Tamarindus indica L. é um componente natural amplamente consumido por humanos, apresentando propriedade antiinflamatória, antidiabética e antihepatotóxica. Além disso, como nós demonstramos previamente, o extrato apresenta atividade hipolipêmica e antioxidante. Neste trabalho, nós demonstramos o efeito do extrato de T. indica sobre mitocôndrias isoladas de fígado de rato. Na presença de Ca2+, o extrato causou um inchamento osmótico mitocondrial concentração-dependente, associado ao aumento da velocidade basal (V4), dissipação do potencial de membrana e liberação do Ca2+ pré-acumulado, os quais foram inibidos por ciclosporina A (CsA) e assim atribuídos a transição de permeabilidade da membrana mitocondrial (TPMM). A indução do inchamento osmótico mitocondrial foi prevenida por EGTA e vermelho de rutênio, indicando a dependência da TPMM de Ca2+. A oxidação de proteínas tiol mitocondrial, um mecanismo bem estabelecido como causador da TPMM, foi detectado. Entretanto, nenhuma alteração significante foi observada no estado redox de GSH. A oxidação de NAD(P)H e o acúmulo de espécies reativas de oxigênio produzidas pela mitocôndria foram observadas, em ambos os casos, foram prevenidas por CsA e/ou EGTA, indicando que são conseqüências da TPMM induzida pelo extrato de T. indica. Portanto, sem aparente envolvimento do estresse oxidativo no processo, sugerindo uma interação direta de compostos do extrato com grupos tiólicos de proteínas da membrana. O extrato de T. indica induziu uma depleção de ATP associada à TPMM, mostrando assim potencial para causar a morte celular por apoptose ou necrose, resultante da indução da TPMM por si ou pela depleção do ATP via TPMM. In vitro, o extrato apresentou capacidade scavenger de radicais livres, verificado pelo ensaio do DPPH, radical superóxido e radical hidroxil; e ainda foi capaz de diminuir a lipoperoxidação mitocondrial. Além disso, em baixas concentrações, o extrato mostrou uma propriedade quelante de Fe2+. / Tamarindus indica L. is a natural dietary component widely consumed by humans, presenting well established anti-inflammatory, anti-diabetic and anti-hepatotoxic properties. In addition, as we have previously demonstrated, extract presents hypolipemic and antioxidant activities. We show here the effects of T. indica extract on isolated rat liver mitochondria. In the presence of Ca2+, the extract caused mitochondrial concentration-dependent swelling, associated to, resting respiration increase (V4), membrane potential dissipation and release of pre-accumulated Ca2+, inhibited by cyclosporine A (CsA) and thus ascribable to mitochondrial permeability transition (MPT). This swelling induction was prevented by EGTA and ruthenium red, indicating strict dependence of MPT on Ca2+. Oxidation of mitochondrial membrane protein thiols, a well established mechanism causing MPT was detected. However, no significant change was observed in the GSH redox state, and the NADPH oxidation and accumulation of mitochondria-generated reactive oxygen species that was observed, were prevented by CsA and/or EGTA, indicating that they are consequence of the MPT induced by T. indica extract. Therefore, no apparent oxidative stress condition is involved as cause of this process suggesting that direct interaction with membrane protein thiol groups of the compounds responsible for MPT induction occurs. T. indica extract led to MPTassociated ATP depletion, thus showing the potential to cause cell death by apoptosis or necrosis resulting from MPT induction per se or from ATP depletion by MPT. In vitro, the extract presented free radical scavenging ability, as assessed by the 2,2-diphenyl-1- picrylhydrazyl (DPPH), superoxide radicals and radical hydroxyl assays, and led to decreased lipid peroxidation in mitochondria, as assessed by the thiobarbituric acid reactive substances (TBARS) assay. In addition, the extract showed an iron chelanting property in low concentrations.

