Differential changes in cyclic adenosine 3'-5' monophosphate (cAMP) effectors and major Ca2+ handling proteins during diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM) is associated with differential and time-specific regulation of ß-adrenergic receptors and cardiac cyclic nucleotide phosphodiesterases with consequences for total cyclic adenosine 3'-5' monophosphate (cAMP) levels. We aimed to investigate whether these changes are associated with downstream impairments in cAMP and Ca2+ signalling in a type 1 diabetes (T1D)-induced DCM model. T1D was induced in adult male rats by streptozotocin (65 mg/kg) injection. DCM was assessed by cardiac structural and molecular remodelling. We delineated sequential changes affecting the exchange protein (Epac1/2), cAMP-dependent protein kinase A (PKA) and Ca2+ /Calmodulin-dependent kinase II (CaMKII) at 4, 8 and 12 weeks following diabetes, by real-time quantitative PCR and western blot. Expression of Ca2+ ATPase pump (SERCA2a), phospholamban (PLB) and Troponin I (TnI) was also examined. Early upregulation of Epac1 transcripts was noted in diabetic hearts at Week 4, followed by increases in Epac2 mRNA, but not protein levels, at Week 12. Expression of PKA subunits (RI, RIIα and Cα) remained unchanged regardless of the disease stage, whereas CaMKII increased at Week 12 in DCM. Moreover, PLB transcripts were upregulated in diabetic hearts, whereas SERCA2a and TnI gene expression was unchanged irrespective of the disease evolution. PLB phosphorylation at threonine-17 was increased in DCM, whereas phosphorylation of both PLB at serine-16 and TnI at serine-23/24 was unchanged. We show for the first time differential and time-specific regulations in cardiac cAMP effectors and Ca2+ handling proteins, data that may prove useful in proposing new therapeutic approaches in T1D-induced DCM.

Phosphodiesterase type 4 anchoring regulates cAMP signaling to Popeye domain-containing proteins.

Cyclic AMP is a ubiquitous second messenger used to transduce intracellular signals from a variety of Gs-coupled receptors. Compartmentalisation of protein intermediates within the cAMP signaling pathway underpins receptor-specific responses. The cAMP effector proteins protein-kinase A and EPAC are found in complexes that also contain phosphodiesterases whose presence ensures a coordinated cellular response to receptor activation events. Popeye domain containing (POPDC) proteins are the most recent class of cAMP effectors to be identified and have crucial roles in cardiac pacemaking and conduction. We report the first observation that POPDC proteins exist in complexes with members of the PDE4 family in cardiac myocytes. We show that POPDC1 preferentially binds the PDE4A sub-family via a specificity motif in the PDE4 UCR1 region and that PDE4s bind to the Popeye domain of POPDC1 in a region known to be susceptible to a mutation that causes human disease. Using a cell-permeable disruptor peptide that displaces the POPDC1-PDE4 complex we show that PDE4 activity localized to POPDC1 modulates cycle length of spontaneous Ca2+ transients firing in intact mouse sinoatrial nodes.

Cardiac Phosphodiesterases Are Differentially Increased in Diabetic Cardiomyopathy.

