Published online before print October 12, 2006, doi: 10.1161/01.RES.0000250046.69918.d5
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Submitted on July 28, 2006
Revised on October 4, 2006
Accepted on October 4, 2006
Cyclic AMP Imaging in Adult Cardiac Myocytes Reveals Far-Reaching 
1-Adrenergic but Locally Confined 2-Adrenergic Receptor-Mediated Signaling
Viacheslav O. Nikolaev ;
From the Institute of Pharmacology and Toxicology (V.O.N., M.B., E.S., M.J.L.), University of Wuerzburg; and Rudolf-Virchow-Center (S.E.), Deutsche Forschungsgemeinschaft-Research Center for Experimental Biomedicine, University of Wuerzburg, Germany.
* To whom correspondence should be addressed. E-mail: stefan.engelhardt{at}virchow.uni-wuerzburg.de.
1- and 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, 8.8±0.4% [2AR] versus 17.3±1.3% [1AR]). 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.
Key words: cAMP • FRET • cardiomyocyte •
-adrenergic receptor
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