L'ischémie-reperfusion (IR) rénale entraîne des lésions de nécrose tubulaire aigue, nécessitant parfois une épuration extra rénale transitoire voir définitive. La mitochondrie joue un rôle important dans la physiopathologie de ces lésions d'IR en entrainant la mort cellulaire. L'étude de l'IR dans la cellule cardiaque a permis de mettre en évidence le rôle central du pore de transition de perméabilité mitochondriale (mPTP) dans le déclenchement de cette mort cellulaire. La ciclosporine (CsA) a été proposée comme thérapeutique pour protéger la cellule des lésions d'IR en retardant l'ouverture de ce mPTP. Cependant la CsA a des effets rénaux vasoconstricteurs aigus, nécessitant une validation expérimentale de sa protection dans l'IR rénale. Au cours de ce travail de thèse, nous avons mis au point un modèle murin d'IR rénale. Ensuite nous avons montré que le post-conditionnement à la CsA, ainsi que le post-conditionnement ischémique, permettent d'améliorer la fonction rénale avec un retard à l'ouverture du mPTP. Dans un deuxième travail, nous montrons que le pré conditionnement à la CsA est dose et temps dépendant, et médié en partie par l'augmentation d'expression d'une protéine chaperonne, l'Heat Shock Protéine 70 (HSP70). L'injection en bolus de CsA permet également d'améliorer la fonction rénale dans ce modèle d'IR avec un retard à l'ouverture du mPTP. Nos résultats ouvrent de nouvelles perspectives dans la protection rénale, notamment dans la réduction des épisodes d'insuffisance rénale aigue après chirurgie aortique ou en transplantation rénale / Ischemia-reperfusion (IR) is a situation encountered in transplantation or during aortic surgery, which can result in renal damages, requiring sometimes transient or definitive dialysis. Mitochondria play a crucial role in the pathophysiology of IR causing cell death. Previous studies of cardiac IR highlighted the role of mitochondrial permeability transition pore (mPTP). Cyclosporin A (CsA) has been proposed as a treatment to protect the kidney from IR by the delay of the opening of the mPTP. However, CsA has acute renal hemodynamic effects and a long-term toxicity, requiring an experimental validation of its protection in the renal IR. In this work, we developed a mouse model of renal IR. In a first study, we showed that the post-conditioning with CsA and ischemic postconditioning improve renal function with a delay of the opening of the mPTP. In a second study, we showed that a high dose of CsA injected just before the ischemia improves renal function and leads to the delay of the opening of mPTP mediated by an increase of HSP70. Our results open new perspectives in renal protection, especially for reducing episodes of acute renal failure in aortic surgery or in renal transplantation

Hypochlorous acid (HOCl) is a powerful oxidant produced by activated phagocytes at sites of inflammation to kill a wide range of pathogens. Yet, it may also damage and kill the neighbouring host cells. The abundance of dead macrophages in atherosclerotic plaques and their colocalization with HOCl-modified proteins implicate HOCl may play a role in killing macrophages, contributing to disease progression. The first part of this research was to investigate the cytotoxic effect and cell death mechanism(s) of HOCl on macrophages. Macrophages require efficient defense mechanism(s) against HOCl to function properly at inflammatory sites. The second part of the thesis was to examine the antioxidative effects of glutathione (GSH) and 7,8-dihydroneopterin (7,8-NP) on HOCl-induced cellular damage in macrophages. GSH is an efficient scavenger of HOCl and a major intracellular antioxidant against oxidative stress, whereas 7,8-NP is secreted by human macrophages upon interferon-γ (IFN-γ) induction during inflammation and can also scavenge HOCl. HOCl caused concentration-dependent cell viability loss in human monocyte derived macrophage (HMDM) cells above a specific concentration threshold. HOCl reacted with HMDMs to cause viability loss within the first 10 minutes of treatment, and it posed no latent effect on the cells afterwards regardless of the HOCl concentrations. The lack of caspase-3 activation, rapid influx of propidium iodide (PI) dye, rapid loss of intracellular ATP and cell morphological changes (cell swelling, cell membrane integrity loss and rupture) were observed in HMDM cells treated with HOCl. These results indicate that HOCl caused HMDM cells to undergo necrotic cell death. In addition to the loss of intracellular ATP, HOCl also caused rapid loss of GAPDH enzymatic activity and mitochondrial membrane potential, indicating impairment of the metabolic energy production. Loss of the mitochondrial membrane potential was mediated by mitochondrial permeability transition (MPT), as blocking MPT pore formation using cyclosporin A (CSA) prevented mitochondrial membrane potential loss. HOCl caused an increase in cytosolic calcium ion (Ca2+) level, which was due to both intra- and extra-cellular sources. However, extracellular sources only contributed significantly above a certain HOCl concentration. Preventing cytosolic Ca2+ increase significantly inhibited HOCl-induced cell viability loss. This suggests that cytosolic Ca2+ increase was associated with HOCl-induced necrotic cell death in HMDM cells, possibly via the activation of Ca2+-dependent calpain cysteine proteases. Calpain inhibitors prevented HOCl-induced lysosomal destabilisation and cell viability loss in HMDM cells. Calpains induced HOCl-induced necrotic cell death possibly by degrading cytoskeletal and other cellular proteins, or causing the release of cathepsin proteases from ruptured lysosomes that also degraded cellular components. The HOCl-induced cytosolic Ca2+ increase also caused mitochondrial Ca2+ accumulation and MPT activation-mediated mitochondrial membrane potential loss. MPT activation, like calpain activation, was also associated with the HOCl-induced necrotic cell death, as preventing MPT activation completely inhibited HOCl-induced cell viability loss. The involvement of both calpain activation and MPT activation in HOCl-induced necrotic cell death in HMDM cells implies a cause and effect relationship between these two events. HMDM cells depleted of intracellular GSH using diethyl maleate showed increased susceptibility towards HOCl insult compared to HMDM cells with intact intracellular GSH levels, indicating that intracellular GSH played an important role in protecting HMDM cells against HOCl exposure. Intracellular GSH level in each HMDM cell preparation directly correlated with HOCl concentration required to kill 50% of population for each cell preparation, indicating intracellular GSH concentrations determine the efficiency of GSH in preventing HOCl-induced damage to HMDM cells. Intracellular GSH and cell viability loss induced by 400 μM HOCl were significantly prevented by 300 μM extracellular 7,8-NP, indicating that added 7,8-NP is an efficient scavenger of HOCl and out-competed intracellular GSH for HOCl. The amount of 7,8-NP synthesized by HMDM cells upon IFN-γ induction was too low to efficiently prevent HOCl-mediated intracellular GSH and cell viability loss. HOCl clearly causes HMDM cells to undergo necrosis when the concentration exceeds the intracellular GSH concentrations. Above this concentration HOCl causes oxidative damage to the Ca2+ ion channels on cell and ER membranes, resulting in an influx of Ca2+ ions into the cytosol and possibly the mitochondria. The rise in Ca2+ ions triggers calpain activation, resulting in the MPT-mediated loss of mitochondrial membrane potential, lysosomal instability and cellular necrosis.

