Search

Cyclic nucleotide phosphodiesterases (PDEs) modulate the neurohormonal regulation of cardiac function by degrading cAMP and cGMP. In cardiomyocytes, multiple PDE isozymes with different enzymatic properties and subcellular localization regulate local pools of cyclic nucleotides and specific functions. This organization is heavily perturbed during cardiac hypertrophy and heart failure (HF), which can contribute to disease progression. Clinically, PDE inhibition has been considered a promising approach to compensate for the catecholamine desensitization that accompanies HF. Although PDE3 inhibitors, such as milrinone or enoximone, have been used clinically to improve systolic function and alleviate the symptoms of acute HF, their chronic use has proved to be detrimental. Other PDEs, such as PDE1, PDE2, PDE4, PDE5, PDE9 and PDE10, have emerged as new potential targets to treat HF, each having a unique role in local cyclic nucleotide signalling pathways. In this Review, we describe cAMP and cGMP signalling in cardiomyocytes and present the various PDE families expressed in the heart as well as their modifications in pathological cardiac hypertrophy and HF. We also appraise the evidence from preclinical models as well as clinical data pointing to the use of inhibitors or activators of specific PDEs that could have therapeutic potential in HF.

BACKGROUNDConstitutive cardiac PDE2 activation was shown to protect against contractile dysfunction and arrhythmia in heart failure (HF). However, it remains unknown whether an acute elevation of PDE2 is efficient to prevent maladaptive remodeling and arrhythmia. In this study we tested whether increasing acutely PDE2A activity in preclinical models of HF using cardiac PDE2 gene transfer could be of therapeutic value.METHODS AND RESULTSC57BL/6 male mice were injected with serotype 9 adeno-associated viruses (AAV9) encoding for PDE2A, or luciferase (LUC). Cardiac function assessed by echocardiography unveiled neither structural change nor dysfunction consecutive to PDE2A overexpression while AAV9 inoculation led to a ≈10-fold rise of PDE2A protein levels. Two weeks after AAV9 injections, mice were implanted with osmotic minipumps delivering NaCl or isoproterenol (Iso) (60 mg/kg/day) or Iso and phenylephrine (Iso+Phe, 30 mg/kg/day each) for 2 weeks. In LUC mice, chronic infusion with Iso increased left ventricular (LV) weight over body weight ratio, promoted fibrosis and decreased ejection fraction, but animals overexpressing PDE2A were protected towards these deleterious effects. Similarly, concomitant treatment with Iso+Phe promoted LV contractile dysfunction, fibrosis and apoptosis in LUC mice, while PDE2A overexpression limited these adverse outcomes. Furthermore, inotropic responses to Iso of ventricular cardiomyocytes isolated from Iso+Phe-LUC mice loaded with 1 µmol/L Fura-2AM and stimulated at 1 Hz to record calcium transients and sarcomere shortening were dampened. Chronic treatment with catecholamines favoured spontaneous calcium waves upon β-AR stimulation at the cellular level and promoted susceptibility to ventricular arrhythmiasin vivoevoked by catheter-mediated ventricular pacing after Iso and atropine injection. However, these adverse effects were blunted by the cardiac gene therapy with PDE2A.CONCLUSIONGene therapy with PDE2A limits cardiac adverse left ventricle remodeling and dysfunction induced by catecholamines as well as ventricular arrhythmias, providing evidence that acutely increasing PDE2A activity could prevent progression towards HF.

International audience

Au départ, la science fiction se révèle beaucoup moins critique socialement que ne l’a été le roman noir et va exalter à ses débuts le bien-fondé absolu de la science pour finir par s’inquiéter d’une technologie qui nous domine. Elle se révèle un sismographe parfait des fantasmes, des peurs et même des névroses qui traversent la société et présente les traits d’une littérature de l’inquiétude. Elle se lancera dans une critique acerbe du consumérisme, de la pollution, des dérives technologiques, d’une urbanisation toujours plus inhumaine. Elle flirte avec la dystopie écologique et catastrophique., Leroy Jérôme. La science fiction nous avait pourtant prévenus !. In: Recherches Internationales, n°119, 2021. De la décroissance à l’effondrement. pp. 99-109.

International audience; Background In cardiomyocytes, phosphodiesterases (PDEs) type 3 and 4 are the predominant enzymes that degrade cAMP generated by β-adrenergic receptors (β-ARs), impacting notably the regulation of the L-type Ca2+ current (ICa,L). Cardiac hypertrophy (CH) is accompanied by a reduction in PDE3 and PDE4, however, whether this affects the dynamic regulation of cytosolic cAMP and ICa,L is not known. Methods and Results CH was induced in rats by thoracic aortic banding over a time period of five weeks and was confirmed by anatomical measurements. Left ventricular myocytes (LVMs) were isolated from CH and sham-operated (SHAM) rats and transduced with an adenovirus encoding a Förster resonance energy transfer (FRET)-based cAMP biosensor or subjected to the whole-cell configuration of the patch-clamp technique to measure ICa,L. Aortic stenosis resulted in a 46% increase in heart weight to body weight ratio in CH compared to SHAM. In SHAM and CH LVMs, a short isoprenaline stimulation (Iso, 100 nM, 15 s) elicited a similar transient increase in cAMP with a half decay time (t1/2off) of ~50 s. In both groups, PDE4 inhibition with Ro-201724 (10 µM) markedly potentiated the amplitude and slowed the decline of the cAMP transient, this latter effect being more pronounced in SHAM (t1/2off ~ 250 s) than in CH (t1/2off ~ 150 s, P

