L’hypertension pulmonaire (HTP) est la principale pathologie de la circulation pulmonaire. Elle secaractérise par une augmentation maintenue de la pression dans les artères intrapulmonaires (AIP) (> à 25mmHg au repos). Cette pression exerce des forces d’étirement au niveau des cellules musculaires lisses desartères intrapulmonaires (CML d’AIP). Au niveau des CML, des canaux mécanosensibles appelés des SAC(« stretch-activated channels ») permettent de transformer un stimulus mécanique d’étirement en uneréponse biologique de contraction : c’est le tonus myogénique. Le Ca2+ est un second messager cellulairequi peut être aussi bien mobilisé depuis le milieu extracellulaire que depuis les réserves calciquesintracellulaires. Une augmentation de sa concentration cytoplasmique induit la contraction des CML. Grâceà des techniques de patch-clamp, de microspectrofluorimétrie, d’immunomarquages et à une approchepharmacologique, nous avons mis en évidence les voies de signalisations calciques qui sont mises en placeà la suite d’un étirement des CML d’AIP. Les expériences ont été réalisées à la fois chez des rats normaux etsur deux modèles de rats présentant une HTP (rats hypoxique chroniques et rats monocrotalines). Lesrésultats montrent que chez les rats normaux un étirement induit un influx de Ca2+ par les SAC. Cet influxcalcique est amplifié par (1) une hyperpolarisation de la membrane plasmique via l’activation de canauxBKCa, (2) une sortie de Ca2+ par les récepteurs à la ryanodine de type 1 (RyR1) du réticulum sarcoplasmique(RS) sous-membranaire. Afin de rétablir l’homéostasie calcique, les mitochondries tamponnent le Ca2+cytosolique. Chez les rats souffrant d’HTP, l’influx de Ca2+ par les SAC et l’amplification calcique par les RyRsont plus importants. Cette amplification est due à une réorganisation des réserves calciquesintracellulaires, notamment chez les rats monocrotalines. De plus, une association fonctionnelle entre lesréserves calciques du RS et les cavéoles conduit à des réponses calciques plus importantes après unétirement chez les rats HTP. Enfin, nous avons mis en évidence la présence de canaux mécanosensiblesPiezo1 dans les AIP de rats. En conclusion, l’organisation spatiale des partenaires calciques au sein des CMLd’AIP est importante pour la signalisation cellulaire et joue un rôle majeur dans l’HTP. / Pulmonary hypertension (PH) is the main disease of the pulmonary circulation. This pathology ischaracterized by an increase of the intrapulmonary arterial (PA) pressure at rest (> 25 mmHg). This pressureexerts stretch forces on pulmonary arterial smooth muscle cells (PASMC). Stretch-activated channels (SAC)are present in PASMC and are able to transform a mechanical stimulus of stretch into a biological responseof contraction, a phenomenon called myogenic tone. Ca2+ is a second messenger that can be mobilizedfrom both the extracellular medium and intracellular Ca2+ stores. An increase of the intracellular Ca2+concentration ([Ca2+]i) leads to PASMC contraction. Using patch-clamp, microspectrofluorimetry,immunostainings and a pharmacological approach, we highlight Ca2+ signaling pathways induced by stretchin PASMC. Experiments were performed in normal rats and in two models of PH (chronically hypoxic ratsand monocrotaline rats). We showed that in normal rats a stretch induces a Ca2+ influx through SAC whichis amplified by (1) a plasma membrane hyperpolarization by BKCa channels and (2) a Ca2+ amplification bysubplasmalemnal ryanodine receptor 1 (RyR) of the sarcoplasmic reticulum (SR). Besides, mitochondria areinvolved in buffering cytoplasmic Ca2+. In PH rats, the Ca2+ influx by SAC and the Ca2+ release by RyR areenhanced due to a reorganization of intracellular Ca2+ stores. Furthermore, a functional associationbetween SR and caveolae conduce to a much greater amplification of the stretch-induced Ca2+ increase inPH rats. Finally, we showed that the mechanosensitive channel Piezo1 is expressed in PA. To conclude, thespatial organization of Ca2+ stores in PASMC is important for cell signaling and plays a casual role in PH.

No description available.

