The Arabidopsis cyclic nucleotide-gated ion channels (AtCNGCs) form a large family consisting of 20 members. It has been suggested that CNGCs contribute to a wide array of biological functions such as pollen tube growth and pathogen defence signalling. However, the precise mechanisms by which AtCNGCs act, and the extent of their biological roles, have yet to be fully elucidated. AtCNGC11/12, the chimeric CNGC that resulted from the fusion of AtCNGC11 and 12, induces a number of pathogen defence related phenotypes in the Arabidopsis mutant cpr22. Spontaneous lesion formation is one such phenotype. Interestingly, when AtCNGC11/12 is transiently expressed in N. benthamiana it causes cell death which was characterized in this study. Also, AtCNGC11/12 was used to investigate the structural features responsible for the proper function and regulation of AtCNGCs. Electron microscopic analysis of the AtCNGC11/12-induced cell death showed similar characteristics to programmed cell death (PCD), such as plasma membrane shrinkage and vesicle formation. Interestingly caspase-1 inhibitors and the silencing of vacuolar processing enzyme, a plant enzyme with caspase-1 activity, suppressed the induction of cell death. Additionally, pharmacological analyses indicated that the AtCNGC11/12-indiced cell death was also dependent on Ca2+. Furthermore, 3 amino acid residues, R190, A225, and G287, were demonstrated to be essential for AtCNGC11/12-induce cell death. Taken together, these results indicate that the cell death that develops in the cpr22 mutant is indeed PCD and that AtCNGC11/12, is at the point of, or up-stream of, the Ca2+ signal necessary for the development of HR. Furthermore, the functionality of AtCNGC11/12 as a model for AtCNGC structure-function analyses was demonstrated by the identification of several amino acids necessary for cell death development. Yoshioka et al. (2006) demonstrated that the loss of AtCNGC11 or 12 results in decreased resistance to avirulent isolates of the oomycete pathogen, H. arabidopsidis. Thus, the present biological role suggested for AtCNGC11 and 12 is in pathogen defence, specifically within effector triggered immunity (ETI). Like AtCNGC11 and 12, AtCNGC2 has been demonstrated to contribute to pathogen defence signalling but has also been implicated in other physiological responses such as ion stress and senescence. To better understand the roles of AtCNGC11 and 12 in both pathogen defence and other Ca2+ dependent signalling processes, I have investigated promoter:GUS reporter lines, as well as, AtCNGC11 and 12 KO and RNAi silenced lines subjected to various treatments. From this work, I have demonstrated that AtCNGC11 and 12 have similar expression patterns during pathogen defence, development, and dark-induced senescence. Additionally, the findings presented here further characterize AtCNGC11 and 12 as contributors to ETI rather than PAMP triggered immunity. Furthermore, I demonstrated that AtCNGC11 and 12 are likely involved in the endogenous movement of Ca2+, contributing to a range of Ca2+ associated signalling pathways including gravitropism and senescence. Taken together, these results have greatly improved the characterization of AtCNGC11 and 12; significantly contributing to the understanding of a large and increasingly important channel family.

The purpose of this thesis work is to model the gate control theory, which explains the modulation of pain signals, with a motivation of finding new possible targets for pain treatment and to find novel control algorithms that can be used in engineering practice. The difference of the current study from the previous modeling trials is that morphologies of neurons that constitute gate control system are also included in the model by which structure-function relationship can be observed. Model of an excitable neuron is constructed and the response of the model for different perturbations are investigated. The simulation results of the excitable cell model is obtained and when compared with the experimental findings obtained by using crayfish, it is found that they are in good agreement. Model encodes stimulation intensity information as firing frequency and also it can add sub-threshold inputs and fire action potentials as real neurons. Moreover, model is able to predict depolarization block. Absolute refractory period of the single cell model is found as 3.7 ms. The developed model, produces no action potentials when the sodium channels are blocked by tetrodotoxin. Also, frequency and amplitudes of generated action potentials increase when the reversal potential of Na is increased. In addition, propagation of signals along myelinated and unmyelinated fibers is simulated and input current intensity-frequency relationships for both type of fibers are constructed. Myelinated fiber starts to conduct when current input is about 400 pA whereas this minimum threshold value for unmyelinated fiber is around 1100 pA. Propagation velocity in the 1 cm long unmyelinated fiber is found as 0.43 m/s whereas velocity along myelinated fiber with the same length is found to be 64.35 m/s. Developed synapse model exhibits the summation and tetanization properties of real synapses while simulating the time dependency of neurotransmitter concentration in the synaptic cleft. Morphometric analysis of neurons that constitute gate control system are done in order to find electrophysiological properties according to dimensions of the neurons. All of the individual parts of the gate control system are connected and the whole system is simulated. For different connection configurations, results of the simulations predict the observed phenomena for the suppression of pain. If the myelinated fiber is dissected, the projection neuron generates action potentials that would convey to brain and elicit pain. However, if the unmyelinated fiber is dissected, projection neuron remains silent. In this study all of the simulations are preformed using Simulink.

