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The mosaic theory of hypertension was advocated by Irvine Page ~80 years ago and suggested that hypertension resulted from the close interactions of different causes. Increasing evidence indicates that hypertension and hypertensive end-organ damage are not only mediated by the proposed mechanisms that result in hemodynamic injury. Inflammation plays an important role in the pathophysiology and contributes to the deleterious consequences of arterial hypertension. Sodium intake is indispensable for normal body function but can be detrimental when it exceeds dietary requirements. Recent data show that sodium levels also modulate the function of monocytes/macrophages, dendritic cells, and different T-cell subsets. Some of these effects are mediated by changes in the microbiome and metabolome due to high-salt intake. The purpose of this review is to propose a revised and extended version of the mosaic theory by summarizing and integrating recent advances in salt, immunity, and hypertension research. Salt and inflammation are placed in the middle of the mosaic because both factors influence each of the remaining pieces.

Dysfunction of the sinoatrial node (SAN), the natural heart pacemaker, is common in heart failure (HF) patients. SAN spontaneous activity relies on various ion currents in the plasma membrane (voltage clock), but intracellular Ca2+ ([Ca2+]i) release via ryanodine receptor 2 (RYR2; Ca2+ clock) plays an important synergetic role. Whereas remodeling of voltage-clock components has been revealed in HF, less is known about possible alterations to the Ca2+ clock. Here, we analyzed [Ca2+]i handling in SAN from a mouse HF model after transverse aortic constriction (TAC) and compared it with sham-operated animals. ECG data from awake animals showed slower heart rate in HF mice upon autonomic nervous system blockade, indicating intrinsic sinus node dysfunction. Confocal microscopy analyses of SAN cells within whole tissue showed slower and less frequent [Ca2+]i transients in HF. This correlated with fewer and smaller spontaneous Ca2+ sparks in HF SAN cells, which associated with lower RYR2 protein expression level and reduced phosphorylation at the CaMKII site. Moreover, PLB phosphorylation at the CaMKII site was also decreased in HF, which could lead to reduced sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) function and lower sarcoplasmic reticulum Ca2+ content, further depressing the Ca2+ clock. The inhibition of CaMKII with KN93 slowed [Ca2+]i transient rate in both groups, but this effect was smaller in HF SAN, consistent with less CaMKII activation. In conclusion, our data uncover that the mechanism of intrinsic pacemaker dysfunction in HF involves reduced CaMKII activation.

Supplemental Digital Content is available in the text., Background: Orai1 is a critical ion channel subunit, best recognized as a mediator of store-operated Ca2+ entry (SOCE) in nonexcitable cells. SOCE has recently emerged as a key contributor of cardiac hypertrophy and heart failure but the relevance of Orai1 is still unclear. Methods: To test the role of these Orai1 channels in the cardiac pathophysiology, a transgenic mouse was generated with cardiomyocyte-specific expression of an ion pore-disruptive Orai1R91W mutant (C-dnO1). Synthetic chemistry and channel screening strategies were used to develop 4-(2,5-dimethoxyphenyl)-N-[(pyridin-4-yl)methyl]aniline (hereafter referred to as JPIII), a small-molecule Orai1 channel inhibitor suitable for in vivo delivery. Results: Adult mice subjected to transverse aortic constriction (TAC) developed cardiac hypertrophy and reduced ventricular function associated with increased Orai1 expression and Orai1-dependent SOCE (assessed by Mn2+ influx). C-dnO1 mice displayed normal cardiac electromechanical function and cellular excitation-contraction coupling despite reduced Orai1-dependent SOCE. Five weeks after TAC, C-dnO1 mice were protected from systolic dysfunction (assessed by preserved left ventricular fractional shortening and ejection fraction) even if increased cardiac mass and prohypertrophic markers induction were observed. This is correlated with a protection from TAC-induced cellular Ca2+ signaling alterations (increased SOCE, decreased [Ca2+]i transients amplitude and decay rate, lower SR Ca2+ load and depressed cellular contractility) and SERCA2a downregulation in ventricular cardiomyocytes from C-dnO1 mice, associated with blunted Pyk2 signaling. There was also less fibrosis in heart sections from C-dnO1 mice after TAC. Moreover, 3 weeks treatment with JPIII following 5 weeks of TAC confirmed the translational relevance of an Orai1 inhibition strategy during hypertrophic insult. Conclusions: The findings suggest a key role of cardiac Orai1 channels and the potential for Orai1 channel inhibitors as inotropic therapies for maintaining contractility reserve after hypertrophic stress.

