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Cyclic nucleotide phosphodiesterases (PDEs) modulate neurohormonal regulation of cardiac function by degrading cAMP and cGMP. In cardiomyocytes, multiple isoforms of PDEs with different enzymatic properties and subcellular locally regulate cyclic nucleotide levels and associated cellular functions. This organisation is severely disrupted during hypertrophy and heart failure (HF), which may contribute to disease progression. Clinically, PDE inhibition has been seen as a promising approach to compensate for the catecholamine desensitisation that accompanies heart failure. Although PDE3 inhibitors such as milrinone or enoximone can be used clinically to improve systolic function and relieve the symptoms of acute CHF, their chronic use has proved detrimental. Other PDEs, such as PDE1, PDE2, PDE4, PDE5, PDE9 and PDE10, have emerged as potential new targets for the treatment of HF, each with a unique role in local cyclic nucleotide signalling pathways. In this review, we describe cAMP and cGMP signalling in cardiomyocytes and present the different families of PDEs expressed in the heart and their modifications in pathological cardiac hypertrophy and HF. We also review results from preclinical models and clinical data indicating the use of specific PDE inhibitors or activators that may have therapeutic potential in CI., (© 2024 médecine/sciences – Inserm.)

Anthracyclines, such as doxorubicin (Dox), are widely used chemotherapeutic agents for the treatment of solid tumors and hematologic malignancies. However, they frequently induce cardiotoxicity leading to dilated cardiomyopathy and heart failure. This study sought to investigate the role of the exchange protein directly activated by cAMP (EPAC) in Dox-induced cardiotoxicity and the potential cardioprotective effects of EPAC inhibition. We show that Dox induces DNA damage and cardiomyocyte cell death with apoptotic features. Dox also led to an increase in both cAMP concentration and EPAC1 activity. The pharmacological inhibition of EPAC1 (with CE3F4) but not EPAC2 alleviated the whole Dox-induced pattern of alterations. When administered in vivo , Dox-treated WT mice developed a dilated cardiomyopathy which was totally prevented in EPAC1 knock-out (KO) mice. Moreover, EPAC1 inhibition potentiated Dox-induced cell death in several human cancer cell lines. Thus, EPAC1 inhibition appears as a potential therapeutic strategy to limit Dox-induced cardiomyopathy without interfering with its antitumoral activity., Competing Interests: MM, AB, MR, DD, AL, ML, TB, PM, MG, FL, JC, CB, CR, FL, BC, JB, MV, RF, AG, CL, EM No competing interests declared, (© 2023, Mazevet et al.)

EPAC1, a cAMP-activated GEF for Rap GTPases, is a major transducer of cAMP signaling and a therapeutic target in cardiac diseases. The recent discovery that cAMP is compartmentalized in membrane-proximal nanodomains challenged the current model of EPAC1 activation in the cytosol. Here, we discover that anionic membranes are a major component of EPAC1 activation. We find that anionic membranes activate EPAC1 independently of cAMP, increase its affinity for cAMP by two orders of magnitude, and synergize with cAMP to yield maximal GEF activity. In the cell cytosol, where cAMP concentration is low, EPAC1 must thus be primed by membranes to bind cAMP. Examination of the cell-active chemical CE3F4 in this framework further reveals that it targets only fully activated EPAC1. Together, our findings reformulate previous concepts of cAMP signaling through EPAC proteins, with important implications for drug discovery., (© 2023. The Author(s).)

