Characterization of the Growth Hormone Secretagogue Receptor in Dilated Cardiomyopathy

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease of skeletal and myocardial degeneration. Eventually, dilated cardiomyopathy develops from ischemia, inflammation and fibrosis. Due to the high mortality rate, there is an emerging need to diagnose DMD cardiomyopathy at early stages. Currently, DMD cardiomyopathy is diagnosed by imaging investigations and detection of circulating biomarkers. However, current imaging strategies detect functional and morphological changes but fall short in detecting molecular changes that underlie this disease. Circulating biomarkers provide information on the molecular level, but they are not cardiac-specific. Therefore, there is an emerging need for a biomarker that is endogenous to cardiac tissues. The growth hormone secretagogue receptor (GHSR) and its ligand, ghrelin are produced by both cardiomyocytes and vascular endothelial cells and could be an indicator of DMD cardiomyopathy. The work described in this thesis sought to characterize GHSR as a cardiac-localized biomarker in DMD cardiomyopathy. Histopathology and confocal imaging using a novel fluorescent ghrelin analog, Cy5-ghrelin(1-19), were used to investigate changes in cardiac tissue architecture and GHSR and inflammatory markers in the mdx:utrn-/- mouse model of DMD. My studies show that GHSR is elevated in mdx:utrn-/- myocardial tissues and correlate strongly with the macrophage marker F4-80 and the pro-inflammatory cytokine IL-6. Interestingly, I also show that both ghrelin and des-acyl ghrelin bind to sites in large cardiac vessels of mdx:utrn-/- which might be an indicator of vascular inflammation. Finally, my project shows the first report of GHSR in cardiac macrophages. In summary, my work suggests that, in dilated cardiomyopathy, elevations in GHSR correlate with the inflammatory phenotype as mediated by both the myocardium and macrophages

Towards Stem Cell Treatment for Duchenne Muscular Dystrophy-Related Cardiomyopathy

Duchenne muscular dystrophy (DMD) is a progressive muscular degenerative disease affecting 1 in 3,500 boys. Cardiomyopathy is observed in 95% of patients aged 18 and older, accounting for 30% of deaths. While innovative treatments have been employed to curb symptoms in skeletal muscle, few show success in the heart, and no cure for DMD exists. Recently, regenerative therapy using cardiac stem cells (CSCs) has shown very promising results, but efficacy in DMD remains undetermined. This dissertation sought to provide preliminary insight into the effects of murine Sca-1+ CSC therapy. Three-dimensional echocardiography was validated and used to gauge cardiomyopathy in the mdx:utrn-/- mouse model. CSCs were isolated from healthy mice and displayed differentiation potential. They were implanted into mdx:utrn-/- mice under ultrasound guidance, and showed preliminary trends towards functional improvement. These results support the notion that stem cells may be an excellent avenue of therapy that should be further investigated

