Multiomics profiling of young and old quiescent skeletal muscle stem cells [GSH RNA-seq]
To uncover new pathways that are important for skeletal muscle stem cell (MuSC) aging, we performed multiomics profiling, including transcriptomics, DNA methylomics, proteomics, and metabolomics on quiescent MuSCs from young and old mice. Our goals were to discover pathways that have been overlooked by isolated profiling approaches and to gain insight into which changes are causal, compensatory, correlational, and consequential. In our work, we found that glutathione metabolism is a key pathway of MuSC that involves a compensatory feedback loop. Follow-up experiments showed that old MuSCs actually form a dichotomy between glutathione-high MuSCs and glutathione-low MuSCs. RNA-Seq showed that glutathione-high old MuSCs are able to synthesize adequate glutathione and thus compensate adequately for oxidative stress with increased glutathione turnover, while glutathione-low old MuSCs have failed to compensate for oxidative stress metabolically and instead show increased inflammatory signaling. Overall design: C57BL/6 male mice were used at two ages: young (4 months) and old (23 months). Quiescent MuSCs were isolated from limb muscles by FACS (CD45- CD31- Sca1- VCAM+). Sorted cells were immediately stained with ThiolTracker Violet (5 uM) to indicate glutathione (GSH) status and resorted by FACS. For young, all cells were recollected (almost all are GSH-high). For old, the population was split into GSH-high and GSH-low collection groups. Each replicate represents the cells from a single animal (young) or pooled two animals (old).