Dilated Cardiomyopathy (DCM) with conduction disease and Atrial Fibrillation (AF) are the two cardiac-specific diseases associated with lamin A/C gene (LMNA) mutations. Protein Kinase C Alpha, (PKCα) functions as a nodal integrator of cardiac contractility by “sensing” intracellular calcium and signal transduction. PKCα has been implicated in heart failure and cardiac hypertrophy. Moreover, abnormal PKCα function results in irregular atrial potassium channel activity associated with chronic AF PKCα is a lamin A/C binding partner. Thus, the deregulation of PKCα signaling can contribute to the development of DCM and AF. Our hypothesis is that the AF (Thr528Met), DCM-associated (Arg541Cys) and (Arg541Gly) and DCM/AF-associated (Tyr481Stop) LMNA variants will disrupt the cellular distribution of PKCα therefore resulting in impaired PKCα function. The first objective was to phenotypically characterise Arg541Cys LMNA variant in murine skeletal myoblasts cell line (C2C12) in comparison to cellular phenotypes induced by LMNA variants associated with AF, DCM and DCM with AF. Arg541Cys lamin A and C variants formed circular and sickle-shaped lamin A/C in the nucleus of C2C12 cells. The second objective was to determine the effect of these lamin variants on cellular distribution of PKCα in C2C12 cells. PKCα mislocalized into the nucleus of C2C12 cells transfected with AF and DCM-associated variants (Thr528Met and Arg541Cys). Colocalization analysis showed significant increase in PKCα in the nucleus of AF (Thr528Met) and DCM (Arg541Cys) variants when lamin A and C, were co-transfected compared to wild-type, DCM (Arg541Gly) and DCM/AF (Tyr481Stop) variants. Densitometry analysis showed statistically significant increase in phosphorylated PKCα, the active form of PKCα, in nuclear and cytoplasmic extracts of C2C12 cells expressing Arg541Cys variant. Densitometry analysis also showed statistically significant increase in non-phosphorylated PKCα in the nuclear extract of Thr528Met variant expressing cells. The third objective was to determine the effect of AF and DCM-associated variants on the activity of PKCα. PKCα activity is quantified by measuring the phosphorylation of a known phosphorylated PKCα substrate. Alpha-6-tubulin phospho (Ser165) is phosphorylated by PKCα. Hence, this was used to quantify PKCα activity. No statistical significance was observed in the level of phosphorylated alpha-6-tubulin at (Ser165) in the C2C12 cells that were transfected with lamin A and C variants compared to wild type. Furthermore, PKCα phosphorylation state is cyclic in nature and this could have had an impact on the phosphorylation state of the chosen substrate in this study. The functional consequence of nuclear translocation of PKCα with respect to laminopathies is unknown. Abnormal activation of the Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK1/2) which are branches of the mitogen-activated protein kinase (MAPK) signalling cascade in hearts of mice, and humans prior to the onset of cardiomyopathy. These findings have been associated to cardiac disease-causing lamin A/C alteration to signal transduction pathways implicated in heart function and cardiomyopathy. Human LMNA cardiomyopathy, could lead to abnormal activation of MAPK signalling pathways via abnormal PKCα activation in cardiomyocytes.

Indiana University-Purdue University Indianapolis (IUPUI) / Atherosclerosis is a chronic disease that happens mostly in aged people, and recently studies have showed many similarities between Hutchinson Gilford Progeria Syndrome (HGPS) cells and aging cells, implicating dysfunctions of lamin A/C in aging process and atherosclerosis, as HGPS is caused by a mutated form of lamin A/C. Blood flow in arteries is generating shear stress that is mostly applied on endothelial cells that align along inner blood vessel wall. At the same time, endothelial cells are also under stretch by periodic arterial pulses. Considering the fact that atherosclerosis is prone to developing at arterial branches with disturbed shear and increased stretch, it is highly possible that laminar flow and proper stretch force are regulating endothelium to function appropriately. In this thesis, the investigation of what effects laminar flow or cyclic stretch can bring to endothelial cells was conducted, and examination of lamin A/C expression under mechanical forces were elaborated and incorporated with cell senescence. Results showed that laminar shear stress and stretch force can regulate lamin A/C expression in different patterns, which were impaired in senescent cells.

