Muscle fiber Myc is dispensable for muscle growth and its forced expression severely perturbs homeostasis

Abstract

The oncogenic transcription factor Myc stimulates many growth processes including cell cycle progression and ribosome biogenesis. Myc expression is low in adult skeletal muscle, but is upregulated upon growth stimuli. Furthermore, muscle fiber Myc overexpression recapitulates many aspects of growth-related gene expression, leading to the hypothesis that Myc mediates pro-growth responses to anabolic stimuli, such as exercise. Here, we test this hypothesis by examining mouse models in which Myc is specifically eliminated or overexpressed in skeletal muscle fibers or muscle stem cells (MuSC). While muscle fiber Myc expression increased during muscle growth and Myc expression in MuSCs was required for successful muscle regeneration, muscle fiber Myc expression was dispensable for post-natal, mechanical overload or PKBα/Akt1-induced muscle growth in mice. Similarly, constitutive Myc expression did not promote skeletal muscle hypertrophy, but instead impaired muscle fiber structure and function within days. These data question the role of Myc in skeletal muscle growth.

Fig. 1. Post-natal muscle growth is not altered in HSA-MycKO mice.

A Digital PCR for transcript levels of Myc and Rpl3 normalized to a housekeeping gene (Hprt) in CON (HSA^wt/wt; Myc^fl/fl) and HSA-MycKO (HSA^Cre/WT; Myc^fl/fl) mice (n = 4). B Representative images of RNA fluorescent in situ hybridization (RNA-FISH) with probes against Myc (white) and Pax7 (yellow; Muscle stem cells) and antibody staining for laminin β1γ1 (magenta). C Body mass for 2-month-old (2 m) males (CON, n = 8; KO, n = 12) and 5- to 7-month-old (5–7 m) males (CON, n = 8, KO, n = 9) and females (CON, n = 12; KO n = 11). D Whole-body measures of %fat (left) and %lean (right) mass (CON, n = 8; KO, n = 9 males, n = 8 females). E Correlations between body mass and TA, TRI, GAS and QUAD muscle mass for CON (n = 20–21 males, n = 19 females) and HSA-MycKO (n = 28 males, n = 20 females) mice. In vitro, EDL force frequency curve and fatigue response to multiple stimulations (F) and representative twitch force traces (G) for 2- (CON, n = 8; KO, n = 10–11) and 5- to 7-month-old (CON, n = 4–5; KO, n = 6) male CON and HSA-MycKO mice. H Principle component analysis of mRNAseq data generated from the GAS muscle of 5- to 7-month-old male mice (n = 4) and (I) heatmap of select downregulated genes (FDR < 0.05) associated with muscle development, the neuromuscular junction (NMJ) and the TGFβ pathway. J Overlap of significantly downregulated genes in HSA-MycKO mice (FDR < 0.05) with a published list of genes upregulated by short-term muscle-specific Myc overexpression. K Scatterplot of log-fold change and -log10 FDR for all ribosomal protein genes from mRNAseq data comparing CON and HSA-MycKO mice. Data are presented as mean ± SEM. Two-way ANOVA with Sidak’s post hoc test (A, C, D, F, G) were used to compare between data. For (I–K), P values were adjusted for multiple comparisons using the Benjamini-Hochberg (FDR) procedure. *, **, and *** denote a significant difference between groups of P  <  0.05, P  <  0.01, and P  <  0.001, respectively.

Fig. 2. Mechanical overload- (MOV) induced muscle growth is not altered in HSA-MycKO mice.

A Schematic of MOV surgery. B Plantaris (PLA) muscle mass normalized to body mass and the mean mass of the contralateral muscle after 3 (CON only; n = 3), 7, 14 and 28 days of MOV in CON (n = 4, 3, 7) and HSA-MycKO (n = 3, 3, 7) mice. C Digital PCR for transcript levels of Myc and Rpl3 normalized to a housekeeping gene (Hprt) in CON (HSA^wt/wt; Myc^fl/fl) and HSA-MycKO (HSA^Cre/wt;Myc^fl/fl) mice after 7 (CON, n = 4; KO, n = 3) and 14 (n = 3) days. D Representative images of RNA fluorescent in situ hybridization (RNA-FISH) with probes against Myc (magenta) and counterstaining with DAPI (blue) and laminin β1γ1 antibodies (white), for 3 (CON only), 7 and 14 days of MOV for CON and HSA-MycKO mice. E Quantification of Myc + (≥3 Myc puncta) nuclei outside laminin-stained muscle fibers (Non-muscle), Pax7+ muscle stem cell nuclei (MuSC) and peripherally (Myofiber) and centrally located (Central nuclei) nuclei within laminin stained muscle fibers, in contralateral and after 7 days of MOV (MOV-7d) in CON (n = 4) and HSA-MycKO (n = 3) mice. F Type-specific (IIA, IIX, IIB) fiber number in complete PLA cross-sections normalized to the contralateral PLA in CON (n = 6) and HSA-MycKO (n = 7) mice after 28 days of MOV and (G) representative images after 14 and 28 days of MOV. H Type-specific (IIA, IIX, IIB) fiber size distribution normalized to contralateral total fiber number (n = 7; or 6 for CON MOV-28d). I Quantification and (J) representative images of Pax7+ nuclei co-stained with <3 (low) 3–5 (Pos) or >5 (High) RNA puncta for Myc (upper) and MyoD (lower) using RNA-FISH in the contralateral muscle and after 7 days MOV (CON, n = 4; KO, n = 3). A Created in BioRender. Ruegg, M. (2025) https://BioRender.com/d74q573. Data are presented as mean ± SEM. Two-way repeated measure ANOVAs (A) or two-way ANOVAs with Sidak’s post hoc test were used to compare between data. *, **, and *** denote a significant difference between groups of P  <  0.05, P  <  0.01, and P  <  0.001, respectively.

