Multicellular senescence impairs skeletal muscle recovery following disuse in aging

Aged skeletal muscle has a diminished capacity to recover after disuse. Although muscle regrowth requires coordinated interactions between immune and progenitor cells, the mechanisms of impaired remodeling in aged skeletal muscle remain poorly understood yet possibly involve the accumulation of senescent cells. We used a flow cytometry approach coupled with scRNAseq to determine the muscle senescent cell identity and transcriptional landscape during skeletal muscle recovery following disuse atrophy. Young and aged mice underwent 14 days of hindlimb unloading followed by reloading (7 or 14 days). At recovery, old mice showed smaller myofibers and abnormal muscle macrophage dynamics corresponding to greater collagen content. These outcomes coincided with elevated markers of muscle senescence (p21 and γH2AX) and increased SPiDER-β-Gal⁺ cells, which inversely correlated with muscle mass. Single-cell resolution of SPiDER⁺ cells unmasked several senescent interstitial muscle vascular and stromal populations. Senescent interstitial cell populations were enriched in aged muscle and displayed a senescence-associated secretory phenotype (SASP) across multiple stromal, vascular, and immune cell types. Senolytic treatment reduced overall senescent cell burden, attenuated macrophage accumulation, and restored muscle mass and function in aged mice following disuse. These findings identify a multicellular senescence environment within the muscle interstitial niche as a hallmark of impaired muscle recovery following disuse.