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During mammalian skeletal muscle development, muscle fibres form in a biphasic manner from the fusion of myoblasts. Primary fibres form first, which subsequently provide a surface for later secondary fibres to form on. The purpose of the present study was to successfully develop new and existing techniques and to employ them in order to study the commitment and behaviour of myoblasts during muscle development in mice. Following part 1; a general introduction into the development of skeletal muscle, the thesis is divided into two subsequent parts giving details of the investigations performed. In the main section (part 2) of this thesis, I investigated the commitment of myoblasts during the foetal development. It has been suggested, that separate populations of myoblasts are present, each committed to producing the different fibre types seen during development. The aim of this study was to see if different populations produced primary and secondary fibres, by seeing if clones of related cells were restricted to fusing with a single type of fibre. Following the injection of replication deficient retroviruses into the hindlimbs of Embryonic day (E)15 and El 7 foetal mice, cells became marked with the lac Z gene encoding for the enzyme [Special character omitted]-galactosidase. The infected cells, their descendants and the fibres they fused with could then be demonstrated histochemically. 83% of the clusters of marked fibres obtained following processing were found to contain both primary and secondary fibres as identified by electron microscopy. The clusters were assumed to be the result of the fusion of a single clone of cells. It was concluded that at these ages, a single population of cells contributes to primary and secondary fibres. Part 3 of the thesis describes a second, shorter study whereby the in vitro behaviour of El 7, El 9 and E21 myoblasts was investigated on artificial grooved substrata. Most cells on grooves with depths of 250nm-6um were found to align parallel with the direction of the grooves. Cells on the shallower grooves (40-140nm) either aligned parallel or perpendicular to the grooves. E21 cells however, orientated randomly on these groove sizes. It was generally concluded however, that myoblasts at the ages studied do align in grooves similar to those formed in vivo by adjacent primary and secondary fibres. It is suggested that grooves such as the ones mentioned may be a possible site for secondary myogenesis. The results of both my studies contribute to the current work being carried out on skeletal muscle development, and may also, in addition, provide useful information towards the development of myoblast transfer therapy, a possible treatment for Duchenne Muscular Dystrophy sufferers.

Duchenne Muscular Dystrophy (DMD) is a disorder caused by mutations in the dystrophin gene, preventing the production of the functional dystrophin protein which assures maintenance of the myofiber integrity throughout muscle contraction. A lack of dystrophin results in severe muscle degeneration and regeneration accompanied by a loss of muscle function. Many pre-clinical and clinical studies are focused on developing strategies to counteract the detrimental effects of DMD; however, there is no cure. One such approach consists of upregulating the endogenous protein utrophin A in dystrophic muscle, which, once highly expressed at the sarcolemma, could functionally compensate for the lack of dystrophin. Recent evidence demonstrates that utrophin A expression is regulated at its 3’ and 5’UTR through post-transcriptional and translational events. Therefore, in the work presented here, we hypothesized that repurposing FDA-approved drugs that target the signaling pathways involved in post-transcriptional and translational regulation of utrophin A will be an efficient approach in rapidly bringing new therapeutic interventions for DMD. In this work, we repurposed four promising FDA-approved drugs able to stimulate utrophin A expression levels in dystrophic muscles: the anti-coagulant drug Heparin, the anti-inflammatory drug Celecoxib, the β-adrenergic receptor blocking agent Betaxolol and the cholesterol-lowering drug Pravastatin. These drugs induce significant improvements in the dystrophic phenotype of mdx mice. This includes amelioration of muscle fiber integrity and muscle function as well as promoting morphological and fiber type changes in mdx mice muscles. Collectively, this thesis describes the potential of a repurposing approach to activate key post-transcriptional and translational pathways involved in utrophin A’s regulation in the hopes of developing new therapeutics for the treatment of DMD.

Myotonic dystrophy is a genetic neurodegenerative disease caused by repeat expansion mutations. Myotonic dystrophy type 1 (DM1) is caused by a CTG repeat expansion in the 3’ UTR of the dystrophia myotonia protein kinase (DMPK) gene, while myotonic dystrophy type 2 (DM2) is caused by a CCTG repeat expansion in intron 1 of the zinc finger protein nine (Znf9) gene. When expressed, these genes produce long CUG/CCUG repeat RNAs that bind and sequester a family of RNA-binding proteins known as muscleblind-like 1, 2 and 3 (MBNL1, MBNL2, MBNL3). Sequestration of these proteins plays a prominent role in pathogenicity in myotonic dystrophy. MBNL proteins regulate alternative splicing, and myotonic dystrophy symptoms are a result of mis-spliced transcripts that MBNL proteins regulate. MBNL proteins bind to a consensus sequence YGCY (Y = pyrimidine), which is found in CUG and CCUG repeats, and cellular RNA substrates that MBNL proteins bind and regulate. CUG and CCUG repeats can form A-form helices, however it is hypothesized that MBNL proteins bind to the helices when they are open and the YGCY binding site is single-stranded in nature. To evaluate this hypothesis, we used structure-stabilizing RNA modifications pseudouridine (Ψ) and 2’-O-methylation to determine if stabilization of CUG and CCUG repeat helices affected MBNL1 binding and toxicity. We also used Ψ to determine if the structure-stabilizing modification affected MBNL binding to single-stranded YGCY RNA. CUG repeats modified with Ψ or 2’-O-methyl groups exhibited enhanced structural stability and reduced affinity for MBNL1. Ψ also stabilized the structure of CCUG repeats and rigidified single-stranded YGCY RNA and inhibited MBNL1 binding to both of these RNAs. Binding data from CCUG repeats and single-stranded YGCY RNA suggest that both pyrimidines in the YGCY motif must be modified for significant inhibition. Molecular dynamics and X-ray crystallography suggest a potential water-bridging mechanism for Ψ-mediated CUG repeat stabilization. Molecular dynamics simulations suggest that Ψ increases base-stacking interactions, and reducing the flexibility of single-stranded RNA leads to reduced MBNL1 binding. Ψ modification rescued mis-splicing in a cellular DM1 model and prevented CUG repeat toxicity in zebrafish embryos. This dissertation includes previously published and unpublished coauthored material.

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