Abstract 192: Roles of Phosphodiesterases 2 and 3 at the T-tubules of Adult Mouse Cardiomyocytes

Abstract

PURPOSE: cAMP is a key regulator of proper heart function. Its functional effects are controlled and regulated by spatial and temporal signal segregation at the subcellular level. In cardiomyocytes, transverse (T)- tubules form a highly organized network of membrane structures which are crucial for normal cAMP signaling. Morphological changes, such as loss of T-tubules, are associated with cardiac disease. This study aimed to gain insight into localized cAMP dynamics and its regulation in the T-tubular microdomain in health and disease. Methods: Generation of a transgenic mouse expressing a targeted Förster resonance energy transfer (FRET)-based biosensor (pmE1). Confocal microscopy for co-localization of pmE1 with T-tubular structures. Histological, morphometric and ultrasound analysis for characterization of heart function. Transverse aortic constriction (TAC) surgery for hypertrophy induction. FRET-based cAMP measurements and PDE activity assay with single freshly isolated adult ventricular myocytes of healthy and diseased mice. Results: The transgene did not alter heart function per se. We performed FRET measurements in freshly isolated cardiomyocytes to compare T-tubular with cytosolic cAMP responses and their regulation by beta1- and beta2-adrenergic receptors (β-ARs) and phosphodiesterase (PDE) isoforms. As expected, T-tubular β2-AR signals were higher than cytosolic ones (>2-fold). Comparison of PDE activity profiles between the two compartments showed marked differences with regard to the β2-cAMP signal. In particular, T-tubular β2-cAMP is mainly controlled by PDE3. Strikingly, in hypertrophied myocytes (8 weeks post-TAC) we could detect a switch from PDE3 to PDE2-dependent regulation of these cAMP signals. Conclusions: We could establish transgenic mice expressing a FRET-biosensor to measure local cAMP levels in the T-tubular compartment and uncover its differential regulation by β1- and β2-ARs and several PDE families, especially by PDE3. The switch from PDE3 to PDE2 regulation of β2-cAMP in diseased cardiomyocytes could represent a novel mechanism of local cAMP signaling and cGMP/cAMP cross-talk in cardiomyocytes.