A vida é um processo dinâmico no tempo e no espaço, resultante da interação celular provida pelas junções comunicantes (JCs), canais formados por conexinas (Cxs) que permitem a passagem de moléculas de até 1kDa, e íons. O foco deste trabalho foi caracterizar a modulação de Cxs no desenvolvimento, adaptação visual e após trauma mecânico localizado na retina, através de técnicas como PCR em tempo real, western blot e imuno-histoquímica. No desenvolvimento, a Cx43 apresentou alta expressão no início (255%, P<0.01), a Cx45 teve alta expressão em todos os estágios, as Cx50 e Cx36 são pouco expressas no início (4-9%, P<0.01), mas muito expressas no final e no adulto. Na adaptação ao escuro houve regulação de 115% do RNam da Cx36 após 24h, evidenciando uma plasticidade do acoplamento frente às condições de iluminação. Na neurodegeneração, a expressão da Cx36 não variou nos diferentes tempos pós-lesão, mas a Cx43 aumentou após 7 dias. O papel do acoplamento na degeneração foi estudado utilizando bloqueadores e abridores de JCs combinados com métodos para avaliar a viabilidade celular (TUNEL, FluoroJade e LDH). Finalmente, o bloqueio das JCs diminuiu a morte celular secundária, mas a abertura gerou um efeito inicial pró-apoptótico que depois de 4h foi diluído no tecido. / Life is a dynamic process in time and space, resulting from an interaction provided by gap junctions (GJs), channels composed of connexins (Cxs) that permits the passage of molecules up to 1kDa and ions. The aim of this study was analyze the modulation of Cxs during development, dark adaptation and after local mechanical trauma in the retina using techniques like real time PCR, western blot and imunohistochemistry. In development, Cx43 was highly expressed in the beginning (255%, P<0.01), Cx45 was highly expressed during all development, Cx50 and Cx36 were virtually absent in the beginning (4-9%, P<0.01), but highly expressed in the end of development. In Dark-adaptation, Cx36 was up-regulated after 24h (115%, P < 0.05) suggesting a plasticity of the neuronal coupling in response to light conditions. In neurodegeneration, Cx36 expression didnt ranged in different days after lesion, but the Cx43 increased after 7 days. The coupling was studied by using GJ blockers and openers combined with methods to evaluating cellular viability (TUNEL, FluoroJade and LDH). Finally, blocking the cellular coupling reflects in a decrease of secondary death. GJ openers seems act allowing the spread of death in the beginning, but this effect be diluted after 4h in the tissue.

Plyšinės jungtys (PJ) yra tarpląstelinę sąveiką užtikrinantys kanalai, svarbūs ląstelių dauginimuisi, diferenciacijai, migracijai bei koordinacijai. Skirtinguose žmogaus organizmo audiniuose atrastas 21 koneksino (Cx) tipas. Tam tikros ligos yra susijusios su Cx mutacijomis. PJ kanalų savybes įtakoja jungties įtampa, viduląstelinis pH (pHi), viduląstelinė dvivalenčių katijonų koncentracija ([Ca2+]i, [Mg2+]i), fosforinimas, įvairios cheminės medžiagos. Disertacinio darbo metu pirmą kartą parodėme, kad plačiai naudojamų skirtingų grupių PJ slopiklių, tokių kaip alkanolių, forano, meflokvino, veikimas priklauso nuo pHi ir Cx tipo. Iškėlėme ir eksperimentiškai patikrinome hipotezę, kad tam tikri PJ slopikliai veikia, sudarydami vandenilinius ryšius su Cx45 baltymo histidinais. Ištyrėme Cx36, Cx40, Cx43, Cx45 ir Cx47 PJ kanalų laidumo priklausomybę nuo pHi. Nustatėme [Mg2+]i įtaką Cx43 ir Cx45 PJ kanalų laidumui bei Cx36 PJ kanalų laidumo priklausomybei nuo pHi, kas iki šiol nebuvo tirta. Cx45 ir Cx36 mutantinių baltymų tyrimai suteikė naujų žinių apie šių baltymų amino rūgštis, kurios gali būti reikšmingos PJ kanalų laidumo reguliavimui viduląsteliniu pH. / Gap junctions (GJ) are intercellular channels, which provide a direct pathway for electrical and metabolic cell-to-cell communication. GJ play an important role in cell proliferation, differentiation, migration, and coordination. The family of connexin (Cx) genes consists of 21 members in the human genome, and a variety of diseases is associated with Cx mutations. Electrical conductance (gj) of gap junctions is regulated by transjunctional voltage, intracellular