Journal: medRxiv

Article Title: A network-based atlas of human skeletal muscle aging

doi: 10.64898/2026.02.15.26346348

Figure Lengend Snippet: A) Fibre type specific gene expression in agreement with Murgia et al 2021. B) Using GeoMX we find a number of genes are differentially regulated in Type I vs Type II fibres, with atrophy – this includes three network module genes, GDNF-SVIL-RBFOX1 C) Merscope profiles before and after human muscle unloading. GDNF (yellow) expression in four ROIs (before and after 2 weeks of disuse). Mature muscle nuclei (MYREM+; blue) muscle satellite cells (MEG3/PAX7+;green) and endothelial cells (APLNR+/ENG+/KDR+/TIE1+; pink). At low magnification fast (ATP2A1, green) and slow muscle (MYH7, blue) fibres are stained. At higher magnification, fast are unstained (ATP2A1 fish channel switched off) and slow fibres are blue (MYH7). Clear examples of GDNF expression in or around mature skeletal muscle nuclei (MYREM+; blue) are show with 4-10 times more GDNF after unloading and consistent with the bulk analyses (Supplemental Table S7)

Article Snippet: Briefly, each fresh frozen sample embedded in the optimal cutting temperature compound (OCT, Tissue-Tek, The Netherlands) was sectioned at a thickness of 10 μm in a cryostat at -20 °C and transferred onto the circular MERSCOPE glass slide (PN 2040001, Vizgen, USA) within the fiducial bead border, and allowed to adhere to the glass for 5 minutes 77,182 .

Techniques: Gene Expression, Expressing, Staining

Journal: medRxiv

Article Title: A network-based atlas of human skeletal muscle aging

doi: 10.64898/2026.02.15.26346348

Figure Lengend Snippet: Merscope ROI QC Data

Article Snippet: Briefly, each fresh frozen sample embedded in the optimal cutting temperature compound (OCT, Tissue-Tek, The Netherlands) was sectioned at a thickness of 10 μm in a cryostat at -20 °C and transferred onto the circular MERSCOPE glass slide (PN 2040001, Vizgen, USA) within the fiducial bead border, and allowed to adhere to the glass for 5 minutes 77,182 .

Techniques:

Journal: medRxiv

Article Title: A network-based atlas of human skeletal muscle aging

doi: 10.64898/2026.02.15.26346348

Figure Lengend Snippet: Using Merscope merfish assays we show rare examples of IL6 (yellow) as well as IL6 receptors (red) in four ROIs (with and without muscle use) from samples with the highest total IL6 count out of 54 examples. A ) Fast (unstained) and slow fibres (MYH7, blue). Mature muscle nuclei (MYREM+; blue) muscle satellite cells (MEG3/PAX7+;green) and endothelial cells (APLNR+/ENG+/KDR+/TIE1+; pink). Overall IL6 is not reliably expressed in human muscle fibres with a single molecule detected in fewer than 1/10 th of fibres, a level we cannot distinguish from indirect contamination/background. B ) In stark contrast, we find isolated examples of numerous IL6 mRNA inside endothelial cells in certain biopsy samples (young healthy muscle). These IL6 expressing endothelial cells are rare so they also cannot be the main source of robust IL6 is qPCR experiments using whole biopsy cDNA libraries – which probably reflects immune cells in the biopsy and these are presumably more abundant in post-exercise tissue due to the increased blood perfusion.

Article Snippet: Briefly, each fresh frozen sample embedded in the optimal cutting temperature compound (OCT, Tissue-Tek, The Netherlands) was sectioned at a thickness of 10 μm in a cryostat at -20 °C and transferred onto the circular MERSCOPE glass slide (PN 2040001, Vizgen, USA) within the fiducial bead border, and allowed to adhere to the glass for 5 minutes 77,182 .

Techniques: Isolation, Expressing

Journal: medRxiv

Article Title: A network-based atlas of human skeletal muscle aging

doi: 10.64898/2026.02.15.26346348

Figure Lengend Snippet: Using the Xenium spatial platform and a 460-plex gene assay (a subset of the Merscope v1 chemistry 960-plex assay) we measure IL6 (yellow) in human muscle. Endothelial cells show specific APLNR, ENG and TIE1 expression (Pink), muscle satellite Cell show MEG3 and PAX7 expression (Green) while mature muscle nuclei expression the noncoding RNA MYREM (Blue). Nuclei are stained with DAPI (show in white as is the cell membrane). Using this second immunofluorescence spatial technology we find no evidence that IL6 is robustly expressed in human muscle cells – with a single IL6 mRNA molecule in one in every ∼20 cells. We cannot rule out these few being non-specific contamination from other cell types. Given the typical signal found using qPCR and cDNA derived from a 20mg tissue biopsy we conclude that IL6 gene expression in muscle tissue is probably derived from immune cells and therefore it is not a myokine.

Article Snippet: Briefly, each fresh frozen sample embedded in the optimal cutting temperature compound (OCT, Tissue-Tek, The Netherlands) was sectioned at a thickness of 10 μm in a cryostat at -20 °C and transferred onto the circular MERSCOPE glass slide (PN 2040001, Vizgen, USA) within the fiducial bead border, and allowed to adhere to the glass for 5 minutes 77,182 .

Techniques: Gene Assay, Plex Assay, Expressing, Staining, Membrane, Immunofluorescence, Derivative Assay, Gene Expression

Journal: medRxiv

Article Title: A network-based atlas of human skeletal muscle aging

doi: 10.64898/2026.02.15.26346348

Figure Lengend Snippet: Using FISH probes for the three main fibres types, we illustrate the variation in Type II hybrid types we observed using the Merscope technology. This clarifies that Type IIx gene expression is found in human muscle

Article Snippet: Briefly, each fresh frozen sample embedded in the optimal cutting temperature compound (OCT, Tissue-Tek, The Netherlands) was sectioned at a thickness of 10 μm in a cryostat at -20 °C and transferred onto the circular MERSCOPE glass slide (PN 2040001, Vizgen, USA) within the fiducial bead border, and allowed to adhere to the glass for 5 minutes 77,182 .

Techniques: Gene Expression

Journal: medRxiv

Article Title: A network-based atlas of human skeletal muscle aging

doi: 10.64898/2026.02.15.26346348

Figure Lengend Snippet: Using the Xenium spatial platform and a 460-plex gene assay (a subset of the Merscope v1 chemistry 960-plex assay) we confirm that some classic cytosolic fibre type specific markers are unsuitable to classify single-nuclei profiles (e.g. MYH7). While MYH7 is, for example, specific to Type I muscle fibres in the cytoplasm, lineage tracing and the present data demonstrate that human muscle nuclei don’t express such genes in a fibre-type specific gene expression pattern and thus can not be utilized to assign snRNAseq profiles to individual fibres types. Only spatial single cell technologies can reliably assign molecular profiles to specific cell types

Article Snippet: Briefly, each fresh frozen sample embedded in the optimal cutting temperature compound (OCT, Tissue-Tek, The Netherlands) was sectioned at a thickness of 10 μm in a cryostat at -20 °C and transferred onto the circular MERSCOPE glass slide (PN 2040001, Vizgen, USA) within the fiducial bead border, and allowed to adhere to the glass for 5 minutes 77,182 .

Techniques: Gene Assay, Plex Assay, Gene Expression, Single Cell