Tamarindus indica L. é um componente natural amplamente consumido por humanos, apresentando propriedade antiinflamatória, antidiabética e antihepatotóxica. Além disso, como nós demonstramos previamente, o extrato apresenta atividade hipolipêmica e antioxidante. Neste trabalho, nós demonstramos o efeito do extrato de T. indica sobre mitocôndrias isoladas de fígado de rato. Na presença de Ca2+, o extrato causou um inchamento osmótico mitocondrial concentração-dependente, associado ao aumento da velocidade basal (V4), dissipação do potencial de membrana e liberação do Ca2+ pré-acumulado, os quais foram inibidos por ciclosporina A (CsA) e assim atribuídos a transição de permeabilidade da membrana mitocondrial (TPMM). A indução do inchamento osmótico mitocondrial foi prevenida por EGTA e vermelho de rutênio, indicando a dependência da TPMM de Ca2+. A oxidação de proteínas tiol mitocondrial, um mecanismo bem estabelecido como causador da TPMM, foi detectado. Entretanto, nenhuma alteração significante foi observada no estado redox de GSH. A oxidação de NAD(P)H e o acúmulo de espécies reativas de oxigênio produzidas pela mitocôndria foram observadas, em ambos os casos, foram prevenidas por CsA e/ou EGTA, indicando que são conseqüências da TPMM induzida pelo extrato de T. indica. Portanto, sem aparente envolvimento do estresse oxidativo no processo, sugerindo uma interação direta de compostos do extrato com grupos tiólicos de proteínas da membrana. O extrato de T. indica induziu uma depleção de ATP associada à TPMM, mostrando assim potencial para causar a morte celular por apoptose ou necrose, resultante da indução da TPMM por si ou pela depleção do ATP via TPMM. In vitro, o extrato apresentou capacidade scavenger de radicais livres, verificado pelo ensaio do DPPH, radical superóxido e radical hidroxil; e ainda foi capaz de diminuir a lipoperoxidação mitocondrial. Além disso, em baixas concentrações, o extrato mostrou uma propriedade quelante de Fe2+. / Tamarindus indica L. is a natural dietary component widely consumed by humans, presenting well established anti-inflammatory, anti-diabetic and anti-hepatotoxic properties. In addition, as we have previously demonstrated, extract presents hypolipemic and antioxidant activities. We show here the effects of T. indica extract on isolated rat liver mitochondria. In the presence of Ca2+, the extract caused mitochondrial concentration-dependent swelling, associated to, resting respiration increase (V4), membrane potential dissipation and release of pre-accumulated Ca2+, inhibited by cyclosporine A (CsA) and thus ascribable to mitochondrial permeability transition (MPT). This swelling induction was prevented by EGTA and ruthenium red, indicating strict dependence of MPT on Ca2+. Oxidation of mitochondrial membrane protein thiols, a well established mechanism causing MPT was detected. However, no significant change was observed in the GSH redox state, and the NADPH oxidation and accumulation of mitochondria-generated reactive oxygen species that was observed, were prevented by CsA and/or EGTA, indicating that they are consequence of the MPT induced by T. indica extract. Therefore, no apparent oxidative stress condition is involved as cause of this process suggesting that direct interaction with membrane protein thiol groups of the compounds responsible for MPT induction occurs. T. indica extract led to MPTassociated ATP depletion, thus showing the potential to cause cell death by apoptosis or necrosis resulting from MPT induction per se or from ATP depletion by MPT. In vitro, the extract presented free radical scavenging ability, as assessed by the 2,2-diphenyl-1- picrylhydrazyl (DPPH), superoxide radicals and radical hydroxyl assays, and led to decreased lipid peroxidation in mitochondria, as assessed by the thiobarbituric acid reactive substances (TBARS) assay. In addition, the extract showed an iron chelanting property in low concentrations.

