faculty, University of Bonn, Bonn, Germany.

In the heart, chronic Gq stimulation can result in pathological modulation of Ca2+ handling and induction of cardiac hypertrophy or heart failure. Our goal is to determine experimentally the threshold of Gq activation for induction of such heart pathologies with a focus on duration, repetition and refractoriness of Gq signaling. For this purpose, we here describe Neuropsin (OPN5) as a new optogenetic receptor enabling precise temporal control of Gq activation. Transgenic mouse embryonic stem cells (mESC, R1 line) were generated to express human OPN5 and differentiated into beating embryonic bodies containing cardiomyocytes. These cells have an embryonic phenotype in which Gq signaling is accelerating the pacemaking machinery. In line with this, illumination with UV light (385 nm) instantaneously accelerated the spontaneous beating rate (delay < 0.1s, time to peak 0.5 ± 0.1 s, n=7). Compared to pharmacological stimulation with 100nM Endothelin1 (time to peak 21.9 ± 4.2 s, n=7), the light-induced increase in beating rate was significantly faster (p=0.002, two-tailed paired t-test) and higher (p=0.0006, two-tailed paired t-test). Importantly, the specific Gq inhibitor FR900359 (1 µM) completely blocked UV light-induced frequency increase. Light intensity-response relationships were analyzed using light pulses with different intensities (from 30 to 3000 µW/mm2) and durations (1 ms to 30 s). Interestingly, we found that human OPN5 activation can be fine-tuned by duration of light pulses as well as light intensity and its light sensitivity is best described by the overall amount of photons applied (product of light duration and intensity). Furthermore, we analyzed which retinal isomer must be supplied for best function of human OPN5 and found similar maximal light-induced frequency increase and light sensitivity when supplementing cells with 11-cis retinal, all-trans retinal, and even without retinal (p=0.34, one-way ANOVA, n=10-12). Finally, we compared the human OPN5 with the mouse OPN5 variant that has a slightly longer c-terminus. Overall human and mouse OPN5 showed no significant differences in light sensitivity (p=0.63, two-tailed unpaired t-test, N=3 clones) and maximal frequency increase (p=0.57, two-tailed unpaired t-test, N=3 clones). However, mESC clones expressing mouse OPN5 showed a much higher variability. In summary, human OPN5 is a well-suited optogenetic tool to investigate Gq signaling with high temporal precision in cardiomyocytes in vitro. In the future, we will use various stimulation protocols to investigate the different timings between physiological and pathophysiological Gq signaling.

Where applicable, experiments conform with Society ethical requirements