EPAC1 inhibition protects the heart from doxorubicin-induced toxicity.

Anthracyclines, such as doxorubicin (Dox), are widely used chemotherapeutic agents for the treatment of solid tumors and hematologic malignancies. However, they frequently induce cardiotoxicity leading to dilated cardiomyopathy and heart failure. This study sought to investigate the role of the exchange protein directly activated by cAMP (EPAC) in Dox-induced cardiotoxicity and the potential cardioprotective effects of EPAC inhibition. We show that Dox induces DNA damage and cardiomyocyte cell death with apoptotic features. Dox also led to an increase in both cAMP concentration and EPAC1 activity. The pharmacological inhibition of EPAC1 (with CE3F4) but not EPAC2 alleviated the whole Dox-induced pattern of alterations. When administered in vivo, Dox-treated WT mice developed a dilated cardiomyopathy which was totally prevented in EPAC1 knock-out (KO) mice. Moreover, EPAC1 inhibition potentiated Dox-induced cell death in several human cancer cell lines. Thus, EPAC1 inhibition appears as a potential therapeutic strategy to limit Dox-induced cardiomyopathy without interfering with its antitumoral activity.

Spatiotemporal AMPKα2 deletion in mice induces cardiac dysfunction, fibrosis and cardiolipin remodeling associated with mitochondrial dysfunction in males only.

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.

Involvement of CFTR in the pathogenesis of pulmonary arterial hypertension.

INTRODUCTION: A 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 OBJECTIVES: We aimed to delineate the role of CFTR in PAH pathogenesis through observational and interventional experiments in human tissues and animal models. METHODS AND RESULTS: Reverse-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. CONCLUSIONS: CFTR 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.

Kcnk3 dysfunction exaggerates the development of pulmonary hypertension induced by left ventricular pressure overload.

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.

Trichloroethylene increases pulmonary endothelial permeability: implication for pulmonary veno-occlusive disease.

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.

Microtubule polymerization state and clathrin-dependent internalization regulate dynamics of cardiac potassium channel: Microtubule and clathrin control of KV1.5 channel.

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 clusterization 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.

Orai1 Channel Inhibition Preserves Left Ventricular Systolic Function and Normal Ca2+ Handling After Pressure Overload.

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.

Right ventricular mitochondrial respiratory function in a piglet model of chronic pulmonary hypertension.

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.

Inducible Cardiac-Specific Deletion of Sirt1 in Male Mice Reveals Progressive Cardiac Dysfunction and Sensitization of the Heart to Pressure Overload.

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 1α (PGC-1α), 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.

Characterization of Kcnk3-Mutated Rat, a Novel Model of Pulmonary Hypertension.

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.

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

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

The BET Bromodomain Inhibitor I-BET-151 Induces Structural and Functional Alterations of the Heart Mitochondria in Healthy Male Mice and Rats.

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.

Bmpr2 Mutant Rats Develop Pulmonary and Cardiac Characteristics of Pulmonary Arterial Hypertension.

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 (Ca2+), 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 Ca2+, decreased [Ca2+] transient amplitude, reduced sarcoplasmic reticulum Ca2+ 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.

Ca2+ handling remodeling and STIM1L/Orai1/TRPC1/TRPC4 upregulation in monocrotaline-induced right ventricular hypertrophy.

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.

NMDA-Type Glutamate Receptor Activation Promotes Vascular Remodeling and Pulmonary Arterial Hypertension.

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. Kv 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.

Loss of KCNK3 is a hallmark of RV hypertrophy/dysfunction associated with pulmonary hypertension.

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.

Standard and Strain Measurements by Echocardiography Detect Early Overloaded Right Ventricular Dysfunction: Validation against Hemodynamic and Myocyte Contractility Changes in a Large Animal Model.

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 (ΔEmax) was assessed by ß-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 < .01), and inotropic responses to dobutamine were attenuated (ΔEmax was 51% vs 193%, P < .05). Echocardiographic measurements of fraction of area change, tricuspid annular plane systolic excursion, tricuspid annular peak systolic velocity (S') and RV free wall longitudinal systolic strain and strain rate were significantly decreased. Strain rate, S', and tricuspid annular plane systolic excursion were correlated with ΔEmax (r = 0.75, r = 0.78, and r = 0.65, respectively, P < .05). These alterations were associated in RV isolated myocytes with the decrease of sarcomere shortening in response to isoproterenol and perturbations of calcium homeostasis assessed by the increase of spontaneous calcium waves. 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.

Basic Hemodynamic Monitoring Using Ultrasound or Electrical Cardiometry During Transportation of Neonates and Infants.

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 < 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.

Atrial natriuretic peptide regulates adipose tissue accumulation in adult atria.

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 WT1CreERT2+/-RosatdT+/- 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)/ß-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.

Repression of CMIP transcription by WT1 is relevant to podocyte health.

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.