Non-invasive evaluation of murine models for genetic muscle diseases

Novel therapeutic approaches are being introduced for genetic muscle diseases such as muscle dystrophies and congenital myopathies, all of them having remained without cure so far. These recent developments have motivated a renewed and augmented interest in non-invasive methods for muscle characterization and monitoring, particularly during and after therapeutic intervention. In this context, animal models are essential to better understand the disease mechanisms and to test new therapies. Recently, significant advances in the non-invasive evaluation of mouse models for genetic muscle diseases have been achieved. Nevertheless, there were still several mouse strains not characterized non-invasively, and it was necessary to develop sensitive methods to identify subtle alterations in the murine affected muscle. The purpose of this thesis was to apply non-invasive techniques in the study of murine models for genetic muscle diseases with variable phenotypes. Three mouse models for muscle dystrophy (mdx, Large_myd, mdx/Large_myd) and one mouse model for congenital myopathy (KI-Dnm2_R465W) were studied with Nuclear Magnetic Resonance (NMR) methods. Two dystrophic strains (Large_myd, mdx/Large_myd) and normal mice after injury were studied through micro-Computed Tomography (micro-CT). On NMR, all affected mouse strains presented increased muscle T2, which could be related to variable features in the histological evaluation, including necrosis and inflammation, but also to clusters of fibers under regeneration or with altered cytoarchitecture. The combination of NMR and texture analyses allowed the unambiguous differential identification of all the dystrophic strains, although it was not feasible when comparing the muscle T2 measurements only. Mdx mice showed functional and morphological alterations of vascular network. In the KI-Dnm2_R465W mice, a pilot study revealed tendencies of functional impairment. Finally, micro-CT images were unable to detect differences in muscle´s content in dystrophic mice. Altogether, these results not only increased the number of murine models for genetic muscle diseases non-invasively characterized, it also demonstrated some degree of specificity of the imaging anomalies, as revealed by texture analysis. It also showed that non-invasive NMR methods can be sensitive enough to identify subtle alterations in murine muscle phenotype, even in early stages. This thesis was developed under an international joint supervision between France and Brazil, and comprised an important transfer of technology, with the first non-invasive studies of murine muscles performed in Brazil.De nouvelles options thérapeutiques sont en cours d'introduction pour les maladies musculaires génétiques telles que les dystrophies musculaires et les myopathies congénitales, maladies jusque là sans traitement causal. Ces développements récents ont suscité un intérêt renouvelé et croissant pour les méthodes atraumatiques en vue de caractériser et de suivre les muscles atteints, en particulier pendant et après une intervention thérapeutique. Dans ce contexte, les modèles animaux sont essentiels pour mieux comprendre les mécanismes des maladies et pour tester des nouvelles thérapies. Récemment, il y a eu des avancées significatives dans l'évaluation atraumatique de modèles murins de maladies musculaires génétiques. Néanmoins, nombre de lignées de souris n'ont pas encore été caractérisées de façon atraumatique et il reste à mettre au point des méthodes plus sensibles pour identifier précocement des altérations subtiles dans le muscle des souris malades. L'objectif de cette thèse est d'appliquer des techniques atraumatiques innovantes à l'étude du muscle de modèles murins de maladies musculaires génétiques avec des phénotypes variés. Trois lignées de souris modèles de dystrophies musculaires (mdx, Large_myd et mdx/Large_myd) et une lignée de souris modèle de la myopathie congénitale (KI-Dnm2_R465W) ont été étudiées par des méthodes de Résonance Magnétique Nucléaire (RMN). Deux lignées dystrophiques (Large_myd et mdx/Large_myd) plus des souris normales après une blessure ont été étudiées par micro-tomographie (micro-CT). En RMN, toutes les souches de souris affectées ont présenté un T2 musculaire augmenté, en relation avec une gamme d'anomalies histologiques, y comprises nécrose et inflammation, mais aussi des groupes de fibres en régénération ou des fibres avec altérations de l'architecture. Avec la combinaison de la RMN et de l'analyse de la texture, il a été possible d'identifier sans ambiguïté toutes les lignées dystrophiques, alors que la seule mesure du T2 ne permettait pas de les différencier. Les souris mdx ont présenté des altérations fonctionnelles et morphologiques du réseau vasculaire musculaire. Pour les souris KI-Dnm2_R465W, des études préliminaires ont révélé une tendance à développer des altérations fonctionnelles musculaires. Finalement, les images de micro-CT n'ont pas pu détecter des différences du contenu musculaire dans les souris dystrophiques. L'ensemble des résultats non seulement enrichit le panel de modèles murins de maladies génétiques musculaires caractérisés de manière atraumatique, il révèle également un certain degré de spécificité des anomalies dans l'imagerie, comme l'a montré l'analyse de texture. Les résultats démontrent aussi que des méthodes de RMN non-invasives peuvent être assez sensibles pour identifier des altérations subtiles dans le phénotype musculaire murin, même à des stades précoces. Cette thèse a été développée dans le cadre d'une co-tutelle internationale entre la France et le Brésil, et elle a comporté un important transfert de compétence, qui a permis de réaliser les premières explorations atraumatiques du muscle murin effectuées au Brésil

Interrogating the Viscoelastic Properties of Tissue Using Viscoelastic Response (VisR) Ultrasound

Affecting approximately 1 in 3,500 newborn males, Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic disorders in humans. Boys with DMD suffer progressive loss of muscle strength and function, leading to wheelchair dependence, cardiac and respiratory compromise, and death during young adulthood. There are currently no treatments that can halt or reverse the disease progression, and translating prospective treatments into clinical trials has been delayed by inadequate outcome measures. Current outcome measures, such as functional and muscle strength assessments, lack sensitivity to individual muscles, require subjective effort of the child, and are impacted by normal childhood growth and development. The goal of this research is to develop Viscoelastic Response (VisR) ultrasound which can be used to delineate compositional changes in muscle associated with DMD. In VisR, acoustic radiation force (ARF) is used to produce small, localized displacements within the muscle. Using conventional ultrasound to track the motion, the displacement response of the tissue can be evaluated against a mechanical model. In order to develop signal processing techniques and assess mechanical models, finite element method simulations are used to model the response of a viscoelastic material to ARF excitations. Results are then presented demonstrating VisR differentiation of viscoelastic changes with progressive dystrophic degeneration in a dog model of DMD. Finally, clinical feasibility of VisR imaging is demonstrated in two boys with DMD.Doctor of Philosoph

Elucidation of Emergent Regional Mechanisms of Heart Muscle Dysfunction in the Mouse Model of Duchenne Muscular Dystrophy