La migration cellulaire possède deux volets antagonistes ; nécessaire à plusieurs processus physiologiques tels que la réponse immunitaire, elle peut également induire la mort d’un organisme en permettant les cellules cancéreuses d’envahir des organes sains. In vivo, la migration s’effectue dans des milieux complexes et confinés qui imposent une forte déformabilité aux cellules migratoires. Récemment, divers études ont montré que le noyau impose la limite de la déformabilité cellulaire lors de la migration en 3D (Wolf et al. JCB, 2013; Harada et al. JCB, 2013). Il a, en effet, été montré que la migration cellulaire peut être augmentée en diminuant la rigidité nucléaire (Wolf et al. JCB, 2013). Cependant, il existe une limite de rigidité nucléaire en dessous de laquelle la migration cellulaire peut être inhibée via l’inhibition de la survie cellulaire (Harada et al. JCB, 2013). Les cellules cancéreuses qui migrent à des vitesses relativement faibles (µm/heure) et ont des noyaux rigides surmontent la limite imposée par la déformation nucléaire en dégradant et élargissant le milieu extracellulaire. Les cellules immunitaires telles que les neutrophiles qui migrent rapidement (10 µm/mn) et ont des noyaux mous sont connus pour mourir aux sites d’infections. Les cellules dendritiques, de la famille des cellules immunitaires, ont une fonction de présentation d’antigènes qui requiert à la fois une grande capacité migratoire et de survie. Elles représentent donc un modèle cellulaire intéressant pour l’étude de la déformation nucléaire chez les cellules qui migrent rapidement et survivent longtemps. Durant mon doctorat, j’ai étudié le mécanisme grâce auquel les cellules dendritiques déforment leurs noyaux afin de migrer de manière efficace en milieux confinés tout en préservant un haut taux de survie. J’ai utilisé un système expérimental nouveau et original consistant en des microcannaux avec des constrictions (Heuzé et al. MMB, 2011). Ces canaux, combinés à des manipulations génétiques et de la video microscopie nous ont permis de montré que les cellules dendritiques possèdent un mécanisme spécifique, indépendant de celui utilisé pour leur migration, leur permettant de déformer leurs noyaux tout en migrant dans des milieux hautement confinés. Ce mécanisme est basé sur la génération d’un réseau d’actin, autour du noyau, nucléé par Arp2/3 et indépendant du moteur Myosin II. Ce réseau d’actine co-localise avec des sites de rupture de la Lamin A/C. De plus, réduire la quantité de Lamin A/C dans les cellules dendritiques inhibe la formation de ce réseau d’actin perinucléaire. Basés sur ces résultats, nous avons proposé un nouveau mécanisme de déformation du noyau lors de la migration en milieux confinés basé sur Arp2/3 qui, en nucléant un réseau d’actine autour du noyau permet de casser la lamin A/C diminuant ainsi la tension de surface nucléaire et permettant le passage noyau. / Cell migration has two opposite faces; necessary for many physiological processes such as immune response, it can also lead to the organism death by allowing metastatic cells to invade new organs. In vivo migration often occurs in complex 3D environments which impose high cellular deformability. Recently, cellular deformability during 3D migration has been shown to be limited by the nucleus (Wolf et al. JCB, 2013). For instance, cell migration can be increased by decreasing nuclear stiffness. However, below a given nuclear stiffness 3D cell migration can be reduced as a result of impaired cell survival (Harada et al. JCB, 2014). Cancer cells which display slow migration and have rather stiff nuclei have been shown to overcome the physical limits of 3D migration through adhesion combined to matrix degradation or high actomyosin contraction (Wolf et al. JCB, 2013). Immune cells such as neutrophils which are fast moving cells with soft nuclei have been reported to die at sites of infection. Interestingly, dendritic cells function as antigen presenting cells requires high migratory ability as well as high survival. They thus constitute an interesting model for studying nuclear deformation in fast moving and long lived cells. During my PhD, I studied the mechanism by which dendritic cells deform their nuclei to achieve proper migration in highly confining space while preserving a high survival rate. I used an original micro fabricated experimental set up (Heuzé et al. MMB, 2011) consisting of microchannels with constrictions to mimic cellular transmigration. Those channels combined with genetic manipulation and live cell imaging followed by image processing were used to assess the mechanism dendritic cells use to deform their nucleus, which we found to be specific and not required for cell motility per se. I showed that dendritic cells overcome the physical limitation imposed by nuclear deformation through small gaps by nucleating an Arp2/3 based actin network around the nucleus. Surprisingly, the formation of this actin network is independent of myosin II based contraction. This actin accumulation around the nucleus co-localized with sites of nuclear Lamin A/C breakage. Moreover, Lamin A/C depletion in dendritic cells leads to the disappearance of this actin ring and the release of the need for Arp2/3 for nuclear deformation. We thus propose a new mechanism of nuclear squeezing through narrow gaps based on an Arp2/3 nucleated actin meshwork which, by transiently breaking the Lamin A/C network, releases the nuclear surface tension and allows nuclear thus cell passage through micrometric constrictions. Lamin A/C repolymerization around the nucleus at the exit of constrictions would then restore nuclear stiffness, allowing cell survival. Interestingly, this actin accumulation around the nucleus was also observed in vivo in migrating macrophages but not in HL-60 derived neutrophils. Taken together, our data suggest that the Arp2/3 based nuclear squeezing mechanism would be a general feature of highly migratory cells which need to survive long enough to accomplish their functions.

