Myostatin-deficiency in mice increases global gene expression at the Dlk1-Dio3 locus in the skeletal muscle

Figures

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  Figure 1. The expression of miRNAs derived from the Dlk1-Dio3 locus is upregulated in myostatin-deficient skeletal muscle (A) A significant increase in miR-411, miR-434-3p, miR-379, and miR-193b expression in myostatin-deficient skeletal muscle at 13 weeks of age was determined by quantitative RT-PCR. (B) Schematic diagram of the Dlk1-Dio3 locus structure. This locus contains imprinting genes; Dlk1, Rtl1, and Dio3 (shown as blue boxes) are paternally expressed, whereas Gtl2, Rtl1as, Rian, Mirg (shown as white boxes), and miRNAs (arrowheads) are maternally expressed. Red arrowheads indicate the miRNAs whose expression was validated by qRT-PCR in Figure 1C. (C) Quantitative RT-PCR analysis shows a significant increase in the expression of miR-337, miR-540-3p, miR-127, miR-434-5p, miR-329, miR-543-3p, miR-376a, miR-300, and miR-381 in myostatin-deficient skeletal muscle at 13 weeks of age. The wild-type mice are the same age as the myostatin knockout mice. The results were normalized to U6 small RNA expression. Data are the mean ± SD (n = 5). *P < 0.05. **P < 0.01. ***P < 0.001.

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  Figure 2. miR-411 and miR-540-3p increase the C2C12 myotube diameters (A) Myostatin knockout mice show a significant increase in the skeletal muscle mass. (B) Confluent C2C12 cells were differentiated into myotubes and transfected with 25 nM of miRNA mimics. Myotubes were transfected with miR-411, miR-434-5p, and miR-540-3p mimics and immunostained with an anti-myosin heavy chain antibody and DAPI. (C and D) Myotube diameter was measured as described in the materials and methods section and was shown as the percent of the control. Transfection of miR-411 and miR-540-3p mimics, but not other miRNAs, significantly increased the myotube diameter. Bar, 250 μm. Error bars indicate the SD of 3 independent experiments. *P < 0.05.

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  Figure 3. Decreased expression of SFRP family genes in myostatin-deficient skeletal muscle Quantification of expression levels of SFRP (Secreted frizzled-related protein) genes, which are secreted inhibitors of Wnt signaling pathway, was performed by qRT-PCR. A significant decrease in the expression of Sfrp1 was observed in myostatin-deficient skeletal muscle at 5 (A) and 13 (B) weeks of age. In addition, a significant decrease in Sfrp5 expression was also detected in myostatin knockout mice at 5 weeks of age (A). The wild-type mice are the same age as the myostatin knockout mice. The results were normalized to the expression of Rpl26. Data are mean ± SD (n = 5). *P < 0.05, ***P < 0.001.

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  Figure 4. Myostatin deficiency increases transcription at the Dlk1-Dio3 locus (A) Quantitative RT-PCR analysis shows a significant increase in the expression of the primary transcript of miR-127 in myostatin-deficient skeletal muscle at 13 weeks of age. (B) Myostatin deficiency increases the expression levels of the maternally expressed Gtl2 and Rian, as well as the paternally expressed Dlk1. The results were normalized to Rpl26 expression. Data are the mean ± SD (n = 5). *P < 0.05. (C) Strand-specific RT-PCR shows increased expression of Rtl1 and Rtl1as in myostatin-deficient skeletal muscle. (D) Quantitative analyses of Rtl1 and Rtl1as expression using Image J software. The wild-type mice are the same age as the myostatin knockout mice. Data are the mean ± SD (n = 3). **P < 0.01, ***P < 0.001.

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  Figure 5. Mice growth decreases the expression levels of imprinting genes in the skeletal muscle Quantitative RT-PCR showing the decreased expression of miR-411 (A) miR-540-3p (B) pri-miR-411 (C) and Gtl2 (D) in both wild-type and myostatin knockout mice with age. (E) The expression level of Igf2, the growth-related imprinting gene, is also decreased in the skeletal muscle as mice grow. (F) The expression of miR-486 that is increased in myostatin knockout mice is not changed with age. The expression level of either U6 or Rpl26 was used to normalize the qRT-PCR results. Data are the mean ± SD (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 6. Methylation status of the IG-DMR and Gtl2-DMR domain in myostatin-deficient skeletal muscle Bisulfite sequencing analysis shows the methylation maps around the IG-DMR (A) and Gtl2-DMR (C) domain. The CpG sites are indicated by circles (black circles indicate methylated and white circles indicate unmethylated cytosine sites, respectively) and each row represents an individual clone. The methylation ratio of the IG-DMR (B) and Gtl2-DMR (D) domain in the myostatin knockout mice is shown as the percentage of that in wild-type mice. (E) Quantitative RT-PCR showing decreased Dnmt3a2 expression in myostatin-deficient skeletal muscle. The wild-type mice are the same age as the myostatin knockout mice. The results were normalized to the expression of Rpl26. Data are the mean ± SD (n = 5). ***P < 0.001.

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  Figure 7. Increased expression of IG-DMR ncRNA in myostatin-deficient skeletal muscle (A) Quantitative RT-PCR analysis shows the increased expression of IG-DMR ncRNA in myostatin-deficient skeletal muscle at 13 weeks of age. (B) Myostatin deficiency does not affect the expression level of IPW lncRNA. The wild-type mice are the same age as the myostatin knockout mice. The results were normalized to the expression of Rpl26. Data are the mean ± SD (n = 5). **P < 0.01.