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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23809号 / 医博第4855号 / 新制||医||1058(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邊 直樹, 教授 浅野 雅秀, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>Ion permeation through voltage gated sodium channels is modulated by many drugs and toxins. However, the atomistic mechanisms of action of most these ligands are poorly understood. This study focuses on three compounds: a steroidal alkaloid batrachotoxin (BTX), a pyrethroid insecticide deltamethrin, and an alkylamide insecticide BTG 502, which bind to distinct but allosterically coupled receptor sites. BTX belongs to the class of the sodium channel agonists (activators), which cause persistent channel activation by inhibiting channel inactivation. Traditionally, BTX is believed to bind at the channel-lipid interface and allosterically modulate ion permeation through the channel. However, in the last decade, amino acid residues that affect BTX action have been found in the pore-facing inner helices of all four domains, suggesting that BTX binds in the channel pore (Tikhonov and Zhorov, <em>FEBS Letters</em> 2005). An alkylamide insecticide BTG 502 reduces sodium currents and antagonizes the action of BTX on cockroach sodium channels, suggesting that it also binds inside the pore. Conversely, pyrethroids bind at the lipid-exposed cavity formed by a short intracellular linker-helix IIS4-S5 and transmembrane helices IIS5 and IIIS6.</p> <p>In this study we first developed a new method of electrostatic-energy calculations, a new protocol of ligand docking, and tested this methodology on 60 ligand-protein complexes of known structure (Garden and Zhorov 2010). We then applied this methodology to rationalize effects of various mutations in the domain III inner helix of the cockroach sodium channel BgNav1.1 on the action of BTX, BTG 502 and deltamethrin. Our collaborators, Dr. Ke Dong et al. from Michigan State University, mutated all residues in the pore-lining helix of domain III (IIIS6) and found several new BTX and BTG 502 sensing residues. Using these data along with other published data on BTX- and deltamethrin-sensing residues as distance constrains, we docked BTX, BTG 502 and deltamethrin in a Kv1.2-based homology model of the open BgNav1.1 channel. We arrived at models, which are consistent with all currently available data on the action of the ligands. In the BTX-binding model, the toxin adopts a “horseshoe” conformation and binds in the channel pore with the horseshoe plane normal to the pore axis. In this binding mode BTX allows would allow ion permeation through the hydrophilic inner face of the horseshoe, and resist the activation-gate closure. Various BTX moieties interact with known BTX sensing residues. In particular, the tertiary ammonium group of BTX is engaged in cation-p interactions with the newly discovered BTX-sensing residue Phe³ⁱ¹⁶. In the BTG 502-binding model, the ligand wraps around IIIS6 making direct contacts with all known BTG 502-sending residues, including buried residues on the IIIS6 helix side, which does not face the pore. Deltamethrin binds within the cavity formed by the linker-helix IIS4-S5, the outer helix IIS5, and the inner helix IIIS6 at the interface between domains II and III, similar to the pyrethroid-binding mode predicted by others (O'Reilly, Khambay et al. 2006). Our study revealed a unique mode of action of BTX in which the agonists enables the ion permeation by forming a “channel within a channel”. We also found that the BTG 502 receptor site overlaps with receptors for BTX and deltamethrin, which are located in different parts of the channel.</p> / Doctor of Philosophy (PhD)

