Search

Background The AMP-activated protein kinase (AMPK) is a major regulator of cellular energetics which plays key role in acute metabolic response and in long-term adaptation to stress. Recent works have also suggested non-metabolic effects. Methods To decipher AMPK roles in the heart, we generated a cardio-specific inducible model of gene deletion of the main cardiac catalytic subunit of AMPK (Ampkα2) in mice. This allowed us to avoid the eventual impact of AMPK-KO in peripheral organs. Results Cardio-specific Ampkα2 deficiency led to a progressive left ventricular systolic dysfunction and the development of cardiac fibrosis in males. We observed a reduction in complex I-driven respiration without change in mitochondrial mass or in vitro complex I activity, associated with a rearrangement of the cardiolipins and reduced integration of complex I into the electron transport chain supercomplexes. Strikingly, none of these defects were present in females. Interestingly, suppression of estradiol signaling by ovariectomy partially mimicked the male sensitivity to AMPK loss, notably the cardiac fibrosis and the rearrangement of cardiolipins, but not the cardiac function that remained protected. Conclusion Our results confirm the close link between AMPK and cardiac mitochondrial function, but also highlight links with cardiac fibrosis. Importantly, we show that AMPK is differently involved in these processes in males and females, which may have clinical implications for the use of AMPK activators in the treatment of heart failure., Highlights AMPK is a metabolic sensor of cellular energy which regulates energy homeostasis.We generated a cardiac-specific inducible deletion of Ampkα2 and demonstrated that this deletion induces mild cardiac dysfunction in male only.Cardiac dysfunction observed in males was associated with cardiac fibrosis and cardiac cardiolipin remodeling that are not seen in females.Although no significant cardiac function alteration was noticed in ovariectomized female Ampkα2ciKO mice, these latter exhibited cardiac fibrosis and mild cardiolipins remodeling.Our results show a higher dependence on AMPK signaling fibrosis and cardiolipin biosynthesis/maturation in males, either due to the absence of female hormones protection or/and to the action of male hormones. This may contribute to the known difference in cardiovascular risk and outcome between sexes.

Objective We aimed to assess the mitochondrial respiratory capacities in the right ventricle in the setting of ventricular remodeling induced by pressure overload. Methods Chronic thromboembolic pulmonary hypertension was induced in 8 piglets over a 12-week period (chronic thromboembolic pulmonary hypertension model). Right ventricular remodeling, right ventricular function, and mitochondrial respiratory function were assessed at 3, 6, and 12 weeks after induction of pulmonary hypertension and were compared with sham animals (n = 5). Right ventricular cardiomyocytes and mitochondrial structure were studied in transmission electronic microscopy after 12 weeks. Results As of 3 weeks, chronic pressure overload induced right ventricular dilatation, right ventricular hypertrophy, and right ventricular dysfunction. Maladaptive remodeling in the chronic thromboembolic pulmonary hypertension model was confirmed by the decrease of right ventricular pulmonary artery coupling and right fractional area change. Mitochondrial functional assays in permeabilized right ventricular myocardial fibers revealed that oxidative phosphorylation capacities (complex I, complex II, and IV of the mitochondrial respiratory chain) were degraded. Furthermore, no change in substrate preference of mitochondria was found in the overloaded right ventricle. There was a good correlation between maximal mitochondrial oxygen consumption rate and right ventricular pulmonary artery coupling (Pearson coefficient r = 0.83). Transmission electronic microscopy analysis showed that the composition of cardiomyocytes was no different between the chronic thromboembolic pulmonary hypertension group and the sham group. However, mitochondrial structure anomalies were significantly increased in the chronic thromboembolic pulmonary hypertension group. Conclusions Mitochondrial respiratory function impairment is involved early in the development of right ventricular dysfunction in a piglet model of chronic thromboembolic pulmonary hypertension. Underlying mechanisms remain to be elucidated.

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.

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.

Right ventricular (RV) failure is a major concern in grown-up congenital heart diseases. Cell-based myocardial repair may be an innovative approach to restore overloaded RV function. A repaired Tetralogy of Fallot pig model was used. After 5 months, cell therapy was surgically administrated using human embryonic stem cell-derived Nkx2.5+ cardiac progenitor cell-seeded collagen patch. A large patch containing 107 cells was engineered to cover the whole RV free wall and applied on the epicardium of 4 pigs (treated group). A control group (n = 4) received a cell-free patch (3) or no patch (1). Myocardial function was assessed by echocardiography using standard and strain parameters before and 2 months after cell graft. The fate of progenitors was tracked using human-and cardiac specific antibodies. Indexed RV diameter and telediastolic RV area significantly decreased in treated group whereas increased in control. The free wall thickness significantly increased in treated group compared to control. In the treated group, RV free wall strain significantly improved by contrast to control (respectively from −13.4 to −15.9% vs. −18.9 to −12.9%, P Fig. 1 ). Although still immature, some cells featured TnT+ actinin+ sarcomeric units. Proliferating small Ki-67+ TnT+ labelled pig myocytes were observed. Expression of collagen genes col1a3, col3a1 monitored in real-Time QPCR reflecting myocardial fibrosis status significantly decreased in the treated group. Human cardiac progenitors migrated and differentiated, contributing together with triggered endogenous mechanism of repair to maintain the RV adaptation to overload and to improve free wall contractility. They also induced a proliferation of host cells supporting potential paracrine effects.

