O escorpião amarelo Tityus serrulatus (Ts) é considerado a espécie mais perigosa do Brasil, e muitas das toxinas de sua peçonha já foram isoladas e caracterizadas. No entanto, as frações XIIA e XIIB, obtidas da cromatografia de troca iônica da peçonha de Ts, possuem várias toxinas de baixa massa molecular ainda não caracterizadas. Através da combinação de técnicas de RP-FPLC em colunas C8 e C18, espectrometria de massas e/ou sequenciamento amino-terminal, foi possível isolar e identificar os componentes destas frações, bem como realizar as caracterizações estrutural, por RMN, e eletrofisiológica, por Two Microelectrode Voltage Clamp, de algumas neurotoxinas isoladas. Foram escolhidas três toxinas de interesse: Ts11, Ts9 e Ts1-G. Nossos resultados mostram que a Ts11 foi capaz de bloquear canais para potássio dependentes de voltagem (Kv): Kv1.2, Kv1.3, Kv4.2, Kv10.1, hERG, e Shaker IR, bloqueando em 25%, 27%, 25%, 15%, 12%, e 10% as correntes de potássio, respectivamente. A Ts11 possui uma estrutura única (estrutura obtida por RMN): ICK scaffold sem os elementos de estrutura secundária (alfa-hélice ou fita-beta). Esta estrutura diferenciada, somada à atividade biológica caracterizada neste estudo, evidencia uma nova subfamília de KTxs, a qual foi denominada como ?-KTx, sendo a Ts11 o primeiro membro desta subfamília: ?-KTx1.1. A caracterização funcional da Ts9 mostra que a mesma não apresenta atividade bloqueadora sobre os canais Kv testados (Kv1.1; Kv1.2; Kv1.3; Kv1.4; Kv1.5; Kv1.6; Kv2.1; Kv3.1; Kv4.2; Kv7.2; Kv10.1 hERG e Shaker IR), na concentração de 100 nM. Apesar da Ts9 não ter bloqueado os canais testados, ela apresenta estrutura e resíduoschave que sugerem sua ação em Kvs e estudo anteriormente publicado mostra que ela é um potente ligante de canais para potássio ativados por cálcio de baixa condutância (SK). Também foi conduzido um estudo comparativo entre a Ts1e sua isoforma precursora Ts1-G nos canais para sódio dependentes de voltagem Nav1.1 - 1.8 e DmNav1, a fim de analisar a importância da amidação C-terminal. A Ts1 madura possui região C-terminal amidada, enquanto que sua isoforma Ts1-G não é amidada, pois apresenta uma Gly na região Cterminal (última etapa de transformação pós-traducional, anterior a ação da enzima ?- amidante). A Ts1-G não apresentou ação nos canais (Nav) na concentração testada (100 nM), enquanto que a Ts1 (100 nM) age como ?- toxina, reduzindo o limiar de excitação dos canais Nav e/ou reduzindo as correntes de sódio, evidenciando que a amidação C-terminal é importante para a atividade biológica da toxina madura. Adicionalmente, nas análises por MALDI/TOF das frações XIIA e XIIB, foram encontrados 45 componentes cujas massas moleculares não correspondem a de toxinas já isoladas, abrindo perspectivas para a identificação de moléculas com potencial uso biotecnológico, visto que toxinas com ação em canais iônicos podem ser ferramentas valiosas para a elucidação das características farmacológicas, fisiológicas e estruturais dos seus alvos. / The yellow scorpion Tityus serrulatus (Ts) is considered the most dangerous species of Brazil, and several toxins present in its venom have been already isolated and characterized. However, fractions XIIA and XIIB obtained from ion exchange chromatography of Ts crude venom, presented many low molecular weight toxins which have not been characterized yet. Through a combination of RP-FPLC technique using C8 and C18 columns, mass spectrometry and / or amino terminal sequencing, it was possible to isolate and identify the components of these fractions, as well as perform structural characterization thru NMR and electrophysiological characterization using Two Microelectrode Voltage Clamp, of some of the neurotoxins isolated. It was choosen three toxins of interest: Ts11, Ts9 and Ts1-G. Our results show that Ts11 was able to block voltage gated potassium channels (Kv): Kv1.2, Kv1.3, Kv4.2, Kv10.1, hERG, and Shaker IR, blocking 25%, 27%, 25% 15%, 12% and 10% of the potassium currents, respectively. Ts11 has an unique structure (structure obtained thru NRM technique): ICK scaffold without the elements of secondary structure (alpha helix or beta-sheet). Additionaly, the differentiated structure and functional characterization, Ts11 shows us an evidence of a new subfamily of toxins, which was named as ?-KTX, and therefore, Ts11 is the first member of this subfamily: ?-KTx1.1. The functional characterization of Ts9 shows that it has no blocking activity on the tested Kv channels (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv1.6, Kv2.1, Kv3.1 , Kv4.2, Kv7.2, Kv10.1, hERG and Shaker IR) at a concentration of 100 nM. Despite the Ts9 have not blocked the tested channels, it presents structure and key-amino acid residues that suggest its action on Kvs and previously published study shows that it is a potent ligant for slow conductance calciumactivated potassium channels (SK). It was also conducted a comparative electrophysiological study between the Ts1and its precursor isoform Ts1-G on voltage gated sodium channels, in order to evaluate the importance of the C-terminal amidation. The mature Ts1 has amidated C-terminal region, whereas Ts1-G isoform is not amidated, and therefore has a Gly at the Cterminal region (last step of post-translational modification, before the action of the enzyme ?