pH (pHi), intracellular divalent cation concentration ([Ca2+]i and [Mg2+]i), phosphorylation and different chemical compounds. In this work we demonstrate for the first time that the potency of widely used GJ inhibitors such as alkanols, forane and mefloquine in GJ channel uncoupling is pHi- and Cx type-dependent. This type of modulation may occur through the formation of hydrogen bonds between uncouplers and histidines of Cx45 protein. We determined gj-pHi dependence of Cx36, Cx40, Cx43, Cx45 and Cx47 GJ channels, and evaluated the influence of [Mg2+]i on the conductance of Cx43 and Cx45 GJs and on the sensitivity of Cx36 GJs to pHi. The investigation of the Cx45 and Cx36 mutants provided with new knowledge on the structural elements, which may be responsible for the sensitivity of Cxs to pHi.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-19T17:30:16Z No. of bitstreams: 1 Fred da Silva Juliao Uso de método de biologia molecular....pdf: 3387831 bytes, checksum: 0efc6396d21a6e8a634b8026665afe7c (MD5) / Made available in DSpace on 2012-07-19T17:30:17Z (GMT). No. of bitstreams: 1 Fred da Silva Juliao Uso de método de biologia molecular....pdf: 3387831 bytes, checksum: 0efc6396d21a6e8a634b8026665afe7c (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A surdez genética é heterogênea em sua base molecular, fenótipo e padrão de herança. Apesar desta heterogeneidade, mutações no gene GJB2 são as principais causas de surdez genética, especialmente entre aquelas com padrão autossômico recessivo (MIM #220290). Neste trabalho investigou-se a origem genética da deficiência auditiva (DA) em pacientes do município de Monte Santo-BA. Oitenta e quatro indivíduos com DA, correspondendo a 38 famílias foram avaliados, responderam questionário clínico-epimediológico e forneceram dados individuoais e familiares para análise genealógica. Foram pesquisadas mutações no gene GJB2, as deleções (GJB6-13S1830) e (GJB6-D13S1854), no gene GJB6 e a mutação A1555G, no gene mitocondrial 12S rRNA. A investigação foi iniciada pela mutação c.35delG no gene GJB2, através de PCR-RFLP com a enzima BstNI, as amostras que não apresentaram essa variante foram genotipadas por sequenciamento do éxon 2 do gene GBJ2. Cerca de 55% dos indivíduos foram do sexo masculino, a idade média foi de 32 anos (variando de 2–70 anos), 33,9% dos indivíduos referiram-se como brancos, houve relato de consanguinidade entre os pais em 52,1% dos casos, DA familial foi relatada em 92,8% dos casos, sendo considerada pré-lingual em 90,9% e bilateral em 98,7%. Oito diferentes mutações foram encontradas no gene GJB2. Cerca de 24% dos indivíduos, correspondendo a 8 famílias, foram homozigotos e 1 (1,2%) foi heterozigoto para mutação c.35delG. Em uma família com 9 afetados foi encontrada a mutação p.R75Q no gene GJB2, que causa DA herdada com padrão dominante, em heterozigose, correspondendo a 14,8% dos pacientes analisados. Encontraram-se ainda 4 mutações relatadas como polimorfismo sem efeito patogênico: p.V27I, p.M34T, c.-15C>T e c.*2C>T, a primeira foi encontrada em 5,8% e as três últimas em 1,2% dos indivíduos e as mutações c.-22-12C>T (5,8%) e p.K168R (1,2%) sem patogenicidade estabelecida. As mutações del (GJB6-D13S1830) e del (GJB6-D13S1854) no gene GJB6, A1555G no gene 12S rRNA não foram detectadas nos pacientes estudados nesta amostra. Foi confirmada etiologia genética em 35,8% dos pacientes e foi observada heterogeneidade alélica e do padrão de herança. Provavelmente mutações em outros genes e/ou fatores ambientais são responsáveis pelos outros casos de DA nessa população. / Genetic deafness is heterogeneous in its molecular basis, phenotype and inheritance pattern. Despite this heterogeneity, mutations in GJB2 are the leading causes of genetic deafness, especially among those with autosomal recessive (MIM # 220290). In this study we investigated the origin of genetic hearing loss (HL) patients from Monte Santo, Bahia. Eighty-four patients with HL, representing 38 families were evaluated, patients answered a questionnaire and clinical-epimediológico individuoais provided data for analysis and family pedigree. We investigated mutations in the GJB2 gene deletions (GJB6-13S1830) and (GJB6-D13S1854) in GJB6 gene and the A1555G mutation in mitochondrial 12S rRNA gene. The investigation was initiated by a mutation in the gene GJB2 c.35delG by PCR-RFLP with the enzyme BstNI, samples that showed no such variant was genotyped by sequencing of exon 2 of gene GBJ2. About 55% of subjects were male, mean age was 32 years (range 20-70 years), 33.9% of subjects reported themselves as white, there were reports of consanguinity between parents in 52.1 % of cases, HL familial was reported in 92.8% of cases being considered pre-lingual in 90.9% and bilateral in 98.7%. Eight different mutations were found in the GJB2 gene. About 24% of individuals, representing eight families, were homozygous and 1 (1.2%) was heterozygous for mutation c.35delG. In a family with nine affected the mutation p.R75Q in heterozygous was found in GJB2 gene, which causes HL inherited with a dominant, corresponding to 14.8% of patients analyzed. There were also four mutations reported as a polymorphism without pathogenic effect: p.V27I, p.M34T, c.-15C> T and c. * 2C> T, the first was found in 5.8% and the last three in a 2% of individuals and mutations c.-22-12C> T (5.8%) and p.K168R (1.2%) without established pathogenicity. Mutations del (GJB6-D13S1830) and del (GJB6-D13S1854) in GJB6 gene, A1555G in the 12S rRNA gene were not detected in the patients studied in this sample. Genetic etiology was confirmed in 35.8% of patients and was observed allelic and the pattern of inheritance heterogeneity. Probably mutations in other genes and / or environmental factors are responsible for other cases of AD in this population.

A vida é um processo dinâmico no tempo e no espaço, resultante da interação celular provida pelas junções comunicantes (JCs), canais formados por conexinas (Cxs) que permitem a passagem de moléculas de até 1kDa, e íons. O foco deste trabalho foi caracterizar a modulação de Cxs no desenvolvimento, adaptação visual e após trauma mecânico localizado na retina, através de técnicas como PCR em tempo real, western blot e imuno-histoquímica. No desenvolvimento, a Cx43 apresentou alta expressão no início (255%, P<0.01), a Cx45 teve alta expressão em todos os estágios, as Cx50 e Cx36 são pouco expressas no início (4-9%, P<0.01), mas muito expressas no final e no adulto. Na adaptação ao escuro houve regulação de 115% do RNam da Cx36 após 24h, evidenciando uma plasticidade do acoplamento frente às condições de iluminação. Na neurodegeneração, a expressão da Cx36 não variou nos diferentes tempos pós-lesão, mas a Cx43 aumentou após 7 dias. O papel do acoplamento na degeneração foi estudado utilizando bloqueadores e abridores de JCs combinados com métodos para avaliar a viabilidade celular (TUNEL, FluoroJade e LDH). Finalmente, o bloqueio das JCs diminuiu a morte celular secundária, mas a abertura gerou um efeito inicial pró-apoptótico que depois de 4h foi diluído no tecido. / Life is a dynamic process in time and space, resulting from an interaction provided by gap junctions (GJs), channels composed of connexins (Cxs) that permits the passage of molecules up to 1kDa and ions. The aim of this study was analyze the modulation of Cxs during development, dark adaptation and after local mechanical trauma in the retina using techniques like real time PCR, western blot and imunohistochemistry. In development, Cx43 was highly expressed in the beginning (255%, P<0.01), Cx45 was highly expressed during all development, Cx50 and Cx36 were virtually absent in the beginning (4-9%, P<0.01), but highly expressed in the end of development. In Dark-adaptation, Cx36 was up-regulated after 24h (115%, P < 0.05) suggesting a plasticity of the neuronal coupling in response to light conditions. In neurodegeneration, Cx36 expression didnt ranged in different days after lesion, but the Cx43 increased after 7 days. The coupling was studied by using GJ blockers and openers combined with methods to evaluating cellular viability (TUNEL, FluoroJade and LDH). Finally, blocking the cellular coupling reflects in a decrease of secondary death. GJ openers seems act allowing the spread of death in the beginning, but this effect be diluted after 4h in the tissue.

