La technologie iPSC (induced pluripotent stem cells) permet l’obtention d’une source cellulaire illimitée pour la modélisation et la thérapie de maladies génétiques, la médecine régénérative, l’étude pharmacologique et récemment la modélisation et l’étude du cancer. La leucémie myéloïde chronique (LMC) est la pathologie modèle du concept de « cellule souche cancéreuse » : des cellules souches LMC (CS-LMC) s’avèrent résistantes aux inhibiteurs de tyrosine kinases (ITK), traitement conventionnel de ce syndrome myéloprolifératif. Elles sont responsables de la persistance d’une maladie résiduelle et de rechutes lors de l’interruption du traitement. Malheureusement, leur isolement est difficile. Nous proposons dans ce travail de modéliser les CS-LMC par les iPSC LMC. Nous montrons pour la première fois dans les iPSC LMC que BCR-ABL1 réprime l’expression des facteurs clés de la pluripotence (OCT4,NANOG, le cluster miR302-367) via les kinases ERK1/2. Les ITK, en bloquant l’activité deBCR-ABL1, entraîne une hausse des niveaux d’expression de ces marqueurs. L’effet « prosouche» des ITK, découvert dans les iPSC LMC, est également observé lors du traitement de CS-LMC primaires. L’agent thérapeutique pourrait ainsi, en maintenant ou renforçant le compartiment LMC immature, participer paradoxalement à la persistance de la maladie résiduelle. Étonnamment, les ITK augmentent également l’expression des marqueurs« souches » dans les iPSC et CS hématopoïétiques normales. Ce résultat suggère un effet« pro-souche » généralisé à plusieurs types cellulaires. / IPSC (induced pluripotent stem cells) offer renewable source of biologically relevant human cells for genetic diseases modelling and therapy, regenerative medicine, pharmacological study and, recently, cancer research. The proof of « cancer stem cell concept » was established in chronic myeloid leukemia (CML) : CML stem cells (CML-SC) are resistant to treatment (tyrosine kinase inhibitors (TKI)). They are involved in residual disease persistence and relapse when treatment is discontinued in the majority of patients. We modelled CML-SC with CML iPSC. We showed, for the first time, that BCR-ABL1 acts as a repressor of key stemness markers (OCT4, NANOG, miR302-367 cluster) via ERK1/2 in human CML iPSC. By blocking BCR–ABL1 activity, TKI increase pluripotency gene expression. Interestingly, a similar pro-stemness effect was observed during CML-SC treatment. By inducing a more primitive stemness phenotype, TKI could promote residual disease and relapse. Interestingly, an increase of stemness gene expression was also observed during TKI treatment of healthy cells (iPSC and hematopoietic stem cells). These data suggest a global TKI pro-stemness effect in other stem cell types.

Vici syndrome is a rare, autosomal recessive disorder with severe dysfunction of multiple organ systems. Mutations in the EPG5 gene encoding ectopic P-granule autophagy protein 5, which lead to defects in the autophagic flux, are found as the genetic cause of Vici syndrome. In around 80% of cases, development of dilated and hypertrophic forms of cardiomyopathies is observed. However, the mechanisms of cardiomyopathy development and possible therapeutic targets remain to be elucidated. Interestingly, previous work in Caenorhabditis elegans showed that targeting the enzyme N-acetylglucosamine transferase (OGT) in EPG5-deficient nematodes restored fully functional autophagy. However, the effect of targeting OGT in a human Vici disease model is unknown. In order to understand more about the mechanisms of Vici cardiomyopathies and the effect of targeting OGT in Vici syndrome, we established a Vici patient-specific in vitro disease model by generating induced pluripotent stem cell-derived cardiomyocytes (iVici iPSC-CMs) from fibroblasts obtained from a Vici patient. The Vici patient carried a homologous EPG5 mutation (c.4952+1G>A), which results in the aberrant splicing of the EPG5 transcript and the removal of exon 28 and introduces a frameshift and a premature stop codon at exon 29. In the iVici iPSC-CMs model, we observed some of the known Vici-associated cardiomyopathy phenotypes including cellular hypertrophy and sarcomere disarray. The aims of this study were to investigate physiological and functional properties of iVici iPSC-CMs and to provide an understanding of the molecular mechanisms driving cardiomyopathy development in Vici patients by investigating the presence of potential molecular defects in iVici iPSC-CMs. In this study we also tested the effect of pharmacological OGT inhibitors in iVici iPSC-CMs. We showed that iVici iPSC-CMs exhibited defects in the autophagosome and lysosome fusion and displayed slower beating frequency and altered beating kinetics possibly resulting from the alterations in calcium handling in iVici iPSC-CMs. We observed the accumulation of fragmented mitochondria and increase in oxidative stress and protein O-GlcNAcylation in iVici iPSC-CMs, which are possible causes of the Vici Syndrome-associated cardiomyopathies. Moreover, we found that the non-specific OGT inhibitor alloxan and the specific OGT inhibitor ST045849 could enhance the fusion between the autophagosome and the lysosome in iVici iPSC-CMs. Taken together, in the current work, we demonstrated that iVici iPSC-CMs model can recapitulate some of the disease phenotypes of Vici syndrome, and at the same time, provided further insight into the underlying mechanisms of Vici cardiomyopathy and possible pharmacological therapeutics. Future studies should focus on further investigation of the feasibility of using OGT inhibition as a therapeutic approach and of the underlying mechanisms to identify new therapeutic targets using iVici iPSC-CMs.