Aims Pulmonary hypertension (PH) is a common complication of left heart disease (LHD, Group 2 PH) leading to right ventricular (RV) failure and death. Several loss-of-function (LOF) mutations in KCNK3 were identified in pulmonary arterial hypertension (PAH, Group 1 PH). Additionally, we found that KCNK3 dysfunction is a hallmark of PAH at pulmonary vascular and RV levels. However, the role of KCNK3 in the pathobiology of PH due to LHD is unknown. Methods and results We evaluated the role of KCNK3 on PH induced by ascending aortic constriction (AAC), in WT and Kcnk3-LOF-mutated rats, by echocardiography, RV catheterization, histology analyses, and molecular biology experiments. We found that Kcnk3-LOF-mutation had no consequence on the development of left ventricular (LV) compensated concentric hypertrophy in AAC, while left atrial emptying fraction was impaired in AAC-Kcnk3-mutated rats. AAC-animals (WT and Kcnk3-mutated rats) developed PH secondary to AAC and Kcnk3-mutated rats developed more severe PH than WT. AAC-Kcnk3-mutated rats developed RV and LV fibrosis in association with an increase of Col1a1 mRNA in right ventricle and left ventricle. AAC-Kcnk3-mutated rats developed severe pulmonary vascular (pulmonary artery as well as pulmonary veins) remodelling with intense peri-vascular and peri-bronchial inflammation, perivascular oedema, alveolar wall thickening, and exaggerated lung vascular cell proliferation compared to AAC-WT-rats. Finally, in lung, right ventricle, left ventricle, and left atrium of AAC-Kcnk3-mutated rats, we found a strong increased expression of Il-6 and periostin expression and a reduction of lung Ctnnd1 mRNA (coding for p120 catenin), contributing to the exaggerated pulmonary and heart remodelling and pulmonary vascular oedema in AAC-Kcnk3-mutated rats. Conclusions Our results indicate that Kcnk3-LOF is a key event in the pathobiology of PH due to AAC, suggesting that Kcnk3 channel dysfunction could play a potential key role in the development of PH due to LHD.

International audience; Trichloroethylene exposure is a major risk factor for pulmonary veno-occlusive disease. We demonstrated that trichloroethylene alters the endothelial barrier integrity, at least in part, through vascular endothelial (VE)-Cadherin internalisation, and suggested that this mechanism may play a role in the development of pulmonary veno-occlusive disease.

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.

The WT1 (Wilm's tumor suppressor) gene is expressed throughout life in podocytes and is essential for the functional integrity of the glomerular filtration barrier. We have previously shown that CMIP (C-Maf inducing protein) is overproduced in podocyte diseases and alters intracellular signaling. Here we isolated the proximal region of the human CMIP promoter and showed by chromatin immunoprecipitation assays and electrophoretic-mobility shift that Wilm's tumor protein (WT1) bound to 2 WT1 response elements, located at positions –290/–274 and –57/–41 relative to transcription start site. Unlike the human CMIP gene, only one Wt1 response element was identified in the mouse Cmip proximal promoter located at position –217/–206. Luciferase reporter assays indicated that WT1 dose-dependently inhibited the transcriptional induction of the CMIP promoter. Transfection of decoy oligonucleotides mimicking the WT1 response elements prevented the inhibition of WT1 on CMIP promoter activity. Furthermore, WT1 silencing promoted Cmip expression. In line with these findings, the abundance of Cmip was early and significantly increased at the transcript and protein level in podocytes displaying a primary defect in Wt1 , including Denys–Drash syndrome and Frasier syndrome. Thus, WT1 is a major repressor of the CMIP gene in physiological situations, while conditional deletion of CMIP in the developing kidney did not affect the development of mature glomeruli.

Ion channel trafficking powerfully influences cardiac electrical activity as it regulates the number of available channels at the plasma membrane. Studies have largely focused on identifying the molecular determinants of the trafficking of the atria-specific KV1.5 channel, the molecular basis of the ultra-rapid delayed rectifier current IKur. Besides, regulated KV1.5 channel recycling upon changes in homeostatic state and mechanical constraints in native cardiomyocytes has been well documented. Here, using cutting-edge imaging in live myocytes, we investigated the dynamics of this channel in the plasma membrane. We demonstrate that the clathrin pathway is a major regulator of the functional expression of KV1.5 channels in atrial myocytes, with the microtubule network as the prominent organizer of KV1.5 transport within the membrane. Both clathrin blockade and microtubule disruption result in channel clusturization with reduced membrane mobility and internalization, whereas disassembly of the actin cytoskeleton does not. Mobile KV1.5 channels are associated with the microtubule plus-end tracking protein EB1 whereas static KV1.5 clusters are associated with stable acetylated microtubules. In human biopsies from patients in atrial fibrillation associated with atrial remodeling, drastic modifications in the trafficking balance occurs together with alteration in microtubule polymerization state resulting in modest reduced endocytosis and increased recycling. Consequently, hallmark of atrial KV1.5 dynamics within the membrane is clathrin- and microtubule-dependent. During atrial remodeling, predominance of anterograde trafficking activity over retrograde trafficking could result in accumulation ok KV1.5 channels in the plasma membrane.

Heart failure is associated with profound alterations of energy metabolism thought to play a major role in the progression of this syndrome. SIRT1 is a metabolic sensor of cellular energy and exerts essential functions on energy metabolism, oxidative stress response, apoptosis, or aging. Importantly, SIRT1 deacetylates the peroxisome proliferator-activated receptor gamma co-activator 1&alpha, (PGC-1&alpha, ), the master regulator of energy metabolism involved in mitochondrial biogenesis and fatty acid utilization. However, the exact role of SIRT1 in controlling cardiac energy metabolism is still incompletely understood and conflicting results have been obtained. We generated a cardio-specific inducible model of Sirt1 gene deletion in mice (Sirt1ciKO) to decipher the role of SIRT1 in control conditions and following cardiac stress induced by pressure overload. SIRT1 deficiency induced a progressive cardiac dysfunction, without overt alteration in mitochondrial content or properties. Sixteen weeks after Sirt1 deletion an increase in mitochondrial reactive oxygen species (ROS) production and a higher rate of oxidative damage were observed, suggesting disruption of the ROS production/detoxification balance. Following pressure overload, cardiac dysfunction and alteration in mitochondrial properties were exacerbated in Sirt1ciKO mice. Overall the results demonstrate that SIRT1 plays a cardioprotective role on cardiac energy metabolism and thereby on cardiac function.

