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Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is an exercise and emotional stress-induced life-threatening inherited heart rhythm disorder, characterized by an abnormal cellular calcium homeostasis. Most reported cases have been linked to mutations in the gene encoding the type 2 ryanodine receptor gene, RYR2. We generated induced pluripotent stem cells (hiPSCs) from peripheral blood mononuclear cells (PBMC) from three CPVT-affected patients, two of them carrying p.R4959Q mutation and one carrying p.Y2476D mutation. These generated hiPSC lines are a useful model to study pathophysiological consequences of RYR2 dysfunction in humans and the molecular basis of CPVT.

Introduction: Recent genetic data suggest that abnormal cardiac development participate to the pathogenesis of Brugada Syndrome (BrS), a rare inherited arrhythmia responsible for sudden cardiac death in young adults. In vitro cardiac differentiation of human induced pluripotent stem cells (hiPSCs) mimics cardiac development at the cellular level up to a prenatal stage. Objective: This study aims at defining whether BrS impairs cardiac differentiation of hiPSCs. Methods & Results: Transcriptomic kinetics (daily bulk 3’RNA-seq from day 0 to day 30 of in vitro cardiac differentiation) were generated in triplicate for 2 control hiPSC lines and 2 BrS-patient hiPSC lines. First, global analysis unveiled that BrS and control kinetics start to diverge as early as day 8, coinciding with the emergence of beating cells. The 500 most differentially expressed genes between BrS and control kinetics revealed 7 main distinct expression profiles. Interestingly, in one of the clusters (Cluster 2), enriched in genes involved in ventricular development ( e.g. IRX4 , NKX2-5 ), the expression levels were higher in BrS as compared to control, starting at day 8. Inversely, another cluster (Cluster 4), enriched in genes involved in atrial development ( e.g. TBX18 , PITX2 ), displayed an opposite expression profile. Cell-type annotation of single-cell RNA-seq data obtained at day 30 of cardiac differentiation for 1 control (n=2; 11,499 cells) and 1 BrS hiPSCs line (n=2; 12,142 cells) confirmed this ventricular-to-atrial imbalance with an average ventricular-to-atrial cell number ratio of 0.97 and 8.27 for control and BrS lines, respectively. Conclusion: This first transcriptomic kinetic study supports the hypothesis of an early developmental defect in BrS. Altogether, our data show that BrS hiPSCs are more prone to ventricular specification as compared to control cells. This suggests that an abnormal cell fate during cardiac differentiation may participate to BrS pathogeny.

Introduction: The transcription factor (TF) Iroquois homeobox 5 (Irx5) is a key patterning and differentiation regulator. In humans, IRX5 regulates the expression of the main cardiac sodium ion channel gene, SCN5A . However, its global mechanistic role in human cardiomyocytes is still to be fully understood. Hamamy syndrome patients, carrying IRX5 mutations, have defects in various organs including the heart and the limbs. Interestingly, heart and limbs defects are also found in Holt-Oram syndrome patients ( TBX5 mutations), suggesting a functional link between TBX5 and IRX5. Moreover, a cooperation of TBX5 with GATA4 and NKX2-5 in cardiac development and functions has already been shown. Hypothesis: We hypothesized that IRX5 may cooperate with the TF complex TBX5/GATA4/NKX2-5 to regulate key cardiac functions in humans. Methods & Results: First, immunoprecipitations in HEK293 cells showed that IRX5 can bind to TBX5, GATA4 and NKX2-5, individually and together as a complex. Using five truncated forms of IRX5 protein, its homeodomain was identified as being the essential protein region for these interactions. Second, we showed by luciferase assays, that each combination of these TFs impacted differently SCN5A expression: e.g. by itself, NKX2-5 has a strong activator effect (increased activity by 10 fold vs . no TF) that is potentialized by IRX5 (12 fold vs . no TF) but inhibited by TBX5 (8 fold vs . no TF). Third, genes bound by IRX5 (n=2253) were identified by ChIP-Seq on cardiomyocytes derived from induced pluripotent stem cells generated from 2 healthy individuals. Analyzing published ChIP-Seq datasets for TBX5, GATA4 or NKX2-5 in the same cellular model, we identified 2990 genes that were bound by these 3 TFs, and associated with cardiac biological pathways, such as muscle contraction. Among these genes, 848 were also bound by IRX5 and were associated with the function of electrical activity and with the fibrosis signaling, suggesting a specific role for IRX5 in these cardiac processes. Conclusions: Overall, our data show new physical and functional interactions between IRX5 and 3 key cardiac TFs (TBX5, GATA4 and NKX2-5), and suggest an unexpected regulatory role for IRX5 in specific human heart functions.

