Loss of full-length dystrophin expression results in major cell-autonomous abnormalities in proliferating myoblasts

Duchenne muscular dystrophy (DMD) affects myofibers and muscle stem cells, causing progressive muscle degeneration and repair defects. It was unknown whether dystrophic myoblasts—the effector cells of muscle growth and regeneration—are affected. Using transcriptomic, genome-scale metabolic modelling and functional analyses, we demonstrate, for the first time, convergent abnormalities in primary mouse and human dystrophic myoblasts. In Dmd(mdx) myoblasts lacking full-length dystrophin, the expression of 170 genes was significantly altered. Myod1 and key genes controlled by MyoD (Myog, Mymk, Mymx, epigenetic regulators, ECM interactors, calcium signalling and fibrosis genes) were significantly downregulated. Gene ontology analysis indicated enrichment in genes involved in muscle development and function. Functionally, we found increased myoblast proliferation, reduced chemotaxis and accelerated differentiation, which are all essential for myoregeneration. The defects were caused by the loss of expression of full-length dystrophin, as similar and not exacerbated alterations were observed in dystrophin-null Dmd(mdx-βgeo) myoblasts. Corresponding abnormalities were identified in human DMD primary myoblasts and a dystrophic mouse muscle cell line, confirming the cross-species and cell-autonomous nature of these defects. The genome-scale metabolic analysis in human DMD myoblasts showed alterations in the rate of glycolysis/gluconeogenesis, leukotriene metabolism, and mitochondrial beta-oxidation of various fatty acids. These results reveal the disease continuum: DMD defects in satellite cells, the myoblast dysfunction affecting muscle regeneration, which is insufficient to counteract muscle loss due to myofiber instability. Contrary to the established belief, our data demonstrate that DMD abnormalities occur in myoblasts, making these cells a novel therapeutic target for the treatment of this lethal disease

Primary mouse myoblast metabotropic purinoceptor profiles and calcium signalling differ with their muscle origin and are altered in mdx dystrophinopathy

Abstract Mortality of Duchenne Muscular Dystrophy (DMD) is a consequence of progressive wasting of skeletal and cardiac muscle, where dystrophinopathy affects not only muscle fibres but also myogenic cells. Elevated activity of P2X7 receptors and increased store-operated calcium entry have been identified in myoblasts from the mdx mouse model of DMD. Moreover, in immortalized mdx myoblasts, increased metabotropic purinergic receptor response was found. Here, to exclude any potential effects of cell immortalization, we investigated the metabotropic response in primary mdx and wild-type myoblasts. Overall, analyses of receptor transcript and protein levels, antagonist sensitivity, and cellular localization in these primary myoblasts confirmed the previous data from immortalised cells. However, we identified significant differences in the pattern of expression and activity of P2Y receptors and the levels of the “calcium signalling toolkit” proteins between mdx and wild-type myoblasts isolated from different muscles. These results not only extend the earlier findings on the phenotypic effects of dystrophinopathy in undifferentiated muscle but, importantly, also reveal that these changes are muscle type-dependent and endure in isolated cells. This muscle-specific cellular impact of DMD may not be limited to the purinergic abnormality in mice and needs to be taken into consideration in human studies

Additional file 3: of Zidovudine ameliorates pathology in the mouse model of Duchenne muscular dystrophy via P2RX7 purinoceptor antagonism

Figure S2. Average weight gain of mice following AZT treatment. (TIFF 756 kb

Additional file 1: of Zidovudine ameliorates pathology in the mouse model of Duchenne muscular dystrophy via P2RX7 purinoceptor antagonism

Figure S1. Human, giant panda and mouse P2RX7 peptide sequence alignments. (PDF 779 kb

Additional file 4: of Zidovudine ameliorates pathology in the mouse model of Duchenne muscular dystrophy via P2RX7 purinoceptor antagonism

Figure S3. Impact of the 2-week AZT treatment on key pathological parameters. (TIFF 6394 kb

Additional file 6: of Zidovudine ameliorates pathology in the mouse model of Duchenne muscular dystrophy via P2RX7 purinoceptor antagonism

Figure S5. 2-Me-AZT treatment impact on the expression of selected inflammatory genes. (TIFF 900 kb

Additional file 2: of Zidovudine ameliorates pathology in the mouse model of Duchenne muscular dystrophy via P2RX7 purinoceptor antagonism

Table S1. Binding Energies for AZT at all of the sites in the 5U1V identified by the site finder in MOE. (DOCX 16 kb