PDE4 and mAKAPβ are nodal organizers of β2-ARs nuclear PKA signalling in cardiac myocytes

International audienceAims: β1- and β2-adrenergic receptors (β-ARs) produce different acute contractile effects on the heart partly because they impact on different cytosolic pools of cAMP-dependent protein kinase (PKA). They also exert different effects on gene expression but the underlying mechanisms remain unknown. The aim of this study was to understand the mechanisms by which β1- and β2-ARs regulate nuclear PKA activity in cardiomyocytes.Methods and results: We used cytoplasmic and nuclear targeted biosensors to examine cAMP signals and PKA activity in adult rat ventricular myocytes upon selective β1- or β2-ARs stimulation. Both β1- and β2-AR stimulation increased cAMP and activated PKA in the cytoplasm. Although the two receptors also increased cAMP in the nucleus, only β1-ARs increased nuclear PKA activity and up-regulated the PKA target gene and pro-apoptotic factor, inducible cAMP early repressor (ICER). Inhibition of phosphodiesterase (PDE)4, but not Gi, PDE3, GRK2 nor caveolae disruption disclosed nuclear PKA activation and ICER induction by β2-ARs. Both nuclear and cytoplasmic PKI prevented nuclear PKA activation and ICER induction by β1-ARs, indicating that PKA activation outside the nucleus is required for subsequent nuclear PKA activation and ICER mRNA expression. Cytoplasmic PKI also blocked ICER induction by β2-AR stimulation (with concomitant PDE4 inhibition). However, in this case nuclear PKI decreased ICER up-regulation by only 30%, indicating that other mechanisms are involved. Down-regulation of mAKAPβ partially inhibited nuclear PKA activation upon β1-AR stimulation, and drastically decreased nuclear PKA activation upon β2-AR stimulation in the presence of PDE4 inhibition.Conclusions: β1- and β2-ARs differentially regulate nuclear PKA activity and ICER expression in cardiomyocytes. PDE4 insulates a mAKAPβ-targeted PKA pool at the nuclear envelope that prevents nuclear PKA activation upon β2-AR stimulation

Imipramine as an alternative to formamide to detubulate rat ventricular cardiomyocytes

International audienceT-tubules are membrane invaginations essential for the excitation-contraction coupling (ECC). Imipramine like other cationic amphiphilic drugs, interfere with the PI(4,5)P2 interactions with proteins maintaining the tubular system connected to cell surface. Our main purpose was to validate imipramine as a new detubulating agent in cardiomyocytes. Staining adult rat ventricular myocytes (ARVMs) with di-4-ANEPPS, we showed that unlike formamide, imipramine induces a complete detubulation with no impact on cell viability. Using the patch-clamp technique, we observed a ~40% decrease in cell capacitance after imipramine pretreatment and a reduction of ICa,L Disclaimer: This is a confidential document

Synergic PDE3 and PDE4 control intracellular cAMP and cardiac excitation-contraction coupling in a porcine model

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Interventricular Differences in β‐Adrenergic Responses in the Canine Heart: Role of Phosphodiesterases

Background RV and LV have different embryologic, structural, metabolic, and electrophysiologic characteristics, but whether interventricular differences exist in β‐adrenergic (β‐AR) responsiveness is unknown. In this study, we examine whether β‐AR response and signaling differ in right (RV) versus left (LV) ventricles. Methods and Results Sarcomere shortening, Ca2+ transients, ICa,L and IKs currents were recorded in isolated dog LV and RV midmyocytes. Intracellular [cAMP] and PKA activity were measured by live cell imaging using FRET‐based sensors. Isoproterenol increased sarcomere shortening ≈10‐fold and Ca2+‐transient amplitude ≈2‐fold in LV midmyocytes (LVMs) versus ≈25‐fold and ≈3‐fold in RVMs. FRET imaging using targeted Epac2camps sensors revealed no change in subsarcolemmal [cAMP], but a 2‐fold higher β‐AR stimulation of cytoplasmic [cAMP] in RVMs versus LVMs. Accordingly, β‐AR regulation of ICa,L and IKs were similar between LVMs and RVMs, whereas cytoplasmic PKA activity was increased in RVMs. Both PDE3 and PDE4 contributed to the β‐AR regulation of cytoplasmic [cAMP], and the difference between LVMs and RVMs was abolished by PDE3 inhibition and attenuated by PDE4 inhibition. Finally LV and RV intracavitary pressures were recorded in anesthetized beagle dogs. A bolus injection of isoproterenol increased RV dP/dtmax≈5‐fold versus 3‐fold in LV. Conclusion Canine RV and LV differ in their β‐AR response due to intrinsic differences in myocyte β‐AR downstream signaling. Enhanced β‐AR responsiveness of the RV results from higher cAMP elevation in the cytoplasm, due to a decreased degradation by PDE3 and PDE4 in the RV compared to the LV

Phosphodiesterase 2 Protects against Catecholamine-induced Arrhythmias and Preserves Contractile Function after Myocardial Infarction

