Angiotensin-Ⅱ-induced Muscle Wasting Is Mediated By 25-Hydroxycholesterol Via GSK3β Signaling Pathway

Objectives:While angiotensin II(ang II)has been implicated in the pathogenesis of cardiac cachexia(CC),the molecules that mediate ang II’s wasting effect have not been identified.It is known that TNF-α level is increased in patients with CC,and TNF-α release is triggered by ang II.Impaired IGF-1 signaling is a common feature in several wasting conditions.Therefore,our study focuses on the role of TNF-αand the key proteins in IGF-1 signaling pathway in the muscle atrophy induced by ang II.Methods:To address whether TNF-α is a mediator of angⅡ induced muscle wasting,we infused wild type C57BL/6 or TNFR1 KO mice with 500ng/kg/min angⅡ or vehicle for 7 days.To understand why loss of muscle mass was blunted in TNFR1 KO mice,we examined key proteins from gastrocnemius involved in IGF-1 signaling pathway,ubiquitin-proteasome pathway and apoptosis.To explore the mechanisms underlying ang II’s wasting effect,we studied the impact of ang II treatment on TNF-α expression using a myoblast line C2C12.We performed microarray analysis in order to identify genes under TNF-α regulation in C2C12 cells cultured in 10%FBS and 2% horse serum.To define the relationship between TNF-α and Ch25 h,we transfected C2C12 cells with a lentivirus over-expressing TNF-α.We then constructed a plasmid over-expressing Ch25 h in order to investigate the effect of Ch25 h expression on muscle wasting.We injected 25-OHC,22-OHC and 20-OHC to C57BL/6 mice to ask if these three sterols could induce muscle wasting.We also examined key proteins from gastrocnemius involved in IGF-1 signaling pathway,ubiquitin-proteasome pathway and apoptosis.To further confirm the role of Ch25 h in ang II induced muscle loss,we injected lentivirus that overexpress 25-hydroxylase gene into TA muscle of C57BL/6 mice.To determine if a therapeutic strategy might be developed to interfere with muscle wasting when GSK3β is activated,we evaluated TDZD-8,a highly selective small molecule inhibitor of GSK3β that binds to the kinase site of GSK3β.Mice were injected with either TDZD-8 or DMSO,and randomly divided into ang II infusion or pair-fed control groups.Results : 1 、 Angiotensin-Ⅱ-induced muscle wasting is mediated by TNF-α signaling pathway.Our data showed that in C57BL/6 mice,ang II infusion led to reduced body weight,grip strength,muscle weight and cross sectional area(CSA)of myofibers,accompanied by elevated serum levels of TNF-α.However,ang II induced body weight reduction and decreased grip strength and muscle loss were prevented in TNFR1 KO mice despite the increase of TNF-α in the sera of the TNFR1 KO mice.Western blot analysis shows that levels of p-Akt and p-GSK3β were decreased in gastrocnemius muscles from ang II infused wild type C57BL/6,but not from TNFR1 KO mice.We next analyzed expression of atrogin-1 and MuRF1,which are ubiquitin ligases implicated in muscle wasting.Western blot results showed increased atrogin-1 and MuRF1 expression in the gastrocnemius muscle of C57BL6,but not in TNFR1 KO mice.These findings suggest that ang II induced reduction of p-Akt and p-GSK3β is mediated by TNF-α.Furthmore these data suggest that ang II induced activation of ubiquitin proteasome pathway is mediated by TNF-α signaling via TNFR1.The expression of cleaved caspase-3 is increased in muscles from ang II infused C57BL/6 mice,but not from TNFR1 KO mice.These results suggest that TNF-α is the mediator of ang II induced apoptosis in skeletal muscle.2、ang II treatment resulted in increased expression of TNF-α,reduced expression of MyoD and inhibition of myogenic differentiation of C2C12 cells.In response to TNF-α treatment,cholesterol 25-hydroxylase(Ch25h)was upregulated 35.6-and 22.2-fold,in C2C12 cells cultured in 10% FBS and 2% horse serum,respectively.Overexpression of TNF-α in C2C12 cells led to inhibition of myogenic differentiation,associated with a significant increase of Ch25 h mRNA expression.The increased mRNA level of Ch25 h in C2C12 cells transfected with Ch25 h plasmid was confirmed by real-time PCR.Over-expression of Ch25 h in C2C12 cells resulted in inhibition of myogenic differentiation and decreased expression of MyoD.Furthermore,the mRNA expression levels of atrogin-1 and MuRF1 are increased in C2C12 cells over-expressing Ch25 h.3、Our data showed that 25-OHC,but not 22-OHC,20-OHC could induce muscle wasting.Western blot analysis revealed the levels of pAkt,pGSK3β,p-BAD were decreased,whereas levels of cleaved caspase-3,atrogin-1,MuRF1 were increased in gastrocnemius muscles from mice received 25-OHC,but not from mice that received 20-OHC,22-OHC or control treatment.Intramuscular injection of lentivirus that overexpress 25-hydroxylase gene into TA muscle of C57BL/6 mice induces muscle atrophy.4、Mice were injected with either TDZD-8 or DMSO,and randomly divided into ang II infusion or pair-fed control groups.After 7 days,both body weights and muscle weight were reduced in ang II infused and DMSO treated mice compared to pair-fed control groups.By contrast,both body weights and muscle weight were preserved in TDZD-8 treated group.Western blot analysis showed that ang II induced upregulation of atrogin-1,MuRF1 and caspase-3 cleavage was prevented by TDZD-8.Conclusions:Ang II infusion led to skeletal muscle wasting in wild type(WT)but not in TNF alpha type 1 receptor knockout(TNFR1KO)mice,suggesting that ang II induced muscle loss is mediated by TNF-α through its type 1 receptor.Microarray analysis identified cholesterol 25-hydroxylase(Ch25h)as the down stream target of TNF-α.Intraperitoneal injection of 25-hydroxycholesterol(25-OHC),the product of Ch25 h,resulted in muscle loss in C57BL/6 mice,accompanied by increased expression of atrogin-1,MuRF1 and suppression of IGF-1/Akt signaling pathway.The identification of 25-OHC as an inducer of muscle wasting has implications for the development of specific treatment strategies in preventing muscle loss.