Mest but Not MiR-335 Affects Skeletal Muscle Growth and Regeneration

<div><p>When skeletal muscle fibers are injured, they regenerate and grow until their sizes are adjusted to surrounding muscle fibers and other relevant organs. In this study, we examined whether <i>Mest</i>, one of paternally expressed imprinted genes that regulates body size during development, and miR-335 located in the second intron of the <i>Mest</i> gene play roles in muscle regeneration. We generated miR-335-deficient mice, and found that miR-335 is a paternally expressed imprinted microRNA. Although both <i>Mest</i> and miR-335 are highly expressed during muscle development and regeneration, only <i>Mest^+/-</i> (maternal/paternal) mice show retardation of body growth. In addition to reduced body weight in <i>Mest^+/-; DMD-null</i> mice, decreased muscle growth was observed in <i>Mest^+/-</i> mice during cardiotoxin-induced regeneration, suggesting roles of Mest in muscle regeneration. Moreover, expressions of <i>H19</i> and <i>Igf2r</i>, maternally expressed imprinted genes were affected in tibialis anterior muscle of <i>Mest^+/-; DMD-null</i> mice compared to <i>DMD-null</i> mice. Thus, Mest likely mediates muscle regeneration through regulation of imprinted gene networks in skeletal muscle.</p></div

Mest is required for skeletal muscle growth during regeneration.

<p>(A) H&E staining of TA muscles under normal condition (top panel) and 14 days after CTX-induced injury (middle and bottom panels). Bottom panel shows the extended images of a part of middle panel. (B) Average cross section areas of TA muscles in WT (n = 3), <i>Mest</i>^<i>+/-</i> (n = 4) and <i>miR-335</i>^<i>+/Neo</i> mice (n = 6) under normal condition and WT (n = 7), <i>Mest</i>^<i>+/-</i> (n = 4) and <i>miR-335</i>^<i>+/Neo</i> mice (n = 6) 14 days after CTX injury. (C) H&E staining of TA muscles of <i>DMD-null</i>, <i>Mest</i>^<i>+/-</i><i>; DMD-null</i> and <i>miR-335</i>^<i>+/Neo</i><i>; DMD-null</i> mice at 11–13 weeks old. (D) Average cross section areas of TA muscles in <i>DMD-null</i> (n = 8), <i>Mest</i>^<i>+/-</i><i>; DMD-null</i> (n = 4) and <i>miR-335</i>^<i>+/Neo</i><i>; DMD-null</i> mice (n = 4) at 11–13 weeks old. Error bars indicate the s.e.m. ^<i>#</i><i>P</i> = 0.0549 compared with WT mice. Scale bar: 100 μm.</p

Mest is required for body and skeletal muscle growth.

<p>(A) Representative images of 4 weeks old mice in individual genotypes. (B and C) Body weights of male littermate WT (n = 3–22), <i>Mest</i>^<i>+/-</i> (n = 6–15), <i>DMD-null</i> (n = 4–25), and <i>Mest</i>^<i>+/-</i><i>; DMD-null</i> mice (n = 4–22) from 1 to 12 (11–13) weeks old. (D) Body weights of WT (n = 15) and <i>miR-335</i>^<i>+/Neo</i> (n = 12) mice at 6 weeks. (E) Body weights of WT (n = 8–15) and <i>miR-335</i>^<i>+/-</i> mice (n = 15–18) from 1 to 6 weeks old. (F) TA muscle weights of male littermate WT (n = 13), <i>Mest</i>^<i>+/-</i> (n = 6), <i>DMD-null</i> (n = 18), and <i>Mest</i>^<i>+/-</i><i>; DMD-null</i> mice (n = 11) at 6 weeks old. (G) TA/Body weights of male littermate WT, <i>Mest</i>^<i>+/-</i>, <i>DMD-null</i>, and <i>Mest</i>^<i>+/-</i><i>; DMD-null</i> mice at 6 weeks old. (H) TA muscle weights of male littermate WT (n = 3), <i>Mest</i>^<i>+/-</i> (n = 6), <i>DMD-null</i> (n = 4), and <i>Mest</i>^<i>+/-</i><i>; DMD-null</i> mice (n = 4) at 11–13 weeks old. (I) TA/Body weights of male littermate WT, <i>Mest</i>^<i>+/-</i>, <i>DMD-null</i>, and <i>Mest</i>^<i>+/-</i><i>; DMD-null</i> mice at 11–13 weeks old. (J and K) The numbers and average cross section areas of TA muscle fibers of male littermate WT (n = 7) and <i>Mest</i>^<i>+/-</i> mice (n = 4) at 6 weeks. Error bars indicate the s.e.m. *<i>P</i> < 0.05, ***<i>P</i> < 0.001. NS = Not significant.</p

<i>Mest</i> and miR-335 are coordinately expressed in skeletal muscle during postnatal development and regeneration.

