A current challenge in cellular signaling is to decipher the complex intracellular spatiotemporal organization that any given cell type has developed to discriminate among different external stimuli acting via a common signaling pathway. This obviously applies to cAMP and cGMP signaling in the heart, where these cyclic nucleotides determine the regulation of cardiac function by many hormones and neuromediators. Recent studies have identified cyclic nucleotide phosphodiesterases as key actors in limiting the spread of cAMP and cGMP, and in shaping and organizing intracellular signaling microdomains. With this new role, phosphodiesterases have been promoted from the rank of a housekeeping attendant to that of an executive officer.

CAMP is a powerful second messenger whose known general effector is protein kinase A (PKA). The identification of a cAMP binding protein, Epac, raises the question of its role in Ca²⁺ signalling in cardiac myocytes. In this study, we analysed the effects of Epac activation on Ca²⁺ handling by using confocal microscopy in isolated adult rat cardiomyocytes. [Ca²⁺]_i transients were evoked by electrical stimulation and Ca²⁺ sparks were measured in quiescent myocytes. Epac was selectively activated by the cAMP analogue 8‐(4‐chlorophenylthio)‐2′‐O‐methyladenosine‐3′,5′‐cyclic monophosphate (8‐CPT). Patch‐clamp was used to record the L‐type calcium current (I_Ca), and Western blot to evaluate phosphorylated ryanodine receptor (RyR). [Ca²⁺]_i transients were slightly reduced by 10 μm 8‐CPT (F/F₀: decreased from 4.7 ± 0.5 to 3.8 ± 0.4, P < 0.05), an effect that was boosted when cells were previously infected with an adenovirus encoding human Epac. I_Ca was unaltered by Epac activation, so this cannot explain the decreased [Ca²⁺]_i transients. Instead, a decrease in the sarcoplasmic reticulum (SR) Ca²⁺ load underlies the decrease in the [Ca²⁺]_i transients. This decrease in the SR Ca²⁺ load was provoked by the increase in the SR Ca²⁺ leak induced by Epac activation. 8‐CPT significantly increased Ca²⁺ spark frequency (Ca²⁺ sparks s⁻¹ (100 μm)⁻¹: from 2.4 ± 0.6 to 6.9 ± 1.5, P < 0.01) while reducing their amplitude (F/F₀: 1.8 ± 0.02 versus 1.6 ± 0.01, P < 0.001) in a Ca²⁺/calmodulin kinase II (CaMKII)‐dependent and PKA‐independent manner. Accordingly, we found that Epac increased RyR phosphorylation at the CaMKII site. Altogether, our data reveal a new signalling pathway by which cAMP governs Ca²⁺ release and signalling in cardiac myocytes.

Steady-state activation of cardiac β-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 ²⁺ 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 ≈5 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 μmol/L Ro 20-1724) increased the amplitude and dramatically slowed down the onset and recovery of cAMP signals, whereas PDE3 blockade (with 1 μmol/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 μmol/L), cAMP signals and I _Ca,L declined with a time constant >10 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 ≈50%) dose-dependent increase in total PDE activity, which was mainly attributable to activation of PDE4. These results reveal temporally distinct β-adrenergic receptor cAMP compartments in ARVMs and shed new light on the intricate roles of PDE3 and PDE4.

β-Adrenergic receptors (β-ARs) enhance cardiac contractility by increasing cAMP levels and activating PKA. PKA increases Ca²⁺-induced Ca²⁺ release via phosphorylation of L-type Ca²⁺ channels (LTCCs) and ryanodine receptor 2. Multiple cyclic nucleotide phosphodiesterases (PDEs) regulate local cAMP concentration in cardiomyocytes, with PDE4 being predominant for the control of β-AR–dependent cAMP signals. Three genes encoding PDE4 are expressed in mouse heart: Pde4a, Pde4b, and Pde4d. Here we show that both PDE4B and PDE4D are tethered to the LTCC in the mouse heart but that β-AR stimulation of the L-type Ca²⁺ current (I_Ca,L) is increased only in Pde4b^–/– mice. A fraction of PDE4B colocalized with the LTCC along T-tubules in the mouse heart. Under β-AR stimulation, Ca²⁺ transients, cell contraction, and spontaneous Ca²⁺ release events were increased in Pde4b^–/– and Pde4d^–/– myocytes compared with those in WT myocytes. In vivo, after intraperitoneal injection of isoprenaline, catheter-mediated burst pacing triggered ventricular tachycardia in Pde4b^–/– mice but not in WT mice. These results identify PDE4B in the Ca_V1.2 complex as a critical regulator of I_Ca,L during β-AR stimulation and suggest that distinct PDE4 subtypes are important for normal regulation of Ca²⁺-induced Ca²⁺ release in cardiomyocytes.