In mesenteric arteries (MAs), aldosterone (ALDO) binds to the endogenous mineralocorticoid receptor (MR) and increases the expression of the voltage-gated L-type Ca

Heart failure (HF) is a complex syndrome in which death rates are >50%. The main causes of death among HF patients are pump failure and ventricular arrhythmias, but severe bradycardia is also a common cause of sudden cardiac death, pointing to sinoatrial node (SAN) dysfunction. SAN pacemaker activity is regulated by voltage-clock and Ca2+-clock mechanisms and, although voltage-clock dysfunction in SAN has been largely proved in HF, Ca2+-clock dysfunction mechanisms in SAN remains unraveled. Here, we used an HF model in mice with transverse aortic constriction (TAC) and, using telemetry, saw slower heart rhythm under autonomic nervous system blockade. Then, by confocal microscopy, we analyzed Ca2+ handling in HF SAN tissue and found that intracellular Ca2+ transients rate were slower together with less frequency of Ca2+ sparks than in SHAM SAN tissue. Next, we studied protein expression of key excitation–contraction coupling proteins and found reduced expression of the Na+/Ca2+ exchanger and reduced phosphorylated status of ryanodine receptor and phospholamban in the CaMKII sites for the SAN in TAC mice. Finally, the application of the CaMKII inhibitor, KN93, caused less effect in slowing the Ca2+ transient rates in HF SAN tissue, confirming the reduced CaMKII activation. In conclusion, our data demonstrates a reduction in CaMKII activation in the Ca2+-clock function of the SAN tissue in a mouse model of HF.

Rationale: The widely used chemotherapeutic agent Doxorubicin (Dox) induces cardiotoxicity leading to dilated cardiomyopathy and heart failure. This cardiotoxicity has been related to ROS generation, DNA intercalation, bioenergetic distress and cell death. However, alternative mechanisms are emerging, focusing on signaling pathways. Objective: We investigated the role of Exchange Protein directly Activated by cAMP (EPAC), key factor in cAMP signaling, in Dox-induced cardiotoxicity. Methods and Results: Dox was administrated in vivo (10 +/- 2 mg/kg, i.v.; with analysis at 2, 6 and 15 weeks post injection) in WT and EPAC1 KO C57BL6 mice. Cardiac function was analyzed by echocardiography and intracellular Ca2+ homeostasis by confocal microscopy in isolated ventricular cardiomyocytes. 15 weeks post-injections, Dox-treated WT mice, developed a dilated cardiomyopathy with decreased ejection fraction, increased telediastolic volume and impaired Ca2+ homeostasis, which were totally prevented in the EPAC1 KO mice. The underlying mechanisms were investigated in neonatal and adult rat cardiac myocytes under Dox treatment (1-10 uM). Flow cytometry, Western blot, BRET sensor assay, and RT-qPCR analysis showed that Dox induced DNA damage and cardiomyocyte cell death with apoptotic features rather than necrosis, including Ca2+-CaMKKβ-dependent opening of the Mitochondrial Permeability Transition Pore, dissipation of the Mitochondrial membrane potential, caspase activation, cell size reduction, and DNA fragmentation. Dox also led to an increase in both cAMP concentration and EPAC1 protein level and activity. The pharmacological inhibition of EPAC1 (CE3F4) but not EPAC2 alleviated the whole Dox-induced pattern of alterations including DNA damage, Mitochondrial membrane potential, apoptosis, mitochondrial biogenesis, dynamic, and fission/fusion balance, and respiratory chain activity, suggesting a crucial role of EPAC1 in these processes. Importantly, while preserving cardiomyocyte integrity, EPAC1 inhibition potentiated Dox-induced cell death in several human cancer cell lines. Conclusion: Thus, EPAC1 inhibition could be a valuable therapeutic strategy to limit Dox-induced cardiomyopathy without interfering with its antitumoral activity.

Summary Diabetes mellitus is a metabolic disorder with a chronic hyperglycaemic state. Cardiovascular diseases are the primary cause of mortality in patients with diabetes. Increasing evidence supports the existence of diabetic cardiomyopathy, a cardiac dysfunction with impaired cardiac contraction and relaxation, independent of coronary and/or valvular complications. Diabetic cardiomyopathy can lead to heart failure. Several preclinical and clinical studies have aimed to decipher the underlying mechanisms of diabetic cardiomyopathy. Among all the co-factors, hyperglycaemia seems to play an important role in this pathology. Hyperglycaemia has been shown to alter cardiac metabolism and function through several deleterious mechanisms, such as oxidative stress, inflammation, accumulation of advanced glycated end-products and upregulation of the hexosamine biosynthesis pathway. These mechanisms are responsible for the activation of hypertrophic pathways, epigenetic modifications, mitochondrial dysfunction, cell apoptosis, fibrosis and calcium mishandling, leading to cardiac stiffness, as well as contractile and relaxation dysfunction. This review aims to describe the hyperglycaemic-induced alterations that participate in diabetic cardiomyopathy, and their correlation with the severity of the disease and patient mortality, and to provide an overview of cardiac outcomes of glucose-lowering therapy.

