Therapeutic targets for duchenne muscular dystrophy
Bahri, Ola Abdulsamad
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Duchenne Muscular Dystrophy (DMD) is a progressive X-linked fatal muscle degenerative disorder caused by the absence of the sarcolemmal associated dystrophin-glycoprotein complex (DGC) that connects the intracellular cytoskeleton to the extracellular matrix. DMD has an incidence of 1:3,500 in males. Females can be carriers of DMD mutations and asymptomatically affected at later ages. This thesis encompasses three main studies focussed on different therapeutic targets for DMD. The first study examines the therapeutic potential of using gene therapy in correcting calcium dysregulation in the pre-clinical mdx mouse model of DMD. Adeno-associated virus 2/9 (AAV2/9) carrying the green fluorescent protein (GFP) showed robust transduction in gastrocnemius of mdx mice. Intramuscular injection of AAV-hSERCA2a (sarco/endoplasmic reticulum Ca2+-ATPase) demonstrated some amelioration in the dystrophic phenotype including a reduction in the percentage of central nucleated fibres (CNFs) and enhanced gene expression of [Ca2+]-buffering proteins. However, striking elevations in the expression of genes encoding SERCA-inhibitors, inflammatory and proteolytic related proteins were also observed. Hence, we conclude that elevation in the aforementioned genes can present a challenge to the benefits of AAV-hSERCA2a gene therapy in mdx mice. The second part of this thesis is mostly dedicated to characterising the dermal associated panniculus carnosus (PC) skeletal muscle in wild-type and mdx mice. Histological and morphological hallmarks of dystrophy were assessed in PC at different age time-points. We next chose to study the myogenicity of the PC by isolating the PC satellite cells (SCs) from wild-type and mdx mice of both genders and differentiated them in vitro. Finally, we examined the hypothesis of Ca2+ dysregulation in the differentiated PC cells. PC muscle demonstrated a high turnover that increased with dystrophy and further augmented with age. We found that PC exemplified gender dimorphism in muscular dystrophy demonstrated by high myogenic activity in male mdx PC-derived cells in contrast to females. In conclusion, we propose that PC is a unique muscle model to study cellular mechanisms impinging on muscle regeneration in healthy and diseased mice that may reveal new therapeutic targets for DMD. Finally, we examined gender dimorphism in muscle regeneration by analysing morphology, myogenic and calcium-handling genes. This was facilitated by studying the histological and morphological hallmarks of dystrophy and measuring mRNA levels of selected genes including sex-hormone receptors, myogenic factors, MyHC isoforms and Ca2+-handling proteins. Demonstrated by the percentage of CNF, dystrophic gastrocnemius muscles regenerated more than dystrophic PC muscles. Male PC muscles of mdx mice demonstrated a higher turnover compared to their female counterparts. In addition, while dystrophic gastrocnemius muscles underwent hypertrophy, dystrophic PC remained at similar size to healthy counterparts. Furthermore, while dystrophy displayed no effect on capillarization in gastrocnemius, PC muscles had variable responses between genders. In summary, we propose that female PC muscles had the highest capability in managing muscular dystrophy and, hence, by further investigating it, this may lead to promising therapeutic approach for DMD.
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