A Study On The Involvement Of UBE2E2 In The Pathogenesis Of Experimental Autoimmune Myocarditis By Regulating Ferroptosis In Cardiomyocytes

Background and objective:Myocarditis is a serious and potentially life-threatening disease that leads to dilated cardiomyopathy(DCM),severe heart failure(HF),and sudden cardiac death,and there is an urgent need for effective therapeutic agents in clinical practice.Myocarditis is characterized by a complex inflammatory response within the heart,and viral infection as a common cause triggers a cardiac-specific autoimmune response leading to autoimmune myocarditis(AM).The pathogenesis of AM is very complex and has not yet been fully elucidated,and there is no uniform and effective clinical treatment protocol.It has been suggested that ferroptosis is involved in regulating the pathology of myocardial infarction,heart failure,myocardial ischemia-reperfusion,and myocardial fibrosis,and whether ferroptosis is involved in the pathogenesis of AM has not been reported in the literature.Previous studies suggest that the ubiquitin-proteasome system may be involved in regulating ferroptosis in pancreatic cancer,ischemia-reperfusion,and tumor stem cells.Whether ubiquitin-conjugating enzyme E2E2(UBE2E2),a member of the ubiquitin-proteasome system family,is involved in the regulation of ferroptosis levels in AM has not been reported in the literature.In this study,we will construct an in vivo and in vitro model of experimental autoimmune myocarditis(EAM)to reveal whether ferroptosis exists in AM and whether UBE2E2 is involved in regulating the level of ferroptosis in AM,and to investigate the molecular mechanism by which UBE2E2 regulates ferroptosis to ameliorate myocardial injury in AM,providing a potential target for clinical treatment of AM.Materials and methods:I.The role of UBE2E2,ferroptosis in in vivo and in vitro models of EAM.1.In vivo(1)The EAM in vivo model was constructed using porcine cardiac myosin,and Balb/c mice were randomly divided into model group(n=8)and control group(n=8).(2)Cardiac hematoxylin-eosin staining(HE)to assess myocardial damage.(3)Real-time quantitative PCR(q RT-PCR)was performed to detect the expression of messenger ribonucleic acid(m RNA)of UBE2E2 in myocardial tissue of EAM model mice.m RNA expression of ferroptosis-related markers in myocardial tissue,such as Glutathione peroxidase 4(GPX4),Ferritin heavy chain(FTH1),Cyclooxygenases-2(COX2),Acyl-Co A synthetase long-chain family member 4(Acyl-Co A synthetase long-chain family member 4(ACSL4).(4)Detection of protein expression of UBE2E2 in myocardial tissue by protein immunoblotting assay(Western blot)and detection of protein expression of ferroptosis-related markers such as GPX4,FTH1,COX2,ACSL4 in myocardial tissue.(5)The level of inflammatory factors tumor necrosis factor alpha(TNF-α)and interleukin-1β(IL-1β)and the level of subferric ions in myocardial tissue were detected by enzyme-linked immunosorbent assay(ELISA).2.In vitro(1)Lipopolysaccharide(LPS)was used to construct an in vitro model of EAM,which was divided into: model group and control group.(2)Western blot and q RT-PCR were used to detect UBE2E2,ferroptosis-related proteins and m RNA expression in cardiomyocytes,such as GPX4,FTH1,COX2,ACSL4.(3)ELISA was performed to detect intracellular ferrous ions in cardiomyocytes and the levels of TNF-α and IL-1β in the supernatant of cardiomyocytes.II.Role of overexpressed UBE2E2 in EAM in vivo and in vitro models1.In vivo(1)The EAM mouse model was constructed using porcine cardiac myosin,and Balb/c mice were randomly divided into four groups: control group(n=8),model group(n=8),model + overexpression null group(n=8),and model + UBE2E2 overexpression group(n=8),in which the model + overexpression null group and model + UBE2E2 overexpression group were overexpressed by adenovirus.(2)Cardiac HE staining was performed to assess cardiac damage.(3)The effects of UBE2E2 overexpression on the structure and function of mouse hearts were assessed by cardiac echocardiography in all groups,including posterior wall-systolic(PW-s),fractional shortening(FS),posterior wall-diastolic(PW-d),and ejection fraction(EF).The effects of UBE2E2 overexpression on the structure and function of mouse heart were evaluated.(4)Western blot and q RT-PCR were performed to detect ferroptosis-related proteins and m RNA expression,such as UBE2E2,GPX4,FTH1,COX2,ACSL4,and HMGB1.