Repeated Disuse Atrophy Imprints a Molecular Memory in Skeletal Muscle: Transcriptional Resilience in Young Adults and Susceptibility in Aged Muscle

Disuse-induced muscle atrophy commonly occurs following illness, injury, or falls and becomes increasingly frequent with ageing. Whether skeletal muscle retains a "memory" of repeated disuse remains unknown. We investigated repeated lower-limb immobilization in young adults and a refined aged rat model, integrating physiological, multi-omic, immunohistochemical, biochemical, and primary human muscle stem cell (MuSC) analyses. To enable robust age comparisons, we integrated previously published young rat data with newly generated aged rat data. In young human muscle, repeated disuse elicited attenuated transcriptional perturbations in oxidative and mitochondrial pathways, suggestive of a protective molecular memory, despite similar atrophy to initial disuse. In contrast, aged muscle exhibited a detrimental memory, characterized by greater atrophy, exaggerated suppression of aerobic metabolism genes despite recovery after initial disuse, NAD⁺ and mitochondrial DNA depletion, and activation of proteasomal, extracellular-matrix, and DNA-damage pathways. Whereas young rats recovered muscle mass after initial disuse, aged rats failed to do so. Across species, repeated disuse induced DNA hypermethylation and downregulation of aerobic metabolism and mitochondrial gene networks. NR4A1 and NR4A3 were among the strongest disuse-suppressed genes; NR4A1 acquired recovery-phase hypermethylation that maintained its transcriptional repression, while NR4A3 was the most downregulated gene after initial atrophy and remained persistently suppressed into recovery. Acetylcholine receptor subunit genes (CHRNA1, CHRND) were epigenetically primed, demonstrating hypomethylation and strong upregulation after disuse, and further amplification after repeated atrophy, while CHRNG was selectively induced after repeated atrophy only. NMRK2, an NAD⁺ biosynthesis gene, was the most downregulated gene across both atrophy periods, and supplementation with its substrate, nicotinamide riboside (NR), improved myotube size in MuSCs derived post-atrophy. Overall, repeated disuse atrophy imprints a molecular memory in skeletal muscle shaping transcriptional resilience in young adults and exaggerated susceptibility in aged muscle.