Activity of the Ryanodine Receptor (RyR2) (aka cardiac Ca2+ release channel) plays a pivotal role in contraction of the heart. S-adenosyl-l-methionine (SAM) is a biological methyl group donor that has close structural similarity to ATP, an important physiological regulator of RyR2. This work provides evidence that SAM can act as a RyR2 regulatory ligand in a manner independent from its recognized role as a biological methyl group donor. RyR2 activation appears to arise from the direct interaction of SAM, via its adenosyl moiety, with the RyR2 adenine nucleotide binding sites. Because uncertainty remains regarding the structural motifs involved in RyR2 modulation by ATP and its metabolites, this finding has important implications for clarifying the structural basis of ATP regulation of RyR2. During the course of this project, direct measurements of single RyR2 activity revealed that SAM has distinct effects on RyR2 conductance. From the cytosolic side of the channel, SAM produced a single clearly resolved subconductance state. The effects of SAM on channel conductance were dependent on SAM concentration and membrane holding potential. A second goal of this work was to distinguish between the two possible mechanisms by which SAM could reduce RyR2 conductance: i) SAM interfering directly with ion permeation via binding within the conduction pathway (pore block), or ii) SAM binding a regulatory (or allosteric) site thereby stabilizing or inducing a reduced conductance conformation of the channel. It was determined that SAM does not directly interact with the RyR2 conduction pathway. To account for these observations an allosteric model for the effect of SAM on RyR2 conductance is proposed. According to this model, SAM binding stabilizes an inherent RyR2 subconductance conformation. The voltage dependence of the SAM related subconductance state is accounted for by direct effects of voltage on channel conformation which indirectly alter the affinity of RyR2 for SAM. Patterns in the transitions between RyR2 conductance states in the presence of SAM may provide insight into the structure-activity relationship of RyR2 which can aid in the development of therapeutic strategies targeting this channel.

The Arabidopsis cyclic nucleotide-gated ion channels (AtCNGCs) form a large family consisting of 20 members. It has been suggested that CNGCs contribute to a wide array of biological functions such as pollen tube growth and pathogen defence signalling. However, the precise mechanisms by which AtCNGCs act, and the extent of their biological roles, have yet to be fully elucidated. AtCNGC11/12, the chimeric CNGC that resulted from the fusion of AtCNGC11 and 12, induces a number of pathogen defence related phenotypes in the Arabidopsis mutant cpr22. Spontaneous lesion formation is one such phenotype. Interestingly, when AtCNGC11/12 is transiently expressed in N. benthamiana it causes cell death which was characterized in this study. Also, AtCNGC11/12 was used to investigate the structural features responsible for the proper function and regulation of AtCNGCs. Electron microscopic analysis of the AtCNGC11/12-induced cell death showed similar characteristics to programmed cell death (PCD), such as plasma membrane shrinkage and vesicle formation. Interestingly caspase-1 inhibitors and the silencing of vacuolar processing enzyme, a plant enzyme with caspase-1 activity, suppressed the induction of cell death. Additionally, pharmacological analyses indicated that the AtCNGC11/12-indiced cell death was also dependent on Ca2+. Furthermore, 3 amino acid residues, R190, A225, and G287, were demonstrated to be essential for AtCNGC11/12-induce cell death. Taken together, these results indicate that the cell death that develops in the cpr22 mutant is indeed PCD and that AtCNGC11/12, is at the point of, or up-stream of, the Ca2+ signal necessary for the development of HR. Furthermore, the functionality of AtCNGC11/12 as a model for AtCNGC structure-function analyses was demonstrated by the identification of several amino acids necessary for cell death development. Yoshioka et al. (2006) demonstrated that the loss of AtCNGC11 or 12 results in decreased resistance to avirulent isolates of the oomycete pathogen, H. arabidopsidis. Thus, the present biological role suggested for AtCNGC11 and 12 is in pathogen defence, specifically within effector triggered immunity (ETI). Like AtCNGC11 and 12, AtCNGC2 has been demonstrated to contribute to pathogen defence signalling but has also been implicated in other physiological responses such as ion stress and senescence. To better understand the roles of AtCNGC11 and 12 in both pathogen defence and other Ca2+ dependent signalling processes, I have investigated promoter:GUS reporter lines, as well as, AtCNGC11 and 12 KO and RNAi silenced lines subjected to various treatments. From this work, I have demonstrated that AtCNGC11 and 12 have similar expression patterns during pathogen defence, development, and dark-induced senescence. Additionally, the findings presented here further characterize AtCNGC11 and 12 as contributors to ETI rather than PAMP triggered immunity. Furthermore, I demonstrated that AtCNGC11 and 12 are likely involved in the endogenous movement of Ca2+, contributing to a range of Ca2+ associated signalling pathways including gravitropism and senescence. Taken together, these results have greatly improved the characterization of AtCNGC11 and 12; significantly contributing to the understanding of a large and increasingly important channel family.

