(P30-029-25) Loss of SHMT2 and Folate Deficiency Impair Protein Levels Involved in Oxidative Phosphorylation, Folate Transport, and Mitochondrial Dynamics in Mouse C2C12 Myoblast Cells

Objectives: Mitochondrial serine hydroxymethyltransferase 2 (SHMT2) is responsible for transferring a one-carbon unit from serine to tetrahydrofolate (THF), yielding glycine and 5-methylTHF. Previous cell models have demonstrated loss of SHMT2 and folate depletion impair mitochondrial respiration, however, no cell model of SHMT2 loss and folate depletion has focused on skeletal muscle or the causes of the mitochondrial dysfunction. Skeletal muscle is a highly oxidative tissue and may be sensitive to changes in SHMT2 expression and folate availability. The objective of this study is to investigate the impact of loss of SHMT2 and folate availability on mitochondrial function, dynamics, and folate transport in mouse C2C12 myoblasts.

Methods: Mouse C2C12 (wild-type) and SHMT2 knockout C2C12 myoblasts were cultured for 4 doublings in 0 nM (depleted) or 25 nM (sufficient) folate modified DMEM medium. Cells were examined for total folate levels, mitochondrial membrane potential, mitochondrial function, and protein levels related to oxidative phosphorylation, folate transport, and mitochondrial dynamics.

Results: Total folate levels were decreased in C2C12 cells exposed to folate-depleted medium. Loss of SHMT2 and folate depletion reduced mitochondrial membrane potential. There was a significant reduction in maximal and basal respiration, and ATP production in SHMT2 knockout cells compared to the wild-type cells. Loss of SHMT2 decreased NDUFB8 (Complex I) and MTCO1 (Complex IV) protein levels more than 60%, while folate depletion decreased SDHB (Complex II) protein levels by 70%. Folate receptor 1 and reduced folate carrier (RFC) protein levels were reduced in SHMT2 knockout cells, while folate depletion decreased RFC protein levels. Furthermore, mitochondrial dynamics proteins, dynamin-related protein 1 and mitofusion 2, increased approximately two-fold in SHMT2 knockout cells, compared to wild-type cells, while folate depletion decreased optic atrophy 1 protein levels.

Conclusions: Our results suggest impairments in oxidative phosphorylation from loss of SHMT2 may be attributed to changes in both nuclear and mitochondrial derived proteins and changes in folate transport in myoblast cells.

Funding Sources: N/A