| Objective: Skeletal muscle is a terminally differentiated tissue,thus,lacks the ability to proliferate.It is maintained by repair and regeneration through a population of satellite cells.Differentiation of primitive myoblasts into mature myotubes requires a metabolic transformation to support the increased energetic demand,which depends on metabolic remodeling of mitochondria.Namely,increasing numbers of mitochondria involved in energy supply during the differentiation of skeletal myoblasts.The Mitochondrial unfolded protein response(UPRmt)is an adaptive transcriptional response that maintains mitochondrial protein balance and cell homeostasis.It also affects cell metabolism.These studies showed that UPRmt plays an important role in myoblast proliferation and differentiation.In this study,the induced differentiation model of C2C12 cells was established.The expression of UPRmt related molecules and the changes of metabolic patterns(glycolysis/aerobic metabolism)during differentiation progression were observed.To investigate the biological role of UPRmt in myogenic differentiation,ATF5 downregulation was induced by the small interfering RNA(si RNA)technique,followed by the induction of C2C12 cells.To observe changes in cell metabolism(glycolysis/aerobic metabolism)and to examine the effects on differentiation.Methods: C2C12 myoblasts were used as the experimental subjects.This study was a two-phase study.In the first phase,differentiation of C2C12 cells was induced and terminated at different time points(0,2,4,6 and 8 days).The experimental groups were 0D,2D,4D,6D and 8D,respectively.Changes in differentiation markers(MHC,MYOG),UPRmt-related molecules(ATF5,Clp P,HSP60,LONP1)and p-AMPK/AMPK expression were examined by WesternBlotting.Glycolysis assay kit was used to detect the change of cell glycolysis,Oxygraph-2K was used to detect the change of cell aerobic metabolism.In the second phase,the expression of ATF5 was reduced by means of si RNA,and the experimental groups were divided into two groups: the interference ATF5 group(si RNA-ATF5)and the control group,and the above indexes were also detected.Then,the data were analyzed using independent t test and one-way ANOVA.Results : Exp.1 Changes of UPRmt and metabolism during C2C12 cell differentiation(1)The Changes of cell metabolism during differentiationCompared with undifferentiated cells,RCR levels were significantly increased at8 days of differentiation(P<0.01);RCR levels were significantly decreased in 4-day differentiated cells compared with 2-day differentiated cells(P<0.05);RCR levels were significantly increased at 8 days of differentiation compared with 6 days of differentiation(P<0.01).Compared with undifferentiated cells,the maximum respiration rate of 8-day differentiated cells was significantly increased(P<0.05).The spare respiratory capacity of 4-day differentiated cells was also significantly increased compared with undifferentiated cells(P<0.05);The spare respiratory capacity of 4-day differentiated cells was also significantly higher than that of undifferentiated cells(P<0.05);Compared with undifferentiated cells,spare respiration levels were significantly increased at 8 days of differentiation(P<0.05).Glycolysis levels were significantly higher in 4-day differentiated cells than in undifferentiated cells(P<0.01);Glycolysis levels were significantly decreased at 6and 8 days of differentiation compared with 4 days of differentiation(P<0.01).(2)Changes in the expression of UPRmt-related molecular and proteins in during differentiationThe expression level of ATF5 protein was significantly higher in 4-day differentiated cells than in undifferentiated cells(P<0.05);The expression level of ATF5 protein was significantly decreased in 6-day differentiated cells compared with4-day differentiated cells(P<0.05);The expression level of ATF5 protein was significantly decreased in 8 day differentiated cells compared with 4 day differentiated cells(P<0.01).Compared with undifferentiated cells,Clp P protein expression levels were significantly increased in 4-day differentiated cells(P<0.01);Compared with undifferentiated cells,Clp P protein expression was significantly increased in 6-day differentiated cells(P<0.01);Compared with undifferentiated cells,Clp P protein expression levels were significantly increased at 8 days of differentiation(P<0.05).HSP60 protein expression level was significantly decreased in 4-day differentiated cells compared with 2-day differentiated cells(P<0.05);HSP60 protein expression was significantly increased in 6-day differentiated cells compared with 4-day differentiated cells(P<0.05);The expression of HSP60 protein was significantly decreased in the 8-day differentiated cells compared with the 6-day differentiated cells(P<0.01).Compared with undifferentiated cells,LONP1 protein expression was significantly increased in 4-day differentiated cells(P<0.05);Compared with 4-day cells,the expression level of LONP1 protein in 6-day cells was significantly increased(P<0.05).(3)Changes in p-AMPK/AMPK protein expression during differentiationp-AMPK protein expression was significantly decreased in 4-day differentiated cells compared with undifferentiated cells(P<0.01);p-AMPK protein expression was significantly decreased in 6-day differentiated cells compared with undifferentiated cells(P<0.05);p-AMPK protein expression was significantly decreased in 8-day differentiated cells compared with undifferentiated cells(P<0.01).The expression of AMPK protein in the 6-day differentiated cells was significantly higher than that in the 4-day differentiated cells.p-AMPK/AMPK protein expression was significantly decreased on day 8 compared with day 6 before differentiation(P<0.05).Effect of interfering ATF5 on expression of myogenic differentiation markers after induction of differentiation Exp.2 Effects of interfering ATF5-mediated UPRmt on myoblastic differentiation and metabolism(1)The effect of ATF5 interference on the expression of differentiation markers after induction of differentiationCompared with the control group,the expression of MHC protein in the interference ATF5 group was significantly decreased(P<0.05),there was no significant difference in MYOG protein expression level.(2)Effects of ATF5 interference on cell metabolism after induction of differentiationCompared with the control group,the level of RCR in the interference ATF5 group was significantly decreased(P<0.05);Compared with the control group,there was no significant difference in the maximum respiratory capacity of the interference ATF5 group.Compared with the control group,the spare respiratory capacity of the interference ATF5 group was significantly reduced(P<0.05);Compared with the control group,the coupling efficiency of interference ATF5 group decreased significantly(P<0.05).Compared with the control group,glycolysis level in the interference ATF5 group was significantly increased(P<0.01).(3)The effect of interference with ATF5 on the expression of UPRmt-related molecular and proteins after induction of differentiationCompared with the control group,the expression level of Clp P protein in the interference ATF5 group was significantly decreased(P<0.05);Compared with the control group,the expression level of LONP1 protein in the interference ATF5 group was significantly increased(P<0.05).(4)The effect of ATF5 interference on p-AMPK/AMPK protein expression after induction of differentiationCompared with the control group,the expression level of p-AMPK protein in the ATF5 interference group was significantly increased(P<0.01);Compared with the control group,there was no significant change in the expression level of AMPK protein in the interference ATF5 group.Compared with the control group,the expression level of p-AMPK/AMPK in the ATF5 interference group was significantly increased(P<0.05).Conclusion : 1.UPRmt was progressively activated with myogenic differentiation and a shift in cellular metabolism from glycolysis to aerobic metabolism.2.Interference with the activation of UPRmt by ATF5-mediated UPRmt,a necessary component of myodifferentiation,impaired myogenic differentiation. |
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