AIM: Diabetic cardiomyopathy (DCM) accomodates a spectrum of cardiac abnormalities. This study aims to investigate whether DCM is associated with changes in cyclic adenosine 3'-5' monophosphate (cAMP) signaling, particularly cyclic nucleotide phosphodiesterases (PDEs). MAIN METHODS: Type 1 diabetes (T1D) was induced in rats by streptozotocin (STZ, 65 mg/kg) injection. Myocardial remodeling, structure and function were evaluated by histology and echocardiography, respectively. We delineated the sequential changes affecting cAMP signaling and characterized the expression pattern of the predominant cardiac PDE isoforms (PDE 1-5) and ß-adrenergic (ß-AR) receptors at 4, 8 and 12 weeks following diabetes induction, by real-time quantitative PCR and Western blot. cAMP levels were measured by immunoassays. KEY FINDINGS: T1D-induced DCM was associated with cardiac remodeling, steatosis and fibrosis. Upregulation of ß1-AR receptor transcripts was noted in diabetic hearts at 4 weeks along with an increase in cAMP levels and an upregulation in the ejection fraction and fraction shortening. However, ß2-AR receptors expression remained unchanged regardless of the disease stage. Moreover, we noted an early and specific upregulation of cardiac PDE1A, PDE2A, PDE4B, PDE4D and PDE5A expression at week 4, followed by increases in PDE3A levels in diabetic hearts at week 8. However, DCM was not associated with changes in PDE4A gene expression irrespective of the disease stage. SIGNIFICANCE: We show for the first time differential and time-specific regulations in cardiac PDEs, data that may prove useful in proposing new therapeutic approaches in T1D-induced DCM.

Selective changes in cytosolic ß-adrenergic cAMP signals and L-type Calcium Channel regulation by Phosphodiesterases during cardiac hypertrophy.

Background In cardiomyocytes, phosphodiesterases (PDEs) type 3 and 4 are the predominant enzymes that degrade cAMP generated by ß-adrenergic receptors (ß-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 20-1724 (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 < 0.01). In contrast, PDE3 inhibition with cilostamide (1 µM) had no effect on the amplitude of the cAMP transient and a minimal effect on its recovery in SHAM, whereas it potentiated the amplitude and slowed the decay in CH (t1/2off ~ 80 s). Iso pulse stimulation also elicited a similar transient increase in ICa,L in SHAM and CH, although the duration of the rising phase was delayed in CH. Inhibition of PDE3 or PDE4 potentiated ICa,L amplitude in SHAM but not in CH. Besides, while only PDE4 inhibition slowed down the decline of ICa,L in SHAM, both PDE3 and PDE4 contributed in CH. Conclusion These results identify selective alterations in cytosolic cAMP and ICa,L regulation by PDE3 and PDE4 in CH, and show that the balance between PDE3 and PDE4 for the regulation of ß-AR responses is shifted toward PDE3 during CH.

Cardiac Overexpression of PDE4B Blunts ß-Adrenergic Response and Maladaptive Remodeling in Heart Failure.

BACKGROUND: The cyclic AMP (adenosine monophosphate; cAMP)-hydrolyzing protein PDE4B (phosphodiesterase 4B) is a key negative regulator of cardiac ß-adrenergic receptor stimulation. PDE4B deficiency leads to abnormal Ca2+ 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 Ca2+ current by whole-cell patch-clamp, and cardiomyocyte shortening and Ca2+ 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 ß-adrenergic receptor stimulation of cardiac inotropy, cAMP, PKA, L-type Ca2+ current, Ca2+ 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 (1012 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.

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

AIMS: Cyclic AMP phosphodiesterases (PDEs) are important modulators of the cardiac response to ß-adrenergic receptor (ß-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 Förster 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 ß-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 ß-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.

PDE2 regulates membrane potential, respiration and permeability transition of rodent subsarcolemmal cardiac mitochondria.

Cyclic adenosine monophosphate (cAMP) production regulates certain aspects of mitochondria function in rodent cardiomyocytes, such as ATP production, oxygen consumption, calcium import and mitochondrial permeability transition (MPT), but how this cAMP pool is controlled is not well known. Here, expression, localization and activity of several cAMP-degrading enzymes, i.e. phosphodiesterases (PDEs), were investigated in isolated rodent cardiac mitochondria. In contrast to the heart ventricle where PDE4 is the major PDE, in cardiac mitochondria, cGMP-stimulated PDE2 activity was largest than PDE3 and PDE4 activities. PDE2 expression was mainly detected in subsarcolemmal mitochondria in association with the inner membrane rather than in interfibrillar mitochondria. PDE2, 3 and 4 activities were further confirmed in neonatal rat cardiomyocytes by real time FRET analysis. In addition, the pharmacological inhibition or the cardiac-specific overexpression of PDE2 modulated mitochondrial membrane potential loss, MPT and calcium import. In mitochondria isolated from PDE2 transgenic mice with a cardiac selective PDE2 overexpression, the oxidative phosphorylation (OXPHOS) was significantly lower than in wild-type mice, but stimulated by cGMP. Thus, cAMP degradation by PDEs represents a new regulatory mechanism of mitochondrial function.

