Spatiotemporal Dynamics of β-Adrenergic cAMP Signals and L-Type Ca ²⁺ Channel Regulation in Adult Rat Ventricular Myocytes

Abstract

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...

Mammalian Cyclic Nucleotide Phosphodiesterases: Molecular Mechanisms and Physiological Functions

“…Immunohistochemistry shows that PDE2 and PDE4 are localized at the sarcomeric Z line suggestive of anchoring to subcellular structures, whereas PDE3 is associated with internal membranes like the sarcoplasmic reticulum (252,253). Live-cell imaging reveals a variable increase in cAMP due to PDE3 and PDE4 inhibition in ventricular myocytes, confirming the existence of distinct subcellular compartments (204). Assuming PDE localization correlates to function, one could hypothesize that PDE3 regulates cAMP-mediated cardiac contractility while PDE4 regulates ␤-AR-sensitive cAMP microdomains.…”

“…The catalytic activities of PDEs provide for breakdown of cNs over a spectrum of concentrations in all cells, and their varied regulatory mechanisms provide for integration and crosstalk with myriad signaling pathways. It has recently been established that particular PDEs are targeted to discrete compartments within cells where they control cN level and sculpt microenvironments for a variety of cN signalosomes that can contain cN-dependent protein kinases, Epacs (exchange proteins activated by cAMP), phosphoprotein phosphatases, and/or cN-gated cation channels (14,30,49,58,70,104,153,158,167,170,204,299,387,391,422,423). Localization of some PDEs to specific areas of a cell is static, whereas that for others involves reversible recruitment (153).…”

“…Likewise, cN-dependent protein kinases, which are major mediators of cN action, are in some instances anchored in certain regions of a cell as well as being found in the cytosol. The physiological importance of particular PDEs and the specific localization or dynamic relocalization of different PDEs has only recently been appreciated (58,104,153,158,159,204,387). However, particular PDEs and their specific locations within cells are key to physiological function.…”

Phosphodiesterase 4B in the cardiac L-type Ca2+ channel complex regulates Ca2+ current and protects against ventricular arrhythmias in mice

“…We previously showed that the β-AR stimulation of I Ca,L is under the control of PDE4 in adult rat ventricular myocytes (18). To determine whether the same was true in mice, I Ca,L was recorded in adult mouse ventricular myocytes (AMVMs) using the whole-cell patch-clamp technique, and the response of this current to a short application of the nonselective β-AR agonist isoprenaline (Iso) was studied.…”

Rôle des phosphodiestérases des nucléotides cycliques de types 3 et 4 dans le couplage excitation-contraction et les arythmies cardiaques

“…Dans la plupart des espèces, l'inhibition des PDE4 n'exerce peu ou pas d'effet inotrope positif direct, mais devient importante lorsque les taux d'AMPc sont augmentés [16,27]. Par exemple, chez le rat, l'inhibition des PDE4 n'a aucun effet sur l'AMPc, I Ca,L et le CEC en conditions basales, mais devient prédominante pour contrôler ces paramètres lors d'une stimulation -AR ou lors de l'inhibition concomitante de la PDE3 [21,28] (D. Mika, J. Leroy, R. Fischmeister, G. Vandecasteele ; résultats non publiés). Chez la souris, l'inhibition des PDE4 n'a pas d'effet sur I Ca,L en conditions basales, mais potentialise fortement les effets d'une stimulation -AR sur I Ca,L et sur la contraction [29].…”