Les cibles moléculaires du récepteur minéralocorticoïde dans le coeur

The mineralocorticoid hormone aldosterone binding its receptor, the mineralocorticoid receptor (MR),regulates the renal reabsorption of sodium. Several studies showed the involvement of the aldosterone/MR complex in cardiovascular diseases, even if the activated signalling pathways are still unclear. This thesis work has been established to increase the knowledge on the mechanisms of thecardiac signalling of MR using two main purposes i) the identification of new molecular targets of the MR in the heart and ii) the understanding of the pathophysiological effects of its activation. Apharmacological approach showed that the diuretic torasemide cannot block the mineralocorticoid signalling in the cell line cardiomyocytes transfected with MR, H9C2-MR+. The study of the MR’s cardiac transactivation activity formed the most important part of this thesis work. We demonstrated with a candidate gene approach that the MR increases the expression of the gene coding the connective tissue growth factor (CTGF) and the aldosterone increases even more this effect in vivo. We foundCTGF specifically expressed in cardiomyocytes and we identified in vitro that the MR binds tohormonal responsive elements on the promoter of the gene coding CTGF. In order to investigate the whole genes differentially expressed by the two ligands of MR in the heart, we treated mice with cardiomyocyte-targeted human MR over expression and their controls with aldosterone or corticosterone. The cardiac transcriptomic analyses show that the majority of aldosterone-regulatedgenes is involved in cell division as Cyclin B1 or Cyclin-dependent kinase 1 (Cdk1). Also, we identified that aldosterone promotes cardiac endothelial cells proliferation.L’hormone minéralocorticoïde aldostérone en se fixant à son récepteur, le récepteur minéralocorticoïde (RM), module la réabsorption de sodium au niveau du rein. De nombreuses étude sont rapporté l’implication du complexe aldostérone/RM dans les pathologies cardiovasculaires, sans que les voies de signalisation activées soient encore entièrement élucidées à ce jour. Ce travail de thèse se propose d’approfondir les connaissances sur les mécanismes de la signalisation cardiaque du RM à travers deux objectifs principaux i) l’identification de nouvelles cibles moléculaires du RM dans lecoeur et ii) la compréhension des effets physiopathologiques de son activation. Par une approche pharmacologique, nous avons montré que le diurétique torasémide n’est pas capable de bloquer la voie de signalisation minéralocorticoïde dans la lignée cellulaire de cardiomyocyte transfectée avec le RM,H9C2-RM+. L’étude de l’activité transcriptionnelle du RM cardiaque a concerné la majeure partie de ce travail de thèse. Par une approche gène candidat, nous avons mis en évidence que l’expression dugène codant pour le facteur de croissance du tissu conjonctif (CTGF, pour connective tissue growthfactor) est augmentée par le RM et que l’aldostérone potentialise cet effet in vivo. Nous avons pu localiser CTGF spécifiquement dans les cardiomyocytes, et une étude in vitro nous a permis d’identifier que le RM se lie au niveau d’éléments de réponse hormonale dans le promoteur du gène codant pour CTGF. Des souris surexprimant le RM humain spécifiquement dans les cardiomyocytes et traitées avec de l’aldostérone ou de la corticostérone ont permis une exploration plus large des gènesdifférentiellement exprimés par les deux ligands du RM dans le coeur. L’analyse des transcriptomes cardiaques de ces souris et de leurs contrôles montre qu’une augmentation modeste de la concentration plasmatique en aldostérone induit dans le coeur l’expression de gènes impliqués dans le cycle cellulaire comme la Cycline B1 ou sa kinase associée Cdk1 (pour Cyclin-dependent kinase 1). Nous avons montré également que l’aldostérone active la prolifération des cellules endothéliales cardiaques