Cyclic nucleotide signalling compartmentation by PDEs in cultured vascular smooth muscle cells.

BACKGROUND AND PURPOSE: Up-regulation of phosphodiesterases (PDEs) is associated with several vascular diseases, and better understanding of the roles of each PDE isoform in controlling subcellular pools of cyclic nucleotides in vascular cells is needed. We investigated the respective role of PDE1, PDE5, and PDE9 in controlling intracellular cAMP and/or cGMP concentrations ([cAMP]i , [cGMP]i ) in cultured rat aortic smooth muscle cells (RASMCs). EXPERIMENTAL APPROACH: We used selective inhibitors of PDE1 (PF-04471141), PDE5 (sildenafil), and PDE9 (PF-04447943) to measure cAMP- and cGMP-PDE activities with a radioenzymatic assay, in RASMC extracts. Real-time [cAMP]i and [cGMP]i were recorded by Förster resonance energy transfer-imaging in single living cells, and cell proliferation was assessed in FBS-stimulated cells. KEY RESULTS: PDE1, PDE5, and PDE9 represented the major cGMP-hydrolyzing activity in RASMCs. Basal PDE1 exerted a functional role in degrading in situ the cGMP produced in response to activation of particulate GC by C-type natriuretic peptide. In high intracellular Ca2+ concentrations, PDE1 also regulated the NO/soluble GC-dependent cGMP response, as well as the ß-adrenoceptor-mediated cAMP response. PDE5 exerted a major role in degrading cGMP produced by NO and the natriuretic peptides. PDE9 only regulated the NO-induced [cGMP]i increase. All three PDEs contributed differently to regulate cell proliferation under basal conditions and upon cGMP-elevating stimuli. CONCLUSIONS AND IMPLICATIONS: Our data emphasize the distinct roles of PDE1, PDE5, and PDE9 in local regulation of [cAMP]i and [cGMP]i , in vascular smooth muscle cells, strengthening the concept of PDEs as key actors in the subcellular compartmentation of cyclic nucleotides.

Cardiac adenylyl cyclase overexpression precipitates and aggravates age-related myocardial dysfunction.

AIMS: Increase of cardiac cAMP bioavailability and PKA activity through adenylyl-cyclase 8 (AC8) overexpression enhances contractile function in young transgenic mice (AC8TG). Ageing is associated with decline of cardiac contraction partly by the desensitization of ß-adrenergic/cAMP signalling. Our objective was to evaluate cardiac cAMP signalling as age increases between 2 months and 12 months and to explore whether increasing the bioavailability of cAMP by overexpression of AC8 could prevent cardiac dysfunction related to age. METHODS AND RESULTS: Cardiac cAMP pathway and contractile function were evaluated in AC8TG and their non-transgenic littermates (NTG) at 2- and 12 months old. AC8TG demonstrated increased AC8, PDE1, 3B and 4D expression at both ages, resulting in increased phosphodiesterase and PKA activity, and increased phosphorylation of several PKA targets including sarco(endo)plasmic-reticulum-calcium-ATPase (SERCA2a) cofactor phospholamban (PLN) and GSK3α/ß a main regulator of hypertrophic growth and ageing. Confocal immunofluorescence revealed that the major phospho-PKA substrates were co-localized with Z-line in 2-month-old NTG but with Z-line interspace in AC8TG, confirming the increase of PKA activity in the compartment of PLN/SERCA2a. In both 12-month-old NTG and AC8TG, PLN and GSK3α/ß phosphorylation was increased together with main localization of phospho-PKA substrates in Z-line interspaces. Haemodynamics demonstrated an increased contractile function in 2- and 12-month-old AC8TG, but not in NTG. In contrast, echocardiography and tissue Doppler imaging (TDI) performed in conscious mice unmasked myocardial dysfunction with a decrease of systolic strain rate in both old AC8TG and NTG. In AC8TG TDI showed a reduced strain rate even in 2-month-old animals. Development of age-related cardiac dysfunction was accelerated in AC8TG, leading to heart failure (HF) and premature death. Histological analysis confirmed early cardiomyocyte hypertrophy and interstitial fibrosis in AC8TG when compared with NTG. CONCLUSION: Our data demonstrated an early and accelerated cardiac remodelling in AC8TG mice, leading to the development of HF and reduced lifespan. Age-related reorganization of cAMP/PKA signalling can accelerate cardiac ageing, partly through GSK3α/ß phosphorylation.