Abstract A non-ischemic etiology of sudden cardiac death (SCD), mostly due to various cardiomyopathies (CMP), accounts for about 20% of all SCDs. Most of the major studies of risk factors for SCD have focused on coronary artery disease (CAD). The aim of the present study was to clarify the characteristics of non-ischemic SCD in Northern Finland. In this study, consecutive victims of SCD (n=2661) were prospectively collected, and among whom post-mortem examinations were performed between 1998 and 2007. Information about the SCD victims was obtained from a combination of available medical records, postmortem examination reports, medication used at the time of SCD, and standardized questionnaire filled out by the closest family members of the victims of SCD. We also screened the candidate genes from a Finnish family in which fatal arrhythmias was first manifestation of a cardiac disease. The collagen content of the myocardium from histological samples in victims of SCD due to idiopathic myocardial fibrosis (IMF) was also evaluated. CAD was the most common cause of death (2082 victims, 78.2%). The prevalence of non-ischemic SCDs was 21.8% of all the SCDs. After sub-grouping the non-ischemic SCDs into various categories, the most common cause of death was CMP related to obesity (23.7%), followed by alcoholic CMP (19.0%), hypertensive CMP (15.5%) and IMF (13.6%). The association of SCD with IMF is notably frequent among victims <40 years old (28.3%). The prevalence of family history of SCD was significantly higher in the victims of ischemic (34.2%) than non-ischemic SCD (13.4%, P<0.001) or controls (17.6%, P<0.001). Lamin A/C gene mutation R541C was found from Finnish SCD family, in which the IMF was predominant pathologic-anatomic finding. Myocardial type I collagen synthesis was increased in victims of SCD due to IMF. In conclusion, the characteristics of non-ischemic SCD in Finland differ from those reported previously. Higher prevalences of CMP-associated SCDs related to obesity, IMF and alcoholic CMP were observed as clinical and/or pathologic bases for non-ischemic SCD. The family history of SCD is not significantly increased in victims of non-ischemic SCD, suggesting a larger role of sporadic occurrence than inherited traits as the cause of non-ischemic SCD. Replacement of cardiac myocytes by fibrosis can be responsible for fatal cardiac arrhythmias in subjects with the lamin A/C gene mutation. The victims of SCD due to IMF have increased myocardial type I collagen synthesis. / Tiivistelmä Ei-iskeeminen sydänperäinen äkkikuolema aiheuttaa noin 20 % kaikista sydänperäisistä äkkikuolemista. Suurin osa ei-iskeemisistä sydänperäisistä äkkikuolemista johtuu erilaisista sydänlihassairauksista, kardiomyopatioista. Useimmat sydänperäisen äkkikuoleman riskitekijöitä kartoittavista