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.
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