Fig. 3. Proliferation is blunted in Myc-depleted MuSCs and muscle regeneration is strongly impaired in Pax7-MyckO mice.

A Schematic and (B) representative images of isolated single fibers from Pax7-eGFP and Pax7-MycKO mice either fixed immediately (0 h) or cultured for 48 h (48 h) and then stained for eGFP, Pax7 and Ki67 (n = 4). C Percentage of eGFP+ MuSC cells expressing Ki67. D Percentage of eGFP+ MuSC cells in cluster sizes of 1, 2, 3-4 and 5+ . E Schematic of cardiotoxin (CTX) experiment in CON and Pax7-MycKO mice. F TA Muscle mass 4, 7 and 14 days after CTX injury in CON (n = 6, 7, 8) and Pax7-MycKO mice (n = 7, 6, 5) normalized to uninjured contralateral TA mass. G Fiber type proportion for eMHC, IIB and unstained fibers (IIA/X) 14 days after CTX injury in CON (n = 4) and Pax7-MycKO (n = 5) TA cross-sections. H Representative images showing immunostaining for embryonic myosin heavy chain (eMHC; magenta), type IIB (green) and laminin (white) at 4, 7 and 14 days post CTX injury and (I) mean minimum fiber feret size at 14 days in CON (n = 4) and Pax7-MycKO (n = 5) TA cross-sections. J Pax7+ and (K) MyoG+ cell number in TA muscle sections, via immunostaining, 4 (n = 4–5), 7 (n = 3-4) and 14 (n = 3–5) days after CTX injury in CON and Pax7-MycKO mice normalized to cross-sectional area. Uninjured samples represent pooled, un-injected contralateral legs from all time points (CON, n = 13–14; KO, n = 11–12). A, E Created in BioRender. Ruegg, M. (2025) https://BioRender.com/d74q573. Data are presented as mean ± SEM. Two-tailed, Student’s independent t tests (I) or two-way ANOVAs with Sidak’s post hoc test were used to compare between conditions. *, **, and *** denote a significant difference between groups of P  <  0.05, P  <  0.01, and P  <  0.001, respectively.

Fig. 4. MOV-induced muscle growth is strongly impaired in Pax7-MycKO mice.

A Muscle mass (CON, n = 4 F and 5 M; KO, n = 5 F and 5 M) and (B) representative images (male), (C) fiber size distribution and (D) total fiber number of plantaris (PLA) muscle cross-sections from the contralateral control limb and after 28 days of MOV in female (CON, n = 4; KO, n = 5 or n = 6 for KO-MOV) and male CON (n = 4) and Pax7-MycKO (n = 5) mice, stained with antibodies against IIA and IIB MHC isoforms. E Quantification of eMHC+ fibers in female and male CON (n = 4 F, 5 M) and Pax7-MycKO (n = 5) PLA muscle cross-sections from the contralateral limb or after 28 days of MOV and (F) representative images of eMHC, IIB and laminin stained sections from female (left) and male (right) muscles 28 days after MOV. Data are presented as mean ± SEM. Mixed-effects (A) or two-way ANOVAs with Sidak’s post hoc test were used to compare between conditions. *, **, and *** denote a significant difference between groups of P  <  0.05, P  <  0.01, and P  <  0.001, respectively.

Fig. 5. Myc depleted MuSCs fail to contribute to muscle regrowth after sciatic nerve injury.