La crête neurale est une population de cellules migratoires multipotentes qui se délaminent du tube neural et se différencient en plusieurs types cellulaires. Des altérations du réseau génique de régulation (GRN) de la CNengendrent des maladies congénitales, peu comprises. Cette thèse a pour but d’approfondir la compréhension du rôle de PAX3, un gène crucial dans le GRN de la CN, pendant le développement normal ou pathologique de la CN. Tout d’abord, nous avons caractérisé deux lignées transgéniques de X. laevis, Pax3:GFP etSox10:GFP qui permettent d’étudier l’induction et la spécification précoce de la CN ou sa migration, respectivement. Ensuite, en utilisant des analyses à large échelle, RNAseq et ChIPseq,nous avons défini le premier CN-GRN centré surPax3 chez X. laevis et avons notamment identifié quatre nouveaux gènes régulés par Pax3 :pcdh8l, ercc1 (directement) et fhl3, mmp14(indirectement). Des analyses par perte et gain de fonction de Pax3 in vivo ont permis de vérifier lapertinence de ces cibles.Puis, nous avons analysé le rôle des cibles, Fhl3,pendant le développement de la CN. Fhl3 s’est avéré être un stimulateur intracellulaire de la voie BMP qui, de manière contrôlée spatio-temporellement,est indispensable pour que les cellules cibles de BMP activent la production de WNT à un niveau suffisant pour le développement de la CN.Finalement, nous avons généré les premières lignées iPSC dérivées de patients atteints du syndrome de Waardenburg de type 1 qui ont un allèle de Pax3 muté et nous avons pu les différencier en CN. L’ensemble de ce travail apporte de nouveaux outils et de nouveaux gènes d’intérêt à étudier la CN tant chez X. laevis que chez l’humain. / The neural crest (NC) is a population of multipotent migratory cells that delaminate from the neural tube and differentiate into several cell types. Alterations in NC regulatory gene network (GRN) result in congenital diseases that are poorly understood. This thesis aims to better understand the role of Pax3, a crucial gene in NC GRN, during the normal orpathological NC development. First, we characterized two transgenic lines of X. laevis,Pax3:GFP and Sox10:GFP that allowed us to study the induction and early specification of NC or its migration, respectively. Then, using large scale analyzes, RNAseq and ChIPseq, we defined the first NC-GRN centered on Pax3 inX. laevis and identified in particular four new genes regulated by Pax3 : pcdh8l, ercc1(directly) and fhl3, mmp14 (indirectly). The relevance of these targets was verified by Pax3loss- and gain-of-function in vivo.Then, we analyzed the role of one target, Fhl3,during NC development. We have shown thatFhl3 is an intracellular stimulator of the BMP pathway, which, in a spatiotemporally controlled manner, is essential for BMP target cells to activate the production of WNT at a sufficient level for the development of NC.Finally, we generated the first iPSC lines derived from Waardenburg syndrome type 1patients with a heterozygous Pax3 loss-of function mutation and we were able to differentiate them into NC. All of this work brings new tools and new genes of interest to study NC in both X. laevis and humans.