Phosphodiesterases (PDE) type 3 and 4 promote vasoconstriction by hydrolysing cAMP. In experimental heart failure (HF), PDE3 makes PDE4 redundant in aorta, but it is not known if this occurs in resistance vessels, such as mesenteric artery. As PDE2 is increased in the failing myocardium, its possible role in the vasculature also needs to be addressed. Here, the function of PDE2, PDE3 and PDE4 in rat mesenteric arteries was characterized in experimental HF. Mesenteric arteries were isolated from rats sacrificed 22 weeks after surgical stenosis of the ascending aorta (HF), or Sham surgery. PDE inhibitors were used to probe isoenzyme contributions in enzymatic and isometric tension assays. PDE2 and PDE4 activities, but not PDE3 activity, facilitate contraction produced by the thromboxane analogue U46619 in Sham arteries, while in HF all three isoenzymes contribute to this response. NO synthase inhibition by L-NAME abolished the action of the PDE2 inhibitor. L-NAME eliminated the contribution of PDE4 in HF, but unmasked a contribution for PDE3 in Sham. PDE3 and PDE4 activities attenuated relaxant response to β-adrenergic stimulation in Sham and HF. PDE2 did not participate in cAMP or cGMP-mediated relaxant responses. PDE3 and PDE4 cAMP-hydrolysing activities were smaller in HF mesenteric arteries, while PDE2 activity was scarce in both groups. Endothelial cells and arterial myocytes displayed PDE2 immunolabelling. We highlight that, by contrast with previous observations in aorta, PDE4 participates equally as PDE3 in contracting mesenteric artery in HF. PDE2 activity emerges as a promoter of contractile response that is preserved in HF., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., (© 2022. Springer Nature Limited.)

Mechanism-based therapy centred on the molecular understanding of disease-causing pathways in a given patient is still the exception rather than the rule in medicine, even in cardiology. However, recent successful drug developments centred around the second messenger cyclic guanosine-3'-5'-monophosphate (cGMP), which is regulating a number of cardiovascular disease modulating pathways, are about to provide novel targets for such a personalized cardiovascular therapy. Whether cGMP breakdown is inhibited or cGMP synthesis is stimulated via guanylyl cyclases or their upstream regulators in different cardiovascular disease phenotypes, the outcomes seem to be so far uniformly protective. Thus, a network of cGMP-modulating drugs has evolved that act in a mechanism-based, possibly causal manner in a number of cardiac conditions. What remains a challenge is the detection of cGMPopathy endotypes amongst cardiovascular disease phenotypes. Here, we review the growing clinical relevance of cGMP and provide a glimpse into the future on how drugs interfering with this pathway may change how we treat and diagnose cardiovascular diseases altogether., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.)

