Spatial transcriptomics maps distinct signatures of human intermuscular adipose expansion in mice

Figures

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  Figure 1. A) Heatmap of human IMAT specific genes differentially expressed between IMAT and muscle/subcutaneous FAT tissue of lean individuals (AVOVA p_fdr < 0.01) B) Representative histological sections of muscle quadriceps in high-fat-diet (HFD) and low-fat-diet (LFD) male mice. Intermuscular adipocytes are indicated by arrows. Scale bar 100um. C) Quantitative digital imaging analysis of histological sections in male mice (n = 7–8) after 84 days of HFD and LFD feeding. Data are presented as mean ± SEM. **P <0,0001 D) OA micrographs (1 mm × 1 mm) obtained from two channels (at 1550, 2856 cm−1) by mid-infrared optoacoustic microscopy (MiROM) (optoacoustic signal – OAS). E to F) OA spectra of lipid droplets (E) and muscle fibers (F) from HFD and LFD quadriceps muscle tissue (randomly selected spectral points; 20 points at lipid droplets and 40 points at muscle fibers) and p-values of t-tests on OA spectra for each wavenumber. The dashed line indicates p=0.05 which is considered as a statistically significant difference threshold. p < 0.05 is considered as a statistically significant difference.

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  Figure 2. A) Immunofluorescence staining of laminin (red), perilipin (green), and DAPI (blue) in murine skeletal muscle cross-sections from low-fat diet (LFD) and high-fat diet (HFD) fed male C57BL/6 mice, acquired on Visium 10x Genomics slides (n = 2). B) Barplot mean and standard deviation of the spotwise perilipin staining percentage. Data are presented as mean ± SEM. *P <0,01 C) Relative spotwise adipocyte content estimated though deconvolution in LFD and HFD fed male mice of panel A. D) Relative spotwise FAP content estimated though deconvolution in LFD and HFD fed male mice of panel A. E) Scatterplot comparing spotwise perilipin intensity (mean) with adipocyte (left) and FAP (right) content for LFD (blue) and HFD (red) fed male mice of panel A. Correlation lines with 95% confidence intervals are shown for both diets.

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  Figure 3. A) UMAP plot of all integrated spots with louvain clustering from low-fat diet (LFD) and high-fat diet (HFD) fed male C57BL/6 mice, acquired on Visium 10x Genomics slides (n = 2). Identifierd clusters are numbered from 0 to 8 and color coded. B) Distribution of adipocyte type proportion over all 9 identified spot clusters from LFD and HFD fed male mice. C) Distribution of FAP type proportion over all 9 identified spot clusters from LFD and HFD fed male mice. D) Spatial distribution of spots belonging to the three selected clusters 4, 6, and 7 for LFD and HFD fed male mice, shown on Visium 10x Genomics slides (n = 2). E) Dot plot for expression levels of the human IMAT specific gene signature over all 9 clusters determined in LFD and HFD fed male mice. F) Expression of the human IMAT specific gene signature between LFD and HFD fed male mice (n = 2). G-H) Scatterplot of directed p-values (p * sign (rho)) comparing IMAT specific correlation between spot wise human IMAT gene expression and cell type proportion for adipocytes (G) and FAPs (H) in LFD fed (x-axis) and HFD (y-axis) fed male mice. Purple dots denote genes significantly correlated in both directions; orange dots denote…

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  Figure 4. A) Expression levels of EBF2, PPARδ, and MYF5 were quantified by RT-PCR in human intermuscular adipose tissue (IMAT) and skeletal muscle (n = 22/30 (IMAT/Muscle). Data are presented as mean ± SEM; statistical significance was determined by unpaired t-test. B) Spatial expression of EBF2 in low-fat diet (LFD) and high-fat diet (HFD) fed male C57BL/6 mice acquired on Visium 10x Genomics slides (n = 2). C) Schematic overview of the experimental design. Human primary myoblasts were transduced with lentiviral vectors encoding EBF2 (HSkMCEBF2) or a control mCherry-GFP (HSkMCmCherry) construct to test whether EBF2 overexpression induces transdifferentiation toward a fat-like phenotype and affects myogenic or adipogenic differentiation. D) Western blotting of EBF2 in HSkMCEBF2 and HSkMCmCherry cells after lentiviral transduction and transdifferentiation with adipogenic media. GAPDH was used as loading control. 5 µg proteins were loaded per sample. Size (in kDa) of molecular weight marker is indicated on the right. E) Comparison between adipocenic transdifferentiated and proliferating HSkMCmCherry and HSkMCEBF2 cells. Western blotting of MHC in HSkMCEBF2 and HSkMCmCherry after 14 days of…