Pathological cardiac stressors, including persistent hypertension or damage from ischemic heart disease, induce a chronic demand for enhanced contractile performance of the heart. The cytosolic calcium (Ca2+) transient that regulates myocyte contraction must be persistently increased in disease states in order to maintain cardiac output to sustain the metabolic requirements of the body. Associated with this enhanced intracellular Ca2+ ([Ca2+]i) state is pathological cardiac myocyte hypertrophy, which results in large part from the activation of Ca2+-dependent activation of calcineurin (Cn)-nuclear factor of activated T cells (NFAT) signaling. The puzzling feature of this hypertrophic signaling is that the cytosolic [Ca2+] that controls contractility appears to be separate from the [Ca2+] which activates Cn-NFAT signaling. The overarching theme of this dissertation is to explore the source and spatial constraints of pathological hypertrophic signaling Ca2+ and to investigate how it is possible that sensitive and finely tuned Ca2+-dependent signaling pathways are regulated in the background of massive Ca2+ fluctuations that oscillate between 100nM and upwards of 1-2&#956;M during each cardiac contractile cycle. L-type Ca2+ channels (LTCCs) are a major source of Ca2+ entry in cardiac myocytes and are known to play an integral role in the initiation of myocyte excitation contraction-coupling (EC-coupling). We performed a number of experiments to show that a small population of LTCCs reside outside of EC-coupling domains within caveolin (Cav-3) signaling microdomains where they provide a local source of Ca2+ to activate Cn-NFAT signaling. We designed a Cav-targeted LTCC blocker that could eliminate Cn-NFAT activation but did not reduce myocyte contractility. The activity of Cav-targeted LTCCs could also be upregulated to enhance hypertrophic signaling without affecting contractility. Therefore, we believe that caveolae-localized LTCCs do not participate in EC-coupling, but instead act locally to control the coordinated activation of Cn-NFAT signaling that drives pathological remodeling. Transient Receptor Potential (TRP) channels are also thought to provide a source of Ca2+ for activation of hypertrophic signaling. The canonical family of TRP channels (TRPC) is expressed at low levels in normal adult cardiac tissue, but these channels are upregulated in disease conditions which implicates them as stress response molecules that could potentially provide a platform for hypertrophic Ca2+ signaling. We show evidence that TRPC channel abundance and function increases in cardiac stress conditions, such as myocardial infarction (MI), and that these channels are associated with hypertrophic responses, likely through a Ca2+ microdomain effect. While we found that TRPC channels housed in caveolae membrane microdomains provides a source of [Ca2+] for induction of cardiac hypertrophy, this effect also requires interplay with LTCCs. We also found that TRPC channels have negative effects on cardiac contractility, which we believe are due to local activation of Ca2+/calmodulin-dependent protein kinase (CaMKII) and subsequent modulation of ryanodine receptors (RyRs). Further, we found that inhibiting TRPC channels in a mouse model of MI led to increased basal myocyte contractility and reduced hypertrophy and cardiac structural and functional remodeling, as well as increased survival. Collectively, the data presented in this dissertation provides comprehensive evidence that Ca2+ regulation of Cn-NFAT signaling and resultant pathological hypertrophy can be coordinated by spatially localized and regulated Ca2+ channels. The compartmentalization of LTCCs and TRPC channels in caveolae membrane microdomains along with pathological hypertrophy signaling effectors makes for an attractive explanation for how Ca2+-dependent signaling pathways are regulated under conditions of continual Ca2+ transients that mediate cardiac contraction during each heart beat. Elucidation of additional Ca2+ signaling microdomains in adult cardiac myocytes will be important in more comprehensively resolving how myocytes differentiate between signaling versus contractile Ca2+. / Molecular and Cellular Physiology

Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2009. / The human ether-à-go-go-related gene (HERG) encodes the protein underlying the cardiac potassium current IKr. Mutations in HERG may produce defective channels and cause Long QT Syndrome (LQTS), a cardiac disease affecting 1 in 2500 people. The disease is characterised by a prolonged QT interval on a surface electrocardiogram and has a symptomatic variability of sudden cardiac death in childhood to asymptomatic longevity. We hypothesised that genetic variation in the proteins that interact with HERG might modify the clinical expression of LQTS. Yeast two-hybrid methodology was used to screen a human cardiac cDNA library in order to identify putative HERG N-terminus ligands. Successive selection stages reduced the number of putative HERG ligandcontaining colonies (preys) from 268 to 8. Putative prey ligands were sequenced and identified by BLAST-search. False positive ligands were excluded based on their function and subcellular location. Three strong candidate ligands were identified: Rhoassociated coiled-coil containing kinase 1 (ROCK1), γ-sarcoglycan (SGCG) and microtubule-associated protein 1A (MAP1A). In vitro co-immunoprecipitation (Co-IP) and mammalian two-hybrid (M2H) analyses were used to validate these proposed interactions, but failed to do so. This should be further investigated. Analysis of confirmed interactions will shed light on their functional role and might contribute to understanding the symptomatic variability seen in LQTS.

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.

O sistema colinérgico possui papel importante na modulação dos processos de aprendizagem e memória. Nesse trabalho, avaliamos a evocação da memória de ratos de 3, 6, 12, 18 e 22 meses em esquiva inibitória e, também, analisamos a densidade dos receptores nicotínicos a7 e da subunidade b2 no hipocampo, além de enzimas colinérgicas, a colina acetiltransferase e a acetilcolinesterase. Os grupos com 18 e 22 meses apresentaram uma redução na evocação da memória de longa duração (MLD) e um aumento na densidade de receptores a7 nas piramidais de CA3 e nas células de PoDG, este apenas em 22 meses. Em contrapartida, os demais marcadores colinérgicos estavam inalterados nas diferentes idades. Assim, a redução na evocação da MLD dos animais de 18 e 22 meses acompanhada do aumento na densidade de receptores a7 pode ser caracterizada como: 1) estratégia adaptativa do envelhecimento (plasticidade colinérgica em neurônios glutamatérgicos) ou 2) modulação nos interneurônios GABAérgicos desencadeando, assim, um bloqueio no processamento da informação e o declínio da memória. / The cholinergic system plays an important role in modulating learning and memory. In this work, we evaluated the memory recall of 3, 6, 12, 18 and 22 months old rats in passive avoidance apparatus and we also evaluated, in the hippocampus, the density of a7 nicotinic receptors, b2 subunit receptor, choline acetiltransferase (ChAT) and acetilcholinesterase (AChE). The 18 and 22 months old showed a deficit in long-term memory (LTM) and an increase in a7 density in CA 3 pyramidals cells (py CA3) and polymorphic cells of Dentate Gyrus (PoDG).On the other hand, the other cholinergic markers were unchanged with aging. Thus, the deficit in LTM observed in 18 and 22 months combined with the increased a7 receptors in PoDG and py CA 3 in the hippocampus could be explained by: 1) as an adaptative strategy of aging (cholinergic plasticity in glutamatergic neurons) or 2) as a modulation in GABA interneurons triggering inhibition in the memory evocation.