tutkimuksista ovat keskittyneet sepelvaltimotautiin. Tämän tutkimuksen tarkoituksena oli selvittää ei-iskeemisen sydänperäisen äkkikuoleman tunnuspiirteitä pohjoissuomalaisessa väestössä. Tutkimuksessa käytettiin potilasaineistona sydänperäiseen äkkikuolemaan menehtyneitä vainajia (n=2661), joille on tehty oikeuslääketieteellinen ruumiinavaus. Tiedot vainajista saatiin saatavilla olevista potilaskertomuksista, ruumiinavauspöytäkirjoista, äkkikuoleman aikaisesta lääkityksestä ja lähiomaisille lähetetystä standardisoidusta kyselylomakkeesta. Kandidaattigeenit tutkittiin pohjoissuomalaisesta perheestä, jossa ensimmäinen oire sydänsairaudesta oli hengenvaarallinen rytmihäiriö. Lisäksi sydänlihaksen kollageenikoostumus analysoitiin histologisista näytteistä potilailta, joiden sydänperäinen äkillinen kuolema johtui idiopaattisesta sydänlihaksen sidekudoskasvusta. Sepelvaltimotauti oli yleisin sydänperäisen äkkikuoleman aiheuttaja (n=2082, 78,2 %). Ei-iskeemisten sydänperäisten äkkikuolemien osuus oli 21,8 % (n=579) kaikista sydänperäisistä äkkikuolemista. Ei-iskeemiset sydänperäiset äkkikuolemat jaettiin alaryhmiin, joista yleisimmät olivat lihavuuteen assosioituva kardiomyopatia (23,7 %), alkoholikardiomyopatia (19,0 %), korkeaan verenpaineeseen assosioituva kardiomyopatia (15,5 %) sekä idiopaattinen sydänlihaksen sidekudoskasvu (13,6 %), joka myös oli yleisin ei-iskeemiseen sydänperäiseen äkkikuolemaan johtava syy alle 40-vuotiailla (28,3 %). Positiivinen sydänperäisen äkkikuoleman sukuhistoria oli tilastollisesti merkitsevästi yleisempää iskeemisillä (34,2 %) kuin ei-iskeemisillä (13,4 %) sydänperäisen äkkikuoleman uhreilla. Lamin A/C – geenin mutaatio löydettiin pohjoissuomalaisesta äkkikuolemaperheestä, jossa idiopaattinen sydänlihaksen sidekudoskasvu todettiin pääasialliseksi patologiseksi löydökseksi. Tyypin I kollageenin synteesi todettiin kohonneeksi idiopaattiseen sydänlihaksen sidekudoskasvuun menehtyneillä vainajilla. Yhteenvetona voidaan todeta, pohjoissuomalaisen väestön ei-iskeemisen sydänperäisen äkkikuoleman tunnuspiirteet eroavat aiemmin raportoiduista; lihavuuteen assosioituva kardiomyopatia, alkoholikardiomyopatia, sekä idiopaattinen sydänlihaksen sidekudoskasvu olivat aiempaa yleisempiä ei-iskeemisen äkkikuoleman aiheuttajia. Positiivinen sydänperäisen äkkikuoleman sukuhistoria ei ollut tilastollisesti merkitsevästi kohonnut ei-iskeemisen sydänperäiseen äkkikuolemaan menehtyneillä. Tämä tarkoittaa, että perinnöllinen syy ei-iskeemisen sydänperäisen äkkikuoleman aiheuttajana on luultua harvinaisempi. Lamin A/C – geenimutaation kantajilla sydänlihassolujen korvautuminen sidekudoksella todettiin hengenvaarallisen rytmihäiriön aiheuttajaksi. Lisäksi, tyypin I kollageenin synteesi todettiin kohonneeksi idiopaattiseen sydänlihaksen sidekudoskasvuun menehtyneillä vainajilla.