Les Troubles du Spectre Autistique (TSA) affectent un individu sur 100 en France et sont caractérisés par des déficits de la communication et des interactions sociales ainsi que par la présence d’intérêts restreints et de comportements répétitifs. Le laboratoire a démontré l’implication de protéines synaptiques dans le développement des TSA et en particulier celle des protéines SHANK. Ces protéines sont des protéines d’échafaudage présentes au niveau de la densité post-synaptique (PSD) des neurones glutamatergiques et interagissant avec différents partenaires. Dans le cadre de mon projet de thèse, nous nous sommes particulièrement intéressés à la protéine SHANK3. Des mutations au sein du gène SHANK3 ont été détectées chez environ 1 à 2% des patients, selon le degré de sévérité du retard mental. Un déficit de SHANK3 altère le fonctionnement synaptique. En effet, des analyses sur modèles de souris invalidées pour le gène SHANK3 ont montré une diminution de la densité des épines dendritiques, de la taille de la densité post-synaptique et de l’expression des partenaires protéiques de SHANK3. Mon modèle principal d’analyse a consisté en la reprogrammation de fibroblastes en cellules pluripotentes induites (iPSC « induced pluripotent stem cells »). Les iPSCs ont ensuite été sélectivement dérivées en neurones corticaux. Nos études se sont focalisées sur l’analyse des conséquences fonctionnelles de mutations de novo du gène SHANK3 retrouvées chez 4 patients à l’état hétérozygote et présentes au sein de l’exon 21. Ces mutations conduisent à un codon stop prématuré. En parallèle, nous avons obtenu des cellules de 4 individus témoins ne présentant aucun trouble psychiatrique identifié. L’analyse a porté d’une part sur des aspects morphologiques et d’autre part sur des aspects fonctionnels. Nous avons étudié l’effet des mutations sur la maturation et les caractéristiques morphologiques des épines dendritiques. Nous avons établi un protocole permettant une analyse détaillée de la morphologie en 3D des épines dendritiques et suivi leur maturation. Un résultat majeur est l’observation d’une diminution de la densité des épines sur les dendrites des neurones pyramidaux issus des patients par rapport aux témoins. Comme attendu, la maturation des épines n’est pas complètement achevée mais varie peu dans son évolution d’un individu à l’autre (témoins vs. patients). Nous avons poursuivi ces études par deux approches fonctionnelles : l’imagerie calcique et des études d’électrophysiologie. Les données électrophysiologiques sont en cours d’analyse. En conclusion, nous avons pu obtenir des cultures de neurones corticaux glutamatergiques et les maintenir en culture durant 40 jours pour effectuer différentes analyses à un stade de maturation suffisant pour la mise en évidence de phénotypes morphologiques et fonctionnels. Nous avons principalement observé une diminution de des densités synaptiques et de maturation des épines dendritiques au sein des neurones issus de patients liée à des altérations d’oscillations calciques spontanées. / Autism Spectrum Disorders (ASD) is a neurodevelopmental disorder affecting 1% of population ; characterised by impairments in social interaction and reciprocal communication as well as repetitive and stereotyped behaviors. The work of the laboratory lead to the identification of several genes associated with ASD, among which genes of the synaptic pathway such as SHANK. The SHANK proteins are scaffolding proteins of the post-synaptic density (PSD) of glutamatergic neurons and interact with several partners. In my thesis project, we were particularly interested in SHANK3 mutations. First, Shank3 mutations represent up to 2.12% of ASD cases with moderate to high ID. A SHANK3 deficit leads to the alteration of the synaptic functioning. Indeed, studies of mice KO for SHANK3 gene showed a decrease of the dendritic spines density, of the PSD size and of the expression of SHANK3 partners. My principal model of analysis consisted in the reprogrammation of fibroblasts into induced pluripotent stem cells (iPSCs). Then, the iPSCs were selectively derived into cortical neurons. Our studies were focus on the analysis of functional consequences of SHANK3 de novo mutations found within 4 patients. These mutations are heterozygous and within the exon 21. They result in a premature stop codon. In parallel, we obtained cells from 4 healthy individuals. The work was about the morphological and functional aspects. We analysed the mutations effects on the maturation and morphological caracteristics of the dendritic spines. We finalized a protocol that enabled a detailed analysis of the spine dendritic 3D morphology and their maturation follow-up. A important result was the observation of a decrease of the spine density on pyramidal neurons dendrites from patients compared to those from controls. Moreover, spines maturation was not fully accomplished but was not much different in its evolution between individuals (controls vs patients). Then, we used two functional skills : calcium imaging and electrophysiological experiments. The electrophysiological data are in progress. To conclude, we succeeded in the obtention of glutamatergic cortical neurons and to maintain them in culture during 40 days in order to realize some analysis at a sufficient maturation stage to observe morphological and functional phenotypes. We mainly observed a decrease of the dendritic spines density and maturation for the neurons from patients, with alterations of the spontaneous calcium oscillations.

