Identification of STIM1 partners in the normal and hypertrophied heart

Nous avons déjà démontré un rôle important de STIM1 dans l induction de l hypertrophie cardiaque, mais l identité des canaux STIM1 dépendants responsables de ce flux calcique pro-hypertrophique dans les cellules ventriculaires du rat reste à déterminer. Dans cette étude, nous avons développé une nouvelle méthode de transfert myocardique de gène non viral, en utilisant l énergie des ultrasons, des liposomes et des injections pressurisées dans le myocarde du rat. Grâce à sa simplicité, son efficacité et sa faible immunogénicité, cette technique a produit un nombre suffisant de cellules transfectées pour effectuer des expériences biochimiques et physiologiques sur cellules isolées. Nous avons ensuite caractérisé le profil d expression des protéines ORAIs et TRPCs dans les cellules ventriculaires normales et hypertrophiées, et avons trouvé une augmentation de l expression de TRPC1 dans l hypertrophie cardiaque. Ensuite nous avons utilisé la méthode de transfert de gènes pour identifier les partenaires canalaires de STIM1 via l ARN interférence par injection de siARN. Nous avons identifié TRPC5 comme un canal calcique non-sélectif qui fonctionne d une façon constitutive en conditions basales, avec une activité accrue dans l hypertrophie cardiaque ; ainsi que ORAI3 qui opère dans deux modes: entrée capacitative de calcium et entrée constitutive en concordance avec TRPC5. Nous avons développé une nouvelle méthode de transfert myocardique de gène non viral que nous avons ensuite utilisée pour identifier TRPC5 et ORAI3 comme nouveaux canaux voltage indépendants et STIM1 dépendants dans les myocytes ventriculaires de ratPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Evidence of a role for fibroblast transient receptor potential canonical 3 Ca2+ channel in renal fibrosis

Abstract: Transient receptor potential canonical (TRPC) Ca -permeant channels, especially TRPC3, are increasingly implicated in cardiorenal diseases. We studied the possible role of fibroblast TRPC3 in the development of renal fibrosis. In vitro, a macromolecular complex formed by TRPC1/TRPC3/TRPC6 existed in isolated cultured rat renal fibroblasts. However, specific blockade of TRPC3 with the pharmacologic inhibitor pyr3 was sufficient to inhibit both angiotensin II- and 1-oleoyl-2-acetyl-snglycerol– induced Ca entry in these cells, which was detected by fura-2 Ca imaging. TRPC3 blockade or Ca removal inhibited fibroblast proliferation and myofibroblast differentiation by suppressing the phosphorylation of extracellular signal-regulated kinase (ERK1/2). In addition, pyr3 inhibited fibrosis and inflammation-associated markers in a noncytotoxic manner. Furthermore, TRPC3 knockdown by siRNA confirmed these pharmacologic findings. In adult male Wistar rats or wild-type mice subjected to unilateral ureteral obstruction, TRPC3 expression increased in the fibroblasts of obstructed kidneys and was associated with increased Ca entry, ERK1/2 phosphorylation, and fibroblast proliferation. Both TRPC3 blockade in rats and TRPC3 knockout in mice inhibited ERK1/2 phosphorylation and fibroblast activation as well as myofibroblast differentiation and extracellular matrix remodeling in obstructed kidneys, thus ameliorating tubulointerstitial damage and renal fibrosis. In conclusion, TRPC3 channels are present in renal fibroblasts and control fibroblast proliferation, differentiation, and activation through Ca -mediated ERK signaling. TRPC3 channels might constitute important therapeutic targets for improving renal remodeling in kidney disease

Evidence of a Role for Fibroblast Transient Receptor Potential Canonical 3 Ca2+ Channel in Renal Fibrosis.

