Oral abstract presentations
Authors: S. M. Davidson, M. M. Duchen, D. M. Yellon, L. Fields, M. Zaccolo, H. Laeremans, E. P. Daskalopoulos, B. J. A. Janssen, W. M. Blankesteijn, J. Tillmanns, D. Hoffmann, Y. Habbaba, D. Fraccarollo, P. Galuppo, J. Bauersachs, S. Novella, G. Segarra, A. P. Dantas, C. Bueno-Beti, L. Novensa, M. Lazaro-Franco, N. Martinez-Gil, P. Medina, C. Hermenegildo, J. Ohanian, A. Liao, A. W. Trafford, V. Ohanian, A. Perez-Moreno, P. Garcia-Canadilla, J. M. Dominguez, F. Crispi, E. Gratacos, I. Amat-Roldan, A. Gotschy, V. Herold, E. Bauer, C. Schrodt, G. Lykowsky, E. Rommel, P. Jakob, W. Bauer, M. Shah, M. B. Sikkel, T. Desplantez, T. P. Collins, P. O'Gara, S. E. Harding, A. R. Lyon, K. T. Macleod, C. Ferrantini, L. Sacconi, I. Lotti, R. Coppini, C. Tesi, P. Yan, L. M. Loew, E. Cerbai, C. Poggesi, F. S. Pavone, D. Franco, H. Daimi, J. N. Dominguez, L. Hove-Madsen, J. Cinca, E. Vazquez, A. E. Aranega, K. L. Poon, B. C. Kirchmaier, T. Schwerte, J. Huisken, C. Winkler, B. C. Jungblut, D. Y. Stainier, T. Brand, D. J. Stuckey, J. L. Tremoleda, S. Mcsweeney, L. Fiedler, M. Harada, C. A. Carr, D. J. Tyler, W. Gsell, K. Clarke, M. D. Schneider, V. Sequeira, J. A. Regan, M. Michels, F. J. Ten Cate, M. A. Van Slegtenhorst, G. J. M. Stienen, C. Dos Remedios, J. Van Der Velden
Journal: Cardiovascular Research (2012)

cAMP generated in response to β-adrenoceptor stimulation modulates excitation-contraction coupling via activation of PKA. It is now widely accepted that cAMP signalling is compartmentalised in the heart and that PDEs, the only cAMP degrading enzyme, play a central role in spatial control of signal propagation. We have previously demonstrated that PKA-I and PKA-II isoforms are selectively activated by spatially confined pools of cAMP leading to phosphorylation of specific downstream targets. It has been suggested that selective deregulation of cAMP/PKA signals in specific subcellular compartment may lead to cardiac pathology. Here we investigate cAMP signalling in an in vitro model of cardiomyocyte hypertrophy and explore the role of different PDE families in the control of the local cAMP response to β-adrenoceptor stimulation. We used a real-time FRET-based imaging approach to monitor cAMP dynamics in adult myocytes. Adenoviral vectors carrying RI or RII Epac were generated by fusing the N-terminus of the cAMP FRET reporter Epac-1 and the dimerisation-docking (DD) domain of the regulatory subunit of PKA-I or PKA-II. Adult rat cardiac myocytes expressing the cAMP reporters were treated overnight with 1μM norepinephrine (NE) as a model of in vitro cardiomyocyte hypertrophy. In control cells, upon stimulation with 100nM isoproterenol, we detected a significantly higher cAMP response in the PKA-II compartment compared to the PKA-I compartment. In NE-treated myocytes, the differences between these compartments were abolished. To investigate which PDE family may be responsible for these changes in cAMP compartmentalisation, experiments were performed in the presence of selective PDEs inhibitors. We found that when PDE2 was inhibited with BAY60-7550 in control cells the resulting increase in cAMP was significantly greater in the PKA-I than in the PKA-II compartment. In hypertrophic myocytes, PDE2 inhibition resulted in a much greater increase in cAMP in the PKA-II compartment compared to control cells. Similar results were found with selective PDE4 inhibition with rolipram. Selective inhibition of PDE3 with cilostamide showed a small but significant rise in cAMP response in the PKA-I compartment in control cells. This was not replicated in hypertrophic myocytes. Our findings indicate that spatial control of cAMP/PKA signalling is altered in this in vitro model of hypertrophy, suggesting that in hypertrophic myocytes mechanisms may be activated resulting in altered localisation and/or activity of PDEs. Further studies will be necessary to fully determine the mechanism responsible for the observed changes.