L’inflammation: Une réponse adaptative du système immunitaire face à une insulte est aujourd’hui reconnue comme une composante essentielle à presque toutes les maladies infectieuses ou autres stimuli néfastes, tels les dommages tissulaires incluant l’infarctus du myocarde et l’insuffisance cardiaque. Dans le contexte des maladies cardiovasculaires, l’inflammation se caractérise principalement par une activation à long terme du système immunitaire, menant à une faible, mais chronique sécrétion de peptides modulateurs, appelés cytokines pro-inflammatoires. En effet, la littérature a montré à plusieurs reprises que les patients souffrant d’arythmies et de défaillance cardiaque présentent des taux élevés de cytokines pro-inflammatoires tels le facteur de nécrose tissulaire alpha (TNFα), l’interleukine 1β (IL-1β) et l’interleukine 6. De plus, ces patients souffrent souvent d’une baisse de la capacité contractile du myocarde. Le but de notre étude était donc de déterminer si un lien de cause à effet existe entre ces phénomènes et plus spécifiquement si le TNFα, l’IL-1β et l’IL-6 peuvent affecter les propriétés électriques et contractiles du cœur en modulant le courant Ca2+ de type L (ICaL) un courant ionique qui joue un rôle primordial au niveau de la phase plateau du potentiel d’action ainsi qu’au niveau du couplage excitation-contraction. Les possibles méchansimes par lesquels ces cytokines exercent leurs effets seront aussi explorés. Pour ce faire, des cardiomyocytes ventriculaires de souris nouveau-nées ont été mis en culture et traités 24 heures avec des concentrations pathophysiologiques (30 pg/mL) de TNFα, IL-1β ou IL-6. Des enregistrements de ICaL réalisés par la technique du patch-clamp en configuration cellule entière ont été obtenus par la suite et les résultats montrent que le TNFα n’affecte pas ICaL, même à des concentrations plus élevées (1 ng/mL). En revanche, l’IL-1β réduisait de près de 40% la densité d’ICaL. Afin d’examiner si le TNFα et l’IL-1β pouvaient avoir un effet synergique, les cardiomyocytes ont été traité avec un combinaison des deux cytokines. Toutefois aucun effet synergique sur ICaL n’a été constaté. En outre, l’IL-6 réduisait ICaL significativement, cependant la réduction de 20% était moindre que celle induite par IL-1β. Afin d’élucider les mécanismes sous-jacents à la réduction de ICaL après un traitement avec IL-1β, l’expression d’ARNm de CaV1.2, sous-unité α codante pour ICaL, a été mesurée par qPCR et les résultats obtenus montrent aucun changement du niveau d’expression. Plusieurs études ont montré que l’inflammation et le stress oxydatif vont de pair. En effet, l’imagerie confocale nous a permis de constater une augmentation accrue du stress oxydatif induit par IL-1β et malgré un traitement aux antioxydants, la diminution de ICaL n’a pas été prévenue. Cette étude montre qu’IL-1β et IL-6 réduisent ICaL de façon importante et ce indépendamment d’une régulation transcriptionelle ou du stress oxydatif. De nouvelles données préliminaires suggèrent que ICaL serait réduit suite à l’activation des protéines kinase C mais des études additionelles seront nécessaires afin d’étudier cette avenue. Nos résultats pourraient contribuer à expliquer les troubles du rythme et de contractilité observés chez les patients souffrant de défaillance cardiaque. / Cytokines are immune system modulators that are secreted in response to an insult. Even though on the short term they play a crucial role in the healing process, the prolonged secretion of pro-inflammatory cytokines, locally or systemically, has many deleterious effects. For almost 20 years reports of alteration in serum cytokine levels have been emerging in patients with various heart failure aetiologies, however it is only recently that the role of inflammation in heart pathologies is being more and more studied. Indeed, several studies have shown that patients suffering from heart failure or arrhythmias have high levels of cytokines. Three particularly of these cytokines in particular are highly present and together they play a central role in the inflammatory response. Tumour Necrosis Factor alpha (TNFα), interleukin 1 beta (IL-1β) and interleukin 6 (IL-6) are secreted chronically by immune cells or the cardiomyocytes themselves and can possibly, as shown by animal studies, induce cardiac remodelling, hypertrophy, apoptosis, fibrosis and generation of highly reactive oxidative species (ROS) among other effects. Furthermore, accumulating evidence suggests that these pro-inflammatory cytokines are not only important mediators of cardiac remodelling that can contribute to worsening of heart failure but they have also been linked to cardiac arrhythmias and prolongation of action potential. Overall, the findings suggests a strong role for pro-inflammatory cytokines in affecting cardiac function and inducing electrical remodelling, thus we hypothesised that high levels of pro-inflammatory