J024 Role du complexe proteique ERM dans la transmission des effets de l’activation de l’echangeur NHE1 en reponse a l’acidification des myocytes cardiaques

Nous avons étudié le rôle du complexe protéique ezrine/radixine/ moésine (ou complexe ERM, relié au cytosquelette) dans la transmission du signal donné par l’augmentation d’activité de l’échangeur Na+/H+, NHE1, en réponse à une acidification intracellulaire. L’étude a été réalisée sur myocytes ventriculaires gauches isolés de coeurs de rats adultes (rats Wistar témoins et rats diabétiques GK). La détection des protéines ERM actives (i.e. phosphorylées) par immuno-marquage a permis de révéler que, dans les conditions basales et en dehors de toute stimulation, ces protéines sont localisées au niveau des disques intercalaires, aussi bien dans les myocytes de rats témoins que de rats GK. L’acidification entraîne une augmentation significative de la phosphorylation des protéines ERM avec remodelage intracellulaire de ces protéines; elles sont alors localisées à proximité des récepteurs de la ryanodine, vraisemblablement au niveau des tubules-T. Ces observations ont été faites aussi bien dans les cardiomyocytes ventriculaires de rats témoins que de rats diabétiques GK, avec un remodelage plus marqué chez ces derniers. Un autre résultat d’importance est l’observation d’une augmentation d’activité de Akt, parallèle à celle du complexe ERM : augmentation avec l’acidification et absence d’augmentation lorsque l’acidification a été induite en présence d’un inhibiteur de NHE1. De plus, l’exploration de deux voies cibles de Akt, la voie mTOR/p70S6K et la voie GSK-3b, a montré que seule la phosphorylation de la GSK-3b est augmentée lors d’une stimulation marquée de l’activité de la voie NHE1/ERM/ Akt. L’ensemble de ce travail, nous a permis de mettre en évidence qu’une activité élevée de l’échangeur NHE1 constitue un signal capable d’enclencher, via le complexe protéique ERM, une cascade d’événements intracellulaires pouvant notamment aboutir, comme montré précédemment1, à une réponse hypertrophique. Ceci nous semble être un résultat fondamental qui met en lumière un rôle important de NHE1, lorsque celui-ci est sollicité de façon excessive (par exemple au cours d’une ischémie chronique), à côté de son rôle reconnu de mécanisme majeur de régulation du pH interne des cellules

Translocation of the Na+/H+ exchanger 1 (NHE1) in cardiomyocyte responses to insulin and energy-status signalling

The Na+/H+ exchanger NHE1 is a highly regulated membrane protein that is required for pH homoeostasis in cardiomyocytes. The activation of NHE1 leads to proton extrusion, which is essential for counteracting cellular acidity that occurs following increased metabolic activity or ischaemia. The activation of NHE1 intrinsic catalytic activity has been well characterized and established experimentally. However, we have examined in the present study whether a net translocation of NHE1 to the sarcolemma of cardiomyocytes may also be involved in the activation process. We have determined the distribution of NHE1 by means of immunofluorescence microscopy and cell-surface biotinylation. We have discovered changes in the distribution of NHE1 that occur when cardiomyocytes are stimulated with insulin that are PI3K (phosphoinositide 3-kinase)-dependent. Translocation of NHE1 also occurs when cardiomyocytes are challenged by hypoxia, or inhibition of mitochondrial oxidative metabolism or electrically induced contraction, but these responses occur through a PI3K-independent process. As the proposed additional level of control of NHE1 through translocation was unexpected, we have compared this process with the well-established translocation of the glucose transporter GLUT4. In immunofluorescence microscopy comparisons, the translocation of NHE1 and GLUT4 to the sarcolemma that occur in response to insulin appear to be very similar. However, in basal unstimulated cells the two proteins are mainly located, with the exception of some co-localization in the perinuclear region, in distinct subcellular compartments. We propose that the mechanisms of translocation of NHE1 and GLUT4 are linked such that they provide spatially and temporally co-ordinated responses to cardiac challenges that necessitate re-adjustments in glucose transport, glucose metabolism and cell pH

Enhanced activity of the myocardial Na+/H+ exchanger contributes to left ventricular hypertrophy in the Goto–Kakizaki rat model of type 2 diabetes: critical role of Akt

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3-(R)-[3-(2 methoxyphenylthio-2-(S)-methylpropyl] alubi-3,4-dihydro-2H-1,5-benzoxathiepine bromhydrate (F 15845) prevents ischemia-induced heart remodeling by reduction of the intracellular Na+ overload.