Orientadores: Anibal Eugênio Vercesi, Helena Coutinho Franco de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Aplicadas / Made available in DSpace on 2018-08-24T17:38:21Z (GMT). No. of bitstreams: 1 Moraes_Audreyde_M.pdf: 2287376 bytes, checksum: 667ac52246453deec2cd72f05541e70c (MD5) Previous issue date: 2014 / Resumo: Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital quando liberada / Abstract: Note: The complete abstract is available with the full electronic document / Mestrado / Fisiopatologia Médica / Mestra em Ciências

Orientadores: Anibal Eugenio Vercesi, Nadja Cristhina de Souza Pinto / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T03:45:12Z (GMT). No. of bitstreams: 1 Ravagnani_FelipeGustavo_D.pdf: 7335132 bytes, checksum: 7043cdada57a4c0242520d7fcb95daeb (MD5) Previous issue date: 2013 / Resumo: O número de indivíduos que sofrem com patologias associadas ao consumo abusivo de etanol tem aumentado significativamente no último século. Como consequência desse fato, os custos associados ao tratamento do alcoolismo, bem como das doenças associadas a ele também têm aumentado, onerando o sistema de saúde e se tornando um problema de saúde pública de grande relevância atualmente. Os mecanismos moleculares que desencadeiam muitas dessas doenças não estão completamente esclarecidos. O tecido hepático é o mais afetado pelo etanol e as mitocôndrias têm sido apontadas como alvos cruciais na toxicidade hepática induzida pelo álcool. Logo, o objetivo desse trabalho foi investigar como o consumo de etanol afeta o estado redox e o metabolismo mitocondriais no fígado. Ratos Wistar machos adultos jovens e de meia-idade receberam ad libitum solução alcoólica 25% (v/v) como única fonte de líquido. Os grupos controle receberam somente água. Ambos os grupos receberam ração ad libitum. Mitocôndrias hepáticas foram isoladas usando técnicas padrão. O consumo de ração e de líquidos foi significativamente menor em animais que ingeriram álcool, resultando em menor ganho de massa corpórea nos protocolos utilizados. As mitocôndrias dos animais que consumiram etanol apresentaram menores níveis de respiração em condição basal e quando energizadas com substratos respiratórios. A atividade e os níveis protéicos de citocromo c oxidase foi menor nos grupos tratados com etanol. Independente da duração do período de tratamento, mitocôndrias hepáticas de animais que ingeriram álcool foram menos susceptíveis à transição de permeabilidade mitocondrial induzida por cálcio, quando comparadas às mitocôndrias dos animais do grupo controle. Esse efeito foi revertido pela adição de oxidantes de nucleotídeos de piridina (acetoacetato, diamida ou tert butil-hidroperóxido) ou em mitocôndrias desacopladas. Também houve aumento em nucleotídeos de piridina na forma reduzida e aumento na razão NAD(P)H/NAD(P)+ em mitôndrias hepáticas de ratos consumidores de etanol. Em concordância a esses dados, houve aumento na capacidade de retenção de cálcio, processo que é dependente do estado redox intramitocondrial. Por outro lado, não houve diferença na produção de espécies reativas de oxigênio entre os grupos controle e tratados com álcool. A atividade de glutationa peroxidase e as quantidades de GSH e de GSSG também não sofreram alterações. Entretanto, houve redução nos níveis de DNA mitocondrial nos tratamentos agudos, porém com tendência para retornar aos níveis normais nos tratamentos crônicos, indicando uma resposta adaptativa à injúria induzida pelo etanol. Em conjunto, nossos resultados indicam que o consumo de etanol modula o estado redox mitocondrial e de sistemas antioxidantes, prevenindo a abertura do poro de transição de permeabilidade mitocondrial. A presença desse xenobiótico no fígado também altera significativamente os níveis de NADP reduzido, agente redutor final para o sistema glutationa redutase/peroxidase que detoxifica H2O2 na matriz mitocondrial. Além disso, a resposta adaptativa ao álcool observada no DNA mitocondrial pode contribuir para compreender melhor os mecanismos envolvidos no reparo de lesões a biomoléculas e os estágios iniciais de adaptação a esse xenobiótico, etapas que precedem a morte celular, hepatite alcoólica ou carcinogênese em tecido hepático exposto cronicamente ao etanol / Abstract: The number of people suffering from alcoholism has increased significantly over the last century. As a result, costs associated with treating the addiction itself as well as the associated pathologies have also increased, such that this is considered as public health issue. Furthermore, the molecular events leading to several of these diseases are not yet clearly understood. Hepatic tissue is the most affected by alcohol, and mitochondria have been suggested to be a crucial target in alcohol-induced liver toxicity. Thus, the aim of our study was to investigate how ethanol consumption affects the redox state and mitochondrial metabolism in the liver. Young adult and middle-aged male Wistar rats were given a 25 % (v/v) ethanol solution as the only source of drinking water. Control groups received water only. Liver mitochondria were isolated using standard techniques. Food and water intake was significantly lower in alcohol-drinking rats, resulting in lower weight gain during the treatment regimes. Mitochondria from the alcohol-drinking group had lower respiration under levels in basal condition, when energized by substrates feeding electrons into complexes I and IV. Cytochrome c oxidase activity and protein levels were lower in the alcohol group as well. Additionally, regardless of the length of the treatment, liver mitochondria from the alcohol-treated animals