cytokines can affect the electrical and subsequently the contractile properties of the heart. Thus, the aim of this project was to help establish the effects of the above mentioned cytokines on the electrical and contractile properties of cardiac myocytes while exploring the mechanisms by which these cytokines mediate their effect. Using cultured intact mouse neonatal ventricular cardiomyocytes which were treated chronically with various cytokines, at a pathophysiological concentration (30 pg/mL), the specific objective of this study was to measure the direct effect of chronic cytokine treatment on the L-type calcium current (ICaL), an important ionic current responsible for the plateau phase of the action potential and in the excitation contraction coupling (ECC) and the current l and subsequently, determine via which pathways cytokines are able to affect the calcium current. Patch-clamp experiments in the whole-cell voltage-clamp configuration were used to measure L-type calcium current and showed that ICaL was not affected by TNFα. Furthermore, no effect at a significantly higher concentration of TNFα (1 ng/mL) could be observed. In contrast, chronic treatment of cardiomyocytes with IL-1β depressed ICaL by up to 40 %. Furthermore, when combining TNFα with IL-1β, two cytokines often reported to act synergistically, no further reduction in ICaL current density compared to IL-1β treatment alone was observed, showing the specificity of IL-1β response. Expression studies using qPCR to quantify the mRNA of CaV1.2, the underlying alpha subunit channel which encodes for ICaL, were conducted in order to determine if the reduction in current is due to a cytokine mediated change in gene expression. We found that none of the cytokines significantly affected levels of CaV1.2 mRNA. A key component of the inflammatory response is the induction of oxidative stress. Indeed, when challenged with cytokines cardiomyocytes exhibited significant increases in ROS level. In an attempt to reverse the depression of ICaL in response to IL-1β, we treated myocytes concurrently with antioxidants and IL-1β. While we observed a significant decrease in intracellular ROS levels, antioxidant therapy failed to restore current density, indicating thus, that ROS produced in response to cytokines does not regulate ion channels. New preliminary data suggests a role for members of the protein kinase C family in regulating the properties of CaV1.2 in response to cytokines. Nonetheless, exploring this avenue will require substantial experimentation and will be the subject of future work. Overall our experiments will help provide a better understanding of the role of cytokines in regulating the electric and contractile properties of cardiomyocytes in the setting of inflammatory cardiomyopathies.

Two classes of ion-channels comprising 22 members were prepared. Three members were linear oligo-esters with terephthalate core designed to span both leaflets of the bilayer; these were prepared in a modular synthesis in three linear steps. 19 half-channels based on cyclodextrins with functionalized primary-rims were prepared by the Huisgen Cu+-catalyzed [3+2]-cyclization; three distinct synthetic protocols were established to be applicable to these substrates. The voltage-clamp experiment was used to characterize the ion transport properties of these 22 compounds as well as 5 oligo-esters previously prepared by solid-phase synthesis. All but two were active in bilayers, with the majority of these compounds showing highly complex conductance activities. Exponentially voltage-dependent currents were observed for two compounds (both terephthalate-derived); exclusive “square-top” activities were observed for one solid-phase–derived compound and one cyclodextrin-based channels; fractal openings were observed for at least two cyclodextrin-based channels. An “activity grid” notation was proposed as an empirical, coarse, but model-free method of treating the complex data. Through an exhaustive analysis of previously published synthetic ion channels, disparate compounds were found to share modes of activity. Supporting software were developed to facilitate the preparation of activity grids from current traces acquired for the aforementioned 27 compounds. Resulting activity grids for individual experiments were collated to generate an activity profile for each compound, from which a structure–activity map was established and could be compared to the literature data. Four core findings emerged. First, the activity grid notation is sufficiently expressive to denote highly complex mixture of activities. Second, systematic application of the notation reduces selection bias in data analysis. Third, many synthetic ion channels share highly sim- ilar activities and suggests the participation of the lipids, water, and ions in pore-formation. Lastly, the cyclodextrin half-channels are generally membrane active, and their activities are clearly modulated by structural variations. / Graduate