A Lineage tracing approach to track MuSC-myofiber fusion events following sciatic nerve crush. B Representative images of eGFP+ fibers, indicating Pax7+ MuSC fusion, in GAS, TA, EDL and SOL muscles of Pax7-eGFP mice, and their absence in Pax7-MycKO mice 28 days after nerve crush and (C) Quantification in TA muscle (n = 3). D Muscle mass 14, 28 and 42 days after sciatic nerve crush normalized to the innervated contralateral limb in CON (n = 13 or 12 for SOL, 16 or 15 for TA and EDL, and 13 or 12 for GAS and EDL) and Pax7-MycKO (n = 9 or 8 for EDL, 11 or 10 for EDL, and 15 or 16 for GAS) mice. E EDL specific muscle force 28 and 42 days after nerve crush and in the contralateral control muscle for CON (28 d, n = 12; 42 d, n = 8) and Pax7-MycKO (28 d, n = 8 or 6 Inn; 42 d, n = 10) mice. F RT-qPCR for Myc mRNA normalized to Gapdh in innervated (CON, n = 13; KO, n = 15) muscle and 14, 28 and 42 days after nerve crush (n = 5). G Representative images and quantification of (H) Pax7+ (I) Pax7+; Ki67+ and (J) Pax7+ ; EdU+ cell number in immunostained whole GAS muscle cross-sections collected 14 d after nerve crush and 24 h after an I.P. injection with 100 mg/kg EdU (n = 5). K Quantification and (L) representative images of eGFP+ and eGFP- fibers with centralized nuclei in TA muscle from Pax7-eGFP and Pax7-MycKO mice (n = 3). A Created in BioRender. Ruegg, M. (2025) https://BioRender.com/d74q573. Data are presented as mean ± SEM. Two-tailed Student’s t tests (C) or two-way ANOVAs with Sidak’s post hoc test (D–F, H–K) were used to compare between conditions. *, **, and *** denote a significant difference between groups of P  <  0.05, P  <  0.01, and P  <  0.001, respectively.

Fig. 6. Muscle fiber Myc expression is not necessary for AktTG-induced muscle hypertrophy.

A Schematic of tamoxifen-inducible AktTG mice crossed with HSA-MycKO mice. Muscle mass for (B) female mice after 3 (CON, n = 4; KO, n = 5) and 14 (CON, n = 8; KO, n = 10) days and (C) male mice after 14 days (CON, n = 3 or 4 for TRI; KO, n = 4) of tamoxifen treatment. CON (n = 13 F, 9 M) and HSA-MycKO (n = 12 F, 11 M) mice treated with tamoxifen for 3 and 14 days but not expressing the PKBα/Akt1 transgene were pooled. D EDL muscle force (males and females; normalized to body mass prior to tamoxifen and force at a stimulation frequency of 200 Hz) for CON (n = 17), HSA-MycKO (n = 19), AktTG (n = 11) and Akt-MycKO (n = 14) mice treated with tamoxifen for 14 days. E Representative images and (F) quantification of type-specific fiber size distribution in TA muscle from female CON (n = 7), MycKO (n = 7), AktTG (n = 7) and Akt-MycKO (n = 6) mice after 14 days of tamoxifen (IIA: orange; IIB green; IIX unstained). G Principle component analysis of mRNAseq data generated from GAS muscle of female CON (n = 4) and HSA-MycKO (n = 4) mice treated with tamoxifen for 3 days or AktTG and Akt-MycKO mice treated for 3 (AktTG, n = 4; Akt-MycKO, n = 3) and 14 days (n = 4). H Differentially expressed genes (FDR < 0.05, Benjamini-Hochberg, or P = 0.02 for 3 d comparison due to low sample number; LFC > 0.5; up in red, down in blue) between CON and HSA-MycKO (baseline, i.e. no PKBα/Akt1) and either 3 or 14 days PKBα/Akt1 activation and between 3 and 14 days PKBα/Akt1 activation for CON and HSA-MycKO mice, as well as between CON and HSA-MycKO mice at baseline, 3 and 14 days. Scatterplot of -Log10(FDR) for (I) upregulated and (J) downregulated gene ontology (GO) terms (Biological process, Molecular function and Cellular component) enriched in AktTG (x-axis) or Akt-MycKO (y-axis) mice after 14 days tamoxifen compared to CON and HSA-MycKO mice, respectively. Top GO terms are labeled, while terms more prominently represented in AktTG than Akt-MycKO are shown in purple. A Created in BioRender. Ruegg, M. (2025) https://BioRender.com/d74q573. Data are presented as mean ± SEM. Two-way ANOVAs with Sidak’s post hoc test were used to compare between conditions. *, **, and *** denote a significant difference between groups of P  <  0.05, P  <  0.01, and P  <  0.001, respectively.

Fig. 7. Muscle fiber Myc expression strongly impairs muscle structure and function.

