Six-hour hypoxia induced protein degradation in M. gastrocnemius of 24-day-old mice by activating FOXO1 and suppressing AKT-mTORC1.

Abstract

Long-term hypoxia has been associated with skeletal muscle atrophy, including increased protein degradation over protein synthesis. This contrasts sharply with muscle hypertrophy and net protein synthesis occurring in developing skeletal muscle of young mice. Here, we aimed to understand the impact of acute, physiologically plausible environmental hypoxia on muscle proteostasis of the M. gastrocnemius of young mice. Fasted prepubertal, 24-day-old male B6JRccHsd(B6J)-Nnt⁺/Wuhap mice with similar body weight and lean mass were exposed to normobaric hypoxia (12% O₂) or normoxia (20.9% O₂) for 6 hours. The transcriptome (n=12) and protein (n=6) response of the M. gastrocnemius were analyzed. A hypoxic response of M. gastrocnemius was confirmed by increased expression of HIF1 (Ankrd37 and Ddit4) and forkhead box-O (FOXO) 1 (Depp1 and Ddit4) target genes. RNA-Seq analysis revealed that hypoxia activated FOXO signaling, which was confirmed by increased FOXO1 protein levels and decreased p-AKT/AKT ratio. Detailed mapping of the FOXO1 pathway suggests a strong FOXO1-mediated hypoxic effect on protein degradation and synthesis. A central role of Atf4 is suggested by the hypoxic-dependent positive correlations with FOXO1, FBXO32, Depp1, Eif4ebp1 and Ddit4. Further analyses showed increased FBXO32, which positively correlated with FOXO1, and decreased p-S6K/S6K and p-4E-BP1/4E-BP1 ratios. Our results showed for the first time that a 6-hour exposure to 12% O₂ normobaric hypoxia in 24-day-old mice activates FOXO1 signaling in M. gastrocnemius, resulting in decreased protein synthesis and increased protein degradation most likely via reduced energy availability, which may be relevant for infant air or high altitude traveling.