Characterization and Functional Analysis of Extracellular Vesicles and Muscle-Abundant miRNAs (miR-1, miR-133a, and miR-206) in C₂C₁₂ Myocytes and <i>mdx</i> Mice

<div><p>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 <i>mdx</i> 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 <i>mdx</i> 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 <i>mdx</i> 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 <i>in vivo</i>, we intraperitoneally injected GW4869 (an inhibitor of exosome secretion) into <i>mdx</i> mice for 5 and 10 days. Levels of miRNAs and creatine kinase in the serum of GW4869-treated <i>mdx</i> mice were significantly downregulated compared with those of controls. The tibialis anterior muscles of the GW4869-treated <i>mdx</i> mice showed a robust decrease in Evans blue dye uptake. Collectively, these results indicate that EVs and myomiRs might protect the skeletal muscle of <i>mdx</i> mice from degeneration.</p></div

Effects of ceramide and S1P on EV secretion from C₂C₁₂ myoblasts.

<p>C₂C₁₂ cells were cultured in growth medium until confluent, and then incubated with serum-depleted medium with or without GW4869 for 72 hr (A), C6-ceramide for 24 hr (B), GW4869 and C6-ceramide (C) or GW4869 and C2-ceramide (D) for 72 hr, ebselen for 48 hr (E), D-erythro-MAPP (deMAPP) for 2 hr (F), and GW4869 or S1P for 48 hr (G). The EVs from these cells were extracted from the culture medium, and the amounts of the released EVs were quantified by measuring AChE activity. Data are represented as means + S.E. of absorbance at 405 nm. *: <i>P</i> < 0.05, **: <i>P</i> < 0.01, ***: <i>P</i> < 0.001. (H) Schematic figure of ceramide biogenesis and metabolism. H₂O₂: hydrogen peroxide; S1PR1/3: S1P receptor 1 or 3; Gαi: a subunit of G protein.</p

Effect of EVs on the survival of C₂C₁₂ cells.

<p>(A) C₂C₁₂ myoblasts (left) and myotubes (right) that were differentiated for 3 days were incubated for the indicated times in serum-depleted medium with low (0.7 μg), medium (2 μg), or high (6 μg) concentrations of EVs that were extracted from the serum of <i>mdx</i> mice. (B,C) C₂C₁₂ myoblasts (B) and myotubes (C) differentiated for 6 days were incubated with or without EVs (0.05 μg, 0.1 μg, 0.2 μg, 0.4 μg, 0.8 μg, or 1.6 μg) (B) or (0.7 μg, 2.0 μg, or 6.0 μg) (C) extracted from the serum of mice subjected for 24 hr to three different conditions; H₂O₂ (10 mM), ethanol (20%), or actinomycin D (0.5 mg/mL). Data represent mean + S.E. of absorbance at 450 nm of CCK-8. *: <i>P</i> < 0.05, **: <i>P</i> < 0.01, ***: <i>P</i> < 0.001. Each independent experiment was repeated at least 3 times.</p

Effect of miRNAs in EVs on C₂C₁₂ cell gene expression.

<p>(A) Myotubes were incubated for 72 hr with 1.0 or 2.0 μg of EVs extracted from C₂C₁₂ cells. Total RNA was extracted from the myotubes. Levels of miR-133a (A) and relative mRNA levels of putative target genes (B) were measured by RT-quantitative PCR. (C) Caspase-3 activity was measured in lysates of C₂C₁₂ myotube cells cultured with EVs (4 μg) for 24 hr. Data represent mean + S.E. *: <i>P</i> < 0.05, **: <i>P</i> < 0.01, ***: <i>P</i> < 0.001 versus the relevant control.</p

Effects of miRNAs in EVs on muscle regeneration <i>in vivo</i>.

<p>(A) Experimental timeline of GW4869 administration into <i>mdx</i> mice. Six-week old <i>mdx</i> mice were injected daily with 100 μL of GW4869 (100 μM) intraperitoneally for 5 or 10 days. After the GW4869 administration period, EBD was injected, and then the next day, whole body blood was collected from the abdominal aorta. miR-1, miR-133a, and miR-206 levels (B) and CK levels (C) in the serum were quantified by RT-quantitative PCR and the Fuji Dri-Chem system, respectively. (D) EBD uptake analyzed in TA muscle and diaphragm sections of <i>mdx</i> mice injected with or without GW4869. (E) Quantification of the area of EBD-positive muscle damage in TA muscles and diaphragms of <i>mdx</i> mice injected with or without GW4869. Data represent mean + S.E. *: <i>P</i> < 0.05, **: <i>P</i> < 0.01, ***: <i>P</i> < 0.001.</p

Effect of miRNAs within EVs on the survival of C₂C₁₂ myotubes.

<p>(A) Schematic representation of the experiment. C₂C₁₂ cells were cultured and transfected with miR-1, miR-133a, miR-206, miR-1/miR-133a, miR-1/miR-206, miR-133a/miR-206, or miR1/miR-133a/miR-206. Their EVs were extracted from the culture medium, and added to C₂C₁₂ cells in serum-depleted medium. (B) Myotubes differentiated for 4 days were incubated in serum-depleted medium with or without 7 μg of EVs extracted from the medium of C₂C₁₂ cells transfected with miR-1, miR-133a, miR-206, miR-1/miR-133a, miR-1/miR-206, miR-133a/miR-206, or miR-1/miR-133a/miR-206 for the indicated times. Data represent mean + S.E. **: <i>Pc</i> < 0.01, ***: <i>Pc</i> < 0.001.</p

Levels of myomiRs and EVs in the sera of DMD patients and <i>mdx</i> mice.

<p>(A) Levels of miR-1, miR-133a, and miR-206 in EVs-containing and EV-depleted supernatants, separated from the sera of wt, <i>mdx</i>, and <i>tg</i> mice (7-weeks old, n = 3, 4, and 4, respectively). Data are represented as means + S.E. *: <i>P</i> < 0.05, **: <i>P</i> < 0.01 for <i>mdx</i> vs wt or <i>mdx</i> vs <i>tg</i>. (B) EVs were extracted and quantified by AChE activity from the sera of wt, <i>mdx</i>, and <i>tg</i> mice at 7, 13, and 27 weeks of age (n = 3, 4, and 4, respectively, *: <i>P</i> < 0.05, **: <i>P</i> < 0.01 vs wt) (left) and DMD patients and healthy controls (right), (n = 5 and 4, respectively, *: <i>P</i> < 0.05). (C-E) miR-1 (C), miR-133a (D), and miR-206 (E) levels in EVs separated by immunoprecipitation with anti-caveolin-3 (cav3), anti-CD63, anti-CD81, anti-flotillin-1 (flot1), or anti-MHC class II (MHC II) antibodies in the sera of DMD patients and controls (n = 5 and 4, respectively). *: <i>P</i> < 0.05 vs controls.</p