International audience

Introduction Recent data suggests that alterations in cardiac development participate to the pathogenesis of Brugada Syndrome (BrS), a rare inherited disorder responsible for sudden cardiac death. In vitro cardiac differentiation of human induced pluripotent stem cells (hiPSCs) can produce numerous cell types found in the fetal heart. This technique is well suited to decipher cell-type specific developmental mechanisms of BrS. Objective This study aims at (1) describing the different cell types obtained after in vitro cardiac differentiation of hiPSCs and (2) identifying cell-type specific molecular alterations associated with BrS. Methods & results Single-cell RNA-seq data were generated at day 30 of in vitro cardiac differentiation of hiPSCs from 1 control subject (n = 2) and 1 BrS patient (n = 2), producing individual transcriptomic expression profile for 29′915 cells. Among those 23′624 (79%) displayed a cardiac transcriptomic profile. Using public database annotations, 8′766 cells and 5′287 cells were assigned to ventricular and atrial cardiomyocytes phenotype, respectively. Focusing on ventricular cardiomyocytes we identified 213 and 153 genes up and down-regulated in BrS samples as compared to control. Interestingly, differentially expressed genes were enriched in genes involved in heart development (e.g. IRX4, NKX2-5, FZD2) and cardiac conduction (e.g. KCNJ5, CASQ2, CACNA1 C) functions. Conclusion In vitro cardiac differentiation of hiPSCs can effectively generate numerous cardiac cell type with a majority (59%) of cardiomyocytes. Comparison of transcriptomic profiles of ventricular cardiomyocytes differentiated from BrS and control hiPSCs revealed alteration of heart development and cardiac conduction functions. Altogether, these data indicate that BrS may have developmental origins and that modeling BrS using cellular level data from in vitro cardiac differentiation of hiPSCs may be key to gain further insight into its molecular mechanisms.

Introduction Transcription factors (TFs), through their gene regulation activity, are known to be powerful regulators of organogenesis, including the heart. IRX5 TF belongs to the Iroquois homeobox (IRX) family and studies in mice have shown that it is expressed from the early stages of cardiac development throughout adulthood. Irx5 has also been shown to play a crucial role in establishing the ventricular repolarization gradient through the modulation of certain cardiac genes: Kcnd2 in mice and SCN5A in humans. However, these studies are gene-candidate based, therefore the global target gene-set of IRX5 and its protein partners in human cardiomyocytes is still to be identified. Objective The aim of this project is to identify all the IRX5 target genes and its protein partners in human cardiomyocytes, and to study its functions during cardiomyocyte differentiation. Methods In order to perform studies in a human context, we used cardiomyocytes derived from induced pluripotent stem cells (iPS-CMs), generated from patients with cardiac defects linked to IRX5 loss of function mutations and healthy individuals. High-throughput transcriptomic experiments as well as immunoprecipitation and luciferase analyses were performed to obtain a global understanding of IRX5 functions and partners. Results We have shown that IRX5 can interact with major cardiac TFs such as GATA4, TBX5 and NKX2-5. More precisely, IRX5 interacts with them as a multi-protein complex to modulate the expression of target genes. We demonstrated the central role of a conserved region of IRX5 in these interactions: its homeodomain. We have also identified misregulated genes between control and IRX5Mut iPS-CMs, associated to the cardiac contraction and fibrosis pathways. Conclusion IRX5 is an important actor of the regulation of human cardiac functions, and these regulations seem to require the establishment of a multiprotein complex with other major cardiac TFs.