Background: The cyclic AMP (adenosine monophosphate; cAMP)-hydrolyzing protein PDE4B (phosphodiesterase 4B) is a key negative regulator of cardiac β-adrenergic receptor stimulation. PDE4B deficiency leads to abnormal Ca 2+ handling and PDE4B is decreased in pressure overload hypertrophy, suggesting that increasing PDE4B in the heart is beneficial in heart failure. Methods: We measured PDE4B expression in human cardiac tissues and developed 2 transgenic mouse lines with cardiomyocyte-specific overexpression of PDE4B and an adeno-associated virus serotype 9 encoding PDE4B. Myocardial structure and function were evaluated by echocardiography, ECG, and in Langendorff-perfused hearts. Also, cAMP and PKA (cAMP dependent protein kinase) activity were monitored by Förster resonance energy transfer, L-type Ca 2+ current by whole-cell patch-clamp, and cardiomyocyte shortening and Ca 2+ transients with an Ionoptix system. Heart failure was induced by 2 weeks infusion of isoproterenol or transverse aortic constriction. Cardiac remodeling was evaluated by serial echocardiography, morphometric analysis, and histology. Results: PDE4B protein was decreased in human failing hearts. The first PDE4B-transgenic mouse line (TG15) had a ≈15-fold increase in cardiac cAMP-PDE activity and a ≈30% decrease in cAMP content and fractional shortening associated with a mild cardiac hypertrophy that resorbed with age. Basal ex vivo myocardial function was unchanged, but β-adrenergic receptor stimulation of cardiac inotropy, cAMP, PKA, L-type Ca 2+ current, Ca 2+ transients, and cell contraction were blunted. Endurance capacity and life expectancy were normal. Moreover, these mice were protected from systolic dysfunction, hypertrophy, lung congestion, and fibrosis induced by chronic isoproterenol treatment. In the second PDE4B-transgenic mouse line (TG50), markedly higher PDE4B overexpression, resulting in a ≈50-fold increase in cardiac cAMP-PDE activity caused a ≈50% decrease in fractional shortening, hypertrophy, dilatation, and premature death. In contrast, mice injected with adeno-associated virus serotype 9 encoding PDE4B (10 12 viral particles/mouse) had a ≈50% increase in cardiac cAMP-PDE activity, which did not modify basal cardiac function but efficiently prevented systolic dysfunction, apoptosis, and fibrosis, while attenuating hypertrophy induced by chronic isoproterenol infusion. Similarly, adeno-associated virus serotype 9 encoding PDE4B slowed contractile deterioration, attenuated hypertrophy and lung congestion, and prevented apoptosis and fibrotic remodeling in transverse aortic constriction. Conclusions: Our results indicate that a moderate increase in PDE4B is cardioprotective and suggest that cardiac gene therapy with PDE4B might constitute a new promising approach to treat heart failure.

Rationale: 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, Ca 2+ -spark analysis in isolated TG cardiomyocytes revealed remarkably reduced Ca 2+ leakage and lower basal phosphorylation levels of Ca 2+ -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.

The cAMP-dependent protein kinase (PKA) consists of two regulatory (R) and two catalytic (C) subunits and comprises two subtypes, PKAI and PKAII, defined by the nature of their regulatory subunits, RIα and RIIα respectively. Whereas PKAII is thought to play a key role in β-adrenergic (β-AR) regulation of cardiac contractility, the function of PKAI is unclear. To address this question, we generated mice with cardiomyocyte-specific and conditional invalidation of the RIα subunit of PKA. Tamoxifen injection in 8 weeks-old mice resulted in a >70% decrease in RIα protein without modification of other PKA subunits, which was associated with ∼2-fold increased basal PKA activity in RIα-KO mice (p2-fold (p

NEW FINDINGS What is the central question of this study? Can imipramine, an antidepressant agent that is a cationic amphiphilic drug that interferes with the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) interactions with proteins maintaining the tubular system, be validated as a new detubulating tool? What is the main finding and its importance? Imipramine was validated as a more efficient and less toxic detubulating agent of cardiomyocytes than formamide. New insights are provided on how PI(4,5)P2 is crucial to maintaining T-tubule attachment to the cell surface and on the cardiotoxic effects of imipramine overdoses. ABSTRACT Cardiac T-tubules are membrane invaginations essential for excitation-contraction coupling (ECC). Imipramine, like other cationic amphiphilic drugs, interferes with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) interactions with proteins maintaining the tubular system connected to the 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 amplitude by ∼72%. These parameters were not affected in atrial cells, excluding direct side effects of imipramine. β-Adrenergic receptor (β-AR) stimulation of the remaining ICa,L with isoproterenol (Iso) was still effective. ECC was investigated in ARVMs loaded with Fura-2 and paced at 1 Hz, allowing simultaneous measurement of the Ca2+ transient (CaT) and sarcomere shortening (SS). Amplitude of both CaT and SS was decreased by imipramine and partially restored by Iso. Furthermore, detubulated cells exhibited Ca2+ homeostasis perturbations. Real-time cAMP variations induced by Iso using a Forster resonance energy transfer biosensor revealed ∼27% decreased cAMP elevation upon β-AR stimulation. To conclude, we validated a new cardiomyocyte detubulation method using imipramine, which is more efficient and less toxic than formamide. This antidepressant agent induces the hallmark effects of detubulation on ECC and its β-AR stimulation. Besides, we provide new insights on how an imipramine overdose may affect cardiac function and suggest that PI(4,5)P2 is crucial for maintaining T-tubule structure.