Transient receptor potential canonical (TRPC) Ca2+-permeant channels, especially TRPC3, are increasinglyimplicated in cardiorenal diseases. We studied the possible role offibroblast TRPC3 in the development ofrenalfibrosis.In vitro, a macromolecular complex formed by TRPC1/TRPC3/TRPC6 existed in isolatedcultured rat renalfibroblasts. However, specific blockade of TRPC3 with the pharmacologic inhibitor pyr3was sufficient to inhibit both angiotensin II- and 1-oleoyl-2-acetyl-sn-glycerol–induced Ca2+entry in thesecells, which was detected by fura-2 Ca2+imaging. TRPC3 blockade or Ca2+removal inhibitedfibroblastproliferation and myofibroblast differentiation by suppressing the phosphorylation of extracellular signal-regulated kinase (ERK1/2). In addition, pyr3 inhibitedfibrosis and inflammation-associated markers in anoncytotoxic manner. Furthermore, TRPC3 knockdown by siRNA confirmed these pharmacologicfind-ings. In adult male Wistar rats or wild-type mice subjected to unilateral ureteral obstruction, TRPC3 ex-pression increased in thefibroblasts of obstructed kidneys and was associated with increased Ca2+entry,ERK1/2 phosphorylation, andfibroblast proliferation. Both TRPC3 blockade in rats and TRPC3 knockout inmice inhibited ERK1/2 phosphorylation andfibroblast activation as well as myofibroblast differentiationand extracellular matrix remodeling in obstructed kidneys, thus ameliorating tubulointerstitial damageand renalfibrosis. In conclusion, TRPC3 channels are present in renalfibroblasts and controlfibroblastproliferation, differentiation, and activation through Ca2+-mediated ERK signaling. TRPC3 channels mightconstitute important therapeutic targets for improving renal remodeling in kidney disease.Fil: Youakim, Saliba. Faculty of Medicine. Pole of Technology and Health. Physiology and Pathophysiology Research Laboratory; LíbanoFil: Karam, Ralph. Faculty of Medicine. Pole of Technology and Health. Physiology and Pathophysiology Research Laboratory; LíbanoFil: Smayra, Viviane. Saint Joseph University. Faculty of Medicine; LíbanoFil: Aftimos, Georges. National Instituteof Pathology. Department of Anatomopathology; LíbanoFil: Abramowitz, Joel. National Institute of Environmental Health Sciences. Laboratory of Neurobiology ; Estados UnidosFil: Birnbaumer, Lutz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. National Institute of Environmental Health Sciences. Laboratory of Neurobiology ; Estados UnidosFil: Farès, Nassi. Faculty of Medicine. Pole of Technology and Health. Physiology and Pathophysiology Research Laboratory; Líban

The Impact of Long-Term Intake of Phenolic Compounds-Rich Grape Pomace on Rat Gut Microbiota

International audienc

Transient Receptor Potential Canonical 3 and Nuclear Factor of Activated T Cells C3 Signaling Pathway Critically Regulates Myocardial Fibrosis

Aims: Cardiac fibroblasts (CFs) are emerging as major contributors to myocardial fibrosis (MF), a final common pathway of many etiologies of heart disease. Here, we studied the functional relevance of transient receptor potential canonical 3 (TRPC3) channels and nuclear factor of activated T cells c3 (NFATc3) signaling in rodent and human ventricular CFs, and whether their modulation would limit MF. Results: A positive feedback loop between TRPC3 and NFATc3 drove a rat ventricular CF fibrotic phenotype. In these cells, polyphenols (extract of grape pomace polyphenol [P.E.]) decreased basal and angiotensin II-mediated Ca 2+ entries through a direct modulation of TRPC3 channels and subsequently NFATc3 signaling, abrogating myofibroblast differentiation, fibrosis and inflammation, as well as an oxidative stress-associated phenotype. N(ω)-nitro-l-arginine methyl ester (l-NAME) hypertensive rats developed coronary perivascular, sub-epicardial, and interstitial fibrosis with induction of embryonic epicardial progenitor transcription factors in activated CFs. P.E. treatment reduced ventricular CF activation by modulating the TRPC3-NFATc3 pathway, and it ameliorated echocardiographic parameters, cardiac stress markers, and MF in l-NAME hypertensive rats independently of blood pressure regulation. Further, genetic deletion (TRPC3 -/- ) and pharmacological channel blockade with N-[4-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-benzenesulfonamide (Pyr10) blunted ventricular CF activation and MF in l-NAME hypertensive mice. Finally, TRPC3 was present in human ventricular CFs and upregulated in MF, whereas pharmacological modulation of TRPC3-NFATc3 decreased proliferation and collagen secretion. Innovation and Conclusion: We demonstrate that TRPC3-NFATc3 signaling is modulated by P.E. and critically regulates ventricular CF phenotype and MF. These findings strongly argue for P.E., through TRPC3 targeting, as potential and interesting therapeutics for MF management.Fil: Saliba, Youakim. Université Saint Joseph; LíbanoFil: Jebara, Victor. Université Saint Joseph; LíbanoFil: Hajal, Joelle. Université Saint Joseph; LíbanoFil: Maroun, Richard. Université Saint Joseph; LíbanoFil: Chacar, Stéphanie. Université Saint Joseph; LíbanoFil: Smayra, Viviane. Université Saint Joseph; LíbanoFil: Abramowitz, Joel. National Institute of Environmental Health Sciences,; Estados UnidosFil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina. National Institute of Environmental Health Sciences,; Estados UnidosFil: Farès, Nassim. Université Saint Joseph; Líban

