The m6A methyltransferase METTL3 regulates muscle maintenance and growth in mice

Abstract

Skeletal muscle serves fundamental roles in organismal health. Gene expression fluctuations are critical for muscle homeostasis and the response to environmental insults. Yet, little is known about post-transcriptional mechanisms regulating such fluctuations while impacting muscle proteome. Here we report genome-wide analysis of mRNA methyladenosine (m⁶A) dynamics of skeletal muscle hypertrophic growth following overload-induced stress. We show that increases in METTL3 (the m⁶A enzyme), and concomitantly m⁶A, control skeletal muscle size during hypertrophy; exogenous delivery of METTL3 induces skeletal muscle growth, even without external triggers. We also show that METTL3 represses activin type 2 A receptors (ACVR2A) synthesis, blunting activation of anti-hypertrophic signaling. Notably, myofiber-specific conditional genetic deletion of METTL3 caused spontaneous muscle wasting over time and abrogated overload-induced hypertrophy; a phenotype reverted by co-administration of a myostatin inhibitor. These studies identify a previously unrecognized post-transcriptional mechanism promoting the hypertrophic response of skeletal muscle via control of myostatin signaling.

Fig. 1. METTL3 and m⁶A remodels with muscle growth.

a Schematic depicting muscle overload-induced hypertrophy of the plantaris muscle created in Adobe Illustrator. b Quantification of m⁶A level relative to total adenosine (m⁶A/A) as determined by ELISA in wild-type baseline and overloaded muscles. c Western blot and d quantification of METTL3 expression normalized to GAPDH in baseline and overloaded muscles. e Schematic of workflow of MeRIP-Seq experiment created in Adobe Illustrator. f Venn diagram of peaks enriched (FC > 1.5) in baseline and overloaded plantaris samples. g Venn diagram of transcripts with m⁶A peaks that were found to be m⁶A-targeted in baseline, overloaded, or common between both. h Peaks detected in baseline and overloaded were plotted via density across mRNA regions (including 5′ UTR, start codon, coding sequence (CDS), stop codon, and 3′ UTR). UTR untranslated region. i Peaks detected in baseline and overloaded samples were plotted via frequency across the indicated mRNA regions. j Gene ontology (GO) analysis of overlapping transcripts or k overload-responsive transcripts enriched with m⁶A peaks in either baseline and overloaded samples. The enrichment score is based on a reference database of protein-coding genes, and all GO categories plotted were found to have a false discovery rate (FDR) < 0.05. Biological animal replicates: n = 3 per group in panel d and f–k; n = 4 (baseline) and 8 (overload) in panel b. Data are presented as the mean ± SEM. *P < 0.05, by two-sided Student’s t test for comparisons between baseline and overloaded muscles. Source data are provided as a Source Data file.

Fig. 2. METTL3 is essential for the growth response of muscle.

a Schematic of muscle-specific METTL3 knock out (M3-mKO) mouse generation. b qPCR analysis of Mettl3 mRNA expression in the plantaris muscles of WT and M3-mKO mice. c Western blot of METTL3 protein expression and GAPDH loading control in WT and M3-mKO muscles. d Body weight, e heart weight, f tibialis anterior (T.A.) weight, g soleus weight, and h plantaris weight in WT and M3-mKO mice 2 weeks post tamoxifen injections to induced muscle-specific deletion of Mettl3. Tibia length (TL) was used to normalize cardiac and skeletal muscle weights. i Plantaris weight, j representative wheat germ agglutinin (WGA; green) stained images and k plantaris fiber size at baseline or 14 days after synergist ablation surgery in WT and M3-mKO mice. Biological animal replicates: n = 5 (WT) and 8 (mKO) in panel b; n = 11 (WT) and 10 (mKO) in panel d and h; n = 8 (WT) and 12 (mKO) in panel e; n = 6 (WT) and 6 (mKO) in panel f and g; n = 11 (WT sham), 10 (mKO sham), 5 (WT overload), and 6 (mKO overload) in panel i; n = 5 (WT sham), 6 (mKO sham), 5 (WT overload), and 7 (mKO overload) in panel k. Data are presented as the mean ± SEM. *P < 0.05, by 2-sided Student’s t test for comparisons between WT and M3-mKO mice, or by 2-way ANOVA with Tukey’s HSD multiple-comparison test for comparison of the means of WT and M3-mKO mice and baseline and during overload. Scale bar = 125 µm. Source data are provided as a Source Data file.