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): ANR (Agence Nationale de la Rercherche) Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a lethal genetic arrhythmia that manifests by syncope or sudden death in children and young adults under stress conditions without obvious cardiac structural abnormality. A novel CPVT mutation located in the RyR2 N terminal portion has been identified in a Spanish family (RyR2R420Q). According to the studies of RyR2 function in HEK293 cell line, this mutation presented gain of function at low cytosolic intracellular Ca2+ concentration ([Ca2+]i) and loss of function at high [Ca2+]i. Moreover, KI mice heterozygous for this mutation presented bradycardia and sino-atrial node (SAN) dysfunction. Here we generated induced pluripotent stem cell (hiPSC) from two brothers (one with mutation, the other without mutation as control) of this family and differentiated them into cardiomyocytes (hiPSC-CM). In order to verify that the differentiated cells were well cardiomyocytes, we did immunofluorescence labelling to detect the α-actinin expression and found that around 90% cells were α-actinin positive in both groups of hiPS-CMs. Then the calcium transient was studied by confocal microscopy and the action potential (AP) by micro-electrode technique. The characteristics of spontaneous AP of mutated cells were mostly similar to that of control cells, but more mutated cells presented proarrhythmic behaviors under adrenergic stimulation. hiPSC-CM are immature cardiomyocytes and contract spontaneously. In order to be able to analyze [Ca2+]i transient characteristics, we paced the cells at a constant rate of 1 Hz by field stimulation through two Pt electrodes. Sarcoplasmic reticulum (SR) Ca2+ load was estimated by rapid caffeine (10 mM) application. hiPSC-CMs from the RyR2R420Q carrier presented smaller SR Ca2+ load than those from the control person, whereas their fractional release (the [Ca2+]i transient normalized by the amount of Ca2+ stored in the SR) was higher than that in control group, indicating a gain-of-function mutation. Even if SR Ca2+ load was smaller in RyR2R420Q cells, they often presented proarrhythmogenic behavior such as Ca2+ waves. The fact was further enhanced during β-adrenergic stimulation, pointing to this model as a valuable tool to study the CPVT disease in human cells.

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agence Nationale de la Recherche Introduction Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is an inherited disease manifested as syncope or sudden death in apparently healthy children or young adults, in the absence of structural cardiac disease. The ryanodine receptor (RyR2) R420Q mutation was identified in a Spanish 14-year-old boy who died suddenly due to emotional stress. We generated human induced-pluripotent (h-iPS) derived cardiomyocytes from two brothers (see Yin’s poster in this meeting). As most of sudden death occurs in males, we had the hypothesis that there might be some sex-dependent differences. Purpose and methods We aim to elucidate the key role of [Ca2+]i in the genesis of cardiac arrhythmia in female RyR2R420Q CPVT patients . We generated here hiPSC-CM from two sisters of the same family, one exhibiting the mutation RyR2R420Q and the other without the mutation used as control. The experiments were conducted at 30-35 days after differentiation into cardiomyocytes. Using confocal microscopy, we assessed intracellular [Ca2+]i handling both spontaneous and during electrical pacing. To record [Ca2+]i transients, h-iPS-CMs were electrically field stimulated by two Pt electrodes at 1Hz. Spontaneous Ca2+ sparks and Ca2+ waves were recorded in quiescent cells, after [Ca2+]i transients recordings. For SR Ca2+ load quantification, h-iPS-CMs were rapidly perfused with 10 mmol/L caffeine. To induce β-adrenergic activation, 100nM isoproterenol (ISO) was added in some experiments. All confocal Ca2+ images analyses were performed by home-made routines using IDL software. Results First, we verify by immunofluorescence the expression of alpha-actinin in the differentiated h-iPS-CMs. Then, by confocal microscopy we found that woman CPVT h-iPS-CMs presented longer cycle length than wt h-iPS-CMs, correlating with the bradycardia observed in CPVT patients. The amplitude of the Ca2+ transients were significantly higher for CPVT h-iPS-CMs compared to wt; however, after ISO perfusion Ca2+ transients did not show a significant increase in CPVT h-iPS-CMs compared to CPVT h-iPS-CMs before ISO. SR Ca2+ load presented no differences between groups but the time decay constant of the caffeine-evoked Ca2+ transients were significantly faster in CPVT h-iPS-CMs whereas it became slower after ISO perfusion. In addition, analysis of Ca2+ pro-arrhythmogenic events were significantly augmented in CPVT h-iPS-CMs compared to wt h-iPS-CMs. After ISO perfusion, pro-arrhythmogenic events in CPVT cells presented no significant difference compared to CPVT cell before ISO perfusion. Although proarrhythmogenic events were similarly higher in both male and female hiPSC-CM, the Ca2+ handling characteristics were slightly different. Conclusion The RyR2R420Q mutation in woman CPVT h-iPS-CMs provides a reliable model to study CPVT in human context.

Rationale: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare disease, manifested by syncope or sudden death in children or young adults under stress conditions. Mutations in the Ca 2+ release channel/RyR2 (type 2 ryanodine receptor) gene account for about 60% of the identified mutations. Recently, we found and described a mutation in RyR2 N-terminal domain, RyR2 R420Q . Objective: To determine the arrhythmogenic mechanisms of this mutation. Methods and Results: Ventricular tachycardias under stress conditions were observed in both patients with catecholaminergic polymorphic ventricular tachycardia and knock-in mice. During action potential recording (by patch-clamp in knock-in mouse cardiomyocytes and by microelectrodes in mutant human induced pluripotent stem cell-derived cardiomyocytes), we observed an increased occurrence of delayed afterdepolarizations under isoproterenol stimulation, associated with increased Ca 2+ waves during confocal Ca 2+ recording in both mouse and human RyR2 R420Q cardiomyocytes. In addition, Ca 2+ -induced Ca 2+ -release, as well as a rough indicator of fractional Ca 2+ release, were higher and Ca 2+ sparks longer in the RyR2 R420Q -expressing cells. At the ultrastructural nanodomain level, we observed smaller RyR2 clusters and widened junctional sarcoplasmic reticulum measured by gated stimulated emission depletion super-resolution and electron microscopy, respectively. The increase in junctional sarcoplasmic reticulum width might be due to the impairment of RyR2 R420Q binding to JPH2 (junctophilin-2), as there were less junctophilin-2 coimmunoprecipitated with RyR2 R420Q . At the single current level, the RyR2 R420Q channel dwells longer in the open state at low intracellular Ca 2+ ([Ca 2+ ] i ), but there is predominance of a subconductance state. The latter might be correlated with an enhanced interaction between the N terminus and the core solenoid, a RyR2 interdomain association that has not been previously implicated in the pathogenesis of arrhythmias and sudden cardiac death. Conclusions: The RyR2 R420Q catecholaminergic polymorphic ventricular tachycardia mutation modifies the interdomain interaction of the channel and weakens its association with JPH2. These defects may underlie both nanoscale disarrangement of the dyad and channel dysfunction.