Aims Cyclic AMP phosphodiesterases (PDEs) are important modulators of the cardiac response to β-adrenergic receptor (β-AR) stimulation. PDE3 is classically considered as the major cardiac PDE in large mammals and human, while PDE4 is preponderant in rodents. However, it remains unclear whether PDE4 also plays a functional role in large mammals. Our purpose was to understand the role of PDE4 in cAMP hydrolysis and excitation-contraction coupling (ECC) in the pig heart, a relevant pre-clinical model. Methods and results Real-time cAMP variations were measured in isolated adult pig right ventricular myocytes (APVMs) using a Forster resonance energy transfer (FRET) biosensor. ECC was investigated in APVMs loaded with Fura-2 and paced at 1 Hz allowing simultaneous measurement of intracellular Ca2+ and sarcomere shortening. The expression of the different PDE4 subfamilies was assessed by Western blot in pig right ventricles and APVMs. Similarly to PDE3 inhibition with cilostamide (Cil), PDE4 inhibition with Ro 20-1724 (Ro) increased cAMP levels and inotropy under basal conditions. PDE4 inhibition enhanced the effects of the non-selective β-AR agonist isoprenaline (Iso) and the effects of Cil, and increased spontaneous diastolic Ca2+ waves (SCWs) in these conditions. PDE3A, PDE4A, PDE4B and PDE4D subfamilies are expressed in pig ventricles. In APVMs isolated from a porcine model of repaired tetralogy of Fallot which leads to right ventricular failure, PDE4 inhibition also exerts inotropic and pro-arrhythmic effects. Conclusions Our results show that PDE4 controls ECC in APVMs and suggest that PDE4 inhibitors exert inotropic and pro-arrhythmic effects upon PDE3 inhibition or β-AR stimulation in our pre-clinical model. Thus, PDE4 inhibitors should be used with caution in clinics as they may lead to arrhythmogenic events upon stress.

International audience; The cyclic nucleotide 3′,5′-cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger of paramount importance in the regulation of the cardiac pump. It is now well established that cAMP is confined in specific subcellular compartments where it modulates various targets associated in signalosomes to control, for example, excitation–contraction coupling or gene transcription. In this review, we summarize notable breakthroughs on how cAMP is synthesized, degraded and compartmentalized. We describe its effectors, including newly described targets with emerging roles in heart. We also briefly discuss innovative methods recently developed to bring new insights on the role of these cAMP microdomains to control cardiac function.

Chronic beta-AR activation is detrimental because it promotes cardiac remodeling and ultimately leads to heart failure (HF). Multiple cyclic nucleotide phosphodiesterases (PDEs) finely tune beta-AR responses by degrading and compartmentalizing cAMP. Since chronic treatment with PDE inhibitors increases mortality in HF, we postulated that decreasing cAMP levels by overexpressing PDE4B in the heart may have therapeutic effects. To test this hypothesis, we explored whether AAV9-mediated cardiac overexpression of PDE4B (1012 viral particles) could prevent maladaptive hypertrophy in mice subjected either to transverse aortic constriction (TAC) for 6 weeks or 2 weeks isoproterenol (Iso) and phenylephrine (Phe) infusion (30μg/g/day each). Cardiac function was assessed by echocardiography. In control mice injected with a Luciferase-AAV9 (LUC), TAC decreased ejection fraction (EF, - 34.2 ± 6%, N = 6, P

International audience; Cyclic nucleotide phosphodiesterases (PDEs) degrade the second messengers cAMP and cGMP, thereby regulating multiple aspects of cardiac function. This highly diverse class of enzymes encoded by 21 genes encompasses 11 families which 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 myocardium, the PDE3 and PDE4 families are predominant to degrade cAMP and thereby regulate cardiac excitation-contraction coupling. PDE3 inhibitors are positive inotropes and vasodilators in human, but their use is limited to acute heart failure and intermittent claudication. PDE5 is particularly important to degrade cGMP in vascular smooth muscle, and PDE5 inhibitors are used to treat erectile dysfunction and pulmonary hypertension. However, these drugs do not seem efficient in heart failure with preserved ejection fraction. There is experimental evidence that these PDEs as well as other PDE families including PDE1, PDE2 and PDE9 may play important roles in cardiac diseases such as hypertrophy and heart failure. After a brief presentation of the cyclic nucleotide pathways in cardiac cells and the major characteristics of the PDE superfamily, this chapter will present their role in cyclic nucleotide compartmentation and the current use of PDE inhibitors in cardiac diseases together with the recent research progresses that could lead to a better exploitation of the therapeutic potential of these enzymes in the future.

PDE3 is considered as the main isoform controlling cardiac function in large mammals. Recently, we showed that PDE4 controls cAMP levels upon β-adrenergic (βAR) regulation in human atrial [1] and canine ventricular cells [2] . However, it was reported that it does not influence contractility of human failing myoardium [3] . Thus, a role for PDE4 to control cardiac excitation-contraction coupling (ECC) in larger mammals is controversial. Here, we investigated the role of PDE4 to control cAMP levels and ECC in a porcine model of repaired tetralogy of Fallot (rToF), which leads to heart failure. PDE4A, 4B and 4D isoforms were detected in the heart, with decreased expression in rToF. Isolated right ventricular myocytes (RVMs) from control (Ctrl) and rToF pigs were loaded with 1 μM Fura-2 and paced at 1 Hz to record simultaneously Ca2+ transients and sarcomere shortening, or infected with an adenovirus encoding the cAMP FRET sensor epacH187 to evaluate intracellular cAMP levels. In Ctrl RVMs, inhibition of PDE3 with cilostamide (Cil, 1 μM) or PDE4 with Ro-201724 (Ro, 10 μM) modestly affected basal cAMP levels while concomitant inhibition of both enzymes had a strong influence (+130 ± 14%, n = 12, P

The cAMP-dependent protein kinase (PKA) consists of two regulatory (R) and two catalytic (C) subunits and comprises two subtypes, PKAI and PKAII, defined by the nature of their regulatory subunits, RIα and RIIα respectively. Whereas PKAII is thought to play a key role in β-adrenergic (β-AR) regulation of cardiac contractility, the function of PKAI is unclear. To address this question, we generated mice with cardiomyocyte-specific and conditional invalidation of the RIα subunit of PKA. Tamoxifen injection in 8 weeks-old mice resulted in a > 70% decrease in RIα protein without modification of other PKA subunits, which was associated with ∼2-fold increased basal PKA activity in RIα-KO mice (P 2-fold (P

Cyclic nucleotide phosphodiesterases (PDEs) degrade the second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), thereby regulating multiple aspects of cardiac and vascular muscle functions. 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 and vascular smooth muscle, the PDE3 and PDE4 families predominate, degrading cAMP and thereby regulating cardiac excitation-contraction coupling and smooth muscle contractile tone. PDE3 inhibitors are positive inotropes and vasodilators in humans, but their use is limited to acute heart failure and intermittent claudication. PDE5 is particularly important for the degradation of cGMP in vascular smooth muscle, and PDE5 inhibitors are used to treat erectile dysfunction and pulmonary hypertension. There is experimental evidence that these PDEs, as well as other PDE families, including PDE1, PDE2 and PDE9, may play important roles in cardiac diseases, such as hypertrophy and heart failure, as well as several vascular diseases. After a brief presentation of the cyclic nucleotide pathways in cardiac and vascular cells, and the major characteristics of the PDE superfamily, this review will focus on the current use of PDE inhibitors in cardiovascular diseases, and the recent research developments that could lead to better exploitation of the therapeutic potential of these enzymes in the future.