The sinoatrial node (SAN) is the natural pacemaker of the heart, producing the electrical impulse that initiates every heart beat. Its activity is tightly controlled by the autonomic nervous system, and by circulating and locally released factors. Neurohumoral regulation of heart rate plays a crucial role in the integration of vital functions and influences behavior and ability to respond to changing environmental conditions. At the cellular level, modulation of SAN activity occurs through intracellular signaling pathways involving cyclic nucleotides: cyclic AMP (cAMP) and cyclic GMP (cGMP). In this Review, dedicated to Professor Dario DiFrancesco and his accomplishements in the field of cardiac pacemaking, we summarize all findings on the role of cyclic nucleotides signaling in regulating the key actors of cardiac automatism, and we provide an up-to-date review on cAMP- and cGMP-phosphodiesterases (PDEs), compellingly involved in this modulation., Competing Interests: Declaration of competing interest The Authors declare no conflicts of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Aim: To obtain a quantitative expression profile of the main genes involved in the cAMP-signaling cascade in human control atria and in different cardiac pathologies., Methods and Results: Expression of 48 target genes playing a relevant role in the cAMP-signaling cascade was assessed by RT-qPCR. 113 samples were obtained from right atrial appendages (RAA) of patients in sinus rhythm (SR) with or without atrium dilation, paroxysmal atrial fibrillation (AF), persistent AF or heart failure (HF); and left atrial appendages (LAA) from patients in SR or with AF. Our results show that right and left atrial appendages in donor hearts or from SR patients have similar expression values except for AC7 and PDE2A. Despite the enormous chamber-dependent variability in the gene-expression changes between pathologies, several distinguishable patterns could be identified. PDE8A, PI3Kγ and EPAC2 were upregulated in AF. Different phosphodiesterase (PDE) families showed specific pathology-dependent changes., Conclusion: By comparing mRNA-expression patterns of the cAMP-signaling cascade related genes in right and left atrial appendages of human hearts and across different pathologies, we show that 1) gene expression is not significantly affected by cardioplegic solution content, 2) it is appropriate to use SR atrial samples as controls, and 3) many genes in the cAMP-signaling cascade are affected in AF and HF but only few of them appear to be chamber (right or left) specific., Topic: Genetic changes in human diseased atria., Translational Perspective: The cyclic AMP signaling pathway is important for atrial function. However, expression patterns of the genes involved in the atria of healthy and diseased hearts are still unclear. We give here a general overview of how different pathologies affect the expression of key genes in the cAMP signaling pathway in human right and left atria appendages. Our study may help identifying new genes of interest as potential therapeutic targets or clinical biomarkers for these pathologies and could serve as a guide in future gene therapy studies., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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 Ca 2+ current (I Ca,L ). Cardiac hypertrophy (CH) is accompanied by a reduction in PDE3 and PDE4, however, whether this affects the dynamic regulation of cytosolic cAMP and I Ca,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 I Ca,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 (t 1/2off ) 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 (t 1/2off  ~ 250 s) than in CH (t 1/2off  ~ 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 (t 1/2off  ~ 80 s). Iso pulse stimulation also elicited a similar transient increase in I Ca,L in SHAM and CH, although the duration of the rising phase was delayed in CH. Inhibition of PDE3 or PDE4 potentiated I Ca,L amplitude in SHAM but not in CH. Besides, while only PDE4 inhibition slowed down the decline of I Ca,L in SHAM, both PDE3 and PDE4 contributed in CH. Conclusion These results identify selective alterations in cytosolic cAMP and I Ca,L regulation by PDE3 and PDE4 in CH, and show that the balance between PDE3 and PDE4 for the regulation of β-AR responses is shifted toward PDE3 during CH., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The concentration of fibroblast growth factor 23 (FGF23) rises progressively in renal failure (RF). High FGF23 concentrations have been consistently associated with adverse cardiovascular outcomes or death, in chronic kidney disease (CKD), heart failure or liver cirrhosis. We identified the mechanisms whereby high concentrations of FGF23 can increase the risk of death of cardiovascular origin. We studied the effects of FGF23 and Klotho in adult rat ventricular cardiomyocytes (ARVMs) and on the heart of mice with CKD. We show that FGF23 increases the frequency of spontaneous calcium waves (SCWs), a marker of cardiomyocyte arrhythmogenicity, in ARVMs. FGF23 increased sarcoplasmic reticulum Ca 2+ leakage, basal phosphorylation of Ca 2+ -cycling proteins including phospholamban and ryanodine receptor type 2. These effects are secondary to a decrease in phosphodiesterase 4B (PDE4B) in ARVMs and in heart of mice with RF. Soluble Klotho, a circulating form of the FGF23 receptor, prevents FGF23 effects on ARVMs by increasing PDE3A and PDE3B expression. Our results suggest that the combination of high FGF23 and low sKlotho concentrations decreases PDE activity in ARVMs, which favors the occurrence of ventricular arrhythmias and may participate in the high death rate observed in patients with CKD.

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.

Numerous mediators and drugs regulate blood flow or arterial pressure by acting on vascular tone, involving cyclic nucleotide intracellular pathways. These signals lead to regulation of several cellular effectors, including ion channels that tune cell membrane potential, Ca 2+ influx and vascular tone. The characterization of these vasocontrictive or vasodilating mechanisms has grown in complexity due to i) the variety of ion channels that are expressed in both vascular endothelial and smooth muscle cells, ii) the heterogeneity of responses among the various vascular beds, and iii) the number of molecular mechanisms involved in cyclic nucleotide signalling in health and disease. This review synthesizes key data from literature that highlight ion channels as physiologically relevant effectors of cyclic nucleotide pathways in the vasculature, including the characterization of the molecular mechanisms involved. In smooth muscle cells, cation influx or chloride efflux through ion channels are associated with vasoconstriction, whereas K + efflux repolarizes the cell membrane potential and mediates vasodilatation. Both categories of ion currents are under the influence of cAMP and cGMP pathways. Evidence that some ion channels are influenced by CN signalling in endothelial cells will also be presented. Emphasis will also be put on recent data touching a variety of determinants such as phosphodiesterases, EPAC and kinase anchoring, that complicate or even challenge former paradigms., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.)