Le récepteur sigma-1 est une protéine chaperonne active dans des tissus lésés. Le récepteur sigma-1 est principalement exprimé dans le cerveau et joue un rôle neuroprotecteur dans l’ischémie ou les maladies neurodégénératives. Le récepteur sigma-1 est également exprimé dans des lignées cellulaires cancéreuses et des travaux récents suggèrent sa participation dans la prolifération et l’apoptose. Cependant, son rôle dans la carcinogenèse reste à découvrir. Les canaux ioniques sont impliqués dans de nombreux processus physiologiques (rythme cardiaque, influx nerveux, …). Ces protéines membranaires émergent actuellement comme une nouvelle famille de cibles thérapeutiques dans les cancers. Au cours de ma thèse, j’ai montré que le récepteur sigma-1 régule l’activité du canal potassique voltage-dépendent hERG et du canal sodique voltage-dépendent Nav1.5 respectivement dans des cellules leucémiques et des cellules issues de cancer du sein. J’ai également montré que le récepteur sigma-1, à travers son action sur l’adressage du canal hERG, augmente l’invasivité des cellules leucémiques en favorisant leur interaction avec le microenvironnement tumoral. Ces résultats mettent en évidence le rôle du récepteur sigma-1 sur la plasticité électrique des cellules cancéreuses et suggèrent l’intérêt de cette protéine chaperonne comme cible thérapeutique potentielle pour limiter la progression tumorale. / The sigma-1 receptor is a chaperone protein active in damaged tissues. The sigma-1 receptor is mainly expressed into brain and have a neuroprotective role in ischemia and neurodegenerative diseases. The sigma-1 receptor is also expressed into cancer cell lines and recent investigations suggest its involvement into proliferation and apoptosis. However, its role in carcinogenesis remains to delineating. Ion channels are involved in numerous physiological processes (heart beating, nervous influx, …). These membrane proteins currently emerge as a new class of therapeutic targets in cancer. During my thesis, I observed that the sigma-1 receptor regulates voltage-dependent potassium channel hERG and voltage-dependent sodium channel Nav1.5 activities respectively into leukemic and breast cancer cell lines. I also demonstrated that the sigma-1 receptor, through its action on hERG channel, increases leukemia invasiveness by promoting interaction with tumor microenvironment. These results highlight the role of the sigma-1 receptor on cancer cell electrical plasticity and suggest this chaperone protein as a potential therapeutic target to limit tumor progression.

L’infarctus de myocarde (IDM) est la première cause de mortalité cardiovasculaire dans le monde. La reperfusion la plus précoce possible est le seul traitement recommandé pour limiter la taille de l’infarctus, déterminant majeur de morbi-mortalité. Cependant, la reperfusion s’accompagne de lésions de reperfusion qui se sur-ajoutent aux lésions d’ischémie et qui sont caractérisées par la mort des cardiomyocytes. Actuellement, il n’existe aucune thérapie ciblant les lésions d’ischémie-reperfusion (IR) afin de limiter l’extension de la zone infarcie. La fréquence cardiaque (FC) est un facteur déterminant de la pathologie cardiaque. La plupart des épisodes ischémiques sont déclenchés par une accélération de la FC provoquant sur le myocarde un déséquilibre entre l’apport et la consommation d’oxygène. Une réduction de la FC se traduit par un allongement de la diastole ventriculaire, permettant une meilleure perfusion coronaire et l’oxygénation optimale du myocarde au repos et durant l’exercice. Ainsi, il semble évident qu’une réduction contrôlée de la FC pourrait limiter l’effort et la vulnérabilité du myocarde pendant l’épisode d’IR, ce qui représenterait un challenge dans le traitement de l’IDM mais aussi un intérêt majeur de santé publique.Les β-bloquants ont été largement étudiés dans ce contexte et ont montré des effets bénéfiques en termes de réduction de la mortalité post-IDM. Cependant, ils exercent en même temps des effets délétères sur le flux sanguin coronarien et la fonction contractile myocardique. Cette constatation met en évidence l’importance de la réduction sélective de la FC par l’inhibition des courants ioniques responsables de l’automatisme cardiaque. Le courant If et les courants calciques ICa,L et ICa,T (portés respectivement par les canaux Cav1.3 et Cav3.1) sont les principaux accélérateurs du rythme cardiaque. Des études cliniques ont montré les effets bénéfiques de la réduction sélective de la FC par l’ivabradine, inhibiteur du courant If, dans le cadre des maladies coronariennes et de l’insuffisance cardiaque. Néanmoins, des études récentes ont montré que le traitement par ivabradine peut induire un risque accru de mortalité cardiovasculaire et d’IDM. Ces résultats soulignent l’intérêt de développer de nouvelles thérapies visant