Rodolphe Fischmeister

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.

AIMSnA 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 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.nnnMETHODS AND RESULTSnExcitation-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 β-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.nnnCONCLUSIONnOur 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.

Phosphodiesterase 2: anti-adrenergic friend or hypertrophic foe in heart disease?

Regardless of the underlying cause, heart failure (HF) patients typically show a hyperactive sympathetic nervous systemwith elevated plasma catecholamine levels and subsequent activation of the β-adrenoceptor (β-AR) pathway (El-Armouche and Eschenhagen 2009). Chronically, this leads to attenuated cardiac β-AR responsiveness mainly as a result of a decrease in β1-AR density, uncoupling from stimulatory G proteins (Gs) through higher β-AR kinase (βARK), deactivation of phosphatase inhibitor 1, and higher levels of inhibitory G proteins (Gi) (Bristow et al. 1982; El-Armouche and Eschenhagen 2009; El-Armouche et al. 2008). In concert, these molecular alterations are assumed to be the basis for the blunted responses to catecholamines in the failing heart, a phenomenon called Bβ-AR desensitization.^ On the one hand, these changes may contribute to the progression of HF by further compromising the contractile performance of the failing heart. On the other hand, β-AR desensitization may protect from cardiotoxic β-AR stimulation, e.g., arrhythmias and myocardial hypertrophy. Accordingly, β-AR blockers are first-line recommended drugs in HF, whereas phosphodiesterase (PDE) inhibitors have been banned from chronic use due to increased mortality (El-Armouche and Eschenhagen 2009; Packer et al. 1991). Recently, we have shed new light on an old but to date somewhat neglected player in cardiac β-AR signaling, PDE2, which is upregulated in failing hearts and fulfills the criteria to be part of the β-AR desensitization machinery in HF (Hartzell and Fischmeister 1986; Mehel et al. 2013). PDEs degrade cyclic nucleotides, among others the second messenger cyclic adenosine monophosphate (cAMP) that is produced upon activation of β-ARs. Thereby, PDEs ensure temporal and spatial confinement of cAMP signals, resulting in distinct intracellular cAMP pools (Fischmeister et al. 2006). Intriguingly, PDE2 is stimulated up to 40-fold by cGMP, establishing a potentially important cellular cross talk to the cardioprotective natriuretic peptide and NO systems (Fig. 1) (Jager et al. 2010). We have shown that PDE2 abundance, activity, and transcript levels are upregulated in human and animal models of HF (Mehel et al. 2013). Using the well-known chronical isoprenaline infusion model in rodents, we showed that pathological β-AR overstimulation alone is sufficient to induce higher PDE2 abundance and activity to a similar extent as observed in human end-stage HF, clearly indicating that PDE2 upregulation is a consequence of pathological β-AR overstimulation. Moreover, adenoviral overexpression of PDE2 dramatically blunted β-AR inotropic responses, cellular hypertrophy, and arrhythmogenic events in isolated ventricular myocytes (Fig. 2) (Mehel et al. 2013). Taken together, greater PDE2 expression and activity in the failing heart is very likely a new player in the desensitization machinery of the β-AR signaling pathway. We postulated that this constitutes a potentially important ANP/BNP/cGMP-triggered defense mechanism during cardiac stress, in particular during excessive β-AR drive. Recently, however, our concept of greater PDE2 as a protective mechanism in the failing heart has been challenged. Zoccarato et al. (2015) suggest in a number of well-designed experiments on isolated cardiomyocytes as well as in vivo that PDE2A regulates a local cAMP pool * Ali El-Armouche Ali.El-Armouche@tu-dresden.de

Carboxy-terminal fragment of fibroblast growth factor 23 induces heart hypertrophy in sickle cell disease.