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 β-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/β 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/β 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/β phosphorylation., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

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 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 Ca 2+ 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 Ca 2+ 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., (Copyright © 2019. Published by Elsevier Ltd.)

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)P 2 ) 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)P 2 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)P 2 ) 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 I Ca,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 I Ca,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 Ca 2+ 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 Ca 2+ homeostasis perturbations. Real-time cAMP variations induced by Iso using a Förster 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)P 2 is crucial for maintaining T-tubule structure., (© 2019 The Authors. Experimental Physiology © 2019 The Physiological Society.)

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., (Copyright © 2019. Published by Elsevier B.V.)

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 Ca 2+ concentrations, PDE1 also regulated the NO/soluble GC-dependent cGMP response, as well as the β-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., (© 2019 The British Pharmacological Society.)

Berries contain bioactive polyphenols, whose capacity to prevent cardiovascular diseases has been established recently in animal models as well in human clinical trials. However, cellular processes and molecular targets of berries polyphenols remain to be identified. The capacity of a polyphenol-enriched diet (i.e., blueberries, blackberries, raspberries, strawberry tree fruits and Portuguese crowberries berries mixture) to promote animal survival and protect cardiovascular function from salt-induced hypertension was evaluated in a chronic salt-sensitive Dahl rat model. The daily consumption of berries improved survival of Dahl/salt-sensitive rats submitted to high-salt diet and normalized their body weight, renal function and blood pressure. In addition, a prophylactic effect was observed at the level of cardiac hypertrophy and dysfunction, tissue cohesion and cardiomyocyte hypertrophy. Berries also protected the aorta from fibrosis and modulated the expression of aquaporin-1, a channel involved in endothelial water and nitric oxide permeability. Left ventricle proteomics analysis led to the identification of berries and salt metabolites targets, including cystein and glycin-rich protein 3 (CSRP3), a protein involved in myocyte cytoarchitecture. In neonatal rat ventricular cardiomyocytes, CSRP3 was validated as a target of a berries-derived polyphenol metabolite, 4-methylcatechol sulfate, at micromolar concentrations, mimicking physiological conditions of human plasma circulation. Accordingly, siRNA silencing of CSRP3 and 4-methylcatechol sulfate pretreatment reversed cardiomyocyte hypertrophy and CSRP3 overexpression induced by phenylephrine. Our systemic study clearly supports the modulation of CSRP3 by a polyphenol-rich berries diet as an efficient cardioprotective strategy in hypertension-induced heart failure., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Metabolic inhibition is a common condition observed during ischemic heart disease and heart failure. It is usually accompanied by a reduction in L-type Ca 2+ channel (LTCC) activity. In this study, however, we show that metabolic inhibition results in a biphasic effect on LTCC current (I CaL ) in human and rat cardiac myocytes: an initial increase of I CaL is observed in the early phase of metabolic inhibition which is followed by the more classical and strong inhibition. We studied the mechanism of the initial increase of I CaL in cardiac myocytes during β-adrenergic stimulation by isoprenaline, a non-selective agonist of β-adrenergic receptors. The whole-cell patch⁻clamp technique was used to record the I CaL in single cardiac myocytes. The initial increase of I CaL was induced by a wide range of metabolic inhibitors (FCCP, 2,4-DNP, rotenone, antimycin A). In rat cardiomyocytes, the initial increase of I CaL was eliminated when the cells were pre-treated with thapsigargin leading to the depletion of Ca 2+ from the sarcoplasmic reticulum (SR). Similar results were obtained when Ca 2+ release from the SR was blocked with ryanodine. These data suggest that the increase of I CaL in the early phase of metabolic inhibition is due to a reduced calcium dependent inactivation (CDI) of LTCCs. This was further confirmed in human atrial myocytes where FCCP failed to induce the initial stimulation of I CaL when Ca 2+ was replaced by Ba 2+ , eliminating CDI of LTCCs. We conclude that the initial increase in I CaL observed during the metabolic inhibition in human and rat cardiomyocytes is a consequence of an acute reduction of Ca 2+ release from SR resulting in reduced CDI of LTCCs.