A doença de Parkinson (DP) caracteriza-se pela perda progressiva de neurônios dopaminérgicos da substância nigra, o que acarreta diversas disfunções motoras. Não há ainda tratamentos capazes de deter ou retardar a degeneração dos neurônios dopaminérgicos, e os medicamentos hoje empregados na clínica apenas amenizam os sintomas, sem alterar a progressão da DP. No presente estudo foi avaliada a atividade neuroprotetora do éster fenetil do ácido cafeico (CAPE), um componente abundante do própolis de abelhas, com atividade anti-inflamatória, antiviral, antioxidante e imunomodulatória. Estudos têm sugerido seus efeitos benéficos contra as doenças neurodegenerativas, incluindo a doença de Parkinson, e alguns mecanismos têm sido propostos; porém muitos dos estudos com CAPE foram feitos apenas em culturas celulares. Este é o primeiro estudo a demonstrar que a administração intraperitoneal do CAPE protege contra a perda neuronal dopaminérgica e a disfunção motora induzidas pela neurotoxina 6-OHDA em ratos, confirmando a capacidade do CAPE de atravessar a barreira hematoencefálica e exercer seus efeitos benéficos no sitema nervoso central (SNC). Adicionalmente foram empregados dois modelos in vitro para o delineamento de possíveis mecanismos de neuroproteção: (i) mitocôndrias isoladas de cérebro de ratos não tratados e (ii) células SH-SY5Y tratadas com 6-OHDA. Os achados in vivo e in vitro sugerem o envolvimento dos seguintes mecanismos: (i) atividade antioxidante (sequestro de ERO, neutralização de radicais livres e quelação de metais); (ii) atividade anti-inflamatória (inibição da ativação do NF-kB, TNF-?, IkK? e Ikk?); (iii) aumento da expressão da conexina 43; (iv) inibição da Transição de Permeabilidade Mitocondrial (TPM); (v) inibição da liberação de citocromo c e (vi) inibição da ativação da caspase-3, executora final da apoptose. Além disso, o estudo também demonstrou que, por si só, o CAPE não interfere nas funções mitocondriais, o que representa uma vantagem com relação a outros inibidores da TPM. Assim, de acordo com nossos achados, o CAPE é um agente neuroprotetor promissor e pode auxiliar em futuras estratégias terapêuticas para as doenças neurodegenerativas, bem como para o melhor entendimento dos mecanismos responsáveis pelo desenvolvimento dessas doenças. / Parkinson\'s disease (PD) is characterized by progressive loss of dopaminergic neurons in the substantia nigra, which causes various motor dysfunctions. There are no treatments able to delay or stop the degeneration of dopaminergic neurons and the therapy employed nowadays only alleviate the symptoms without altering the progression of PD. In the present study we evaluated the neuroprotective activity of caffeic acid phenethyl ester (CAPE), a compound abundant in honeybees\' propolis, with anti-inflammatory, antiviral, antioxidant and immunomodulatory activities. Its beneficial effects against neurodegenerative diseases, including Parkinson\'s disease, have been suggested and some mechanisms have been proposed; however, many of the studies with CAPE have been performed only in cell cultures. This is the first study to demonstrate that the intraperitoneal administration of CAPE protects against the dopaminergic neuronal loss and the motor dysfunction induced by the neurotoxin 6-OHDA in rats, confirming the ability of CAPE to cross the blood brain barrier and exert its beneficial effects on the central nervous system (CNS). Additionally two in vitro models were used to delineate the possible mechanisms of neuroprotection: (i) mitochondria isolated from the brain of non-treated rats and (ii) cells SH-SY5Y treated with 6-OHDA. The in vivo and in vitro findings suggest the involvement of the following mechanisms: (i) antioxidant activity (scavenger of ROS, free radicals neutralization and metal chelation); (ii) anti-inflammatory activity (inhibition of activation of NF-kB, TNF-?, IkK? and Ikk?); (iii) increased expression of connexin-43; (iv) Inhibition of Mitochondrial Permeability Transition (MPT); (v) inhibition of cytochrome c release (vi) inhibition of the activation of caspase-3, the final executioner of apoptosis. Furthermore, the study also showed that CAPE alone does not interfere with mitochondrial functions, which represents an advantage in relation to other inhibitors of MPT. Therefore, according to our findings, CAPE is a promising neuroprotective agent and may be useful for future therapeutic strategies for neurodegenerative diseases, as well as to better understand the mechanisms responsible for the development of these diseases.

