Methylome-transcriptome integration reveals molecular signatures of intrinsic aerobic capacity in aged skeletal muscle.

BACKGROUND: Intrinsic aerobic capacity is a critical determinant of metabolic health and healthy aging, yet its epigenomic and transcriptomic features in aged skeletal muscle, and whether these intrinsic differences are accompanied by distinct exercise-related molecular responses, remain unclear. METHODS: Soleus muscle from aged selectively bred high- and low-running capacity rats (HCR and LCR; 23-24 months) was profiled by reduced representation bisulfite sequencing (RRBS) and RNA sequencing (RNA-seq). Differentially methylated regions (DMRs) were annotated, functionally enriched, integrated with differentially expressed genes (DEGs), and correlated with maximal oxygen uptake (VO₂max). An age-comparable voluntary-running cohort was further analyzed by RNA-seq and selected protein profiling to assess exercise adaptation. RESULTS: RRBS identified broad baseline methylome remodeling, including 7196 significant DMRs, with a higher proportion of hypermethylated regions in HCR muscle. These DMRs were mainly localized to open-sea CpGs, while gene-associated DMRs were predominantly intronic and exonic, and showed context-dependent functional enrichment. Baseline RNA-seq identified 322 DEGs between HCR and LCR muscle. Methylome-transcriptome integration revealed 72 DMR-DEG pairs representing 53 unique genes, mostly located in open-sea, intronic, and exonic regions. Exploratory VO2max analysis identified 63 DMRs associated with aerobic capacity. Chronic voluntary running induced more DEGs in HCR than in LCR muscle, while pathway-level responses in both lines converged on mitochondrial and oxidative metabolism. Selected protein profiling further revealed mainly baseline LCR-HCR differences, with limited additional exercise-associated changes. CONCLUSION: Intrinsic high aerobic capacity in aged skeletal muscle is associated with hypermethylation-enriched methylome remodeling, feature-dependent functional enrichment, distinct transcriptional signatures, and oxidative-metabolic protein differences. These findings provide a multi-layer molecular framework for understanding intrinsic aerobic-capacity divergence in aged skeletal muscle and highlight candidate regulatory regions for future functional validation.