Molecular targets of the mineralocorticoïd receptor in the heart

L’hormone minéralocorticoïde aldostérone en se fixant à son récepteur, le récepteur minéralocorticoïde (RM), module la réabsorption de sodium au niveau du rein. De nombreuses étude sont rapporté l’implication du complexe aldostérone/RM dans les pathologies cardiovasculaires, sans que les voies de signalisation activées soient encore entièrement élucidées à ce jour. Ce travail de thèse se propose d’approfondir les connaissances sur les mécanismes de la signalisation cardiaque du RM à travers deux objectifs principaux i) l’identification de nouvelles cibles moléculaires du RM dans lecoeur et ii) la compréhension des effets physiopathologiques de son activation. Par une approche pharmacologique, nous avons montré que le diurétique torasémide n’est pas capable de bloquer la voie de signalisation minéralocorticoïde dans la lignée cellulaire de cardiomyocyte transfectée avec le RM,H9C2-RM+. L’étude de l’activité transcriptionnelle du RM cardiaque a concerné la majeure partie de ce travail de thèse. Par une approche gène candidat, nous avons mis en évidence que l’expression dugène codant pour le facteur de croissance du tissu conjonctif (CTGF, pour connective tissue growthfactor) est augmentée par le RM et que l’aldostérone potentialise cet effet in vivo. Nous avons pu localiser CTGF spécifiquement dans les cardiomyocytes, et une étude in vitro nous a permis d’identifier que le RM se lie au niveau d’éléments de réponse hormonale dans le promoteur du gène codant pour CTGF. Des souris surexprimant le RM humain spécifiquement dans les cardiomyocytes et traitées avec de l’aldostérone ou de la corticostérone ont permis une exploration plus large des gènesdifférentiellement exprimés par les deux ligands du RM dans le coeur. L’analyse des transcriptomes cardiaques de ces souris et de leurs contrôles montre qu’une augmentation modeste de la concentration plasmatique en aldostérone induit dans le coeur l’expression de gènes impliqués dans le cycle cellulaire comme la Cycline B1 ou sa kinase associée Cdk1 (pour Cyclin-dependent kinase 1). Nous avons montré également que l’aldostérone active la prolifération des cellules endothéliales cardiaques.The mineralocorticoid hormone aldosterone binding its receptor, the mineralocorticoid receptor (MR),regulates the renal reabsorption of sodium. Several studies showed the involvement of the aldosterone/MR complex in cardiovascular diseases, even if the activated signalling pathways are still unclear. This thesis work has been established to increase the knowledge on the mechanisms of thecardiac signalling of MR using two main purposes i) the identification of new molecular targets of the MR in the heart and ii) the understanding of the pathophysiological effects of its activation. Apharmacological approach showed that the diuretic torasemide cannot block the mineralocorticoid signalling in the cell line cardiomyocytes transfected with MR, H9C2-MR+. The study of the MR’s cardiac transactivation activity formed the most important part of this thesis work. We demonstrated with a candidate gene approach that the MR increases the expression of the gene coding the connective tissue growth factor (CTGF) and the aldosterone increases even more this effect in vivo. We foundCTGF specifically expressed in cardiomyocytes and we identified in vitro that the MR binds tohormonal responsive elements on the promoter of the gene coding CTGF. In order to investigate the whole genes differentially expressed by the two ligands of MR in the heart, we treated mice with cardiomyocyte-targeted human MR over expression and their controls with aldosterone or corticosterone. The cardiac transcriptomic analyses show that the majority of aldosterone-regulatedgenes is involved in cell division as Cyclin B1 or Cyclin-dependent kinase 1 (Cdk1). Also, we identified that aldosterone promotes cardiac endothelial cells proliferation

Remodeling of Ion Channel Trafficking and Cardiac Arrhythmias

International audienceBoth inherited and acquired cardiac arrhythmias are often associated with the abnormal functional expression of ion channels at the cellular level. The complex machinery that continuously traffics, anchors, organizes, and recycles ion channels at the plasma membrane of a cardiomyocyte appears to be a major source of channel dysfunction during cardiac arrhythmias. This has been well established with the discovery of mutations in the genes encoding several ion channels and ion channel partners during inherited cardiac arrhythmias. Fibrosis, altered myocyte contacts, and post-transcriptional protein changes are common factors that disorganize normal channel trafficking during acquired cardiac arrhythmias. Channel availability, described notably for hERG and KV1.5 channels, could be another potent arrhythmogenic mechanism. From this molecular knowledge on cardiac arrhythmias will emerge novel antiarrhythmic strategies

