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

Background Duchenne muscular dystrophy (DMD) is the most common inherited muscle disease that leads to severe disability and death in young men. DMD is caused by out-of-frame mutations in the largest known gene, which encodes dystrophin. The loss of DMD gene expression manifests in progressive degeneration and wasting of striated muscles aggravated by sterile inflammation. Current conventional treatments are palliative only, whereas experimental therapeutic approaches focus on the re-expression of dystrophin in myofibers. However, recent studies established that DMD pathology begins already in prenatal development prior to myofiber formation while, in adult muscle, it affects satellite (stem) cells and the proper development of myofibers. Regeneration defects that exacerbate muscle degeneration appear to be a good therapeutic target, as maintaining regeneration would counteract muscle wasting. It is also the only feasible treatment in advanced stages of the disease. Yet, it is unknown whether dystrophic myoblasts, the intermediary between satellite cells and myofibers and effectors of muscle growth and repair, are also affected. Therefore, we investigated whether DMD myoblasts show a dystrophic phenotype.

Methods and Findings Using a combination of transcriptomic, molecular, biochemical, and functional analyses we demonstrate, to our knowledge for the first time, convergent cell-autonomous abnormalities in primary mouse and human dystrophic myoblasts. In Dmd^mdx mouse myoblasts lacking full-length dystrophin transcripts, 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 increased proliferation (p=3.0e⁻³), reduced migration 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 Dmd^mdx-βgeo myoblasts. Furthermore, corresponding abnormalities were identified in human DMD primary myoblasts and in an established dystrophic mouse muscle (SC5) cell line, confirming universal, cross-species and cell-autonomous nature of this defect.

Conclusions These results, for the first time, demonstrate the disease continuum: DMD defects in satellite cells cause myoblast dysfunctions diminishing muscle regeneration, which is essential to counteract myofiber degeneration. Full-length dystrophins play a critical role in these processes. Contrary to the established belief, our data identify myoblasts as a novel and important therapeutic target for treatment of this lethal disease.