[Heart rate: when macrophages hit the note]. / Fréquence cardiaque - Quand les macrophages battent la mesure.

Macrophages regulate cardiac homeostasis under pathological and physiological conditions. Recent studies have elegantly substantiated the presence of specific subset of macrophages residing within the distal atrioventricular node in mice and humans. These macrophages directly couple with cardiomyocytes via connexin-43-containing gap junctions and increase atrioventricular conduction by accelerating cardiomyocyte repolarization. Conditional deletion of connexin-43 in macrophages or congenital lack of macrophages delay nodal conduction and foster progressive atrioventricular block. Exhaustive understanding of the role of tissue-resident macrophages in normal and aberrant cardiac conduction could initiate the development of therapeutic strategies focused on the modulation of macrophage functions in heart arrhythmia.

PDE4 and mAKAPß are nodal organizers of ß2-ARs nuclear PKA signalling in cardiac myocytes.

Aims: ß1- and ß2-adrenergic receptors (ß-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 ß1- and ß2-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 ß1- or ß2-ARs stimulation. Both ß1- and ß2-AR stimulation increased cAMP and activated PKA in the cytoplasm. Although the two receptors also increased cAMP in the nucleus, only ß1-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 ß2-ARs. Both nuclear and cytoplasmic PKI prevented nuclear PKA activation and ICER induction by ß1-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 ß2-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 mAKAPß partially inhibited nuclear PKA activation upon ß1-AR stimulation, and drastically decreased nuclear PKA activation upon ß2-AR stimulation in the presence of PDE4 inhibition. Conclusions: ß1- and ß2-ARs differentially regulate nuclear PKA activity and ICER expression in cardiomyocytes. PDE4 insulates a mAKAPß-targeted PKA pool at the nuclear envelope that prevents nuclear PKA activation upon ß2-AR stimulation.

Heterologous desensitization of cardiac ß-adrenergic signal via hormone-induced ßAR/arrestin/PDE4 complexes.