Fig. 3. METTL3 is pro-hypertrophic in skeletal muscle.

a Schematic of overexpression of Myc-control or Myc-tagged METTL3 plasmid through DNA electroporation in overloaded WT muscles created in Adobe Illustrator. b qPCR analysis of human METTL3 expression in the plantaris muscles of Myc-control (Ctrl) or Myc-tagged METTL3 (M3-OE). c Quantification of m⁶A level relative to total adenosine (m⁶A/A) as determined by ELISA in electroporated muscles. d Plantaris weight, e representative wheat germ agglutinin (WGA; green) stained images, and f plantaris fiber size 14 days after synergist ablation surgery in control (Ctrl) or Myc-tagged METTL3 (M3-OE) plantaris muscles. g Schematic of overexpression through AAV9-Ctrl or AAV9-METTL3 injections into neonatal WT mice and experimental endpoint created in Adobe Illustrator. h qPCR analysis of Mettl3 expression in the muscles of AAV9-Ctrl or AAV9-M3 muscles. i Plantaris weight, and j plantaris fiber size 8 weeks after AAV injection in AAV9-Ctrl or AAV9-M3 animals. k Schematic of overexpression through AAV9-Control (AAV9 Ctrl) or AAV9-METTL3 (AAV9 M3) intramuscular injections into the tibialis anterior (T.A.) of 5-month-old WT mice and experimental endpoint created in Adobe Illustrator. l qPCR analysis of Mettl3 expression, m T.A. weight, and n T.A. myofiber size of AAV9 Ctrl or AAV9 M3 mice following 8 weeks of injection. Biological animal replicates: n = 7 (Ctrl) and 6 (M3-OE) in panel b; n = 7 (Ctrl) and 7 (M3-OE) in panel c; n = 11 (Ctrl) and 8 (M3-OE) in panel d; n = 6 (Ctrl) and 5 (M3-OE) in panel f; n = 3 per group in panel h, j, and n; n = 6 per group in panel i; n = 4 (Ctrl) and 5 (AAV9-M3) in panel l; and n = 3 (Ctrl) and 4 (AAV9-M3) in panel m. Data are presented as the mean ± SEM. *P < 0.05, by two-sided Student’s t test for comparisons between 2 groups. Scale bar = 125 µm. Source data are provided as a Source Data file.

Fig. 4. Chronic deletion of METTL3 drives muscle atrophy.

a Schematic of M3-mKO chronic deletion study. b Body weight, c heart weight, d gastrocnemius (gastroc) weight, e quadriceps (quad) weight, f tibialis anterioris (T.A.) weight, g plantaris weight, and h soleus weight of WT and M3-mKO mice. Tibia length (TL) was used to normalize cardiac and skeletal muscle weights. i Representative wheat germ agglutinin (WGA; green) stained images of WT and M3-mKO mice at 14 months of age. j T.A., k plantaris, and l soleus fiber size at in WT and M3-mKO mice following chronic deletion. m In vivo muscle twitch and tetanic torque measurements in 14 months old WT and M3-mKO mice. n Maximal running distance and o maximal running oxygen consumption following a graded maximal exercise test in WT and M3-mKO mice at 14 months. Biological animal replicates: n = 6 (WT) and 7 (mKO) in panel b, c, e, f, n, and o; n = 5 (WT) and 7 (mKO) in panel d, g, h, and m; n = 4 (WT) and 4 (mKO) in panel j; n = 5 (WT) and 5 (mKO) in panel k and l. Data are presented as the mean ± SEM. *P < 0.05, by two-sided Student’s t test for comparisons between WT and M3-mKO mice. Scale bar = 275 µm. Source data are provided as a Source Data file.