Cyclic 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.

Cyclic 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.

Objectives This study was designed to examine whether a cyclic adenosine monophosphate (cAMP) phosphodiesterase (PDE), PDE4, is expressed in human atrium and contributes to the control of electrical stability. Background Atrial fibrillation is accompanied by a profound remodeling of membrane receptors and alterations in cAMP-dependent regulation of Ca2+ handling. Being responsible for cAMP hydrolysis, PDEs are likely to play a role in this setting. In the rodent heart, PDE4 contributes up to 60% of total cAMP-hydrolytic activity. However, its role in the human heart remains controversial. Methods L-type Ca2+ current and spontaneous Ca2+ release were recorded in isolated human atrial myocytes. Intracellular cAMP was measured by live cell imaging using a fluorescence resonance energy transfer-based sensor. Contractile force and arrhythmias were recorded in human atrial trabeculae. PDE activity was measured in human atrial tissue from patients in sinus rhythm and permanent atrial fibrillation. Results PDE4 is expressed in human atrial myocytes and accounts for approximately 15% of total PDE activity. PDE4D represents the major PDE4 subtype. PDE4 inhibition increased intracellular cAMP and L-type Ca2+ current and dramatically delayed their decay after a brief beta-adrenergic stimulation. PDE4 inhibition also increased the frequency of spontaneous Ca2+ release at baseline, as well as the contractile response and the incidence of arrhythmias in human atrial strips during beta-adrenergic stimulation. Total PDE activity decreased with age, and the relative PDE4 activity was lower in patients with permanent atrial fibrillation than in age-matched sinus rhythm controls. Conclusions PDE4 is critical in controlling cAMP levels and thereby Ca2+ influx and release in human atrial muscle, hence limiting the susceptibility to arrhythmias. (J Am Coll Cardiol 2012;59:2182-90) (C) 2012 by the American College of Cardiology Foundation

The role(s) of the novel stargazin-like γ-subunit proteins remain controversial. We have shown previously that the neuron-specific γ7 suppresses the expression of certain calcium channels, particularly CaV2.2, and is therefore unlikely to operate as a calcium channel subunit. We now show that the effect of γ7 on CaV2.2 expression is via an increase in the degradation rate of CaV2.2 mRNA and hence a reduction of CaV2.2 protein level. Furthermore, exogenous expression of γ7 in PC12 cells also decreased the endogenous CaV2.2 mRNA level. Conversely, knockdown of endogenous γ7 with short-hairpin RNAs produced a reciprocal enhancement of CaV2.2 mRNA stability and an increase in endogenous calcium currents in PC12 cells. Moreover, both endogenous and expressed γ7 are present on intracellular membranes, rather than the plasma membrane. The cytoplasmic C terminus of γ7 is essential for all its effects, and we show that γ7 binds directly via its C terminus to a heterogeneous nuclear ribonucleoprotein (hnRNP A2), which also binds to a motif in CaV2.2 mRNA, and is associated with native CaV2.2 mRNA in PC12 cells. The expression of hnRNP A2 enhances CaV2.2IBa, and this enhancement is prevented by a concentration of γ7 that alone has no effect onIBa. The effect of γ7 is selective for certain mRNAs because it had no effect on α2δ-2 mRNA stability, but it decreased the mRNA stability for the potassium-chloride cotransporter, KCC1, which contains a similar hnRNP A2 binding motif to that in CaV2.2 mRNA. Our results indicate that γ7 plays a role in stabilizing CaV2.2 mRNA.

CaVbeta subunits of voltage-gated calcium channels contain two conserved domains, a src-homology-3 (SH3) domain and a guanylate kinase-like (GK) domain with an intervening HOOK domain. We have shown in a previous study that, although Gbetagamma-mediated inhibitory modulation of CaV2.2 channels did not require the interaction of a CaVbeta subunit with the CaValpha1 subunit, when such interaction was prevented by a mutation in the alpha1 subunit, G protein modulation could not be removed by a large depolarization and showed voltage-independent properties (Leroy et al., J Neurosci 25:6984-6996, 2005). In this study, we have investigated the ability of mutant and truncated CaVbeta subunits to support voltage-dependent G protein modulation in order to determine the minimal domain of the CaVbeta subunit that is required for this process. We have coexpressed the CaVbeta subunit constructs with CaV2.2 and alpha2delta-2, studied modulation by the activation of the dopamine D2 receptor, and also examined basal tonic modulation. Our main finding is that the CaVbeta subunit GK domains, from either beta1b or beta2, are sufficient to restore voltage dependence to G protein modulation. We also found that the removal of the variable HOOK region from beta2a promotes tonic voltage-dependent G protein modulation. We propose that the absence of the HOOK region enhances Gbetagamma binding affinity, leading to greater tonic modulation by basal levels of Gbetagamma. This tonic modulation requires the presence of an SH3 domain, as tonic modulation is not supported by any of the CaVbeta subunit GK domains alone.

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.

