Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching β1-adrenergic but locally confined β2-adrenergic receptor-mediated signaling

beta(1) and beta(2)-adrenergic receptors (beta 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 cardiornyocytes. 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 beta(1)AR-mediated cAMP signals were entirely dependent on PDE4 activity, whereas beta(2)AR-mediated cAMP was under control of multiple PDE isoforms. beta(1)AR subtype-specific stimulation yielded approximate to 2-fold greater cAMP responses compared with selective beta(2)-subtype stimulation, even on treatment with the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) (Delta FRET, 17.3 +/- 1.3% [beta(1)AR] versus 8.8 +/- 0.4% [beta(2)AR]). Treatment with pertussis toxin to inactivate G(i) did not affect cAMP production. Localized beta(1)AR stimulation generated a cAMP gradient propagating throughout the cell, whereas local beta(2)AR stimulation did not elicit marked cAMP diffusion. Our data reveal that in adult cardiac myocytes, beta(1)ARs induce far-reaching cAMP signals, whereas beta(2)AR-induced cAMP remains locally confined.