-amidante). The Ts1-G showed no action on Nav channels at the concentration tested (100 nM), whereas Ts1 (100 nM) acts as ?-toxin, lowering the excitation threshold of Nav channels and/or reducing sodium currents, evidencing that the C-terminal amidation is important for the biological activity of the mature toxin. Additionally, the analyses by MALDI/TOF of the fractions XIIA and XIIB showed several molecules whose molar masses do not match the toxins already identified, opening prospects for the identification of new molecules with potential biotechnological use, considering that toxins that act on ion channels can be valuable tools for the elucidation of pharmacological, physiological and structural characteristics of their targets.

A ocorrência de status epileticus (SE) desencadeia algumas alterações no sistema nervoso central. O giro denteado (GD) do hipocampo sofre com modificações na expressão gênica dos canais iônicos das células granulares (CGs) e essas células sofrem alterações morfológicas. Essas alterações se manifestam com o brotamento de fibras musgosas, redução no número de espinhas dendríticas, encurtamento e estreitamento da arborização dendrítica. As modificações na expressão gênica dos canais iônicos afetam suas densidades máximas de condutância. Este estudo utilizou 40 modelos computacionais realistas para simular alterações nas condutâncias de canais iônicos e seus efeitos sobre dois grupos de CGs do GD. Os modelos foram construídos com base em reconstruções tridimensionais de 20 CGS com morfologia alterada após SE induzido por pilocarpina (CG-PILO) e 20 de morfologia normal (CG-controle). Foram dotados dos canais iônicos de sódio rápido (Na), canal de potássio de retificação tardia rápido (fKdr), canal de potássio de retificação tardia lento (fKdr), canal de potássio de tipo A (KA), canal de potássio dependente de cálcio e de voltagem de alta condutância (BK), canal de potássio dependente de cálcio de baixa condutância (SK) e canais de cálcio dos tipos T, N e L. As simulações foram realizadas no software Neuron. Foram realizados test t para detectar se ocorre diferenças significativas entre os grupos CG-controle e CG-PILO As alterações nas densidades máximas de condutância provocaram mudanças nos parâmetros de excitabilidade dos grupos CG-PILO e CG- controle, alterando valores de frequência de disparos, reobase e cronaxia. Os grupos apresentam respostas significativamente diferentes para as médias de reobase para a maioria dos valores de densidade máxima de condutância,, porém para cronaxia a maioria dos grupo não apresentou diferenças significativas. O grupo CG-controle apresentou médias maiores de frequência de disparos que o CG-PILO e o grupo CG-PILO apresentou valores de reobase maior para as alterações de densidade de condutância da maioria dos canais, sendo essas diferenças significativas. / The occurrence of status epilepticus (SE) triggers some changes in the central nervous system. The dentate gyrus (DG) of the hippocampus suffers from changes in gene expression of ion channels of granule cells (GCs) and these cells undergo morphological changes. These changes manifest themselves with mossy fiber sprouting, reduction in the number of dendritic spines, shortening and narrowing of dendritic branching. Changes in gene expression of ion channels affect their maximum densities of conductance. This study used 40 realistic computer models to simulate changes in conductance of ion channels and its effect on two groups of CGs of the GD. The models were built based on three-dimensional reconstructions of 20 CGS with morphology changed after pilocarpine-induced SE (CG-PILO) and 20 normal morphology (CG-control). The models were equipped with the ion channels of fast sodium (Na), fast delayed rectifying potassium channel (fKDR), slow delayed rectifying potassium channel (fKdr), potassium channel type A (KA), potassium channel dependent calcium and high voltage conductance (BK), potassium channel dependent calcium low conductance (SK) and the calcium channel types T, N and L. The simulations were performed at Neuron software.T tests were performed to p-values <0.05 for detecting significant differences between the GC-control group and GC-PILO. Changes in maximum densities conductance caused changes in excitability parameters CG-PILO and GC- control groups, by changing frequency values of spikes, rheobase and chronaxie. The groups have significantly different responses to the averages for the most rheobase maximum density values of conductance, but these differences were shortly found for chronaxie values. The CG-control group had higher average frequency of spikes than the CG-PILO group. The CG-PILO group had rheobase values higher for conductance density changes the most channels. These differences are significant.