Introduction Kv11.1 channel (hERG) and the corresponding IKr current is a major component of cardiac repolarization and sinoatrial pacemaking. Dysfunctions in hERG channels are associated with cardiac arrhythmias and remains a driving force to study the structure, biophysics and pharmacology of this channel. Several compounds that block hERG channels have been developed. In this study, we focused on BeKm-1, a toxin which is highly selective for the hERG channel. Objective To study hERG channels at high temporal and spatial resolution, we developed a photo-protected analog of BeKm-1 that enables control of hERG using light. Method We developed a photo-protected BeKm-1 analog (BeKm-1-Lys18Nvoc) which is cleaved by UV-light at 365 nm. Using a hERG stable cell line and high-throughput automated patch-clamp (Syncropatch 384, Nanion) we validated several properties of the BeKm-1 analog blockage. To demonstrate spatial control of cardiomyocyte function, hiPS-derived cardiomyocytes were plated on CardioExcyte96 to record extrafield potentials. Results We first validated physico-chemical properties of BeKm-1-Nvoc compared to BeKm-1 using HPLC analyses. Patch-clamp experiments displayed no inhibition of hERG current by BeKm-1-Nvoc at concentrations for which wild-type Bekm-1 is maximally inhibiting. Illumination of BeKm-1-Nvoc (100 nM) at 365 nm allows cleavage of the protecting group and strong inhibition of hERG current. The block of hERG is reversible after washes. Illumination of BeKm-1-Nvoc on hiPS-derived cardiomyocytes induces prolongation of extrafied potential which reflects an increase in repolarization time. Study of effects of photoactivation of BeKm-1 on mouse and zebrafish heart rates are ongoing. Conclusion We developed for the first time a photo-sensitive blocker of endogenous hERG channel which allows control of its activity with the spatiotemporal precision of light. Studies of implication of hERG on arrhythmias using this compound are ongoing.

Background Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac disease exposing afflicted patients to sudden cardiac death. Most CPVT cases have been linked to mutations of the gene encoding ryanodine receptor type 2 (RYR2). RyR2 is a calcium channel located in the membrane of the sarcoplasmic reticulum (SR). It mediates release of calcium stocks from the SR to the cytosol, playing a major role in excitation-contraction coupling. Although several studies have investigated mechanisms underlying CPVT in mice or in vitro models, little has been done concerning functional assessment of CPVT physiopathology in human cardiomyocytes. Objective We aimed at characterizing, at cellular level, CPVT phenotype of patients carrying two new RyR2 mutations. Method Human induced pluripotent stem cells (hiPSCs) were derived from three CPVT patients. Two patients belonging to the same family carry the R4959Q RyR2 pore mutation. The third patient harbors the Y2476D mutation located in the cytosolic domain of the protein. hiPSCs were differentiated into cardiomyocytes for functional studies. Action potentials were recorded using patch-clamp technique. Calcium handling and contractility were investigated using Ionoptix and CardioExcyte technologies. Results hiPS-derived cardiomyocytes (hiPS-CMs) from CPVT patients displayed impaired beating properties. Calcium handling properties were also disrupted with changes in calcium transients, accompanied with increased diastolic calcium leak. At last, CPVT hiPS-CMs showed characteristics changes in action potential and arrhythmic events. Coherently with the patients’ phenotype, arrhythmic events were more frequent under beta-adrenergic stress. Conclusion This work enabled us to characterize two novel CPVT mutations, providing more insight into functional mechanisms involved in this pathology, and further validating the use of human pluripotent stem cells for investigating complex cardiac rhythm disorders.

Introduction In the heart, several transcription factors (TF) are powerful regulators of cardiac cell fate and organogenesis. Less well studied are the roles of TF in physiological functions. Several studies on animal models have shown that the Iroquois homeobox (Irx) TFs are key patterning and differentiation regulators that confer cell type-specific properties to specialized cardiac lineages. Importantly, studies in mice have shown that IRX5 is expressed very early during cardiogenesis and maintained in adult heart where it regulates ventricular electrophysiological properties, the repolarizing gradient. It has also been shown that IRX5 target-genes regulation occurs through recruitment of histone modifiers. Objective/Method In order to study IRX5 in human, we used cardiomyocytes differentiated from induced pluripotent stem cells (hiPS-CMs) generated from patients carrying loss of function mutations in IRX5 (IRX5Mut) and control individuals. As we detected IRX5 protein as early as in the pluripotent state, we also analyzed cells at hiPS stage in our experiments. High-throughput transcriptomic and epigenetic analyses were performed to obtain a global understanding of IRX5 function. Results The transcriptomic experiments allowed us to determine the entire set of differentially expressed genes in hiPS and hiPS-CMs IRX5Mut cells as compared to control cells. The epigenetic experiments, performed by ChIPseq, suggested that IRX5 binds to already open chromatin, and preferentially, to enhancer rich regions rather than proximal promoters. Combining these high-throughput experiments, we found direct target genes of IRX5 at hiPS-CM stage, and some of them are relevant to cardiac function. Notably, we showed that this TF regulates the expression of several actors of electrical conduction, such as SCN5A, at hiPS-CM stage. Conclusion IRX5 seems to be an important factor for the regulation of human cardiac function, and more specifically of its electrical function.

