Interplay Of Endoplasmic Reticulum Stress And Autophagy Is Involved In The Cardiomyocyte Apoptosis Induced By β₁-adrenoceptor Autoantibodies

Heart failure is the final stage of various cardiovascular diseases,In recent years,heart failure as a cardiovascular syndrome with increasing morbidity and mortality,and it has become a major threat to public health.Among them,cardiomyocyte apoptosis is the key factor of the occurrence and development of heart failure.Some studies have found that cell loss caused by persistent cardiomyocyte apoptosis may cause the gradual deterioration of cardiac function,and then lead to end-stage heart failure,therefore,it is particularly important to explore the underlying mechanism of cardiomyocyte apoptosis.Clinical studies have showed that the serum levels ofβ₁-adrenergic receptor autoantibody（β₁-AA）were increased in 40-60%of patients with heart failure.β₁-AA can induce cardiomyocyte apoptosis through specific activation theβ₁-adrenergic receptor（β₁-AR）,and then participates in the development of heart failure.However,the specific mechanism is not yet clear.Several studies have shown that appropriate level of autophagy is essential for maintaining homeostasis.Autophagy dysfunction precedes cell apoptosis,and it play a vital role in the development of cardiovascular diseases through regulating apoptosis.Our previous research found thatβ₁-AA could promote apoptosis by inducing autophagy dysfunction in cardiomyocytes,and the upregulation of autophagy by rapamycin can partially alleviate cardiomyocyte apoptosis induced byβ₁-AA.While the mechanism of autophagy dysfunction induced byβ₁-AA is still unclear.Numerous studies have shown that endoplasmic reticulum stress（ERS）is closely relatedtoautophagy.ERScouldmaintainsintracellularhomeostasisby eliminating some unfavorable elements through inducing the unfolded protein reaction（UPR）.Under stress conditions,mild ERS maintain homeostasis by activating autophagy in cells,and upregulated autophagy induce cell-survival signaling by activing ERS-related mechanisms,On the contrary,continuous and excessive activation of ERS prevents cells from adapting to external stimuli and induce a significant reduction of autophagy level,and decreased autophagy level can further activate ERS-related apoptosis.It can be concluded that the interaction between ERS and autophagy is crucial in the maintenance of intracellular homeostasis.So,whether the interaction between ERS and autophagy is exist in cardiomyocytes and whether the interaction is important as an mechanism of the cardiomyocytes apoptosis induced byβ₁-AA.This is the overall focus of our study.In this study,male SD rats were actively immunized with the peptide of the second extracellular loop of theβ₁-AR,and the serum level ofβ₁-AA were measured by SA-ELISA,thenβ₁-AA was purified by affinity chromatography 8 weeks post immunization for the in vitro experiment.Then the level of autophagy and ERS of myocardial tissue and H9c2 myocardial cells were detected by using real time-PCR,westernblot,autophagicdouble-labeled（mRFP-GFP-LC3）adenovirusand immunohistochemistry;The apoptosis in rat myocardium and H9c2 myocardial cells were mesured by TUNEL assay,Annexin-V FITC/PI apoptosis double staining and Caspase-3activity;The survival rate of myocardial cells was detected using CCK-8 method.The results showed thatβ₁-AA could induce a significant reduction in autophagy level and continuous activation of ERS in myocardial tissue and H9c2 myocardial cells.Upregulating autophagy level by Rapa or inhibiting ERS by 4-PBA both could partly alleviate the increase of apoptosis induced byβ₁-AA,suggested that both autophagy inhibition and ERS activation are involved in the apoptosis of cardiomyocytes induced byβ₁-AA.Furthermore,4-PBA was used to inhibit ERS and observe its effect on autophagy of cardiomyocytes,the results showed thatβ₁-AA could induce autophagy dyfunction by activating the ERS of cardiomyocytes;Using 3-methyladenine（3MA）and Rapa to inhibit or up-regulate cell autophagy,and observe the effect of autophagy on ERS in cardiomyocytes,the results showed that the decrease of autophagy could further active ERS in turn.Thus,the interaction between ERS and autophagy is an important mechanism of cardiomyocytes apoptosis induced byβ₁-AA.In view of the interaction between ERS and autophagy in the process of cardiomyocyte apoptosis induced byβ₁-AA,this study further used combined pretreatment of 4-PBA and Rapa to observe the changes of cardiomyocyte apoptosis induced byβ₁-AA,aim to provide a theoretical basis for improving the progress and prognosis ofβ₁-AA-positive heart failure patients.Part 1 Decreased autophagy participates in the increased cardiomyocyte apoptosis induced byβ₁-AAObjective:Active immunization rat models were successfully established and theβ₁-AA was purified for the study of cardiomyocyte.To investigate the role