The giant protein titin is the third myofilament in the cardiac sarcomere. It is responsible for generating passive forces in stretched myocardium and maintaining sarcomere structure. The force generation properties of titin are determined by titin's elastic springlike elements, and this dissertation focuses on the determination of the physical properties of these springlike elements using atomic force microscopy. The primary project of this dissertation investigates the link between a single point mutation in one of titin's subdomains and arrhythmogenic cardiomyopathy.

La cardiomyopathie/dysplasie arythmogène du ventricule droit (ARVC/D) est un désordre d’origine génétique caractérisé par le remplacement du myocarde par du tissus fibro-adipeux dans le ventricule droit. Ce désordre est responsable d’un grand pourcentage de mort subite, spécialement chez les plus jeunes. ARVC/D est difficile à diagnostiquer avec les outils cliniques actuels. Elle est causée en grande majorité par des mutations dans les protéines desmosomales. ARVC/D a donc des implications d’une grande importance chez les membres de la famille, qui peuvent sans le savoir, être aussi à risque de mort subite. Dans le but d’améliorer le diagnostique, un nouvel outil, le test génétique, est de plus en plus utilisé. Hypothèses: Dans le but d’évaluer la valeur du test génétique en complément du test clinique classique chez ARVC/D nous avons effectué une investigation clinique et génétique chez 23 cas-index atteints. Méthodes: Les cas-index sont diagnostiqué après une mort subite dans la famille ou après un examen clinique poussé pour arythmies. Le diagnostique d’ARVC/D a été fait avec les outils cliniques selon les critères. L’analyse génétique des protéines desmosomales associées à la maladie a été effectuée en séquençant leurs exons ainsi que les régions introniques nécessaires à l’épissage alternatif. Résultats: Le diagnostique clinique était clair dans 18/23 et incertain dans 5/23 des individus. Nous avons identifié 15 différentes mutations chez 10 cas-index. 64% des mutations n’avaient jamais été décrites. De plus, nous avons observé la présence de double ou triple mutant dans 40% des cas-index positifs. Les individus avec mutations sont plus jeunes et ont plus de symptômes que les individus sans mutation. Conclusion: Les tests génétiques sont positifs dans 43% des patients avec ARVC/D. L’utilisation de la technologie génétique basée sur l’identification de mutations connues a une valeur limitée vu le haut pourcentage des mutations nouvelles dans la maladie. La présence de double, même de triple mutant n’est pas associé avec un phénotype plus sévère, mais renforce l’idée de la nécessité d’un test génétique pour tous les gènes. Le test génétique est un outil fort utile à ajouter aux tests cliniques pour le diagnostique des patients qui ne remplissent pas tous les critères cliniques de la maladie. Mots clés: génétique, ARVC/D, mort subite, desmosome / Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is a genetic disorder characterized by the presence of fibro-fatty replacement of the myocardium in the right ventricle. The disease is thought responsible for an important percentage of sudden cardiac death in the young. Hence the disease is usually difficult to diagnose with present clinical tools. ARVC/D is it caused in greater part by mutations in desmosomal proteins. The diagnosis of the genetic carriers bears important implications in family members, who unknowingly may be at risk for sudden death. In order to improve the diagnosis, a new tool, genetic testing, is increasingly being used. Hypothesis: In order to assess the value of genetic testing in complementing clinical testing in ARVC/D, we undertook the project to collect and perform clinical and genetic investigation in 23 probands with the disease. Methods: The probands were usually identified either after the death of a family member or after their clinical investigation for arrhythmias. The diagnosis of ARVC was made with clinical tools according to accepted criteria. Genetic analysis of desmosomal proteins previously associated with the disease was performed by sequencing the exons and intron-exon boundaries. Results: The clinical diagnosis was clear in 18/23 and suspicious in 5/23 individuals. We identified 15 different mutations in 10 probands. 64% of the mutations were not previously described. Interestingly we also observed the presence of double or triple mutants in 40% of the positive individuals. Individuals with mutations were younger and had more symptoms than individuals with no mutation. Conclusion: Genetic testing is useful in 43% of patients with ARVC. The use of mutation-based genetic technology has a very limited value due to the high percentage of previously unknown mutations in this disease. The presence of double and even triple mutants is not associated with a more severe phenotype but it indicates the need to have genetic testing performed for all genes for familial screening. Genetic testing is a useful tool to add to the clinical testing for the diagnosis of patients who do not completely fulfill the clinical criteria for the disease. Key words: genetic, ARVC/D, sudden death, desmosome

No description available.

