Transient MYC Mimicking the Exercise Response Orchestrates Multifaceted Skeletal Muscle Adaptations

Figures

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  Figure 1.. Acute MYC induction rapidly remodels the soleus transcriptome. (A) Experimental design schematic depicting acute MYC induction protocol and bulk RNA sequencing on MYC-induced (n = 3) and control (n = 3) mice. (B) Principal component analysis of DESeq2-normalized soleus transcriptome. (C) Volcano plot of differentially expressed genes in MYC-induced versus control soleus. Genes meeting thresholds of adj. p < 0.05 and |log2 fold change| > 1 are colored red (upregulated, n = 1,817) or blue (downregulated, n = 1,150); representative genes are labeled. (D-E) Heatmaps of the top 25 upregulated (D) and downregulated (E) differentially expressed genes ranked by adjusted p-value, displaying row Z-scored expression across individual control and MYC-induced samples. (F-G) Gene ontology biological process (GO:BP) gene set enrichment analysis of downregulated (F) and upregulated (G) programs in MYC-induced soleus. Point size reflects gene set size and point color reflects −log10 adjusted p-value; pathways with adj. p<0.05 are shown.

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  Figure 2.. A primary myotube MYC induction model recapitulates in vivo growth and transcriptional remodeling. (A) Experimental design schematic for primary myogenic cell isolation and in vitro cell culture experiment. (B) GFP fluorescence during primary myotube differentiation, showing progressive activation of the HSA-driven reporter as myoblasts fuse and mature from 24 to 96 hours. (C) Representative western blot and quantification of MYC protein abundance expressed as fold change relative to control. (D-E) Representative myotube immunofluorescence (D) and quantification of myotube area normalized to myonuclear number (E). (F) Volcano plot of differentially expressed genes in MYC-induced versus control primary myotubes. Genes with Benjamini-Hochberg adjusted p<0.05 are colored red for upregulated genes (n = 225) or blue for downregulated genes (n = 167); representative genes are labeled. (G) Curated pathway-level transcriptional shifts in MYC-induced primary myotubes organized into ribosome biogenesis/rRNA processing, anabolic/biosynthetic, ECM/extracellular remodeling, and patterning/signaling suppression modules. Dot size reflects −log10 adjusted p-value and dot color reflects log2 fold…

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  Figure 3.. The nucleus type-specific response to a single pulse of MYC in the soleus. (A) Experimental design schematic for acute single-nucleus RNA-seq of soleus muscle following a single 48-hour doxycycline exposure and 12-hour washout in MYC-induced and control mice. (B) Bioinformatic workflow for snRNA-seq processing. (C) UMAP of integrated soleus nuclei colored by annotated nucleus type. (D) Feature plots showing human MYC transgene expression overlaid on the integrated UMAP in acute control, acute MYC 12-hour washout, and acute MYC 24-hour washout conditions. (E) Volcano plot of pseudobulk differential gene expression in myonuclei from acute MYC-induced versus control soleus. Genes meeting thresholds of adjusted p<0.05 and |log2 fold change| > 0.30 are colored red for upregulated genes (n = 2,694) or blue for downregulated genes (n = 1,077); representative genes are labeled. (F-G) Top 10 downregulated (F) and upregulated (G) GO Biological Process pathways enriched in acute MYC-induced myonuclei. Pathways are plotted by normalized enrichment score (NES), with point size reflecting gene set size and point color reflecting −log10 adjusted p-value; pathways shown meet adj. p<0.05. (H)…

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  Figure 4.. Repeated transient MYC induction over four weeks produces persistent myonuclear transcriptional remodeling and a shift in myonuclear proportion toward MyHC 1. (A) Experimental design schematic for snRNA-seq of soleus muscle following four weeks of repeated pulsatile MYC induction and 24-hour washout in MYC-induced and control mice. (B) UMAP feature plots showing human MYC transgene expression after a 24-hour washout following acute induction and repeated four-week induction. (C) Stacked bar plots showing acute-specific, repeated-specific, and shared differentially expressed genes across myonuclear and non-myonuclear populations. DEGs were defined using adj. p<0.05 and |log2 fold change|>0.30. (D) Conserved upregulated myonuclear GO:BP pathways across acute (12-hour washout) and repeated MYC induction, organized into ribosome biogenesis and translation/transcription machinery modules. Circles represent acute induction and triangles represent repeated induction; point color reflects normalized enrichment score (NES) and point size reflects −log10 adjusted p-value. (E) Nucleus-type composition in repeated MYC-induced and control soleus, expressed as the percentage of total nuclei…

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  Figure 5.. Repeated MYC induces a fast-to-slow fiber type transition in the soleus. (A) Experimental design schematic for 8-week repeated MYC induction and endpoint soleus phenotyping. (B) Soleus mass normalized to body weight in control and MYC-induced mice. (C) Soleus fiber-type composition from MyHC immunofluorescence. (D) Representative soleus cross-sections stained for MyHC isoforms, showing the fiber-type distribution in control and MYC-induced mice. (E) Quantification of SDH staining intensity categories, expressed as the proportion of low-, medium-, and high-SDH fibers relative to total SDH-positive fibers. (F) Representative soleus SDH histochemistry images from control and MYC-induced mice. Bar graphs show mean ± SEM with individual biological replicates overlaid.

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  Figure 6.. Repeated MYC induction causes coordinated RNA and protein remodeling in 8-week soleus. (A) Volcano plot of bulk RNA-seq differential gene expression in 8-week MYC-induced versus control soleus. Significantly upregulated genes are shown in red (n = 3,171) and significantly downregulated genes are shown in blue (n = 2,972); non-significant genes are shown in gray and representative genes are labeled. (B) Principal component analysis of DESeq2-normalized bulk RNA-seq data from 8-week MYC-induced and control soleus samples, with PC1 and PC2 explaining 35.7% and 11.4% of total variance, respectively. (C) Principal component analysis of mass spectrometry-based protein abundance from 8-week MYC-induced and control soleus samples, with PC1 and PC2 explaining 44.1% and 11.7% of total variance, respectively. (D) Venn diagrams comparing significantly upregulated and downregulated genes from acute soleus RNA-seq with significantly differentially abundant proteins from 8-week soleus proteomics. (E) Venn diagrams comparing significantly upregulated and downregulated genes/proteins from sample matched 8-week soleus RNA-seq and proteomics. Overlap analyses used adjusted p<0.05 and |logFC| > 0.1.…

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  Figure 7.. Repeated MYC induction remodels metabolic protein programs toward biosynthetic pathways and enhances oxidative metabolism. (A) Relative mitochondrial protein abundance in 8-week soleus proteomics, expressed as mitochondrial enrichment factor (MEF). (B) Summed abundance of proteins assigned to each oxidative phosphorylation (OXPHOS) complex (Complexes I-V), expressed as percent of the maximal signal within each complex. (C) Curated proteomic violin plots showing normalized abundance of metabolic proteins altered by repeated MYC induction. Adjusted p-values from differential abundance analysis are shown above each comparison. (D) Schematic summary of fatty acid uptake, activation, mitochondrial entry, β-oxidation, and phospholipid synthesis pathways altered in eight-week MYC soleus proteomics. Upward and downward arrows indicate the direction of significant protein abundance changes with repeated MYC induction from the proteomics data. (E) Schematic summary of glucose uptake, pentose phosphate pathway activity, NADPH-linked metabolism, and nucleotide synthesis proteins altered in eight-week MYC soleus proteomics. Upward arrows indicate increased protein abundance with repeated MYC…