Este estudo teve como objetivo avaliar a expressão hipocampal de proteínas e de RNAm das Cx43 e Cx36 no modelo de epilepsia do lobo temporal (ELT) induzido por pilocarpina. Além disso, os efeitos do bloqueador de canais de junções comunicantes (CJC), carbenoxolona (CBX), foram avaliados por eletrofisiologia durante o período de status epilepticus. Os dados referentes à Cx43 demonstraram redução dos níveis proteicos no período latente (p<0,05) e aumento no período crônico do modelo (p<0,01). Os níveis de RNAm de Cx43 não sofreram alterações. Tanto os níveis proteicos quanto os de RNAm de Cx36 não se alteraram. Os dados eletrofisiológicos mostraram redução da potência na banda de frequência entre 15 e 30 Hz no eletrocortigrama, além de redução da amplitude relativa dos potenciais epileptiformes. Foi observado ainda que o grupo tratado com CBX passou a apresentar períodos flat antecipadamente. Os dados deste estudo sugerem um importante papel dos CJC na ELT induzida por pilocarpina, contribuindo para o conhecimento da regulação destes canais na epilepsia. / In this study, the hippocampal protein and mRNA levels of Cx43 and Cx36 were investigated in the pilocarpine model of temporal lobe epilepsy (TLE). In addition, the effects of a gap junction (GJ) blocker (carbenoxolone-CBX) on pilocarpine-induced status epilepticus (SE) were also evaluated by electrophysiological recordings. Our results on Cx43 showed reduction of protein levels in the latent period (p<0.05) and increase in the chronic period of the model (p<0.01), whereas no changes were observed in the mRNA levels. Both protein and mRNA levels of Cx36 showed no changes. The electrophysiological recordings indicated that CBX promoted a marked reduction of power in the 15-30 Hz electrocorticographic frequency. Decrease in the amplitude of the epileptiform potentials was also seen, in addition to anticipation of occurrence of flat periods in the group treated with CBX. Data obtained from this study suggest an important role for GJ channels in the pilocarpine-induced TLE, contributing to a greater understanding of the regulation of these channels in the epilepsy.

Atherosclerosis is an inflammatory disease which develops focally in regions of the vasculature where there is dysfunction of endothelial cells modulated in part by shear stress from flowing blood. To address the clinical crisis of atherosclerosis, tissue engineering has focused on development of a living blood vessel substitute for use as a vascular graft in bypass surgery. Despite substantial progress in understanding the biological basis and developing clinical treatments for cardiovascular disease, critical challenges remain. As a novel strategy to improve understanding of basic human vascular biology and develop superior tissue engineered grafts, this dissertation combines the scientific and clinical approaches by using a tissue engineered blood vessel as a more physiologic in vitro model to study endothelial cell biology. Through the use of transcriptional profiling, results demonstrate significant changes in endothelial cell gene expression using the tissue engineered blood vessel model. Furthermore, the presence of a more physiologic substrate alters the cellular response to shear stress which is a critical mediator of vascular pathology. A case study of endothelial cell function in this system focuses on cell-cell communication through gap junctions. Endothelial cell connexins which form gap junctions are shown to be differentially regulated by substrate and shear stress. Moreover, gap junction communication between endothelial cells is modulated by the mechanical environment. Studies using RNA interference to knockdown expression of individual connexin isotypes demonstrate integrated regulation of connexins yet unique roles in endothelial cell function. Collectively, results exemplify the sensitivity of endothelial cell phenotype to substrate and shear stress and underline the importance of using more physiologic models in the study of basic cell biology.

L'hypothalamus est fortement impliqué dans la régulation nerveuse de l'homéostasie énergétique. Il existe dans cette structure des neurones spécialisés (gluco-sensibles) qui détectent notamment l’hyperglycémie puis déclenchent des réponses adaptées comme le maintien de la glycémie, en stimulant la sécrétion d’insuline ou encore le rassasiement. Les astrocytes sont suspectés de participer à la détection neuronale du glucose. Dans l’ensemble du cerveau, il existe un couplage métabolique entre astrocytes et neurones. Le lactate, issu de la métabolisation du glucose par les astrocytes, est transporté par les neurones par des transporteurs aux monocarboxylates (MCTs). De plus, il a récemment été montré que les jonctions gap (GJ), à l’origine de la formation de réseaux au sein des astrocytes sont indispensables au passage du glucose de la circulation sanguine vers les neurones en activité. Ces GJ astrocytaires sont formées majoritairement de connexines 43 et 30 (Cxs).Mon travail de thèse s’est orienté suivant deux axes, qui ont visé à étudier le rôle des astrocytes dans la détection hypothalamique du glucose et du lactate. Dans un premier temps, nous avons montré que le lactate, comme le glucose, est détecté au niveau central et induit une sécrétion d’insuline. Dans un modèle de rat hyperglycémique pendant 48h (qui présente aussi une hyperlactatémie), nous avons montré que la détection du glucose et du lactate est altérée. Ces modifications ne sont pas dues à une variation de l’expression protéique des MCTs astrocytaires ou neuronale de l’hypothalamus.Dans un deuxième