Spermidine-eIF5A axis is essential for muscle stem cell activation via translational control (Ribo-Seq)

Adult skeletal muscle stem cells, also known satellite cells (SCs), are quiescent and activate in response to injury. However, the activation mechanisms of quiescent SCs (QSCs) remain largely unknown. Here, we investigated the metabolic regulation of SC activation by identifying regulatory metabolites that promote SC activation. Using targeted metabolomics, we found that spermidine acts as a regulatory metabolite to promote SC activation and muscle regeneration in mice. Mechanistically, spermidine activates SCs via generating hypusinated eIF5A. Using SC-specific eIF5A-knockout (KO) and Myod-KO mice, we further found that eIF5A is required for spermidine-mediated SC activation by controlling MyoD translation. More significantly, depletion of eIF5A in SCs results in impaired muscle regeneration in mice. Together, the findings of our study define a novel mechanism that is essential for SC activation and acts via spermidine-eIF5A-mediated MyoD translation. Our findings suggest that the spermidine-eIF5A axis represents a promising pharmacological target in efforts to activate endogenous SCs for the treatment of muscular disease. Overall design: Primary myoblasts were isolated from the hind-limb muscles of adult (3-week-old) mice by digesting the muscle chops with 2 U/ml dispase and 0.2% collagenase for 45 min. The cells were filtered through 70-µm cell strainers and washed in PBS. Then the cells were resuspended in culture medium. Cells were treated CHX at 100 µg/ml for 1 min and collected, and 5 x 10 6 cells were lysed in lysis buffer (5 mM MgCl2, 10% Triton X-100, Tris-HCl pH 7.5, and 150 mM NaCl) supplemented with 1 U/µl DNase ?, 50 µg/ml CHX, and 100 mM dithiothreitol (DTT) for 30 minutes on ice. Cell lysates were centrifuged at 18,000 x g at 4 ? for 15 min and the supernatant was digested with RNase ?.Ribosome-protected RNA fragments (RPF) were obtained using MicroSpin S-400 column resin; the was column enriched for ribosome-mRNA complexes and the targeted RNA fragments were purified by PAGE. The purified RNA fragments were end-repaired and A-tailed, and 5' and 3' joints were directly added to the ends. RNA reverse transcriptase was used to synthesize cDNA, and additional steps of PCR enrichment and PAGE-based screening of target fragments were used to obtain a library containing target fragments. The library was verified by quality control screening and sequencing.