Introduction Mitochondrial dysfunction plays a major role in the Heart Failure (HF) pathophysiology.The AMP activated protein kinase (AMPK) is activated by a high AMP-ADP/ATP ratio and regulates a number of metabolic pathways. Many studies have highlighted a protective role of AMPK in HF, but its relevance to cardiac tissue, its metabolic part and its sex specificity are not well established. Purpose Then, the aim of this study is to determine the role of AMPK in the healthy and failing heart in male and female mice. Methods We developed and validated a mouse strain with an adult-inducible cardiac-specific deletion of AMPKα2, the major cardiac isoform, using the Cre-Lox system (40mg/kg tamoxifen injection on two consecutive days at adult age). At four months after the deletion, cardiac contractility, morphology and metabolism were studied in control and KO mice from both sexes. Results We observed only in male KO mice a decrease of left ventricular ejection fraction (−10%), an increase of the total fibrosis (+64%) and defects in mitochondrial structures. Male KO mice also showed a reduced (−28%) mitochondrial respiration via complex I associated with a different cardiolipin species distribution. Conclusion Our results reveal in adult healthy hearts, a sex-specificity in the effects of AMPKα2 deletion, leading to impaired contractile function related to metabolic and non-metabolic alterations only in male mice.

Rationale: Pulmonary arterial hypertension is a severe lethal cardiopulmonary disease. Loss of function mutations in KCNK3 (potassium channel subfamily K member 3) gene, which encodes an outward rectifier K + channel, have been identified in pulmonary arterial hypertension patients. Objective: We have demonstrated that KCNK3 dysfunction is common to heritable and nonheritable pulmonary arterial hypertension and to experimental pulmonary hypertension (PH). Finally, KCNK3 is not functional in mouse pulmonary vasculature. Methods and Results: Using CRISPR/Cas9 technology, we generated a 94 bp out of frame deletion in exon 1 of Kcnk3 gene and characterized these rats at the electrophysiological, echocardiographic, hemodynamic, morphological, cellular, and molecular levels to decipher the cellular mechanisms associated with loss of KCNK3. Using patch-clamp technique, we validated our transgenic strategy by demonstrating the absence of KCNK3 current in freshly isolated pulmonary arterial smooth muscle cells from Kcnk3 -mutated rats. At 4 months of age, echocardiographic parameters revealed shortening of the pulmonary artery acceleration time associated with elevation of the right ventricular systolic pressure. Kcnk3 -mutated rats developed more severe PH than wild-type rats after monocrotaline exposure or chronic hypoxia exposure. Kcnk3 -mutation induced a lung distal neomuscularization and perivascular extracellular matrix activation. Lungs of Kcnk3 -mutated rats were characterized by overactivation of ERK1/2 (extracellular signal–regulated kinase1-/2), AKT (protein kinase B), SRC, and overexpression of HIF1-α (hypoxia-inducible factor-1 α), survivin, and VWF (Von Willebrand factor). Linked with plasma membrane depolarization, reduced endothelial-NOS expression and desensitization of endothelial-derived hyperpolarizing factor, Kcnk3 -mutated rats presented predisposition to vasoconstriction of pulmonary arteries and a severe loss of sildenafil-induced pulmonary arteries relaxation. Moreover, we showed strong alteration of right ventricular cardiomyocyte excitability. Finally, Kcnk3 -mutated rats developed age-dependent PH associated with low serum-albumin concentration. Conclusions: We established the first Kcnk3 -mutated rat model of PH. Our results confirm that KCNK3 loss of function is a key event in pulmonary arterial hypertension pathogenesis. This model presents new opportunities for understanding the initiating mechanisms of PH and testing biologically relevant therapeutic molecules in the context of PH.

Pulmonary arterial hypertension is a severe lethal cardiopulmonary disease. Loss of function mutations inWe have demonstrated that KCNK3 dysfunction is common to heritable and nonheritable pulmonary arterial hypertension and to experimental pulmonary hypertension (PH). Finally, KCNK3 is not functional in mouse pulmonary vasculature.Using CRISPR/Cas9 technology, we generated a 94 bp out of frame deletion in exon 1 ofWe established the first

Background: Monoallelic mutations in the gene encoding bone morphogenetic protein receptor 2 ( Bmpr2 ) are the main genetic risk factor for heritable pulmonary arterial hypertension (PAH) with incomplete penetrance. Several Bmpr2 transgenic mice have been reported to develop mild spontaneous PAH. In this study, we examined whether rats with the Bmpr2 mutation were susceptible to developing more severe PAH. Methods: The zinc finger nuclease method was used to establish rat lines with mutations in the Bmpr2 gene. These rats were then characterized at the hemodynamic, histological, electrophysiological, and molecular levels. Results: Rats with a monoallelic deletion of 71 bp in exon 1 (Δ 71 rats) showed decreased BMPRII expression and phosphorylated SMAD1/5/9 levels. Δ 71 Rats develop age-dependent spontaneous PAH with a low penetrance (16%–27%), similar to that in humans. Δ 71 Rats were more susceptible to hypoxia-induced pulmonary hypertension than wild-type rats. Δ 71 Rats exhibited progressive pulmonary vascular remodeling associated with a proproliferative phenotype and showed lower pulmonary microvascular density than wild-type rats. Organ bath studies revealed severe alteration of pulmonary artery contraction and relaxation associated with potassium channel subfamily K member 3 (KCNK3) dysfunction. High levels of perivascular fibrillar collagen and pulmonary interleukin-6 overexpression discriminated rats that developed spontaneous PAH and rats that did not develop spontaneous PAH. Finally, detailed assessments of cardiomyocytes demonstrated alterations in morphology, calcium (Ca 2+ ), and cell contractility specific to the right ventricle; these changes could explain the lower cardiac output of Δ 71 rats. Indeed, adult right ventricular cardiomyocytes from Δ 71 rats exhibited a smaller diameter, decreased sensitivity of sarcomeres to Ca 2+ , decreased [Ca 2+ ] transient amplitude, reduced sarcoplasmic reticulum Ca 2+ content, and short action potential duration compared with right ventricular cardiomyocytes from wild-type rats. Conclusions: We characterized the first Bmpr2 mutant rats and showed some of the critical cellular and molecular dysfunctions described in human PAH. We also identified the heart as an unexpected but potential target organ of Bmpr2 mutations. Thus, this new genetic rat model represents a promising tool to study the pathogenesis of PAH.