La dystrophie musculaire d'Emery-Dreifuss (EDMD) est caractérisée par des retractions précoces, une faiblesse et atrophie musculaire lentement progressive, et une atteinte cardiaque. Les mutations des gènes EMD et LMNA sont respectivement responsables de formes liées à l'X et de formes autosomiques de l'EDMD. Ces deux gènes codent pour des protéines de l'enveloppe nucléaire, l'émerine et les lamines A/C. Les mutations du gène FHL1 ont été impliquées dans d'autres cas d'EDMD liée à l'X. FHL1 codent pour FHL1A, FHL1B et FHL1C, protéines jamais décrites comme localisées à l'enveloppe nucléaire. Nous avons cherché à enrichir les connaissances sur la distribution subcellulaire des différentes isoformes de FHL1 dans les muscles squelettiques humains sains et malades. Nous avons mis en évidence que les isoformes FHL1 présentent à la fois une localisation cytoplasmique et nucléaire dans les myoblastes humains. Au noyau, FHL1B est fortement accumulé au niveau de l'enveloppe nucléaire où il interagit avec les lamines A/C et l'émerine. Cette localisation à l'enveloppe nucléaire est indépendante de l'expression de l'émerine ou des lamines A/C. La différenciation des myoblastes entraîne une forte réduction de l'expression de FHL1B et de son exclusion progressive du noyau, n'impliquant pas la protéine CRM-1. Nous avons mis en évidence l'augmentation de l'expression de FHL1B dans les myoblastes de deux patients atteints d'EDMD, l'un porteur d'une mutation dans le gène LMNA, l'autre dans le gène FHL1. En conclusion, la localisation spécifique de FHL1B et sa modulation dans les myoblastes de patients confirment les cas d'EDMD liés à FHL1 comme des pathologies de l'enveloppe nucléaire. / Emery-Dreifuss muscular dystrophy (EDMD) is characterized by the triad of early contractures, slowly progressive muscle wasting and weakness, and cardiac disease. Mutations in EMD and LMNA, encoding for the nuclear envelope (NE) proteins emerin and lamin A/C, are associated with X-linked and autosomal form of EDMD, respectively. The discovery that FHL, encoding FHL1A, FHL1B and FHL1C, is implicated in the pathogenesis of EDMD, raises the question of how a non-NE protein can be linked to emerin and lamin A/C. We aimed to provide knowledge of the subcellular distribution and expression of the various FHL1 isoforms in healthy and diseased human skeletal muscle. We found that FHL1 isoforms display a dual cytoplasmic and nuclear localization in human myoblasts. In addition, FHL1B strongly accumulated at the NE where it interacted with both lamin A/C and emerin. NE localization of FHL1B was independent of emerin and lamin A/C expression. Myoblast differentiation resulted in greatly reduced FHL1B protein expression and in the progressive nuclear exclusion of FHL1 protein isoforms. We have shown that chromosome region maintenance 1 (CRM1)-mediated nuclear export was not involved in the progressive decrease of nucleoplasmic FHL1B. Finally, we detected increased FHL1B protein levels in myoblasts of two patients with LMNA- and FHL1-related EDMD. Altogether, the specific localization of FHL1B and its modulation in disease-patient’s myoblasts confirmed FHL1-related EDMD as a NE disease.

No description available.

La forme du noyau peut varier significativement au cours du développement ou lors de processus pathologiques en raison des forces mécaniques émanant du microenvironnement ou générées par le cytosquelette. L’impact de la morphologie nucléaire sur la machinerie transcriptionnelle n’est cependant pas connu. En utilisant plusieurs approches afin de manipuler la morphologie nucléaire, nous avons observé que des changements de forme de l’enveloppe nucléaire régulent l’activité de AP1 et TEAD. Nous avons montré que l’aplatissement du noyau augmente la phosphorylation de c-Jun et la translocation de YAP, conduisant à une augmentation de la transcription des gènes cibles de AP1 et TEAD. Nous avons également observé que l’aplatissement du noyau se produit au cours du cycle cellulaire et favorise la prolifération via l’activation de TEAD et AP1 qui stimulent la progression de la phase G1 à la phase S. / .The shape of the cell nucleus can vary considerably during developmental and pathological processes as a consequence of the mechanical forces emanating from the microenvironment or generated by the cytoskeleton. However the impact of nuclear morphology on the transcriptional machinery is not known. Using a combination of tools to manipulate the nuclear morphology, we observed that changes in nuclear shape regulate the activity of AP1 and TEAD. We showed that nuclear flattening increases c-Jun phosphorylation and YAP nuclear translocation, leading to transcriptional induction of AP1 and TEAD-target genes. Surprisingly, we found that nuclear compression is necessary and sufficient to mediate c-Jun and YAP activation in response to cell- generated contractility or cell spreading. We additionally observed that nuclear flattening occurs during the cell cycle and promotes proliferation via TEAD and AP1- dependent G1 to S progression.

Overall, this study enhances our understanding of the role of c-Myc activation in chromosome 11 repositioning in mouse PreB v-abl/myc cells and a possible interaction between telomeres, TRF2, and lamin A/C underlying this phenomenon. Additionally, the importance of human 17q25.3 is confirmed as a potential region involved in NSCLC tumorigenesis. A utilizationof the 3D telomeric organization profiles is demonstrated a tendency to categorize NSCLC patients into different prognostic subgroups, underscoring a potential future value of its clinical application. / February 2017