Identification of Muscle-Specific MicroRNAs in Serum of Muscular Dystrophy Animal Models: Promising Novel Blood-Based Markers for Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder caused by mutations in the dystrophin gene, which encodes a cytoskeletal protein, dystrophin. Creatine kinase (CK) is generally used as a blood-based biomarker for muscular disease including DMD, but it is not always reliable since it is easily affected by stress to the body, such as exercise. Therefore, more reliable biomarkers of muscular dystrophy have long been desired. MicroRNAs (miRNAs) are small, ∼22 nucleotide, noncoding RNAs which play important roles in the regulation of gene expression at the post-transcriptional level. Recently, it has been reported that miRNAs exist in blood. In this study, we hypothesized that the expression levels of specific serum circulating miRNAs may be useful to monitor the pathological progression of muscular diseases, and therefore explored the possibility of these miRNAs as new biomarkers for muscular diseases. To confirm this hypothesis, we quantified the expression levels of miRNAs in serum of the dystrophin-deficient muscular dystrophy mouse model, mdx, and the canine X-linked muscular dystrophy in Japan dog model (CXMDJ), by real-time PCR. We found that the serum levels of several muscle-specific miRNAs (miR-1, miR-133a and miR-206) are increased in both mdx and CXMDJ. Interestingly, unlike CK levels, expression levels of these miRNAs in mdx serum are little influenced by exercise using treadmill. These results suggest that serum miRNAs are useful and reliable biomarkers for muscular dystrophy

Roles of miR-1, miR-133a, and miR-206 in calcium, oxidative stress, and NO signaling involved in muscle diseases: DOI: 10.14800/rd.558

miR-1, miR-133a, and miR-206 are abundantly expressed in skeletal muscle and regulate the post-transcriptional expression of target genes. These miRNAs are upregulated in sera of DMD, BMD, LGMD, and FSHD patients, as well as mdx mice and CXMDj dogs, suggesting that the serum miRNAs may substitute for CK levels as be novel biomarkers for muscle disorders. These miRNAs are released into the extracellular environment in  vesicular structures called exosomes, by mechanisms that are regulated by calcium, oxidative stress, and NO signaling. In this review, we will highlight the relationship between calcium, oxidative stress, and NO signaling and the release of miRNAs via exosomes as well as discuss the functions of these miRNAs

Therapeutic Application of Extracellular Vesicles-Capsulated Adeno-Associated Virus Vector via nSMase2/Smpd3, Satellite, and Immune Cells in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is caused by loss-of-function mutations in the dystrophin gene on chromosome Xp21. Disruption of the dystrophin–glycoprotein complex (DGC) on the cell membrane causes cytosolic Ca2+ influx, resulting in protease activation, mitochondrial dysfunction, and progressive myofiber degeneration, leading to muscle wasting and fragility. In addition to the function of dystrophin in the structural integrity of myofibers, a novel function of asymmetric cell division in muscular stem cells (satellite cells) has been reported. Therefore, it has been suggested that myofiber instability may not be the only cause of dystrophic degeneration, but rather that the phenotype might be caused by multiple factors, including stem cell and myofiber functions. Furthermore, it has been focused functional regulation of satellite cells by intracellular communication of extracellular vesicles (EVs) in DMD pathology. Recently, a novel molecular mechanism of DMD pathogenesis—circulating RNA molecules—has been revealed through the study of target pathways modulated by the Neutral sphingomyelinase2/Neutral sphingomyelinase3 (nSMase2/Smpd3) protein. In addition, adeno-associated virus (AAV) has been clinically applied for DMD therapy owing to the safety and long-term expression of transduction genes. Furthermore, the EV-capsulated AAV vector (EV-AAV) has been shown to be a useful tool for the intervention of DMD, because of the high efficacy of the transgene and avoidance of neutralizing antibodies. Thus, we review application of AAV and EV-AAV vectors for DMD as novel therapeutic strategy

Comparative analysis of DNA aberrated by irradiation using PCR and simulation methods

International symposium on innovative technology for radiation risk study 200

Application of Real Time PCR for the Quantitative Detection of Radiation-induced Genomic DNA Strand Breaks

The frequency of single strand breaks(SSBs) occurring on both strands of the pBR322 plasmid DNA region flanked by a pair of primers used for polymerase chain reaction(PCR) amplifications was determined after irradiation with 137Cs gamma rays. We verified that real time PCR is suitable for the detection and quantitative analysis of SSBs caused by gamma irradiation. The utility of this approach was also supported by the comparison of the practical experimental data with the Monte Carlo simulation. The potential application of this PCR method for the detection of genomic DNA damage was also confirmed

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 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

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