Study On The Mechanism Of Rotenone-induced Mitochondrial Damage And Inhibition Of FNDC5 Synthesis In Myoblast

Objective: Irisin is a muscle factor discovered in 2012.When muscle contracts,it enters tissues and organs of the whole body through endocrine,which has an important impact on the physiological activities and development of target tissues/organs.So far,hundreds of literatures have studied irisin’s measurement and linked it with diseases and physiological conditions,but there are few reports on the mechanism related to its formation.The synthesis of Irisin precursor FNDC5 is regulated by PGC-1α,which plays an important role in the formation of mitochondria.Mitochondria are essential organelles in all cells except a few mammalian cell types,and they have many functions.Among them,the important function is to produce ATP needed by cell activities and ROS as signal molecules to promote antioxidant capacity.ROS mainly passes through:1)Affect the activity of mitochondrial respiratory chain complex;2)The permeability of mitochondrial membrane is changed;3)Interfering with the transcription and translation of mt DNA;4)Direct attack on cellular biological macromolecules leads to the decrease of intracellular antioxidant enzyme levels and other aspects,resulting in mitochondrial damage.It has been reported that the content of Irisin skeletal muscle and serum of aging body decreased significantly,and it was positively correlated with the energy metabolism level of skeletal muscle mitochondria.However,it is not known whether the mitochondrial homeostasis affects the synthesis of FNDC5.Methods: In this study,firstly,C2C12 myoblasts were induced by rotenone.After24 hours of culture,their mitochondrial function was tested.The expressions of proteins related to energy synthesis pathway and oxidative stress pathway,PGC-1αand FNDC5 proteins were detected by Western Blotting.The influence mechanism of mitochondrial damage on FNDC5 synthesis was discussed from energy synthesis sensitive pathway and oxidative stress sensitive pathway.Subsequently,the expression of PGC-1α and FNDC5 protesi RNA was detected by Western Blotting,and the role of ATF5 in the synthesis of FNDC5 was explored.Finally,C2C12 myoblasts were transfected with ATF5 overexpression plasmid.Results:(1)Compared with the control group,the expression of FNDC5,SIRT1 and p38MAPK in C2C12 myoblasts was significantly decreased after 24 hours of incubation with rotenone with the concentration of 1μM(p<0.01);PGC-1α,GCN5,ATF5 and HSP60 were significantly decreased(p<0.05).NF-κB increased significantly(p<0.05).(2)Compared with the control group,C2C12 myoblasts transfected with si RNA interfered with ATF5 display,and PGC-1α and FNDC5 decreased significantly(p<0.05);(3)After the overexpression of ATF5,C2C12 myoblasts were incubated with 1μM rotenone.Compared with group C,the expression of PGC-1α in ROT group decreased significantly(p<0.01).Compared with ATF5-OE+ROT group,the expression of PGC-1α decreased significantly(p<0.05).Compared with ROT group,the expression of PGC-1α in ATF5-OE+ROT group decreased,but there was no significant difference.Compared with group C,the expression of FNDC5 in ROT group decreased significantly(p<0.01).Compared with ATF5-OE+ROT group,the expression of PGC-1α decreased,but there was no significant difference.Compared with ROT group,the expression of FNDC5 in ATF5-OE+ROT group also decreased,but there was no significant difference.Research conclusions:(1)Rotenone can increase ROS,inhibit p38 MAPK,activate NF-κB,then inhibit PGC-1α,and then reduce the expression of FNDC5.(2)Rotenone decreased the mitochondrial membrane potential by SIRT1 and GCN5,which further inhibited PGC-1α and decreased the expression of FNDC5.(3)Overexpression of ATF5 can resist the decrease of PGC-1α and FNDC5 expression induced by rotenone through UPRmt.(4)Interfering with ATF5 can partially inhibit the expression of PGC-1α and FNDC5,suggesting that ATF5 mediates PGC-1α through UPRmt,and then regulates FNDC5.