Interest has increased in the use of exogenous stem cells to optimize lung repair and serve as carriers of a therapeutic gene for genetic airway diseases such as cystic fibrosis. We investigated the survival and engraftment of exogenous stem cells after intratracheal injection, in a murine model of acute epithelial airway injury already used in gene therapy experiments on cystic fibrosis. Embryonic stem cells and mesenchymal stem cells were intratracheally injected 24 hr after 2% polidocanol administration, when epithelial airway injury was maximal. Stem cells were transfected with reporter genes immediately before administration. Reporter gene expression was analyzed in trachea-lungs and bronchoalveolar lavage, using nonfluorescence, quantitative, and sensitive methods. Enzyme-linked immunosorbent assay quantitative results showed that 0.4 to 5.5% of stem cells survived in the injured airway. Importantly, no stem cells survived in healthy airway or in the epithelial lining fluid. Using 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside staining, transduced mesenchymal stem cells were detected in injured trachea and bronchi lumen. When the epithelium was spontaneously regenerated, the in vivo amount of engrafted mesenchymal stem cells from cell lines decreased dramatically. No stem cells from primary culture were located within the lungs at 7 days. This study demonstrated the feasibility of intratracheal cell delivery for airway diseases with acute epithelial injury.

Alors meme que les fonctions publiques de la plupart des pays de l’OCDE se sont engagees dans un processus de modernisation de leurs pratiques, nous questionnons la pertinence de l’introduction de remunerations au merite, notamment en regard des effets le plus souvent negatifs sur la motivation au travail des fonctionnaires. La fonction publique francaise ayant recemment fait le choix de remunerer une partie de ses agents en fonction de leur performance, nous montrons en quoi ces pratiques salariales individualisees peuvent a terme nuire aux motivations de service public qu’eprouvent certains fonctionnaires. Nous appuyons notre raisonnement sur les resultats empiriques obtenus aux Etats-Unis, en Angleterre mais aussi en France, ainsi que sur les developpements des theories de la motivation intrinseque, combines a ceux de la theorie des motivations de service public.Remarques a l’intention des praticiensAlors meme que le recours a la remuneration au merite comme outil de motivation s’est largement diffuse au sein de la fonction publique, cet article revient sur les difficultes propres a ces pratiques salariales. Nous soulignons plus particulierement les effets negatifs de la remuneration au merite sur les motivations de service publics des fonctionnaires. Ces motivations specifiques pouvant etre evincees, il conviendrait de recourir a des outils de gestion des ressources humaines qui encouragent le developpement de la motivation intrinseque. L’elargissement et l’enrichissement des tâches ou encore la mise en œuvre de methodes de management participatives en sont des exemples.

At a time when the civil services of most OECD countries have embarked on a process of modernization of their practices, we are questioning the relevance of introducing performance-related pay systems, particularly in view of the, more often than not, negative effects on the work motivation of civil servants. The French civil service has recently decided to pay a part of its public officials on the basis of their performance, and we show how these individualized remuneration practices can, in the long term, undermine the public service motivations that drive some civil servants. Our reasoning is supported by the empirical results of studies conducted in the United States, in England and also in France, as well as on the developments of intrinsic motivation theories, combined with the developments of the public service motivation theory. Points for the practitioners Whereas the implementation of performance-related pay as an instrument of motivation has become widespread within the civil service, this article focuses on the difficulties inherent to these compensation practices. We underline in particular the negative effects of performance-related pay on the public service motivations of civil servants. As these specific motivations can be pushed aside, human resource management tools should be adopted that encourage the development of intrinsic motivation, such as task enlargement or enrichment or the implementation of participative management methods.