Desmosomen sind hochorganisierte Zell-Zell-Verbindungen. Aufgrund ihrer hohen Adhäsivität sind sie für die mechanische Kopplung und strukturelle Stabilität stark beanspruchter Gewebe von essentieller Bedeutung. Die adhäsive interzelluläre Kernstruktur der Desmosomen wird durch die transmembranen Cadherine des Desmocollin und Desmoglein-Typs gebildet. Deren extrazelluläre Domänen stellen den Kontakt zwischen zwei benachbarten Zellen her. Dsc2 ist neben Dsg2 die prädominante Isoform und wird in allen Desmosomen-bildenden Geweben wie auch dem Herzen exprimiert. Ziel der Arbeit war es, die Rolle von Dsc2 bei der Etablierung und Aufrechterhaltung der desmosomalen Adhäsivität und Gewebeintegrität zu untersuchen. Hierfür wurde ein klassisches Knockout-Mausmodell für Dsc2 etabliert und sowohl basal als auch unter Belastungsbedingungen charakterisiert. Unsere Daten demonstrieren, dass der ubiquitäre Knockout keinen Einfluss auf die Morphogenese des Embryos und die postnatale Entwicklung hat. Dsc2-/- -Mäuse waren lebensfähig und wiesen keinen pathologischen Phänotyp auf. Zudem ließen sich in den Herzen adulter Tiere strukturell unveränderte Desmosomen nachweisen. Dahingegen konnte eine verminderte mechanische Beanspruchbarkeit der Dsc2-/- -Herzen aufgezeigt werden. Unter erhöhter Belastung zeigte sich bereits nach wenigen Tagen eine signifikante Reduktion der kardialen Funktion.Die vorliegende Arbeit zeigt somit erstmalig in vivo, dass Dsc2 entgegen der Lehrmeinung nicht essentiell für die Embryonalentwicklung und die Bildung strukturell intakter Desmosomen ist. Anhand des Funktionsverlustes der adulten Knockout-Herzen unter Belastungsbedingungen lässt sich jedoch eine mögliche Rolle von Dsc2 für die Adhäsivität der Desmosomen postulieren. / Desmosomes are highly organized adhesive intercellular junctions providing mechanical strength and structural stability to several tissues such as skin and heart. The adhesive core of desmosomes is formed by the transmembrane glycoproteins desmocollins (Dsc) and desmogleins (Dsg) which link neighbouring cells via interaction with their extracellular cadherin domains. Dsc2 and Dsg2 are the predominant isoforms ubiquitously expressed in all desmosome bearing tissues including the heart. To elucidate the role of Dsc2 for establishment and maintenance of desmosome adhesion and tissue integrity, we generated a constitutive knockout model of the mouse. The effect of gene inactivation was characterized under basal as well as under stress conditions, using two different stress models. Our data demonstrate that Dsc2 is not required for pre- and postnatal development. Dsc2-/- mice were viable and showed no pathological alterations at embryonic or adult stages. Consistently, Dsc2 deficient cardiomyocytes exhibited distinct and ultrastructural well organized desmosomes. However, mutant hearts displayed a decreased stress resistance. Increased mechanical pressure led to a significant reduction of cardiac function in Dsc2-/- animals. In summary, our results demonstrate for the first time in vivo that Dsc2 is not essential for embryonic development and for the establishment and maintenance of distinct and well organized desmosomes. However, the reduced cardiac function in stressed knockout-mice suggests a crucial importance of Dsc2 for desmosomal adhesive strength.

