Repeated Disuse Atrophy in Aged Rat Skeletal Muscle (Reduced Representation Enzymatic Seq / Methylome)

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. Overall design: Ethical approval was obtained, and experimental procedures were conducted with the permissions within a project license granted (PA693D221, Jonathan Jarvis, Liverpool John Moores University, UK) under the British Home Office Animals (Scientific Procedures) Act 1986. Aged (23 ± 1 months) male Fischer 344 rats (Charles River) weighing 354 ± 54 g were housed at 20°C and 45% relative humidity on an alternating 12 hr light and 12 hr dark cycle, with food and water available ad libitum. Animals were assigned to 1 of 4 groups (n = 3 per group) including a control group (sham control) and 3 experimental groups (atrophy, recovery and repeated atrophy). All experimental groups received tetrodotoxin (TTX) to the common peroneal nerve (CPN), preventing contraction of the ankle dorsiflexor muscles. TTX binds to the voltage-gated sodium channel in the nerve cell membranes and so prevents the conduction of action potentials. Therefore muscles innervated by the blocked CPN cannot be activated in order to contract. This induces disuse atrophy of the hind limb dorsiflexor muscles i.e., the tibialis anterior (TA) and extensor digitorum longus (EDL), and is advantageous vs. denervation or hindlimb unloading as it allows normal conduction in the tibial nerve to achieve plantarflexion, and therefore animals can undertake locomotion for daily living. The atrophy and recovery groups received a single dose of TTX (6-7 d) followed by 9 d of TTX abstinence to recover (e.g., TTX and recovery), whereas the repeated atrophy group received a second TTX dose (5-6 d) following recovery (e.g., TTX - recovery - repeated TTX). The sham control group consisted of n = 1 for each timepoint (TTX/recovery/repeated TTX) that received the same surgical procedures as the experimental groups but in the absence of TTX administration. Note: Samples 364_L and 364_R were identified as PCA outliers after initial sequencing, despite DNA quantity and quality being comparable to other samples. These were re-sequenced (364_L_rpt, 364_R_rpt) along with two PCA-clustered samples from the original run (370_R, 369_L; re-sequenced as 370_R_rpt, 369_L_rpt). Resequencing yielded consistent performance, so analyses were pooled, effectively increasing technical replicates. Differential methylation analyses, as reported in https://doi.org/10.1101/2025.10.16.681134, were performed for each pairwise comparison on (i) the original sequencing data and (ii) combined original and resequenced replicates. Both approaches identified largely overlapping significant differentially methylated regions (DMRs; FDR = 0.05), with a small number of unique yet significant DMRs in each. Pooling DMRs from (i) and (ii) was therefore justified to maximize sensitivity and capture both shared and unique relevant regions, while maintaining rigor since all DMRs met stringent significance thresholds. This approach reduces false negatives, improves confidence in true signals, and ensures that potentially meaningful loci are not excluded from downstream discovery analyses.