The effect of leucine supplementation on extracellular matrix remodeling in aged male mice at the onset of muscle regeneration

Skeletal muscle regeneration is a crucial biological process that restores muscle tissue following injury. However, aging significantly diminishes the capacity for muscle repair, leading to delayed recovery in older individuals. This diminished regenerative capacity is associated with dysregulation of the extracellular matrix (ECM), characterized by increased expression of genes related to collagen deposition. Leucine, a branched-chain amino acid, has shown potential in enhancing muscle regeneration. However, its specific effects on ECM remodeling during skeletal muscle regeneration in aged mice remain underexplored. Our previous work demonstrated that muscle injury, independent of leucine supplementation, induces ECM remodeling in aged female mice. We hypothesize that leucine supplementation will attenuate gene expression leading to ECM remodeling in aged male mice during the early phases of muscle regeneration. Experiments were conducted exclusively on aged male C57/BL6 mice (24 months old), which were divided into four groups: uninjured without leucine (UNL), uninjured with leucine (UL), injured without leucine (INL), and injured with leucine (IL). Muscle injury was induced in the tibialis anterior (TA) muscles using bupivacaine injections, while uninjured groups received phosphate-buffered saline (PBS). Leucine supplementation (1.5%) was administered ad libitum via drinking water. TA muscles were harvested three days post-injection, and quantitative PCR was performed to assess gene expression changes. Data was analyzed by two-way ANOVA and significance was established at p < 0.05. There were no differences in body mass between UNL, INL, UL, and IL (p>0.05). There was no difference in the TA mass across the 4 groups (46.2 mg ± 2.6, 51.7 mg ± 1.7, 51.5 mg ± 1.2, 49.2 mg ± 1.0) (p>0.05). There was a main effect of injury to increase fibronectin and collagen-3 mRNA abundance ~9-10 fold (p < 0.05) independent of leucine supplementation. There was no significant change in the MMP9 mRNA abundance (p>0.05). In conclusion, leucine was insufficient to alter gene expression to remodel the ECM at the onset of muscle regeneration in aged male mice. Injury significantly increased the mRNA abundance of fibronectin and collagen, independent of leucine supplementation. These findings highlight the persistent role of injury in driving ECM remodeling and suggest that leucine alone may not be sufficient to counteract age-associated dysregulation of ECM dynamics during muscle regeneration. Further research could explore combinatorial approaches or later time points in the regeneration process. This study was funded by the Arkansas Biological Institute, the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.