were more resistant to Ca2+-induced mitochondrial permeability transition (MPT), when compared to mitochondria from control animals. This effect was abrogated by oxidizing agents of pyridine nucleotides (acetoacetate, diamide or tert butylhydroperoxide) or in uncoupled mitochondria. We also found that liver mitochondria from the alcohol-drinking rats had a more reduced pyridine nucleotide pool and higher NAD(P)H/NAD(P)+ ratios. In addition, Nampt (an enzyme of the NAD+ synthetic pathway) protein levels did not differ after alcohol consumption. Accordingly, the calcium retention capacity of the isolated mitochondria, which is dependent upon intramitochondrial redox state, was higher in the alcohol group. On the other hand, levels of reactive oxygen species showed no differences between the control and alcohol groups, both in mitochondria and in splenic lymphocytes. Glutathione peroxidase activity and the amounts of GSH and GSSG were also not changed. However, mitochondrial DNA levels were decreased in the short term treatments, but tended to go back up to normal levels in the chronic treatments, indicating an adaptative response to ethanol-induced injury. Together, our results indicate that ethanol consumption modulates the mitochondrial redox state and the antioxidant systems, protecting against Ca2+-induced mitochondrial pore transition permeability opening. The presence of this xenobiotic can significantly change the levels of reduced NADP, the ultimate reducing agent in the gluthatione reductase/peroxidase system that detoxifies H2O2 in the mitochondrial matrix. In addition, the adaptative response to ethanol, seen in mitochondrial DNA, may contribute to further understand the mechanisms related to lesions in biomolecules and the initial steps that preceed cell death, alcoholic hepatitis or carcinogenic process in hepatic tissue exposed chronically to ethanol / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Fisiopatologia Medica

Orientadores: Tiago Rezende Figueira, Roger Frigério Castilho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T03:45:16Z (GMT). No. of bitstreams: 1 Chweih_Hanan_M.pdf: 1684363 bytes, checksum: edae156378f90e7315bca30c16544071 (MD5) Previous issue date: 2015 / Resumo: Algumas das características das mitocôndrias, incluindo as suas funções de transporte de Ca2+, podem apresentar dimorfismo sexual e especificidades teciduais. No entanto, as mensurações do transporte de Ca2+ em mitocôndrias isoladas estão sujeitas a artefatos secundários a abertura do poro de transição de permeabilidade mitocondrial (PTP) induzido pelo acúmulo excessivo de Ca2+ nesta organela. Neste estudo, o objetivo inicial foi avaliar se a inibição do PTP pela ciclosporina A (CsA) afeta a mensuração de diversas variáveis que descrevem o transporte de Ca2+por mitocôndrias isoladas de fígado de rato. Os resultados obtidos indicam que as concentrações de estado estável do Ca2+ externo a mitocôndria e as taxas deefluxo mitocondrial de Ca2+através de trocadores seletivos foram superestimados em até 4 vezes quando o PTP não foi inibido farmacologicamente pela CsA. O objetivo subsequente foi analisar o transporte de Ca2+ em mitocôndrias isoladas de fígado, de músculo esquelético, de coração e de cérebro de ratos machos e fêmeas sob condições experimentais específicas (i.e. meio de incubação contendo inibidores TPM, substratos energéticos ligados a NAD e níveis relevantes de Ca2+, Mg2+e Na+). Os dados indicaram que a taxa de influxo de Ca2+em mitocôndrias de fígado foi ~4 vezes superior a dos outros tecidos, as quais foram semelhantes entre si. Em contrapartida, as taxas de efluxo de Ca2+ apresentaram uma maior diversidade entre tecidos, especialmente na presença de Na+. Curiosamente, o efluxo de Ca2+na ausência de Na+foi significativamente mais elevado nas mitocôndrias cardíacas (~4nmol/mg/min) em relação às taxas observadas nos outros tecidos, contrariando a concepção de que o efluxo de Ca2+de mitocôndrias de coração é dependente, quase que exclusivamente, de um trocador que requer Na+. A especificidade em relação ao sexo só foi observada em dois índices relacionados a homeostase mitocondrial de Ca2+(i.e. cinética geral normalizada da captação de Ca2+ e a concentração de estado estável do Ca2+ externo a mitocôndria) em mitocôndrias isoladas de coração (mais lentos ou maiores na fêmea) e na respiração estimulada por ADP em mitocôndrias de fígado (~20% maior na fêmea). O presente estudo demonstrou a importância metodológica de se prevenir a abertura do PTP para a análise das propriedades e da variabilidade fisiológica do transporte de Ca2+por mitocôndrias isoladas. Adicionalmente, concluímos que sob as condições experimentais aqui utilizadas, o efluxo de Ca2+ mitocondrial apresenta grandes especificidades teciduais e que alguns achados desafiam conceitos estabelecidos em estudos anteriores sob condições arguivelmente menos controladas / Abstract: The characteristics of mitochondria, including their Ca2+ transport functions, may exhibit tissue specificity and sex dimorphism. Because the measurements of the Ca2+ handling by isolated mitochondria may be biased by dysfunction secondary to Ca2+-induced mitochondrial permeability transition (MPT) pore opening, this study evaluates the extent to which MPT inhibition by cyclosporine-A affects the measurement of Ca2+ transport in isolated rat liver mitochondria. The results indicate that the steady-state levels of external Ca2+ and the rates of mitochondrial Ca2+ efflux through the selective pathways can be overestimated by up to 4-fold if MPT pore opening is not prevented. Then, we analyzed the Ca2+ transport