<p>(A) qRT-PCRs for <i>Mest</i> mRNA and miR-335 were performed with TA muscles of P0, 6 weeks, and 12 weeks old WT mice (n = 3 per time point). (B) qRT-PCRs for <i>Mest</i> mRNA and miR-335 were performed with TA muscles of 3 months old WT (n = 4) and <i>DMD–null</i> mice (n = 7). (C) qRT-PCRs for <i>Mest</i> mRNA and miR-335 were performed with TA muscles from day 0 to day 10 after CTX injection (n = 3 per time point). (D) A schematic diagram of the Mest and miR-335 genomic region on chromosome 6 in mouse. (E) qRT-PCR for miR-335 was performed in TA muscles of WT and <i>Mest</i>^<i>+/-</i> mice (n = 3 per genotype). Expression of <i>Mest</i> and that of miR-335 are normalized to <i>Gapdh</i> and snoRNA-202, respectively. Error bars indicate the s.e.m. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001 compared with P0 (A), WT (B and E), and day 0 (C).</p

Generation of miR-335 deficient mice.

<p>(A) Design of constructs used for generation of miR-335 deficient mice. The miR-335 genomic locus was replaced by a floxed neomycin-resistance cassette (loxP-Neo-loxP) to obtain <i>miR-335</i>^<i>+/Neo</i> mice. <i>miR-335</i>^<i>+/-</i> mice (+/-) were generated by crossing male <i>miR-335</i>^<i>+/Neo</i> mice with female CAG-<i>cre</i> transgenic mice. (B) Southern blot analysis of WT and G418 resistant ES clones with 5’ and 3’ probes. †: Non-specific band. (C and D) PCR analysis for targeted allele with genomic DNA in tails of WT (+/+), <i>miR-335</i>^<i>+/Neo</i> (+/Neo), and <i>miR-335</i>^<i>+/-</i> mice (+/-). In (C), an insertion and a deletion of a floxed neomycin-resistance cassette in genomic DNA of <i>miR-335</i>^<i>+/Neo</i> (+/Neo) and <i>miR-335</i>^<i>+/-</i> mice (+/-), respectively, were detected with PCRs amplified with primers shown in Fig 2A (Orange and Green arrows). In (D), a PCR analysis to distinguish alleles for WT (+/+), <i>miR-335</i>^<i>+/-</i> (+/-), and <i>miR-335</i>^<i>-/-</i> mice (-/-) was shown. The WT allele-specific (280 bp) and the mutant allele-specific (306 bp) bands were amplified with the primers shown in Fig 2A (Green arrow). (E, F and G) qRT-PCR for miR-335 was performed in TA muscles isolated from WT, <i>miR-335</i>^<i>+/Neo</i>, and <i>miR-335</i>^<i>+/-</i> or <i>miR-335</i>^<i>-/+</i> mice (n = 3 per genotype). (H and I) qRT-PCR for <i>Mest</i> mRNA was performed in TA muscles of WT, <i>miR-335</i>^<i>+/-</i>, and <i>miR-335</i>^<i>+/Neo</i> mice (n = 3 per genotype). Expression of <i>Mest</i> mRNA and that of miR-335 are normalized to <i>Gapdh</i> and snoRNA-202, respectively. Error bars indicate the s.e.m. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

FMPs and SCs show similar myogenic differentiation potential in vitro.

<p>(<b>A,B</b>) Immunocytochemistry of FMPs and SCs for MyoD (A) and Myogenin (B) during culture (days 3 and 5). DAPI was used for to stain nuclei. Scale bar = 50 µm. (<b>C,D</b>) Quantitative analysis of MyoD (C) and Myogenin (D) in cultured FMPs and SCs. Data are reported as mean and s.d. of 500–1000 cells per staining from three independent experiments. P-values indicated on figures are <0.01 (**). (<b>E,F</b>) Phase contrast micrographs of FMPs (E) and SCs (F) after 7 days in culture. (<b>G,H</b>) Immunocytochemistry of FMPs (G) and SCs (H) shown in (E,F) for Troponin T and DAPI. Scale bar = 50 µm. FMPs, fetal skeletal muscle progenitors; SCs, satellite cells.</p

MyoD-positive FMPs have more regenerative ability than MyoD-negative FMPs.