TRPC3 regulates islet beta-cell insulin secretion

Abstract: Insulin release is tightly controlled by glucose-stimulated calcium (GSCa) through hitherto equivocal pathways. This study investigates TRPC3, a non-selective cation channel, as a critical regulator of insulin secretion and glucose control. TRPC3’s involvement in glucose-stimulated insulin secretion (GSIS) is studied in human and animal islets. TRPC3-dependent in vivo insulin secretion is investigated using pharmacological tools and Trpc3−/− mice. TRPC3’s involvement in islet glucose uptake and GSCa is explored using fluorescent glucose analogue 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose and calcium imaging. TRPC3 modulation by a small-molecule activator, GSK1702934A, is evaluated in type 2 diabetic mice. TRPC3 is functionally expressed in human and mouse islet beta cells. TRPC3-controlled insulin secretion is KATP-independent and primarily mediated by diacylglycerol channel regulation of the cytosolic calcium oscillations following glucose stimulation. Conversely, glucose uptake in islets is independent of TRPC3. TRPC3 pharmacologic inhibition and knockout in mice lead to defective insulin secretion and glucose intolerance. Subsequently, TRPC3 activation through targeted small-molecule enhances insulin secretion and alleviates diabetes hallmarks in animals. This study imputes a function for TRPC3 at the onset of GSIS. These insights strengthen one’s knowledge of insulin secretion physiology and set forth the TRPC3 channel as an appealing candidate for drug development in the treatment of diabetes

Hypothyroidism and Its Rapid Correction Alter Cardiac Remodeling

<div><p>The cardiovascular effects of mild and overt thyroid disease include a vast array of pathological changes. As well, thyroid replacement therapy has been suggested for preserving cardiac function. However, the influence of thyroid hormones on cardiac remodeling has not been thoroughly investigated at the molecular and cellular levels. The purpose of this paper is to study the effect of hypothyroidism and thyroid replacement therapy on cardiac alterations. Thirty Wistar rats were divided into 2 groups: a control (n = 10) group and a group treated with 6-propyl-2-thiouracil (PTU) (n = 20) to induce hypothyroidism. Ten of the 20 rats in the PTU group were then treated with L-thyroxine to quickly re-establish euthyroidism. The serum levels of inflammatory markers, such as C-reactive protein (CRP), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL6) and pro-fibrotic transforming growth factor beta 1 (TGF-β1), were significantly increased in hypothyroid rats; elevations in cardiac stress markers, brain natriuretic peptide (BNP) and cardiac troponin T (cTnT) were also noted. The expressions of cardiac remodeling genes were induced in hypothyroid rats in parallel with the development of fibrosis, and a decline in cardiac function with chamber dilation was measured by echocardiography. Rapidly reversing the hypothyroidism and restoring the euthyroid state improved cardiac function with a decrease in the levels of cardiac remodeling markers. However, this change further increased the levels of inflammatory and fibrotic markers in the plasma and heart and led to myocardial cellular infiltration. In conclusion, we showed that hypothyroidism is related to cardiac function decline, fibrosis and inflammation; most importantly, the rapid correction of hypothyroidism led to cardiac injuries. Our results might offer new insights for the management of hypothyroidism-induced heart disease.</p></div

The LT4 treatment reversed cardiac dilation and improved systolic function.

<p><b>A</b>: Heart weight to tibia length ratio (mg/cm) in the different treatment groups. Statistical analysis was performed with Kruskal-Wallis One-way ANOVA on rank tests followed by post hoc Dunn's multiple comparison tests. **<i>p</i><0.01 <i>vs.</i> control and reverse. <b>B</b> and <b>C</b>: Echocardiographic measurements of the ejection fraction (%) and fractional shortening (%) in the control, PTU-treated and LT4-treated rats at different time points. Statistical analysis was performed with two-way ANOVA tests followed by post hoc Tukey's multiple comparison tests. *<i>p</i><0.05 <i>vs.</i> control as well as 7 and 12 weeks reverse; ^#<i>p</i><0.05 <i>vs.</i> PTU. <b>D</b>: M-mode echocardiographic tracings from the control, PTU and reverse groups. Echocardiography scale bars: 0.25 s and 0.5 mm. Data are represented as the mean ± SEM. n = 10 animals for each group.</p

Serum levels of fT3 (A), fT4 (A) and TSH (B) in the different rat groups at different times after the PTU or LT4 treatments and sacrifice.

<p>Data are represented as the mean ± SEM. n = 10 animals for each group. Statistical analysis was performed with two-way ANOVA tests followed by post hoc Tukey's multiple comparison tests. *<i>p</i><0.001 <i>vs.</i> control; ^#<i>p</i><0.001 <i>vs.</i> control and reverse; ^$<i>p</i><0.001 <i>vs</i>. 7 and 12 weeks reverse and 12 weeks PTU; ^§<i>p</i><0.001 <i>vs.</i> 7 and 12 weeks reverse; ⁺<i>p</i><0.001 <i>vs</i>. 7 and 12 weeks reverse and 7 weeks PTU.</p