in isolated mitochondria from the liver, skeletal muscle, heart and brain of male and female rats under incubation conditions containing MPT inhibitors, NAD-linked substrates and relevant levels of free Ca2+, Mg2+ and Na+. Except for the liver mitochondria displaying values4-fold higher, the Ca2+ influx rates were similar among the other tissues. In contrast, the Ca2+ efflux rates exhibited more tissue diversity, especially in the presence of Na+. Interestingly, the Na+-independent Ca2+ efflux was highest in the heart mitochondria (~4 nmol/mg/min), thus challenging the view that heart mitochondrial Ca2+ efflux relies almost exclusively on a Na+-dependent pathway. Sex specificity was only observed in two kinetic indexes (i.e. the normalized overall kinetics of Ca2+ uptake and the steady-state levels of external Ca2+) of heart mitochondrial Ca2+ homeostasis (slower or higher in female)and in the ADP-stimulated respiration of liver mitochondria (~20% higher in females). The present study shows the methodological importance of preventing MPT when measuring the properties and the physiological variability of the Ca2+ handling by isolated mitochondria. Moreover, we conclude that mitochondrial Ca2+ efflux exhibits great tissue specificity under our conditions, which may challenge some concepts raised in previous studies that employed experimental conditions that are arguably not well controlled / Mestrado / Fisiopatologia Médica / Mestra em Ciências

L’AMPc est un messager important de la régulation neurohormonale du cœur. En activant ses effecteurs, l’AMPc régule de nombreuses fonctions cellulaires telles que l'expression de gènes, le couplage excitation-contraction et le métabolisme cellulaire. Chez les mammifères, l'AMPc est produit par une famille d’adénylate cyclases au sein de plusieurs compartiments subcellulaires solubles ou membranaires. L'existence et le rôle de la signalisation des nucléotides cycliques dans les mitochondries ont été postulés, mais n'ont pas encore été démontrés. De plus, son implication dans la régulation de la mort cellulaire est encore inconnue. Dans cette thèse, nous avons démontré l'expression locale de plusieurs acteurs de la signalisation de l'AMPc dans les mitochondries cardiaques, à savoir une forme tronquée soluble AC (sACt) et la protéine d'échange directement activées par AMPc 1 (Epac1). Nous avons montré un rôle protecteur pour sACt contre la mort cellulaire, l'apoptose, ainsi que la nécrose de cardiomyocytes primaires. Lors de la stimulation par du bicarbonate (HCO3-) et du Ca2+, la sACt produit de l’AMPc, qui à son tour stimule la consommation d'oxygène, une augmentation du potentiel mitochondrial de membrane (ΔΨm) et la production d'ATP. L’AMPc est limitant pour l’entrée matricielle de Ca2+ via l’uniport calcique mitochondrial (MCU) et, en conséquence, prévient la transition de perméabilité mitochondriale (MPT). En outre, dans les mitochondries isolées de cœurs de rats défaillants, la stimulation de la voie de l'AMPc par le HCO3- prévient la sensibilisation des mitochondries au Ca2+. Nous avons également constaté que les familles de phosphodiestérases (PDE), PDE2, 3 et 4, sont exprimées dans les mitochondries cardiaques régulant le taux d’AMPc. Ainsi, ces protéines forment une voie de signalisation locale dans la matrice régulant la fonction mitochondriale cardiaque. Finalement, notre étude a permis d’identifier un lien entre l'AMPc mitochondrial, le métabolisme, certaines PDEs et la mort cellulaire dans le cœur, qui est indépendant de la signalisation AMPc cytosolique. Ceci pourrait constituer un nouveau mécanisme cardioprotecteur via la préservation de la fonction mitochondriale dans un contexte physiopathologique. / CAMP is an important messenger in neurohormonal regulation of the heart. By activating its effectors, cAMP regulates many cellular functions such as gene expression, excitation-contraction coupling and cellular metabolism. In mammals, cAMP is produced by a family of adenylyl cyclase with various subcellular locations and membrane anchorage. The existence and role of cyclic nucleotide signaling in mitochondria has been postulated, but has not yet been demonstrated. Moreover, its implication in the regulation of cell death is still unknown. In this thesis, we demonstrated the local expression of several actors of cAMP signaling within cardiac mitochondria, namely a truncated form of soluble AC (sACt) and the exchange protein directly activated by cAMP 1 (Epac1) and showed a protective role for sACt against cell death, apoptosis as well as necrosis, in primary cardiomyocytes. Upon stimulation with bicarbonate (HCO3-) and Ca2+, sACt produces cAMP, which in turn stimulates oxygen consumption, increased the mitochondrial membrane potential (∆Ψm) and ATP production. cAMP is rate-limiting for matrix Ca2+ entry via the mitochondrial calcium uniporter (MCU) and, as a consequence, prevented mitochondrial permeability transition (MPT). In addition, in mitochondria isolated from failing rat hearts, stimulation of the mitochondrial cAMP pathway by HCO3- rescued the sensitization of mitochondria to Ca2+-induced MPT. We also found that PDE2, 3 and 4 families are located in cardiac mitochondria. They form a local signaling pathway with soluble AC in the matrix, which regulates cardiac mitochondrial functions. Thus, our study identifies a link between mitochondrial cAMP, mitochondrial metabolism, some PDEs and cell death in the heart, which is independent of cytosolic cAMP signaling. This might constitute a novel cardioprotective mechanism through mitochondrial function preservation in pathophysiological conditions.

