The Role Of Autophagy In The Cardiomyocyte Apoptosis Induced By β₁-adrenoceptor Autoantibodies

A large number of clinical data shows that cardiac dysfunction is the end-stage of various cardiovascular diseases,such as coronary heart disease,myocardial infarction,angina,arrhythmia and other cardiovascular diseases,and the therapeutic effect of advanced patients is limited,suggesting that we should prevent and treat cardiovascular disease in the early stage.It has been found that there is a wide range of myocardial cell apoptosis in the early stage of heart failure,and it can effectively prevent or reduce cardiac dysfunction as long as by suppressing cell apoptosis.It can be seen that myocardial cells apoptosis play an important role in the occurrence and development of cardiac dysfunction.Some clinical investigations showed that β₁-adrenergic receptor autoantibodies（β₁-AA）can be detected in the serum of 40%-60%heart failure patients,and it was found that the apoptosis of cardiomyocytes could be induced by activating Pi-adrenergic receptor（β₁-AR）.However,it is not clear how β₁-AA induces apoptosis in cardiomyocytes.Autophagy is an important mechanism for maintaining cellular homeostasis,which can promote self-renewal and material cycling,and provide energy for cells.Our previous studies have found that β₁-AA can induce the decrease of autophagy level in myocardial cells.It has been found that autophagy and apoptosis are ubiquitous in a cell,meanwhile autophagy occurs earlier than apoptosis,and is involved in the regulation of apoptosis.In some cases,autophagy,as a stress response,can protect cells from death by inhibiting apoptosis.Therefore,autophagy may play an important role in the regulation of apoptosis.However,whether autophagy can affect β₁-AA-induced cardiomyocyte apoptosis remains unknown.Therefore,in this study,we used β₁-AR-ECII antigen peptide to purify β₁-AA in 8 weeks male Wistar rats.The purified β₁-AA was used to treat H9c2 cells and primary rat neonatal cardiomyocytes.Use Caspase-3 activity assay,Annexin V-FITC/PI staining,flow cytometry and Hoechst 33258 staining to reflect myocardial cell apoptosis,and detect the myocardial cell death by CCK-8 method,our results showed that myocardial cell apoptosis induced by β₁-AA involves in the myocardial cell death.In addition,the expression of autophagy related protein LC3,Beclin 1 and P62 was detected by western blot and real-time PCR.The results indicated that β₁-AA could induce the transient increase of autophagy in the early stage,and then decreased significantly.In order to further observe the effect of the decrease of autophagy level on the apoptosis of myocardial cells induced by β₁-AA,this experiment adopts the autophagy inhibitor 3-Methyladenine（3-MA）to inhibit autophagy and mTOR pathway inhibitor Rapamycin（Rapa）which can upregulate the autophagy and then to observe the changes of myocardial cell apoptosis,the results found that the decrease of autophagy promoting the apoptosis of myocardial cells induced by β₁-AA,and we further explored the possible mitochondrial mechanism.The aim of this study was to investigate the possible mechanism of β₁-AA induced apoptosis from the perspective of autophagy,and to provide some ideas and experimental basis for improving the prognosis of patients with cardiac insufficiency.Objective:In this study,we investigated the role of autophagy in the apoptosis of cardiomyocytes induced by β₁-AA by using the β₁-AA cell model.Methods:（1）Preparation of antibody:Use the company’s synthesis of β₁-AR extracellular peptide as the immune antigen peptide to active immune 8 weeks Wistar rats.The rats were randomly divided into two groups:①Immunization group:The rats was immunized with β₁-AR-ECII antigen peptide in the dose of 0.4 μg/g.②Vehicle group or pseudo immunity group.After the first immunization,enhanced immunization once every two weeks,blood samples were taken from abdominal aorta after 8 weeks of immunity,and SA-ELISA method was used to detect the antibody titers in blood.The purified β₁-AA was purified by affinity chromatography.（2）In vivo:The cells（H9c2 myocardial cells and primary neonatal rat cardiomyocytes）were divided into β₁-AA group and negative IgG control group.The β₁-AA group was treated with different time in the final concentration of 1 μM β₁-AA,and the control group was treated with the same dose of negative IgG.The survival rate of myocardial cells was detected using CCK-8 method,the apoptosis of myocardial cells using Caspase-3 kit and Annexin V-FITC/PI double staining flow cytometry and Hoechst 33258 staining,and myocardial cell autophagy related genes and proteins were detected by using real time-PCR and western blot.Using Broad-spectrum Caspase inhibitor Z-VAD-FMK inhibited apoptosis,classical autophagy inhibitor,3-MA,and mTOR pathway inhibitor,Rapa,inhibited and upregulated the autophagy,and observed the changes of myocardial cell death after reducing the apoptosis and changing the level of autophagy.Use 3-MA and Rapa pretreatment in myocardial cells to downregulate and upregulate autophagy and observe myocardial cell apoptosis by Annexin V-FITC/PI double staining,flow cytometry,Hoechst 33258 staining,Caspase-3 activity and JC-1 staining.Results:1.Cardiomyocyte apoptosis induced β₁-AA is involved in myocardial cell death1.1 H9c2 cells could be used for the following experiments in the routine recovery and subcultureRapid melt frozen liquid and eccentric cell at a speed of 800 r/min,abandoned the freezing medium,use a new culture medium to Suspension cells in a cell culture bottle,Incubator culture