à contrôler sélectivement la FC sans effets indésirables sur le myocarde et sur le pronostic des patients.L’objectif de ce travail de thèse est d’étudier le rôle de la FC au cours de l’IR myocardique et de valider sur des modèles murins de bradycardie l’hypothèse que la réduction de la FC est capable de limiter les lésions d’IR. Notre stratégie de travail est basée sur une approche génétique avec l’utilisation de souris knock-out pour les canaux calciques Cav1.3 et Cav3.1. Dans un premier temps, la réduction pharmacologique de la FC par l’ivabradine induit une diminution de la zone infarcie chez des souris contrôles, validant le concept dans notre modèle murin d’IR in vivo. Ensuite, les souris mutantes, dont la FC est plus lente, présentent aussi une réduction de la taille de l’infarctus suite au protocole d’IR, ce qui confirme l’implication de la FC dans la cardioprotection. Il existe une relation proportionnelle directe entre la taille de l’infarctus et la FC pendant chaque étape de l’IR et indépendamment de la souris modèle. De façon intéressante, cette relation est supprimée lorsque le système nerveux parasympathique est rendu inactif. A l’inverse, l’accélération de la FC dans un modèle ex vivo d’IR cardiaque engendre des effets délétères sur le myocarde, se traduisant par une augmentation de la taille de l’infarctus et une réduction du flux coronarien lors de la reperfusion.L’ensemble de ces résultats confirme l’effet cardioprotecteur de la réduction de la FC dans les lésions d’IR et nous permet d’envisager une application clinique pour la prise en charge des maladies ischémiques. / Acute myocardial infarction (AMI) is the major cause of cardiovascular mortality worlwide. Early reperfusion is the only treatment recommended to reduce infarct size, a major determinant of morbidity and mortality. However, reperfusion leads to reperfusion injury that precipitates in death the cells that survived the ischemic insult. To date, there is no therapy targeting ischemia-reperfusion (IR) injury to limit the extent of the infarcted area. Heart rate (HR) is a main determinant of cardiac pathology. Most ischemic episodes are triggered by an increase in HR inducing an imbalance between myocardial oxygen delivery and consumption. HR reduction results in a lengthening of the ventricular diastole, allowing better coronary perfusion and optimal oxygenation of the myocardium at rest and during exercise. Thus, it seems clear that a controlled reduction of HR may limit the effort and the vulnerability of the myocardium during the whole IR episode, which would represent a challenge in the treatment of AMI but also a major interest to public health.β-blockers have been extensively studied in this context and have shown beneficial effects in terms of reduction of post-AMI mortality. However, they also exert deleterious effects on myocardial coronary blood flow and contractile function. This finding highlights the importance of selective HR reduction through inhibition of ion currents responsible for cardiac automatism. The If current and the ICa,L and ICa,T calcium currents (mediated by Cav1.3 and Cav3.1 channels, respectively) are important accelerators of cardiac rhythm. Clinical trials have shown the beneficial effects of selective HR reduction by ivabradine, an inhibitor of the If current, in the context of coronary artery disease and heart failure. However, recent studies have shown that treatment with ivabradine may involve an increased risk of cardiovascular mortality and infarct. These results underscore the need to develop new therapies aimed to selectively control HR without adverse effects on the myocardium and prognosis of patients.The goal of this thesis is to study the role of HR during myocardial IR and to validate in murine models of bradycardia the hypothesis that HR reduction is able to limit IR injury. Our work strategy is based on a genetic approach with the use of knockout mice for Cav1.3 and Cav3.1 calcium channels. In a first time, pharmacological HR reduction by ivabradine induces infarct size decrease in control mice, validating the concept in our in vivo mouse model of IR. Genetically-modified mice in which these channels have been ablated have reduced heart rate and show a reduction in infarct size after the IR protocol, confirming the involvement of HR in cardioprotection. There is a direct relationship between infarct size and HR during each phase of IR and independently of the animal model. Surprisingly, this relation is suppressed when the parasympathetic nervous system is inactivated. Secondly, HR acceleration in an ex vivo model of cardiac IR generates deleterious effects on the myocardium, including infarct size increase and coronary flow reduction during reperfusion.These results confirm the cardioprotective effect of HR reduction against IR injury and allow us to consider a clinical application in the treatment of ischemic diseases.