Le zinc est un métal, membre des éléments trace dont le rôle protecteur contre l’ischémie-reperfusion myocardique a été suggéré. Dans ce travail de thèse, nous avons étudié le mécanisme par lequel le zinc procure cette protection. Nous avons utilisé des cultures cellulaires de cardiomyoblastes de rat, des cardiomyocytes isolés de rat, des mitochondries isolées ainsi qu’un modèle de cœur isolé perfusé de rat. Nous avons montré que l'administration de zinc au début de la reperfusion permet de diminuer la mort cellulaire sur un modèle d’ischémie-reperfusion simulée. Nous avons montré que l’administration de zinc entraine l’activation de la cascade des kinases de survie (PI-3K/Akt), l’inhibition de GSK-3β et celle de l’ouverture du pore de transition de perméabilité mitochondrial. Nous avons ensuite montré qu’en présence de zinc, l’activation d’Akt provenait d’une inhibition de certaines phosphatases et d’une activation du domaine tyrosine kinase du récepteur IGF-1. Nos derniers travaux ont eu pour but de préciser le rôle du zinc suite à l'activation des récepteurs à adénosine. / Zinc is a member of the family of the metal trace elements and its role in the protection against lethal reperfusion injury has been strongly suggested. The aim of the present work was to determine the molecular mechanisms involved in zinc cardioprotection. We have used cultured rat cardiomyoblasts, isolated rat cardiomyocytes, isolated mitochondria and isolated perfused rat heart. We demonstrated that zinc given at reperfusion, limits cell necrosis in a model of simulated ischemia-reperfusion. Administration of zinc results in activation of the reperfusion-injury salvage kinases pathway (PI-3K/Akt), inhibition of GSK-3β and of the opening of the mitochondrial permeability transition pore. We then showed that, in the presence of zinc, activation of Akt is mediated by the inhibition of various phosphatases and by activation of the tyrosine kinase domain of the IGF-1 receptor. Our latest experiments report the action of zinc following activation of adenosine receptors

La maladie coronaire d’origine ischémique reste l’une des principales causes de mortalité dans le monde industrialisé.Le traitement de l’ischémie aiguë du myocarde est entré dans une nouvelle ère où la mortalité peut être diminuée de moitié en utilisant des procédures qui permettent un retour rapide du débit sanguin dans la zone ischémique du myocarde: la revascularisation.Cette reperfusion entraîne des complications appelées lésions de la reperfusion qui ont été décrites pour la 1ère fois par Jennings et al., en 1960. Le développement de stratégies cardioprotectrices associées à la reperfusion constitue un besoin majeur en clinique afin d’améliorer la fonction myocardique, de diminuer l'incidence des arythmies, de retarder l'apparition de la mort cardiomyocytaire et de limiter la taille de l’infarctus du myocarde lors de l'ischémie/reperfusion (I/R).La découverte de 2 formes principales de mécanismes cardioprotecteurs endogènes, a encouragé la recherche de nouveaux moyens pharmacologiques capables de protéger le myocarde ischémié/reperfusé et a développé nos connaissances sur les bases moléculaires des lésions et de la survie cellulaire au cours des processus d’I/R.L’étude des mécanismes responsables de l’induction de la mort cellulaire a permis de mettre en évidence le rôle joué par la mitochondrie et l’augmentation de la perméabilité de ses membranes, induite par la formation/ouverture d’un pore au niveau des points de contacts entre les membranes mitochondriales;ce pore a été appelé « pore de transition de la perméabilité mitochondriale » (mPTP).L’inhibition de l’ouverture de ce pore apparaît comme une stratégie privilégiée pour protéger le myocarde.Des études ont montré que les espèces réactives d’oxygène (EROs) jouent un rôle majeur dans les lésions de l’I/R et dans l’ouverture du mPTP.Il existe peu d’informations claires sur le seuil et la période de production (ischémie et/ou reperfusion) des EROs qui conduisent à l’ouverture du mPTP.Nous avons mis au point un modèle cellulaire d’hypoxie/réoxygénation (H/R) afin d’établir une relation causale entre la production d’EROs, l’ouverture du mPTP et la mort cellulaire tout en explorant le rôle de différents types d’EROs.Ce modèle d’H/R nous a permis de mesurer en temps réel et simultanément la production des EROs, l’ouverture du mPTP et la mort cellulaire.Nous avons montré que la production des EROs débute pendant la période d’hypoxie et qu’elle est directement liée à l’augmentation du temps d’hypoxie.Cette production d’EROs à l’hypoxie, plus particulièrement de radicaux hydroxyles et de peroxyde d’hydrogène, a été directement relié, à l’ouverture du mPTP et à la mort cellulaire lors de l’H/R.Nous avons utilisé ce modèle pour étudier le mécanisme d’action de deux stratégies pharmacologiques cardioprotectrices, un nouveau ligand de la protéine translocatrice mitochondriale (TSPO), le TRO 40303, et l’activation de la voie RISK par la morphine. Nous avons ainsi