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Outcomes of 38 patients with PFIC3: Impact of genotype and of response to ursodeoxycholic acid therapy

Background & Aims: Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare liver disease caused by biallelic variations in ABCB4. Data reporting on the impact of genotype and of response to ursodeoxycholic acid (UDCA) therapy on long-term outcomes are scarce. Methods: We retrospectively describe a cohort of 38 patients with PFIC3 with a median age at last follow-up of 19.5 years (range 3.8–53.8). Results: Twenty patients presented with symptoms before 1 year of age. Thirty-one patients received ursodeoxycholic acid (UDCA) therapy resulting in serum liver test improvement in 20. Twenty-seven patients had cirrhosis at a median age of 8.1 years of whom 18 received a liver transplant at a median age of 8.5 years. Patients carrying at least one missense variation were more likely to present with positive (normal or decreased) canalicular MDR3 expression in the native liver and had prolonged native liver survival (NLS; median 12.4 years [range 3.8-53.8]). In contrast, in patients with severe genotypes (no missense variation), there was no detectable canalicular MDR3 expression, symptom onset and cirrhosis occurred earlier, and all underwent liver transplantation (at a median age of 6.7 years [range 2.3–10.3]). The latter group was refractory to UDCA treatment, whereas 87% of patients with at least one missense variation displayed an improvement in liver biochemistry in response to UDCA. Biliary phospholipid levels over 6.9% of total biliary lipid levels predicted response to UDCA. Response to UDCA predicted NLS. Conclusions: Patients carrying at least one missense variation, with positive canalicular expression of MDR3 and a biliary phospholipid level over 6.9% of total biliary lipid levels were more likely to respond to UDCA and to exhibit prolonged NLS. Impact and implications: In this study, data show that genotype and response to ursodeoxycholic acid therapy predicted native liver survival in patients with PFIC3 (progressive familial intrahepatic cholestasis type 3). Patients carrying at least one missense variation, with positive (decreased or normal) immuno-staining for canalicular MDR3, and a biliary phospholipid level over 6.9% of total biliary lipids were more likely to respond to ursodeoxycholic acid therapy and to exhibit prolonged native liver survival

∆4-3-oxo-5β-reductase deficiency: favorable outcome in 16 patients treated with cholic acid

Abstract Background Oral cholic acid therapy is an effective therapy in children with primary bile acid synthesis deficiencies. Most reported patients with this treatment have 3β-hydroxy-Δ5-C27-steroid oxidoreductase deficiency. The aim of the study was the evaluation of cholic acid therapy in a cohort of patients with the rarer Δ4-3-oxosteroid 5β-reductase (Δ4-3-oxo-R) deficiency. Methods Sixteen patients with Δ4-3-oxo-R deficiency confirmed by AKR1D1 gene sequencing who received oral cholic acid were retrospectively analyzed. Results First symptoms were reported early in life (median 2 months of age), with 14 and 3 patients having cholestatic jaundice and severe bleeding respectively. Fifteen patients received ursodeoxycholic acid before diagnosis, with partial improvement in 8 patients. Four patients had liver failure at the time of cholic acid initiation. All 16 patients received cholic acid from a median age of 8.1 months (range 3.1–159) and serum liver tests normalized in all within 6–12 months of treatment. After a median cholic acid therapy of 4.5 years (range 1.1–24), all patients were alive with their native liver. Median daily cholic acid dose at last follow-up was 8.3 mg/kg of body weight. All patients, but one, had normal physical examination and all had normal serum liver tests. Fibrosis, evaluated using liver biopsy (n = 4) or liver elastography (n = 9), had stabilized or improved. Cholic acid therapy enabled a 12-fold decrease of 3-oxo-∆4 derivatives in urine. Patients had normal growth and quality of life. The treatment was well tolerated without serious adverse events and signs of hepatotoxicity. Conclusions Oral cholic acid therapy is a safe and effective treatment for patients with Δ4-3-oxo-R deficiency

Roles of extracellular nucleotides and P2 receptors in ectodomain shedding

Ectodomain shedding of integral membrane receptors results in the release of soluble molecules and modification of the transmembrane portions to mediate or modulate extracellular and intracellular signalling. Ectodomain shedding is stimulated by a variety of mechanisms, including the activation of P2 receptors by extracellular nucleotides. This review describes in detail the roles of extracellular nucleotides and P2 receptors in the shedding of various cell surface molecules, including amyloid precursor protein, CD23, CD62L, and members of the epidermal growth factor, immunoglobulin and tumour necrosis factor families. This review discusses the mechanisms involved in P2 receptor-mediated shedding, demonstrating central roles for the P2 receptors, P2X7 and P2Y2, and the sheddases, ADAM10 and ADAM17, in this process in a number of cell types