O escorpião amarelo Tityus serrulatus (Ts) é considerado a espécie mais perigosa do Brasil, e muitas das toxinas de sua peçonha já foram isoladas e caracterizadas. No entanto, as frações XIIA e XIIB, obtidas da cromatografia de troca iônica da peçonha de Ts, possuem várias toxinas de baixa massa molecular ainda não caracterizadas. Através da combinação de técnicas de RP-FPLC em colunas C8 e C18, espectrometria de massas e/ou sequenciamento amino-terminal, foi possível isolar e identificar os componentes destas frações, bem como realizar as caracterizações estrutural, por RMN, e eletrofisiológica, por Two Microelectrode Voltage Clamp, de algumas neurotoxinas isoladas. Foram escolhidas três toxinas de interesse: Ts11, Ts9 e Ts1-G. Nossos resultados mostram que a Ts11 foi capaz de bloquear canais para potássio dependentes de voltagem (Kv): Kv1.2, Kv1.3, Kv4.2, Kv10.1, hERG, e Shaker IR, bloqueando em 25%, 27%, 25%, 15%, 12%, e 10% as correntes de potássio, respectivamente. A Ts11 possui uma estrutura única (estrutura obtida por RMN): ICK scaffold sem os elementos de estrutura secundária (alfa-hélice ou fita-beta). Esta estrutura diferenciada, somada à atividade biológica caracterizada neste estudo, evidencia uma nova subfamília de KTxs, a qual foi denominada como ?-KTx, sendo a Ts11 o primeiro membro desta subfamília: ?-KTx1.1. A caracterização funcional da Ts9 mostra que a mesma não apresenta atividade bloqueadora sobre os canais Kv testados (Kv1.1; Kv1.2; Kv1.3; Kv1.4; Kv1.5; Kv1.6; Kv2.1; Kv3.1; Kv4.2; Kv7.2; Kv10.1 hERG e Shaker IR), na concentração de 100 nM. Apesar da Ts9 não ter bloqueado os canais testados, ela apresenta estrutura e resíduoschave que sugerem sua ação em Kvs e estudo anteriormente publicado mostra que ela é um potente ligante de canais para potássio ativados por cálcio de baixa condutância (SK). Também foi conduzido um estudo comparativo entre a Ts1e sua isoforma precursora Ts1-G nos canais para sódio dependentes de voltagem Nav1.1 - 1.8 e DmNav1, a fim de analisar a importância da amidação C-terminal. A Ts1 madura possui região C-terminal amidada, enquanto que sua isoforma Ts1-G não é amidada, pois apresenta uma Gly na região Cterminal (última etapa de transformação pós-traducional, anterior a ação da enzima ?- amidante). A Ts1-G não apresentou ação nos canais (Nav) na concentração testada (100 nM), enquanto que a Ts1 (100 nM) age como ?- toxina, reduzindo o limiar de excitação dos canais Nav e/ou reduzindo as correntes de sódio, evidenciando que a amidação C-terminal é importante para a atividade biológica da toxina madura. Adicionalmente, nas análises por MALDI/TOF das frações XIIA e XIIB, foram encontrados 45 componentes cujas massas moleculares não correspondem a de toxinas já isoladas, abrindo perspectivas para a identificação de moléculas com potencial uso biotecnológico, visto que toxinas com ação em canais iônicos podem ser ferramentas valiosas para a elucidação das características farmacológicas, fisiológicas e estruturais dos seus alvos. / The yellow scorpion Tityus serrulatus (Ts) is considered the most dangerous species of Brazil, and several toxins present in its venom have been already isolated and characterized. However, fractions XIIA and XIIB obtained from ion exchange chromatography of Ts crude venom, presented many low molecular weight toxins which have not been characterized yet. Through a combination of RP-FPLC technique using C8 and C18 columns, mass spectrometry and / or amino terminal sequencing, it was possible to isolate and identify the components of these fractions, as well as perform structural characterization thru NMR and electrophysiological characterization using Two Microelectrode Voltage Clamp, of some of the neurotoxins isolated. It was choosen three toxins of interest: Ts11, Ts9 and Ts1-G. Our results show that Ts11 was able to block voltage gated potassium channels (Kv): Kv1.2, Kv1.3, Kv4.2, Kv10.1, hERG, and Shaker IR, blocking 25%, 27%, 25% 15%, 12% and 10% of the potassium currents, respectively. Ts11 has an unique structure (structure obtained thru NRM technique): ICK scaffold without the elements of secondary structure (alpha helix or beta-sheet). Additionaly, the differentiated structure and functional characterization, Ts11 shows us an evidence of a new subfamily of toxins, which was named as ?-KTX, and therefore, Ts11 is the first member of this subfamily: ?