Androgen receptor regulates the differentiation of myoblasts under cyclic mechanical stretch and its upstream and downstream signals

Our previous studies have demonstrated the important roles of androgen receptor (AR) in myoblast proliferation regulated by 15 % (mimic appropriate exercise) and 20 % (mimic excessive exercise) mechanical stretches. Except for myoblast proliferation, differentiation is also an important factor affecting muscle mass and strength. But the role of AR in stretch-regulated myoblast differentiation and AR's upstream and downstream signals remain unknown. In the present study, firstly the differences of myogenic differentiation between C2C12 (with AR expression) and L6 (without AR expression) myoblasts induced by 15 % and 20 % mechanical stretches were compared; secondly, AR antagonist flutamide and AR agonist GTx-007 were used in 15 % and 20 % stretched myoblasts respectively to confirm AR's roles in stretch-regulated myoblast differentiation; thirdly, RNA-seq, molecular dynamic simulation (MD) and co-immunoprecipitation were performed to screen the downstream and upstream molecules of AR during stretches. We found that (1) 15 % stretch increased while 20 % stretch decreased myotube number in differentiating C2C12 and L6 myoblasts, with more significant changes in C2C12 cells than L6 cells; (2) in stretched C2C12 myoblasts, AR antagonist flutamide inhibited 15 % stretch-promoted differentiation while AR agonist GTx-007 reversed 20 % stretch-inhibited differentiation (reflected by changes in myotube number, MHC contents of fast-twitch and slow-twitch fiber, and the levels of myogenic regulatory factors (MRFs) such as MyoD and myogenin); (3) KEGG analysis of RNA-seq showed that the differently expressed genes (DEGs) in C2C12 cells induced by 15 % stretch were enriched in FoxO and JAK-STAT signaling pathways, while DEGs by 20 % stretch were enriched in FoxO and MAPK signaling pathways; (4) MD and co-immunoprecipitation showed that β1 integrin could interact with AR and influence AR's activity in C2C12 cells. In conclusion, AR plays important roles in myoblast differentiation promoted by 15 % stretch while inhibited by 20 % stretch, which was fulfilled through FoxO-MRFs. In addition, α7β1 integrin may be a bridge linking mechanical stretch and AR. This study is beneficial to deeply understand the roles and mechanisms of AR in stretch-regulated muscle mass and strength; and reports firstly that myoblasts sense mechanical stimulus and transmit into intracellular AR via α7β1 integrin.