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Rationale: Cardiac tissue cohesion relying on highly ordered cardiomyocytes (CM) interactions is critical because most cardiomyopathies are associated with tissue remodeling and architecture alterations. Objective: Eph/ephrin system constitutes a ubiquitous system coordinating cellular communications which recently emerged as a major regulator in adult organs. We examined if eph/ephrin could participate in cardiac tissue cyto-organization. Methods and Results: We reported the expression of cardiac ephrin-B1 in both endothelial cells and for the first time in CMs where ephrin-B1 localized specifically at the lateral membrane. Ephrin-B1 knock-out (KO) mice progressively developed cardiac tissue disorganization with loss of adult CM rod-shape and sarcomeric and intercalated disk structural disorganization confirmed in CM-specific ephrin-B1 KO mice. CMs lateral membrane exhibited abnormal structure by electron microscopy and notably increased stiffness by atomic force microscopy. In wild-type CMs, ephrin-B1 interacted with claudin-5/ZO-1 complex at the lateral membrane, whereas the complex disappeared in KO/CM-specific ephrin-B1 KO mice. Ephrin-B1 deficiency resulted in decreased mRNA expression of CM basement membrane components and disorganized fibrillar collagen matrix, independently of classical integrin/dystroglycan system. KO/CM-specific ephrin-B1 KO mice exhibited increased left ventricle diameter and delayed atrioventricular conduction. Under pressure overload stress, KO mice were prone to death and exhibited striking tissue disorganization. Finally, failing CMs displayed downregulated ephrin-B1/claudin-5 gene expression linearly related to the ejection fraction. Conclusions: Ephrin-B1 is necessary for cardiac tissue architecture cohesion by stabilizing the adult CM morphology through regulation of its lateral membrane. Because decreased ephrin-B1 is associated with molecular/functional cardiac defects, it could represent a new actor in the transition toward heart failure.

International audience; We examined the role of sphingosine kinase-1 (SphK1), a critical regulator of the ceramide/sphingosine 1-phosphate (S1P) biostat, in the regulation of death and survival of SH-SY5Y neuroblastoma cells in response to amyloid beta (Abeta) peptide (25-35). Upon incubation with Abeta, SH-SY5Y cells displayed a marked down-regulation of SphK1 activity coupled with an increase in the ceramide/S1P ratio followed by cell death. This mechanism was redox-sensitive; N-acetylcysteine totally abrogated the down-regulation of SphK1 activity and strongly inhibited Abeta-induced cell death. SphK1 overexpression impaired the cytotoxicity of Abeta, whereas SphK1 silencing by RNA interference mimicked Abeta-induced cell death, thereby establishing a critical role for SphK1. We further demonstrated that SphK1 could mediate the well established cytoprotective action of insulin-like growth factor (IGF-I) against Abeta toxicity. A dominant-negative form of SphK1 or its pharmacological inhibition not only abrogated IGF-I-triggered stimulation of SphK1 but also hampered IGF-I protective effect. Similarly to IGF-I, the neuroprotective action of TGF-beta1 was also dependent on SphK1 activity; activation of SphK1 as well as cell survival were impeded by a dominant-negative form of SphK1. Taken together, these results provide the first illustration of SphK1 role as a critical regulator of death and survival of Abeta-treated cells.

International audience; Functional selectivity of G protein-coupled receptor (GPCR) ligands toward different downstream signals has recently emerged as a general hallmark of this receptor class. However, pleiotropic and crosstalk signaling of GPCRs makes functional selectivity difficult to decode. To look from the initial active receptor point of view, we developed new, highly sensitive and direct bioluminescence resonance energy transfer-based G protein activation probes specific for all G protein isoforms, and we used them to evaluate the G protein-coupling activity of [(1)Sar(4)Ile(8)Ile]-angiotensin II (SII), previously described as an angiotensin II type 1 (AT(1)) receptor-biased agonist that is G protein independent but β-arrestin selective. By multiplexing assays sensing sequential signaling events, from receptor conformations to downstream signaling, we decoded SII as an agonist stabilizing a G protein-dependent AT(1A) receptor signaling module different from that of the physiological agonist angiotensin II, both in recombinant and primary cells. Thus, a biased agonist does not necessarily select effects from the physiological agonist but may instead stabilize and create a new distinct active pharmacological receptor entity.

Background Ephrin-B1 is a ligand from Eph/Ephrin family involved in cell-cell interactions. If the role of ephrine molecules is well known in embryonic tissue, their expressions/functions in adult remain unclear. Aim The aim of the present work is to characterize the cardiac phenotype of two-months old ephrin-B1 knockout mice. Results Western-blot analysis demonstrated specific expression of ephrin-B1 in total heart protein extracts from WT mice that was lost in KO mice. Further IF studies demonstrated broad expression of ephrin-B1 protein throughout all heart compartments (right ventricle, septum, left ventricle) with different cellular localizations (cardiomyocytes and micro/macrocirculation). HE stained-paraffin-embedded heart sections from KO mice revealed loss of organized cardiac tissue characterized by the presence of wavy cardiomyocytes in the myocardium from septum and right/left ventricles. Modification of cardiomyocytes’ morphology correlates with a loss of their apparent lateral junctions and a significant reduction in cytoskeleton proteins (α-actinin/β-actin levels), consistent with a reduced size of the mycoytes. Ultrastructural analysis (electronic microscopy) revealed significant loss of electronic density of Z-disks and a highly packed intercalated disk (ID) with no modifications in the ID space, probably as a consequence of myocytes’ lateral junction's loss. Functional analysis of KO-mice with echocardiography revealed a significant increase in systolic and diastolic diameters of the left ventricle. Consistent with disruption of myocardial architecture, electrocardiograms demonstrated a first degree AV block together with a significant reduction of heart frequency. Conclusion This is the first report providing evidence for the presence of ephrin molecules in the adult heart tissue. This study using ephrin-B1 KO mouse identified ephrin-B1 as a new ultrastructural protein of the cardiomyocyte whose deletion leads to a dilated cardiomyopathy.

