Abstract 177: Beta1- and Beta2-Adrenergic Receptors Differentially Regulate Spatial cAMP-Signaling in Adult Cardiomyocytes
Beta1- and beta2-adrenergic receptors (bARs) are known to differentially regulate cardiomyocyte contraction and growth. These divergent effects have been attributed to differential PKA-dependent phosphorylation of various target proteins. We tested the hypothesis that these differences are due to spatial compartmentation of the second messenger cAMP. We used a fluorescent resonance energy transfer (FRET)- based approach, to directly monitor the spatial and temporal distribution of cAMP in adult cardiomyocytes. We developed a new cAMP-FRET sensor (termed HCN2-camps) based on a single cAMP binding domain of the hyperpolarization-activated cyclic nucleotide gated channel 2 (HCN2). The cytosolic distribution, the high dynamic range and a sensitivity optimized to determine intracellular cAMP-concentrations render HCN2-camps exquisitely well suited to monitor subcellular distribution of cardiomyocyte cAMP. We generated HCN2-camps transgenic mice and studied freshly isolated cardiomyocytes. Whole cell superfusion of isolated cardiomyocytes with isoproterenol showed a moderate elevation of cAMP. Application of various phosphodiesterase (PDE)-inhibitors revealed stringent control of cardiomyocyte cAMP through several PDE-isoforms. beta1-AR subtype specific stimulation yielded significantly greater cAMP-responses compared to selective beta2-subtype stimulation. The beta1-mediated cAMP signal was largely dependent on PDE4-activity, whereas beta2-mediated cAMP was under the control of multiple PDE-isoforms. Treatment with pertussis toxin to inactivate Gi did not affect cAMP-generation. Localized beta1AR-stimulation generated a cAMP-gradient propagating throughout the cell in a PDE-dependent manner. In contrast, local beta2AR-stimulation did not elicit marked cAMP-diffusion, even in the presence of PDE-inhibitors. Our data reveal, that beta1- and beta2-adrenergic receptor subtypes lead to distinct cAMP-responses, which are under differential control by various PDE-isoforms. In addition to control by PDE-activity, compartmentation of beta2AR-mediated cAMP signals involves PDE and Gi-independent mechanisms.