Ephrine-B1 : une nouvelle protéine structurante des membranes latérales des cardiomyocytes adultes

Ephrine-B1 est exprimée au niveau de la membrane latérale (ML) du cardiomyocyte (CM). Sa délétion (KO général et ciblé dans les CMs) entraine une désorganisation architecturale du tissu cardiaque, corrélée à une perte de la morphologie en brique du CM et de l'ultrastructure de la ML. Ces animaux démontrent une hypersensibilité au stress barométrique caractérisée par une mortalité élevée et une exacerbation de la désorganisation tissulaire. Dans un modèle d'insuffisance cardiaque (IC), l'expression génique d'éphrine-B1dans les CMs est diminuée de 50%. Ce travail suggère qu'éphrine-B1, en stabilisant la structure de la ML et la morphologie du CM, pourrait représenter un nouvel acteur participant à la mise en place de l'IC. Par l'utilisation de la Microscopie de Force Atomique nous avons montré que chez des souris saines, la surface du CM présente une architecture périodique en creux / crêtes ; les crêtes étant corrélées à la présence de mitochondries subsarcolemmales (SSM). Les CMs de souris présentant une IC post ischémiques présentent une désorganisation générale de la topographie de surface du sarcolemme caractérisée par une perte de la périodicité creux/crêtes. Ces modifications sont associées à une mort des SSM, rendant compte de surfaces lisses du CM. Un stress osmotique (formamide) indique que la mort des SSM précède la déstructuration des Tubules-T. Cette mort des SSM pourrait donc représenter un événement initiateur de l'évolution vers l'IC. L'étude du rôle d'éphrine-B1 dans la structuration du sarcolemme ouvre donc un nouvel axe de recherche et soulève l'importance des altérations de la ML des CMs dans l'apparition de l'IC.Ephrin-B1 is expressed at the lateral membrane (ML) of the cardiomyocyte (CM). Its deletion (KO General and targeted in CMs) causes an architectural disorganization of the heart tissue, correlated with a loss of the rode shape of CMs and ultrastructure of the ML. These animals show hypersensitivity to barometric stress characterized by high mortality and exacerbation of tissue disorganization. In a model of heart failure (HF), gene expression of ephrin - B1dans CMs is reduced by 50 %. This work suggests that Ephrine-B1, stabilizing the structure of the ML and morphology of CM could be a new player involved in the development of the HF. Using atomic force microscopy we have shown that in healthy mice, the surface of the CM has a periodic architecture hollow / crests, peaks are correlated to the presence of subsarcolemmal mitochondria (SSM). The CMs of mice with ischemic post HF have a general disorganization of the surface topography of the sarcolemma characterized by a loss of hollow / crests frequency. These changes are associated with death SSM, reporting CM smooth surfaces. Osmotic stress ( formamide ) indicates that the death of SSM before the breakdown of T - tubules . The death of SSM could therefore represent an initiating event of changes to the HF. The study of the ephrin -B1 role in structuring the sarcolemma thus opens a new research and raises the importance of alterations in the ML of CMs in the development of the HF

Ultra-strong coupling of molecular materials: spectroscopy and dynamics

We report here a study of light–matter strong coupling involving three molecules with very different photo-physical properties. In particular we analyze their emission properties and show that the excitation spectra are very different from the static absorption of the coupled systems. Furthermore we report the emission quantum yields and excited state lifetimes, which are self-consistent. The above results raise a number of fundamental questions that are discussed and these demonstrate the need for further experiments and theoretical studie

Ephrine-B1 (une nouvelle protéine structurante des membranes latérales des cardiomyocytes adultes)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Le réveil des cardiomyocytes adultes résidents

Les pathologies cardiaques, et en particulier l’infarctus du myocarde, conduisent inéluctablement à la mort des cardiomyocytes adultes favorisant le développement de l’insuffisance cardiaque. Les efforts en médecine cardiaque régénératrice se sont concentrés jusqu’à présent sur l’utilisation des cellules souches, laissant de côté les cardiomyocytes préexistants, considérés comme étant dans un état postmitotique. Néanmoins, des données récentes obtenues chez le poisson zèbre et les mammifères relancent le débat sur la capacité proliférative de ces cellules. Dans cette revue, nous proposons un état des lieux des connaissances de la capacité proliférative et régénératrice des cardiomyocytes résidents, et discutons certains aspects mécanistiques. Dans le futur, l’identification précise des mécanismes moléculaires permettant à ces cellules de reprendre leur prolifération devrait permettre d’ouvrir de nouvelles perspectives thérapeutiques en régénération cardiaque

