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Ca2+ Handling in a Mice Model of CPVT Yueyi Wang

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Ca2+ Handling in a Mice Model of CPVT Yueyi Wang Ca2+ handling in a mice model of CPVT Yueyi Wang To cite this version: Yueyi Wang. Ca2+ handling in a mice model of CPVT. Cardiology and cardiovascular system. Université Paris Saclay (COmUE), 2016. English. NNT : 2016SACLS156. tel-01552304v2 HAL Id: tel-01552304 https://tel.archives-ouvertes.fr/tel-01552304v2 Submitted on 6 Jul 2017 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. NNT : 2016SACLS156 THESE DE DOCTORAT DE L’UNIVERSITE PARIS-SACLAY PREPAREE A L’UNIVERSITE PARIS SUD EN CHATENAY-MALABRY ECOLE DOCTORALE N° 569 Innovation Thérapeutique : du fondamental à l’appliqué Physiopathologie Moléculaire et Cellulaire Par Mme YueYi WANG Ca2+ handling in a mouse model of Catecholaminergic Polymorphic Ventricular Tachycardia Thèse présentée et soutenue à Châtenay-Malabry, le 7 juillet 2016 : Composition du Jury : M Pous Christian, Professeur, Université Paris-Saclay, Châtenay-Malabry, France, Président du Jury M Mangoni Matteo E., Directeur de Recherche, CNRS, Montpellier, France, Rapporteur M Egger Marcel, Professeur, University of Bern, Suisse, Rapporteur M Charpentier Flavien, Directeur de Recherche, Inserm, Nantes, Examinateur Mme Zorio Esther, MD, Hospital Universitario y Politécnico La Fe, Valencia, Spain, Examinatrice Mme Gomez Ana-Maria, Directrice de Recherche, Inserm, Châtenay-Malabry, France, Directeur de thèse Acknowledgment My PhD is an amazing experience and it would not have been possible without the support and guidance that I received from many people. I would like to express my deepest gratitude to my supervisor Dr. Ana Maria Gomez for her professional guidance, patience, constant encouragement and support. Her guidance helped me in all the time of research and writing of this thesis. Her advices on research, my career and my personal life have been priceless. I have learnt a great deal from her and I could not imagine having a better supervisor. I sincerely thank Dr. Jean-Pierre Benitah and Dr. Rodolphe Fischmeister, who opened the door and accepted me into this big family about five years ago, making all these life-changing experiences possible. I also thank Dr. Jean-Pierre Benitah for the guidance, valuable comments and suggestions for my research, and help that he kindly offered during my PhD. I greatly appreciate the contribution to my project by the authors in my paper. I thank Pietro Mesirca and Matteo Mangoni for the patch-clamp experiments and analysis, Elena Marqués-Sulé for the ECG arrhythmia analysis, Alexandra Zahradnikova Jr for the first ECGs implant surgery, recording and analysis, Olivier Villejouvert for some ECG recording and analysis, Esther Zorio for the clinical data and arrhythmia analysis, Jean-Pierre Benitah for supervising the work, and Ana Maria Gomez for supervising the work and writing the IDL programs. I also thank all the authors for edited the manuscript of the paper. I am grateful to Alexandra Zahradnikova Jr and Ana Llach for their help in my experiments and my life. I am also grateful to Riccardo Rizzetto, Elena Marqués-Sulé, Olivier Villejouvert and Thassio Mesquita for their support and being great friends. I also thank Francoise Boussac for administrative assistance. Many thanks to all my wonderful colleagues for offering precious help and letting my PhD life enjoyable. I I gratefully acknowledge the financial support of China Scholarship Council (CSC) for the past four years. Last but not the least, I would like to thank my family: my Mum Guirong Li, my Dad Xingxu WANG, my husband Yongchao XU, my mother-in-law Dongfeng HUANG and my father-in-law Jinxiang Xu. II Ca2+ manutention dans un model de souris de CPVT La tachycardie ventriculaire catécholergique (CPVT) est une maladie héréditaire qui se manifeste par des syncopes voire une mort subite, lors de stress émotionnel ou physique en absence d’altération morphologique du cœur. Bien qu’étant une maladie rare, qui touche 1 à 5 personnes sur 10 000 en Europe, cette maladie n’est malheureusement diagnostiquée qu’après le décès d’un enfant ou jeune adulte dans une famille avec des causes inconnues. Une syncope ou la mort subite peuvent apparaitre dès l’âge de 7 ans. Pour 10-20% des patients, la première manifestation est la mort subite. Les arythmies les plus fréquentes sont la tachycardie ventriculaire bidirectionnelle, mais aussi la tachycardie supraventriculaire, la fibrillation atriale, et la dysfonction sinusale. Au repos, ces patients ont un électrocardiogramme normal, mais une tendance plus importante à la bradycardie. Des mutations dans 4 gènes ont été identifiées comme