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(Circulation Research. 2006;99:1084.)
© 2006 American Heart Association, Inc.

Cellular Biology

Cyclic AMP Imaging in Adult Cardiac Myocytes Reveals Far-Reaching ß₁-Adrenergic but Locally Confined ß₂-Adrenergic Receptor–Mediated Signaling

Viacheslav O. Nikolaev, Moritz Bünemann, Eva Schmitteckert, Martin J. Lohse, Stefan Engelhardt

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.

Correspondence to Stefan Engelhardt, MD PhD, Rudolf-Virchow-Center/DFG-Research Center for Experimental Biomedicine and Martin J. Lohse, MD, Institute of Pharmacology, University of Wuerzburg, Versbacher Strasse 9, Wuerzburg 97078, Germany. E-mail stefan.engelhardt{at}virchow.uni-wuerzburg.de and lohseatoxi.uni-wuerzburg.de

ß₁- and ß₂-adrenergic receptors (ßARs) 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 ß₁AR-mediated cAMP signals were entirely dependent on PDE4 activity, whereas ß₂AR-mediated cAMP was under control of multiple PDE isoforms. ß₁AR subtype–specific stimulation yielded 2-fold greater cAMP responses compared with selective ß₂-subtype stimulation, even on treatment with the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) (FRET, 17.3±1.3% [ß₁AR] versus 8.8±0.4% [ß₂AR]). Treatment with pertussis toxin to inactivate G_i did not affect cAMP production. Localized ß₁AR stimulation generated a cAMP gradient propagating throughout the cell, whereas local ß₂AR stimulation did not elicit marked cAMP diffusion. Our data reveal that in adult cardiac myocytes, ß₁ARs induce far-reaching cAMP signals, whereas ß₂AR-induced cAMP remains locally confined.

Key Words: cAMP • FRET • cardiomyocyte • ß-adrenergic receptor

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