A Distrofia Muscular de Duchenne (DMD) é uma patologia neuromuscular causada pela mutação ou deleção do gene da distrofina, localizado no cromossomo X, levando a degeneração muscular ao longo da vida do paciente. A doença também tem sido associada a déficit cognitivo e falta de habilidade comportamental. Pesquisas com células neurais de pacientes com DMD poderiam ajudar a elucidar os sintomas neurológicos associados. Neste trabalho, através de células-tronco pluripotentes induzidas (iPSC) derivadas da polpa de dente decíduo esfoliado (SHED) de pacientes com DMD modelamos a DMD produzindo células neurais vivas in vitro. A expressão da distrofina foi verificada durante e após a diferenciação neuronal e nos ensaios de imunofluorescência, mostrando que essa proteína está presente em células do SNC. Na análise gênica através do qPCR, a Dp71 e a Dp140, isoformas da distrofina, apresentavam uma expressão menor do que os controles. Além disso, as análises das sinapses baseada na colocalização de marcadores pré e pós-sinápticos (Sinapsina1 e Homer 1) revelaram que os neurônios dos pacientes com DMD tinham menor quantidade de sinapses que os controles, reforçando o papel da distrofina no SNC. Logo, a expressão de genes relacionados a plasticidade sináptica revelou 10 genes alterados nos neurônios dos pacientes DMD, sugerindo que a mutação no gene da distrofina possivelmente altera a plasticidade sináptica e pode estar envolvida na habilidade cognitiva destes pacientes. Desta forma, com base nos nossos achados, a modelagem neuronal de DMD é factível e pode auxiliar a elucidar os mecanismos da fisiopatologia da doença / The Duchenne muscular dystrophy (DMD) is a neuromuscular disorder caused by a mutation or deletion of the dystrophin gene located on the X chromosome, leading to muscle degeneration throughout the patient\'s life. The disease has also been associated with cognitive impairment and lack of behavioral skill. Research on neural cells from patients with DMD could help to elucidate the neurological symptoms associated. In this work, through induced pluripotent stem cells (iPSC) derived from dental pulp exfoliated (SHED) of patients with DMD model the DMD producing living neural cells in vitro. The dystrophin expression was observed during and after neuronal differentiation and immunofluorescence assays, showing that this protein is present in CNS cells. In gene analysis by qPCR, the Dp71 and Dp140, isoforms of dystrophin, had a lower expression than controls. Furthermore, based on analysis of synapses colocalization pre and postsynaptic markers (Synapsin1 and Homer 1) showed that neurons of DMD patients had lower number of synapses controls, supporting a role for dystrophin in the CNS. Finally, the expression of synaptic plasticity related genes wasfound in 10 genes altered in neurons of DMD patients, suggesting that the mutation of the dystrophin gene possibly alters synaptic plasticity and may be involved in cognitive ability of these patients. Finally, based on our findings, neuronal modeling DMD is feasible and may help elucidate the mechanisms of pathophysiology of the disease

A Distrofia Muscular de Duchenne (DMD) é uma patologia neuromuscular causada pela mutação ou deleção do gene da distrofina, localizado no cromossomo X, levando a degeneração muscular ao longo da vida do paciente. A doença também tem sido associada a déficit cognitivo e falta de habilidade comportamental. Pesquisas com células neurais de pacientes com DMD poderiam ajudar a elucidar os sintomas neurológicos associados. Neste trabalho, através de células-tronco pluripotentes induzidas (iPSC) derivadas da polpa de dente decíduo esfoliado (SHED) de pacientes com DMD modelamos a DMD produzindo células neurais vivas in vitro. A expressão da distrofina foi verificada durante e após a diferenciação neuronal e nos ensaios de imunofluorescência, mostrando que essa proteína está presente em células do SNC. Na análise gênica através do qPCR, a Dp71 e a Dp140, isoformas da distrofina, apresentavam uma expressão menor do que os controles. Além disso, as análises das sinapses baseada na colocalização de marcadores pré e pós-sinápticos (Sinapsina1 e Homer 1) revelaram que os neurônios dos pacientes com DMD tinham menor quantidade de sinapses que os controles, reforçando o papel da distrofina no SNC. Logo, a expressão de genes relacionados a plasticidade sináptica revelou 10 genes alterados nos neurônios dos pacientes DMD, sugerindo que a mutação no gene da distrofina possivelmente altera a plasticidade sináptica e pode estar envolvida na habilidade cognitiva destes pacientes. Desta forma, com base nos nossos achados, a modelagem neuronal de DMD é factível e pode auxiliar a elucidar os mecanismos da fisiopatologia da doença / The Duchenne muscular dystrophy (DMD) is a neuromuscular disorder caused by a mutation or deletion of the dystrophin gene located on the X chromosome, leading to muscle degeneration