The archetypal store-operated Ca2+ channels (SOCs), Orai1, which are stimulated by the endo/sarcoplasmic reticulum (ER/SR) Ca2+ sensor stromal interaction molecule 1 (STIM1) upon Ca2+ store depletion is traditionally viewed as instrumental for the function of non-excitable cells. In the recent years, expression and function of Orai1 have gained recognition in excitable cardiomyocytes, albeit controversial. Even if its cardiac physiological role in adult is still elusive and needs to be clarified, Orai1 contribution in cardiac diseases such as cardiac hypertrophy and heart failure (HF) is increasingly recognized. The present review surveys our current arising knowledge on the new role of Orai1 channels in the heart and debates on its participation to cardiac hypertrophy and HF.

Aim Deletion of QKP1507-1509 amino-acids in SCN5A gene product, the voltage-gated Na+ channel Nav1.5, has been associated with a large phenotypic spectrum of type 3 long QT syndrome, conduction disorder, dilated cardiomyopathy and high incidence of sudden death. The aim of this study was to develop and characterize a novel model of type 3 long QT syndrome to study the consequences of the QKP1507–1509 deletion. Methods and results We generated a knock-in mouse presenting the delQKP1510–1512 mutation (Scn5a+/ΔQKP) equivalent to human deletion. Scn5a+/ΔQKP mice showed prolonged QT interval, conduction defects and ventricular arrhythmias at the age of 2 weeks, and, subsequently, structural defects and premature mortality. The mutation increased Na+ window current and generated a late Na+ current. Ventricular action potentials from Scn5a+/ΔQKP mice were prolonged. At the age of 4 weeks, Scn5a+/ΔQKP mice exhibited a remodeling leading to [Ca2+]i transients with higher amplitude and slower kinetics, combined with enhanced SR Ca2+ load. SERCA2 expression was not altered. However, total phospholamban expression was higher whereas the amount of Ca2+-calmodulin-dependent kinase II (CaMKII)-dependent T17-phosphorylated form was lower, in hearts from 4-week-old mice only. This was associated with a lower activity of CaMKII and lower calmodulin expression. In addition, Scn5a+/ΔQKP cardiomyocytes showed larger Ca2+ waves, correlated with the presence of afterdepolarizations during action potential recording. Ranolazine partially prevented action potential and QT interval prolongation in 4-week-old Scn5a+/ΔQKP mice and suppressed arrhythmias. Conclusion The Scn5a+/ΔQKP mouse model recapitulates the clinical phenotype of mutation carriers and provides new and unexpected insights into the pathological development of the disease in patients carrying the QKP1507–1509 deletion.

Background Right ventricular (RV) function is the most important prognostic factor for pulmonary arterial hypertension (PAH) patients. The progressive increase of pulmonary vascular resistance induces RV hypertrophy (RVH) and at term RV failure (RVF). However, the molecular mechanisms of RVH and RVF remain understudied. In this study, we gained insights into cytosolic Ca2+ signaling remodeling in ventricular cardiomyocytes during the pathogenesis of severe pulmonary hypertension (PH) induced in rats by monocrotaline (MCT) exposure, and we further identified molecular candidates responsible for this Ca2+ remodeling. Methods and results After PH induction, hypertrophied RV myocytes presented longer action potential duration, higher and faster [Ca2+]i transients and increased sarcoplasmic reticulum (SR) Ca2+ content, whereas no changes in these parameters were detected in left ventricular (LV) myocytes. These modifications were associated with increased P-Ser16-phospholamban pentamer expression without altering SERCA2a (Sarco/Endoplasmic Reticulum Ca2+-ATPase) pump abundance. Moreover, after PH induction, Ca2+ sparks frequency were higher in hypertrophied RV cells, while total RyR2 (Ryanodine Receptor) expression and phosphorylation were unaffected. Together with cellular hypertrophy, the T-tubules network was disorganized. Hypertrophied RV cardiomyocytes from MCT-exposed rats showed decreased expression of classical STIM1 (Stromal Interaction molecule) associated with increased expression of muscle-specific STIM1 Long isoform, glycosylated-Orai1 channel form, and TRPC1 and TRPC4 channels, which was correlated with an enhanced Ca2+-release-activated Ca2+ (CRAC)-like current. Pharmacological inhibition of TRPCs/Orai1 channels in hypertrophied RV cardiomyocytes normalized [Ca2+]i transients amplitude, the SR Ca2+ content and cell contractility to control levels. Finally, we showed that most of these changes did not appear in LV cardiomyocytes. Conclusions These new findings demonstrate RV-specific cellular Ca2+ cycling remodeling in PH rats with maladaptive RVH and that the STIM1L/Orai1/TRPC1/C4-dependent Ca2+ current participates in this Ca2+ remodeling in RVH secondary to PH.