-KTx1.1. The functional characterization of Ts9 shows that it has no blocking activity on the tested Kv channels (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv1.6, Kv2.1, Kv3.1 , Kv4.2, Kv7.2, Kv10.1, hERG and Shaker IR) at a concentration of 100 nM. Despite the Ts9 have not blocked the tested channels, it presents structure and key-amino acid residues that suggest its action on Kvs and previously published study shows that it is a potent ligant for slow conductance calciumactivated potassium channels (SK). It was also conducted a comparative electrophysiological study between the Ts1and its precursor isoform Ts1-G on voltage gated sodium channels, in order to evaluate the importance of the C-terminal amidation. The mature Ts1 has amidated C-terminal region, whereas Ts1-G isoform is not amidated, and therefore has a Gly at the Cterminal region (last step of post-translational modification, before the action of the enzyme ?-amidante). The Ts1-G showed no action on Nav channels at the concentration tested (100 nM), whereas Ts1 (100 nM) acts as ?-toxin, lowering the excitation threshold of Nav channels and/or reducing sodium currents, evidencing that the C-terminal amidation is important for the biological activity of the mature toxin. Additionally, the analyses by MALDI/TOF of the fractions XIIA and XIIB showed several molecules whose molar masses do not match the toxins already identified, opening prospects for the identification of new molecules with potential biotechnological use, considering that toxins that act on ion channels can be valuable tools for the elucidation of pharmacological, physiological and structural characteristics of their targets.

A ocorrência de status epileticus (SE) desencadeia algumas alterações no sistema nervoso central. O giro denteado (GD) do hipocampo sofre com modificações na expressão gênica dos canais iônicos das células granulares (CGs) e essas células sofrem alterações morfológicas. Essas alterações se manifestam com o brotamento de fibras musgosas, redução no número de espinhas dendríticas, encurtamento e estreitamento da arborização dendrítica. As modificações na expressão gênica dos canais iônicos afetam suas densidades máximas de condutância. Este estudo utilizou 40 modelos computacionais realistas para simular alterações nas condutâncias de canais iônicos e seus efeitos sobre dois grupos de CGs do GD. Os modelos foram construídos com base em reconstruções tridimensionais de 20 CGS com morfologia alterada após SE induzido por pilocarpina (CG-PILO) e 20 de morfologia normal (CG-controle). Foram dotados dos canais iônicos de sódio rápido (Na), canal de potássio de retificação tardia rápido (fKdr), canal de potássio de retificação tardia lento (fKdr), canal de potássio de tipo A (KA), canal de potássio dependente de cálcio e de voltagem de alta condutância (BK), canal de potássio dependente de cálcio de baixa condutância (SK) e canais de cálcio dos tipos T, N e L. As simulações foram realizadas no software Neuron. Foram realizados test t para detectar se ocorre diferenças significativas entre os grupos CG-controle e CG-PILO As alterações nas densidades máximas de condutância provocaram mudanças nos parâmetros de excitabilidade dos grupos CG-PILO e CG- controle, alterando valores de frequência de disparos, reobase e cronaxia. Os grupos apresentam respostas significativamente diferentes para as médias de reobase para a maioria dos valores de densidade máxima de condutância,, porém para cronaxia a maioria dos grupo não apresentou diferenças significativas. O grupo CG-controle apresentou médias maiores de frequência de disparos que o CG-PILO e o grupo CG-PILO apresentou valores de reobase maior para as alterações de densidade de condutância da maioria dos canais, sendo essas diferenças significativas. / The occurrence of status epilepticus (SE) triggers some changes in the central nervous system. The dentate gyrus (DG) of the hippocampus suffers from changes in gene expression of ion channels of granule cells (GCs) and these cells undergo morphological changes. These changes manifest themselves with mossy fiber sprouting, reduction in the number of dendritic spines, shortening and narrowing of dendritic branching. Changes in gene expression of ion channels affect their maximum densities of conductance. This study used 40 realistic computer models to simulate changes in conductance of ion channels and its effect on two groups of CGs of the GD. The models were built based on three-dimensional reconstructions of 20 CGS with morphology changed after pilocarpine-induced SE (CG-PILO) and 20 normal morphology (CG-control). The models were equipped with the ion channels of