Abstract 17326: Phoshodiesterase Type 3 (PDE3) Controls cAMP Produced by Adenylyl Cyclase Type 5 (AC5) but Not AC6 in Adult Mouse Cardiomyocytes
Background: The two enzyme families involved in the spatiotemporal control of intracellular cAMP are adenylyl cyclases (AC) which account for its synthesis and phosphodiesterases (PDE) which account for its degradation. In adult cardiomyocytes, AC5 and AC6, and PDE3 and PDE4 are the major isoforms of these two enzyme families, respectively. In addition to their role in modulating cardiac contractile function, AC5 and AC6 are differentially regulated in response to different cardiac stress. However the role of each cyclase in cardioprotection is still controversial. Dynamic inter-regulation between ACs and PDEs has never been investigated yet. In this study, we aimed to determine the effect of AC5 and AC6 overexpression on submembrane PDE3 related cAMP hydrolysis in response to β-adrenergic receptor stimulation.
Methods and Results: Mice with cardiac specific overexperssion of AC5 (AC5Tg) and AC6 (AC6Tg) were produced. The spatiotemporal dynamics of cAMP were determined on both AC5Tg and AC6Tg mouse lines with the same maximal AC activity (10-11 times higher than WT). Intramyocardial in vivo injection of Lyn-Epac2-camps adenovirus (2x109 pfu) resulted in an average of 20% of infected cardiac myocytes which were isolated 5 days later and immediately used to monitor on line cAMP changes. Concentration response-curves for the effects of isoproterenol (Iso) on cAMP showed a similar potency in the 3 groups, with an EC50 of ∼20 nM and with 100 nM Iso producing 80-90% maximal response. The peak response of cAMP to 100 nM Iso, as evidenced by the percentage increase in FRET ratio, was significantly higher in AC6Tg than in WT (13.8±0.9 vs. 9.9±0.5%, p<0.05) whereas no difference was observed between AC5Tg (9.9±0.5%) and WT. Specific inhibition of PDE3 by 1 µM Cilostamide resulted in a significant increase (p<0.05) in cAMP level in WT (13.0±0.65%) and AC5Tg (13.1±1.08%) but not in AC6Tg.
Conclusions: These results indicate a difference in the subsarcolemmal organization of the cAMP compartments generated by the two major cardiac adenylyl cyclase isoforms, AC5 and AC6. When AC6 is overexpressed, the pool of cAMP produced in response to β-adrenergic stimulation circumvents the control by PDE3.
- © 2012 by American Heart Association, Inc.