AIMS: Cardiac ß-adrenergic receptor (ßAR) signalling is susceptible to heterologous desensitization by different neurohormonal stimuli in clinical conditions associated with heart failure. We aim to examine the underlying mechanism of cross talk between ßARs and a set of G-protein coupled receptors (GPCRs) activated by hormones/agonists. METHODS AND RESULTS: Rat ventricular cardiomyocytes were used to determine heterologous phosphorylation of ßARs under a series of GPCR agonists. Activation of Gs-coupled dopamine receptor, adenosine receptor, relaxin receptor and prostaglandin E2 receptor, and Gq-coupled α1 adrenergic receptor and angiotensin II type 1 receptor promotes phosphorylation of ß1AR and ß2AR at putative protein kinase A (PKA) phosphorylation sites; but activation of Gi-coupled α2 adrenergic receptor and activation of protease-activated receptor does not. The GPCR agonists that promote ß2AR phosphorylation effectively inhibit ßAR agonist isoproterenol-induced PKA phosphorylation of phospholamban and contractile function in ventricular cardiomyocytes. Heterologous GPCR stimuli have minimal to small effect on isoproterenol-induced ß2AR activation and G-protein coupling for cyclic adenosine monophosphate (cAMP) production. However, these GPCR stimuli significantly promote phosphorylation of phosphodiesterase 4D (PDE4D), and recruit PDE4D to the phosphorylated ß2AR in a ß-arrestin 2 dependent manner without promoting ß2AR endocytosis. The increased binding between ß2AR and PDE4D effectively hydrolyzes cAMP signal generated by subsequent stimulation with isoproterenol. Mutation of PKA phosphorylation sites in ß2AR, inhibition of PDE4, or genetic ablation of PDE4D or ß-arrestin 2 abolishes this heterologous inhibitory effect. Ablation of ß-arrestin 2 or PDE4D gene also rescues ß-adrenergic stimuli-induced myocyte contractile function. CONCLUSIONS: These data reveal essential roles of ß-arrestin 2 and PDE4D in a common mechanism for heterologous desensitization of cardiac ßARs under hormonal stimulation, which is associated with impaired cardiac function during the development of pathophysiological conditions.

[Cyclic nucleotide phosphodiesterases: role in the heart and therapeutic perspectives]. / Phosphodiestérases des nucléotides cycliques : rôle dans le cœur et potentiel thérapeutique.

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 in heart and vessels: A therapeutic perspective.

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.

Calmodulin kinase II inhibition limits the pro-arrhythmic Ca2+ waves induced by cAMP-phosphodiesterase inhibitors.

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 ß-adrenergic receptor (ß-AR) stimulation, hence enhancing Ca(2+) 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 Ca(2+) and sarcomere shortening. The PDE4 inhibitor Ro 20-1724 (Ro) promoted the inotropic effects of the non-selective ß-AR agonist isoprenaline (Iso) and also spontaneous diastolic Ca(2+) waves (SCWs). PDE4 inhibition potentiated RyR2 and PLB phosphorylation at specific PKA and CaMKII sites increasing sarcoplasmic reticulum (SR) Ca(2+) load and SR Ca(2+) leak measured in a 0Na(+)/0Ca(2+) solution ± tetracaine. PKA inhibition suppressed all the effects of Iso ± Ro, whereas CaMKII inhibition prevented SR Ca(2+) 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 Ca(2+) 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.

Interventricular differences in ß-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 ß-adrenergic (ß-AR) responsiveness is unknown. In this study, we examine whether ß-AR response and signaling differ in right (RV) versus left (LV) ventricles. METHODS AND RESULTS: Sarcomere shortening, Ca(2+) 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 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 ß-AR stimulation of cytoplasmic [cAMP] in RVMs versus LVMs. Accordingly, ß-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 ß-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 ß-AR response due to intrinsic differences in myocyte ß-AR downstream signaling. Enhanced ß-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.

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

AIMS: The cAMP-dependent protein kinase (PKA) mediates ß-adrenoceptor (ß-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. ß-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 ß-AR stimulation in cardiac myocytes. This may have important implications in the physiological and pathological hypertrophic response to ß-AR stimulation.

cAMP-PKA inhibition of SK3 channel reduced both Ca2+ entry and cancer cell migration by regulation of SK3-Orai1 complex.