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

The bromodomain and extra-terminal domain family inhibitors (BETi) are a promising new class of anticancer agents. Since numerous anticancer drugs have been correlated to cardiomyopathy, and since BETi can affect non-cancerous tissues, we aimed to investigate in healthy animals any ultrastructural BETi-induced alterations of the heart as compared to skeletal muscle. Male Wistar rats were either treated during 3 weeks with I-BET-151 (2 or 10 mg/kg/day) (W3) or treated for 3 weeks then allowed to recover for another 3 weeks (W6) (3-weeks drug washout). Male C57Bl/6J mice were only treated during 5 days (50 mg/kg/day). We demonstrated the occurrence of ultrastructural alterations and progressive destruction of cardiomyocyte mitochondria after I-BET-151 exposure. Those mitochondrial alterations were cardiac muscle-specific, since the skeletal muscles of exposed animals were similar in ultrastructure presentation to the non-exposed animals. I-BET-151 decreased the respiration rate of heart mitochondria in a dose-dependent manner. At the higher dose, it also decreased mitochondrial mass, as evidenced by reduced right ventricular citrate synthase content. I-BET-151 reduced the right and left ventricular fractional shortening. The concomitant decrease in the velocity-time-integral in both the aorta and the pulmonary artery is also suggestive of an impaired heart function. The possible context-dependent cardiac side effects of these drugs have to be appreciated. Future studies should focus on the basic mechanisms of potential cardiovascular toxicities induced by BETi and strategies to minimize these unexpected complications.

IntroductionA reduction in pulmonary artery relaxation is a key event in the pathogenesis of pulmonary arterial hypertension (PAH). Cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction in airway epithelial cells plays a central role in cystic fibrosis; CFTR is also expressed in pulmonary arteries and has been shown to control endothelium-independent relaxation.Aim and objectivesWe aimed to delineate the role of CFTR in PAH pathogenesis through observational and interventional experiments in human tissues and animal models.Methods and resultsReverse-transcriptase quantitative PCR, confocal imaging and electron microscopy showed that CFTR expression was reduced in pulmonary arteries from patients with idiopathic PAH (iPAH) and in rats with monocrotaline-induced pulmonary hypertension (PH). Moreover, using myography on human, pig and rat pulmonary arteries, we demonstrated that CFTR activation induces pulmonary artery relaxation. CFTR-mediated pulmonary artery relaxation was reduced in pulmonary arteries from iPAH patients and rats with monocrotaline- or chronic hypoxia-induced PH. Long-term in vivo CFTR inhibition in rats significantly increased right ventricular systolic pressure, which was related to exaggerated pulmonary vascular cell proliferation in situ and vessel neomuscularisation. Pathologic assessment of lungs from patients with severe cystic fibrosis (F508del-CFTR) revealed severe pulmonary artery remodelling with intimal fibrosis and medial hypertrophy. Lungs from homozygous F508delCftr rats exhibited pulmonary vessel neomuscularisation. The elevations in right ventricular systolic pressure and end diastolic pressure in monocrotaline-exposed rats with chronic CFTR inhibition were more prominent than those in vehicle-exposed rats.ConclusionsCFTR expression is strongly decreased in pulmonary artery smooth muscle and endothelial cells in human and animal models of PH. CFTR inhibition increases vascular cell proliferation and strongly reduces pulmonary artery relaxation.

Purpose Although inflammatory features have been reported in the pressure-overloaded right ventricle, prior proteomic studies of pulmonary arterial hypertension (PAH) have mainly focused on identifying circulating biomarkers associated with pulmonary vascular disease severity or outcomes. This study sought to determine the circulating proteomic immune profile associated with right heart maladaptive phenotype (RHMP) in PAH. Methods In this observational study of PAH patients enrolled at Stanford University from 2008-2011 (n=121, discovery cohort) and from 2011-2014 (n=76, validation cohort), we carried out plasma proteomic profiling using Luminex® multiplex immunoassay (48-plex of interleukins, chemokines and growth factors). RHMP was defined by the Mayo right heart score (based on echocardiographic right ventricular RV strain, NYHA class and NT-proBNP) and Stanford right heart score (based on RV end-systolic remodeling index, NYHA class and NT-proBNP). The association between cytokines and RHMP was assessed by partial least square regression (PLS-R). To further validate the circulating markers associated with RHMP, we confirmed tissue-level expression in RV biopsies from PAH patients. Results Median age was 50 [39-59] years in the discovery cohort, where the majority of patients was female (74%) and PAH was most commonly associated with connective tissue disease (33%). Patients from the validation cohort had more severe features (lower six minute walk test distance, lower cardiac index and higher levels of NT-proBNP) than patients from the discovery cohort, with similar resistance levels and right heart echocardiography metrics. High levels of hepatic growth factor (HGF), stem cell growth factor beta (SCGFβ), nerve growth factor (NGF) and SDF1 were significantly associated with worst Mayo and Stanford scores but not with pulmonary vascular resistance or mean pulmonary arterial pressure, in both cohorts. In the right ventricle of deceased PAH patients, we confirmed expression of HGF and its receptor cMet using immunofluorescence. Conclusion High plasma levels of HGF, SCGFβ, NGF and SDF1 are associated with RHMP in PAH, independent of pulmonary vascular disease severity. The HGF-cMet pathway warrants further exploration to investigate a potential RV specific therapeutic target.

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.