<p>(<b>A</b>) Representative fluorescence-activated cell sorting profiles for (MyoD)RFP- and (MyoD)RFP+ cells from <i>Pax3^GFP/+;MyoD-Cre;R26R^RFP</i> fetus. (<b>B,C</b>) A fluorescence stereomicroscope view of RFP fluorescence in TA muscles of <i>DMD-null</i> mice transplanted with (MyoD)RFP- (B) and (MyoD)RFP+ FMPs (C). Scale bar = 2.5 mm. (<b>D–I</b>) Immunohistochemistry for RFP (D,E), dystrophin (E,F), and merged with DAPI (F,I) in (MyoD)RFP- (D–F) and (MyoD)RFP+ (G–I) FMP engrafted TA muscles. Scale bar = 20 µm. (<b>J</b>) Quantification of dystrophin+ fibers in TA muscles engrafted with (MyoD)RFP- and (MyoD)RFP+ FMPs. Error bars represent the mean and s.e.m. from 3 engrafted mice. P-value indicated on the figure is <0.01 (**). FMPs, fetal skeletal muscle progenitors; TA, tibialis anterior.</p

FMPs express MyoD in developing limbs.

<p>(<b>A–F</b>) Immunohistochemistry of FMP cells on longitudinal sections of limbs at E16.5 for GFP (A,D), MyoD (B,E) and merged with DAPI (C,F). MyoD-positive (A–C) and -negative (D–F) FMPs are shown. Scale bar = 10 µm. (<b>G</b>) Percentage of MyoD-positive and MyoD-negative cells in (Pax3)GFP+ cells in limbs at E16.5 with data reported as the mean and s.d. of three fetuses. (<b>H–M</b>) Immunocytochemistry of freshly isolated FMPs (H–J) and SCs (K–M) for GFP (H,K), MyoD (I,L), and merged with DAPI (J,M). Arrows in H–J indicate MyoD-positive FMPs. Scale bar = 20 µm. (<b>N</b>) Percentage of MyoD-positive and MyoD-negative cells of freshly isolated FMPs and SCs (<i>n = </i>100 cells par condition). FMPs, fetal skeletal muscle progenitors; SCs, satellite cells.</p

SCs are more primed into the myogenic program than FMPs.

<p>(<b>A</b>) CD34 expression of (MyoD)RFP- FMPs, (MyoD)RFP+ FMPs, and SCs. Flow cytometry histograms show a control staining profile (gray line) and CD34 (red line). The percentage of cells expressing CD34 is indicated. (<b>B</b>) Relative amounts of the indicated transcripts determined by quantitative reverse transcription polymerase chain reaction among (MyoD)RFP- FMPs, (MyoD)RFP+ FMPs, and SCs. Data is normalized to the expression of transcripts for <i>Rpl13A</i>. Data is reported as the mean and s.e.m. (<i>n</i> = 3). P-values indicated on figures are no significant difference (NS), <0.01 (**), and <0.001 (***). FMPs, fetal skeletal muscle progenitors; SCs, satellite cells.</p

Recovery of (Pax3)GFP+ cells from FMP- and SC-engrafted TA muscles.

<p>(<b>A,B</b>) A representative fluorescence-activated cell sorting profile for (Pax3)GFP+ cells from control (A) and FMP-engrafted (B) TA muscle. The percentage of cells that express GFP is indicated. (<b>C–F</b>) Immunocytochemistry for Pax7 (D) and GFP (E) of freshly isolated (Pax3)GFP+ cells from FMP-engrafted TA muscles. Scale bars = 5 µm. (<b>G,H</b>) Phase contrast micrographs (G) and immunocytochemistry with anti-Troponin T antibodies (H) of isolated (Pax3)GFP+ cells cultured in proliferation and differentiation conditions for 6 days. Scale bar = 50 µm. (<b>I–P</b>) Immunostaining for laminin, dystrophin, and GFP in TA muscles of <i>DMD-null</i> mice injected with FMPs (I–L) and SCs (M–P) 2 weeks after intramuscular engraftment. Arrows indicate (Pax3)GFP+ cells underneath laminin and outside of dystrophin. Scale bar = 10 µm. (<b>Q</b>) Quantification of (Pax3)GFP+ cells underneath laminin and outside of dystrophin in TA engrafted with FMPs (<i>n</i> = 5 recipient mice) and SCs (<i>n</i> = 5 recipient mice). Data is reported as the mean and s.e.m. from all engrafted mice. P-value indicated on the figure is <0.05 (*). FMPs, fetal skeletal muscle progenitors; SCs, satellite cells; TA, tibialis anterior.</p