SK3 channel mediates the migration of various cancer cells. When expressed in breast cancer cells, SK3 channel forms a complex with Orai1, a voltage-independent Ca(2+) channel. This SK3-Orai1 complex associates within lipid rafts where it controls a constitutive Ca(2+) entry leading to cancer cell migration and bone metastases development. Since cAMP was found to modulate breast cancer cell migration, we hypothesized that this could be explained by a modulation of SK3 channel activity. Herein, we study the regulation of SK3 channel by the cAMP-PKA pathway and the consequences for SK3-dependent Ca(2+) entry and cancer cell migration. We established that the beta-adrenergic receptor agonist, isoprenaline, or the direct adenylyl cyclase activator forskolin alone or in combination with the PDE4 inhibitor, CI-1044, decreased SK3 channel activity without modifying the expression of SK3 protein at the plasma membrane. Forskolin and CI-1044 reduced the SK3-dependent constitutive Ca(2+) entry and the SK3-dependent migration of MDA-MB-435s cells. PKA inhibition with KT 5720 reduced: (1) the effect of forskolin and CI-1044 by 50 % on Ca(2+) entry and (2) SK3 activity by inhibiting the serine phosphorylation of SK3. These cAMP-elevating agents displaced Orai1 protein outside lipid rafts in contrast to SK3, which remained in the lipid rafts fractions. All together, these results show that activation of the cAMP-PKA pathway decreases SK3 channel and SK3-Orai1 complex activities, leading to a decrease in both Ca(2+) entry and cancer cell migration. This work supports the potential use of cAMP-elevating agents to reduce cancer cell migration and may provide novel opportunities to address/prevent bone metastasis.

Inactivation of the Carney complex gene 1 (PRKAR1A) alters spatiotemporal regulation of cAMP and cAMP-dependent protein kinase: a study using genetically encoded FRET-based reporters.

Carney complex (CNC) is a hereditary disease associating cardiac myxoma, spotty skin pigmentation and endocrine overactivity. CNC is caused by inactivating mutations in the PRKAR1A gene encoding PKA type I alpha regulatory subunit (RIα). Although PKA activity is enhanced in CNC, the mechanisms linking PKA dysregulation to endocrine tumorigenesis are poorly understood. In this study, we used Förster resonance energy transfer (FRET)-based sensors for cAMP and PKA activity to define the role of RIα in the spatiotemporal organization of the cAMP/PKA pathway. RIα knockdown in HEK293 cells increased basal as well as forskolin or prostaglandin E1 (PGE1)-stimulated total cellular PKA activity as reported by western blots of endogenous PKA targets and the FRET-based global PKA activity reporter, AKAR3. Using variants of AKAR3 targeted to subcellular compartments, we identified similar increases in the response to PGE1 in the cytoplasm and at the outer mitochondrial membrane. In contrast, at the plasma membrane, the response to PGE1 was decreased along with an increase in basal FRET ratio. These results were confirmed by western blot analysis of basal and PGE1-induced phosphorylation of membrane-associated vasodilator-stimulated phosphoprotein. Similar differences were observed between the cytoplasm and the plasma membrane in human adrenal cells carrying a RIα inactivating mutation. RIα inactivation also increased cAMP in the cytoplasm, at the outer mitochondrial membrane and at the plasma membrane, as reported by targeted versions of the cAMP indicator Epac1-camps. These results show that RIα inactivation leads to multiple, compartment-specific alterations of the cAMP/PKA pathway revealing new aspects of signaling dysregulation in tumorigenesis.

Phosphodiesterase-2 is up-regulated in human failing hearts and blunts ß-adrenergic responses in cardiomyocytes.

OBJECTIVES: This study investigated whether myocardial phosphodiesterase-2 (PDE2) is altered in heart failure (HF) and determined PDE2-mediated effects on beta-adrenergic receptor (ß-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 ß-AR stimulation via catecholamine infusions in rats enhanced PDE2 expression ~2-fold and cAMP hydrolytic activity ~4-fold, which correlated with blunted cardiac ß-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 ß-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 ß-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 ß-AR stimulation. This may constitute an important defense mechanism during cardiac stress, for example, by antagonizing excessive ß-AR drive. Thus, activating myocardial PDE2 may represent a novel intracellular antiadrenergic therapeutic strategy in HF.