Stereo-cell coupled with single-cell transcriptomics identifies a transitional metabolic state in maturing skeletal muscle myofibers

Figures

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  FIGURE 1. Generation of a single-cell transcriptomic atlas of mouse skeletal muscle (A) Schematic of the single-cell/nucleus RNA-seq workflow for mouse skeletal muscle. (B) UMAP visualization of cell type annotations from scRNA-seq of mouse skeletal muscle. (C) Expression of marker genes across cell types. Color intensity represents expression level, and bubble size indicates the proportion of cells expressing the gene. (D) Proportions of cell types across different sequencing technologies and skeletal muscles. (E) UMAP distribution of cells across different sequencing technologies and skeletal muscles.

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  FIGURE 2. Transcriptomic features, developmental trajectories, and metabolic pathway differences among muscle fiber subtypes. (A) UMAP visualization of muscle fiber subtype annotations. (B) Expression of marker genes across muscle fiber subtypes. Color intensity represents expression level, and bubble size indicates the proportion of cells expressing the gene. (C) Proportions of muscle fiber subtypes across single-cell and single-nucleus sequencing data from different skeletal muscles. (D) Bubble plot of GO enrichment analysis for DEGs across muscle fiber subtypes. (E) Developmental trajectory and pseudotime analysis of mouse skeletal muscle fiber subtypes. (F) Dynamic expression of key functional genes across pseudotime in different muscle fiber subtypes. (G) Differences in glycolysis and oxidative phosphorylation pathway scores across muscle fiber subtypes in the total dataset and individual skeletal muscles.

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  FIGURE 3. Spatial distribution of muscle fiber subtypes in mouse skeletal muscle revealed by Stereo‐cell (A) Experimental workflow for capturing single muscle fibers using Stereo‐cell. (B) Image processing pipeline to generate a gene expression matrix of muscle fiber ROIs. (C) Spatial expression distribution of muscle fiber subtype marker genes in EDL and SOL muscles. In the EDL Myh1 panel, colored ellipses mark three expression patterns in Myh4+ (type IIB) fibers (n = 19): red (no/low Myh1, 57.9%), yellow (terminal enrichment, 31.6%), green (diffuse expression, 10.5%).

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  FIGURE 4. Oxidative phosphorylation and glycolysis exhibit distinct metabolic partitioning along MF_IIB muscle fibers (A) Spatial distribution differences in glycolysis and oxidative phosphorylation pathway gene expression along muscle fibers. (B) K-means clustering of muscle fiber ROIs based on metabolic pathway scores. (C) Heterogeneity in glycolysis and oxidative phosphorylation metabolic activity across clusters. (D) Expression differences in key glycolysis and oxidative phosphorylation pathway genes across clusters. (E) RCTD deconvolution of MF_IIB subtypes in Stereo-cell data. (F) Statistical differences in oxidative phosphorylation and glycolysis pathway scores between MF_IIB subtypes. *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001.