International audienceRationale: Phosphodiesterase 2 is a dual substrate esterase, which has the unique property to be stimulated by cGMP, but primarily hydrolyzes cAMP. Myocardial phosphodiesterase 2 is upregulated in human heart failure, but its role in the heart is unknown.Objective: To explore the role of phosphodiesterase 2 in cardiac function, propensity to arrhythmia, and myocardial infarction.Methods and Results: Pharmacological inhibition of phosphodiesterase 2 (BAY 60–7550, BAY) led to a significant positive chronotropic effect on top of maximal β-adrenoceptor activation in healthy mice. Under pathological conditions induced by chronic catecholamine infusions, BAY reversed both the attenuated β-adrenoceptor–mediated inotropy and chronotropy. Conversely, ECG telemetry in heart-specific phosphodiesterase 2-transgenic (TG) mice showed a marked reduction in resting and in maximal heart rate, whereas cardiac output was completely preserved because of greater cardiac contraction. This well-tolerated phenotype persisted in elderly TG with no indications of cardiac pathology or premature death. During arrhythmia provocation induced by catecholamine injections, TG animals were resistant to triggered ventricular arrhythmias. Accordingly, Ca2+-spark analysis in isolated TG cardiomyocytes revealed remarkably reduced Ca2+ leakage and lower basal phosphorylation levels of Ca2+-cycling proteins including ryanodine receptor type 2. Moreover, TG demonstrated improved cardiac function after myocardial infarction.Conclusions: Endogenous phosphodiesterase 2 contributes to heart rate regulation. Greater phosphodiesterase 2 abundance protects against arrhythmias and improves contraction force after severe ischemic insult. Activating myocardial phosphodiesterase 2 may, thus, represent a novel intracellular antiadrenergic therapeutic strategy protecting the heart from arrhythmia and contractile dysfunction

Phosphodiestérases des nucléotides cycliques : rôle dans le coeur et potentiel thérapeutique

International audienceCyclic nucleotide phosphodiesterases (PDEs) degrade the second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), thereby regulating multiple aspects of cardiac function. This highly diverse class of enzymes encoded by 21 genes encompasses 11 families that are not only responsible for the termination of cyclic nucleotide signalling, but are also involved in the generation of dynamic microdomains of cAMP and cGMP, controlling specific cell functions in response to various neurohormonal stimuli. In the myocardium, the PDE3 and PDE4 families predominate, degrading cAMP and thereby regulating cardiac excitation-contraction coupling. PDE3 inhibitors are positive inotropes and vasodilators in humans, but their use is limited to acute heart failure and intermittent claudication. PDE5 inhibitors, which are used with success to treat erectile dysfunction and pulmonary hypertension, do not seem efficient in heart failure with preserved ejection fraction. There is experimental evidence however that these PDE, as well as other PDE families including PDE1, PDE2 and PDE9, may play important roles in cardiac diseases, such as hypertrophy and heart failure (HF). After a brief presentation of the cyclic nucleotide pathways in cardiac myocytes and the major characteristics of the PDE superfamily, this review will focus on the potential use of PDE inhibitors in HF, and the recent research developments that could lead to a better exploitation of the therapeutic potential of these enzymes in the future.Les phosphodiestérases des nucléotides cycliques (PDE) dégradent les seconds messagers AMPc et GMPc qui constituent des régulateurs majeurs de la fonction cardiaque. Cette classe d’enzymes très diversifiée, codée par vingt et un gènes, englobe onze familles qui sont responsables de la terminaison des signaux transmis par les nucléotides cycliques. Ces PDE sont également impliquées dans la génération de microdomaines dynamiques d’AMPc et de GMPc, contrôlant des fonctions spécifiques des cellules en réponse à divers stimuli neuro-hormonaux. Dans le myocarde, les PDE3 et PDE4 sont prédominantes pour dégrader l’AMPc et régulent le couplage excitation-contraction cardiaque. Les inhibiteurs de PDE3 sont inotropes positifs et vasodilatateurs chez l’homme, mais leur utilisation est limitée au traitement de l’insuffisance cardiaque aiguë et de la claudication intermittente. Les inhibiteurs de PDE5, utilisés avec succès pour traiter la dysfonction érectile et l’hypertension pulmonaire, ne semblent pas efficaces dans l’insuffisance cardiaque à fraction d’éjection préservée. Des travaux expérimentaux suggèrent néanmoins que ces PDE ainsi que d’autres, en particulier les PDE1, PDE2 et PDE9, jouent un rôle important dans l’hypertrophie et l’insuffisance cardiaque. Après un bref aperçu des voies des nucléotides cycliques dans les myocytes cardiaques et des principales caractéristiques des PDE, cette revue fera le point sur les travaux de recherche récents susceptibles de conduire à une meilleure exploitation du potentiel thérapeutique de ces enzymes pour le traitement futur de l’insuffisance cardiaque

PEGylated isoprenaline reveals distinct functions of cardiac β-adrenergic receptors located in the T-tubule vs . outer surface membrane

