RUNX1‐Mediated Hypoxic Signaling Impairs Myogenesis and Promotes Fibrosis in Myopathies

Figures

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  FIGURE 1. Single‐nucleus atlas of CON and WB muscle. (A) UMAP visualization showing cell clusters identified by unsupervised clustering. (B) The proportional abundance of each cell population. (C) Expression patterns of marker genes used for cell type annotation. (D) Heatmap of DEGs in each cell cluster and the top 3 significantly enriched GO terms for key cell clusters.

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  FIGURE 2. Transcriptional profiles of myonuclei and structural alterations of myofibers in CON and WB muscle. (A) UMAP visualization showing the Myonuclei clusters from CON and WB muscle. (B‐C) GSEA plot for upregulated and downregulated genes in the Myonuclei 2 cluster. (D) Representative images of ultrastructural and Masson staining of CON and WB muscle. Scale bars: 1 µm and 100 µm. (E, F) GO analysis of upregulated and downregulated genes between CON and WB muscle. (G) Heatmap showing the expression levels of representative factors synthesized and secreted by the Myonuclei in CON and WB muscle.

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  FIGURE 3. Transcriptional profiling of satellite cells (SCs) in CON and WB muscle. (A) UMAP visualization of SCs clusters in CON and WB muscle. (B) Violin plots showing the expression of marker genes in each SCs cluster. (C) The relative proportions of cell nuclei in each SCs cluster. (D) GSEA analysis of DEGs between the Act and Qui clusters. (E) UMAP visualization of SCs grouped by cell cycle marker genes. (F) The relative proportions of different cell cycle states between the CON group and WB group. (G) Bubble plot of GO enrichment analysis for DEGs between SCs from CON and WB muscle. (H) Bubble plot of the top five highly expressed genes in SCs from CON and WB muscle. (I) Pseudotime trajectory analysis of SCs. (J) Dynamic expression changes of highly expressed transcription factors and SCs marker genes. (K) Violin plot shows the expression levels of highly expressed transcription factors in each SCs cluster.

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  FIGURE 4. Alterations in the transcriptional profile of fibroblasts between CON and WB muscle. (A, B) Bubble plot of KEGG enrichment analysis for upregulated and downregulated genes between fibroblasts from CON and WB muscle. (C) Pseudotime trajectory analysis of fibroblasts. (D) Representative images of Sirius Red staining in CON and WB muscle (scale bar = 50 µm, n = 8 birds per group). Collagen fibers are stained in red, while muscle fibers appear yellow. Quantification of the collagen‐positive area (%) is shown on the right. (E, F) Heatmap showing the dynamic changes of representative upregulated and downregulated genes in fibroblasts. (G) Dynamic changes in representative upregulated genes and fibroblast differentiation marker genes. Data are expressed as the mean ± SEM. Error bars represent the SEM of biological replicates. Statistical significance was assessed by unpaired two‐tailed Student's t‐test. * p < 0.05, ** p < 0.01, *** p < 0.001.

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  FIGURE 5. Effects of RUNX1 on satellite cell (SC) proliferation and differentiation. (A) Interference efficiency detection of the three designed siRNAs in SCs (n = 3). (B) Relative mRNA expression levels of proliferation‐related genes (n = 3). (C) Representative immunofluorescence images of EdU staining and quantitative analysis of EdU‐positive cells 24 h after RUNX1 knockdown in SCs (scale bar = 50 µm, n = 3). (D) CCK‐8 assay showing cell viability 24 h after RUNX1 knockdown in SCs (3 biological replicates, with 3 technical replicates per biological replicate). (E) Western blot analysis of MyOG and MyOD protein expression 48 h after induction of SC differentiation following RUNX1 knockdown (n = 3). (F) Relative mRNA expression levels of myogenic differentiation marker genes in SCs following RUNX1 knockdown (n = 3). (G) Representative immunofluorescence images and bright‐field images of MyHC (left) and quantitative analysis of the myotube fusion index (right) 48 h after inducing SCs differentiation (scale bar = 50 and 25 µm, n = 3). (H) Detection of RUNX1 overexpression efficiency in SCs (n = 3). (I) Relative mRNA expression levels of proliferation‐related genes (n = 3). (J) Representative…