Fig. 5. METTL3-mediated m⁶A modifications regulate the myostatin pathway.

a Schematic of the myofiber-specific Ribo-Tag WT and M3-mKO mice. b Western blot of HA expression and GAPDH control in muscles of Ribo-Tag expressing WT and M3-mKO mice or mice not expressing the tag (negative control; neg. ctrl). c Schematic of Ribo-seq protocol to capture muscle-specific ribosome-bound RNAs in WT and M3-mKO mice created in Adobe Illustrator. d Venn diagram showing the number of total enriched transcripts in myofiber ribosomes from METTL3 WT and mKO baseline muscle (big circles) and the number of differentially translated transcripts that also contain m⁶A at baseline (small circles). e Gene ontology (GO) analysis of Ribo-enriched, and m⁶A containing, as determined from baseline MeRIP-Seq samples. The enrichment score is based on a reference database of protein-coding genes; plotted GO categories have a false discovery rate (FDR) < 0.05. f Integrative Genomics View (IGV) of input and immunoprecipitation overlays on the Acvr2a gene from the MeRIP-seq baseline data set, and the Ribo-seq data sets for Ribo-Tag M3-mKO and WT. g Relative m⁶A enrichment, determined by qPCR analysis, following m⁶A immunoprecipitation in WT and M3-mKO plantaris muscles. h Relative ribosome occupancy enrichment, determined by qPCR analysis, of Acvr2a following Ribo-Tag immunoprecipitation in Ribo-Tag WT and M3-mKO plantaris muscles. i Immunofluorescence analysis of ACVR2A expression (red) in plantaris sections from WT and M3-mKO mice. j qPCR from RNA immunoprecipitation of Acvr2a mRNA in muscle using antibody against YTHDF1 (Y1), YTHDF2 (Y2), YTHDF3 (Y3), or normal IgG negative control (NC). k qPCR for Acvr2a post actinomycin treatment for the indicated times in 3T3 cells transfected with plasmids encoding for Myc-tagged Mettl3 (Myc-M3) or Myc alone control (Myc). Biological animal replicates: n = 3 per group in panel d–f, and i; n = 4 per group in panel g and h. Biological cell replicates: n = 3 per group in panels j and k. Data are presented as the mean ± SEM. *P < 0.05, by two-sided Student’s t test for comparisons between WT and M3-mKO animals, or by two-way ANOVA with Tukey’s HSD multiple-comparison test for comparison of the mean of WT and M3-mKO animal inputs and immunoprecipitations. Scale bar = 125 µm. Source data are provided as a Source Data file.

Fig. 6. Hypertrophy defects in METTL3-mKO mice are rescued with myostatin inhibition.

a Relative m⁶A enrichment, determined by qPCR analysis, following m⁶A immunoprecipitation in WT plantaris muscles following 7 days of sham (base) or overload (overl) surgeries. b Western blot of p-SMAD3 (p = phospho), total SMAD3 and GAPDH expression in muscles of WT and M3-mKO mice at baseline or 7 days following muscle overload. c qPCR analysis of Murf1 (muscle-specific ring finger protein 1) and d Mafbx (muscle-atrophy F-box protein) expression in WT and M3-mKO muscles. e Schematic of myostatin inhibition experimental plan created in Adobe Illustrator. f Plantaris weight, g representative wheat germ agglutinin (WGA; green) stained images, and h fiber size in day 14 overloaded WT and M3-mKO muscles treated with and without myostatin inhibitor ACE-031. i Descriptive figure of working model created in Adobe Illustrator. As muscle undergoes a hypertrophic response to overload, the METTL3 complex allows for the distribution of m⁶A to the Acvr2a transcript, which prevents its translation, and allows for normal muscle growth. In M3-mKO animals, there is no METTL3 to distribute m⁶A on the Acvr2a transcript, thus promoting Acvr2a translation, myostatin (MSTN) activity, and atrophic signaling through SMAD3 phosphorylation (p). Biological animal replicates: n = 4 (baseline) and 5 (overload) in panel a; n = 4 per group in panel c and d; and n = 4 per group in panel f and h. Data are presented as the mean ± SEM. *P < 0.05, by two-sided Student’s t test for comparisons between WT and M3-mKO animals, or by two-way ANOVA with Tukey’s HSD multiple-comparison test for comparison of the mean of WT and M3-mKO overloaded animal at with and without inhibitor ACE-031. Scale bar = 125 µm. Source data are provided as a Source Data file.