ATF3 as a molecular nexus linking ferroptosis regulation to sarcopenia pathogenesis via PI3K/Akt pathway activation

PURPOSE: Sarcopenia, characterized by progressive skeletal muscle loss and weakness, has unclear pathogenesis and lacks targeted therapies. Emerging evidence implicates ferroptosis in sarcopenia progression, though its regulatory mechanisms remain undefined. This study investigates the ferroptosis-sarcopenia interplay, identifies core regulators and elucidates their molecular basis. METHODS: The experimental design combined in vivo and in vitro approaches using SAMP8 mice and C2C12 myoblast. Sarcopenia phenotypes were systematically characterized through functional assessments of mice, histomorphological analysis of gastrocnemius muscle, and quantification of iron deposition. Bioinformatics cross-analysis was performed by intersecting the sarcopenia-related gene expression dataset (GSE175495) with ferroptosis-associated genes from the FerrDb database, identifying ATF3 as a hub gene. Validation was conducted through Western blot (WB) and quantitative real-time PCR (qPCR). For mechanistic exploration, ferroptosis was induced in C2C12 cells using ferric ammonium citrate (FAC, 500 μM, 48 h), followed by lentivirus-mediated ATF3 overexpression. The regulatory role of ATF3 in ferroptosis was assessed via reactive oxygen species (ROS) assay, malondialdehyde (MDA) quantification, and FerroOrange fluorescent probe for intracellular iron detection. Transcriptome sequencing of ATF3-dysregulated cell lines was performed, and GO/ KEGG enrichment analyses were applied to identify critical signaling pathways. Functional validation was further conducted using pathway-specific inhibitors. RESULTS: Aged SAMP8 mice exhibited hallmark sarcopenia characteristics including 31 % reduction in grip strength, 42 % decrease in muscle fiber cross-sectional area, and 2.1-fold elevation in intramuscular iron content. Molecular analysis revealed age-dependent downregulation of ATF3 expression (57 % protein decrease, 63 % mRNA reduction). ATF3 overexpression in C2C12 cells significantly attenuated ferroptosis, evidenced by 45-52 % reductions in ROS/MDA levels and reversal of atrophy-related protein expression. Transcriptomic profiling identified 773 differentially expressed genes functionally enriched in PI3K/Akt signaling, with ATF3 overexpression inducing 3.2-fold activation of p-Akt. Crucially, pharmacological PI3K inhibition completely abolished ATF3-mediated ferroptosis suppression, establishing pathway dependency. CONCLUSION: These findings demonstrate that ATF3 serves as a critical molecular nexus linking ferroptosis regulation to sarcopenia pathogenesis through PI3K/Akt pathway activation.