High-Intensity Interval Training Combined with Glycine Supplementation Drives Ferroptosis Resistance to Counteract Skeletal Muscle Atrophy in Aging Mice

ABSTRACT Objective To investigate the molecular mechanisms underlying the improvement of aged skeletal muscle atrophy by High-intensity Interval Training (HIIT) combined with glycine supplementation. Methods Male C57BL/6 J mice aged 19 months (n = 16) were randomly assigned to Old Sedentary (OSED), HIIT, OSED+Glycine (O-Gly), and HIIT+Glycine (H-Gly) groups for an 8-week intervention. Maximum grip strength and running speed were assessed. Myocyte apoptosis was detected by TUNEL staining; myofiber cross-sectional area was measured by laminin staining; reactive oxygen species (ROS) in myocytes were detected by dihydroethidium staining. Western blot and RT-qPCR were used to measure protein and gene expression levels related to senescence, apoptosis, and ferroptosis in myocytes. Chemical methods were employed to detect changes in Malondialdehyde (MDA), Lipid peroxide (LPO), glutathione (GSH), Glutathione-oxidized (GSSG), and total glutathione (T-GSH) contents. RNA-seq technology was utilized to screen for key differentially expressed genes. AutoDockTools software was used for molecular docking predictions between glycine and key differential proteins. Results H-Gly group mice showed an improved maximum grip strength and muscle fiber cross-sectional area, with a significant reduction in TUNEL-positive cells. RNA-seq analysis revealed a high correlation between ferroptosis pathway genes and Slc25a25 ion transport-related genes, which was further validated by the detection of ferroptosis-related markers. Molecular docking indicated that glycine has binding sites with Slc25a25, with the highest binding energy of -3.7 kcal/mol. Conclusions Glycine supplementation has a significant synergistic effect with HIIT in increasing muscle mass and grip strength in aged muscle. The mechanism might be associated with the decrease of Slc25a25-mediated ferroptosis.