throughout the patient\'s life. The disease has also been associated with cognitive impairment and lack of behavioral skill. Research on neural cells from patients with DMD could help to elucidate the neurological symptoms associated. In this work, through induced pluripotent stem cells (iPSC) derived from dental pulp exfoliated (SHED) of patients with DMD model the DMD producing living neural cells in vitro. The dystrophin expression was observed during and after neuronal differentiation and immunofluorescence assays, showing that this protein is present in CNS cells. In gene analysis by qPCR, the Dp71 and Dp140, isoforms of dystrophin, had a lower expression than controls. Furthermore, based on analysis of synapses colocalization pre and postsynaptic markers (Synapsin1 and Homer 1) showed that neurons of DMD patients had lower number of synapses controls, supporting a role for dystrophin in the CNS. Finally, the expression of synaptic plasticity related genes wasfound in 10 genes altered in neurons of DMD patients, suggesting that the mutation of the dystrophin gene possibly alters synaptic plasticity and may be involved in cognitive ability of these patients. Finally, based on our findings, neuronal modeling DMD is feasible and may help elucidate the mechanisms of pathophysiology of the disease

In adult CMs, ryanodine receptor 2 (RYR2) is an indispensable Ca2+ release channel that ensures the integrity of excitation-contraction (E-C) coupling, which is fundamental for every heartbeat. However, the role and importance of RYR2 during human embryonic cardiac development are still poorly understood. In this study, after the knockout of RYR2 gene (RYR2–/–), induced pluripotent stem cells (iPSCs) were able to differentiate into cardiomyocytes (CMs) with an efficiency similar to control iPSCs (Ctrl-iPSCs); however, the survival of iPSC-CMs was markedly affected by the lack of functional RYR2. While Ctrl-iPSC-CMs exhibited regular Ca2+ handling, significantly reduced frequency and intense abnormalities of Ca2+ transients were observed in RYR2–/–-iPSC-CMs. Ctrl-iPSC-CMs displayed sensitivity to extracellular calcium ([Ca2+]o) and caffeine in a concentration-dependent manner, while RYR2–/–-iPSC-CMs showed inconsistent reactions to [Ca2+]o and were insensitive to caffeine, indicating there is no RYR2-mediated Ca2+ release from the sarcoplasmic reticulum (SR). Instead, the compensatory mechanism for Ca2+ handling in RYR2–/–-iPSC-CMs is partially mediated by the Inositol 1,4,5-trisphosphate receptor (IP3R). Similar to Ctrl-iPSC-CMs, SR Ca2+ refilling in RYR2–/–-iPSC-CMs is mediated by sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA). Additionally, RYR2–/–-iPSC-CMs showed a decreased beating rate and a reduced L-type Ca2+ current (ICaL) density. These findings demonstrate that RYR2 is not required for CM lineage commitment but is important for CM survival and contractile function. IP3R-mediated Ca2+ release is one of the major compensatory mechanisms for Ca2+ cycling in human CMs with the RYR2 deficiency. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a life-threatening inherited arrhythmogenic disorder. RYR2 mutations are the genetic cause of CPVT Type 1. So far, the pathogenic mechanism of how RYR2 mutations remodel cardiac rhythm remains controversial, because all existing hypotheses cannot independently and universally represent the mechanism behind CPVT. Patient-specific iPSCs offer a unique opportunity for CPVT modeling and investigation in vitro. In this study, the effects of four different RYR2 mutations (R420W, A2254C, E4076K, and H4742Y) on cardiac Ca2+ handling were examined individually. The R420W mutation in CPVTa-iPSC-CMs showed no effect on the amplitude of paced Ca2+ transients but led to an increased Ca2+ leak and a decreased SR Ca2+ content. Moreover, CPVTa-iPSC-CMs presented an enhanced sensitivity to [Ca2+]o and caffeine but a lower ICaL density. Compared to Ctrl cells, CPVTb-iPSC-CMs carrying the A2254V mutation displayed Ca2+ transients with smaller amplitude and higher frequency. More importantly, CPVTb-iPSC-CMs showed remarkably severe Ca2+ leak and unaltered SR Ca2+ content. The A2254V mutation also enhanced the sensitivity of iPSC-CMs to [Ca2+]o and caffeine. Interestingly, the ICaL density was found higher in CPVTb-iPSC-CMs. As for the E4076K mutation, it caused a reduction in both amplitude and frequency of Ca2+ transients in CPVTc-iPSC-CMs. In addition, the sensitivity to [Ca2+]o was diminished in CPVTc-iPSC-CMs, while the caffeine sensitivity and ICaL density were not changed. Regarding the H4742Y mutation, it led to a reduction of Ca2+ transient amplitude. In addition, CPVTd-iPSC-CMs manifested unique SR Ca2+ leak, which was resistant to tetracaine, suggesting a conformational remodeling of the H4742Y-mutated RYR2. Furthermore, CPVTd-iPSC-CMs also showed enhanced sensitivity to [Ca2+]o and caffeine, although the ICaL