title: Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching beta1-adrenergic but locally confined beta2-adrenergic receptor-mediated signaling.
abstract: Beta(1)- and beta(2)-adrenergic receptors (betaARs) 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 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 approximately 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) (DeltaFRET, 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.
authors.affiliation: Institute of Pharmacology and Toxicology, University of Wuerzburg, Germany.
authors.forename: Viacheslav O
authors.initials: VO
authors.lastname: Nikolaev
authors.forename: Moritz
authors.initials: M
authors.lastname: Bünemann
authors.forename: Eva
authors.initials: E
authors.lastname: Schmitteckert
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Lohse
authors.forename: Stefan
authors.initials: S
authors.lastname: Engelhardt
mesh_terms: D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods; D000242:Cyclic AMP / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008958:Models, Molecular; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D015221:Potassium Channels; D017434:Protein Structure, Tertiary; D018342:Receptors, Adrenergic, beta-1 / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D000318:Adrenergic beta-Agonists; C578710:HCN2 protein, human; C578711:Hcn2 protein, mouse; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D007527:Isoenzymes; D015221:Potassium Channels; D018342:Receptors, Adrenergic, beta-1; D018343:Receptors, Adrenergic, beta-2; D000242:Cyclic AMP; D010727:Phosphoric Diester Hydrolases