Asymmetric Fitness of Second-Generation Interspecific Hybrids Between Ciona robusta and Ciona intestinalis

Reproductive isolation is central to speciation, but interspecific crosses between two closely related species can produce viable and fertile hybrids. Two different species of tunicates in the same ascidian genus, Ciona robusta and Ciona intestinalis, can produce hybrids. However, wild sympatric populations display limited gene flow, suggesting the existence of obstacles to interspecific reproduction that remain unknown. Here, we took advantage of a closed culture system to cross C. robusta with C. intestinalis and established F1 and F2 hybrids. We monitored post-embryonic development, survival, and sexual maturation to characterize the genetic basis of simple traits, and further probe the physiological mechanisms underlying reproductive isolation. Partial viability of first and second generation hybrids suggested that both pre- and postzygotic mechanisms contributed to genomic incompatibilities in hybrids. We observed asymmetric fitness, whereby the C. intestinalis maternal lines fared more poorly in our system, pointing to maternal origins of species-specific sensitivity. We discuss the possibility that asymmetrical second generation inviability and infertility emerge from interspecific incompatibilities between the nuclear and mitochondrial genomes, or other maternal effect genes. This work paves the way to quantitative genetic approaches to study the mechanisms underlying genomic incompatibilities and other complex traits in the genome-enabled Ciona model

Nanostructured Dense Collagen-Polyester Composite Hydrogels as Amphiphilic Platforms for Drug Delivery

International audienceAssociating collagen with biodegradable hydrophobic polyesters constitutes a promising method for the design of medicated biomaterials. Current collagen‐polyester composite hydrogels consisting of pre‐formed polymeric particles encapsulated within a low concentrated collagen hydrogel suffer from poor physical properties and low drug loading. Herein, an amphiphilic composite platform associating dense collagen hydrogels and up to 50 wt% polyesters with different hydrophobicity and chain length is developed. An original method of fabrication is disclosed based on in situ nanoprecipitation of polyesters impregnated in a pre‐formed 3D dense collagen network. Composites made of poly(lactic‐co‐glycolic acid) (PLGA) and poly(lactic acid) (PLA) but not polycaprolactone (PCL) exhibit improved mechanical properties compared to those of pure collagen dense hydrogels while keeping a high degree of hydration. Release kinetics of spironolactone, a lipophilic steroid used as a drug model, can be tuned over one month. No cytotoxicity of the composites is observed on fibroblasts and keratinocytes. Unlike the incorporation of pre‐formed particles, the new process allows for both improved physical properties of collagen hydrogels and controlled drug delivery. The ease of fabrication, wide range of accessible compositions, and positive preliminary safety evaluations of these collagen‐polyesters will favor their translation into clinics in wide areas such as drug delivery and tissue engineering

Cleavage of mer tyrosine kinase (MerTK) from the cell surface contributes to the regulation of retinal phagocytosis