temps, nous nous sommes intéressés au rôle des Cxs astrocytaires. La Cx43 est très exprimée autour des micro-vaisseaux sanguins de l’hypothalamus médio-basal (MBH), un site présentant de nombreux neurones gluco-sensibles. L’expression de la Cx30 est plus diffuse dans cette structure. Nous montrons également que l’expression protéique des Cxs astrocytaires varie très rapidement suite à des modifications du statut métabolique (jeûne, réalimentation, hyperglycémie). Afin d’évaluer l’implication de la Cx43 astrocytaire (majoritaire) dans la détection hypothalamique du glucose, nous avons inhibé son expression dans le MBH, in vivo, en injectant des siRNA permettant d’inhiber la synthèse de cette protéine. L’inhibition de la Cx43 (30% à 72h) induit une diminution de la prise alimentaire sans modification du poids, de la glycémie et de l’insulinémie comparée aux témoins. Suite à l’injection carotidienne de glucose (censée mimer une hyperglycémie), la sécrétion d’insuline est fortement inhibée chez les animaux siCx43. De même, l’effet satiétogène du glucose semble inhibé chez ces animaux lors de la réalimentation après un jeûne.Ces résultats montrent pour la première fois, de façon intégrée, l’importance des connexines, et probablement des réseaux astrocytaires, lors de la détection hypothalamique du glucose. Ces nouvelles données renforcent l’importance du rôle métabolique des astrocytes lors de fonctions neuronales précises / The hypothalamus plays a pivotal role in the nervous control of glucose homeostasis. This area contains gluco-sensitive neurons. Some of them detect increases in glucose levels and regulate glucose homeostasis by stimulating insulin secretion or inhibiting food intake. It is widely accepted that astrocytes are metabolically coupled to neurons. Lactate, resulting from the metabolism of glucose by astrocytes, is transported via the monocarboxylate transporters (MCTs). In addition, gap junctions (GJ), that form networks within astrocytes, are essential to transfer glucose from the bloodstream to the active neurons. These astroglial GJ mainly consist of connexins 43 and 30 (Cxs).The aims of my thesis are twofold: first, to show that an intracarotid lactate injection toward the brain, as for glucose, triggers insulin secretion and, second, to investigate the role of astroglial Cxs.Our results demonstrate that lactate and glucose sensing are altered in 48h hyperglycemic rats (accompanied by high blood lactate level). These alterations are not due to changes in protein expression of astroglial or neuronal MCTs in the hypothalamus. We then show that Cx43 is highly expressed in astrocytic end-feet enwraping blood vessels, in medio-basal hypothalamus (MBH) where many gluco-sensitive neurons are present. The Cx30 expression is more diffuse in this structure. We also show that the protein expression of astroglial Cxs varies very rapidly due to changes in metabolic status (fasting, refeeding and hyperglycemia). To evaluate the involvement of astroglial Cx43 (the major isoform) in the hypothalamic glucose sensing, we silenced its expression in the MBH in vivo by injecting specific siRNA. A 30% diminution in protein levels (after 72h) induced a decrease in food intake without changes in weight, blood glucose and insulin levels compared to vehicle treated animals. The central response to glucose is drastically inhibited in terms of insulin secretion in siCx43 animals. Similarly, an intracarotid injection of glucose towards the brain does not reduce refeeding in siRNA treated animals.These results demonstrate for the first time in vivo, the importance of connexins and astroglial networks in hypothalamic glucose sensing mechanism. These new data reinforce the importance of the metabolic role of astrocytes in specific neuronal functions

La fibrillation auriculaire (FA) est l’arythmie cardiaque soutenue la plus fréquente et elle peut mener à des conséquences médicales sévères, comme les accidents vasculaires cérébraux (AVC). Des remodelages électrophysiologiques, c’est-à-dire dans les courants ioniques, ainsi que des remodelages structurels, comme l’hypertrophie et la fibrose, ont été associés avec la fibrillation auriculaire. Ces remodelages peuvent affecter la conduction auriculaire, augmentant les risques de fibrillation auriculaire. Plusieurs facteurs de risque ont été associés avec la fibrillation auriculaire, parmi ceux-ci, on compte les niveaux élevés d’angiotensine II. L’angiotensine II est l’effecteur principal du système rénine-angiotensine (SRA) et ses effets néfastes sont médiés par le récepteur de type I à l’angiotensine II (AT1R). La suractivation du SRA, par l’action d’AT1R peut entraîner, entre autres, l’hypertension, l’insuffisance cardiaque, ainsi que des arythmies, comme la fibrillation auriculaire. Cependant, les mécanismes par lesquelles l’angiotensine II affectent directement le cœur et prédispose à la fibrillation auriculaire sont encore peu connus. L’objectif de ce projet de recherche consiste à étudier les remodelages électriques et structurels chez des souris transgéniques surexprimant le récepteur AT1 de manière cardiomyocyte-spécifique. Ces souris, nommées souris AT1R, permettent de voir l’effet de la suractivation du SRA uniquement au niveau cardiaque, sans modification hémodynamique. Les souris AT1R ont été utilisés à deux âges afin de distinguer les effets directs d’AT1R sur l’électrophysiologie auriculaire (50 jours), de ceux induit par la présence de remodelage structurel (6 mois). Ceci a été validé par la quantification de la fibrose auriculaire par marquage histologique de type rouge Sirius. L’utilisation