Introdução: As extrassístoles ventriculares e supraventriculares (EV e ESSV) são frequentes e muitas vezes sintomáticas. O íon magnésio (Mg) desempenha um papel importante na fisiologia do potencial de ação transmembrana celular e do ritmo cardíaco. Objetivo: Avaliar se a administração do Pidolato de Magnésio (PMg) em pacientes com EV e ESSV é superior ao placebo (P) na melhora dos sintomas e densidade das extrassístoles(DES). Métodos: Estudo duplo-cego, randomizado, com 90 pacientes sintomáticos consecutivos, com mais de 240/EV ou ESSV ao Holter de 24 horas e selecionados para receber P ou PMg. Para avaliar a melhora da sintomatologia, foi feito um questionário categórico e específico de sintomas relacionado às extrassístoles. Foi considerada significante uma redução de mais de 70% na DES por hora após o tratamento. A dose do PMg foi de 3,0g/dia por 30 dias, equivalente a 260mg do elemento Mg. Nenhum paciente tinha cardiopatia estrutural ou insuficiência renal. Resultados: Dos 90 pacientes estudados, 49 eram do sexo feminino (54,4%). A faixa etária variou de 16 a 70 anos. No grupo PMg, 77,8% dos pacientes tiveram redução maior que 70%, 6,7% deles entre 50% a 70% e, somente 13,3% dos pacientes com redução menor que 50% na DES . No grupo P, 44,4% dos pacientes tiveram melhora de apenas 30% na frequência de extrassístoles (p<0,001). A melhora dos sintomas foi alcançada em 91,1% dos pacientes do grupo PMg, comparada com somente 15,6% do grupo P(p<0,001). Conclusão: A suplementação de Mg por via oral reduziu a DES, resultando em melhora dos sintomas. Estudos clínicos e moleculares são necessários para avaliar o Mg intracelular e orientar quanto às necessidades diárias deste íon, evidenciar as prováveis deficiências e esclarecer melhor como prevenir e tratar pacientes com extrassístoles sintomáticas e sem cardiopatia estrutural. / Introduction: Premature ventricular and supraventricular complexes (PVC and PsVC) are frequent and often symptomatic. The magnesium (Mg) ion plays a role in the physiology of cell membranes and cardiac rhythm. Objective: We evaluated whether the administration of Mg Pidolate (MgP) in patients with PVC and PsVC is superior to placebo (P) in improving symptoms and arrhythmia frequency. Methods: Randomized double-blind study with 90 consecutive symptomatic patients with more than 240 PVC or PsVC on 24-hour Holter monitoring who were selected to receive placebo or MgP. To evaluate symptom improvement, a categorical and a specific questionnaire for symptoms related to PVC and PsVC was made. Improvement in premature complex density (PCD) per hour was considered significant if percentage reduction was >=70% after treatment. The dose of MgP was 3.0 g/day for 30 days, equivalent to 260mg of Mg element. None of the patients had structural heart disease or renal failure. Results: Of the 90 patients, 49 were female (54,4%). Ages ranged from 16 to 70 years old. In the MgP group, 77.8% of patients had a PCD reduction >70%, 6,7% of them from 50% to 70%, and only 13.3% <50%. In the P group, 44,4% showed slight improvement, <30%, in the premature complexes frequency (p<0.001). Symptom improvement was achieved in 91.1% of patients in the MgP group, compared with only 15.6% in the P group (p<0.001). Conclusions: Oral Mg supplementation decreases PCD, resulting in symptom improvement. Clinical and molecular studies are needed to evaluate intracellular Mg and develop better targets for the daily needs of this ion, show probable deficiencies, and explain how to prevent and better treat patients with symptomatic premature ventricular, and supraventricular complexes and no apparent heart disease.