of decreased autophagy in cardiomyocyte apoptosis induced byβ₁-AA in vivo and in vitro.Methods:（1）Establishment of active immunization rat models:Male SD rats were actively immunized withβ₁-AR-ECII antigen peptide.The rats were randomly divided into immunization and pseudo immunity group,the antigen peptide was injected subcutaneously into the back of rats at a dose of 0.4 ug/g,and immunization was strengthened every two weeks,in pseudo immunity group,the antigen peptide was replaced by Na₂CO₃ solution.Blood was taken from the tail vein of the rat,SA-ELISA was used to detect the production ofβ₁-AA in serum,and to confirm the successful establishment of the active immunization rat models.If the serum levels ofβ₁-AA in rats was increased significantly and maintain a higher level,and then theβ₁-AA could be purified for further experiments by affinity chromatography.（2）Real-time PCR was used to detect the mRNA expression of autophagy-related genes Beclin1 and LC3 in myocardial tissue and myocardial cells with the existence ofβ₁-AA;（3）Western blot was used to detect the protein expression of autophagy-related proteins Beclin1,LC3 and p62 in myocardial tissue and myocardial cells with the existence ofβ₁-AA;（4）autophagic double-labeled（mRFP-GFP-LC3）adenovirus was used to detect the changes in autophagic flux of H9c2 myocardial cells after the treatment ofβ₁-AA;（5）Rats were intraperitoneally injected Rapa to up-regulate autophagy in myocardium,and TUNEL staining was used to detect the apoptotic rate of myocardium induced byβ₁-AA;（6）Inhibite autophagy by 3MA and upregulate autophagy by Rapa in H9c2 myocardial cells,and Annexin V-FITC/PI flow cytometry was used to detect the apoptotic rate of myocardium induced byβ₁-AA;Results:（1）Active immunization rat models were established successfully:compared with the vehicle group,The OD value ofβ₁-AA in the serum from rats 2 weeks after active immunization were increased significantly,and theβ₁-AA levels increased gradually with the prolongation of active immunization time.theβ₁-AA levels reached a high level at 6weeks and lasted until 8 weeks,suggested that the active immunization rat models were established successfully;（2）β₁-AA caused myocardial tissue autophagy level decreased significantly:The real-time PCR and western blot results showed that compared with the vehicle group,the relative mRNA and protein expression of Beclin1 and LC3 was significantly decreased 2weeks after the active immunization,and it continued to decrease with the prolongation of active immunization time,then dropped to the lowest level after 8 weeks.The level of autophagic substrate p62 was increased gradually 2 weeks after the active immunization and lasted until 8 weeks;（3）β₁-AA induced H9c2 cardiomyocytes autophagy level decreased:Real-time PCR and western blot results suggested that the relative protein expression of Beclin1 and LC3 was significantly decreased 6 h afterβ₁-AA treatment and lasted until 24 h,While the expression of autophagic substrate p62 was increased significantly 12 h afterβ₁-AA treatment;H9c2 cardiomyocytes were infected by autophagy double-labeled adenovirus（mRFP-GFP-LC3）,the results showed that the numbers of autophagosomes and autophagic lysosomes were decreased significantly 6 h after the treatment withβ₁-AA,suggested that autophagic flux was decreased with the existence ofβ₁-AA and lasted until24 h;（4）Autophagy dyfunctin is the important reason for cardiomyocytes apoptosis:TUNEL staining showed that intraperitoneally injection of Rapa could partially alleviate the increase of apoptosis in myocardium induced byβ₁-AA,and Rapa pretreatment could also partially alleviated the cardiomyocytes apoptosis induced byβ₁-AA,While 3MA induced further increase of cell apoptosis.Conclusion:Decreased autophagy participates in the increase of cardiomyocyte apoptosis induced byβ₁-AA.Part 2 Endoplasmic reticulum stress participates in the reduction of autophagy induced by β1-AA in cardiomyocytesObjective:To measure how change in the rule of endoplasmic reticulum stress in myocardial tissue and H9c2 cardiomyocytes with the existence of β1-AA and its role in the reduction of autophagy.Methods:（1）Real-time PCR was used to detect the m RNA expression of endoplasmic reticulum stress-related genes GRP78,CHOP and Caspase-12 in myocardial tissue and myocardial cells with the existence of β1-AA;（2）Western blot was used to detect the expression of endoplasmic reticulum stress-related proteins GRP78,CHOP and Caspase-12 in myocardial tissue and myocardial cells with the existence of β1-AA;（3）Immunohistochemical was used to detect the in situ expression of endoplasmic reticulum stress-related proteins GRP78,CHOP and Caspase-12 in myocardial tissue and myocardial cells with the existence of β1-AA;（4）Real-time PCR,western blot and m RFP-GFP-LC3 adenovirus were used to detect the autophagy level after pretreatment