METTL3-Mediated m6A Methylation Regulates Muscle Stem Cells and Muscle Regeneration by Notch Signaling Pathway

Abstract

The Pax7+ muscle stem cells (MuSCs) are essential for skeletal muscle homeostasis and muscle regeneration upon injury, while the molecular mechanisms underlying muscle stem cell fate determination and muscle regeneration are still not fully understood. N6-methyladenosine (m⁶A) RNA modification is catalyzed by METTL3 and plays important functions in posttranscriptional gene expression regulation and various biological processes. Here, we generated muscle stem cell-specific METTL3 conditional knockout mouse model and revealed that METTL3 knockout in muscle stem cells significantly inhibits the proliferation of muscle stem cells and blocks the muscle regeneration after injury. Moreover, knockin of METTL3 in muscle stem cells promotes the muscle stem cell proliferation and muscle regeneration in vivo. Mechanistically, METTL3-m⁶A-YTHDF1 axis regulates the mRNA translation of Notch signaling pathway. Our data demonstrated the important in vivo physiological function of METTL3-mediated m⁶A modification in muscle stem cells and muscle regeneration, providing molecular basis for the therapy of stem cell-related muscle diseases.

Figure 1

METTL3 is required for muscle regeneration in vivo. (a) Schematic outline of CTX injection in tamoxifen-treated CTL and cKO littermates. (b–d) Immunofluorescence staining and Western blot analysis of TA muscle after injection for 5 days for METTL3. (e) Hematoxylin and eosin (H&E) staining of TA muscles of METTL3 cKO mice and control littermates 2, 5, and 8 days after CTX injection. (f) Number of muscle fibers. (g) Area of regenerating centronuclear fibers. (h) Masson's Trichrome staining of TA muscles of METTL3 cKO and control littermates. (i) Western blot analysis of Pax7, myosin, myogenin, and METTL3 protein levels in control TA muscle at day 0, day 2, day 5, and day 8. (j) Immunofluorescence staining of TA muscles from METTL3 cKO and control mice detected by myogenin. (k) Quantifications of myogenin staining. (l) Western blot analysis of myogenin. (m) Immunohistochemical staining of myosin. (n) Quantifications of myosin staining. (o) Western blot analysis of myosin. N = 10. ^∗∗∗∗p < 0.0001; ^∗∗∗p < 0 .001; ^∗∗p < 0.01; ^∗p < .05. CTX: cardiotoxin; TAM: tamoxifen; TA: tibialis anterior; CTL: control; cKO: METTL3 conditional knockout.

Figure 2

METTL3 regulates muscle stem cell proliferation in vivo. (a–d) Immunohistochemical analysis of TA muscle samples from METTL3 cKO and control by Pax7 and Ki67 antibodies, respectively. Quantifications of Pax7 and Ki67 staining were shown on the right. (e) EdU of TA muscles and (f) images of cell numbers 5 days after isolation from injured muscles of METTL3cKO and control mice. ^∗∗∗∗p < 0.0001; ^∗∗∗p < 0.001. CTL: control; cKO: METTL3 conditional knockout.

Figure 3

METTL3 conditional knockin promotes muscle regeneration in vivo. (a) Schematic outline of CTX injection in TAM-treated control and METTL3 cKI littermates at the age of 8–9 weeks. (b) H&E staining of representative tibialis muscle sections from METTL3 cKI, control mice treated 2, 5, and 8 days after CTX injection. (c) Quantified of muscle fibers. (d) The upregulation effect of METTL3 was verified at protein levels by Western blot. (e, h, k) Immunohistochemical and (n) immunofluorescence staining of TA muscle from METTL3 cKI and control detected by Pax7, Ki67, myogenin, and myosin antibodies. (f, i, l, o) Quantifications of Pax7+, Ki67+, myogenin+, and myosin+ cells were shown on the right. (g, j, m, p) Western blot detected the protein expression levels of Pax7+, Ki67+, myogenin, and myosin. Data represent mean ± SEM. N = 5. ^∗∗∗∗p < 0.0001, ^∗∗p < 0.01, ^∗p < 0.05. CTX: cardiotoxin; TAM: tamoxifen; TA: tibialis anterior; CTL: control; cKI: METTL3 conditional knockin.

Figure 4

METTL3 regulates m⁶A modification of Notch signaling pathway components. (a) Schematic representation of m⁶A MeRIP-Seq. (b) m⁶A motif identified in the MeRIP-Seq. (c) Enriched KEGG molecular function pathways identified from differentially m⁶A-enriched genes in METTL3 cKO vs. CTL D2 samples. (d) Notch signaling pathway identified in the MeRIP-Seq. (e, f) qRT-PCR and Western blot analyzed of Notch signaling pathway. CTL: control; cKO: METTL3 conditional knockout.

Figure 5

Deletion of METTL3 affects C2C12 myoblast progenitor cell proliferation and differentiation. (a) Western blot detection of METTL3 expression. (b) Proliferation ability significantly decreased after METTL3 knockout determined by cell counting Kit-8 at indicated days. (c) Western blot analysis of the expression of early differentiation marker myogenin and late differentiation marker myosin in C2C12 cells. (d) Myosin staining (red) was performed 3 days later using an anti-myosin antibody, and the nuclei were counterstained with DAPI. (e) Differentiation index in the METTL3-depleted and control cells. Data represent mean ± SEM. ^∗∗∗p < 0.001. NC: negative control; sh-1: METTL3 shRNA-1; sh-2: METTL3 shRNA-2.

Figure 6

METTL3 regulates the mRNA translation efficiency of Notch signaling pathway components. (a) The Notch pathway genes with m⁶A modification. (b) The protein levels of Notch signaling pathway in the METTL3-depleted and control cells. (c) mRNA expression of Notch signaling pathway analyzed by q-PCR. (d) Polyribosome-bound mRNA-qPCR was used to detect translation efficiency. (e) The downstream gene expression of Notch signaling pathway was detected by qRT-PCR. ^∗∗∗∗p < 0.0001; ^∗∗∗p < 0.001; ^∗∗p < 0.01. NC: negative control; sh-1: METTL3 shRNA-1; sh-2: METTL3 shRNA-2.

Figure 7

YTHDF1 regulates the mRNA translation efficiency of Notch signaling pathway components. (a) The protein levels of Notch signaling pathway in the METTL3 overexpressed or (d) depleted and control cells. (b, e) mRNA expression of Notch signaling pathway analyzed by q-PCR. (c, f) Polyribosome-bound mRNA-qPCR was used to detect translation efficiency. (g) Working model of METTL3 regulates muscle stem cells by Notch signaling pathway. ^∗∗∗∗p < 0.0001; ^∗∗∗p < 0.001; ^∗∗p < 0.01. NC: negative control; OE: YTHDF1 overexpressed; si: YTHDF1 siRNA.