montré que (1) les propriétés cardioprotectrices du TRO40303 sont associées à une inhibition de l’ouverture du mPTP, ce qui n’avait pas pu être démontré au moyen d’expériences réalisées ex vivo et (2) l’activation de la voie RISK par la morphine, qui aboutit à une limitation de la taille d’infarctus associée à une amélioration des fonctions respiratoires mitochondriales, entraîne également une inhibition de l’ouverture du mPTP et un retard de la mort cellulaire des cardiomyocytes isolés soumis à une H/R.La suite de ce travail sera de rechercher si l’inhibition du stress oxydant peut constituer un mécanisme commun aux deux stratégies pharmacologiques cardioprotectrices en utilisant notre modèle d’H/R.Il serait possible d’étendre notre modèle à des animaux génétiquement modifiés pour appréhender les phénomènes impliqués dans cette activité antioxydante.A plus long terme, il sera nécessaire d’approfondir nos connaissances sur la production d’EROs pendant l’I/R en recherchant plus spécifiquement l’origine de cette production. / Ischemic coronary artery disease remains one of the main causes of mortality in the industrialized countries. The treatment of acute myocardial ischemia entered a new era where mortality can be reduced by 50% using revascularization procedures that allow a rapid return of blood flow to the ischemic area. However, this reperfusion leads to complications known as lethal reperfusion induced injury that have been described for the first time by Jennings et al., in 1960. It became crucial to develop cardioprotective strategies in combination with early reperfusion in order to improve myocardial function, to reduce the incidence of arrhythmias, to delay the onset of cardiomyocytes death and to limit the extension of infarct size following reperfusion. The discovery of two major forms of endogenous cardioprotective mechanisms, which consist of the realization of short cycles of ischemia/reperfusion (I/R) prior to a long period of ischemia (ischemic preconditioning) or before reperfusion after the long period of ischemia (Ischemic Postconditioning), encouraged the search for new pharmacological tools to protect the ischemic myocardium to develop our knowledge on the molecular mechanisms of lethal reperfusion injury and cell survival in the I/R process.The study of cell death mechanisms has highlighted the crucial role of the mitochondria and more specifically the increase in mitochondrial membrane permeability following I/R.One reason for increasing permeability is the formation/opening of a pore at mitochondrial membranes contact sites at reperfusion.This pore has been called "the mitochondrial permeability transition pore" (mPTP). Inhibition of this pore opening has been presented as a main strategy to protect the myocardium.Many studies have shown that reactive oxygen species (ROS) play a major role in I/R injury and mPTP opening, but there is very few information to date about the threshold and the period of ROS production (ischemia and/or reperfusion) that lead to mPTP opening.We designed a cellular model of hypoxia/reoxygenation (H/R) to establish a causal relationship between ROS production, mPTP opening and cell death while exploring the role of different types of ROS.This H/R model used freshly isolated adult rat cardiomyocytes and allowed us to measure online and simultaneously ROS production, mPTP opening and cell death. We have demonstrated that ROS production starts during the period of hypoxia and thisproduction is directly linked to the increase in the duration of hypoxia.This ROS production during hypoxia has been, for the first time, directly related to mPTP opening and cell death following H/R.We used this model to study the mechanism of action of two cardioprotective strategies, a new ligand of the mitochondrial translocator protein (TSPO), TRO 40303 and a RISK (Reperfusion Injury Salvage Kinase) pathway activator, morphine. We have shown that (1) the cardioprotective properties of TRO40303 were associated with inhibition of mPTP opening, a mechanism that could not be demonstrated using ex vivo experiments and (2) morphine that provoked infarct size limitation associated with an improvement of mitochondrial respiratory functions through RISK pathway activation, also inhibited mPTP opening and delayed cell death of isolated cardiomyocytes subjected to H/R.Finally, a question comes into sight whether the inhibition of oxidative stress may be a common mechanism to both cardioprotective pharmacological strategies that we have described using our H/R model. To do this, it would be possible to extend our model to genetically modified animals specifically adapted to understand the phenomena involved in antioxidant activity.On long-term, it will be necessary to develop our knowledge on ROS production during I/R by looking for the origin of this production, more precisely the role of the mitochondria and the effect of other reactive species in order to target the treatment and to develop new cardioprotective strategies.