Key points Leptin, is a 16 kDa pleiotropic peptide not only primarily secreted by adipocytes, but also produced by other tissues, including the heart. Controversy exists regarding the adverse and beneficial effects of leptin on the heart We analysed the effect of a non-hypertensive dose of leptin on cardiac function, [Ca2+ ]i handling and cellular electrophysiology, which participate in the genesis of pump failure and related arrhythmias, both in control mice and in mice subjected to chronic pressure-overload by transverse aorta constriction. We find that leptin activates mechanisms that contribute to cardiac dysfunction under physiological conditions. However, after the establishment of pressure overload, an increase in leptin levels has protective cardiac effects with respect to rescuing the cellular heart failure phenotype. These beneficial effects of leptin involve restoration of action potential duration via normalization of transient outward potassium current and sarcoplasmic reticulum Ca2+ content via rescue of control sarcoplasmic/endoplasmic reticulum Ca2+ ATPase levels and ryanodine receptor function modulation, leading to normalization of Ca2+ handling parameters. Abstract Leptin, is a 16 kDa pleiotropic peptide not only primary secreted by adipocytes, but also produced by other tissues, including the heart. Evidence indicates that leptin may have either adverse or beneficial effects on the heart. To obtain further insights, in the present study, we analysed the effect of leptin treatment on cardiac function, [Ca2+ ]i handling and cellular electrophysiology, which participate in the genesis of pump failure and related arrhythmias, both in control mice and in mice subjected to chronic pressure-overload by transverse aorta constriction (TAC). Three weeks after surgery, animals received either leptin (0.36 mg kg-1 day-1 ) or vehicle via osmotic minipumps for 3 weeks. Echocardiographic measurements showed that, although leptin treatment was deleterious on cardiac function in sham, leptin had a cardioprotective effect following TAC. [Ca2+ ]i transient in cardiomyocytes followed similar pattern. Patch clamp experiments showed prolongation of action potential duration (APD) in TAC and leptin-treated sham animals, whereas, following TAC, leptin reduced the APD towards control values. APD variations were associated with decreased transient outward potassium current and Kv4.2 and KChIP2 protein expression. TAC myocytes showed a higher incidence of triggered activities and spontaneous Ca2+ waves. These proarrhythmic manifestations, related to Ca2+ /calmodulin-dependent protein kinase II and ryanodine receptor phosphorylation, were reduced by leptin. The results of the present study demonstrate that, although leptin treatment was deleterious on cardiac function in control animals, leptin had a cardioprotective effect following TAC, normalizing cardiac function and reducing arrhythmogeneity at the cellular level.

Introduction Anticancer treatments (chemo- and radiation therapies), such as Doxorubicin (Dox), used to treat many types of cancer are associated with many side effects such as cardiotoxicity mainly leading to a dilated cardiomyopathy (DCM) in chronic. Objectives There is a need for new treatment options and strategies aiming at reducing Doxorubicin side effects in the heart. Among these mechanisms Epac (exchange protein directly activated by cAMP) signaling could be worth investigating. Methods We have investigated the time/dose-dependent Dox effect on Epac signaling in both in vivo mice model (C57Bl63/Knock-out Epac1 mice, iv injections, 12 mg/kg cumulative dose) and in vitro using primary culture of neonatal rat cardiomyocytes (NRVM, 24 h, Dox 1 μM). Then we investigated the effect of Epac inhibition on human cancer cell lines (24 h, Dox 1-10 μM). Results Dox-treated mice developed a DCM associated with Ca2+ homeostasis dysfunction. In vitro, as measured by flow cytometry and western blot, Dox induced DNA damages and cell death. This cell death is associated with apoptotic features including mitochondrial membrane permeabilization, caspase activation, cell size reduction and relative plasma membrane integrity. The inhibition of Epac1 (ESI09, CE3F4), but not Epac2 (ESI05), prevented DNA/TopIIβ complexes, decreased Dox-induced DNA damage, loss of mitochondrial membrane potential, apoptosis and finally cardiomyocyte death. These results were confirmed in vivo since Dox-induced cardiotoxicity was prevented in Epac1 KO mice as evidenced by unaltered cardiac function (no DCM) 15 weeks post-treatment. Interestingly, the protection conferred by Epac1 inhibition was not transferred to human cancer cell lines treated by Dox. Conclusion Inhibition of Epac1 may thus represents a novel strategy to prevent Anthracyclines-induced cardiotoxicity. Moreover, Epac1 inhibition seems to be a valuable therapeutic strategy to treat cancer by potentiating Dox toxicity.