(5)ELISA detects serum levels of myocardial injury markers such as Creatine kinase-MB(CK-MB)and Aspartate aminotransferase(AST);levels of inflammatory factors TNF-α and IL-1β;levels of heart failure markers Brain Natriuretic Peptide(BNP);levels of Anti-cardiac myosin antibody(AMA);and levels of ferrous ions in myocardial tissue.2.In vitro(1)LPS was used to construct a myocarditis cell model,and the specific groups were control group,model group,model + overexpression null group,and model +UBE2E2 overexpression group.(2)Cell Counting Kit-8(CCK8)was used to detect changes in cell proliferation in each group.(3)Flow cytometric assays were performed to detect changes in reactive oxygen species(ROS)in cardiomyocytes of each group.(4)Measurement of mitochondrial membrane potential by mitochondrial membrane potential immunofluorescence(JC-1 method).(5)Detection of mitochondrial morphological changes by transmission electron microscopy.(6)Western blot and q RT-PCR were performed to detect the expression of ferroptosis-related proteins and m RNAs,that includes UBE2E2,GPX4,FTH1,COX2,ACSL4,and HMGB1.(7)Measurements of ferrous ions in cardiomyocytes and levels of TNF-α and IL-1β in cardiomyocyte supernatants by ELISA.III.Investigating the molecular mechanism of UBE2E2 inhibition of ferroptosis in cardiomyocytes1.The interaction between UBE2E2 and HGMB1 was analyzed by immunoprecipitation(Co-Immunoprecipitation,Co-IP)and protein profiling techniques.2.Protein expression of HGMB1,ubiquitinated Ubiquitin was analyzed by CO-IP assay.3.The mouse cardiomyocytes HL-1 were divided into control group,control group + MG-132 treatment group,UBE2E2 overexpression group and UBE2E2 overexpression + MG-132 treatment group,and the cells were transfected with UBE2E2 overexpression plasmid and the corresponding null plasmid according to the above groups,after which the cells were treated with 5u M of MG-132,and the expression of the target protein HMGB1 in each group was detected by Western blot after 24 h.Results:I.The role of UBE2E2,ferroptosis in in vivo and in vitro models of EAM1.In vivo(1)HE staining: Myocardial fibers in control mice were neatly arranged without obvious lesions;HE staining in model mice suggested that inflammatory cell infiltration in myocardial tissue increased,myocardial cells showed necrotic disintegration and edematous degeneration,and some myocardial fractures and myocardial fibrosis appeared.(2)QRT-PCR/Western blot:UBE2E2 m RNA and protein expression were significantly down-regulated in myocardial tissues of mice in the model group compared with normal controls(p < 0.05),making it clear that UBE2E2 is lowly expressed in autoimmune myocarditis.(3)QRT-PCR/Western blot: The m RNA and protein expression of GPX4 and FTH1,the negative regulators of ferroptosis,were significantly down-regulated(p <0.05),while the m RNA and protein expression of COX2 and ACSL4,the positive regulators of ferroptosis,were significantly up-regulated(p < 0.05)in the myocardial tissue of mice in the model group compared with the normal control group.(4)ELISA: The level of ferrous ions in the myocardial tissue of mice in the model group was increased(p < 0.05),and the levels of TNF-α and IL-1β in the serum were increased(p < 0.05)compared with those in the normal control group.2.In vitro(1)Western blot and q RT-PCR: Compared with the normal cell group,GPX4,FTH1,UBE2E2 protein and m RNA expression were down-regulated(p < 0.05)and COX2,ACSL4 protein and m RNA expression were up-regulated(p < 0.05)in the model group cardiomyocytes.(2)ELISA:Compared with the normal cell group,the levels of intracellular iron ions,TNF-α and IL-1β in the cell supernatant were increased in the model group cardiomyocytes(p < 0.05).II.Role of overexpressed UBE2E2 in EAM in vivo and in vitro models1.In vivo(1)HE staining: In the control group,myocardial fibers were neatly arranged without obvious lesions;in the model group,myocardial fibers were disordered,myocardial cells were damaged,cytoplasmic lysis and light staining;in the overexpression null group,myocardial fibers were disordered,inflammatory cells were infiltrated and fibrosis was present;compared with the model group,the structure of myocardial fibers in the UBE2 overexpression group was improved and inflammatory infiltration was significantly better.(2)Cardiac echocardiography: Compared with normal control mice,LV PW-s,PW-d,EF and FS were significantly decreased in the model group(p <0.05);compared with mice in the model group,LV EF,FS,PW-s and PW-d were significantly increased in the UBE2E2 overexpression group(p <0.05).