Cardiac remodeling is frequently observed in patients with sickle cell disease (SCD), and contributes to cardiac dysfunction and premature death. Cardiac dysfunction in SCD has been related to chronic anemia and low oxygen saturation. However, the mechanisms whereby they induce cardiac dysfunction are not completely understood and additional factors still have to be identified; a potential candidate is the fibroblast growth factor 23 (FGF23). FGF23 is secreted by bone cells and controls calcitriol and serum phosphate concentrations by acting on the kidney, which is its main physiological target. The intact form of FGF23 (iFGF23) is the only form that circulates under physiological conditions and mediates its physiological effects. iFGF23 can be cleaved in an Nand a C-terminal fragment and it is not certain whether these fragments have any biological effects. Two different immunoassays can be used to measure iFGF23 or C-terminal FGF23 fragment (cFGF23) concentrations. In patients with chronic kidney disease (CKD), plasma FGF23 concentration progressively increases when glomerular filtration rate (GFR) declines. Elevated FGF23 concentration has been associated with cardiac hypertrophy and mortality in CKD patients and in the general population. Injection of iFGF23 in the heart of rats induces cardiac hypertrophy. In 5 of 6 nephrectomized rats (which have elevated iFGF23 plasma concentrations), treatment with an FGF receptor (FGFR) antagonist attenuated cardiac hypertrophy. The mechanism whereby iFGF23 can induce heart hypertrophy is still not completely understood but does not require the expression of the FGF23 co-receptor aKlotho. To determine whether FGF23 could contribute to cardiac remodeling in SCD patients, we measured plasma iFGF23 and cFGF23 concentrations in 77 young adult SCD patients and 172 healthy control subjects of the same ethnic background. A local ethic committee approved the study protocol (Comité de Protection des Personnes Ile de France II number: 2011531-RCEB). The main characteristics of the SCD patients and the control subjects are presented in Online Supplementary Table S1. Among the SCD patients, 53 had hemoglobin SS genotype (HbSS), 15 had hemoglobin SC (HbSC), 8 had sickle cell hemoglobin S-β thalassemia (HbS β), and one had hemoglobin SD (HbSD). Patients with SCD were younger, had lower body mass index (BMI), lower blood pressure, higher estimated GFR and lower hemoglobin concentration than controls. SCD patients had significantly higher cFGF23 plasma concentration than controls whatever their phenotypes (SS or non-SS) (Figure 1A). Fifty-eight (75.3%) SCD patients versus 18 (10.5%) controls had values above normal (150 RU/mL) (P<10). Patients with SS genotype had significantly higher cFGF23 concentration than non-SS patients (Figure 1A). Forty-five of the SS patients (86.5%) and 13 of the non-SS patients (52%) had cFGF23 concentration above the upper normal value (P=0.0013). We also measured FGF23 concentration with an assay that measures only iFGF23 in 50 SCD patients (33 with SS genotype and 17 with non-SS genotype). In this subgroup, cFGF23 concentration was above normal values in 39 patients (78%). By contrast, iFGF23 concentration was normal (<50 pg/mL) in all but 5 of these 50 SCD patients. Furthermore, in SCD patients, at variance with our observations in non-SCD subjects, iFGF23 and cFGF23 levels were not correlated (Online Supplementary Figure S1) suggesting that mainly cleaved FGF23 was present in the plasma of SCD patients. cFGF23 concentration correlated negatively with hemoglobin levels in SCD patients (r=0.187, P<0.0001). We observed a negative correlation between hemoglobin levels and GFR (r=0.062, P=0.0293) and a positive correlation between cFGF23 and GFR (r=0.058, P=0.0343). There was no difference in

Treatments targeting inotropy

Acute heart failure (HF) and in particular, cardiogenic shock are associated with high morbidity and mortality. A therapeutic dilemma is that the use of positive inotropic agents, such as catecholamines or phosphodiesterase-inhibitors, is associated with increased mortality. Newer drugs, such as levosimendan or omecamtiv mecarbil, target sarcomeres to improve systolic function putatively without elevating intracellular Ca2+. Although meta-analyses of smaller trials suggested that levosimendan is associated with a better outcome than dobutamine, larger comparative trials failed to confirm this observation. For omecamtiv mecarbil, Phase II clinical trials suggest a favourable haemodynamic profile in patients with acute and chronic HF, and a Phase III morbidity/mortality trial in patients with chronic HF has recently begun. Here, we review the pathophysiological basis of systolic dysfunction in patients with HF and the mechanisms through which different inotropic agents improve cardiac function. Since adenosine triphosphate and reactive oxygen species production in mitochondria are intimately linked to the processes of excitation-contraction coupling, we also discuss the impact of inotropic agents on mitochondrial bioenergetics and redox regulation. Therefore, this position paper should help identify novel targets for treatments that could not only safely improve systolic and diastolic function acutely, but potentially also myocardial structure and function over a longer-term.

Contribution of BKCa channels to vascular tone regulation by PDE3 and PDE4 is lost in heart failure

AimsnRegulation of vascular tone by 3,5-cyclic adenosine monophosphate (cAMP) involves many effectors including the large conductance, Ca2+-activated, K+ (BKCa) channels. In arteries, cAMP is mainly hydrolyzed by type 3 and 4 phosphodiesterases (PDE3, PDE4). Here, we examined the specific contribution of BKCa channels to tone regulation by these PDEs in rat coronary arteries, and how this is altered in heart failure (HF).nnnMethods and resultsnConcomitant application of PDE3 (cilostamide) and PDE4 (Ro-20-1724) inhibitors increased BKCa unitary channel activity in isolated myocytes from rat coronary arteries. Myography was conducted in isolated, U46619-contracted coronary arteries. Cilostamide (Cil) or Ro-20-1724 induced a vasorelaxation that was greatly reduced by iberiotoxin (IBTX), a BKCa channel blocker. Ro-20-1724 and Cil potentiated the relaxation induced by the β-adrenergic agonist isoprenaline (ISO) or the adenylyl cyclase activator L-858051 (L85). IBTX abolished the effect of PDE inhibitors on ISO but did not on L85. In coronary arteries from rats with HF induced by aortic stenosis, contractility and response to acetylcholine were dramatically reduced compared with arteries from sham rats, but relaxation to PDE inhibitors was retained. Interestingly, however, IBTX had no effect on Ro-20-1724- and Cil-induced vasorelaxations in HF. Expression of the BKCa channel α-subunit, of a 98u2009kDa PDE3A and of a 80u2009kDa PDE4D were lower in HF compared with sham coronary arteries, while that of a 70u2009kDa PDE4B was increased. Proximity ligation assays demonstrated that PDE3 and PDE4 were localized in the vicinity of the channel.nnnConclusionnBKCa channels mediate the relaxation of coronary artery induced by PDE3 and PDE4 inhibition. This is achieved by co-localization of both PDEs with BKCa channels, enabling tight control of cAMP available for channel opening. Contribution of the channel is prominent at rest and on β-adrenergic stimulation. This coupling is lost in HF.