Whereas cardiac TRPC (transient receptor potential canonical) channels and the associated store-operated Ca2+ entry (SOCE) are abnormally elevated during cardiac hypertrophy and heart failure, the mechanism of this upregulation is not fully elucidated but might be related to the activation of the mineralocorticoid pathway. Using a combination of biochemical, Ca2+ imaging, and electrophysiological techniques, we determined the effect of 24-h aldosterone treatment on the TRPCs/Orai-dependent SOCE in adult rat ventricular cardiomyocytes (ARVMs). The 24-h aldosterone treatment (from 100 nM to 1 µM) enhanced depletion-induced Ca2+ entry in ARVMs, as assessed by a faster reduction of Fura-2 fluorescence decay upon the addition of Mn2+ and increased Fluo-4/AM fluorescence following Ca2+ store depletion. These effects were prevented by co-treatment with a specific mineralocorticoid receptor (MR) antagonist, RU-28318, and they are associated with the enhanced depletion-induced N-[4-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP2)-sensitive macroscopic current recorded by patch-clamp experiments. Molecular screening by qRT-PCR and Western blot showed a specific upregulation of TRPC1, TRPC5, and STIM1 expression at the messenger RNA (mRNA) and protein levels upon 24-h aldosterone treatment of ARVMs, corroborated by immunostaining. Our study provides evidence that the mineralocorticoid pathway specifically promotes TRPC1/TRPC5-mediated SOCE in adult rat cardiomyocytes.

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

Introduction Doxorubicin (Dox) is an anthracycline commonly used to treat many types of cancer; unfortunately this chemotherapeutic agent induces many side effects such as cardiotoxicity leading to dilated cardiomyopathy (DCM). The cardiotoxicity of Dox has been related to reactive oxygen species generation, DNA intercalation, topoisomerase II inhibition and bioenergetics alterations leading to cardiomyocyte death. Objective Nowadays the challenge is to find new treatment options aiming at reducing Dox cardiotoxicity. Epac (exchange protein directly activated by cAMP) signaling could be worth investigating as Epac activates small G proteins which are known to be involved in Dox-induced cardiotoxicity. Methods We investigated the time/dose-dependent Dox effect on Epac signaling in both in vivo mice model (C57Bl63/ Knock-out Epac1 mice, iv injections, 12mg/kg cumulative dose) and in vitro (primary culture of neonatal rat cardiomyocytes (NRVM, 24h, Dox 1μM). Results In vivo, Dox-treated mice developed a DCM associated with Ca2+ homeostasis dysfunction (increase of Ca2+ waves and Ca2+ leaks). In vitro, as measured by flow cytometry and western blot, Dox (1μM) induced DNA damages and cell death in NRVM. This cell death is associated with apoptotic features including mitochondrial membrane permeabilization, caspase activation and cell size reduction. The inhibition of Epac1 (ESI09, CE3F4) decreased Dox-induced DNA damage, loss of mitochondrial membrane potential, apoptosis and finally cardiomyocyte death. Moreover, in vivo, Epac1 KO mice were protected against Dox-induced cardiotoxicity by unaltered cardiac function (no DCM) and calcium homeostasis at 15 weeks post-treatment. Conclusion Inhibition of Epac1 could be a valuable therapeutic strategy to limit Dox-induced cardiomyopathy during cancer chemotherapy. Indeed, preliminary data show also that preventing Dox-induced cardiotoxicity, the inhibition of Epac1 can also potentiate cancerous cells death. Acknowledgement/Funding Labex Lermit (ANR 0033), Torino and Inserm

Introduction Doxorubicin (Dox) is an Anthracycline commonly used to treat many types of cancer; unfortunately this chemotherapeutic agent often induces side effects such as cardiotoxicity leading to cardiomyocyte death and dilated cardiomyopathy (DCM). This cardiotoxicity has been related to reactive oxygen species generation, DNA intercalation, topoisomerase II inhibition and bioenergetics alterations resulting in DNA damages leading to cardiomyocyte death. Objectives There is a need for new therapeutic agents and strategies aiming at reducing Dox side effects in the heart. Among these mechanisms, the role of Epac (exchange protein directly activated by cAMP) could be worth investigating. Methods Therefore we have investigated the time/dose-dependent Dox effect on Epac signaling in both in vivo mice model (C57BL/6 vs. Epac1 KO mice, iv injections, 12 mg/kg) and in vitro (primary culture of neonatal rat cardiomyocytes (NRVM), Dox 1 μM). Results In vivo, Dox-treated mice developed a DCM associated with Ca2+ homeostasis dysfunction. In vitro, as measured by flow cytometry and western blot, Dox induced DNA damage and cell death. This cell death is associated with apoptotic features including mitochondrial membrane permeabilization, caspase activation, cell size reduction and relative plasma membrane integrity. We also observed that Dox led to a modification of the protein level of Epac1 and Epac2 isoforms. The inhibition of Epac1 (ESI09, CE3F4), but not of Epac2 (ESI05), prevented DNA/TopIIβ complexes, decreased Dox-induced DNA damage, loss of mitochondrial membrane potential, apoptosis and finally cardiomyocyte death. These results were confirmed in vivo since Dox-induced cardiotoxicity was prevented in Epac1 KO mice as evidenced by unaltered cardiac function (no DCM) 15 weeks post-treatment. Conclusion Inhibition of Epac1 could be a valuable therapeutic strategy to limit Dox-induced cardiomyopathy during cancer chemotherapy.

