The Role Of Cell Death In Myocardial Injury Induced By Autoimmune Myocarditis Rats

Part one To establish the model of experimental autoimmune myocarditis and investigate the change of necroptosis,apoptosis andautophagy in EAM Objective:The aim of this study was to establish an animal model of experimental autoimmune myocarditis and investigate the change of necroptosis,apoptosis and autophagy in acute stage and chronic phase of EAM model.Methods:Eighteen male Lewis rats,8 weeks,specific pathogen free,were randomly divided into control group(3 weeks)(Control-3W),control group(8 weeks)(Control-8W),experimental autoimmune myocarditis group(3 weeks)(EAM-3W)and experimental autoimmune myocarditis group(8 weeks)(EAM-8W).The Lewis rats of EAM group were induced by porcine cardiac myosin(PCM)on day 1 and day8.Rats were sacrificed respectively on 3 weeks and 8 weeks after immunization.Echocardiogram was adopted to assess cardiac function.Hematoxylin-eosin(HE)staining and immunohistochemical(IHC)was performed to evaluate the histopathological changes of myocardium.The expression of RIP1/RIP3/MLKL,Caspase-3/Bax/Bcl-2 and LC3 were detected by q RT-PCR and western blot.Results:(1)On 3 weeks and 8 weeks after immunization,cardiac function decreased obviously in EAM group compared with control group,left ventricular end-systolic diameter(LVIDs)increased both in EAM-3W and EAM-8W(P<0.05),left ventricular end-diastolic diameter(LVIDd)increased in EAM-3W(P<0.05)and EAM-8W(P>0.05),ejection fraction(EF)and fractional shortening(FS)decreased both in EAM-3W and EAM-8W(P<0.05).(2)Histopathological examination showed an dense inflammatory infiltrates with myocardial cell degeneration and necrosis in EAM-3W group,obviousmyocardial fibrosis and inflammatory infiltrates in EAM-8W group compared with control group.(3)According to the result of immunohistochemistry,q RT-PCR and western blot,we found that on 3 weeks and 8 weeks after immunization,RIP1/RIP3/MLKL,caspase-3/Bax/Bcl-2 and LC-3 is mainly located in cytoplasm of myocardial cell,the m RNA and protein expression level of RIP1/RIP3/MLKL/Caspase 3/Bax in EAM group increased significantly(P<0.05),and the level of Bcl-2 decreased obviously in the EAM group compared with control group(P<0.05).In addition,the m RNA expression level of LC-3II in EAM group was higher than control group on 3 weeks and 8 weeks after immunization(P<0.05),and the expression level of LC-3II protein was higher than control group on 3 weeks(P<0.05)and there was no statistical difference on 8 weeks(P>0.05).Conclusion:(1)Experimental autoimmune myocarditis(EAM)model was successfully established induced by porcine cardiac myosin(PCM).(2)Cardiac function of experimental autoimmune myocarditis decreased obviously during the acute phase.(3)Necroptosis and apoptosis are activated and induce myocardial injury in experimental autoimmune myocarditis in vivo,and are upregulated;In addition,autophagy possibly participates only in the acute phase of experimental autoimmune myocarditis.Part two Influence of necroptosis,apoptosis and autophagyinhibitor on cardiac function in experimental autoimmunemyocarditis and the role of cell death in the process Objective:According to the part one,we used Nec-1,z VAD-fmk,3-Ma respectively to evaluate the effect of necroptosis,apoptosis and autophagy inhibitor on cardiac function in acute phase of experimental autoimmune myocarditis,and the role of cell death.Methods:Twenty-nine male Lewis rats,8 weeks,specific pathogen free,were randomly divided into control group and experimental group.The experimental autoimmune myocarditis model were induced by porcine cardiac myosin(PCM).The experimental group were randomly divided into five groups: EAM group,EAM+Nec-1 group intraperitoneal injection of Nec-1(0.6mg/kg,dissolved in 0.1% DMSO,2 weeks),EAM+z VAD-fmk group intraperitoneal injection of z VAD-fmk(1mg/kg,dissolved in0.1% DMSO,2 weeks),EAM+3-Ma group intraperitoneal injection of 3-Ma(15mg/kg,dissolved in 0.1% DMSO,2 weeks),EAM+DMSO group intraperitoneal injection of same dose 0.1% DMSO on day 8 after first immunization.Rats were sacrificed respectively on 3 weeks after immunization.Echocardiogram was adopted to assess cardiac function.Hematoxylin-eosin(HE)staining and immunohistoche-mical(IHC)was performed to evaluate the histopathological changes of myocardium.The expression of RIP1/RIP3/MLKL,Caspase-3/Bax/Bcl-2 and LC3 were detected by q RT-PCR and western blot.Results:(1)On 3 weeks after immunization,cardiac function decreased obviously in EAM group and EAM+DMSO group compared with control group,LVIDs and LVIDd increased obviously(P<0.05),EF and FS decreased significantly(P<0.05).Cardiac function in EAM+Nec-1group,EAM+z VAD-fmk group and EAM+3-Ma group was significantly improved,LVID;d and LVID;s decreased,EF,FS increased obviously than EAM+DMSO group(P<0.05).(2)Histopathological examination showed an dense inflammatory infiltrates in EAM group and EAM+DMSO group compared with control group.EAM+Nec-1group,EAM+z VAD-fmk group and EAM+3-Ma group was significantly improved in myocardial pathology change compared with EAM+DMSO group.(3)According to the result of immunohistochemistry,q RT-PCR and western blot,we found that RIP1/RIP3/MLKL,caspase-3/Bax/Bcl-2 and LC-3 is mainly located in cytoplasm of myocardial cell,The m RNA and protein level of RIP1/RIP3/MLKL/Caspase 3/Bax/LC-3II increased significantly,and the expression of Bcl-2decreased in the EAM group compared with control group on 3 weeks and 8 weeks after immunization(P<0.05).(4)In EAM+Nec-1 group,The m RNA and protein level of RIP1/RIP3/MLKL decreased(P<0.01),Caspase-3 and Bax elevated(P<0.01),the level of Bcl-2 was lower than EAM+DMSO group(P<0.01),and LC-3II had no significantly difference compared with EAM+DMSO group(P>0.05).(5)In EAM+z VAD-fmk group,the m RNA and protein level of RIP1,RIP3 and MLKL increased obviously(P<0.01),Caspase-3,Bax and LC-3II was lower than EAM+DMSO group(P<0.01),the level of Bcl-2 was higher than EAM+DMSO group(P<0.01).(6)In EAM+3-Ma group,the m RNA and protein level of RIP1,RIP3,MLKL,Caspase-3 and Bax was higher than EAM+DMSO group(P<0.01),while the expres-sion of LC-3II was lower than EAM+DMSO group(P<0.01),and the level of Bcl-2had no significantly difference compared with EAM+DMSO group(P>0.05).Conclusion:(1)Necroptosis,apoptosis and autophagy inhibitors respectively improved cardiac function in acute phase of experimental autoimmune myocarditis.(2)Necrosis inhibitor necrostatin-1 prompt apoptosis,and had no significant effect on autophagy in autoimmune myocarditis in vivo.(3)Apoptosisinhibitor z VAD-fmk dramatically enhanced necroptosis and inhibited autophagy in autoimmune myocarditis in vivo.(4)Autophagy inhibitor 3-methyl adenine(3-MA)triggers necroptosis,and prompt apoptosis in autoimmune myocarditis in vivo.