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 Ca(2+) signalling in cardiac myocytes. In this study, we analysed the effects of Epac activation on Ca(2+) handling by using confocal microscopy in isolated adult rat cardiomyocytes. [Ca(2+)](i) transients were evoked by electrical stimulation and Ca(2+) 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). [Ca(2+)](i) transients were slightly reduced by 10 microm 8-CPT (F/F(0): 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 [Ca(2+)](i) transients. Instead, a decrease in the sarcoplasmic reticulum (SR) Ca(2+) load underlies the decrease in the [Ca(2+)](i) transients. This decrease in the SR Ca(2+) load was provoked by the increase in the SR Ca(2+) leak induced by Epac activation. 8-CPT significantly increased Ca(2+) spark frequency (Ca(2+) sparks s(-1) (100 microm)(-1): from 2.4 +/- 0.6 to 6.9 +/- 1.5, P < 0.01) while reducing their amplitude (F/F(0): 1.8 +/- 0.02 versus 1.6 +/- 0.01, P < 0.001) in a Ca(2+)/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 Ca(2+) release and signalling in cardiac myocytes.

International audience; Aims: A major concern of using phosphodiesterase (PDE) inhibitors in heart failure is their potential to increase mortality by inducing arrhythmias. By diminishing cyclic adenosine monophosphate (cAMP) hydrolysis, they promote protein kinase A (PKA) activity under β-adrenergic receptor (β-AR) stimulation, hence enhancing Ca2+ cycling and contraction. Yet, cAMP also activates CaMKII via PKA or the exchange protein Epac, but it remains unknown whether these pathways are involved in the pro-arrhythmic effect of PDE inhibitors.Methods and results: Excitation–contraction coupling was investigated in isolated adult rat ventricular myocytes loaded with Fura-2 and paced at 1 Hz allowing coincident measurement of intracellular Ca2+ and sarcomere shortening. The PDE4 inhibitor Ro 20-1724 (Ro) promoted the inotropic effects of the non-selective β-AR agonist isoprenaline (Iso) and also spontaneous diastolic Ca2+ waves (SCWs). PDE4 inhibition potentiated RyR2 and PLB phosphorylation at specific PKA and CaMKII sites increasing sarcoplasmic reticulum (SR) Ca2+ load and SR Ca2+ leak measured in a 0Na+/0Ca2+ solution ± tetracaine. PKA inhibition suppressed all the effects of Iso ± Ro, whereas CaMKII inhibition prevented SR Ca2+ leak and diminished SCW incidence without affecting the inotropic effects of Ro. Inhibition of Epac2 but not Epac1 diminished the occurrence of SCWs. PDE3 inhibition with cilostamide induced an SR Ca2+ leak, which was also blocked by CaMKII inhibition.Conclusion: Our results show that PDE inhibitors exert inotropic effects via PKA but lead to SCWs via both PKA and CaMKII activation partly via Epac2, suggesting the potential use of CaMKII inhibitors as adjuncts to PDE inhibition to limit their pro-arrhythmic effects.

BackgroundRight ventricular (RV) dysfunction is a major determinant of long-term survival in congenital heart diseases. Early echocardiographic detection of RV failure is mandatory, but recent parameters need to be validated.AimsObjectives were to: (1) validate standard and strain echocardiographic parameters for evaluation of RV systolic function, compared to hemodynamic parameters; (2) assess the accuracy of these parameters for early detection of RV failure.MethodsCombined RV overload as observed in repaired tetralogy of Fallot was surgically reproduced in 2-month-old piglets (n=6). Age-matched piglets were used as controls (n=4). RV function was evaluated at baseline and 4 months of follow-up by standard and strain echocardiographic parameters, compared to hemodynamic (conductance catheter). Sarcomere shortening and calcium transients were recorded in RV isolated myocytes (IonOptix). Contractile reserve was assessed by in-vivo (dobutamine 5奯kg) and ex-vivo (isoprenaline 100nM) ?-adrenergic stimulation.Results4 months after surgery, hemodynamic RV ejection fraction (FEVD) was significantly decreased (29.7% [26.2-34] vs 42.9% [40.7-48.6], p

β-adrenoceptors (β-AR) stimulation increases cardiac function by rising cAMP that activates protein kinase A (PKA) and enhances Ca2+-induced Ca2+- release by phosphorylating proteins such as ryanodine receptors and phospholamban, which are also targets of the Ca2+/Calmodulin Kinase II (CaMKII). Any dysregulation of the β-AR pathway promotes arrhythmias. Local cAMP concentration in heart is mainly regulated by phosphodiesterases (PDE) type 3 and 4. Here, we investigated the proarrhythmic effects of PDE3 and PDE4 inhibition and evaluated the relative contribution of PKA and CaMKII to these mechanisms. An IonOptix system was used to record intracellular Ca2+ transients in isolated adult rat and pig ventricular myocytes (ARVMs and APVMs) loaded with 1μM Fura-2AM and paced at 1Hz. In ARVMs, PDE4 inhibition with Ro20-1724 (Ro, 10μM) potentiated the inotropic effect of the β-AR agonist isoproterenol (Iso, 1nM) but induced spontaneous Ca2+ waves (SCWs) recorded during 10s pacing pauses (2.3±0.2 SCWs/10s, n=15; p

The CaVβ subunits of voltage-gated calcium channels regulate these channels in several ways. Here we investigate the role of these auxiliary subunits in the expression of functional N-type channels at the plasma membrane and in the modulation by G-protein-coupled receptors of this neuronal channel. To do so, we mutated tryptophan 391 to an alanine within the α-interacting domain (AID) in the I-II linker of CaV2.2. We showed that the mutation W391 virtually abolishes the binding of CaVβ1b and CaVβ2a to the CaV2.2 I-II linker and strongly reduced current density and cell surface expression of both CaV2.2/α2δ-2/β1b and/β2a channels. When associated with CaVβ1b, the W391A mutation also prevented the CaVβ1b-mediated hyperpolarization of CaV2.2 channel activation and steady-state inactivation. However, the mutated CaV2.2W391A/β1b channels were still inhibited to a similar extent by activation of the D2dopamine receptor with the agonist quinpirole. Nevertheless, key hallmarks of G-protein modulation of N-type currents, such as slowed activation kinetics and prepulse facilitation, were not observed for the mutated channel. In contrast, CaVβ2a was still able to completely modulate the biophysical properties of CaV2.2W391A channel and allow voltage-dependent G-protein modulation of CaV2.2W391A. Additional data suggest that the concentration of CaVβ2a in the proximity of the channel is enhanced independently of its binding to the AID by its palmitoylation. This is essentially sufficient for all of the functional effects of CaVβ2a, which may occur via a second lower-affinity binding site, except trafficking the channel to the plasma membrane, which requires interaction with the AID region.