<p>  </p> <p>Calcium influx into pancreatic beta-cells is required for proper beta-cell growth and function. While the functional significance of calcium influx into the beta-cells is known, the significance of release of calcium from intracellular stores is less understood. Calcium-induced calcium release (CICR) is a process by which calcium influx into the cell through voltage-gated calcium channels activated release of calcium from intracellular stores. The functional significance of CICR is well understood in cardiac and vascular muscle cells in regard to excitation-contraction coupling. However, the functional significance of CICR in beta-cells in not well understood. </p> <p>To investigate the role of RyR2 in pancreatic beta-cell function, we utilized CRISPR-Cas9 gene editing to delete RyR2 from the rat insulinoma INS-1 cell line. we found that RyR2KO cells displayed an enhanced glucose-stimulated Ca2+ integral (area under the curve; AUC) which was sensitive to inhibition by the IP3R antagonist, xestospongin C. Loss of RyR2 also resulted in a reduction in IRBIT protein levels. Therefore, we deleted IRBIT from INS-1 cells (IRBITKO) and found that IRBITKO cells also displayed an increased Ca2+ AUC in response to glucose stimulation. RyR2 KO and IRBIT KO cells had reduced glucose-stimulated insulin secretion and insulin content. RT-qPCR revealed that <em>INS2</em> transcript levels were reduced in both RyR2KO and IRBITKO. Nuclear localization of AHCY were increase in both the RyR2KO and IRBITKO cells, corresponding with increased levels of insulin gene methylation. Proteomic analysis revealed that deletion of RyR2 or IRBIT resulted in differential regulation of 314 and 137 proteins, respectively, with 41 in common. Our results suggest that RyR2 and IRBIT activity regulate insulin content, insulin secretion, and regulate the proteome in INS-1 cells</p> <p>We next sought to assess the consequences on cellular Ca2+ handling in the absence of RyR2 and IRBIT in INS-1 cells. Store-operated Ca2+ entry (SOCE) stimulated with thapsigargin was reduced in RyR2KO cells compared to controls, but this was not different in IRBITKO cells. STIM1 protein levels were not different between the three cell lines. Basal and carbachol stimulated phospholipase C (PLC) activity was reduced specifically in RyR2KO cells and not IRBITKO cells. However, basal PIP2 levels were elevated in both RyR2KO and IRBITKO cells. Insulin secretion stimulated by tolbutamide was reduced in RyR2KO and IRBITKO cells compared to controls, but this was still potentiated by an EPAC-selective cAMP analog in all three cell lines. Cortical f-actin is known to regulate insulin secretion, and levels were markedly reduced in RyR2KO cells compared to control INS-1 cells. Whole-cell Cav channel current density was reduced in RyR2KO cells compared to controls, and Ba2+ current was significantly reduced by PIP2 depletion preferentially in RyR2KO cells over control INS-1 cells. Action potentials stimulated by 18 mM glucose were more frequent in RyR2KO cells compared to controls, and insensitive to the SK channel inhibitor apamin. Taken together, these results suggest that RyR2 plays a critical role in regulating PLC activity and PIP2 levels via regulation of SOCE. RyR2 also regulates beta-cell electrical activity by controlling Cav current density, via regulation of PIP2 levels, and SK channel activation.</p> <p>Lastly, we investigated the role of PDE subtypes cAMP in INS-1 cells and human islets. We utilized subtype selective inhibitors of PDE1, PDE3 and PDE8 to assess the potential of these PDEs as potential therapeutic targets. We found that PDE1 is the primary subtype in INS-1 cells, whereas PDE3 appears to be required in human pancreatic β-cells by cAMP measurements. PDE1 inhibition potentiated glucose-stimulated to the greatest extent in both INS-1 cells and human islets. PDE1 inhibition potentiated CREB phosphorylation to the greatest extent and was also capable of mitigating lipotoxicity in INS-1 cells. Collectivity, this work highlights the role of cAMP compartmentalized signaling in pancreatic β-cells, and this has drastic effects on pancreatic beta-cell function and survival.</p>

