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

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.

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.

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.

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.