Expression of the calcium channel Ca(V)2.2 is markedly suppressed by coexpression with truncated constructs of Ca(V)2.2. Furthermore, a two-domain construct of Ca(V)2.1 mimicking an episodic ataxia-2 mutation strongly inhibited Ca(V)2.1 currents. We have now determined the specificity of this effect, identified a potential mechanism, and have shown that such constructs also inhibit endogenous calcium currents when transfected into neuronal cell lines. Suppression of calcium channel expression requires interaction between truncated and full-length channels, because there is inter-subfamily specificity. Although there is marked cross-suppression within the Ca(V)2 calcium channel family, there is no cross-suppression between Ca(V)2 and Ca(V)3 channels. The mechanism involves activation of a component of the unfolded protein response, the endoplasmic reticulum resident RNA-dependent kinase (PERK), because it is inhibited by expression of dominant-negative constructs of this kinase. Activation of PERK has been shown previously to cause translational arrest, which has the potential to result in a generalized effect on protein synthesis. In agreement with this, coexpression of the truncated domain I of Ca(V)2.2, together with full-length Ca(V)2.2, reduced the level not only of Ca(V)2.2 protein but also the coexpressed alpha2delta-2. Thapsigargin, which globally activates the unfolded protein response, very markedly suppressed Ca(V)2.2 currents and also reduced the expression level of both Ca(V)2.2 and alpha2delta-2 protein. We propose that voltage-gated calcium channels represent a class of difficult-to-fold transmembrane proteins, in this case misfolding is induced by interaction with a truncated cognate Ca(V) channel. This may represent a mechanism of pathology in episodic ataxia-2.

There is growing evidence that Ca v 2.3 (α1E, E-type) transcripts may encode the ion-conducting subunit of a subclass of R-type Ca 2+ channels, a heterogeneous group of channels by definition resistant to blockers of L-, N-, and P/Q-type Ca 2+ channels. To understand whether splice variation of Ca v 2.3 contributes to the divergence of R-type channels, individual variants of Ca v 2.3 were constructed and expressed in HEK-293 cells. With Ba 2+ as charge carrier, the tested biophysical properties were similar. In Ca 2+ , the inactivation time course was slower and the recovery from short-term inactivation was faster; however, this occurred only in variants containing a 19-amino-acid-long insertion, which is typical for neuronal Ca v 2.3 Ca 2+ channel subunits. This different Ca 2+ sensitivity is not responsible for the major differences between various R-type channels, and future studies might clarify its importance for in vivo synaptic or dendritic integration and the reasons for its loss in endocrine Ca v 2.3 splice variants.

The effects of 10 mM caffeine (CAF) on intramembrane charge movements (ICM) were studied in isolated guinea-pig ventricular heart cells with the whole-cell patch-clamp technique. In the presence of CAF, the properties (voltage dependence, maximum QON[Qmax], availability with voltage) of QON charge activated from −110 mV were barely affected. Following a 100 ms prepulse to −50 mV to decrease the participation of charges originating from Na channels, the voltage dependence of QON was shifted by 5 mV (negative component) and by 10 mV (positive component) towards negative potentials, and Qmax was depressed by 16.5%. CAF drastically reduced in a time- and voltage-dependent manner QOFF on repolarization to −50 mV, the effects being greater at positive potentials. CAF-induced QOFF immobilization could be almost entirely removed by repolarization to voltages as negative as −170 mV. In these conditions, the voltage-dependence of QOFF (repolarization to +30 to −170 mV) was shifted by 17 mV (negative component) and 30 mV (positive component) towards negative potentials, suggesting an interconversion into charge 2. Most of CAF effects were suppressed when the sarcoplasmic reticulum (SR) was not functional or when the cells were loaded with BAPTA-AM. We conclude that CAF effects on ICM are likely due to Ca2+ ions released from the SR, and which accumulate in the subsarcolemmal fuzzy spaces in the vicinity of the Ca channels. Because CAF effects were more pronounced on QOFF than on QON the channels have likely to open before Ca2+ ions could affect their gating properties. It is speculated that such an effect on gating charges might contribute to the Ca-induced inactivation of the Ca current. British Journal of Pharmacology (2002) 135, 721–734; doi:10.1038/sj.bjp.0704520

Background Early detection of right ventricular (RV) failure is required to improve the management of patients with congenital heart diseases. The aim of this study was to validate echocardiography for the early detection of overloaded RV dysfunction, compared with hemodynamic and myocyte contractility assessment. Methods Using a porcine model reproducing repaired tetralogy of Fallot, RV function was evaluated over 4 months using standard echocardiography and speckle-tracking compared with hemodynamic parameters (conductance catheter). Sarcomere shortening and calcium transients were recorded in RV isolated myocytes. Contractile reserve (ΔE max ) was assessed by β-adrenergic stimulation in vivo (dobutamine 5 μg/kg) and ex vivo (isoproterenol 100 nM). Results Six operated animals were compared with four age- and sex-matched controls. In the operated group, hemodynamic RV efficient ejection fraction was significantly decreased (29.7% [26.2%–34%] vs 42.9% [40.7%–48.6%], P max was 51% vs 193%, P max ( r = 0.75, r = 0.78, and r = 0.65, respectively, P Conclusions In this porcine model, both standard and strain echocardiographic parameters detected early impairments of RV function and cardiac reserve, which were associated with cardiomyocyte excitation-contraction coupling alterations.