Introduction Stromal interaction molecules, STIM1 and STIM2, are ER Ca2+ sensors that initiate store-operated Ca2+ entry (SOCE). It has been recently identified three STIM2 splice variants (STIM2.1, 2.2 and 2.3), STIM2.2 being SOCE activator whereas STIM2.1 will be SOCE inhibitor. The role of STIM1-mediated SOCE has been highlighted in cardiac pathobiology but the one of STIM2.1 and STIM2.2 is unknown. Objective To assess whether and how STIM2.1 and STIM2.2 regulate SOCE in neonatal rat ventricular cardiomyocytes (NRVMs). Method To analyze the function of STIM2.1 and STIM2.2 variants, the NRVMs were transfected with STIM2.2 or STIM2.1 in combination or not with STIM1 or dn-Orai1106A mutant plasmids or with a mixture of 4 STIM2 siRNAs. Results At rest, both native STIM2 and exogenous STIM2.2 proteins present a reticular organization. Live cell confocal imaging in STIM2.2 overexpressing cells showed that STIM2.2 is aggregated and translocated towards the sarcolemma following the SR Ca2+ store depletion. With Ca2+ imaging, we found that STIM2.2 overexpression significantly increases SOCE, similarly to STIM1 overexpression, but the STIM1 and STIM2.2 co-expression has no additional effect. Importantly, the STIM2.2-enhanced SOCE is suppressed by functional Orai1 inhibition, either by co-expressing STIM2.2 with the dn-Orai1106A mutant or with the selective Orai1 inhibitor, JPIII. In addition, the JPIII-sensitive Isoc current is also higher in STIM2.2 overexpressing cells. Nonetheless, silencing of endogenous STIM2 also increases SOCE. By co-immunoprecipitation, we found that STIM2, STIM1 and Orai1 interact together suggesting that all these proteins form a complex and constitute functional SOCE machinery. Conclusion We demonstrated that, in overexpressing system, STIM2.2 functions together with STIM1 as a positive regulator of Orai1-mediated SOCE and that, in endogenous system, the STIM2.1 variant may be the predominant variant that negatively regulates SOCE in NRVMs.

Store-operated Ca(2+) entry (SOCE) has emerged as an important mechanism in cardiac pathology. However, the signals that up-regulate SOCE in the heart remain unexplored. Clinical trials have emphasized the beneficial role of mineralocorticoid receptor (MR) signaling blockade in heart failure and associated arrhythmias. Accumulated evidence suggests that the mineralocorticoid hormone aldosterone, through activation of its receptor, MR, might be a key regulator of Ca(2+) influx in cardiomyocytes. We thus assessed whether and how SOCE involving transient receptor potential canonical (TRPC) and Orai1 channels are regulated by aldosterone/MR in neonatal rat ventricular cardiomyocytes. Molecular screening using qRT-PCR and Western blotting demonstrated that aldosterone treatment for 24 h specifically increased the mRNA and/or protein levels of Orai1, TRPC1, -C4, -C5, and stromal interaction molecule 1 through MR activation. These effects were correlated with a specific enhancement of SOCE activities sensitive to store-operated channel inhibitors (SKF-96365 and BTP2) and to a potent Orai1 blocker (S66) and were prevented by TRPC1, -C4, and Orai1 dominant negative mutants or TRPC5 siRNA. A mechanistic approach showed that up-regulation of serum- and glucocorticoid-regulated kinase 1 mRNA expression by aldosterone is involved in enhanced SOCE. Functionally, 24-h aldosterone-enhanced SOCE is associated with increased diastolic [Ca(2+)]i, which is blunted by store-operated channel inhibitors. Our study provides the first evidence that aldosterone promotes TRPC1-, -C4-, -C5-, and Orai1-mediated SOCE in cardiomyocytes through an MR and serum- and glucocorticoid-regulated kinase 1 pathway.

Introduction Epidemiological and preclinical studies have pointed out a correlation between hyperglycemia and increasing risk of heart failure. In cardiomyocytes, hyperglycemia has been shown to alter Ca2+ signaling via CaMKII a downstream effector of Epac2, a key player in Ca2+ mishandling. However the role of Epac2 in high-glucose (HG) mediated Ca2+ dysregulation is unclear. Objective To test the involvement of Epac2 in hyperglycemia-mediated cardiac Ca2+ mishandling. Method Ca2+ signaling was measured by confocal microscopy in Fluo-4 loaded ventricular cardiomyocytes isolated from C57BL6 and Epac2-KO mice and treated with different glucose concentrations. Results HG (500 mg/dL) enhanced the occurrence of Ca2+ sparks and pro-arrhythmic events in WT mice but not in Epac2 KO. This increase was prevented by Epac2 pharmacological inhibition (ESI-05 10 μM) (Ca2+ sparks frequency [#/s/100 μm]: 1.07 ± 0.39 for HG vs. 0.16 ± 0.06 for normal glucose [NG, 100 mg/dL], P Conclusion Our work shows an Epac2 O-GlcNAcylation by HG responsible for pro-arrhythmic SR Ca2+ leak in cardiomyocytes, a new mechanism that might be involved in diabetic cardiomyopathy.