Introduction: Cardiac cell-cell junctions play important roles in maintaining cardiac integrity linking single cardiomyocytes into a single functioning syncytium. There are three main types of cell junctions in the heart: gap junctions (GJ), desmosomes (D) and adherens junctions (AJ). Mutations in the proteins which make-up these junctions are known to cause arrhythmogenic right ventricular cardiomyopathy (ARVC). Pathological features include progressive replacement of right ventricular cardiac muscle with fibrofatty tissue. This can lead to heart failure and life threatening arrhythmias. During normal development of the mammalian heart, protein components of AJ and D gradually fuse to form composite junctions at the intercalated discs, also called areae compositae (singular, area composita, AC). In contrast, the adult heart of lower vertebrates, including the zebrafish, may have few or no AC type junctions. The detailed structure of cardiomyocyte cell-cell junctions in the adult zebrafish heart remain poorly defined and their role in normal development, growth and response to injury have yet to be studied. This thesis will examine the hypothesis that localisation and distribution of myocardial cell-cell junction proteins are crucial in normal myocardial development and in endogenous cardiac regeneration and repair following injury. This will be achieved by understanding the normal development of cell-cell junction proteins in zebrafish from embryonic to adulthood. These findings will then be analysed in comparison to cell-cell junction proteins localisation and distribution in early and late mammalian (mouse and human) myocardium. Once a normal pattern of cell-cell junction proteins will be established, the localisation of cell-cell junction proteins in plakologbin mutant zebrafish model for cardiomyopathy will be studied to understand the distribution and localisation of these proteins in disease manifestation. This model will then be used to test if localisation of cell-cell junction proteins plays an important in cardiac repair following injury by using embryonic laser injury model, this will be further tested by drug intervention study to investigate underlying pathways such as Wnt signalling pathway. Methods: Myocardial cell-cell junctions were assessed using immunohistochemistry in embryonic, juvenile and adult zebrafish hearts and in foetal and adult human hearts. The Plakoglobin mutant zebrafish line (UAS:Gal-4:Plakoglobin Naxos; named as PGNx) was characterised using various functional and morphological assessments including histology, echocardiography and MRI scanning. Similar studies were undertaken in PGNx mutants at different developmental stages. A pharmacological intervention study, using a GSK-3 inhibitor, was carried out in PGNx mutants followed by cardiac structural and functional assessments. Laser-induced cardiac trauma was used to assess the response to injury and repair in normal and PGNx embryos following treatment with the GSK3 inhibitor drug. Results: Cell-cell junction patterning in the embryonic, juvenile and adult zebrafish heart shows a characteristic pearl string appearance of desmoplakin and β-catenin labelled distinct disc shaped AJ. Human foetal heart showed small distinct D and AJ, while the adult human heart had features consistent with AC type junctions. PGNx fish showed reduced ventricle ejection fraction, dilatation of the atrium, reduced amplitude of wall motion and ventricle relaxation velocity compared to age-matched controls. Echocardiography and MRI imaging confirmed severe atrial dilatation and restrictive ventricle physiology in adult fish. The cell-cell junction proteins were over-expressed in the zebrafish PG mutant (PGNx) hearts compared to age-matched controls. Drug studies using a GSK-3β inhibitor showed complete recovery of cardiac function and partial recovery of heart structure. Cardiac injury studies, using laser, showed failure of repair in PGNx embryos compared to age-matched controls. The GSK3 inhibitor failed to improve the functional response following heart laser injury. Conclusions: Cell-cell junctions are distributed abundantly around cardiomyocytes in the zebrafish heart during early development and into adulthood. In contrast to previous studies in adult mammalian heart, there was no evidence of AC type junctions in adult zebrafish cardiomyocytes. The mutant zebrafish line showed restrictive cardiac physiology and abnormal cardiac structure confirming the key role played by plakoglobin in the normal heart development. This is further supported by evidence showing failure of repair in PGNx mutant embryos after injury. Drug treatment with a GSK-3 inhibitor highlights a potentially novel therapeutic pathway for treatment of ARVC involving Wnt signalling.