SIRT3 Inhibits The Transdifferentiation Of Cardiac Fibroblasts Induced By ANG ? Via ?-catenin/PPAR? Signaling

Background:Cardiac fibrosis,which is characterized by disorder of extracellular matrix(ECM),is one of widely pathological processes existing in various cardiovascular diseases,including hypertension,diabetic cardiomyopathy and so on.Cardiac fibroblasts(CFs)play a key role in the process of cardiac fibrosis.CFs can transdifferentiate to myofibroblasts following a variety of stimuli.Myofibroblast is one of the pivotal sources of ECM.Hence,It's very important to inhibit the proliferation and transdifferentiation of CFs.Sirtuin is a kind of deacetylase depended on NAD+,including SIRT1-7.SIRT3 is mainly located in mitochondria,and plays an important role in regulating metabolism,oxidative stress,etc.SIRT3 can protect against cardiac hypertrophy induced by oxidative stress.Recently researches indicated that SIRT3-KO mice led to cardiac fibrosis spontaneously;what' more,resveratrol could activate SIRT3 to ameliorate cardiac fibrosis via TGF-?/SMAD signaling.All these demonstrated that SIRT3 could protect against cardiac fibrosis,however,the mechanism needs further study.Peroxisome proliferator activated receptor y(PPARy)is a member of nuclear receptor superfamily,and can regulate insulin sensitivity,the differentiation of adipocyte and inhibit the transdifferentiation of CFs induced by TGF-?,etc.Additionally,PPARy could be regulated by acetylation.A study has reported that both SIRT1 and rosiglitazone could increase the deacetylation level of PPARy to promote its activation.?-catenin is a pivotal factor in Wnt signaling pathway to promote the transdifferentiation of CFs.Previous study showed that SIRT3 could deacetylate and activate GSK-3? and then suppress the phosphorylation of ?-catenin to accelerate the degration of ?-catenin.Besides,more and more researches have found that PPARy could interact with p-catenin.All these studies revealed that SIRT3 plays a key role in suppressing cardiac fibrosis via ?-catenin/PPARy signaling.This study intended to induce the transdifferentiation of CFs by ANG II and explore the effect of SIRT3 in this process,and clarify its possible mechanism.Objective:1.To explore the role of SIRT3 in the transdifferentiation of CFs induced by ANG II.2.To investigate the effect of PPARy in the proliferation and transdifferentiation of CFs induced by ANG II.3.To clarify the possible mechanism and signaling pathway of SIRT3 for inhibition of cell transdifferentiation.Methods:1.Cardiac fibroblasts isolation and culture Cardiac fibroblasts were extracted from new-bore SD rats.CFs were cultured in high glucose DMEM containing 10%fetal bovine serum and penicillin-streptomycin in an incubator with 5%CO2 at 37?.2.Treatments for cardiac fibroblasts CFs were treated with ANG II(100nmol/L)for 48h as an in vitro model.Virus and siRNA transfections were used to overexpress or silence SIRT3.Pioglitazone and GW9662 were adopted to activate or inhibit the expression of PPARy.XAV939 was applied for suppressing the expression of ?-catenin.3.Western blot analysisTotal proteins or nuclear proteins extracted from CFs werev separated by 10%SDS-PAGE and transferred into a PVDF membrane with a wet transfer apparatus.The blots were blocked with 5%non-fat milk,and then incubated with the primary antibodies overnight at 4?.After incubated with the secondary antibodies,the blots were detected by AI600 chemiluminescence reader.4.Quantitative real-time PCR RNA were extracted from CFs with Trizol according to the manufacture's instructions,and then reverse transcribed to cDNA.Quantitative real-time PCR was detected on a real-time system using SYBR green kit.5.Immunofluorescence CFs were fixed for 30min in immune fixed liquid,punched for 25min with 0.5%Triton,blocked by 1%BS A for 30min,and then incubated with the primary antibodies overnight at 4?.After incubated with the secondary antibodies and DAPI staining for 5min,the results were detected by fluorescence microscope.6.CCK-8 analysis CCK-8 analysis was applied for detecting the proliferation of CFs.7.Statistical analysis GraphPad Prime6 software was used to analyse the data.All data were presented as mean± SEM.Data analysis were performed with one way-ANOVA or t-test.P<0.05 was considered as stastically significant.Results:1.SIRT3 inhibited the transdifferentiation of CFs induced by ANG ?.Overexpression of SIRT3 decreased the protein level and immunofluorescence intensity of a-SMA while silencing of SIRT3 exerted the opposite effect following the treatment of ANG ?,which confirmed that SIRT3 could inhibit the transdifferentiation of CFs induced by ANG II.2.Activation of PPAR? suppressed the proliferation and transdifferentiation of CFs.The mRNA and protein levels of PPARy were increased when SIRT3 was overexpressed.What's more,the acetylation level of PPARy was decreased.In order to evaluate the effect of PPAR?,we activated PPAR? by its agonist,pioglitazone.Cells were divided into control group,ANG ? group and PIO+ANG ? group.Compared with the ANG ? group,the PIO+ANG ? group showed lower mRNA and protein expressions and immunofluorescence intensity of a-SMA.Besides,CCK-8 analysis indicated that the proliferation of CFs was restrained by using pioglitazone.All the data identified that PPAR? could ameliorate the cardiac fibroblasts-to myoblasts differentiation,and the activation of PPAR? may attribute to SIRT3.3.The inhibition of PPAR? suppressed the anti-fibrotic effect of SIRT3.To explore the underlying mechanism for SIRT3 to regulate the activation of PPAR?,we used GW9662 to inhibit the expression of PPAR?.CFs were divided into control group,ANG ? group,SIRT3-overexpression+ANG ? group and SIRT3-overexpression+GW9662+ANG ? group.The results revealed that compared with the SIRT3-overexpression+ANG ? group,the SIRT3-overexpression+GW9662+ANG ? group showed higher mRNA and protein expressions and immunofluorescence intensity of a-SMA.These data confirmed that the anti-fibrotic effect of SIRT3 partially depended on the activation of PPAR?.4.SIRT3 inhibited the transdifferentiation of CFs via ?-catenin/PPAR? signaling.SIRT3 is mainly located in mitochondrial while PPAR? is located in nuclear,so we performed further investigation how SIRT3 regulated the activation of PPAR?.Previous and our researches found that the expression of ?-catenin was higher when SIRT3 was silenced.Meantime,we found that the expression of?-catenin was lower when PPAR? was activated by pioglitazone.To explore the role of ?-catenin on the regulation of PPAR? by SIRT3,we inhibited the expression of ?-catenin by XAV939.Data demonstrated that the inhibition of?-catenin promoted the level of PPAR?.Conclusively,we confirmed that SIRT3 inhibited the transdifferentiation of CFs via ?-catenin/PPAR? signaling.Conclusion:1.SIRT3 inhibited the transdifferentiation of CFs induced by ANG ?.2.PPAR? inhibited the transdifferentiation of CFs induced by ANG II.3.SIRT3 inhibited the transdifferentiation of CFs via ?-catenin/PPARy signaling.