with 4-PBA.Results:（1）β1-AA caused myocardial tissue endoplasmic reticulum stress level increased significantly: Real-time PCR,western blot and immunohistochemical results showed that compared with the vehicle group,the relative m RNA and protein expression of GRP78 was significantly increased 2 weeks after the active immunization,while the relative m RNA and protein expressions of CHOP and Caspase-12 was significantly increased 4weeks after the active immunization,and it continued to increase with the prolongation of active immunization time;（2）β1-AA caused H9c2 cardiomyocytes endoplasmic reticulum stress level increased significantly: Real-time PCR,western blot and immunohistochemical results suggested that endoplasmic reticulum stress wass markedly activated 6 h after β1-AA treatment and lasted until 24 h;（3）Endoplasmic reticulum stress participates in the reduction of autophagy induced byβ1-AA in H9c2 cardiomyocytes: Real-time PCR,western blot and m RFP-GFP-LC3 adenovirus results suggested that inhibiting endoplasmic reticulum stress by 4-PBA could significantly increase the expression of Beclin1 and LC3,decrease the level of p62,and increase the autophagic flux.Conclusion:Endoplasmic reticulum stress participates in the reduction of autophagy induced byβ1-AA in cardiomyocytes.Part 3 Autophagy dyfunction induced by β1-AA active the endoplasmic reticulum stress in cardiomyocytesObjective:To investigate whether the reduction of autophagy induced by β1-AA could active the endoplasmic reticulum stress in cardiomyocytes,and to clarify the role of interaction between endoplasmic reticulum stress and autophagy in the apoptosis of cardiac myocytes induced by β1-AA.Methods:（1）Western blot was used to detect the expression of autophagy-related protein Beclin1,LC3,p62 and endoplasmic reticulum stress-related protein GRP78,CHOP,Caspase-12 among the vehicle group,β1-AA group（24 h）,3MA+β1-AA group and Rapa+β1-AA group;（2）Inhibit endoplasmic reticulum stress by 4-PBA in cardiomyocytes,and to detect the apoptotic rate of cardiac myocytes induced by β1-AA using Annexin V-FITC/PI flow cytometry;（3）Inhibit endoplasmic reticulum stress by 4-PBA in cardiomyocytes,and to detect the survival rate of cardiac myocytes induced by β1-AA using CCK-8;（4）Real-time PCR was used to detect the m RNA expression of autophagy-related genes Beclin1 and LC3 among vehicle group,β1-AA group;4-PBA pretreatment group,Rapa pretreatment group and 4-PBA+Rapa pretreatment group;（5）Western blot was used to detect the expression of autophagy-related protein Beclin1,LC3 and p62 among vehicle group,β1-AA group;4-PBA pretreatment group,Rapa pretreatment group and 4-PBA+Rapa pretreatment group;（6）Autophagy double-labeled adenovirus（m RFP-GFP-LC3）was used to detect the autophagic flux among vehicle group,β1-AA group;4-PBA pretreatment group,Rapa pretreatment group and 4-PBA+Rapa pretreatment group;（7）Annexin V-FITC/PI flow cytometry and Caspsae-3 activity detection were used to detect apoptotic rate of H9c2 cardiomyocyte among vehicle group,β1-AA group;4-PBA pretreatment group,Rapa pretreatment group and 4-PBA+Rapa pretreatment group.Results:（1）Reduction of autophagy induced by β1-AA could active endoplasmic reticulum stress in H9c2 cardiomyocytes: Compared with the β1-AA group,the expression of GRP78,CHOP and Caspase-12 protein could be further up-regulated by autophagy inhibitor 3MA,and significantly alleviated by Rapa;（2）Inhibit endoplasmic reticulum stress can partly alleviate the increase of apoptosis and the decrease of survival rate in cardiomyocytes induced by β1-AA: Annexin V-FITC/PI flow cytometry and Caspsae-3 activity results showed that inhibit endoplasmic reticulum stress by 4-PBA could significantly alleviate the increase of apoptosis and the decrease of survival rate induced by β1-AA,but not completely;（3）Combined pretreatment with 4-PBA and Rapa could further increase the autophagy level of cardiomyocytes: Real-time PCR,western blot and autophagy double-labeled adenovirus results showed that compared with 4-PBA and Rapa-alone pretreatment group,4-PBA+Rapa combined pretreatment could further increase the level of Beclin1,LC3 protein and autophagic flux,and decrease the expression of p62;（4）Combined pretreatment with 4-PBA and Rapa could further alleviate apoptosis of cardiomyocytes induced by β1-AA: Annexin V-FITC/PI flow cytometry and Caspsae-3activity results showed that inhibit endoplasmic reticulum stress by 4-PBA or up-regulate autophagy by Rapa alone only partially alleviated the increase of apoptosis induced byβ1-AA,compared with 4-PBA and Rapa-alone pretreatment group,4-PBA+Rapa combined pretreatment could further alleviated the increase of apoptosis induced byβ1-AA,and this effect was most obvious at 12 h with the existence of β1-AA.Conclusion:Reduction of autophagy induced by β1-AA could active the endoplasmic reticulum stress in cardiomyocytes.