Background: Excessive proliferation and apoptosis resistance in pulmonary vascular cells underlie vascular remodeling in pulmonary arterial hypertension (PAH). Specific treatments for PAH exist, mostly targeting endothelial dysfunction, but high pulmonary arterial pressure still causes heart failure and death. Pulmonary vascular remodeling may be driven by metabolic reprogramming of vascular cells to increase glutaminolysis and glutamate production. The N -methyl- d -aspartate receptor (NMDAR), a major neuronal glutamate receptor, is also expressed on vascular cells, but its role in PAH is unknown. Methods: We assessed the status of the glutamate-NMDAR axis in the pulmonary arteries of patients with PAH and controls through mass spectrometry imaging, Western blotting, and immunohistochemistry. We measured the glutamate release from cultured pulmonary vascular cells using enzymatic assays and analyzed NMDAR regulation/phosphorylation through Western blot experiments. The effect of NMDAR blockade on human pulmonary arterial smooth muscle cell proliferation was determined using a BrdU incorporation assay. We assessed the role of NMDARs in vascular remodeling associated to pulmonary hypertension, in both smooth muscle-specific NMDAR knockout mice exposed to chronic hypoxia and the monocrotaline rat model of pulmonary hypertension using NMDAR blockers. Results: We report glutamate accumulation, upregulation of the NMDAR, and NMDAR engagement reflected by increases in GluN1-subunit phosphorylation in the pulmonary arteries of human patients with PAH. K v channel inhibition and type A-selective endothelin receptor activation amplified calcium-dependent glutamate release from human pulmonary arterial smooth muscle cell, and type A-selective endothelin receptor and platelet-derived growth factor receptor activation led to NMDAR engagement, highlighting crosstalk between the glutamate-NMDAR axis and major PAH-associated pathways. The platelet-derived growth factor-BB-induced proliferation of human pulmonary arterial smooth muscle cells involved NMDAR activation and phosphorylated GluN1 subunit localization to cell-cell contacts, consistent with glutamatergic communication between proliferating human pulmonary arterial smooth muscle cells via NMDARs. Smooth-muscle NMDAR deficiency in mice attenuated the vascular remodeling triggered by chronic hypoxia, highlighting the role of vascular NMDARs in pulmonary hypertension. Pharmacological NMDAR blockade in the monocrotaline rat model of pulmonary hypertension had beneficial effects on cardiac and vascular remodeling, decreasing endothelial dysfunction, cell proliferation, and apoptosis resistance while disrupting the glutamate-NMDAR pathway in pulmonary arteries. Conclusions: These results reveal a dysregulation of the glutamate-NMDAR axis in the pulmonary arteries of patients with PAH and identify vascular NMDARs as targets for antiremodeling treatments in PAH.

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

Background— Cardiovascular diseases are the major cause of mortality among both men and women with a lower incidence in women before menopause. The clinical use of doxorubicin, widely used as an antineoplastic agent, is markedly hampered by severe cardiotoxicity. Even if there is a significant sex difference in incidence of cardiovascular disease at the adult stage, it is not known whether a difference in doxorubicin-related cardiotoxicity between men and women also exists. The objective of this work was to explore the cardiac side effects of doxorubicin in adult rats and decipher whether signaling pathways involved in cardiac toxicity differ between sexes. Methods and Results— After 7 weeks of doxorubicin (2 mg/kg per week), males developed major signs of cardiomyopathy with cardiac atrophy, reduced left ventricular ejection fraction and 50% mortality. In contrast, no female died and their left ventricular ejection fraction was only moderately affected. Surprisingly, neither global oxidation levels nor the antioxidant response nor the apoptosis signaling pathways were altered by doxorubicin. However, the level of total adenosine monophosphate–activated protein kinase was severely decreased only in males. Moreover, markers of mitochondrial biogenesis and cardiolipin content were strongly reduced only in males. To analyze the onset of the pathology, maximal oxygen consumption rate of left ventricular permeabilized fibers after 4 weeks of treatment was reduced only in doxorubicin-treated males. Conclusions— Altogether, these results clearly evidence sex differences in doxorubicin toxicity. Cardiac mitochondrial dysfunction and adenosine monophosphate–activated protein kinase seem as critical sites of sex differences in cardiotoxicity as evidenced by significant statistical interactions between sex and treatment effects.

Aims Mutations in the KCNK3 gene, which encodes for an outward-rectifier K+ channel, have been identified in patients suffering from pulmonary arterial hypertension (PAH), and constitute the first described channelopathy in PAH. In human PAH and experimental pulmonary hypertension (PH), we demonstrated that KCNK3 expression and function are severely reduced in pulmonary vascular cells, promoting PH-like phenotype at the morphologic and haemodynamic levels. Since KCNK3 channel is also expressed in both the human and rodent heart, we aimed to elucidate the pathophysiological role of KCNK3 channel in right ventricular (RV) hypertrophy (RVH) related to PH. Methods and results Using whole-cell Patch-clamp technique, we demonstrated that KCNK3 is predominantly expressed in adult rat RV cardiomyocytes compared to the left ventricle cardiomyocytes and participates in the repolarizing phase of the RV action potential. We revealed a reduction in KCNK3 function prior to development of RVH and the rise of pulmonary vascular resistance. KCNK3 function is severely reduced in RV cardiomyocytes during the development of RVH in several rat models of PH (exposure to monocrotaline, chronic hypoxia, and Sugen/hypoxia) and chronic RV pressure overload (pulmonary artery banding). In experimental PH, we revealed a reduction in KCNK3 function before any rise in pulmonary vascular resistance and the development of RVH. KCNK3 mRNA level is also reduced in human RV tissues from PAH patients compared to non-PAH patients. In line with these findings, chronic inhibition of KCNK3 in rats with the specific inhibitor (A293) induces RV hypertrophy which is associated with the re-expression of foetal genes, RV fibrosis, RV inflammation, and subsequent loss of RV performance as assessed by echocardiography. Conclusion Our data indicate that loss of KCNK3 function and expression is a hallmark of the RV hypertrophy/dysfunction associated with PH.

International audience; The abundance of epicardial adipose tissue (EAT) is associated with atrial fibrillation (AF), the most frequent cardiac arrhythmia. However, both the origin and the factors involved in EAT expansion are unknown. Here, we found that adult human atrial epicardial cells were highly adipogenic through an epithelial-mesenchymal transition both in vitro and in vivo. In a genetic lineage tracing the WT1(CreERT2+/-)Rosa(tdT+/-)mouse model subjected to a high-fat diet, adipocytes of atrial EAT derived from a subset of epicardial progenitors. Atrial myocardium secretome induces the adipogenic differentiation of adult mesenchymal epicardium-derived cells by modulating the balance between mesenchymal Wingless-type Mouse Mammary Tumor Virus integration site family, member 10B (Wnt10b)/beta-catenin and adipogenic ERK/MAPK signaling pathways. The adipogenic property of the atrial secretome was enhanced in AF patients. The atrial natriuretic peptide secreted by atrial myocytes is a major adipogenic factor operating at a low concentration by binding to its natriuretic peptide receptor A (NPRA) receptor and, in turn, by activating a cGMP-dependent pathway. Hence, our data indicate cross-talk between EAT expansion and mechanical function of the atrial myocardium.