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, Ca 2+ transients, I Ca,L and I Ks 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 Ca 2+ ‐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 I Ca,L and I Ks 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 /dt max ≈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.

β-adrenergic receptor (β-AR) stimulation increases cardiac function by increasing cAMP levels and activating protein kinase A (PKA). PKA enhances Ca2+-induced Ca2+-release by phosphorylating L-type Ca2+ channels, ryanodine receptors and phospholamban (PLB) which are also targets of the Ca2+/Calmodulin Kinase II (CaMKII). Any dysregulation in the β-adrenergic pathway leads to cardiac arrhythmias. Multiple cyclic nucleotide phosphodiesterases (PDEs) regulate local concentrations of cAMP, among which the PDE4 family is overriding in rodent heart. We investigated the proarrhythmic effects of PDE4 inhibition and evaluated the relative contribution of PKA and CaMKII to this mechanism. Action potentials were recorded at a frequency of 1Hz in isolated adult rat ventricular myocytes using the patch-clamp technique. Delayed afterdepolarizations (DADs) observed upon application of the non selective β-AR agonist Isoproterenol (Iso 1nM) after cessation of electrical stimulation during 10s were potentiated by the PDE4 inhibitor Ro20-1724 (Ro 10µM). These DADs correlated with spontaneous calcium waves (SCWs), recorded in myocytes loaded with Fura-2AM (1µM) using an Ionoptix system. Ro potentiated the inotropic and lusitropic effects of Iso and furthered sarcoplasmic reticulum (SR) Ca2+ load leading to SR Ca2+ leak measured in a 0Na+,0Ca2+ solution ±1mM tetracaine. Upon PDE4 inhibition, PLB was phosphorylated not only by PKA but also by CaMKII demonstrating that both kinases were activated. PKA inhibition with H-89 (10µM) suppressed the SCWs and the inotropic and lusitropic effects of Iso±Ro. CaMKII inhibition with KN-93 (10µM) diminished SCWs incidence by 72% without affecting the inotropic effects of Ro. Thus, upon β-AR stimulation, PDE4 inhibition exerts positive inotropic effects via PKA but induces SCWs and DADs via both PKA and CaMKII activation, suggesting the potential use of CaMKII inhibitors as an adjunct to PDE inhibition in cardiac diseases to limit arrhythmias.

Objectives This study investigated whether myocardial phosphodiesterase-2 (PDE2) is altered in heart failure (HF) and determined PDE2-mediated effects on beta-adrenergic receptor (β-AR) signaling in healthy and diseased cardiomyocytes. Background Diminished cyclic adenosine monophosphate (cAMP) and augmented cyclic guanosine monophosphate (cGMP) signaling is characteristic for failing hearts. Among the PDE superfamily, PDE2 has the unique property of being able 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 Immunoblotting, radioenzymatic- and fluorescence resonance energy transfer–based assays, video edge detection, epifluorescence microscopy, and L-type Ca2+ current measurements were performed in myocardial tissues and/or isolated cardiomyocytes from human and/or experimental HF, respectively. Results 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, which correlated with blunted cardiac β-AR responsiveness. In diseased cardiomyocytes, higher PDE2 activity could be further enhanced by stimulation of cGMP synthesis via nitric oxide donors, whereas specific PDE2 inhibition partially restored β-AR responsiveness. Accordingly, PDE2 overexpression in healthy cardiomyocytes reduced the rise in cAMP levels and L-type Ca2+ current amplitude, and abolished the 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 up-regulated in failing hearts and desensitizes against acute β-AR stimulation. This may constitute an important defense mechanism during cardiac stress, for example, by antagonizing excessive β-AR drive. Thus, activating myocardial PDE2 may represent a novel intracellular antiadrenergic therapeutic strategy in HF.

Cyclic nucleotide phosphodiesterases (PDE) represent a superfamily of enzymes specialised in the degradation of cAMP and cGMP. In heart, PDE3 and PDE4 are the two major families involved in the regulation of cAMP levels and the control of inotropism. Both families are encoded by several genes, and the recent analysis of the cardiac phenotype of mice lacking these different genes provided new insights into the way they regulate excitation-contraction coupling (ECC). In particular, these studies emphasize the local character of ECC regulation by PDE, as well as the role of these PDE in maintaining calcium homeostasis and preventing cardiac arrhythmias.

International audience; In cardiac cells, the L-type calcium channel (LTCC) current or ICa,L underlies the plateau phase of the action potential. Upon depolarisation, ICa,L reflects calcium influx via the CaV1.2 channels. This current initiates cardiac contraction by gating the ryanodine receptor, therefore triggering the calcium release from the sarcoplasmic reticulum. 1 Among several regulatory pathways of this current, the best described is the β-adrenergic stimulation which contributes to the positive inotropic effects of catecholamines. To date, three β-adrenergic receptors (β-AR), respectively β1-AR, β-AR and β3-AR, have been cloned 2 and this major achievement has led to the 2012 Nobel prize award to Robert Lefkowitz and Brian Kobilka who paved the road to our current understanding of their structures and functions. The classical pathway for β-AR receptor signaling is activation of adenylyl cyclases via Gαs, resulting in increased intracellular cAMP levels. The primary target of cAMP is the cAMP-dependent protein kinase (PKA) that in turn phosphorylates the CaV1.2 channels among other key proteins of the excitation-contraction coupling (figure 1). While direct modulation by G proteins of LTCCs was first suspected to partially mediate the upregulation of ICa,L upon β-AR activation, it has been clearly established that a cAMP phosphorylation mediated by PKA is responsible for this increase. 3,4 This chapter reviews the literature on the β-AR regulation of LTCC, with emphasis on recent informations on the molecular mechanisms of PKA regulation of CaV1.2 channels

