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

Disuse-induced muscle atrophy is common after 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 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, characterised 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 did not. Repeated disuse induced DNA hypermethylation and downregulation of aerobic metabolism and mitochondrial genes across species. NR4A1 remained suppressed into recovery, while AChR subunit genes (CHRNA1, CHRND) were epigenetically primed. NMRK2, an NAD biosynthesis gene, was among the most downregulated after both atrophy periods, and nicotinamide riboside improved myotube size in MuSCs post-atrophy. Repeated disuse atrophy imprints a molecular memory in skeletal muscle shaping transcriptional resilience in young adults and exaggerated susceptibility in aged muscle.