until the density reaches 80%,then use 0.25%trypsin to digeste cells and passage,conventional cell culture is convenient for subsequent experiments.1.2 The primary cultured neonatal rat cardiomyocytes had good activity,high purity and sensitive drug reactionUsing 0.4%trypan blue solution to detect the survival rate of freshly isolated cell.The newly isolated cell survival rate reached 90%;When cells grow to 4 days later,the myocardial cell marker a sarcomeric actin（a-actin）and cardiac troponin I（cTnI）on the isolated cells were identified by immunofluorescence staining,the result showed that the purity of isolated cardiomyocytes was up to 92.64%.1.3 Serum β₁-AA purified from the active immunization rats can increase the beating freq uency of neonatal rat cardiomyocytesSA-ELISA was used to measure β₁-AA levels（OD value）in the serum from rats 6 weeks after active immunization.The results showed that the OD value was up to 1.93± 0.26,and the concentration up to 6.4 mg/mL after purification.Cultured neonatal rat cardiomyocytes for 4 days,and then treat with β₁-AA,the results showed that myocardial cells beat frequency increased significantly after the 1 μM β₁-AA treatment for 24 h and 48 h,indicated that the purified β₁-AA was active,which could increase the myocardial cell beating frequency.1.4 β₁-AA can reduce the survival rate of myocardial cellsCCK-8 method is used to reflect the β₁-AA effects on myocardial cells survival rate.The result showed that the livability of myocardial cells was significantly decreased after the 48 h stimulated by β₁-AA.1.5 β₁-AA can induce myocardial cell apoptosisUsing Caspase-3 activity detection,Annexin V-FITC/PI flow cytometry and Hoechst 33258 to detected the cell apoptosis.Testing results of three methods are similar,showed a significant increase in myocardial cell apoptosis after the treatment of Pi-AA for 6 h,suggesting that Pi-AA could induce cardiomyocyte apoptosis.1.6 Caspase broad-spectrum inhibitors Z-VAD-FMK can inhibit apoptosis and partly reverse myocardial cell death induced β₁-AAWith a broad spectrum of Caspase inhibitor Z-VAD-FMK preprocessing myocardial cell for 30 min,use the β₁-A A stimulating myocytes,the results showed that Z-VAD-FEK can partially inhibit Caspase-3 activity increases induced by β₁-AA,and inhibition of apoptosis can reduce myocardial cell death induced by β₁-AA.2.The time course of myocardial autophagy induced by β₁-AA and its significance in myocardial cell death2.1 β₁-AA cause cardiomyocyte autophagy level increased first and then decreasedUsing real time PCR and western blot method to detect the LC3 mRNA level as well as LC3,Beclin 1 and P62 protein expression after β₁-AA treatment at 0 h,3 h,6 h,12 h,24 h in H9c2 cardiomyocytes;Separate the primary neonatal rat cardiomyocytes,usingβ₁-AA stimulating at 0 min,1 min,3 min,5 min,10 min,20 min,30 min,1 h,3 h,6 h,12 h,24 h and 36 h,48 h and 72 h,and then detect LC3 mRNA level and LC3,Beclin 1 and P62 protein expression.Results suggested that β₁-AA could induce the autophagy level increasing first and then decreasing.2.2 The reduce of autophagy levels is the important reason for myocardial cell deathUse the 3-MA and Rapa preprocessing myocardial cells,found that 3-MA pretreatment deregulated the autophagy level of myocardial cells,and compared with theβ₁-AA treated alone group,the myocardial viability declined further;After Rapa pretreatment,the autophagy level of myocardial cells were upregulated and the survival rate of myocardial cells was restored partially,suggesting that the decrease of autophagy is involved in the death of myocardial cells induced by Pi-AA.3.Study on the role of decreased autophagy in the apoptosis of myocardial cells induced by Pi-AA3.1 Using Rapa and 3-MA to upregulate or downregulate autophagy can significantly change the apoptosis of myocardial cells induced by β₁-AAApoptosis was detected by Caspase-3 activity assay,Annexin V-FITC/PI double staining flow cytometry and Hoechst 33258 staining.The results showed that the apoptosis rate of 3-MA+β₁-AA group was higher than that of β₁-AA alone group,which indicated that inhibition of autophagy could increase the apoptosis of myocardial cells.However,compared with the β₁-AA treated alone group,the apoptosis rate of Rapa+β₁-AA group was significantly decreased,which indicated that the upregulation of autophagy could decrease the apoptosis of primary myocardial cells.These results suggested that the decrease of autophagy is involved in the apoptosis of myocardial cells induced by β₁-AA.3.2 Reduced autophagy can promote the mitochondrial pathway apoptosis induced byβ₁-AAThe intensity of red and green fluorescence of JC-1 fluorescence staining and the change of Caspase-9 activity were closely related to mitochondrial pathway apoptosis.Results of the two methods showed that compared with the β₁-AA treatment alone group,Caspase-9 activity increased further in 3-MA pretreatment group,JC-1 staining showed a significant increase in ratio of green fluorescence and red fluorescence,indicating that mitochondrial depolarization,mitochondrial pathway of apoptosis increased significantly;Compared to β₁-AA treatment alone group,the activity of Caspase-9 of Rapa pretreatment group was decreased,and JC-1 staining showed that cells with Rapa pretreatment maintaining the basic mitochondrial membrane potential,suggested that that the decrease of autophagy induced β₁-AA may lead to the increase of myocardial apoptosis through the mitochondrial apoptotic pathway.Conclusion:Autophagy decline participates in the myocardial apoptosis induced by β₁-AA.