International audience; Apoptosis is a highly organized, energy-dependent program by which multicellular organisms eliminate damaged, superfluous, and potentially harmful cells. Although caspases are the most prominent group of proteases involved in the apoptotic process, the role of lysosomes has only recently been unmasked. This study investigated the role of the lysosomal serine protease CLN2 in apoptosis. We report that cells isolated from patients affected with late infantile neuronal ceroid lipofuscinosis (LINCL) having a deficient activity of CLN2 are resistant to the toxic effect of death ligands such as tumor necrosis factor (TNF), CD95 ligand, or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not to receptor-independent stress agents. CLN2-deficient cells exhibited a defect in TNF-induced Bid cleavage, release of cytochrome c, and caspase-9 and -3 activation. Moreover, extracts from CLN2-overexpressing cells or a CLN2 recombinant protein were able to catalyze the in vitro cleavage of Bid. Noteworthy, correction of the lysosomal enzyme defect of LINCL fibroblasts using a medium enriched in CLN2 protein enabled restoration of TNF-induced Bid and caspase-3 processing and toxicity. Conversely, transfection of CLN2-corrected cells with small interfering RNA targeting Bid abrogated TNF-induced cell death. Altogether, our study demonstrates that genetic deletion of the lysosomal serine protease CLN2 and the subsequent loss of its catalytic function confer resistance to TNF in non-neuronal somatic cells, indicating that CLN2 plays a yet unsuspected role in TNF-induced cell death.

Tumor necrosis factor (TNF) alpha-induced neutral sphingomyelinase-mediated generation of ceramide, a bioactive lipid molecule, is transduced by the adaptor protein FAN, which binds to the intracellular region of the CD120a TNFalpha receptor. FAN-deficient mice do not exhibit any gross abnormality. To further explore the functions of FAN in vivo and because CD120a-deficient mice are resistant to endotoxin-induced liver failure and lethality, we investigated the susceptibility of FAN-deficient animals to lipopolysaccharide (LPS). We show that after d-galactosamine sensitization, FAN-deficient mice were partially resistant to LPS- and TNFalpha-induced lethality. Although LPS challenge resulted in a hepatic ceramide content lower in mutant mice than in control animals, it triggered similar histological alterations, caspase activation, and DNA fragmentation in the liver. Interestingly, LPS-induced elevation of IL-6 (but not TNFalpha) serum concentrations was attenuated in FAN-deficient mice. A less pronounced secretion of IL-6 was also observed after LPS or TNFalpha treatment of cultured peritoneal macrophages and embryonic fibroblasts isolated from FAN-deficient mice, as well as in human fibroblasts expressing a mutated FAN. Finally, we show that d-galactosamine-sensitized IL-6-deficient mice were partially resistant to endotoxin-induced liver apoptosis and lethality. These findings highlight the role of FAN and IL-6 in the inflammatory response initiated by endotoxin, implicating TNFalpha.

Background Ephrin-B1 is a ligand from Eph/Ephrin family involved in cell-cell interactions. If the role of ephrine molecules is well known in embryonic tissue, their expressions/functions in adults remain unclear and to date, no study have paid attention to their potential implication in heart physiopathology. Aim To characterize the cardiac phenotype of ephrin-B1 knockout mice. Methods Two months old-mice were analyzed. Heart tissue was fixed (formaldehyde 10 %) and embedded in paraffin for immunohistochemistry or frozen for western-blot (WB) analysis. Immunofluorescence (IF) studies were performed on heart cryosections. ECG was recorded (PowerLab, DSI) under isoflurane anaesthesia and heart rate spectral variability (HRV) was performed (FFT) in low frequency (LF: 0.15-1.5 Hz) and high frequency (HF: 1.5-5 Hz) ranges ; LH/HF ratio was also calculated. Results We first assessed expression of Ephrin-B1 by WB in heart from WT animals. A specific band around 47 kDa was detected in WT heart total protein extracts that was lost in KO mice. Further IF studies demonstrated broad expression of Ephrin-B1 protein throughout all heart compartments with different cellular localizations (cardiomyocytes and micro/macrocirculation). Hematoxylin-eosin (HE) staining of paraffinembedded heart sections from KO mice revealed loss of organized cardiac tissue characterized by the presence of wavy cardiomyocytes in both septum and ventricles. Myocytes intersected at various angles with bundles wavy appearance. No inflammation, interstitial fibrosis or necrosis were noticed. These pathological observations correlated well with the lack of stiffness of hearts from KO mice compared with controls. When we examined ANS-dependent heart rate variability, LFHRV was significantly reduced in KO mice (16.3±1.2 %) when compared to controls (48.5±6.2 %) without any change in HF, suggesting a specific loss of cardiac sympathetic innervation in these animals. LF/HF ratio was lower in KO mice (0.6±0.2 vs 1.3±0.2 in controls). Conclusion This study provides the first evidence for the presence of ephrin molecules in the adult heart tissue with a specific expression of Ephrin-B1 ligand. The use of ephrin-B1 genetic mouse model highly suggests a role for ephrin-B1 in heart tissue architecture and in sympathetic control of heart rate variability.