Abstract
Duchenne muscular dystrophy (DMD) is a genetic disease evoked by a mutation in the dystrophin gene. It is associated with progressive muscle degeneration and increased inflammation. Up to this date, mainly anti-inflammatory treatment is available for patients suffering from DMD. miR-146a is known to diminish inflammation and fibrosis in different tissues by downregulating the expression of proinflammatory cytokines. However, its role in DMD has not been studied so far.In our work, we have generated mice globally lacking both dystrophin and miR-146a (miR-146a−/−mdx) and examined them together with wild-type, single miR-146a knockout and dystrophic (mdx—lacking dystrophin) mice in a variety of aspects associated with DMD pathophysiology (muscle degeneration, inflammatory reaction, muscle satellite cells, muscle regeneration, and fibrosis).We have shown that miR-146a level is increased in dystrophic muscles in comparison to wild-type mice. Its deficiency augments the expression of proinflammatory cytokines (IL-1β, CCL2, TNFα). However, muscle degeneration was not significantly worsened in mdx mice lacking miR-146a up to 24 weeks of age, although some aggravation of muscle damage and inflammation was evident in 12-week-old animals, though no effect of miR-146a deficiency was visible on quantity, proliferation, and in vitro differentiation of muscle satellite cells isolated from miR-146a−/−mdx mice vs. mdx. Similarly, muscle regeneration and collagen deposition were not changed by miR-146a deficiency. Nevertheless, the lack of miR-146a is associated with decreased Vegfa and increased Tgfb1.Overall, the lack of miR-146a did not aggravate significantly the dystrophic conditions in mdx mice, but its effect on DMD in more severe conditions warrants further investigation.
Highlights
Duchenne muscular dystrophy (DMD) is an X chromosome-associated monogenic disease, caused by mutations in a gene encoding dystrophin, leading to the lack of functional protein [1, 2]
Results miR-146a is elevated in dystrophic muscles and its lack increases expression of proinflammatory genes To confirm the miR-146a deficiency of miR-146a−/−mdx mice generated in our lab, qRT-PCR was performed (Fig. 1a). miR-146a−/− and miR-146a−/−mdx animals lack the expression of miR-146a (Fig. 1a)
MiR-146a deficiency does not significantly aggravate muscle degeneration and inflammatory reaction in dystrophic muscles Degeneration of skeletal muscle was measured basing on markers released to blood (Fig. 2a) and determination of the percentage of necrotic fibres (Fig. 2b)
Summary
Duchenne muscular dystrophy (DMD) is an X chromosome-associated monogenic disease, caused by mutations in a gene encoding dystrophin, leading to the lack of functional protein [1, 2]. Persistent membrane instability and proinflammatory cytokines induce the expression of major histocompatibility complex (MHC I and II) on muscle cells, and afterwards recruitment of Th and Tc lymphocytes, that further contribute to muscle damage [11, 14] by secretion of tumour necrosis factor-α (TNFα) and interferon-γ (IFNγ) cytokines that induce proinflammatory phenotype in macrophages [13,14,15]. In response to the repetitive primary and secondary damage of muscle tissue, the process of muscle regeneration is induced [5, 7]. It is strictly dependent on the muscle satellite cells (SCs)—progenitors of skeletal muscle tissue that became activated upon injury and give rise to myoblasts [17, 18]. Its lack in SCs of dystrophic muscles results in the impaired polarity of SCs, loss of asymmetric division, reduced generation of myogenic progenitors, and impaired muscle regeneration [20]
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