A peçonha de anêmonas do mar é uma fonte conhecida de compostos bioativos, incluindo peptídeos, que atuam em canais voltagem-dependentes. Foram descritos 4 tipos de neurotoxinas de anêmonas do mar, que atuam em canais NaV e 4 tipos que atuam em canais KV. Essas toxinas têm permitido discriminar subtipos de canais voltagem-dependentes, estreitamente relacionados, e constituem poderosas ferramentas para estudar o funcionamento e estrutura desses canais. Neste estudo, foram isolados e caracterizados três peptídeos da fração neurotóxica da anêmona do mar Anthopleura cascaia. Esses peptídeos foram nomeados como AcaIII1425, AcaIII2970 e AcaIII3090, onde Aca faz referência a espécie e os números seguem os resultados obtidos nas etapas de purificação. A peçonha foi extraída por meio de estímulos elétricos e purificada por gel-filtração (Sephadex G-50) e fase reversa por HPLC (C-18). As massas moleculares foram obtidas por meio de MALDI-TOF, apresentando 3337,4 Da para a AcaIII1425, 4881,7 Da para a AcaIII2970 e 4880,5 Da para AcaIII3090. Através da técnica de voltage-clamp, esses peptídeos foram testados em diferentes subtipos de canais NaV e KV expressados em ovócito de Xenopus. As toxinas AcaIII2970 e AcaIII3090 retardam, de maneira seletiva, a inativação rápida dos subtipos rNaV1.3, mNaV1.6 e hNaV1.5, enquanto que as outras isoformas testadas permaneceram inalteradas. É importantemente salientar que, a AcaIII2970 e AcaIII3090 também foram examinadas no canal de inseto DmNaV1, revelando uma clara \"filo-seletividade\" na eficácia da atividade das toxinas. A AcaIII2970 e AcaIII3090 inibem fortemente a inativação do canal NaV de inseto, resultando em um aumento na amplitude do pico da corrente e removendo completamente a inativação rápida. Para quantificarmos essa \"filo-seletividade\", foram construídas curvas da dependência da concentração no retardo da inativação induzida pelas toxinas AcaIII2970 e AcaIII3090 nos canais em que apresentaram maior eficácia. Os IC50 foram obtidos após a plotagem dos dados em uma curva sigmoidal. Para a AcaIII2970, os seguintes valores de IC50 foram obtidos: DmNaV1 = 162,19 ± 11,22 nM, mNaV1.6 = 645,92 ± 18,52 nM, rNaV1.3 = 572,56 ± 44,96 nM. Para a AcaIII3090, os seguintes valores de IC50 foram obtidos: DmNaV1 = 99,03 ± 9,25 nM, mNaV1.6 = 158,30 ± 33,86 nM, rNaV1.3 = 371,60 ± 6,48 nM. A AcaIII1425 atua, bloqueando, de modo seletivo os subtipos rKV1.1, rKV1.6 e rKV4.3, enquanto que as outras isoformas testadas permaneceram inalteradas. Devido à maior especificidade da toxina AcaIII1425 pelos subtipos rKV1.1 e rKV1.6, foram realizados ensaios de bloqueio da corrente do canal em função da concentração da toxina (curva dose-resposta). Os valores de IC50 para os subtipos rKV1.1 e rKV1.6 são de 7642,98 ±1601,65 nM e 241,65 ±4,27 nM, respectivamente. Desta forma, a AcaIII1425 é cerca de 32 vezes mais potente em canais do subtipo rKV1.6 do que em relação aos canais do subtipo rKV1.1. A estrutura primária das toxinas foram determinadas por degradação de Edman. A sequência parcial da AcaIII2970 e AcaIII3090 revelou que estas são similares a toxinas de canal de sódio do tipo1 de anêmonas do mar. A sequência completa da AcaIII1425 não apresenta similaridade com toxinas de anêmonas do mar, mas é similar a toxinas de Conus e aranha que possuem um motivo estrutural conhecido como ICK. Dessa forma, propomos que a AcaIII1425 seja um novo grupo de toxinas de anêmonas do mar que bloqueiam KV. Dado o ineditismo da toxina AcaIII1425, foram feitos experimentos in silico para obtermos um maior refinamento do mecanismo de interação entre a toxina e o canal rKV1.6. Estes experimentos indicaram que diferentes regiões dos canais KV são importantes para a seletividade e potência da toxina, corroborando com as propostas que vem sendo descritas / The venom of sea anemones is a known source of bioactive compounds, including peptides that act on voltage-gated ion channels. Four types of neurotoxins from sea anemones, acting on NaV channels, and four types acting on KV channels, have been reported. These toxins have developed the ability to discriminate closely related subtypes of voltage-gated channels, making them powerful tools to studying the function and structure of these channels. In this study, we isolated and characterized three peptides of the neurotoxic fraction from the venom of the sea anemone Anthopleura cascaia. These peptides were named as AcaIII1425, and AcaIII2970 AcaIII3090, where Aca refers to the species and the following numbers refer to results obtained in the purification steps. The venom was milked by electric shock and purified by molecular exclusion (Sephadex G-50) and reverse phase HPLC (C-18). Their molecular weights are 3337.4 Da to AcaIII1425, 4881.7 Da to AcaIII2970 and 4880.5 Da to