Aims: Regulation 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)., Methods and Results: Concomitant 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 98 kDa PDE3A and of a 80 kDa PDE4D were lower in HF compared with sham coronary arteries, while that of a 70 kDa PDE4B was increased. Proximity ligation assays demonstrated that PDE3 and PDE4 were localized in the vicinity of the channel., Conclusion: BKCa 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.

Cardiac failure is a common complication in cancer survivors treated with anthracyclines. Here we followed up cardiac function and excitation-contraction (EC) coupling in an in vivo doxorubicin (Dox) treated mice model (iv, total dose of 10 mg/Kg divided once every three days). Cardiac function was evaluated by echocardiography at 2, 6 and 15 weeks after the last injection. While normal at 2 and 6 weeks, ejection fraction was significantly reduced at 15 weeks. In order to evaluate the underlying mechanisms, we measured [Ca 2+ ] i transients by confocal microscopy and action potentials (AP) by patch-clamp technique in cardiomyocytes isolated at these times. Three phases were observed: 1/depression and slowing of the [Ca 2+ ] i transients at 2 weeks after treatment, with occurrence of proarrhythmogenic Ca 2+ waves, 2/compensatory state at 6 weeks, and 3/depression on [Ca 2+ ] i transients and cell contraction at 15 weeks, concomitant with in-vivo defects. These [Ca 2+ ] i transient alterations were observed without cellular hypertrophy or AP prolongation and mirrored the sarcoplasmic reticulum (SR) Ca 2+ load variations. At the molecular level, this was associated with a decrease in the sarcoplasmic reticulum Ca 2+ ATPase (SERCA2a) expression and enhanced RyR2 phosphorylation at the protein kinase A (PKA, pS2808) site (2 and 15 weeks). RyR2 phosphorylation at the Ca 2+ /calmodulin dependent protein kinase II (CaMKII, pS2814) site was enhanced only at 2 weeks, coinciding with the higher incidence of proarrhythmogenic Ca 2+ waves. Our study highlighted, for the first time, the progression of Dox treatment-induced alterations in Ca 2+ handling and identified key components of the underlying Dox cardiotoxicity. These findings should be helpful to understand the early-, intermediate-, and late- cardiotoxicity already recorded in clinic in order to prevent or treat at the subclinical level., (Copyright © 2018. Published by Elsevier Ltd.)

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

Aims: Quantitative real-time RT-PCR (RT-qPCR) has become the method of choice for mRNA quantification, but requires an accurate normalization based on the use of reference genes showing invariant expression across various pathological conditions. Only few data exist on appropriate reference genes for the human heart. The objective of this study was to determine a set of suitable reference genes in human atrial and ventricular tissues, from right and left cavities in control and in cardiac diseases., Methods and Results: We assessed the expression of 16 reference genes (ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, IPO8, PGK1, POLR2A, PPIA, RPLP0, TBP, TFRC, UBC, YWHAZ, 18S) in tissues from: right and left ventricles from healthy controls and heart failure (HF) patients; right-atrial tissue from patients in sinus rhythm with (SRd) or without (SRnd) atrial dilatation, patients with paroxysmal (pAF) or chronic (cAF) atrial fibrillation or with HF; and left-atrial tissue from patients in SR or cAF. Consensual analysis (by geNorm and Normfinder algorithms, BestKeeper software tool and comparative delta-Ct method) of the variability scores obtained for each reference gene expression shows that the most stably expressed genes are: GAPDH, GUSB, IPO8, POLR2A, and YWHAZ when comparing either right and left ventricle or ventricle from healthy controls and HF patients; GAPDH, IPO8, POLR2A, PPIA, and RPLP0 when comparing either right and left atrium or right atria from all pathological groups. ACTB, TBP, TFRC, and 18S genes were identified as the least stable., Conclusions: The overall most stable reference genes across different heart cavities and disease conditions were GAPDH, IPO8, POLR2A and PPIA. YWHAZ or GUSB could be added to this set for some specific experiments. This study should provide useful guidelines for reference gene selection in RT-qPCR studies in human heart., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For Permissions, please email: journals.permissions@oup.com.)

