Lactate Is a Metabolic–Epigenetic Signal That Links HIIT to miRNA-Centered Remodeling of the Skeletal-Muscle Methylome and Transcriptome

Lactate, a major exercise-derived metabolite, has been proposed to couple cellular metabolism to gene regulation, yet its direct impact on skeletal muscle remains unclear. Male mice underwent six-week interventions (Control, lactate, HIIT, MCT1/2 inhibition, or HIIT+inhibition). We profiled gastrocnemius DNA methylation, mRNA-seq and miRNA-seq, and assessed signaling proteins, metabolites, and running performance. Lactate and HIIT each induced broad, site-specific remodeling of the methylome and transcriptome with substantial overlap at promoter CpGs and among DEGs. Promoter methylation changes showed weak coupling to steady-state mRNA, whereas integrative analyses revealed robust anti-directional miRNA–mRNA networks and enriched for chromatin/epigenetic regulators, identifying a lactate-driven miRNA axis as a proximate regulator of transcriptional output. At the protein level, lactate selectively increased TET2 and DNMT3A and activated PAX7, VEGF, and AKT–S6 signaling, consistent with integrative miRNA–mRNA findings. HIIT increased TET1/2 and DNMT3A with DNMT3B reduction and uniquely enhanced mitochondrial/antioxidant signaling. Blocking MCT1/2 abrogated HIIT-induced methylome and miRNA remodeling and blunted transcriptomic and protein adaptation, demonstrating that intact lactate flux is required for exercise-evoked epigenetic and transcriptomic reprogramming. Despite molecular convergence, chronic lactate did not improve running performance, indicating that lactate is necessary but not sufficient for the full physiological adaptation of training. These findings position a lactate–miRNA axis linking metabolic perturbation to epigenetic and transcriptional remodeling in skeletal muscle. Overall design: Small RNA sequencing of gastrocnemius muscle from male BALB/c mice (~2 months old) after 6-week interventions. Groups: Control (PBS), Lactate administration (500 mg/kg i.p.), High-intensity interval training (HIIT, treadmill protocol), MCT1/2 inhibitor (AR-C155858, i.p.), and HIIT + inhibitor.