Endophilin-A2 dependent VEGFR2 endocytosis promotes sprouting angiogenesis

International audienceEndothelial cell migration, proliferation and survival are triggered by VEGF-A activation of VEGFR2. However, how these cell behaviors are regulated individually is still unknown. Here we identify Endophilin-A2 (ENDOA2), a BAR-domain protein that orchestrates CLATHRIN-independent internalization, as a critical mediator of endothelial cell migration and sprouting angiogenesis. We show that EndoA2 knockout mice exhibit postnatal angiogenesis defects and impaired front-rear polarization of sprouting tip cells. ENDOA2 deficiency reduces VEGFR2 internalization and inhibits downstream activation of the signaling effector PAK but not ERK, thereby affecting front-rear polarity and migration but not proliferation or survival. Mechanistically, VEGFR2 is directed towards ENDOA2-mediated endocytosis by the SLIT2-ROBO pathway via SLIT-ROBO-GAP1 bridging of ENDOA2 and ROBO1. Blocking ENDOA2-mediated endothelial cell migration attenuates pathological angiogenesis in oxygen-induced retinopathy models. This work identifies a specific endocytic pathway controlling a subset of VEGFR2 mediated responses that could be targeted to prevent excessive sprouting angiogenesis in pathological conditions

Cardiac fibroblasts regulate sympathetic nerve sprouting and neurocardiac synapse stability.

International audienceSympathetic nervous system (SNS) plays a key role in cardiac homeostasis and its deregulations always associate with bad clinical outcomes. To date, little is known about molecular mechanisms regulating cardiac sympathetic innervation. The aim of the study was to determine the role of fibroblasts in heart sympathetic innervation. RT-qPCR and western-blots analysis performed in cardiomyocytes and fibroblasts isolated from healthy adult rat hearts revealed that Pro-Nerve growth factor (NGF) and pro-differentiating mature NGF were the most abundant neurotrophins expressed in cardiac fibroblasts while barely detectable in cardiomyocytes. When cultured with cardiac fibroblasts or fibroblast-conditioned medium, PC12 cells differentiated into/sympathetic-like neurons expressing axonal marker Tau-1 at neurites in contact with cardiomyocytes. This was prevented by anti-NGF blocking antibodies suggesting a paracrine action of NGF secreted by fibroblasts. When co-cultured with cardiomyocytes to mimic neurocardiac synapse, differentiated PC12 cells exhibited enhanced norepinephrine secretion as quantified by HPLC compared to PC12 cultured alone while co-culture with fibroblasts had no effect. However, when supplemented to PC12-cardiomyocytes co-culture, fibroblasts allowed long-term survival of the neurocardiac synapse. Activated fibroblasts (myofibroblasts) isolated from myocardial infarction rat hearts exhibited significantly higher mature NGF expression than normal fibroblasts and also promoted PC12 cells differentiation. Within the ischemic area lacking cardiomyocytes and neurocardiac synapses, tyrosine hydroxylase immunoreactivity was increased and associated with local anarchical and immature sympathetic hyperinnervation but tissue norepinephrine content was similar to that of normal cardiac tissue, suggesting depressed sympathetic function. Collectively, these findings demonstrate for the first time that fibroblasts are essential for the setting of cardiac sympathetic innervation and neurocardiac synapse stability. They also suggest that neurocardiac synapse functionality relies on a triptych with tight interaction between sympathetic nerve endings, cardiomyocytes and fibroblasts. Deregulations of this triptych may be involved in pathophysiology of cardiac diseases

Neurotrophic factor gene expression profiling in isolated adult cardiomyocytes and cardiac fibroblasts.

