microRNA-501 controls myogenin⁺/CD74⁺ myogenic progenitor cells during muscle regeneration

Objective: Skeletal muscle regeneration is markedly impaired during aging. How adult muscle stem cells contribute to this decrease in regenerative capacity is incompletely understood. We investigated mechanisms of age-related changes in myogenic progenitor cells using the tissue-specific microRNA 501.

Methods: Young and old C57Bl/6 mice were used (3 months or 24 months of age, respectively) with or without global or tissue-specific genetic deletion of miR-501. Muscle regeneration was induced using intramuscular cardiotoxin injection or treadmill exercise and analysed using single cell and bulk RNA sequencing, qRT-PCR and immunofluorescence. Muscle fiber damage was assessed with Evan`s blue dye (EBD). In vitro analysis was performed in primary muscle cells obtained from mice and humans.

Results: Single cell sequencing revealed myogenic progenitor cells in miR-501 knockout mice at day 6 after muscle injury that are characterized by high levels of myogenin and CD74. In control mice these cells were less in number and already downregulated after day 3 of muscle injury. Muscle from knockout mice had reduced myofiber size and reduced myofiber resilience to injury and exercise. miR-501 elicits this effect by regulating sarcomeric gene expression through its target gene estrogen-related receptor gamma (Esrrg). Importantly, in aged skeletal muscle where miR-501 was significantly downregulated and its target Esrrg significantly upregulated, the number of myog⁺/CD74⁺ cells during regeneration was upregulated to similar levels as observed in 501 knockout mice. Moreover, myog⁺/CD74⁺-aged skeletal muscle exhibited a similar decrease in the size of newly formed myofibers and increased number of necrotic myofibers after injury as observed in mice lacking miR-501.

Conclusions: miR-501 and Esrrg are regulated in muscle with decreased regenerative capacity and loss of miR-501 is permissive to the appearance of CD74⁺ myogenic progenitors. Our data uncover a novel link between the metabolic transcription factor Esrrg and sarcomere formation and demonstrate that stem cell heterogeneity in skeletal muscle during aging is under miRNA control. Targeting Esrrg or myog⁺/CD74⁺ progenitor cells might improve fiber size and myofiber resilience to exercise in aged skeletal muscle.