Phagocytosis of apoptotic cells by macrophages and spent photoreceptor outer segments (POS) by retinal pigment epithelial (RPE) cells requires several proteins, including MerTK receptors and associated Gas6 and protein S ligands. In the retina, POS phagocytosis is rhythmic, and MerTK is activated promptly after light onset via the αvβ5 integrin receptor and its ligand MFG-E8, thus generating a phagocytic peak. The phagocytic burst is limited in time, suggesting a down-regulation mechanism that limits its duration. Our previous data showed that MerTK helps control POS binding of integrin receptors at the RPE cell surface as a negative feedback loop. Our present results show that a soluble form of MerTK (sMerTK) is released in the conditioned media of RPE-J cells during phagocytosis and in the interphotoreceptor matrix of the mouse retina during the morning phagocytic peak. In contrast to macrophages, the two cognate MerTK ligands have an opposite effect on phagocytosis and sMerTK release, whereas the integrin ligand MFG-E8 markedly increases both phagocytosis and sMerTK levels. sMerTK acts as a decoy receptor blocking the effect of both MerTK ligands. Interestingly, stimulation of sMerTK release decreases POS binding. Conversely, blocking MerTK cleavage increased mostly POS binding by RPE cells. Therefore, our data suggest that MerTK cleavage contributes to the acute regulation of RPE phagocytosis by limiting POS binding to the cell surface.The Institut de la Vision is funded by INSERM, Université Pierre et Marie Curie-Paris6, CNRS, and Départment de Paris

Cleavage of Mer Tyrosine Kinase (MerTK) from the Cell Surface Contributes to the Regulation of Retinal Phagocytosis

International audienc

The Diuretic Torasemide Does Not Prevent Aldosterone-Mediated Mineralocorticoid Receptor Activation in Cardiomyocytes

International audienceAldosterone binds to the mineralocorticoid receptor (MR) and exerts pleiotropic effects beyond enhancing renal sodium reabsorption. Excessive mineralocorticoid signaling is deleterious during the evolution of cardiac failure, as evidenced by the benefits provided by adding MR antagonists (MRA) to standard care in humans. In animal models of cardiovascular diseases, MRA reduce cardiac fibrosis. Interestingly diuretics such as torasemide also appear efficient to improve cardiovascular morbidity and mortality, through several mechanisms. Among them, it has been suggested that torasemide could block aldosterone binding to the MR. To evaluate whether torasemide acts as a MRA in cardiomyocytes, we compared its effects with a classic MRA such as spironolactone. We monitored ligand-induced nuclear translocation of MR-GFP and MR transactivation activity in the cardiac-like cell line H9C2 using a reporter gene assay and known endogenous aldosterone-regulated cardiac genes. Torasemide did not modify MR nuclear translocation. Aldosterone-induced MR transactivation activity was reduced by the MRA spironolactone, not by torasemide. Spironolactone blocked the induction by aldosterone of endogenous MR-responsive genes (Sgk-1, PAI-1, Orosomucoid-1, Rgs-2, Serpina-3, Tenascin-X), while torasemide was ineffective. These results show that torasemide is not an MR antagonist; its association with MRA in heart failure may however be beneficial, through actions on complementary pathways

Torasemide does not act as a MR antagonist for the regulation of endogenous genes in H9C2-MR cells in the presence of 10⁻⁸ M aldosterone.

<p>10⁻⁸ M aldosterone (Aldo) increased expression of the aldosterone-targets genes Sgk-1, PAI-1, Orosomucoid-1, Rgs-2, Serpina-3 and Tenascin-X. Addition of increasing doses of spironolactone (A+S) inhibited aldosterone-induced gene expression. In contrast, increasing concentrations of torasemide (A+T) had no antagonistic effect. Mean ± SEM (n = 4). *<i>p</i><0.05 <i>vs</i> control (Ctrl); # <i>p</i><0.05 <i>vs</i> aldosterone.</p

In vivo kinetics of nuclear translocation of MR in COS-7 cells.

<p>COS-7 cells transfected with GFP-MR were treated with 10⁻⁸ M aldosterone (Aldo), 10⁻⁸ M aldosterone +10⁻⁶ M torasemide (Aldo+ Tora), 10⁻⁸ M aldosterone +10⁻⁶ M spironolactone (Aldo+Spiro), 10⁻⁶ M spironolactone (Spiro) or 10⁻⁶ M torasemide (Tora), starting at time 0. Individual points represent the average percentage fluorescent intensity of the nucleus vs. total cellular fluorescence (Fn/Ft) measured in individual cells over the indicated period of time (± SE, n = 16). Data points were fitted to a sigmoid curve.</p