fast sodium (Na), fast delayed rectifying potassium channel (fKDR), slow delayed rectifying potassium channel (fKdr), potassium channel type A (KA), potassium channel dependent calcium and high voltage conductance (BK), potassium channel dependent calcium low conductance (SK) and the calcium channel types T, N and L. The simulations were performed at Neuron software.T tests were performed to p-values <0.05 for detecting significant differences between the GC-control group and GC-PILO. Changes in maximum densities conductance caused changes in excitability parameters CG-PILO and GC- control groups, by changing frequency values of spikes, rheobase and chronaxie. The groups have significantly different responses to the averages for the most rheobase maximum density values of conductance, but these differences were shortly found for chronaxie values. The CG-control group had higher average frequency of spikes than the CG-PILO group. The CG-PILO group had rheobase values higher for conductance density changes the most channels. These differences are significant.

Thèse en cotutelle avec Université de Nantes - Pays de La Loire - France (2005-2010) / Les maladies cardio-vasculaires sont la première cause de mortalité dans le monde. L’hypertrophie cardiaque est un processus de remodelage provoqué par une surcharge de travail du muscle cardiaque afin de mieux répondre à la demande de l’organisme. Bien que bénéfique à court terme, une hypertrophie trop accentuée conduira à long terme, à une insuffisance cardiaque. L’hypertrophie est associée à un remodelage électrique qui conduit généralement à un allongement du potentiel d’action, une des causes des arythmies ventriculaires et de la mort subite. Généralement, le mécanisme causal est la fibrillation ventriculaire, un trouble du rythme irréversible dont les mécanismes sont complexes et méconnus. Si les conséquences fonctionnelles in vitro des mutations génétiques ou du remodelage ionique sont relativement simples à étudier ou à prévoir, leur rôle dans les mécanismes des troubles du rythme in vivo sont plus difficiles à appréhender. Parmi les nombreux modèles animaux développés pour la recherche sur les troubles du rythme, la souris est de plus en plus utilisée en raison de notre capacité à muter, invalider ou sur-exprimer les gènes d'intérêt chez ces animaux. L'objectif de mon travail de thèse était de mieux comprendre le rôle des canaux ioniques en physiopathologie cardiaque, en particulier dans la survenue des troubles du rythme in vivo. Ces travaux ont permis d'améliorer notre connaissance du rôle des anomalies génétiques impliquant des canaux ioniques et du remodelage ionique dans la physiopathologie des troubles du rythme et pourrait ainsi ouvrir de nouvelles perspectives thérapeutiques dans le traitement anti-remodelage cardiaque et la prévention de la mort subite. / Cardiovascular disease is the leading cause of death in the world each year. If no action is taken to improve cardiovascular health and current trends continue, WHO estimates that 25% more healthy life years will be lost to cardiovascular disease globally by 2020. Cardiac hypertrophy is the consequence of an excessive workload of the heart muscle leading to cardiac remodeling process. As the workload increases, the ventricular walls grow thicker, lose elasticity and eventually may fail to pump with as much force as a healthy heart. Furthermore, hypertrophied myocardium is not physiologically normal and may confer a predisposition to potentially fatal arrhythmias. Generally, the causal mechanism is ventricular fibrillation, a cardiac rhythm disorder which is irreversible but the pathophysiological mechanisms are complex and poorly understood. The functional consequences of mutations or ionic remodeling are relatively simple to study in vitro, but their role in the pathophysiology of arrhythmias in vivo is more difficult to grasp. Among the different animal models developed in cardiac arrhythmias research, the mouse is increasingly used because of our ability to mutate, knock-out or over-express genes of interest. The objective of my thesis was to study the role of ion channels in physiology as well as cardiac pathophysiology, particularly in the involvement of the occurrence of cardiac arrhythmias in vivo. This thesis will improve our understanding of the role of genetic abnormalities involving ionic remodeling in the pathogenesis of the heart and may also open new therapeutic perspectives in the treatment of cardiac remodeling as well as sudden cardiac death.

