Subcellular control of cAMP microdomain signaling using targeted optogenetics

Abstract

In cardiomyocytes, cAMP/PKA-dependent phosphorylation of LTCC and RyR2 increases Ca²⁺ transient and contractile force. Due to the close proximity of both proteins, it is still unclear whether their cAMP/PKA microdomains are functionally separated or cross-talk. To investigate differences in selective phosphorylation, we generated cAMP microdomains at the LTCC and the RyR2 using a novel optogenetic approach that subcellularly targets photo-activated adenylate cyclase from <em>Turneriella parva</em> (TpPAC). TpPAC was targeted together with EYFP or mCitrine to RyR2 by fusion with the high-affinity protein FKBP12.6 and to the LTCC ß-subunit by fusion with a specific nanobody (nb.F3). TpPAC-EYFP was used as a control for global cytosolic cAMP generation. Intact light-dependent cAMP generation by TpPAC fusion proteins was detected in HEK293 cells co-expressing the cAMP-sensitive GloSensor, where blue light increased cAMP levels dose-dependently. <br/> After expression in nCMs, TpPAC-EYFP showed a homogeneous distribution. TpPAC-mCitrine-FKBP12.6 localized near the Z-discs, indicating RyR2 targeting, while nb.F3-TpPAC-EYFP showed cell membrane targeting. However, both targeted TpPACs also exhibited some background cytosolic expression. Brief flashes of blue light induced a transient increase in spontaneous beating rate in all constructs in a light dose-dependent manner. The global production of cAMP in the cytosol or the local production at RyR2 by brief light pulses of 250 ms resulted in a long-lasting, constant plateau (>50 s) of the increased beating rate effect. In contrast, the effect of cAMP generation locally at the LTCC declined after its peak, without a clear plateau, but with a longer-lasting, small effect. Ca²⁺ imaging of electrically stimulated cardiomyocytes (0.75-1 Hz) with the red-shifted dye Cal630 revealed a light-induced increase in the Ca²⁺ transient height with all three constructs. Interestingly, the local generation of cAMP at RyR2 increased diastolic Ca²⁺ levels, incontrast to local LTCC or global cAMP generation. Furthermore, PKA phosphorylation intensity analysis showed that TpPAC-mCitrine-FKBP12.6 had already reached 85% of its maximum at low illumination protocols. In contrast, TpPAC-EYFP reached only 45% and TpPAC-nb.F3 43% of its maximum, indicating a higher phosphorylation sensitivity of the RyR2 to low light in response to cAMP generation.<br/> The kinetic differences suggest the light-induced generation of a "large" cAMP pool in the cytosol and RyR2 microdomain and a high PDE expression or activation of a feedback mechanism in the LTCC microdomain. Furthermore, the higher increase of diastolic Ca²⁺ by cAMP in RyR2 microdomains highlights the significance of PKA-dependent Ca²⁺ leak from the SR. Hereby, the higher sensitivity to low cAMP increase may be an important mechanism in developing cardiac arrhythmias. In a second preliminary approach, both globally and locally, the inhibition of PKA with the optogenetic LOV-PKI protein was analyzed. A decrease in beating rate and phosphorylation levels was observed upon illumination. This presents another novel tool for the further investigation into the intricate regulatory mechanisms of cAMP microdomains.