(OR17-05-26) Methylmalonyl-CoA Mutase and Protein Succinylation in Skeletal Muscle Myogenesis

Objectives: Nutrient dynamics play a critical role in skeletal muscle regeneration (i.e., myogenesis). Evidence from our lab suggests that anaplerosis of substrates into the TCA cycle impact succinyl-CoA and subsequent succinylation as well as cell state transition during myogenesis. Methylmalonyl-CoA mutase (MUT), which metabolizes methylmalonyl-CoA to succinyl-CoA, is dynamically expressed during myogenesis; however, the function of MUT and protein succinylation in myogenic cell states is unknown. This study was designed to address two hypotheses: (1) MUT is involved in regulating nutrient dynamics during myogenesis, and (2) post-translational modifications (PTMs), specifically succinylation, impact anaplerosis of TCA cycle metabolites.

Methods: Using immortalized C2C12 muscle progenitor cells (MPCs), cultured in growth (proliferation) or differentiation media (days 1, 3, and 6 of differentiation), we measured the media and cell pellets for methylmalonic acid (MMA, indirect measurement of MUT activity); identified changes in the succinylation profile and proteome using succinyl and untargeted proteomics, respectively; and identified branched-chain amino acid (BCAA) carbons in TCA cycle intermediates using stable isotope tracing. Key findings are validated in primary MPCs and with immunoblot.

Results: We identified significantly higher levels of extracellular (media) MMA at late differentiation (vs. proliferation), suggesting impaired MUT activity with differentiation. This observation corresponded with an overall decrease in protein succinylation with MPC differentiation. Differentially succinylated proteins were enriched in BCAA catabolism pathways, with differentiation. We demonstrated increased protein levels for BCAA (isoleucine and valine) catabolic enzymes with differentiation; however, stable isotope tracing revealed no carbon enrichment in TCA cycle intermediates from isoleucine and valine catabolism.

Conclusions: Our findings suggest that dynamic changes in MUT activity correspond with myogenesis cell state changes and protein succinylation. Whether MUT activity or its byproduct, MMA, directly regulate MPC state will be examined next, recognizing the importance of vitamin B12 (cofactor for MUT) for skeletal muscle biology.

Funding Sources: CDIB