Membrane proteins are present in both cardiac T-tubule (TTM) and outer surface membrane (OSM), although at a different density. Classical pharmacology does not allow to explore the function of a membrane protein separately in OSM vs. TTM. Here, we developed a technology based on size exclusion to explore the function of β-adrenergic receptors (β-ARs) located in the OSM. We synthetized a PEG-Iso molecule by covalent linking between isoprenaline (Iso) and a 5000 Da PolyEthylene-Glycol (PEG). Using confocal microscopy, we show that PEGylation constrains molecules outside the T-tubule network. PEG-Iso produced similar effects as Iso on I ca,L , sarcomere shortening and Ca 2+ transients. However, PEG-Iso increased [cAMP] i with a lower efficacy than Iso, produced a much lower stimulation of nuclear PKA activity than Iso but a larger stimulation of cytosolic PKA at equivalent levels of [cAMP] i . Our results show that activation of OSM β-ARs is sufficient to activate cytosolic PKA and excitation-contraction coupling, but insufficient to activate nuclear PKA or nuclear protein phosphorylation for which additional activation of TTM β-ARs is needed

Control of cytoplasmic and nuclear protein kinase A by phosphodiesterases and phosphatases in cardiac myocytes

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Phosphodiesterase-2 Is Up-Regulated in Human Failing Hearts and Blunts β-Adrenergic Responses in Cardiomyocytes

International audienceObjectives: We investigated whether myocardial phosphodiesterase 2 (PDE2) is altered in heart failure (HF) and determined PDE2-mediated effects on β-adrenoceptor (AR) signaling in healthy and diseased cardiomyocytes.Background: Diminished cAMP- and augmented cGMP-signaling is characteristic for failing hearts. Among the PDE superfamily, PDE2 has the unique property to be stimulated by cGMP, thus leading to a remarkable increase in cAMP hydrolysis mediating a negative cross-talk between cGMP- and cAMP-signaling. However, the role of PDE2 in HF is poorly understood.Methods and Results: Immunoblotting and radioenzymatic assay revealed that myocardial PDE2 expression and activity were ~2-fold higher in advanced human HF. Chronic β-AR stimulation via catecholamine infusions in rats enhanced PDE2 expression 2-fold and cAMP hydrolytic activity 4-fold as determined by FRET-based sensors, which correlated with blunted cardiac β-AR responsiveness. Notably, in diseased cardiomyocytes the higher PDE2 activity could be further enhanced by stimulation of cGMP synthesis via NO donors whereas specific PDE2 inhibition partially restored β-AR responsiveness. Accordingly, PDE2 overexpression in healthy cardiomyocytes reduced the rise in cAMP levels and ICa,L amplitude and abolished the positive inotropic effect following acute β-AR stimulation, without affecting basal contractility. Importantly, PDE2-overexpressing cardiomyocytes showed marked protection from norepinephrine-induced hypertrophic responses.Conclusions: PDE2 is markedly upregulated in failing hearts and desensitizes against acute β-AR stimulation. This may constitute an important defence mechanism during cardiac stress, e.g. by antagonizing excessive β-AR drive. Thus, activating myocardial PDE2 may represent a novel intracellular anti-adrenergic therapeutic strategy in HF

Control of cytoplasmic and nuclear protein kinase A by phosphodiesterases and phosphatases in cardiac myocytes

AIMS: The cAMP-dependent protein kinase (PKA) mediates β-adrenoceptor (β-AR) regulation of cardiac contraction and gene expression. Whereas PKA activity is well characterized in various subcellular compartments of adult cardiomyocytes, its regulation in the nucleus remains largely unknown. The aim of the present study was to compare the modalities of PKA regulation in the cytoplasm and nucleus of cardiomyocytes. METHODS AND RESULTS: Cytoplasmic and nuclear cAMP and PKA activity were measured with targeted fluorescence resonance energy transfer probes in adult rat ventricular myocytes. β-AR stimulation with isoprenaline (Iso) led to fast cAMP elevation in both compartments, whereas PKA activity was fast in the cytoplasm but markedly slower in the nucleus. Iso was also more potent and efficient in activating cytoplasmic than nuclear PKA. Similar slow kinetics of nuclear PKA activation was observed upon adenylyl cyclase activation with L-858051 or phosphodiesterase (PDE) inhibition with 3-isobutyl-1-methylxantine. Consistently, pulse stimulation with Iso (15 s) maximally induced PKA and myosin-binding protein C phosphorylation in the cytoplasm, but marginally activated PKA and cAMP response element-binding protein phosphorylation in the nucleus. Inhibition of PDE4 or ablation of the Pde4d gene in mice prolonged cytoplasmic PKA activation and enhanced nuclear PKA responses. In the cytoplasm, phosphatase 1 (PP1) and 2A (PP2A) contributed to the termination of PKA responses, whereas only PP1 played a role in the nucleus. CONCLUSION: Our study reveals a differential integration of cytoplasmic and nuclear PKA responses to β-AR stimulation in cardiac myocytes. This may have important implications in the physiological and pathological hypertrophic response to β-AR stimulation