Abstract 353: Distinct cGMP Compartmentalization by Membrane Guanylate Cyclases

Abstract

Introduction: In adult cardiomyocytes (CM), cGMP produced by two membrane guanylate cyclases (GC-A and GC-B) play distinct roles. Using Förster Resonance Energy Transfer (FRET) and Scanning Ion Conductance Microscopy (SICM)-based approaches, cGMP compartmentation by GCs and phosphodiesterases (PDEs) were assessed. Methods: cGMP was measured in CM isolated from transgenic mice expressing membrane targeted cGMP sensor (red cGES-DE5). SICM was used to define the t-tubules across sarcolemma and its nano-pipette filled with natriuretic peptides (CNP and ANP) was used to locally stimulate GC in t-tubules and crest. Results: CNP activating GC-B induced maximum cGMP increase, while ANP activating GC-A did not increase cGMP. Pre-inhibiting PDE2 before ANP stimulation massively increased cGMP production to functionally significant levels as measured by PLN phosphorylation in western blot. cGMP in unstimulated cells was regulated mostly by PDE3. Local ANP stimulation in t-tubules, but not in crest, induced cGMP increase and CNP induced cGMP increase in both t-tubule and crest. After methyl-β-cyclodextrin treatment, ANP induced cGMP in both t-tubules and crest suggesting a role for lipid rafts in trapping GC-A in t-tubules. In transfected HEK293 cells, Fluorescence Recovery After Photobleeching (FRAP) showed that membrane diffusion of YFP-tagged GC-A was decreased after methyl-β-cyclodextrin treatment. Conclusions: GC-A exclusively located in T-tubules produce cGMP locally, which is tightly regulated by PDE2, while GC-B is expressed across the sarcolemma and produces global cGMP.