(P22-028-25) Essential Amino Acid Regulation of tRNA Synthetases to Support Skeletal Muscle Protein Synthesis With Age

Objectives: Skeletal muscle (SM) is vital for both long term health and quality of life. Although results from in vitro studies model how essential amino acids (EAA) can regulate muscle protein synthesis (MPS), the relevance of these models to muscle protein breakdown (MPB), and the presence of physiological plasma amino acid concentrations (PAA) remain to be established. Therefore, the objective of this study was to determine the effects of EAA with (0.2, 1.0, and 3.0 x PAA) in a young (passages 2-10) and aging (passages 16-24) C2C12 murine muscle cell model. We hypothesized that increased levels of EAA will regulate MPS through tRNA synthesis in aging cells and suppress MPB via the mTORC1 pathway when compared to young cells.

Methods: Myoblasts were seeded (1x10⁵) into 6-well plates and differentiated into myotubes when they reached 80% confluency. Myotubes were serum and amino acid (AA) starved for 24 hours before receiving one of the following treatments: control (CON), 0.2 x PAA, 1.0 x PAA, 3.0 x PAA with or without rapamycin (100 nm; RAP) for 24 hours. All treatments were performed in triplicate and each experiment was repeated three times, yielding nine wells per treatment. Proteins and genes related to sensing of AA, tRNA synthesis, and protein synthesis and degradation were assessed using Western Blot and real-time PCR. Data was analyzed using one-way ANOVA (p < 0.05 was considered significant).

Results: There was no effect of cell age, AA concentration, or rapamycin on tRNA synthetase genes (WARS, MARS, RARS, FARBS, IARS, HARS, VARS, KARS, TARS, LARS). There was also no effect of AA treatment on phosphorylation of 4EBP1 and p70S6K1 after 24 hours. There was no change in gene expression of MURF1 with age, AA concentration, or rapamycin. However, GCN2 expression was higher (p < 0.05) in old versus young cells, with no effect of AA concentration. MAFbx expression was lower (p < 0.05) in old versus young cells, with no effect of AA concentration or rapamycin. Amino acid transporter LAT1 was lower (p < 0.05) in old versus young cells and was inhibited (p < 0.05) with rapamycin.

Conclusions: While age had a significant impact on the expression of GCN2, MAFbx, and LAT1, there were no notable effects of AA concentration or rapamycin treatment on the examined genes or phosphorylation markers.

Funding Sources: Arkansas Biosciences Institute