Background: Inula viscosa L. (Asteraceae) is a medicinal plant widely used as a folk medicine in oriental Morocco, to treat hypertension. The antihypertensive effect of the methanolic extract obtained from I. viscosa leaves was evaluated in hypertensive L-NAME rats. Systolic blood pressure (SBP) was measured using a non-invasive indirect tail-cuff plethysmographic method. Four groups of rats were used: a control group; a hypertensive group treated with L-NAME (32mg/kg/day); a positive control group treated with L-NAME plus enalapril (15mg/kg/day) as a reference antihypertensive agent; and a group treated with L-NAME plus MeOH-extract (40mg/kg/day)., Methods: Treatment with L-NAME alone caused a progressive increase in SBP. After 4 weeks, the value of SBP reached 160±2mmHg which shows the installation of hypertension. Enalapril prevented the increase in SBP, which remained normal at 123±1mmHg after 4 weeks of treatment. The administration of MeOH-extract along with L-NAME prevented the increase in SBP as well, which remained constant at 115±1mmHg after 4 weeks of treatment. In ex-vivo models, MeOH-extract produced a relaxation of pre-contracted ring aorta (54 ± 2% of relaxation at 3g/L). But, when the rings were denuded, MeOH-extract failed to relax pre-contracted rings of aorta. Phytochemical study of I. viscosa MeOH-extract revealed the presence of phenolic compounds, such as cynarin and chlorogenic acid., Results: The present results suggest that I. viscosa MeOH-extract has an antihypertensive, predominantly mediated by an endothelium-dependent vasodilatory effect. Cynarin and chlorogenic acid, which have a strong vasorelaxant effect may be involved in the antihypertensive effect of the plant extract. The bioinformatic POM analysis confirms the therapeutic potential of cynarin and chlorogenic acids as inhibitors of various biotargets. Based on the results, the coordination of these phytochemicals with calcium and transition metals should be studied, for better scope at antihypertensive drug development., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Metabolic stress evoked by myocardial ischemia leads to impairment of cardiac excitation and contractility. We studied the mechanisms by which metabolic inhibition affects the activity of L-type Ca2+ channels (LTCCs) in frog ventricular myocytes. Metabolic inhibition induced by the protonophore FCCP (as well as by 2,4- dinitrophenol, sodium azide or antimycin A) resulted in a dose-dependent reduction of LTCC current (ICa,L) which was more pronounced during β-adrenergic stimulation with isoprenaline. ICa,L was still reduced by metabolic inhibition even in the presence of 3 mM intracellular ATP, or when the cell was dialysed with cAMP or ATP-γ-S to induce irreversible thiophosphorylation of LTCCs, indicating that reduction in ICa,L is not due to ATP depletion and/or reduced phosphorylation of the channels. However, the effect of metabolic inhibition on ICa,L was strongly attenuated when the mitochondrial F1F0-ATP-synthase was blocked by oligomycin or when the cells were dialysed with the non-hydrolysable ATP analogue AMP-PCP. Moreover, increasing the intracellular pH buffering capacity or intracellular dialysis of the myocytes with an alkaline solution strongly attenuated the inhibitory effect of FCCP on ICa,L. Thus, our data demonstrate that metabolic inhibition leads to excessive ATP hydrolysis by the mitochondrial F1F0-ATP-synthase operating in the reverse mode and this results in intracellular acidosis causing the suppression of ICa,L. Limiting ATP break-down by F1F0-ATP-synthase and the consecutive development of intracellular acidosis might thus represent a potential therapeutic approach for maintaining a normal cardiac function during ischemia.