Cyclic Nucleotide Phosphodiesterases and Compartmentation in Normal and Diseased Heart

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.

Influence of cell confluence on the cAMP signalling pathway in vascular smooth muscle cells

The influence of cell confluence on the β-adrenoceptor (β-AR)/cAMP/phosphodiesterase (PDE) pathway was investigated in cultured rat aortic smooth muscle cells (RASMCs). Cells were plated either at low density (LD: 3·103cells/cm2) or high density (HD: 3·104cells/cm2) corresponding to non-confluent or confluent cells, respectively, on the day of experiment. β-AR-stimulated cAMP was monitored in real-time using the fluorescence resonance energy transfer (FRET)-based cAMP sensor, Epac2-camps. A brief application (15s) of the β-AR agonist isoprenaline (Iso) induced a typical transient FRET signal, reflecting cAMP production followed by its rapid degradation. The amplitude of this response, which increased with the concentration of Iso (10 or 100nM), was higher in HD than in LD cells, whatever the Iso concentration used. However, activation of adenylyl cyclase by L-858051 (100μM) induced a similar saturating response in both LD and HD cells. A β1-AR antagonist (CGP 20712A, 100nM) reduced the Iso (100nM) response in HD but not LD cells, whereas a β2-AR antagonist (ICI 118,551, 5nM) reduced this response in HD cells and almost abolished it in LD cells. Competitive [125I]-ICYP binding experiments with betaxolol, a β-AR ligand, identified two binding sites in HD cells, corresponding to β1- and β2-ARs with a proportion of 11% and 89%, respectively, but only one binding site in LD cells, corresponding to β2-ARs. Total cAMP-PDE activity (assessed by a radioenzymatic assay) was increased in HD cells compared to LD cells. This increase was associated with a rise in mRNA expression of five cAMP-PDEs subtypes (PDE1A, 3A, 4A, 4B and 7B) in HD cells, and a decrease in basal [cAMP]i (assessed by an EIA assay). PDE4 inhibition with Ro-20-1724 (10μM) strongly prolonged the Iso response in LD and HD cells, whereas PDE3 inhibition with cilostamide (1μM) slightly prolonged Iso response only in LD cells. Interestingly, inhibition of PDE4 unmasked an effect of PDE3 in HD cells. Our results show that in cultured RASMCs, the β-AR/cAMP/PDE signalling pathway is substantially modulated by the cell density. In HD cells, Iso response involves both β1- and β2-AR stimulation and is mainly controlled by PDE4, PDE3 being recruited only after PDE4 inhibition. In LD cells, Iso response involves only β2-AR stimulation and is controlled by PDE4 and to a lower degree by PDE3. This low density state is associated with an absence of membrane expression of the β1-AR, a lower cAMP-PDE activity and a higher basal [cAMP]i. This study highlights the critical role of the cellular environment in controlling the vascular β-AR signalling.

Vendredi 3 avril 2009, de 11 h 00 à 12 h 30J014 Simultaneous recordings of cell shortening and camp or calcium transients reveal differential regulation of cardiac contractility by specific phosphodiesterases

Multiple cyclic nucleotide phosphodiesterases (PDEs) belonging to four families (PDE1 to PDE4) hydrolyze cAMP in cardiac cells, but the functional significance of this diversity is not well understood. The goal of this study was to characterize the involvement of different PDEs in excitation-contraction coupling in cardiomyocytes. For this, sarcomere shortening and Ca2+ transients were recorded simultaneously in rat ventricular myocytes field stimulated at 0.5 Hz with an IonOptix system. Selective inhibition of PDE2 with Bay 60-7550 (Bay, 100xa0nM) or PDE4 with Ro-201724 (Ro, 10μM) had no effect on basal cell contraction, whereas selective inhibition of PDE3 with cilostamide (Cil, 1μM) or β-adrenergic stimulation with isoprenaline (Iso, 1nM) increased myocyte shortening. Inhibition of PDE4 potentiated the response to Cil and Iso, showing that PDE4 becomes important when cAMP is prestimulated. Similar results were obtained on Ca2+ transients. cAMP measurements by FRET in beating cardiomyocytes indicate that Iso strongly increases cAMP levels. Effects of selective PDE inhibitors are under investigation. These results show that PDE2, PDE3 and PDE4 differentially regulate excitation-contraction coupling in cardiomyocytes.