AC5 and 6 are the two major cardiac adenylyl cyclase isoforms. Their involvement in the spatiotemporal regulation of intracellular cAMP ([cAMP]i) in cardiomyocytes has never been studied. Here, we used transgenic mice with a cardiac AC6 overexpression (AC6 Tg) to measure membrane [cAMP]i in response to β-AR stimulation and its regulation by cardiac phosphodiesterases (PDEs).Material and Methods
3 months old AC6 Tg males or their wild type littermates (WT) underwent surgery to delive, by multiple injection sites, an adenovirus encoding for the membrane-bound cAMP sensor Lyn-Epac2-camps (2x109 pfu). Five to 7 days following surgery, cardiac myocytes were freshly isolated and used to measure real time subsarcolemmal [cAMP]I variations using FRET-technology. Membrane [cAMP]i was measured in response to isoproterenol (Iso, 15 s pulse application), in the presence and absence of inhibitors of the two main cardiac PDEs: cilostamide (Cil, 1 μM) for PDE3; Ro-201724 (Ro, 10 μM) for PDE4.Results
Direct intramyocardial injection of Lyn-Epac2-camps adenovirus results in a reasonable percentage of infected cardiac myocytes. Dose-response curves for the effect of Iso on [cAMP]i have been established for each genotype. AC6 overexpression leads to an increase in membrane [cAMP]i at all tested Iso concentrations. At a submaximal concentration of 100 nM Iso, the percentage of maximal increase in FRET signal was 40% higher in AC6 Tg than in WT (p < 0.001) indicating that the AC6 transgene was functional and well coupled to the up-stream part of the β-AR signaling system. PDE4 inhibition with Ro produced a significant (p < 0.05) and similar relative increase of the effect of Iso on [cAMP]i in both WT (15.8 ± 1.1%, n=45 vs. 9.9 ± 0.5%, n=14, in Iso alone) and AC6 Tg mice (18.2 ± 0.7%, n=29 vs. 13.8 ± 0.9%, n=29 in Iso alone). However, PDE3 inhibition with Cil increased [cAMP]i concentration in WT (13.0 ± 0.6%, n=15 vs. 9.9 ± 0.5%, n=14, in Iso alone, p < 0.001) but had no effect in AC6 Tg mice (13.3 ± 0.4%, n=30 vs. 13.8 ± 0.9, n=29, in Iso alone).Conclusion
When overexpressed, AC6 controls a pool of cAMP which is controlled by PDE4 but not by PDE3. This indicates a different subsarcolemmal organization of the cAMP compartments generated by the two cardiac AC isoforms.