Phosphodiesterase 2 (PDE2) is a dual substrate enzyme, hydrolyzing both cAMP and cGMP. We showed that myocardial PDE2 is upregulated in human and experimental heart failure (HF) while PDE3 and PDE4 are reduced. To explore the pathophysiological consequences of enhanced PDE2 activity, transgenic mice with a heart-specific overexpression of the PDE2A3 isoform (PDE2-TG) were generated.PDE2 activity was measuredin heart extractsby radioenzymatic assay. Sarcomere shortening, Ca2+ transients and L-type Ca2+ current (ICa,L) were recorded in adult ventricular cardiomyocytes from wild-type (WT) and PDE2-TG mice. SR Ca2+ leak was estimated with tetracaine (RyR blocker) and spontaneous Ca2+ waves (SCW) were recorded during 30s pacing pause. Intracellular cAMP level was measured with FRET. Phosphorylation level of excitation-contraction coupling (ECC) proteins was determined by Western Blot. Heart function was investigated by echocardiography and ECG-telemetry. Isoprenaline (ISO) was used to compare β-adrenergic (β-AR) response of all parameters in WT and PDE2-TG mice.cAMP and cGMP-PDE2 activity was strongly increased in PDE2-TG as compared to WT mice. Resting and maximal heart rate was markedly reduced in PDE2-TG mice. The β-AR stimulation of cell contractility, Ca2+ transient, ICa,L and [cAMP]i was severely blunted. PDE2-TG mice showed reduced SR Ca2+ leak and SCW (both at the cellular and in vivo levels), without changes in Ca2+ load as compared to WT during β-AR activation. PDE2-TG mice showed a lower basal phosphorylation of ECC proteins and have a longer lifespan than their WT littermates. All effects of PDE2 overexpression were reversed by PDE2 inhibitor, Bay-607550.Our results demonstrate that PDE2 plays a critical role in the regulation of cardiac ECC. PDE2 overexpression appears to protect the cardiomyocytes by reducing Ca2+-leakage and arrhythmias during β-AR stimulation. PDE2 may represent a new therapeutic strategy in heart failure.

Background— Phosphoinositide 3-kinase γ (PI3Kγ) signaling engaged by β-adrenergic receptors is pivotal in the regulation of myocardial contractility and remodeling. However, the role of PI3Kγ in catecholamine-induced arrhythmia is currently unknown. Methods and Results— Mice lacking PI3Kγ (PI3Kγ −/− ) showed runs of premature ventricular contractions on adrenergic stimulation that could be rescued by a selective β 2 -adrenergic receptor blocker and developed sustained ventricular tachycardia after transverse aortic constriction. Consistently, fluorescence resonance energy transfer probes revealed abnormal cAMP accumulation after β 2 -adrenergic receptor activation in PI3Kγ −/− cardiomyocytes that depended on the loss of the scaffold but not of the catalytic activity of PI3Kγ. Downstream from β-adrenergic receptors, PI3Kγ was found to participate in multiprotein complexes linking protein kinase A to the activation of phosphodiesterase (PDE) 3A, PDE4A, and PDE4B but not of PDE4D. These PI3Kγ-regulated PDEs lowered cAMP and limited protein kinase A–mediated phosphorylation of L-type calcium channel (Ca v 1.2) and phospholamban. In PI3Kγ −/− cardiomyocytes, Ca v 1.2 and phospholamban were hyperphosphorylated, leading to increased Ca 2+ spark occurrence and amplitude on adrenergic stimulation. Furthermore, PI3Kγ −/− cardiomyocytes showed spontaneous Ca 2+ release events and developed arrhythmic calcium transients. Conclusions— PI3Kγ coordinates the coincident signaling of the major cardiac PDE3 and PDE4 isoforms, thus orchestrating a feedback loop that prevents calcium-dependent ventricular arrhythmia.

International audience; Reçu le 25 novembre 2011 Résumé-La voie de l'AMP cyclique (AMPc) joue un rôle majeur dans la régulation neuro-hormonale de la fonction cardiaque. Les travaux effectués ces dernières années ont montré que ce second messager n'est pas uniformément distribué à l'intérieur des myocytes cardiaques, et que cette compartimentation est nécessaire pour la définition des signaux AMPc générés par des récepteurs distincts au niveau de la membrane plasmique. La localisation des signaux AMPc est liée à celle des adénylate-cyclases, responsables de sa synthèse, mais aussi à sa diffusion dans le cytoplasme. La diffusion de l'AMPc peut être limitée par des structures intracellulaires mais aussi et surtout par les phosphodiestérases (PDE), qui sont à l'origine de sa dégradation. Cette revue fait le point sur le rôle des PDE dans la formation de microdomaines dynamiques d'AMPc impliqués dans la spécificité des hormones agissant sur le coeur. Mots clés-Régulation neuro-hormonale cardiaque / AMP cyclique / phosphodiestérases / compartimentation / insuffisance cardiaque Abstract-Role of cyclic nucleotide phosphodiesterases in the cAMP compartmentation in cardiac cells In the light of the knowledge accumulated over the years, it becomes clear that intracellular cAMP is not uniformly distributed within cardiomyocytes and that cAMP compartmentation is required for adequate processing and targeting of the information generated at the membrane. Localized cAMP signals may be generated by interplay between discrete production sites and restricted diffusion within the cytoplasm. In addition to specialized membrane structures that may limit cAMP spreading, degradation of the second messenger by cyclic nucleotide phosphodiesterases (PDEs) appears critical for the formation of dynamic microdomains that confer specificity of the response to various hormones. This review summarizes the main findings that support the cAMP compartmentation hypothesis in cardiac cells, with a special emphasis on PDEs. The respective roles of the four main cardiac cAMP-PDE families (PDE1 to PDE4) in the organization of cAMP microdomains and hormonal specificity in cardiac cells are reviewed. The evidence that these PDEs are modified in heart failure is summarized, and the implication for the progression of the disease is discussed. Finally, the potential benefits that could be awaited from the manipulation of specific PDE subtypes in heart failure are presented.