International audience; 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 [Ca2+]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 [Ca2+]i transients at 2 weeks after treatment, withoccurrence of proarrhythmogenic Ca2+ waves, 2/ compensatory state at 6 weeks, and 3/ depression on [Ca2+]i transients and cell contraction at 15 weeks, concomitant with in vivo defects. These [Ca2+]i transient alterations were observed without cellular hypertrophy or AP prolongation and mirrored the sarcoplasmic reticulum (SR) Ca2+ load variations. At the molecular level, this was associated with a decrease in the sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) expression and enhanced RyR2 phosphorylation at the protein kinase A (PKA, pS2808) site (2 and 15 weeks). RyR2 phosphorylation at the Ca2+/calmodulin dependent protein kinase II (CaMKII, pS2814) site was enhanced only at 2 weeks, coinciding with the higher incidence of proarrhythmogenic Ca2+ waves. Our study highlighted, for the first time, the progression of Dox treatment-induced alterations in Ca2+ 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.

Rationale: The MR (mineralocorticoid receptor) antagonists belong to the current therapeutic armamentarium for the management of cardiovascular diseases, but the mechanisms conferring their beneficial effects are poorly understood. Part of the cardiovascular effects of MR is because of the regulation of L-type Ca v 1.2 Ca 2+ channel expression, which is generated by tissue-specific alternative promoters as a long cardiac or short vascular N-terminal transcripts. Objective: To analyze the molecular mechanisms by which aldosterone, through MR, modulates Ca v 1.2 expression and function in a tissue-specific manner. Methods and Results: In primary cultures of neonatal rat ventricular myocytes, aldosterone exposure for 24 hours increased in a concentration-dependent manner long cardiac Ca v 1.2 N-terminal transcripts expression at both mRNA and protein levels, correlating with enhanced concentration-, time-, and MR-dependent P1-promoter activity. In silico analysis and mutagenesis identified MR interaction with both specific activating and repressing DNA-binding elements on the P1-promoter. The relevance of this regulation is confirmed both ex and in vivo in transgenic mice harboring the luciferase reporter gene under the control of the cardiac P1-promoter. Moreover, we show that this cis -regulatory mechanism is not limited to the heart. Indeed, in smooth muscle cells from different vascular beds, in which the short vascular Cav1.2 N-terminal transcripts is normally the major isoform, we found that MR signaling activates long cardiac Ca v 1.2 N-terminal transcripts expression through P1-promoter activation, leading to vascular contractile dysfunction. These results were further corroborated in hypertensive aldosterone/salt rodent models, showing notably a positive correlation between blood pressure and cardiac P1-promoter activity in aorta. This new vascular long cardiac Ca v 1.2 N-terminal transcripts molecular signature reduced sensitivity to the Ca 2+ channel blocker, nifedipine, in aldosterone-treated vessels. Conclusions: Our results reveal that MR acts as a transcription factor to translate aldosterone signal into specific cardiac P1-promoter activation that might influence the therapeutic outcome of cardiovascular diseases.

Deletion of QKP1507-1509 amino-acids in SCN5A gene product, the voltage-gated NaWe generated a knock-in mouse presenting the delQKP1510-1512 mutation (Scn5aThe Scn5a

Right ventricular (RV) function is the most important prognostic factor for pulmonary arterial hypertension (PAH) patients. The progressive increase of pulmonary vascular resistance induces RV hypertrophy (RVH) and at term RV failure (RVF). However, the molecular mechanisms of RVH and RVF remain understudied. In this study, we gained insights into cytosolic CaAfter PH induction, hypertrophied RV myocytes presented longer action potential duration, higher and faster [CaThese new findings demonstrate RV-specific cellular Ca

Introduction Epidemiological and preclinical studies have pointed out a correlation between hyperglycemia and increasing risk of heart failure and cardiac death. In cardiomyocytes, hyperglycemia has been shown to alter Ca2 + signalling via CaMKII. Yet, the underlying mechanisms are still unclear. Objective To determine the molecular basis of high glucose-mediated Ca2 + mishandling. Methods Ventricular cardiomyocytes were isolated from control and Epac2-KO mice. Parallel experiments were repeated in human cardiomyocytes differentiated from induced pluripotent stem cells (h-iPSC-CM). Ca2 + signaling was studied in cells loaded with a fluorescent Ca2 + dye and treated with high glucose (HG) ± ESI-05 (Epac2 inhibitor). We assessed ryanodine receptor (RyR2) phosphorylation state by western blot and measured single RyR2 channel activity incorporated into bilayers. Results HG-mediated SR Ca2 + leak and pro-arrhythmic events were prevented with Epac2 blocker (ESI-05) in normal mice cells and with Epac2 deletion (Epac2-KO) (Ca2 + sparks frequency N/100 μm/s: 0.23 ± 0.15 for HG + ESI-05, 0.09 ± 0.04 for Epac2-KO + HG vs. 0.16 ± 0.06 for normal glucose, P = N.S.). HG treatment increased RyR2 phosphorylation at the CaMKII site (S2814, ∼ 29% increase, P Conclusion Our data suggests that HG alters Ca2 + signalling via Epac2–mediated pro-arrhythmic events due a CaMKII-dependent increase of RyR2 activity. Overtime, this newly described mechanism lowers systolic Ca2 + release as seen in diabetic-associated cardiomyopathy.