(3)Western blot and q RT-PCR:Compared with normal control mice,myocardial tissues of mice in the model group showed low expression of UBE2E2,GPX4,FTH1 protein and m RNA(p < 0.05)and high expression of HMGB1,COX2,ACSL4 protein and m RNA(p < 0.05);compared with mice in the model group,the model +UBE2E2 overexpression group showed low expression of UBE2E2,GPX4,FTH1 protein and m RNA were highly expressed(p < 0.05),and HMGB1,COX2,ACSL4 protein and m RNA were lowly expressed(p < 0.05)in the model+UBE2E2overexpression group compared to the model mice.(4)ELISA: In comparison with normal control mice,the levels of subferric ions,CK-MB,AST,TNF-α,IL-1β,BNP and AMA were increased in the model group mice(p < 0.05);compared with the model group mice,the levels of subferric ions,CK-MB,AST,TNF-α,IL-1β,BNP and AMA were decreased in the UBE2E2 overexpression group mice(p < 0.05).2.In vitro(1)CCK-8: The proliferation viability of cardiomyocytes was decreased in the model group compared with the normal control group(p < 0.05);the proliferation viability of cells in the UBE2E2 overexpression group was increased compared with the model group(p < 0.05).(2)Flow Cytometry: The level of ROS in the cells of the model group was significantly increased compared with the normal control group(p < 0.05);the level of ROS in the cardiomyocytes of the UBE2E2 overexpression group was significantly decreased compared with the model group(p < 0.05).(3)Results of the JC-1 assay for mitochondrial membrane potential: Compared with the normal control group,the mitochondrial membrane potential level of cells in the model group decreased(p < 0.05);compared with the model group,the mitochondrial membrane potential level of cells in the UBE2E2 overexpression group increased(p < 0.05).(4)Transmission Electron Microscope: In the control group,the mitochondrial structure was clear,the mitochondrial cristae were intact and more numerous;compared with the control group,the mitochondrial number in the model group decreased,the mitochondrial morphology became smaller,and the cristae were broken or even disappeared;there was no significant difference between the overexpression null group and the model group,the mitochondria were reduced,and there were mitochondrial cristae broken and disappeared;the mitochondrial number in the UBE2E2 overexpression group increased compared with the model group,the morphology tended to be normal,and the mitochondrial ridges were clearly visible.(5)Western blot 和 q RT-PCR: Compared with the control group,UBE2E2,GPX4,FTH1 protein and m RNA were lowly expressed(p < 0.05),HMGB1,COX2,ACSL4 protein and m RNA were highly expressed(p < 0.05)in the model group cardiomyocytes;compared with the model group cardiomyocytes,UBE2E2,GPX4,FTH1 protein and m RNA were highly expressed in the model+UBE2E2overexpression group(p < 0.05),and HMGB1,COX2,ACSL4 proteins and m RNA were lowly expressed(p < 0.05).(6)ELISA:Elevated levels of ferrous ions,TNF-α and IL-1β in the model group compared with the normal cell group(p < 0.05);decreased levels of ferrous ions,TNF-α and IL-1β in the model + UBE2E2 overexpression group compared with the model group(p < 0.05)..III.Investigating the molecular mechanism of UBE2E2 inhibition of ferroptosis in cardiomyocytes1.The product of UBE2E2 protein pull-down was screened by mass spectrometry means,and after analysis,it was found that UBE2E2 could target binding to high mobility group protein B1(High mobility group box1,HMGB1).2.When compared with the control group,the protein expression of HGMB-1was down-regulated and ubiquitinated Ubiquitin expression was up-regulated in the UBE2E2 overexpression group Input.assay results in Co-IP showed that the expression of HGMB1 was down-regulated and ubiquitinated Ubiquitin expression was up-regulated after UBE2E2 overexpression.3.Compared with the control group,HMGB1 protein expression was higher in the control + MG132-treated cells and lower in the UBE2E2 overexpression group(p< 0.05);compared with the UBE2E2 overexpression group,HMGB1 protein expression was higher in the UBE2E2 overexpression + MG132-treated group(p <0.05).Conclusion1.Ferroptosis exists in EAM.2.UBE2E2 can target binding to HMGB1.3.UBE2E2 overexpression can degrade HMGB1 through ubiquitination,regulate ferroptosis-related protein expression,inhibit the ferroptosis process in cardiomyocytes,attenuate myocardial immune damage,and delay cardiac diastolic dysfunction,thus exerting a protective effect on cardiomyocytes in EAM.