OBJECTIVES: Electrical cardiometry and heart ultrasound might allow hemodynamic evaluation during transportation of critically ill patients. Our aims were 1) to test feasibility of stroke volume monitoring using electrical cardiometry or ultrasound during transportation and 2) to investigate if transportation impacts on electrical cardiometry and ultrasound reliability. DESIGN: Prospective, pragmatic, feasibility cohort study. SETTING: Mobile ICUs specialized for neonatal and pediatric transportation. PATIENTS: Thirty hemodynamically stable neonates and infants. INTERVENTIONS: Patients enrolled underwent paired stroke volume measurements (180 before/after and 180 during the transfer) by electrical cardiometry (SVEC) and ultrasound (SVUS). MEASUREMENTS AND MAIN RESULTS: No problems or malfunctioning occurred neither with electrical cardiometry nor with ultrasound. Ultrasound lasted on average 90 (10) seconds, while 45 (15) seconds were needed to instigate electrical cardiometry monitoring. Coefficient of variation was higher for SVUS (before/after: 0.57; during: 0.66) than for SVEC (before/after: 0.38; during: 0.36). Correlations between SVEC and SVUS before/after and during the transfer were r equal to 0.57 and r equal to 0.8, respectively (p always \textless 0.001). Bland-Altman analysis showed that stroke volume tends to be higher if measured by electrical cardiometry. SVEC measured before (5.5 [2.4] mL), during (5.4 [2.4] mL), and after the transfer (5.4 [2.3] mL) are similar (p = 0.955); same applies for SVUS before (2.6 [1.5] mL), during (2.4 [2] mL), and after (2.9 [2] mL) the transfer (p = 0.268). CONCLUSIONS: Basic hemodynamic monitoring is feasible during pediatric and neonatal transportation both with electrical cardiometry and ultrasound. These two techniques show comparable reliability, although stroke volume was higher if measured by electrical cardiometry. The transportation itself does not affect the reliability of stroke volume measurements.

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

Background Early and easy to do detection of left ventricular (LV) failure is crucial to improve following and outcomes of patients with right ventricular (RV) overload in congenital heart diseases. Electrical cardiometry (Osypka medical) is easy handling, even in medical office or in pre-hospital condition, and can provide cardiac output, and a new contractility index (ICON) supposed to be independent from load conditions. ICON have never been previously challenged to our knowledge. Objectives We aim to compare ICON with the only contractility parameter independent from load conditions: the elastance slope (Emax). Methods Using porcine models of Fallot repaired and pulmonary hypertension (PH), we assess LV function using conductance catheter and electrical cardiometry devices over 4 months after surgery. We measured ICON, Emax, Contractile reserve (ΔEmax) and VIC (respiratory variations of ICON) at basal state and after adrenergic stimulation (Dobutamine). Results Three animals of each group were compared with 6 controls. Non parametric correlation (spearman) hightlights at basal state a non significant and low correlation between ICON and Emax and ΔEmax (r = 0.5). However after Dobutamine, correlation is important and strong with r = 0.98 between ICON/Emax (0.05) and 0.89 between VIC/Emax. We did not find strong correlation between ΔEmax and VIC or ΔICON. Conclusion These results obtain on a small in vivo/animal cohort highlight than electrical cardiometry device could be a useful and easy handling (4 skin patchs) tool for LV failure and loss of contractility early screening, specially after adrenergic stimulation and stress conditions. It could provide precious help in patients following.

Introduction Mitochondrial dysfunction plays a major role in the Heart Failure (HF) pathophysiology. The AMP activated protein kinase (AMPK) is activated by a high AMP-ADP/ATP ratio and regulates a number of metabolic pathways. Many studies have highlighted a protective role of AMPK in HF, but its relevance to cardiac tissue, its metabolic part and its sex-specificity are not well established. Objective The aim of this study is to determine the role of AMPK in the healthy and failing heart in male and female mice. Method We developed and validated a mouse strain with an adult-inducible cardiac-specific deletion of AMPKα2, the major cardiac isoform, using the Cre-Lox system (40 mg/kg tamoxifen injection on two consecutive days at adult age). At four months after the deletion, cardiac contractility, morphology and metabolism were studied in control and KO mice from both sexes. Results We observed only in male KO mice a decrease of left ventricular ejection fraction (− 10%), an increase of the total fibrosis (+ 64%) and defects in mitochondrial structures. Male KO mice also showed a reduced (− 28%) mitochondrial respiration via complex I associated with a different cardiolipin species distribution. Conclusion Our results reveal in adult healthy hearts, a sex-specificity in the effects of AMPKα2 deletion, leading to impaired contractile function related to metabolic and non-metabolic alterations only in male mice.

Membranous nephropathy (MN) is a glomerular disease characterized by a nephrotic syndrome without infiltration of inflammatory cells or proliferation of resident cells. Although the cause of the disease is unknown, the primary pathogenic mechanism responsible for the accumulation of immune deposits on the outer aspect of the glomerular basement membrane involves the generation of autoantibodies against antigen targets expressed at the membrane surface of podocyte. The molecular mechanisms of nephrotic proteinuria, which reflects a profound disorder of podocyte function, remain unclear. We show here that induction of c-mip in passive type Heymann nephritis (PHN) coincides with the occurrence of proteinuria. c-mip expression is not detectable in the glomeruli of PHN rats receiving a single dose of anti-megalin polyclonal antibody, yet the immune complexes are readily present but without triggering of proteinuria. Rats reinjected with anti-megalin develop a few days later heavy proteinuria concomitantly with c-mip overproduction in podocytes. We show that overexpression of c-mip is associated with downregulation of synaptopodin in human MN, PHN rats and c-mip transgenic mice, while the abundance of death-associated protein kinase (DAPK) and integrin linked kinase (ILK) is increased. Finally, cyclosporine treatment reduces significantly proteinuria in PHN rats, concomitantly with downregulation of c-mip abundance in podocytes. These results suggest that c-mip plays an active role in podocyte disorders of MN.

