Cyclic AMP Imaging in Adult Cardiac Myocytes Reveals Far-Reaching

1and 2-adrenergic receptors ( ARs) are known to differentially regulate cardiomyocyte contraction and growth. We tested the hypothesis that these differences are attributable to spatial compartmentation of the second messenger cAMP. Using a fluorescent resonance energy transfer (FRET)-based approach, we directly monitored 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 potassium channel 2 (HCN2). Its cytosolic distribution, high dynamic range, and sensitivity make HCN2-camps particularly well suited to monitor subcellular localization of cardiomyocyte cAMP. We generated HCN2-camps transgenic mice and performed single-cell FRET imaging on freshly isolated cardiomyocytes. Whole-cell superfusion with isoproterenol showed a moderate elevation of cAMP. Application of various phosphodiesterase (PDE) inhibitors revealed stringent control of cAMP through PDE4 PDE2 PDE3. The 1AR-mediated cAMP signals were entirely dependent on PDE4 activity, whereas 2AR-mediated cAMP was under control of multiple PDE isoforms. 1AR subtype–specific stimulation yielded 2-fold greater cAMP responses compared with selective 2-subtype stimulation, even on treatment with the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) ( FRET, 17.3 1.3% [ 1AR] versus 8.8 0.4% [ 2AR]). Treatment with pertussis toxin to inactivate Gi did not affect cAMP production. Localized 1AR stimulation generated a cAMP gradient propagating throughout the cell, whereas local 2AR stimulation did not elicit marked cAMP diffusion. Our data reveal that in adult cardiac myocytes, 1ARs induce far-reaching cAMP signals, whereas 2AR-induced cAMP remains locally confined. (Circ Res. 2006;99:1084-1091.)