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  FIGURE 6. Effects of RUNX1 on fibroblast differentiation. (A–C) Effects of RUNX1 knockdown on fibroblasts. (A) Representative immunofluorescence images of α‐SMA staining (left) and quantification of mean fluorescence intensity of α‐SMA in RUNX1‐knockdown cells (right, scale bar = 25 µm, n = 3). (B) Representative Western blot analysis of RUNX1 and the myofibroblast‐associated proteins α‐SMA and Collagen I after TGF‐β1‐induced differentiation (n = 3). (C) Relative mRNA expression levels of differentiation marker genes (n = 3). (D–F) Effects of RUNX1 overexpression on fibroblasts. (D) Representative immunofluorescence images of α‐SMA staining (left) and quantification of mean fluorescence intensity of α‐SMA (right, scale bar = 25 µm, n = 3). (E) Representative Western blot analysis of RUNX1, α‐SMA, and Collagen I after TGF‐β1‐induced differentiation (n = 3). (F) Relative mRNA expression levels of differentiation marker genes (n = 3). For panels (A and D), each biological replicate was quantified from 3 random fields per condition. n = 3 independent biological replicates (independent batch); error bars represent the SEM of biological replicates. Data are expressed as the mean ± SEM.…

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  FIGURE 7. Association between hypoxia‐related indicators and RUNX1 expression in wooden breast (WB) muscle. (A) Relative expression levels of RUNX1 mRNA in CON and WB muscles (n = 8 birds per group). (B) Relative protein expression levels of RUNX1 in CON and WB muscles (n = 8 birds per group). (C, D) Representative laser speckle flow imaging and quantitative histograms of blood flow results in CON and WB muscles (n = 3 birds per group). (E, F) Comparative analysis of oxygen partial pressure (pO2) and oxygen saturation (sO2) in wing‐vein blood gas measurements from CON and WB broilers (n = 8 birds per group). (G, H) Relative protein expression levels of HIF‐1α and RUNX1 in satellite cells 24 h after hypoxia induction (n = 3). n = 3 represents three independent biological replicates, with each replicate generated by pooling SCs isolated from 2 to 3 embryos. (I, J) Relative protein expression levels of HIF‐1α and RUNX1 in fibroblasts 24 h after hypoxia induction (n = 3). n = 3 independent biological replicates (independent batches). Error bars represent the SEM of biological replicates. Data are expressed as the mean ± SEM. Statistical significance was assessed by an unpaired two‐tailed…

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  FIGURE 8. Comparison of cell communication between normal and wooden breast (WB) muscle. (A, B) The number and strength of inferred communication links between normal and WB muscle. (C) Differential interaction analysis identifying prominently altered signaling sources and targets in CON and WB muscle. x‐axis and y‐axis are the total outgoing or incoming communication probability associated with each cell cluster. (D) Comparisons of significant interactions between ligand‐receptor pairs between CON and WB muscle. Dot color reflects communication probabilities, and dot size represents computed p‐values. Empty space means the communication probability is zero. p‐values are computed from a one‐sided permutation test. (E) Relative mRNA expression levels of TGFβ1 and TGFβ3 in fibroblasts after 24 h of hypoxia treatment (n = 3). n = 3 independent biological replicates (independent batches). (F) Relative mRNA expression levels of TGFBR1 and TGFBR2 in satellite cells after 24 h of treatment with conditioned medium collected from hypoxia‐treated fibroblasts (n = 3). n = 3 represents three independent biological replicates, with each replicate generated by pooling SCs isolated from 2 to 3 embryos.…

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  FIGURE 9. Integrated analysis of human and mouse skeletal muscle datasets. For (A–D), snRNA‐seq datasets for human and mouse skeletal muscle cells were obtained and modified from the interactive tools of the UCSC Cell Browser (https://cells.ucsc.edu/) and the CNSknowall website (https://www.cnsknowall.com/). (A) Dot plot showing the expression levels of the three RUNX family members (RUNX1, RUNX2, and RUNX3) and HIF1A across the annotated cell clusters. (B, C) Summary of RUNX1 expression in skeletal muscle from mouse models of muscle disease (B) and human patients with various muscle diseases (C), based on transcriptome sequencing and microarray datasets obtained from the DiSignAtlas (http://www.inbirg.com/disignatlas). (D) Dot plot showing the expression pattern of RUNX1 and HIF1A under 3 conditions (CTRL, IBM, and IMNM). (E) UMAP showing the distribution of myogenic cells under the 3 conditions. (F) Feature plot showing the expression pattern of RUNX1 mapped onto the UMAP. IBM: Inclusion Body Myositis; IMNM: Immune‐Mediated Necrotizing Myopathy.