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Stimulation of Gq-protein coupled receptors activates phospholipase C (PLC) which produces inositol-1,4,5-trisphosphate (IP3) and diacylglycerol. The Gq-signaling cascade is involved in many fundamental cellular processes including cardiac pacemaking and arrhythmogenesis.Gq-proteins can be activated by agonists with low spatio-temporal precision. To overcome this limitation, we use melanopsin, a directly light-sensitive Gq-coupled receptor (Qiu et al., Nature 2005; 433:745-9).In melanopsin-expressing HEK293 cells cytosolic Ca2+-transients could be induced by brief light-pulses and biochemical analyses revealed IP3-production upon illumination.Embryonic stem cells with stable expression of melanopsin under control of the chicken β-actin promoter showed repetitive light-induced Ca2+-transients. Cardiomyocytes were differentiated from these embryonic stem cells by generation of spontaneously contracting embryoid bodies (EBs). Immunofluorescence staining revealed clear melanopsin expression in α-actinin-positive cardiomyocytes.Brief illumination (60 s) of spontaneously contracting EBs caused an acceleration of beating rate. A slight and delayed increase (to 117±3% of control frequency, n=12) was observed at low light-intensities of 9.1 nW/mm2. In contrast stimulation with 166.7 nW/mm2 led to an instantaneous acceleration of frequency to 301±19% with subsequent decline to a constant plateau of 211±13% (n=11). Similar to dose-response-curves of receptor agonists, the acceleration of frequency showed a sigmoid dependence on light-intensity. Addition of the PLC-blocker U-73122 or the IP3-receptor-blocker 2-aminoethoxydiphenyl borate attenuated the light-induced acceleration of beating. Interestingly we observed a higher rate of arrhythmic contractions during and after light-stimulation of melanopsin.The effect of local Gq-activation was analyzed using micro-electrode arrays to identify the leading pacemaker site in EBs. Stimulation of a small (O180 μm) region with focused light led to pacemaker activity or arrhythmias in the illuminated area indicating the importance of Gq-signaling for pacemaking.In summary optogenetic Gq-activation in cardiomyocytes using melanopsin induces pacemaking and arrhythmia and will enable the investigation of Gq-signaling with high spatio-temporal precision.

Numerous neurological diseases lead to paralysis of intact skeletal muscles. Electrical stimulation of these to restore contraction is problematic due to side effects, the lack of cellspecificity and simultaneous activation of antagonist muscles.Optogenetic stimulation of skeletal muscles expressing the light-sensitive cation channel Channelrhodopsin2 (ChR2) could overcome these limitations. Therefore we have generated a transgenic mouse line with expression of ChR2 fused to EYFP in skeletal muscles under the control of the chicken-β-actin promotor. Soleus muscles showed membrane bound EYFP fluorescence and light induced force was analyzed by isometric force measurements upon illumination (470nm) with different durations, intensities and repetition rates. Using single light pulses, the highest force generation (52.7±3.4mN, sd, n=4) was reached by 100ms long pulses with a light intensity of 0.51mW/mm2. For tetanus stimulation we found that 5ms pulses at 30Hz and 1.4mW/mm2 were most efficient and led to the force of 69.1±7.7mN (sd,n=4). This was ∼70% of the force generated by electrical tetanus stimulation.Bilateral laryngeal paralysis is a severe disease resulting in immobility of the vocal cords and life-threatening dyspnea. In this disease electrical stimulation is problematic due to electrode corrosion, recognition of the stimuli and non-selective stimulation of agonist and antagonist muscles (Zealer DL et al., Laryngoscope 2003,113:1149-1156). We therefore tested the feasibility of selective optogenetic stimulation of the posterior cricoarytenoid muscle, which is the only intralaryngeal muscle opening the vocal cords. Larynges were isolated from ChR2-transgenic mice and locally stimulated with a light guide (O400μm,NA=0,48) in the postarytenoid region. Application of light pulses (470nm,50ms,35.7mW/mm2) led to a repetitive opening of the vocal cords. The distance between vocal cords during illumination was 230±38% of baseline (sd,n=3).Thus optogenetic muscle stimulation is a promising method for therapy of nerve lesions and might be useful to treat bilaryngeal paralysis.

Stimulation of GS-protein coupled receptors leads to an increase of the second messenger cyclic adenosine monophosphate (cAMP). In cardiomyocytes the GS-signaling cascade is involved in positive regulation of chronotropy and contractility but chronic GS stimulation can also induce cardiac hypertrophy or arrhythmia.Experimentally, the GS-signaling cascade can be activated by s-receptor agonists but diffusion of drugs does not allow the precise control of location and timing. To overcome these limitations we used the optogenetic protein JellyOp, a directly GS-coupled, light-sensitive receptor (Bailes et al. PloS One, 2012) to activate GS-signaling by light.Illumination of JellyOp expressing HEK 293 cells resulted in elevation of cAMP levels without detectable dark activity. Cardiomyocytes were differentiated within embryoid bodies (EBs) from transgenic mouse embryonic stem cells that express JellyOp under control of the ubiquitous chicken s-actin promoter. Spontaneously beating EBs were analyzed at day 13 of differentiation by infrared video microscopy. Brief illumination (20 sec, 470 nm, 166.7 nW/mm2) increased beating frequency to 1239±349% of baseline (n=3) which returned to baseline after termination of illumination. The lowest effective light intensity was of 9.1 nW/mm2 resulting in frequency acceleration to 428±131% of baseline and the shortest effective illumination was 1 sec. Similar to dose-response-curves of receptor agonists, light-induced frequency acceleration showed a sigmoid dependence on light-intensity with a half maximal light intensity of 33.5 nW/mm2. Direct comparison showed that the rate of frequency increment was much faster using illumination (13.9±3.1%/s) than using perfusion with the s-receptor agonist isoprenaline (2.7±1.5%/s) but both stimulations led to a similar response in frequency elevation.In summary optogenetic JellyOp activation in cardiomyocytes enables the stimulation of the GS-signaling pathway with high temporal precision and will be useful to investigate temporal and site-specific effects of physiological and pathophysiological GS-activation.