International audience; ac‐ cepted for publication February 01, 2016; available online without sub‐ scription through the open access option. ©AlphaMed Press 1066‐5099/2016/$30.00/0 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typeset‐ ting, pagination and proofreading process which may lead to differ‐ ences between this version and the Version of Record. Please cite this article as 1,2,3,4 Key words. house dust mite asthma  mesenchymal stem cells  M2 macro‐ phage  airway hyper‐responsiveness  phagocytosis  airway smooth muscle contraction. ABSTRACT Mesenchymal stem cell (MSC) immunosuppressive functions make them attractive candidates for anti‐inflammatory therapy in allergic asthma. However the mechanisms by which they ensure therapeutic effects remain to be elucidated. In an acute mouse model of house dust mite (Der f)‐ induced asthma, one i.v. MSC injection was sufficient to normalize and stabilize lung function in Der f‐sensitized mice as compared to control mice. MSC injection decreased in vivo airway responsiveness and de‐ creased ex vivo carbachol‐induced bronchial contraction, maintaining bronchial expression of the inhibitory type 2 muscarinic receptor. To eval‐ uate in vivo MSC survival, MSCs were labelled with PKH26 fluorescent marker prior to i.v. injection, and 1 to 10 days later total lungs were digest‐ ed to obtain single‐cell suspensions. 91.5 ± 2.3% and 86.6 ± 6.3% of the recovered PKH26 + lung cells expressed specific macrophage markers in control and Der f mice respectively, suggesting that macrophages had phagocyted in vivo the injected MSCs. Interestingly, only PKH26 + macro‐ phages expressed M2 phenotype, while the innate PKH26 ‐ macrophages expressed M1 phenotype. Finally, the remaining 0.5% PKH26 + MSCs ex‐ pressed 10 to 100 fold more COX‐2 than before injection, suggesting in vivo MSC phenotype modification. Together, the results of this study indicate that MSCs attenuate asthma by being phagocyted by lung macrophages, which in turn acquire a M2 suppressive phenotype. STEM CELLS 2016; 00:000–000 SIGNIFICANCE STATEMENT In a model of asthma, injected mesenchymal stem cells (MSCs) are in vivo phagocyted by lung macrophages in the next 24 hours following i.v. injec‐ tion. Lung macrophages that have phagocyted MSCs, in turn, acquire an im‐ munosuppressive phenotype, responsible for MSC anti‐inflammatory in vivo efficacy.

International audience; We have shown recently that some indigestible carbohydrate (short-chain fructo-oligosaccharides [sc-FOS]) reduced colon tumor incidence in Apc+/Min mice, and that this effect depended on a functional local immune system. In addition, IL-15 mRNA was concomitantly modulated in the mucosa. Since intraepithelial lymphocytes (IELs) are in close contact with intestinal epithelial cells, these cells are the candidates most likely to be involved in early cancer immunosurveillance. The present study documents the effects of sc-FOS on large intestine IELs (LI-IELs) from Apc+/+ or Apc+/Min mice by analyzing markers related to their phenotype, their activation status, and the cell surface IL-15/IL-5R alpha. In the colons of Apc+/Min mice, fewer LI-IELs expressed surface IL-15/IL-15R alpha. In addition, a lower number of CD4+ LI-IELs expressed CD25, although more LI-IELs expressed CD69, as compared to normal mice. The sc-FOS enriched diet caused a decrease in the proportion of CD25+ LI-IELs and an increase in the percentage of LI-IELs bearing surface IL-15/IL-15R alpha, independently of the Apc gene status. The IL-15/IL-15R alpha increase was, however, higher in Min mice, and returned to a level very similar to that of Apc+/+ mice when the latter mice were fed a low-fiber diet. The sc-FOS-enriched diet specifically induced an increase in CD69+ cells in Apc+/+ mice, and a decrease in the proportion of CD4+ CD25+ LI-IELs in Apc+/Min mice. Some of these modulations could contribute to the development of a better immune anticancer response in the early steps of cancer development.

Face a l'echec relatif des dispositifs legislatifs en matiere d'egalite professionnelle, les pratiques de RSE peuvent apparaitre comme une solution pour reduire les inegalites que les femmes subissent encore quant a leurs conditions d'emploi. Nous nous interrogeons sur ce point en mobilisant les travaux propres a l'institutionnalisme americain, et notamment ceux de T. Veblen et de J.R. Commons. Ainsi, si les pratiques de RSE peuvent contribuer a davantage d'egalite, nous montrons que le role des pouvoirs publics demeure determinant.

Adenomatous polyposis coli (APC) functions are involved in the heterotypic interactions occurring between intestinal epithelial cells (IECs) and intra-epithelial lymphocytes (IELs). These interactions may be of interest in cancer prevention, since recent data provide evidence for lymphocyte mediated immunosurveillance of epithelial cancers. The present study attempts to determine if APC inactivation induces changes in the cross-talk between IEC and large intestine IEL (LI-IEL) through intercellular adhesion molecule (ICAM-1)/leukocyte function-associated (LFA-1) interactions. Mouse Apc+/+ and Apc+/Min colonocytes were co-cultivated with LI-IEL. When co-cultured with LI-IEL Apc+/Min IEC but not Apc+/+ IEC expressed high levels of ICAM-1. The presence of ICAM-1 was linked to TNFalpha production in both co-cultures and TNFR expression only in co-cultivated Apc+/Min IEC. Finally, butyrate enhanced the expression of ICAM-1 in Apc+/Min IEC co-cultured with LI-IEL, and the secretion of TNFalpha by both types of co-cultures. These events could participate in determining the Apc+/Min IEC immunogenicity under different in vivo conditions.