Cardiac dysfunction is a primary cause of mortality in Duchenne Muscular Dystrophy (DMD), potentially related to elevated cytosolic calcium. However, the regional versus global functional consequences of cellular calcium mishandling have not been defined in the whole heart. Here, we elucidate, for the first time, loci- and age-dependencies between calcium mishandling and myocardial sheet function as a manifestation of dystrophin-deficient cardiomyopathy. We also map calcium transients to illustrate the regional dependence of ion flux disturbances in the dystrophin-deficient (mdx) mouse heart. Furthermore, we elucidate abnormalities in autophagic processes that can be corrected with nanoparticle therapeutics delivering rapamycin to heart tissues to improve ventricular function in affected older mice with incipient cardiomyopathy. We conclude that the rapid reversibility of functional defects by reducing cytosolic calcium or by impacting impaired autophagy points to the significance of regional mechanical factors in the progression of the disease

Myocardial Fat Imaging

The presence of intramyocardial fat may form a substrate for arrhythmias, and fibrofatty infiltration of the myocardium has been shown to be associated with sudden death. Therefore, noninvasive detection could have high prognostic value. Fat-water–separated imaging in the heart by MRI is a sensitive means of detecting intramyocardial fat and characterizing fibrofatty infiltration. It is also useful in characterizing fatty tumors and delineating epicardial and/or pericardial fat. Multi-echo methods for fat and water separation provide a sensitive means of detecting small concentrations of fat with positive contrast and have a number of advantages over conventional chemical-shift fat suppression. Furthermore, fat and water–separated imaging is useful in resolving artifacts that may arise due to the presence of fat. Examples of fat-water–separated imaging of the heart are presented for patients with ischemic and nonischemic cardiomyopathies, as well as general tissue classification

Angiogenic Therapy in a Fibrotic Murine Model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disorder that affects approximately 1 in 5000 males. Vascular-targeted therapy has been proposed as a treatment for DMD to reduce ischemia and enhance endogenous repair. Additionally, a more vascularized environment may enhance regenerative approaches currently under investigation. Vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANG1) are two of the most studied pro-angiogenic factors for this approach. To date, little is known regarding the effect of these pro-angiogenic factors on muscle function and whether they may exacerbate fibrosis in a relevant murine model of DMD. The first aim of this thesis was to determine the murine model that is best suited for assessing vascular therapy. We demonstrate the dystrophin null, utrophin heterozygous mouse (mdx/utrn+/-) develops more collagen deposition at an earlier age than the commonly used mdx mouse and is therefore a superior choice for assessing therapeutic effects on fibrosis. Next, we investigated the effect of exogenous VEGF treatment on fibroblasts derived from severely affected diaphragm and mildly affected gastrocnemius muscles of mdx/utrn+/- mice. VEGF treatment induced differentiation into myofibroblasts in both cell types, suggesting induction of a fibrotic response. The final aim of the thesis was to assess the effect of VEGF alone or in combination with ANG1 on functional perfusion as assessed non-invasively using dynamic contrast-enhanced computed tomography. A combination of VEGF and ANG1, but not VEGF alone, slowed progression of ischemia in the mdx/utrn+/- hind limb. Increased vessel maturation, as assessed histologically, validated the imaging findings. The combination treatment also decreased fibrosis and leukocyte infiltration, consistent with decreased vascular permeability following ANG1 treatment. Overall, the research in this thesis highlights the drawbacks to use of VEGF as a treatment for ischemia associated with DMD and reveals considerations for future use of vascular therapy in murine models of the disease

Effect of novel ryanodine receptor modulators in mouse and human models of Duchenne muscular dystrophy

157 p.La distrofia muscular de Duchenne (DMD) es una enfermedad hereditaria ligada al cromosoma X queafecta a uno de cada 3500 varones. Esta enfermedad se caracteriza por la degeneración progresiva de lasfibras musculares que conlleva debilidad muscular, incapacidad y muerte prematura.Desafortunadamente, actualmente no existe terapia eficaz para el tratamiento de estos pacientes. La DMDestá producida por mutaciones en el gen que codifica la distrofina una proteína de membrana que actúacomo amortiguador y a su vez sirve de anclaje para diversas proteínas de señalización que son esencialespara el correcto funcionamiento muscular. A su vez, se ha visto que en la DMD los receptores derianodina (RyR) sufren modificaciones post-transcripcionales que afectan su unión a la calstabina (Calst),y generan una pérdida de calcio desde el retículo sarcoplásmico, lo que conlleva al aumento de losniveles de calcio intracelulares. En el presente trabajo, los compuestos AHK que han sido diseñados paraactuar sobre esta prometedora diana terapéutica en colaboración con el grupo del Dr Aizpurua de laUniversidad del País Vasco, han demostrado aumentar significativamente la interacción RyR-Calstmejorando el fenotipo distrófico en ratones mdx, modelo animal de DMD. A su vez, el efecto de loscompuestos AHK ha sido testado in vitro en un modelo humano de DMD, donde han demostrado sueficacia aumentando la interacción RyR1-Calst1 y provocando la reducción de los niveles de calciointracelular. Por todo ello, se concluye que los compuestos AHK podrían consolidar una alternativaterapéutica de gran utilidad para el tratamiento de la DMD