Les maladies cardio-vasculaires sont la première cause de mortalité dans le monde. L’hypertrophie cardiaque est un processus de remodelage provoqué par une surcharge de travail du muscle cardiaque afin de mieux répondre à la demande de l’organisme. Bien que bénéfique à court terme, une hypertrophie trop accentuée conduira à long terme, à une insuffisance cardiaque. L’hypertrophie est associée à un remodelage électrique qui conduit généralement à un allongement du potentiel d’action, une des causes des arythmies ventriculaires et de la mort subite. Généralement, le mécanisme causal est la fibrillation ventriculaire, un trouble du rythme irréversible dont les mécanismes sont complexes et méconnus. Si les conséquences fonctionnelles in vitro des mutations génétiques ou du remodelage ionique sont relativement simples à étudier ou à prévoir, leur rôle dans les mécanismes des troubles du rythme in vivo sont plus difficiles à appréhender. Parmi les nombreux modèles animaux développés pour la recherche sur les troubles du rythme, la souris est de plus en plus utilisée en raison de notre capacité à muter, invalider ou sur-exprimer les gènes d'intérêt chez ces animaux. L'objectif de mon travail de thèse était de mieux comprendre le rôle des canaux ioniques en physiopathologie cardiaque, en particulier dans la survenue des troubles du rythme in vivo. Ces travaux ont permis d'améliorer notre connaissance du rôle des anomalies génétiques impliquant des canaux ioniques et du remodelage ionique dans la physiopathologie des troubles du rythme et pourrait ainsi ouvrir de nouvelles perspectives thérapeutiques dans le traitement anti-remodelage cardiaque et la prévention de la mort subite. / Cardiovascular disease is the leading cause of death in the world each year. If no action is taken to improve cardiovascular health and current trends continue, WHO estimates that 25% more healthy life years will be lost to cardiovascular disease globally by 2020. Cardiac hypertrophy is the consequence of an excessive workload of the heart muscle leading to cardiac remodeling process. As the workload increases, the ventricular walls grow thicker, lose elasticity and eventually may fail to pump with as much force as a healthy heart. Furthermore, hypertrophied myocardium is not physiologically normal and may confer a predisposition to potentially fatal arrhythmias. Generally, the causal mechanism is ventricular fibrillation, a cardiac rhythm disorder which is irreversible but the pathophysiological mechanisms are complex and poorly understood. The functional consequences of mutations or ionic remodeling are relatively simple to study in vitro, but their role in the pathophysiology of arrhythmias in vivo is more difficult to grasp. Among the different animal models developed in cardiac arrhythmias research, the mouse is increasingly used because of our ability to mutate, knock-out or over-express genes of interest. The objective of my thesis was to study the role of ion channels in physiology as well as cardiac pathophysiology, particularly in the involvement of the occurrence of cardiac arrhythmias in vivo. This thesis will improve our understanding of the role of genetic abnormalities involving ionic remodeling in the pathogenesis of the heart and may also open new therapeutic perspectives in the treatment of cardiac remodeling as well as sudden cardiac death. / Thèse en cotutelle avec Université de Nantes - Pays de La Loire - France (2005-2010)

Introduction : La dysfonction primaire du greffon (DPG) post-transplantation pulmonaire est la principale cause de décès en phase péri-opératoire. Sa physiopathologie n’est pas encore totalement élucidée mais les lésions d’Ischémie/Reperfusion (I/R) pourraient constituer un facteur important de son développement. L’I/R et la DPG sont caractérisées par des dommages de l’endothélium vasculaire et de l’épithélium alvéolaire, un œdème pulmonaire et une réaction inflammatoire exacerbée. La résorption de l’œdème dépend du rétablissement de l’intégrité fonctionnelle alvéolaire, dont la capacité à réabsorber les ions Na+ (via les canaux ENaC), et secondairement le liquide par les cellules alvéolaires. Nous avons émis l’hypothèse que la dysfonction épithéliale alvéolaire, causée par l’I/R, présente