Abstract
Duchenne muscular dystrophy (DMD) affects myofibers and muscle stem cells (SC), causing progressive muscle degeneration and repair defects. It was not known whether dystrophic myoblasts—the effector cells of muscle growth and regeneration—are affected. Using a combination of transcriptomic, molecular, functional analyses, and genome-scale metabolic modelling, we demonstrate, for the first time, convergent cell-autonomous abnormalities in primary mouse and human dystrophic myoblasts. In Dmdmdx mouse myoblasts lacking full-length dystrophin transcripts, the expression of 170 other genes was significantly altered. Myod1 (p=2.9e-21) and key muscle genes controlled by MyoD (Myog, Mymk, Mymx, epigenetic regulators, ECM interactors, calcium signalling and fibrosis genes) were significantly downregulated. Gene ontology enrichment analysis indicated significant alterations in genes involved in muscle development and function. These transcriptomic abnormalities translated into functional alterations such as increased proliferation (p=3.0e-3), reduced chemotaxis towards both sera-rich (p=3.8e-2) and cytokine-containing medium (p=1.0e-2), and significantly accelerated differentiation in 3D organotypic cultures. These altered myoblast functions are essential for muscle regeneration. The defects were caused by the loss of expression of full-length dystrophin, as strikingly similar and not exacerbated alterations were also observed in dystrophin-null Dmdmdx-βgeo myoblasts. Corresponding abnormalities were identified in an established dystrophic mouse muscle (SC5) cell line and human DMD primary myoblasts, confirming universal, cross-species and cell-autonomous nature of these defects. The genome-scale metabolic analysis in human DMD myoblasts indicated significant alteration in the rate of glycolysis/gluconeogenesis (log2FC = 4.8), leukotriene metabolism (log2FC = 4.754), mitochondrial beta-oxidation of branched-chain, odd-chain, and di-unsaturated fatty acids (n-6) (log2FC = -1.187, log2FC = -0.8295 and log2FC = -0.655). These results demonstrate the disease continuum: DMD defects in satellite cells cause myoblast dysfunctions affecting muscle regeneration, which is essential to counteract myofiber loss. Contrary to the established belief, our data demonstrate that typical DMD alterations occur in myoblasts, making these cells a novel therapeutic target for the treatment of this lethal disease.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
In the revised manuscript we have included additional genome-scale metabolic analysis in human DMD myoblasts, which revealed significant alteration in specific metabolic pathways. These are consistent with the metabolic alterations found previously in dystrophic muscle and brain, thus confirming the commonality of dystrophic defects found here in myoblasts and described before in other tissues. We have elaborated on why the mdx mouse is a good model of DMD. We clearly linked transcriptomic alterations described in this paper to the published functional data on the abnormalities in human myoblasts, thus demonstrating that data in this paper provide a molecular underpinning for these previously reported abnormalities. Cell homogeneity across genotypes was already confirmed by sample-based hierarchical clustering, clearly segregating transcripts into groups corresponding to genotypes. For completeness, in the revised manuscript, we describe cell characterisation using a myoblast marker, excluding varying contamination with non-myogenic cells as a factor significantly influencing our results.
https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-10322/