des deux groupes est importante puisque la fibrose est connue pour affecter l’électrophysiologie cardiaque et altérer la conduction auriculaire. L’hypothèse de ce projet de recherche est que la surexpression d’AT1R induit un remodelage électrique auriculaire qui altère la conduction et mène au développement de fibrillation auriculaire. La présence de remodelage structurel amplifierait ces effets néfastes, favorisant davantage la survenue de fibrillation auriculaire. Les données obtenues montrent une diminution du courant sodique d’environ 60% comparativement aux souris contrôles (CTL) dès l’âge de 50 jours. Cette diminution est associée avec une augmentation de l’expression protéique sarcolemmale de la PKCα, une protéine kinase capable de phosphoryler le canal Nav1.5 menant à la réduction du courant sodique. L’expression génique du canal Nav1.5 (encodé par le gène Scn5a) n’était pas modifiée à 50 jours, suggérant une absence de régulation transcriptionnel pour ce canal. De plus, une diminution de l’expression génique des connexines 40 et 43 a été observée chez les souris AT1R dès 50 jours. Une prolongation de la durée de l’onde P, qui correspond au temps nécessaire à la dépolarisation des oreillettes, est observée à 50 jours sur les électrocardiogrammes des souris AT1R par rapport aux CTL. À 6 mois, la présence de remodelage structurel n’aggrave pas les remodelages au niveau du courant sodique et des connexines, mais prolonge encore davantage la durée de l’onde P. La susceptibilité à la fibrillation auriculaire est légèrement augmentée à 50 jours et nettement plus à 6 mois. En conclusion, la surexpression d’AT1R induit directement des remodelages électriques qui affectent la conduction dans l’oreillette, et ce indépendamment de la présence de remodelage structurel. Le remodelage structurel affecte la conduction auriculaire, mais n’amplifie pas les altérations du courant sodique et des connexines observées dans ce projet. Ainsi, ce projet apporte des connaissances sur les mécanismes par lesquels l’angiotensine II peut altérer la conduction auriculaire et mener à la fibrillation auriculaire, indépendamment des effets hémodynamiques. / Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and is associated with an increased risk of strokes and other morbidities. Atrial fibrillation has been associated with ionic currents remodeling, as well as structural remodeling, such as fibrosis and hypertrophy. These atrial remodeling can slow down atrial conduction velocity increasing the risk of atrial fibrillation. Several risk factors have been associated with atrial fibrillation and elevated levels of angiotensin II are one of them. Angiotensin II is the primary effector of the renin-angiotensin system (RAS). Angiotensin II type 1 receptors (AT1R) are responsible for the several pathologies induced by RAS overactivity, such as hypertension, heart failure and atrial fibrillation. However, the direct effects of angiotensin II on the heart and its role in atrial fibrillation pathophysiology remain largely unexplored. The objective of this research project is to study electrical and structural remodeling in transgenic mice overexpressing AT1R specifically in cardiomyocytes. These mice, identified as AT1R mice, do not have hemodynamic changes and are therefore suitable for studying the direct effect of RAS overactivation on the heart. Two age groups were used to distinguish the direct effects of AT1R on atrial electrophysiology (50-day-old mice) from those induced by structural remodeling (6-month-old mice). The presence of atrial fibrosis solely in 6 months old AT1R mice was confirmed using Picrosirius Red histological method. Characterizing electrical remodeling with and without structural remodeling is important since fibrosis is known to modulate cardiac electrophysiology and impair atrial conduction. The hypothesis of this project is that AT1R overexpression induces atrial electrical remodeling which alters conduction, leading to atrial fibrillation. Structural remodeling is expected to worsen conduction defects, further increasing the risk of atrial fibrillation. In this project, we measured an approximately 60% decrease in sodium current in 50 days old AT1R mice compared to controls (CTL). This change in sodium current was associated with an increase of PKCα protein expression in the sarcolemmal fraction. PKCα is a protein kinase able to phosphorylate Nav1.5 channels, resulting in a decrease in its function. Scn5a gene expression, encoding for Nav1.5 channels, was not changed at 50 days, suggesting an absence of transcriptional regulation of the sodium current. Moreover, a decrease in connexins 40 and 43 gene expression was observed in AT1R mice from the age of 50 days. Accordingly, a prolongation in P wave duration, which corresponds to atrial depolarization, is observed at 50 days in AT1R mice compared to CTL. At 6 months, structural remodeling did not worsen the reduction in sodium current or connexins induced by AT1R overexpression but was associated with a further prolongation of P wave duration. Susceptibility to AF was slightly increased in 50 days old AT1R mice and even more at 6 months. In conclusion, AT1R overexpression directly induces electrical remodeling, which slows down atrial conduction, independently of structural remodeling. Structural remodeling further alters atrial conduction without changes in sodium current or connexins expression. This project provides knowledge on mechanisms by which angiotensin II alters atrial conduction and leads to atrial fibrillation, even in absence of angiotensin II-induced hemodynamic changes.