<p><b>A</b> NGF, NT3, NT4, CNTF and BDNF gene expression were measured by real time qPCR using mRNA extracted from cardiomyocytes (CM) and cardiac fibroblasts (Fb) isolated from adult control heart. Values represent the mean ±SEM. (***, <i>p<0.001</i> vs. CM; **<i>p<0.001</i> vs. CM; <i>ND</i>: no detectable; n = 3–5). <b>B</b> NGF protein expression was evaluated by Western-blot experiments on primary cultures of adult cardiomyocytes (CM) and adult cardiac fibroblasts (Fb). Lower panel: Ponceau Red staining to control for equal protein loading between CM and Fb samples used for NGF blotting (upper).</p

Myocardial infarction is associated with NGF over-expression by myofibroblasts which promotes PC12 cells axonal neuritogenesis.

<p>Photomicrographs and quantitative analysis of myocardial sections stained with Masson's trichrome (MT) (<b>A</b>), α-smooth muscle actin (α-SMA) (<b>B</b>). (<b>C</b>) Quantification of NGF gene expression in cardiac fibroblasts isolated from sham-operated rats (Fb) and in cardiac myofibroblasts isolated from the ischemic region of infarcted heart (Myofb). **, p<0.001 vs. Fb (n = 3–4 per rat group). (<b>D</b>) Neurite outgrowth quantification in PC12 cells, 48 and 96 hours after co-culture with myofibroblasts isolated from ischemic region of infarcted heart (PC12+Myofb). ***, p<0.001 vs. PC12, #, p<0.05 vs. PC12+Myofb 48 hours (n = 4).</p

Cardiac fibroblasts promote PC12 cells axonal/neuritogenesis through NGF paracrine action.

<p>Representative images (<b>A</b>) and quantification (<b>B</b>) of PC12 cells cultured alone (PC12), PC12 treated with NGF (70 ng/ml) as a positive control for differentiated PC12 (P12+NGF), PC12 cultured in the presence of an anti-NGF antibody and treated with NGF (70 ng/ml) (PC12+NGF+anti-NGF), PC12 co-cultured with cardiac fibroblasts (PC12+Fb), PC12 cultured with cardiac fibroblast conditioned media (PC12+<i>C</i>Fb), PC12 co-cultured with cardiac fibroblast conditioned media in the presence of an anti-NGF antibody (PC12+<i>C</i>Fb+anti-NGF), 48 or 96 hours following supplementation. (n = 4–6 per group). Scale bars  = 50 µm. <b>C</b> Immunofluorescent co-staining for Tau-1 (axonal marker) and MAP-2 (dendritic marker) in PC12 cells cultured alone (upper) or co-cultured with cardiac fibroblasts (lower) (arrow: fibroblast). Scale bar  = 20 µm.</p

Myocardial infarction is associated with immature sympathetic heart hyperinnervation.

<p>(<b>A</b>) Representative Western-blot analysis of NGF expression in the ischemic area of infarcted area (MI), compared to sham-operated rats (Ctl). (<b>B</b>) Tyrosine hydroxylase-positive nerve fibers (TH) in sham-operated animals (Ctl) and within the ischemic area of infarcted heart 15 days following myocardial infarction (MI). Values are mean ±SEM. ***, <i>p</i><0.001 vs. Ctl (n = 3–6 per rat lineage group). Scale bars  = 50 µm. (<b>C</b>) Immunofluorescent staining for PSA-NCAM (immature neurons marker) in myocardial sections of sham-operated animals (Ctl) and ischemic area (<i>I-area</i>) of infarcted hearts 15 days following myocardial infarction (MI). Scale bars  = 100 µm. (<b>D</b>) Cardiac tissue norepinephrine (NE) levels after myocardial infarction. Cardiac tissue norepinephrine was quantified by HPLC in sham-operated rats (Ctl) and in non-ischemic or ischemic areas of infarcted heart (MI), 15 days after myocardial infarction (n = 4–5 per rat group). (<b>E</b>) Representative and quantitative Western-blot analysis of cardiac tyrosine hydroxylase (TH) protein expression in sham-operated rats (Ctl) and in ischemic areas 15 days after myocardial infarction (MI). Values are means ±SEM. (n = 6 per rat group). *, p<0.05 vs. Ctl.</p