Sestrin2 is Induced Upon Cellular Stress but Has No Effect on Myotube Size or Amino Acid Sensing in C2C12 Myotubes

Sestrins are a stress-inducible family of proteins that function in cell survival and nutrient sensing through their regulation of mTORC1. Muscle wasting is associated with cellular stress, but to date, there is limited in vitro research investigating sestrins in skeletal muscle cells. Here we use C2C12 myotubes to understand how sestrin proteins (sestrin 1-3) are regulated by different forms of cellular stress linked to muscle wasting conditions. Furthermore, since sestrin2 is a well-characterised protein but is lowly expressed in muscle tissue in the absence of stress, we also aimed to determine if silencing this protein impacted parameters of muscle growth or nutrient sensing by mTORC1 under basal conditions. Incubating C2C12 myotubes with the endoplasmic reticulum (ER) stress-inducing agent tunicamycin, or a high concentration (1000 µM) of hydrogen peroxide (H₂O₂), increased sestrin2 protein levels with no change in sestrins 1 or 3. This increase was temporally associated with increased ER stress markers Ddit3 mRNA and ATF4 protein levels, and could be blocked by approximately half when myotubes were co-incubated with H₂O₂ and the ER-stress inhibitor 4-Phenylbutyrate. siRNA silencing of sestrin2 blunted the phosphorylation of the mTORC1 effector S6K1, but did not acutely influence protein synthesis or myotube size. Similarly, silencing sestrin2 did not affect mTORC1 signalling in response to nutrient deprivation. These data indicate that sestrin2 is stress-inducible and may play a role in protecting skeletal muscle from ER stress, but is less important in regulating mTORC1 and nutrient sensing in unstressed/basal conditions.