Absence of the dystrophin protein in Duchenne muscular dystrophy : effects on transcriptome, proteome and microRNA secretome

Duchenne muscular dystrophy (DMD) is a classical monogenic disorder characterised by severe muscle weakness. Progressive muscle loss ultimately culminates in fatality due to failure of the heart and diaphragm muscles, typically in the 2nd-4th decade of life. The genetic cause of DMD is disruption of the DMD gene encoding for the dystrophin protein. Dystrophin is important for cytoskeletal structure and normal muscle function, and it plays a vital role in maintaining membrane stability and signalling functions. However, the downstream molecular events that follow dystrophin loss are incompletely understood. Moreover, pharmacodynamic biomarkers that can inform on disease progression are severely lacking. This thesis aimed to identify the effects of the dystrophin protein absence on the cardiac transcriptome, skeletal muscle proteome and extracellular microRNA (miRNA) secretome with pathological progression. To improve the understanding of the dystrophic cardiomyopathic advancement in the various anatomical regions of the heart, transcriptomic arrays were performed on RNA taken from the distinct chambers to identify novel pathways involved in pathology. Analysis identified early molecular changes in the right atrium and ventricle, mostly related to cell migration and cardiac remodelling. Furthermore, Sfrp2 (secreted Frizzled-related protein 2), a major regulator of the Wnt signalling pathway, was found to be highly differentially expressed between control and dystrophic hearts. It was observed that expression of the non-canonical c-Jun N-terminal kinase (JNK)-arm of the Wnt cascade was reduced. Interestingly, cellular fractionation demonstrated decreased levels in the cytoplasmic/extracellular region and increased expression surrounding the nucleus, which suggests a potential mislocalisation of the sFRP2 protein in dystrophic hearts. Protein expression in skeletal muscle was characterised by high-resolution mass spectrometry-based proteomics in two dystrophic mouse models over time. Analysis of the two models at different time points allowed for protein identification irrespective of mutation or age. The results highlighted the involvement of many proteins in DMD, such as Rho GTPases, SLC25A24, RICTOR, CAV3 and MVP. Expression of major vault protein (MVP) increased during differentiation of myoblasts in vitro and interestingly, Mvp knockdown was observed to potentially influence Dmd expression levels, suggesting a novel role for MVP in DMD pathology. The miRNA secretome was examined to investigate the relationship between skeletal muscle dystrophin expression and extracellular miRNA abundance. Interestingly, while muscle-related miRNAs (myomiRs, indicative of muscle turnover) were not restored to wild-type levels in mdx-XistΔhs mice, which express low levels of dystrophin from birth (~8-44%), other miRNAs such as miR-193b-3p, miR-370-3p and miR-483-3p did exhibit a decrease in extracellular abundance. In contrast, exon skipping treatment achieving similar dystrophin re-expression levels was able to completely restore serum miRNA levels. The disparity between the mdx-XistΔhs and mdx mice treated with exon skipping was attributed to uneven dystrophin distribution at the sarcolemma in the former. The observed patchy, within-fibre mosaicism was unable to restore extracellular myomiR abundance, and by extension muscle turnover. These results are directly relevant to the development of therapeutic strategies for dystrophin re-expression. In conclusion, this work has identified novel molecular pathways involved in cardiac and skeletal muscle dystrophic pathology. Furthermore, the results have demonstrated the importance of uniform dystrophin distribution at the sarcolemma for pathological correction. Together, these findings provide new insights into the molecular pathology of DMD with implications for therapy.