Study On The Mechansim Of Amphiregulin Enhancing Cardiac Fibrosis After Myocardial Infarction

ObjectiveCardiac fibrosis(CF),a main process of ventricular remodeling after myocardial infarction(MI),plays a crucial role in the patheogenesis of heart failure(HF)post MI.It is known that amphiregulin(AR)is involved in fibrosis of several organs.However,the expression of AR and its role post MI are yet to be determined.This study aimed to investigate the impact of AR on CF post MI and related mechanisms.Methods and results1.AR is secreted by cardiac macrophage post MI.1)MI model was established in mice.The results of ELISA,WB,RT-PCR and immunofluorescence(IF)showed that AR levels in the infarct border zone and blood were significantly enhanced at day 7 post MI,and AR was mainly secreted by cardiac macrophages;2)The vitro study demonstrated that TGF-?1 induced AR secretion in BMDMs was mainly mediated through MAPK signaling pathway.2.AR induces macrophage M2 polarization.1)BMDMs were treated with AR in vitro and then changed into M2 polarization.Also,the enhanced TGF-?1 secretion was majorly originated from macrophages post AR stimulation.In addtion,the result of WB showed that EGFR signaling pathway was activated by AR stimulation and the secretion of TGF-?1 were significantly abrogated by EGFR inhibitors;2)The culture supernatant of BMDMs post AR treatment was collected,and then the cardiac fibroblasts were cultured with the collected culture supernatant.The results of WB,RT-PCR and Transwell showed that fibroblasts were activated by collected culture supernatant.3.AR enhances CF and aggravates cardiac dysfunction in mice post MI partly through activating EGFR-dependent pathway.1)The fibroblasts were activated after AR treatment in vitro.In addtion,the EGFR signaling pathway of fibroblast was also activated after AR stimulation.After inhibiting EGFR expression,fibroblast activation induced by AR was significantly attenuated and the EGFR signaling pathway was significantly retarded;2)MI model was established.Solution containing lentivirus AR shRNA(shAR)or scambled shRNA(shNC)was injected into the anterior and lateral part of the visibly ischemic zone.Four groups were included in the in vivo experiment: Sham group,MI group,MI-shNC group and MI-shAR group.(1)The GFP of lentivirus in heart was detected by IF.The expression of AR was also detected by WB and RT-PCR.The results showed that AR expression level in infarct border zone at day 7 post MI was significantly reduced after shAR injection;(2)The survival rate up to 28 days post MI was significantly higher in MI-shAR group than in MI group;(3)The cardiac function was enhanced in MI-shAR mice at the 28 th day post MI by small animal ultrasound system and the heart failure index of the MI-shAR mice decreased;(4)28 days post MI,the expression levels of ECMs and cardiac fibrosis in infarct border zone were all significantly reduced in MI-shAR mice compared to MI mice by using RT-PCR,WB,IF,Picrosirius Red staining and Masson's trichrome staining;(5)At day 7 post MI,the apoptotic cardiomyocytes were similar among MI mice,MI-shAR and MI-shNC in all three areas by TUNEL assay.In addtion,the result of WB showed that aggravated CF induced by AR was at least partly mediated though activating the EGFR signaling pathway.ConclusionsOur results show that AR is mainly secreted by macrophages post MI,subsequently,AR could promote the CF by stimulating TGF-?1 secretion in macrophages.On the other hand,activation EGFR signaling pathway of fibroblasts also contributes the process of CF after MI.This study revealed the relationship between AR and CF post MI and explored the specific mechanisms.Targeting AR might thus be a potential novel therapeutic target for reducing CF after MI.