La fibrillation auriculaire (FA) est l’arythmie cardiaque la plus fréquente et elle peut entraîner des complications médicales sévères, notamment des accidents vasculaires cérébraux. On observe des différences sexuelles importantes dans la présentation clinique de la FA. Son incidence est 1,5 à 2 fois plus élevée chez les hommes, tandis que les femmes tendent à développer de la FA plus tardivement et plus sévèrement. Malheureusement, on ignore toujours les causes de ces différences sexuelles. La FA est une pathologie multifactorielle généralement causée par un débalancement des propriétés électrophysiologiques et/ou structurelles des oreillettes favorisant l’initiation et/ou le maintien de cette arythmie. Le but de ce projet est de déterminer s’il existe des différences sexuelles parmi les mécanismes impliqués dans la pathogenèse de la FA chez la souris. Sachant que les hormones sexuelles peuvent avoir un impact considérable sur l’électrophysiologie cardiaque, un objectif complémentaire de ce projet est de déterminer la contribution des hormones sexuelles dans les différences observées. Au cours de ce projet, nous avons découvert que la prédisposition masculine à la FA est également retrouvée chez la souris mâle. Nous avons identifié des différences sexuelles dans la régulation du calcium intracellulaire favorisant l’initiation de la FA chez les mâles. Celles-ci sont reliées à une expression et une activité plus élevée de l’échangeur Na+-Ca2+ chez les mâles. Nous avons également observé que le maintien de la FA était favorisé par des oreillettes de plus grande taille et par une latéralisation plus prononcée des connexines chez les mâles. L’orchiectomie réduit la susceptibilité des mâles à la FA en diminuant la latéralisation des connexines ainsi que la taille des cardiomyocytes auriculaires, suggérant un rôle des androgènes. À terme, ce projet permettra de mieux comprendre les mécanismes impliqués dans les différences sexuelles dans la pathogenèse de la FA. Une meilleure compréhension de ces mécanismes pourrait mener à une approche thérapeutique mieux adaptée au sexe des patients, pour une meilleure prise en charge de ceux-ci. / Atrial fibrillation (AF) is the most common type of cardiac arrhythmia, and it can lead to severe medical complications, including stroke. There are significant sex differences in the clinical presentation of AF. Its incidence is 1.5- to 2-fold higher in men, whereas women tend to develop AF later and more severely. Unfortunately, the mechanisms underlying these sex differences remain unknown. AF is a multifactorial pathology generally caused by an imbalance in electrophysiological and/or structural properties of the atria that promote AF initiation and/or maintenance. The aim of this project is to determine whether there are sex differences among the mechanisms involved in the pathogenesis of AF in mice.. Knowing that sex hormones can have a considerable impact on cardiac electrophysiology, a complementary objective of this project was to explore the contribution of sex hormones in the sex differences we observed. We first discovered that the male predisposition to AF is also found in mice. We have identified major sex differences in the regulation of intracellular calcium that promote AF initiation in males. These differences are related to a higher Na+-Ca2+ exchanger expression and function in males. We have also observed that AF maintenance was favoured by larger atria and more pronounced lateralization of connexins in males. Orchiectomy reduced AF susceptibility of males by reducing connexin lateralization and the dimensions of atrial myocytes, suggesting a role for androgens. Ultimately, this project will provide a better understanding of the mechanisms underlying sex differences in the pathogenesis of AF. This information could lead to a therapeutic approach better adapted to the sex of the patients, for a better management of AF.