AcaIII3090, obtained through a MALDI-TOF. Using the voltage-clamp technique, we have assayed the effects of these peptides on different subtypes of NaV and KV channels expressed in Xenopus oocytes. AcaIII2970 and AcaIII3090 toxins selectively slow down the fast inactivation of rNaV1.3, mNaV1.6 and hNaV1.5 subtypes, while the other mammalian isoforms remained unaffected. Importantly, AcaIII2970 and AcaIII3090 were also examined in insect DmNaV1 channel, revealing a clear phyla-selectivity with regards to the efficacy of the toxin. AcaIII2970 and AcaIII3090 strongly inhibit the inactivation of the insect NaV channel, resulting in an increase in the amplitude of the peak current, and complete removal of the fast and steady-state inactivation. In order to quantify this \"phyla-selectivity\", curves of the concentration dependence of the delayed inactivation induced by AcaIII2970 and AcaIII3090 toxins channels with higher efficacy, were built. After plotting the data on a sigmoidal curve the IC50 values were obtained. For AcaIII2970, the following IC50 values were obtained: DmNaV1 = 162.19 ± 11.22 nM, mNaV1.6 = 645.92 ± 18.52 nM and rNaV1.3 = 572.56 ± 44.96 nM. For AcaIII3090, the following IC50 values were obtained: DmNaV1 = 99.03 ± 9.25 nM, mNaV1.6 = 158.30 ± 33.86 nM and rNaV1.3 = 371.60 ± 6.48 nM. AcaIII1425 acts, selectively, blocking rKV1.1, rKV1.6 and rKV4.3 subtypes, while the others isoforms tested remained unaltered. Due the higher specificity of AcaIII1425 to rKV1.1 and rKV1.6 subtypes, assays were performed to evaluate the blocking channel current versus toxin concentration (dose-response curve). IC50 values for the subtypes rKV1.6 and rKV1.1 are 7642.98 ± 1601.65 nM and 241.65 ± 4.27 nM, respectively. Thus, AcaIII1425 is about 32 times more potent in the rKV1.6 than in the rKV1.1 channel. The primary structure of the toxins was determined by the Edman degradation. The partial sequence of AcaIII2970 and AcaIII3090 revealed that these toxins are similar to the type 1 sodium channel sea anemones neurotoxins. The complete sequence of AcaIII1425 has no similarity with other sea anemone toxins, but is similar to the Conus and spider neurotoxins which have a structural motif known as ICK. Thus, we propose that AcaIII1425 comprises a new group of sea anemones toxins that block KV channels. Given the unprecedented nature of the toxin AcaIII1425, in silico assays were carried out in order to further refining the proposed mechanism underlying the interaction between the toxin and the rKV1.6 channel. The results indicate that, in agreement to what has been proposed elsewhere, different regions of the KV channels are important for the toxin selectivity and potency

Os efeitos inibitórios do eugenol (EUG) em canais para Na+ ativados por voltagem (NaV) mostrados anteriormente não são totalmente compatíveis com nossos resultados. Nós estudamos os efeitos do EUG em correntes macroscópicas de Na+ e os comparamos aos da lidocaína, um anestésico local, para referência. O EUG bloqueou, rápida e reversivelmente, correntes de Na+ mistas (TTX-S+TTX-R) assim como as correntes de Na+ TTX-R. As IC50 para a inibição das correntes mistas e TTX-R pelo EUG foram de 2,28 e 2,27 mmol/L, respectivamente. O bloqueio depende da freqüência de despolarizações. Nas correntes mistas, o EUG desloca a curva de ativação para a direita, a de inativação para a esquerda, não altera a cinética de inativação e retarda a recuperação da inativação, rápida e lenta, dos canais. Nas correntes TTX-R, o efeito é semelhante, exceto na curva de ativação, que não é deslocada. Nós concluímos que o EUG bloqueia os NaV por se ligar a estados conformacionais de repouso e inativados, rápido e lento. Os efeitos são semelhantes, mas não idênticos aos da lidocaína. / The previously described inhibitory effects of eugenol (EUG) on voltage-activated Na+ channels (Nav) are not compatible with our results. We have studied the effects of EUG on macroscopic Na+ currents and compared them to the effects of lidocaine, a local anesthetic. EUG blocked both mixed (TTX-S and TTX-R) and TTX-R Na+ currents in a fast and reversible manner. The values of IC50 for the inhibition of mixed and TTX-R currents were 2.28 and 2.27 mmol/L respectively. The blockade depends on frequency of depolarizing pulses. In mixed currents EUG displaced the activation curve to the right, the inactivation curve to the left, does not alter the inactivation kinetics and retards the recovery from inactivation, fast and slow, of the Na+ channels. In TTX-R currents, EUG effects were similar, except on the activation curve, which was not shifted. In conclusion, EUG blocks Nav by binding to the resting and inactivated conformational states of channels, fast and slow. EUG effects resembles lidocaine ones, but are not identical.