Phosphodiesterases (PDEs) regulate the local concentration of 3',5' cyclic adenosine monophosphate (cAMP) within cells. cAMP activates the cAMP-dependent protein kinase (PKA). In patients, PDE inhibitors have been linked to heart failure and cardiac arrhythmias, although the mechanisms are not understood. We show that PDE4D gene inactivation in mice results in a progressive cardiomyopathy, accelerated heart failure after myocardial infarction, and cardiac arrhythmias. The phosphodiesterase 4D3 (PDE4D3) was found in the cardiac ryanodine receptor (RyR2)/calcium-release-channel complex (required for excitation-contraction [EC] coupling in heart muscle). PDE4D3 levels in the RyR2 complex were reduced in failing human hearts, contributing to PKA-hyperphosphorylated, "leaky" RyR2 channels that promote cardiac dysfunction and arrhythmias. Cardiac arrhythmias and dysfunction associated with PDE4 inhibition or deficiency were suppressed in mice harboring RyR2 that cannot be PKA phosphorylated. These data suggest that reduced PDE4D activity causes defective RyR2-channel function associated with heart failure and arrhythmias.

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β1- and β2-adrenergic receptors (βARs) are highly homologous, yet they play clearly distinct roles in cardiac physiology and pathology. Myocyte contraction, for instance, is readily stimulated by β1AR but not β2AR signaling, and chronic stimulation of the two receptors has opposing effects on myocyte apoptosis and cell survival. Differences in the assembly of macromolecular signaling complexes may explain the distinct biological outcomes. Here, we demonstrate that β1AR forms a signaling complex with a cAMP-specific phosphodiesterase (PDE) in a manner inherently different from a β2AR/β-arrestin/PDE complex reported previously. The β1AR binds a PDE variant, PDE4D8, in a direct manner, and occupancy of the receptor by an agonist causes dissociation of this complex. Conversely, agonist binding to the β2AR is a prerequisite for the recruitment of a complex consisting of β-arrestin and the PDE4D variant, PDE4D5, to the receptor. We propose that the distinct modes of interaction with PDEs result in divergent cAMP signals in the vicinity of the two receptors, thus, providing an additional layer of complexity to enforce the specificity of β1- and β2-adrenoceptor signaling.

Abstract-Steady-state activation of cardiac ␤-adrenergic receptors leads to an intracellular compartmentation of cAMP resulting from localized cyclic nucleotide phosphodiesterase (PDE) activity. To evaluate the time course of the cAMP changes in the different compartments, brief (15 seconds) pulses of isoprenaline (100 nmol/L) were applied to adult rat ventricular myocytes (ARVMs) while monitoring cAMP changes beneath the membrane using engineered cyclic nucleotide-gated channels and within the cytosol with the fluorescence resonance energy transfer-based sensor, Epac2-camps. cAMP kinetics in the two compartments were compared to the time course of the L-type Ca 2ϩ channel current (I Ca,L ) amplitude. The onset and recovery of cAMP transients were, respectively, 30% and 50% faster at the plasma membrane than in the cytosol, in agreement with a rapid production and degradation of the second messenger at the plasma membrane and a restricted diffusion of cAMP to the cytosol. I Ca,L amplitude increased twice slower than cAMP at the membrane, and the current remained elevated for Ϸ5 minutes after cAMP had already returned to basal level, indicating that cAMP changes are not rate-limiting in channel phosphorylation/dephosphorylation. Inhibition of PDE4 (with 10 mol/L Ro 20-1724) increased the amplitude and dramatically slowed down the onset and recovery of cAMP signals, whereas PDE3 blockade (with 1 mol/L cilostamide) had a minor effect only on subsarcolemmal cAMP. However, when both PDE3 and PDE4 were inhibited, or when all PDEs were blocked using 3-isobutyl-l-methylxanthine (300 mol/L), cAMP signals and I Ca,L declined with a time constant Ͼ10 minutes. cAMP-dependent protein kinase inhibition with protein kinase inhibitor produced a similar effect as a partial inhibition of PDE4 on the cytosolic cAMP transient. Consistently, cAMP-PDE assay on ARVMs briefly (15 seconds) exposed to isoprenaline showed a pronounced (up to Ϸ50%) dose-dependent increase in total PDE activity, which was mainly attributable to activation of PDE4. These results reveal temporally distinct ␤-adrenergic receptor cAMP compartments in ARVMs and shed new light on the intricate roles of PDE3 and PDE4. (Circ Res. 2008;102:1091-1100.) Key Words: cAMP Ⅲ L-type calcium current Ⅲ 5Ј-3Ј cyclic nucleotide phosphodiesterases Ⅲ ␤-adrenergic receptors Ⅲ compartmentation C AMP is an ubiquitous second messenger regulating a myriad of cellular functions. In the heart, cAMP mediates the positive inotropic and lusitropic effects of ␤-adrenergic receptor (␤-AR) stimulation by activating the cAMP-dependent protein kinase (PKA), thereby promoting the phosphorylation and activation of key components of the excitation-contraction coupling process. These include the sarcolemmal L-type Ca 2ϩ channels (Ca V 1.2), which are responsible for the initial Ca 2ϩ influx; the ryanodine receptors, which allow Ca 2ϩ release from the sarcoplasmic reticulum (SR); troponin I, which controls myofilament sensitivity to Ca 2ϩ ; and phospholamban, which regulates Ca 2ϩ withdrawal from t...