dans les greffons donneurs (GD), jouerait un rôle clef dans le développement de la DPG chez les receveurs. Notre but était d’identifier de biomarqueurs, associés à la dysfonction épithéliale des GD et au développement de DPG chez les receveurs. Méthodes : L’impact d’un protocole mimant une I/R a d’abord été évalué sur des cultures primaires de cellules alvéolaires de rats. Puis, nous avons étudié l’impact de l’I/R in vivo grâce à des modèles de stress inflammatoire par infusion de LPS ou transplantation unilatérale chez le porc. Finalement, des biopsies de tissus de GD ont été recueillies durant les transplantations pulmonaires. Après détermination du grade de DPG chez les receveurs, nous avons étudié les facteurs et les altérations alvéolaires associés. Résultats : Une baisse d’expression des protéines de jonctions serrées (ZO-1), des canaux ioniques ENaC et CFTR ainsi qu’une réduction de la résistance transépithéliale et de la capacité de réparation suite aux lésions ont été observées suite au protocole mimant l’I/R dans le modèle de cultures primaires de cellules alvéolaires. Un traitement avec un activateur du canal K+ KvLQT1 (R L3) a permis d’améliorer la vitesse de réparation, l’intégrité de la barrière épithéliale et l’expression d’ENaC et CFTR. Dans nos modèles animaux, nous avons observé une réponse pro-inflammatoire et une altération des protéines ZO-1, ENaC et CFTR. Nos données préliminaires indiquent aussi une infiltration inflammatoire et une baisse d’ENaC, CFTR et ZO-1, déjà présentes dans les GD ayant subits une I prolongée, chez les receveurs ayant ensuite développés une DPG. Conclusion : Nos résultats soutiennent notre hypothèse du développement d’une dysfonction épithéliale alvéolaire, caractérisée par une altération de biomarqueurs de fonctionnalité et d’intégrité (ENaC, CFTR et ZO-1), en lien avec l’I/R et la DPG. / Background: Primary graft dysfunction (PGD) after lung transplantation is the first cause of death in the perioperative phase. The PGD pathophysiology is not fully elucidated, but Ischemia/Reperfusion (I/R) injury might be an important factor. I/R and PGD both feature endothelial/ epithelial damage, lung edema and inflammation. Edema resorption then depends on the restoration of the alveolar functional integrity, especially the ability of alveolar epithelial cells to reabsorb Na+ (through ENaC channels) and fluid. We hypothesized that alveolar epithelial dysfunction (related to I/R), observed within donor grafts, then plays a key role in the development of PGD in lung recipients. Our goal was to identify novel biomarkers, associated with epithelial dysfunction within donor’s grafts, and then PGD development in recipients. Methods: The impact of a protocol mimicking hypothermic ischemia and reperfusion was first tested on primary rat alveolar epithelial cell cultures. Then, the impact of I/R was studied in vivo using models of inflammatory stress induced by LPS infusion or after unilateral transplantation in pigs. Finally, lung biopsies from donor grafts were collected during lung transplantations. After defining PGD scores within the recipients, associated factors and alveolar alterations were finally analyzed. Results: In primary cell cultures, the protocol mimicking hypothermic I/R induced a decrease in tight junction proteins (ZO-1), transepithelial resistance, wound repair capacity as well as ENaC and CFTR channel expression. Treatment with a KvLQT1 K+ channel activator (R-L3) accelerated the repair rates and enhanced barrier integrity (ZO-1 staining) as well as ENaC and CFTR protein expressions. In the porcine models, an exacerbated inflammatory response was observed along with alveolar damage, lung edema and decreased ZO-1, ENaC and CFTR expressions. Our preliminary data using human samples collected during lung transplantations also indicate an inflammatory response and reduced ENaC, CFTR and ZO-1 expressions, already observed within lung grafts, submitted to longer cold ischemia duration, among lung recipients then developing a PGD. Conclusion: Altogether these data support our hypothesis of an alveolar epithelial dysfunction, featuring an alteration of functionality and barrier integrity biomarkers (ENaC, CFTR and ZO-1), associated with I/R and DPG.