Rationale: BMPR2 mutations are the main genetic risk factor for heritable pulmonary arterial hypertension (hPAH) with an incomplete penetrance of 20%. Transgenic mice have been created but develop no or mild PAH. Aim: To create Bmpr2 mutated rats and to characterize their phenotype. Methods: Rats with heterozygous mutations in Bmpr2 were obtained using zinc finger nuclease technique. We looked for spontaneous PAH at hemodynamic and histologic level. Mutation-related molecular alterations were studied by qPCR and Western blot. Pulmonary artery vasoreactivity was assessed by organ bath studies, and K + currents were recorded by Patch-clamp on pulmonary smooth muscle cells (PASMC). Results : Two rat lines with a deletion of 71 bp ( Bmpr2 Δ 71Ex1/+ ) and 140 bp ( Bmpr2 Δ 140Ex1/+ ) in the exon 1 of Bmpr2 were selected. Both deletions are responsible of a decrease in half of phosphorylated SMAD1/5/8 (BMPR-II signaling). A spontaneous increase in right ventricular systolic pressure (RVSP) was observed in 19% (n=26) and in 36% (n=11) Bmpr2 Δ 71Ex1/+ rats aged of 6 months and 1 year respectively. RVSP was not correlated with right ventricle hypertrophy and pulmonary vascular remodeling. Bmpr2 Δ 140Ex1/+ rats developed a significant remodeling of small pulmonary vessel at 6 months (p=0.015) and 7% (n=13) develop spontaneous increase of RVSP at 1 year. Organ bath studies showed that Bmpr2 Δ 71Ex1/+ rats were more prone to vasoconstriction induced by KCl and K + currents (crucial determinant of PASMC contractile activity) were significantlyreduced in both strains as compared to controls. Conclusion: Bmpr2 mut rats recapitulate important characteristics of human PAH genesis and development. These models represent a promising tool in hPAH understanding.

Background: Inactivating mutations in the KCNK3 gene (K + channel) have been identified in heritable forms of pulmonary arterial hypertension (PAH). We discovered that KCNK3 dysfunction contributes actually to the development of both heritable and non-heritable PAH, and to experimental pulmonary hypertension (PH) ( Antigny et al. Circulation 2016 ). Methods and results: We generated Kcnk3 deficient rats using CRISPR-Cas9 technology and characterized at electrophysiological, hemodynamics, morphological and molecular levels the first genetically modified rat model linked to kcnk3 mutation: Kcnk3 Δ 94Ex1/+ (94pb deletion in exon 1 of Kcnk3 gene). Using a patch–clamp technique in freshly isolated pulmonary artery smooth muscle cells (PASMCs), we found that KCNK3-current is absent in heterozygous and homozygous Kcnk3 mutated rats and that isolated PASMC are significantly depolarized compared to WT rats. Kcnk3 mutation induced distal neomuscularization, abnormal pulmonary arteries vasoreactivity, elevated mean right ventricular systolic pressures and upregulation of MAP kinase signaling pathways. Heterozygous Kcnk3 -mutated rats developed more severe pulmonary hypertension than WT littermate under hypoxic condition. Conclusions: In this study we established the first Kcnk3 deficient rat model. We demonstrated that a genetic inactivation of KCNK3 in rats promoted the early signs of pulmonary hypertension, confirming that KCNK3 loss of function is a key event in PAH pathogenesis. The development and characterization of the first Kcnk3 deficient PH-rats model open new opportunities for testing relevant therapeutics molecules in context of heritable PH.

Background and aims The accumulation of the adipose tissue (AT) in the sup- and subepicardium of the atrial myocardium is associated with a high risk of atrial fibrillation. Here we addressed the question of the cellular origin of atrial AT. Methods Human right atrial specimen obtained during cardiac surgery were used for histological, biochemical studies and to harvest epicardial progenitors. Cells were characterized using flow cytometry, proteomic and genic expression assays and maintained in culture conditions, in some experiments they were co cultured with human atrial myocytes using a Transwell system. Results In atrial section, epicardial progenitors expressing the pre-adipocyte factor 1 (Pref-1) were detected in the epicardial and sub-epicardial layer which contained a number of progenitor cells referred as epicardic progenitorderived cells (EPDCs). Some EPDCs expressed Pref-1 suggesting that they could engaged in the adipocyte pathways. This was tested in vitro using EPDCs harvested from human right atrial samples (n=20). From 1 st to 5-6 cells passages, EPDCs underwent an epithelial-to-mesenchymal transition (EMT), acquired mesenchymal phenotypes and could differentiate into osteocyte or chondrocyte. When cultured using an adipogenic medium, around 40±6% of EPDCs cells showed lipid droplet colored with oil red and expressed mature adipocyte perilipin marker. Culture medium enriched with the secretome of human atria too induced the differentiation of EPDCs in adipocytes (n=6). A shift from pre- to mature adipocytes was indicated by the detection of Pref-1 at 7 but not 30 days of culture, it was the opposite for perilipin. Human atrial myocyte could be the source of pro-adipogenic factor. In fact, after 7 days in coculture, EPDCs are differentiated into adipocytes (n=6). Conclusion Human EPDCs from atria can differentiate into adipocyte; factors secreted by atrial myocytes regulate this differentiation process. The author hereby declares no conflict of interest

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

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

Aims Ezrin, radixin, and moesin (ERM) proteins have been implicated in regulating signalling molecules. The aim of the present study was to investigate the activity and subcellular distribution of ERM proteins in cardiac myocytes from both Wistar and diabetic Goto-Kakizaki (GK) rats, and the role of these proteins in mediating the downstream effects of the cardiac sarcolemmal Na+/H+ exchanger (NHE1) activation in response to cell acidification. Methods and results Immunofluorescence microscopy revealed that activated ERM proteins were localized predominantly at the intercalated disc regions in left ventricular (LV) myocytes of both Wistar and GK rats under basal conditions. After acid loading, profound changes in activated ERM distribution were observed in both groups of myocytes, with immunolabelling detected in regions corresponding to the transverse tubules. This correlated with a marked increase in phospho-ERM levels in both groups, which was higher in GK myocytes and blocked by NHE1 inhibitor treatment. Levels of phospho-Akt paralleled those of phospho-ERM under the various experimental conditions used; in particular, the marked acid-induced increase in both phospho-ERM and phospho-Akt in GK myocytes was abolished by an NHE1 inhibitor treatment. Moreover, the pattern of glycogen synthase kinase-3β (GSK-3β) phosphorylation in these myocytes was strikingly similar to that observed for Akt activity under the conditions used. Conclusion Activated ERM proteins mediate the effects of acid-induced NHE1 activation in LV myocytes. Akt is a downstream effector in the cascade activated by NHE1–ERM interaction. In addition, GSK-3β phosphorylation is required for downstream effects of NHE1/ERM-Akt signalling.

