Combining cGMP biosensors and scavengers to decipher cardiac compartmented signaling

The natriuretic peptides ANP, BNP and CNP activate transmembrane guanylyl cyclases (GC) that producecyclic GMP (cGMP). We have previously employed targeted FRET-based biosensors to demonstrate that thenatriuretic peptides have differential effects in cardiomyocytes and intact heart, where CNP activates GC-B thatincreases cGMP near troponin I and phospholamban (PLB), enhancing relaxation, while activation of GC-A withANP/BNP modestly increases cGMP only near PLB and do not enhance relaxation, suggesting spatiallyrestricted cGMP signaling from GC-A and GC-B. Using a biosensor targeted to the outer mitochondrialmembrane (OMM), we have found that GC-A and GC-B increase cGMP at the OMM and reduce cardiomyocyteapoptosis. To understand this spatial cGMP signaling from GC-A and GC-B, we combined targeted variants ofthe cGMP scavenger SponGee with our targeted FRET-based cGMP biosensors in cardiac H9c2 cells.Activation of GC-B increased cytosolic cGMP, which was modestly reduced by the lipid raft (Lyn-SponGee),non-raft (SponGee-Kras) or OMM-targeted (OMM-SponGee) cGMP scavengers compared to the untargetedSponGee. At the OMM, cGMP increase from GC-B was reduced in content and kinetics only by the OMMSponGee,while cGMP increase from GC-A was reduced by SponGee-Kras and the untargeted SponGee.Our results indicate that GC-A and GC-B are differentially organized on the plasma membrane and that cGMPreaching the OMM could have different origin. Using cGMP scavengers can therefore be used to deciphersignaling from different areas of the plasma membrane to various subcellular locations in cardiac cells.