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24878号 / 医博第5012号 / 新制||医||1068(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 萩原 正敏, 教授 湊谷 謙司, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Myocardial infarction (MI) leads to heart failure (HF) and premature death. The respective roles of myocyte death and depressed myocyte contractility in the induction of HF after MI have not been clearly defined. Cardiac ryanodine receptor (RyR2) has been linked to cardiac arrhythmias and HF. It has been controversial that protein kinase A (PKA) hyperphosphorylation of the RyR2 at a single residue, Ser-2808 is a critical mediator of progressive cardiac dysfunction after MI. We developed two mouse models. In one model with beta2a (LTCC subunit) overexpression we could prevent depressed myocyte contractility after MI and use it to test the idea that preventing depression of myocyte Ca2+ handling defects could avert post MI cardiac pump dysfunction. In the other model, mice with Ser2808 in RyR2 replaced by alanine (S2808A) to prevent the phosphorylation at this site were used to determine whether loss of functional PKA phosphorylation site at Ser2808 could protect against cardiac dysfunction progression after MI. beta2a myocytes had increased Ca2+ current; contraction and Ca2+ transients (versus controls) and beta2a hearts had increased performance before MI. After MI, ventricular dilation, myocyte hypertrophy, and depressed cardiac pump function was greater in beta2a versus control hearts. There was also an increased rate of myocyte death in beta2a hearts after MI and survival was significantly reduced in these animals. We concluded that maintaining myocyte contractility after MI, by increasing Ca2+ influx, depresses rather than improves cardiac pump function. Baseline cardiac function was similar in wild type (WT) and RyR-S2808A mice before MI. After MI, there was no significant difference between WT and RyR-S2808A mice in EF and FS at 4 weeks. ICa-L &euro; in WT and RyR-S2808A myocytes was not significantly different. There were significant ISO responses in all myocytes, and no appreciable differences in responsiveness were found. Contractions and Ca2+ transients were not significantly different in WT and RyR-S2808A myocytes after MI. In conclusion, preventing PKA phosphorylation of RyR at Ser2808 after MI does not protect the heart or its myocytes. The role of RyR phosphorylation at other sites on abnormal Ca2+ handling in diseased hearts is yet to be defined. / Physiology

Status epilepticus (SE) is a major clinical emergency resulting in continuous seizure activity that can cause brain injury and many molecular and pathophysiologic changes leading to neuronal plasticity. The neuronal plasticity following SE-induced brain injury can initiate epileptogenesis and lead to the ultimate expression of acquired epilepsy (AE), characterized clinically by spontaneous, recurrent seizures. Epileptogenesis is the process wherein healthy brain tissue is transformed into hyperexcitable neuronal networks that produce AE. Understanding these alterations induced by brain injury is an important clinical challenge and can lend insight into possible new therapeutic targets to halt the development of AE. Currently there are no means to prevent epileptogenesis following brain injury; thus, the elucidation of mechanisms of epileptogenesis will be useful in preventing the long-term clinical sequela. It has been demonstrated in vivo that calcium (Ca2+) dynamics are severely altered during SE and that elevations in intracellular Ca2+ ([Ca2+]i) in hippocampal neurons are maintained well past the duration of the injury itself (Ca2+ plateau). Here we report that similar changes in [Ca2+]i are observed in the hippocampal neuronal culture model of SE-induced AE. As an important second messenger, the maintenance of a Ca2+ plateau following injury can lead to several changes in gene expression, neurotransmitter release, and overall, neuronal plasticity. Thus, changes in post-SE [Ca2+]i and Ca2+ homeostasis may be important in understanding epileptogenesis and eventually preventing the progression to chronic epilepsy. This dissertation examines the development and maintenance of the Ca2+ plateau after SE and demonstrates the novel finding that pharmacological modulation of [Ca2+]i following SE may inhibit epileptogenesis in vitro.