Background: The NMDA receptor (NMDAR), a glutamate receptor, is a crucial component of neuronal synapses, but it is also described in vascular cells where its role remains elusive. Aims: 1) Highlight components of glutamatergic communication in pulmonary vascular cells. 2) Search for dysegulation of this pathway in pulmonary arterial hypertension (PAH). 3) Study the contribution of pulmonary arterial smooth muscle cell (PASMC) NMDAR to PH. 4) Assess the effects of the NMDAR antagonist MK-801 on established PH. Methods: Immunohistochemistry and mass spectrometry imaging were used to detect components of glutamatergic communication in human lungs and vascular cells. Enzymatic assays were used to detect glutamate release from vascular cells. PASMC proliferation was measured using BrdU incorporation. NMDAR knock out mice in PASMC were exposed to hypoxia for 3 weeks. MK-801 (3mg/kg/d) was administrated to monocrotaline (MCT) rats. Results: 1) Pulmonary vascular cells express NMDAR subunits, vesicular glutamate transporters, and release glutamate in a calcium-dependent way. 2) Remodeled arteries from iPAH patients contain higher level of glutamate compared to control arteries. Beside, Phospho896-GluN1 subunit of NMDAR, involved in its trafficking to the cell membrane, is present in vascular lesions from iPAH patients but not in controls. 3) Endothelin-1 triggers glutamate release from PASMC and NMDAR activation participates to PASMC proliferation. Knocking out NMDAR in PASMC decreases PH in hypoxic mice. 4) Administration of MK-801 reverses PH in MCT rats. Conclusion: Glutamatergic signaling through pulmonary vascular NMDAR occurs in PAH pathophysiology and may represent a new therapeutic target.

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

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

Heart failure is still a major cause of hospitalization and mortality in developed countries. Many clinical trials have tested the use of multipotent stem cells as a cardiac regenerative medicine. The benefit for the patients of this therapeutic intervention has remained limited. Herein, we review the pluripotent stem cells as a cell source for cardiac regeneration. We more specifically address the various challenges of this cell therapy approach. We question the cell delivery systems, the immune tolerance of allogenic cells, the potential proarrhythmic effects, various drug mediated interventions to facilitate cell grafting and, finally, we describe the pathological conditions that may benefit from such an innovative approach. As members of a transatlantic consortium of excellence of basic science researchers and clinicians, we propose some guidelines to be applied to cell types and modes of delivery in order to translate pluripotent stem cell cardiac derivatives into safe and effective clinical trials.

Background— Mutations in the KCNK3 gene have been identified in some patients suffering from heritable pulmonary arterial hypertension (PAH). KCNK3 encodes an outward rectifier K + channel, and each identified mutation leads to a loss of function. However, the pathophysiological role of potassium channel subfamily K member 3 (KCNK3) in PAH is unclear. We hypothesized that loss of function of KCNK3 is a hallmark of idiopathic and heritable PAH and contributes to dysfunction of pulmonary artery smooth muscle cells and pulmonary artery endothelial cells, leading to pulmonary artery remodeling: consequently, restoring KCNK3 function could alleviate experimental pulmonary hypertension (PH). Methods and Results— We demonstrated that KCNK3 expression and function were reduced in human PAH and in monocrotaline-induced PH in rats. Using a patch-clamp technique in freshly isolated (not cultured) pulmonary artery smooth muscle cells and pulmonary artery endothelial cells, we found that KCNK3 current decreased progressively during the development of monocrotaline-induced PH and correlated with plasma-membrane depolarization. We demonstrated that KCNK3 modulated pulmonary arterial tone. Long-term inhibition of KCNK3 in rats induced distal neomuscularization and early hemodynamic signs of PH, which were related to exaggerated proliferation of pulmonary artery endothelial cells, pulmonary artery smooth muscle cell, adventitial fibroblasts, and pulmonary and systemic inflammation. Lastly, in vivo pharmacological activation of KCNK3 significantly reversed monocrotaline-induced PH in rats. Conclusions— In PAH and experimental PH, KCNK3 expression and activity are strongly reduced in pulmonary artery smooth muscle cells and endothelial cells. KCNK3 inhibition promoted increased proliferation, vasoconstriction, and inflammation. In vivo pharmacological activation of KCNK3 alleviated monocrotaline-induced PH, thus demonstrating that loss of KCNK3 is a key event in PAH pathogenesis and thus could be therapeutically targeted.

Background— The vascular remodeling responsible for pulmonary arterial hypertension (PAH) involves predominantly the accumulation of α-smooth muscle actin–expressing mesenchymal-like cells in obstructive pulmonary vascular lesions. Endothelial-to-mesenchymal transition (EndoMT) may be a source of those α-smooth muscle actin–expressing cells. Methods and Results— In situ evidence of EndoMT in human PAH was obtained by using confocal microscopy of multiple fluorescent stainings at the arterial level, and by using transmission electron microscopy and correlative light and electron microscopy at the ultrastructural level. Findings were confirmed by in vitro analyses of human PAH and control cultured pulmonary artery endothelial cells. In addition, the mRNA and protein signature of EndoMT was recognized at the arterial and lung level by quantitative real-time polymerase chain reaction and Western blot analyses. We confirmed our human observations in established animal models of pulmonary hypertension (monocrotaline and SuHx). After establishing the first genetically modified rat model linked to BMPR2 mutations (BMPR2 Δ140Ex1/+ rats), we demonstrated that EndoMT is linked to alterations in signaling of BMPR2, a gene that is mutated in 70% of cases of familial PAH and in 10% to 40% of cases of idiopathic PAH. We identified molecular actors of this pathological transition, including twist overexpression and vimentin phosphorylation. We demonstrated that rapamycin partially reversed the protein expression patterns of EndoMT, improved experimental PAH, and decreased the migration of human pulmonary artery endothelial cells, providing the proof of concept that EndoMT is druggable. Conclusions— EndoMT is linked to alterations in BPMR2 signaling and is involved in the occlusive vas cular remodeling of PAH, findings that may have therapeutic implications.