étant à l’origine de la CPVT. La plupart des mutations autosomiques dominantes identifiées (~60%, CPVT1) concernent le récepteur de la 2+ ryanodine type 2 (RyR2), canal de libération du Ca du réticulum sarcoplasmique (RS) cardiaque. D’autres mutations concernent des gènes codant pour des protéines régulatrices du RyR2 : la calséquestrine (CPVT2, autosomique récessive, qui apparait dans environ 2% de cas), la triadine (autosomique récessive) et la calmoduline1 (autosomique dominante). R420Q Nos collaborateurs ont identifié la mutation RyR2 chez une famille espagnole atteinte de CPVT. Ces patients ont une dysfonction du pacemaker (nœud sinusal, NSA) avec une bradycardie. Nous avons construit une souris portant cette mutation, et étudié l’activité du NSA en analysant le calcium intracellulaire, afin d’élucider les mécanismes impliqués dans cette bradycardie. Toutes les expériences ont été faites sur des souris, mâles et femelles, d’environ 6 mois, hétérozygotes pour la mutation (KI), et sur leurs frères et sœurs ne portant pas la mutation (WT). Tout d’abord, pour caractériser les souris, des électrocardiogrammes (ECG) ont été enregistrés en I télémétrie. Des capteurs ont été implantés chez la souris en subcutanée sous anesthésie sous isofluorane. Les enregistrements ont été faits au moins une semaine après la chirurgie, pour que les animaux récupèrent de l’opération. Une injection d'épinéphrine et de caféine (β+1β0 mg/Kg) en intra-péritonéal a été réalisée, afin de vérifier leur phénotype. Ceci a induit une tachycardie bidirectionnelle chez toutes les souris KI et aucune chez les WT, validant ainsi le modèle. Le rythme cardiaque était similaire pendant la période d’activité (nuit) chez les souris KI et WT, mais plus lent pendant la période de repos (jour) chez les femelles KI. Une stimulation sympathique avec injection d’isoprénaline (ISO 1 mg/kg) a accéléré le rythme de toutes les souris, mais de manière plus importante chez les souris KI. D’autre part, la stimulation parasympathique avec injection de carbachol (CCH 0,25 mg/kg) ralentit le rythme cardiaque de façon similaire chez toutes les souris. En présence d’ISO, un plus grand nombre de souris présentent des échappements jonctionnels, indiquant que l’absence d’activité du NSA durant ces battements. Afin d’analyser les mécanismes, nous avons disséqué le NSA et l'avons chargé avec un indicateur fluorescent du Ca2+ (le fluo-4) puis analysé les mouvements calciques spontanés par microscopie confocale. Cette technique, développée dans le laboratoire, est très délicate mais nous permet d’étudier les mouvements du Ca2+ dans la cellule du NSA dans son contexte naturel, sans dissocier le tissu. Ces données in-vitro ont montré une différence basale du rythme cardiaque entre mâles et 2+ femelles, avec une fréquence de transitoires [Ca ]i plus lente chez les femelles. La présence de la 2+ mutation ralentie la fréquence des transitoires [Ca ]i chez les deux sexes (WT : mâle 0,66 ± 0,03 s ; femelle 0,76 ± 0,03 s ; KI : mâle 0,76 ± 0,04 s ; femelle 0,94 ± 0,04 s). De plus, ces transitoires 2+ [Ca ]i ont une plus faible amplitude et une prolongation du temps au pic (amplitude : WT 3,82 ± 0,11 ; KI 3,55 ± 0,10 ; temps au pic : WT 46,95 ± 1,63 ms ; KI 52,41 ± 1,50 ms). 2+ Afin d’évaluer l’activité du RyR2, nous avons analysé les sparks Ca , qui sont des évènements élémentaires produits par l’activation d’un cluster de RyR2s. Nos analyses en microscopie confocale sur des NSA disséqués ont montré que la fréquence des sparks Ca2+ n'était que légèrement augmentée (WT : mâle 5,92 ± 1,33 sparks×s-1×100µm-1; femelle 6,30 ± 1,07 sparks×s-1×100µm-1; II KI : mâle 13,07 ± 2,32 sparks×s-1×100µm-1; femelle 8,03 ± 1,26 sparks×s-1×100µm-1). Par contre, la durée de ces sparks est fortement prolongée dans les cellules KI par rapport aux sparks Ca2+ enregistrés dans les cellules WT (WT : mâle 42,69 ± 0,47 ms ; femelle 36,52 ± 0,37 ms ; KI : mâle 52,98 ± 0,38 ms ; femelle 58,17 ± 0,47 ms). Ceci produit une libération plus importante de Ca2+ dans chaque spark en moyenne. Ainsi, les cellules KI ont une fuite de Ca2+ par unité du temps pendant la diastole plus importante que les cellules WT (WT : mâle 1749,06 ± 28,01 Ca2+ release ×s-1×100µm-1; femelle 1679,65 ± 25,09 Ca2+ release ×s-1×100µm-1; KI : mâle 5091,91 ± 56,82 Ca2+ release ×s-1×100µm-1; femelle 3300,64 ± 36,91 Ca2+ release ×s-1×100µm-1). Pour analyser la conséquence que cette augmentation de la libération du Ca2+ peut avoir, nous avons évalué la charge calcique du RS, qui était réduite, ceci pouvant ainsi réduire l’automatisme. De plus, les canaux calciques de type L du sarcolemme, aussi impliqués dans l’automatisme, sont inactivés par le calcium intracellulaire.
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