L’escarre est une pathologie liée à l’immobilité des patients, accidentelle ou associée à des comorbidités. Les premières lésions apparaissent dans le muscle avant de se développer en plaie cutanée sans que les mécanismes physiopathologiques de cette atteinte ne soient encore connus. L’objectif principal de cette thèse était d’identifier des voies de signalisation intervenant de manière précoce dans le développement des escarres au travers d’une étude transversale. Nous formulons l’hypothèse qu’une compression musculaire induit une altération de l’homéostasie calcique musculaire par atteinte des canaux calciques du réticulum sarcoplasmique (les récepteur de la ryanodine de type 1, RyR1) conduisant à la lésion du tissu musculaire et une inflammation du tissu sous-cutané.Sur un modèle animal de compression de 2 heures, à 100 mmHg, nous avons identifié une initiation des voies apoptotiques et une augmentation du stress oxydant des muscles de la paroi abdominale. Le RyR1 y est hyper-nitrosylé et hyper-oxidé et sa protéine régulatrice, calstabin1 se dissocie sous l’action de ce remodelage, ce qui entraîne une fuite calcique du réticulum sarcoplasmique vers le cytosol. Cette dysfonction n’est pas réversible à 3 jours post-compression mais il est possible de la prévenir en traitant les souris avec un rycal qui bloque la déplétion de la calstabin1. En clinique, chez une cohorte de patients paraplégiques, porteurs d’escarres, nous avons identifiés un remodelage du RyR1 dans les muscles paralysés (comparaison intra patient avec une biopsie saine) et une hypoxie des tissus sous la lésion médullaire. La dissociation de la calstabin1 au RyR1 a pu être corrélée à la pression moyenne et maximale exercée sur la peau de la zone sacrée du patient allongé en regard du muscle biopsié.Ce travail de thèse a permis de préciser les voies de signalisation intervenant de manière précoce dans le développement des escarres dans le muscle squelettique. Une compression mécanique induit une augmentation du stress oxydant, un remodelage du RyR1 et une dysfonction du canal à cause de la perte de l’interaction RyR1/calstabin1. Ces résultats ouvrent des perspectives intéressantes sur des traitements préventifs pharmacologiques et de suivi non-invasif qui permettront de retarder l’apparition des premières lésions musculaires. / Pressure ulcer is a pathology related to patient immobility, which can be either accidental or incidental to comorbidities. The first damage are located in muscle tissue before developing in cutaneous breakdown per an unclear pathophysiology. The core objective of my PhD was to identify the early signaling pathways involved in pressure ulcer development, through a transversal study. We hypothesized that muscle compression will induce a calcium imbalance in muscles by a dysfunction of calcium channels from the sarcoplasmic reticulum (Ryanodine receptor isoform 1, RyR1) which will lead to muscle damage and sub-cutaneous inflammation.Mice model of a 100 mmHg, 2 hours compression of abdominal muscles was used to identify the apoptotic pathway initiation and a rise of oxidative stress. RyR1 is hyper-nitrosylated and hyper-oxydated thus this remodeling induces depletion of RyR1 stabilizing protein, calstabin1, and the resulting leaky phenotype increases intracellular calcium concentration. This channel functional impairment was not reversible up to 3 days post-compression but it was possible to prevent it through rycal treatment, protecting the binding calstabin1/RyR1. In a clinical trial, we identified from a paraplegic population with existing pressure ulcers, a RyR1 remodeling in paralyzed muscles (intra patient comparison with a healthy muscle biopsy) and a hypoxia of tissues below the spinal cord injury. Calstabin1 dissociation was correlated to the mean and peak pressure intensity of interface pressure applied over the sacrum skin of the bedridden patient directly above the biopsy location.This thesis project focused on early signaling pathways participating in pressure ulcer in skeletal muscle. A mechanical strain induces an increase of the intracellular redox state, post translational RyR1 modifications and a channel dysfunction because of calstabin1 depletion. The significance of my work is to propose both pharmacology and non-invasive monitoring solutions to prevent first muscle damage in pressure ulcer development.

The tightly regulated openings of the cardiac ryanodine receptor (RyR2) help to ensure that intracellular Ca2+- release from the sarcoplasmic reticulum (SR) can only occur when heart contractions are required. Usually this process is self-regulatory, where Ca2+ both activates and inhibits release of further Ca2+ from the SR. In the progression of heart failure some of this control is lost and in rest periods Ca2+ can leak from the SR into the cytosol. Recent evidence has suggested that Zn2+- dyshomeostasis may contribute to SR Ca2+- leak but the underlying mechanism is unclear. Using single channel electrophysiological studies in combination with live cell imaging of HEK 293 and fibroblasts, this study reveals that Zn2+, along with Ca2+ and the inhibitor Mg2+, plays a physiological role in the grading of Ca2+- release via RyR2. Importantly the data reveal that pathophysiological concentrations of Zn2+ (> 100pM) within the cytosol remove the requirement of Ca2+ to activate RyR2, resulting in irregular channel activity even in the presence of Mg2+. This increase in channel open probability due to Zn2+ is known to be associated with increased Ca2+- release events such as Ca2+ sparks suggesting that Zn2+ is a regulator of the SR Ca2+-leak current. A potential source of releasable Zn2+, which could modulate RyR2 activity in cardiomyocytes, are the acidic organelles (endosomes and lysosomes). This study provides key evidence that the two pore channels (TPCs), which are expressed on the surface of these organelles, are candidate channels for ligand-gated release of Zn2+. Importantly this research demonstrates that dysregulated Zn2+ homeostasis, resulting in elevated Zn2+ within the lysosome, has severe consequences upon cellular Ca2+- release from fibroblasts, which is primarily the result of Zn2+ acting as a pore blocker of TPC2. Together these data reveal a key role of Zn2+ as a second messenger which can regulate intracellular Ca2+- release in both health and disease.

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.