density was comparable to Ctrl-iPSC-CMs. In summary, the A2254V variation presented a typical gain-of-function mutation, rendering the RYR2 hyperactive, while the E4076K variation was identified as a loss-of-function mutation, leading to hypoactive RYR2. R420W and H4742Y mutations did not enhance or suppress the activity of RYR2. However, by destabilizing the N-terminal domain (NTD) of RYR2, the R420W mutation caused Ca2+ leak via the mutant channel, which could be blocked by RYR2 inhibitor. As for the H4742Y mutation, it led to a consistent and inhibitor-resistant Ca2+ leak via RYR2, suggesting a structural remodeling of RYR2 that disturbs complete closure of the channel. These results confirmed the importance of RYR2 on the maintenance of Ca2+ handling and gained evidence for the theory that the underlying mechanisms of CPVT caused by mutations in RYR2 should be mutation-specific rather than unified. This study suggests hiPSC-CMs as a suitable platform for modeling cardiac arrhythmogenic disease, interpreting potential molecular and pathophysiological mechanisms, testing new therapeutic compounds, and guiding mechanism-specific therapy.:Abbreviations V List of figures VIII List of tables X 1 Introduction 1 1.1 Human induced pluripotent stem cells 1 1.1.1 Generation and characteristics of human induced pluripotent stem cells 1 1.1.2 Differentiation of hiPSCs into cardiomyocytes 3 1.1.3 Modeling of inherited cardiac disease with hiPSCs 4 1.2 Catecholaminergic polymorphic ventricular tachycardia 7 1.2.1 Clinical characteristics and diagnosis of CPVT 7 1.2.2 Genetic background of CPVT 8 1.2.3 Clinical descriptions of CPVT patients recruited in this study 10 1.2.4 Patient-specific iPSC-CMs recapitulate the phenotypes of CPVT in vitro 10 1.3 Cardiac excitation-contraction coupling 11 1.3.1 Cardiac action potential 12 1.3.2 Ca2+ homeostasis in cardiomyocytes 14 1.3.2.1 Ca2+ influx via L-type Ca2+ channel 14 1.3.2.2 Initiation and termination of SR Ca2+ release 15 1.3.2.3 Ca2+ removal from cytosol 17 1.3.3 Cardiomyocyte contraction 20 1.4 Cardiac ryanodine receptor 21 1.4.1 Distribution and classification of RYRs 22 1.4.2 Regulation of RYR2 23 1.4.2.1 Cytosolic Ca2+ 23 1.4.2.2 Luminal Ca2+ 24 1.4.2.3 Phosphorylation by PKA and CaMKII 25 1.4.2.4 Calmodulin 27 1.4.2.5 Caffeine 27 1.4.3 Pathophysiological mechanisms of CPVT associated with RYR2 mutations 28 2 Aims of this study 33 3 Materials and methods 34 3.1 Materials 34 3.1.1 Cells 34 3.1.2 Laboratory devices and experimental hardware 34 3.1.3 Disposable items 36 3.1.4 Chemicals, solutions, and buffers for physiological and molecular experiment 36 3.1.5 Antibodies 40 3.1.6 Primers 41 3.1.7 Chemicals, media and solutions used for cell culture 42 3.1.8 Software 44 3.2 Methods 44 3.2.1 Cell culture 44 3.2.1.1 Preparation of glass coverslips for cell culture 44 3.2.1.2 Coating of plates and dishes 44 3.2.1.3 Cultivation of iPSCs with feeder-free method 45 3.2.1.4 Cryopreservation and thawing of iPSCs 45 3.2.1.5 Spontaneous differentiation of iPSCs in vitro 45 3.2.1.6 Directed differentiation of iPSCs into cardiomyocytes 46 3.2.1.7 First digestion of iPSC-CMs 46 3.2.1.8 Cryopreservation and thawing of iPSC-CMs 46 3.2.1.9 Time-dependent proliferation analysis of iPSC-CMs 47 3.2.1.10 Second digestion of iPSC-CMs 47 3.2.1.11 Collection of cell pellets for molecular experiment 47 3.2.2 Cell viability assay 48 3.2.3 Gene expression analyses 48 3.2.3.1 RNA isolation 48 3.2.3.2 Reverse transcription reaction 48 3.2.3.3 Polymerase chain reaction 49 3.2.3.4 Agarose gel electrophoresis 49 3.2.4 Protein expression analyses 49 3.2.4.1 Western blot 49 3.2.4.1.1 Lysis of cultured cells 49 3.2.4.1.2 SDS-polyacrylamide gel electrophoresis 50 3.2.4.1.3 Transfer and detection of proteins 50 3.2.4.2 Flow cytometry 51 3.2.4.3 Immunofluorescence staining 51 3.2.5 Calcium imaging 51 3.2.5.1 Measurement of spontaneous Ca2+ transients 52 3.2.5.2 Evaluation of diastolic SR Ca2+ leak and SR Ca2+ content 52 3.2.5.3 Assessment of iPSC-CM sensitivity to [Ca2+]o 53 3.2.5.4 Quantification of iPSC-CM response to caffeine 53 3.2.6 Patch-clamp 53 3.2.6.1 Preparation of agar salt bridge 53 3.2.6.2 Assessment of liquid junction 53 3.2.6.3 Measurement of action potential and L-type calcium current 54 3.2.7 Statistical analysis 54 4 Results 55 4.1 IP3R-mediated SR Ca2+ release partially restores the impaired Ca2+ handling in iPSC-CMs with RYR2 deficiency 55 4.1.1 Loss of RYR2 does not alter the pluripotency of RYR2–/–-iPSCs 55 4.1.2 Loss of RYR2 leads to increased death of RYR2–/–-iPSC-CMs 56 4.1.3 Loss of RYR2 does not affect the expression of IP3R in iPSC-CMs 58 4.1.4 RYR2–/–-iPSC-CMs show abnormal Ca2+ transients 60 4.1.5 The sensitivity of RYR2–/–-iPSC-CMs to [Ca2+]o and caffeine is changed 62 4.1.6 IP3R is critical for the generation Ca2+ transients in RYR2–/–-iPSC-CMs 63 4.1.7 SERCA-mediated SR Ca2+ uptake is crucial for the Ca2+ handling in both Ctrl- and