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Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG) Deutsche Zentrum für Herz-Kreislauf-Forschung (DZHK) Background Gq signalling plays an imperative role in cardiac physiology as well as pathophysiologies such as arrhythmias, hypertrophy, and heart failure. However, the underlying kinetics and the transition from physiological to pathological signalling is yet to be investigated. Optogenetics provides an unprecedented spatial and temporal precision as well as cell specificity, but up to now, selective control of Gq signalling in the intact heart with light has not been achieved. Aim To prove the potential of human Neuropsin (OPN5) to selectively control Gq signalling in the adult heart. Methods The biophysical behaviour of OPN5 in HEK293 cells was characterized by measuring IP1 accumulation, Ca2+ imaging, and patch-clamp analysis of GIRK current activity. We generated a new transgenic mouse model expressing OPN5 in fusion with eYFP under the control of the chicken-β-actin promotor and analysed the expression pattern in the whole heart after tissue clearing with light sheet microscopy and the expression rate after single cell dissociation. Adult isolated cardiomyocytes were examined to measure the effect of OPN5 activation on contractility with a novel custom-made software for on-line contraction analysis by pixel tracking. In Langendorff-perfused hearts, illumination of the sinus node region with UV light (385 nm, 10 s, 1 mW/mm2) was used to measure the influence on the heart rate in electrogram recordings. The light-induced effects and basal ECG parameters as well as the heart-weight-to-femur-length ratio of OPN5 hearts were compared to hearts from non-expressing siblings as well as from CD1 wild-type. Results We prove the selective activation of the Gq proteins by UV light-induced increase in IP3 production and induction of Ca2+ transients in HEK293 cells because both effects were abolished after applying the Gq protein specific blocker FR900359. Promiscuous activation of Gi proteins was excluded by light-induced inhibition of GIRK channels instead of activation. Within two different mouse lines, we found an OPN5/eYFP expression rate of ∼70% within the cardiomyocytes targeted to the plasmamembrane. UV light induced a significant increase in contraction velocity in ventricular cardiomyocytes, and this effect could be abolished by blocking Gq proteins. In the right atrium HCN4 positive sinus nodal cells expressed OPN5/ eYFP and illumination of this region resulted in OPN5 expressing hearts in an average increase of 5.9 ± 0.97% (n= 20, in founder line 1) in the heart rate, which was significant lager than control hearts (all groups &amp;lt;2.4% increase, n≥17). Importantly we could not find any differences in basal ECG parameters or the heart weight-to-femur-length between OPN5 expressing and control.  Conclusion We herein prove the specificity of OPN5 for Gq protein activation and demonstrate its use in cardiomyocytes as well as the whole heart. Thus OPN5 is the first optogenetic tool allowing to control Gq signalling in the whole heart precisely.

OPN5 a new optogenetic receptor to study Gq signalling in the heart with light