A Schematic of tamoxifen-inducible, muscle stem cell-specific transgenic Myc expression mouse model and time course of tissue collection after an intramuscular cardiotoxin (CTX) injection. B Muscle mass loss (%) in CON and Pax7-MycTG mice 4 (CON, n = 4; TG, n = 5), 7 (CON, n = 6; TG, n = 4) and 14 (CON, n = 7; TG, n = 5) days after intramuscular CTX injection. C Representative images of eMHC, IIA/X (unstained) and IIB fiber type proportion in TA cross-sections 4, 7 and 14 days after intramuscular CTX injection and the contralateral control muscle (Uninj) and (D) quantification at 14 days (n = 4). E Schematic of HSA-MycTG mouse model and tamoxifen treatment regime. Representative western blots (F) and quantification (G) for Myc, RPS6 and RPS14, normalized to α-actinin (CON, n = 9; TG, n = 7). H Body mass immediately before the first tamoxifen injection and 4 or 10 days later for CON (n = 7) and HSA-MycTG (4 d, n = 11; 10 d, n = 8) mice. I Force frequency curve and fatigue response to repeated 200 Hz stimulations in EDL muscle from CON (n = 10) and HSA-MycTG mice at 4 (n = 6) or 10 d (n = 8) time points. J Representative images of H&E (left) and IgG (right) staining in TA cross-sections of CON and 10 d HSA-MycTG mice. K Muscle mass for CON (n = 14) and 4 d (n = 11) and 10 d (n = 8) HSA-MycTG mice. A, E Created in BioRender. Ruegg, M. (2025) https://BioRender.com/d74q573. Data are presented as mean ± SEM. Two-way ANOVAs (B, D, I, K) and repeated measures ANOVAs (H) with Sidak’s post hoc test or Students t test (G) were used to compare between conditions. *, **, and *** denote a significant difference between groups of P  <  0.05, P  <  0.01, and P  <  0.001, respectively.

Fig. 8. Muscle fiber Myc overexpression promotes growth-related gene expression signatures, but even a minority of Myc overexpressing fibers impairs muscle regrowth after nerve injury.

A Principle component analysis of mRNAseq data generated from GAS muscle of male CON and HSA-MycTG mice 4 (CON, n = 3; TG, n = 4) or 10 (n = 4) days after the first of 5 tamoxifen injections. 4 d and 10 d CON group samples were pooled. B Overlap of differentially expressed genes (FDR < 0.05; LFC > 0.5) between CON and either 4 d or 10 d HSA-MycTG groups and C Scatterplot of log-fold change and –log10 FDR between CON and 4 d HSA-MycTG for all detected ribosomal protein genes. D Pairwise Venn diagram comparisons of genes differentially expressed for acute (3 d AktTG and 4 d HSA-MycTG) and chronic (14 d AktTG and 10 d HSA-MycTG) activation of AKT and overexpression of Myc. Tan (MycTG) and lilac (AktTG) colored arrows indicate direction of change. E Gene ontology (Biological process) terms associated with overlapping genes between 14 d AktTG and 10 d HSA-MycTG (D, right). F Lineage tracing approach to track MuSC-myofiber fusion events following sciatic nerve crush in Pax7-MycTG mice. G Representative images and quantification of eGFP+ fiber number (H) in TA muscle cross-sections 28 d after nerve crush in Pax7-eGFP (n = 3), and Pax7-MycTG (n = 4) mice and in contralateral TA muscles from Pax7-MycTG. White arrowhead indicates an eGFP+ MuSC (eGFP image intensity increased compared to fibers). Fiber size distribution of eGFP+ and eGFP- muscle fibers 28 d after nerve crush in TA muscle from (I) Pax7-eGFP (n = 3) and (J) Pax7-MycTG (n = 4) mice. K Representative image of eGFP+ fibers in longitudinal sections from Pax7-MycTG mice 28 d after nerve crush. L Muscle mass 28 days after sciatic nerve crush normalized to the innervated contralateral limb in control (CON, n = 14) and Pax7-MycTG (n = 13) mice. M In vitro specific force in EDL muscles 28 d after nerve crush (and innervated contralateral) in CON (Inn, n = 13; 28 d, n = 14) and Pax7-MycTG (n = 13) mice. F Created in BioRender. Ruegg, M. (2025) https://BioRender.com/d74q573. Data are mean ± SEM. Two tailed, independent (I, J) and paired (H) Student’s t tests, two-way repeated measures (L) or two-way ANOVAs with Sidak’s post hoc test (M) were used to compare between conditions. For (B–D), P values were adjusted for multiple comparisons using the Benjamini-Hochberg (FDR) procedure. *, **, and *** denote a significant difference between groups of P  <  0.05, P  <  0.01, and P  <  0.001, respectively.