RYR2–/–-iPSC-CMs 65 4.1.8 RYR2–/–-iPSC-CMs display abnormal action potentials 66 4.2 Investigation of the impaired function of RYR2 in CPVTa-iPSC-CMs 69 4.2.1 The R420W mutation leads to increased SR Ca2+ leak and decreased SR Ca2+ content 69 4.2.2 The R420W mutation leads to an enhanced sensitivity of iPSC-CMs to [Ca2+]o 70 4.2.3 CPVTa-iPSC-CMs shows increased sensitivity to caffeine 71 4.2.4 CPVTa-iPSC-CMs show reduced ICaL density 72 4.3 Investigation of the impaired function of RYR2 in CPVTb-iPSC-CMs 74 4.3.1 CPVTb-iPSC-CMs show abnormal Ca2+ transients 74 4.3.2 The A2254V mutation intensifies the SR Ca2+ leak in iPSC-CMs 75 4.3.3 The A2254V mutation enhances the sensitivity of iPSC-CMs to [Ca2+]o 76 4.3.4 The A2254V mutation increases the sensitivity of iPSC-CMs to caffeine 78 4.3.5 CPVTb-iPSC-CMs show increased ICaL density 78 4.4 Investigation of the impaired function of RYR2 in CPVTc-iPSC-CMs 79 4.4.1 CPVTc-iPSC-CMs show abnormal Ca2+ transients 79 4.4.2 The E4076K mutation shows no effect on the SR Ca2+ leak and content 80 4.4.3 The E4076K mutation diminishes the sensitivity of iPSC-CMs to [Ca2+]o 81 4.4.4 The E4076K mutation shows almost no effect on the response of iPSC-CMs to caffeine 82 4.4.5 The E4076K mutation does not alter the ICaL density in iPSC-CMs 83 4.5 Investigation of the impaired function of RYR2 in CPVTd-iPSC-CMs 84 4.5.1 CPVTd-iPSC-CMs show abnormal Ca2+ transients 84 4.5.2 The H4742Y mutation leads to a tetracaine-resistant Ca2+ leak in iPSC-CMs 84 4.5.3 The H4742Y mutation improves the sensitivity of iPSC-CMs to [Ca2+]o 86 4.5.4 The H4742Y mutation enhances the response of iPSC-CMs to caffeine 87 4.5.5 The H4742Y mutation alters the gating properties of LTCC in iPSC-CMs 88 5 Discussion 90 5.1 IP3R-mediated compensatory mechanism for Ca2+ handling in iPSC-CMs with RYR2 deficiency 90 5.2 Pathophysiological mechanisms of RYR2 mutation-related CPVT are mutation-specific 93 5.2.1 Dysfunctional Ca2+ handling caused by RYR2-R420W mutation 94 5.2.2 Dysfunctional Ca2+ handling caused by RYR2-A2254V mutation 96 5.2.3 Dysfunctional Ca2+ handling caused by RYR2-E4076K mutation 99 5.2.4 Dysfunctional Ca2+ handling caused by RYR2-H4742Y mutation 101 5.3 Conclusions and future perspectives 104 6 Summary 106 7 Zusammenfassung 108 8 References 111 9 Acknowledgements 131 10 Declaration 132

Background and aims: Leptin resistance or leptin signalling deficiency are associated with increased risk of diabetic cardiomyopathy and heart failure, which is a leading cause of obesity- and diabetes type 2 (T2DM)-related morbidity and mortality. Various metabolic disturbances are involved in this pathogenesis, such as elevated glucose and fatty acid levels, insulin resistance and altered myocardial substrate utilization. Rodent models provided useful insights into the underlying molecular mechanisms of obese- and T2DM-associated cardiometabolic diseases, however, they cannot fully recapitulate the disease phenotype of obese or T2DM patients. The aims of this study were to study the effect of leptin receptor (LEPR) mutations on the leptin-mediated signalling pathways in human cardiomyocytes, and to investigate glucose and fatty acid metabolism in the heart under (patho)physiological conditions. Methods and results: To study the role of LEPR in human cardiomyocytes (CMs), human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were used as a model. In the first part of this study, LEPR expression and function was investigated in wild type (WT)-iPSC-CMs by PCR and Western Blot. LEPR protein expression was almost not detectable in iPSCs and during early cardiac differentiation stages, however mRNA LEPR expression was comparable in the different steps of cardiac development. Importantly, LEPR protein expression was observed in WT-iPSC-CMs at the maturation stages, indicating that LEPR plays an important role in matured CMs. Thanks to CRISPR/Cas9 technology, LEPR mutations were introduced into iPSCs. Among the several clones obtained, 1B2 LEPRΔ/Δ-iPSC line was fully characterized and showed normal capacity to differentiate into spontaneously beating CMs. Although the B27 medium represents a well-established medium to cultivate iPSC-CMs, it has limitations for studying CM metabolism due to its high concentration of insulin and glucose, but low concentration of fatty acids. Physiological medium condition (F2) including physiological range of glucose, insulin and fatty acids was found to be fundamental to study LEPR signalling pathway in iPSC-CMs. Western blot analysis showed functional LEPR downstream pathway activation in WT-iPSC-CMs, while the absence of LEPR function was demonstrated in LEPRΔ/Δ-iPSC-CMs cultured in F2 medium. Moreover, improved medium condition, offered by the F2 medium, ameliorates insulin sensitivity as result of increased insulin-dependent AKT phosphorylation in WT-iPSC-CMs, while loss of LEPR function was associated with downregulation of