Compartmentalization of Protein Kinase A Signaling in Cardiac Myoblast (H9c2) Cells Using a Photoactivatable Adenylyl Cyclase

Abstract ID 87610 Poster Board 262 Aim: Cardiac cAMP signaling is coordinated in distinct intracellular domains where PKA protein kinase A (PKA) localization occurs. This signaling axis plays a functional role in cardiac homeostasis and disease progression. However, the use of traditional pharmacological tools that stimulate global activation fails to capture the unique contribution of spatially specific PKA signaling. Photoactivatable adenylyl cyclase is a potential means to study cAMP-dependent PKA signaling with spatiotemporal resolution. This study tests the hypothesis that compartmentalized generation of cAMP by photoactivatable adenylyl cyclase will achieve spatially specific PKA signaling in cardiac cells. Methods: Cytosolically diffuse (bPACCy), plasma membrane (bPACPM), and outer mitochondrial membrane (bPACOMM) anchored photoactivatable adenylyl cyclase from beggiotoa (bPAC) were transfected into H9c2 cardiac myoblast cells. The cells were exposed to blue light stimulus and cAMP signaling was assessed by the protein kinase A phosphorylation of a genetically encoded sensor derived from vasodilator-stimulated phosphoprotein (VASP). The phosphorylation of endogenous VASP was used to validate cAMP generation in response to light. PM, and OMM anchored VASP reporters were used to map light induced cAMP signaling at distinct intracellular domains. Western blot analysis was conducted to determine the extent of protein phosphorylation. Results: Pharmacological cAMP stimulation using forskolin (100 uM, 30 m) leads to the phosphorylation of endogenous VASP as well as the PM and OMM VASP reporters. Cytosolic bPACCy increases phosphorylation of OMM VASP reporter in response to light, while having no impact on PM localized VASP reporter or endogenous VASP. bPACPM increased endogenous VASP phosphorylation in response to light but had no effect on OMM VASP reporter. bPACOMM did not phosphorylate endogenous VASP, but increased phosphorylation of PM VASP reporters. Conclusion: bPAC can be used as a tool to differentially produce cAMP in response to light. The optogenetic constructs activated either endogenous, PM, and OMM-anchored VASP preferentially in light, highlighting the use of bPAC to study PKA stimulation with spatiotemporal control. The optogenetic control of cAMP production allows for the ability to study microdomain specific signaling that characterizes cardiac signaling in health and disease. This work is supported by NIH grant R01 HL159194 (L.H.) and the Morehead-Cain Foundation.