Abstract
Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disorder. Here, we show that the CD63 antigen, which is located on the surface of extracellular vesicles (EVs), is associated with increased levels of muscle-abundant miRNAs, namely myomiRs miR-1, miR-133a, and miR-206, in the sera of DMD patients and mdx mice. Furthermore, the release of EVs from the murine myoblast C2C12 cell line was found to be modulated by intracellular ceramide levels in a Ca2+-dependent manner. Next, to investigate the effects of EVs on cell survival, C2C12 myoblasts and myotubes were cultured with EVs from the sera of mdx mice or C2C12 cells overexpressing myomiRs in presence of cellular stresses. Both the exposure of C2C12 myoblasts and myotubes to EVs from the serum of mdx mice, and the overexpression of miR-133a in C2C12 cells in presence of cellular stress resulted in a significant decrease in cell death. Finally, to assess whether miRNAs regulate skeletal muscle regeneration in vivo, we intraperitoneally injected GW4869 (an inhibitor of exosome secretion) into mdx mice for 5 and 10 days. Levels of miRNAs and creatine kinase in the serum of GW4869-treated mdx mice were significantly downregulated compared with those of controls. The tibialis anterior muscles of the GW4869-treated mdx mice showed a robust decrease in Evans blue dye uptake. Collectively, these results indicate that EVs and myomiRs might protect the skeletal muscle of mdx mice from degeneration.
Highlights
Duchenne muscular dystrophy (DMD; OMIM #310200) is an X-linked recessive, severe and progressive muscle disease with a prevalence of 1 in 3,500 live male births, and is caused by PLOS ONE | DOI:10.1371/journal.pone.0167811 December 15, 2016Characterization and Functional Analysis of Extracellular Vesicles and Muscle-Abundant miRNAs mutations in the dystrophin gene [1,2]
Expression levels of myomiRs and extracellular vesicles (EVs) contents in sera of mdx mice and DMD patients We previously showed that myomiRs in the serum of mice were upregulated by cardiotoxininduced muscle injury [25]
To determine the levels of the three myomiRs in the two fractions from the sera of tg, mdx, and wt mice, sera were separated by centrifugation followed by miRNA extraction. qRT-PCR demonstrated that the levels of the three myomiRs in these fractions were significantly downregulated in tg mice compared with mdx mice, in which the expression levels of the three myomiRs were significantly increased compared with wt mice (Fig 1A)
Summary
Characterization and Functional Analysis of Extracellular Vesicles and Muscle-Abundant miRNAs mutations in the dystrophin gene [1,2]. This disorder is usually first recognized by muscular weakness from two to five years of age. Patients lose their ability to ambulate before 12 years of age, and eventually experience failure of respiratory and cardiac functions owing to degeneration of the diaphragm and cardiac muscles [3].
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