insulin pathway activation. Additionally, leptin direct effect was observed on the regulation of glucose metabolism in WT-iPSC-CMs by reducing glycolytic fluxes, which was not observed in LEPRΔ/Δ-iPSC-CMs, as measured by 13C-isotope-assisted glucose metabolic flux. These data indicate that the signalling interaction between insulin and leptin is important in regulation of glucose metabolism and is abolished in LEPRΔ/Δ-iPSC-CMs. The matured WT-iPSC-CMs in F2 medium display adult CM-like metabolic phenotype such as enhanced mitochondrial respiration and glycolytic function, as measured by Seahorse analyser, compared to the same group cultured in the B27 medium. The mutation generated in LEPRΔ/Δ-iPSC-CMs caused an “energy starvation” status which led to increased AMPK phosphorylation compared to the WT group in B27 medium, which was associated with lower mitochondrial oxygen consumption rate (OCR) linked basal respiration and ATP production. In the next part of this study, the long-term leptin treatment of iPSC-CMs under physiological medium conditions in the presence of physiological range of insulin, glucose, and fatty acids (F2+) influenced LEPR downstream pathway activation such as JAK2 and AMPK suggesting a leptin-dependent role in fatty acid uptake and oxidation in WT-iPSC-CMs. On the contrary, leptin did not affect JAK2 and AMPK activation in LEPRΔ/Δ-iPSC-CMs. Culturing of (WT)-iPSC-CMs in F2+ medium demonstrated no significant difference in mitochondrial oxygen consumption, while slightly lower glycolysis and glycolytic capacity was observed. However, a leptin effect on fatty acid and glucose metabolism was observed in LEPR∆/∆-iPSC-CMs, which is independent from LEPR downstream regulation. To study the effect of high leptin levels, a medium mimicking some of the diabetic hallmarks, such as high glucose, high insulin, and high leptin levels, was used. Metabolic flexibility was observed in WT-iPSC-CMs in F3+ medium as showed by no difference in mitochondrial function in WT-iPSC-CMs in the presence or absence of high leptin. In contrast, LEPRΔ/Δ-iPSC-CMs in F3+ medium demostrated higher OCR compared to F2 medium, which is accompanied by lower glycolysis and glycolytic capacity, indicating the incapability of LEPRΔ/Δ-iPSC-CMs to use glucose as energy source, as measured by Seahorse analysis. Conclusion and outlook: Taken together, this study demonstrates the importance of leptin and LEPR at the late stage of CM maturation and the fundamental role of metabolic medium condition including physiological range of glucose and fatty acid to study the role of leptin in iPSC-CMs. In addition, LEPRΔ/Δ-iPSC-CMs in diabetic condition (F3+) represent a suitable model to investigate leptin-dependent cardiac metabolism, resulting in increased mitochondrial oxygen consumption and decreased glycolytic function, resembling the condition known in obesity-related T2DM patients. Further studies should focus on the regulation of the metabolic switch between glucose and fatty acid utilization in the absence of a functional LEPR. Understanding the contribution of leptin/LEPR signalling in human CM metabolism will shed light on novel therapeutic approaches to treat diabetic cardiomyopathy.

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MITOCHONDRIAL GENE EXPRESSION IN HUMAN MONONUCLEAR CELLS By Monika D. Ruchała, M.S. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University. Virginia Commonwealth University, 2014. Director: Dr. James P. Bennett Jr, M.D., Ph.D., Bemiss Professor Departments of Neurology, Psychiatry and Physiology and Biophysics Adult neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), have been intensively studied in recent years in pursuit of mechanisms responsible for origin and progression. One emerging theme is mitochondrial energetic deficiency as a mechanism of neuronal death. Recent descriptions of protocols to generate induced pluripotent stems cells (iPSCs) from living patients offer the potential to create unique disease models. This model can potentially lead to crucial advances in developing treatment options for a wide variety of neurodegenerative diseases. In this thesis, we attempt to induce iPSCs from mononuclear cells (MNC) in peripheral blood acquired from patients with ALS and healthy control (CTL) subjects, and analyze their mitochondrial genomes. The reprogramming of MNC to yield iPSC was done by nucleofection of an episomal plasmid pEB­ C5, expressing OriP sequences of the Epstein­Barr and five reprogramming transgenes Oct4, Sox2, Klf4, c­Myc and Lin28. We investigated the expression of mitochondrial DNA genes, ND2, ND4, COXIII and 12s rRNA in the ALS and CTL MNC before and after their culturing. The results implicate deregulated mitochondrial bioenergetics as a characteristic of ALS. Future work will establish whether these abnormalities in mitochondrial bioenergetics persist in iPSC’s and iPSC-derived neurons from ALS subject