The Role And Mechanism Of Autophagy Rhythm Disturbance In β₁-AA-induced Cardiac Dysfunction

As the final stage of the development of various cardiovascular diseases,cardiac dysfunction has become a serious public health problem.Studies have shown that myocardial cell death is an important factor in the process of cardiac dysfunction.Even a small amount of myocardial cell death can cause disturbances in electrical or mechanical activities of the heart,leading to cardiac dysfunction.However,the reasons and mechanisms of inducing cardiomyocyte death need to be further clarified. β₁-adrenergic receptor can cause cardiomyocyte death when it is over-activated.Studies have shown that more than 50%of patients with cardiac dysfunction can detect the β₁-adrenergic receptor autoantibodies（ β₁-AA）produced against the second loop antigen peptide of β₁-AR in the serum. β₁-AA can cause continuous activation of β₁-AR,which promotes the death of cardiomyocytes and the decline of heart function.However,the mechanism of β₁-AA causing cardiomyocyte death has not been fully elucidated.As a self-protection mechanism of cells,autophagy can digest and degrade damaged,denatured or senescent proteins and organelles in cells,recycle long-lived proteins or damaged organelles,and maintain cell homeostasis.Once the autophagy process is destroyed,damaged proteins and organelles cannot be cleared in time and will accumulate in the cells,affecting the normal function and structure of cardiomyocytes.Studies have shown that reducing the level of autophagy can induce the death of cardiomyocytes,which in turn leads to left ventricular dilation and contractile dysfunction.It can be seen that the decline of autophagy is a key factor that causes the death of cardiomyocytes and promotes cardiac dysfunction.However,in recent years,studies have found that after Beclin1+/-heterozygous mice or myocardium-specific overexpression of m TOR inhibits myocardial autophagy,myocardium protects against pressure overload-induced cardiac dysfunction,suggesting that in some cases,Inhibition of myocardial autophagy can play a role in myocardial protection.From this point of view,there is a contradiction in the effect of reduced autophagy on the death of cardiomyocytes.Therefore,autophagy itself is not the only mechanism that affects the death of cardiomyocytes.In recent years,studies have found that autophagy also has circadian rhythm fluctuations in mammalian SCN,heart,liver,kidney,skeletal muscle and other tissues.The rhythm of autophagy maintains the homeostasis by regulating the time interval of its packaging,degradation,and release of proteins and organelles.The previous Beclin1+/-heterozygous mice or myocardial-specific over-expressing m TOR mouse models simply inhibited autophagy and did not affect the myocardial autophagy rhythm.Therefore,we speculate that the autophagy rhythm disorder may be caused by the death of cardiomyocytes.However,there is no research to show whether β₁-AA inhibits autophagy function in cardiomyocytes and whether it affects the rhythm of autophagy,and whether changes in the rhythm of autophagy have an effect on the death of cardiomyocytes.Therefore,this study intends to prove the effect of β₁-AA on myocardial autophagy rhythm and its role in myocardial cell death,and to find a new mechanism of myocardial cell death in the process of cardiac dysfunction.Part 1 β₁-adrenergic receptor autoantibodies could induce cardiac dysfunctionObjective:Use bioinformatics analysis to verify whether the β₁adrenergic receptor signaling pathway in the left ventricle of patients with cardiac dysfunction is abnormal;construct an active immunization mouse model to explore the effect of β₁-AA on mouse heart function;use β₁-AA to induce cardiomyocytes,explore whether it will cause the death of cardiomyocytes.Methods:1.Download data and visualize processing:Download high-throughput sequencing information of normal left ventricular samples and left ventricular samples of patients with cardiac dysfunction in the GEO database;2.Enrichment analysis through GO,KEGG:Enrichment analysis through R language;3.Synthetic antigen peptide:synthesized by Shanghai Jier Biochemical Co.,Ltd.;4.Establish an animal model for β₁-AA active immunity:Inject β₁-AR-EC_IIinto the back of C57BL/6 mice and SD rats;5.SA-ELISA:Detect the β₁-AA content in the serum of C57BL/6 mice and SD rats after active immunization;6.Affinity chromatography:Purify β₁-AA in rat serum;7.Mouse cardiac ultrasound:the cardiac function of mice in different groups was tested;8.CCK8 method:the survival of cardiomyocytes is tested after β₁-AA is applied.Results:1.The differentially expressed genes in the left ventricular myocardial tissue samples of normal people and patients with cardiac dysfunction were screened out by bioinformatics analysis,and enrichment analysis of these differential genes by KEGG revealed that the adrenergic receptor signaling pathway in myocardial tissues of patients with cardiac dysfunction Gene Ratio is higher;2.GO enrichment analysis found that β₁ adrenergic receptor signaling pathway related genes play a role in the process of cardiac dysfunction;3.The results of small animal ultrasound show that β₁-AA can cause a significant decrease in the systolic and diastolic functions of the mice’s heart;4.CCK8 results show that β₁-AA can cause a significant increase in the death rate of H9c2 cardiomyocytes.Conclusion:1.The genomic information of patients with cardiac dysfunction is rearranged,and the rearranged genome results show that the genes related to the β₁adrenergic recept or signaling pathway are significantly abnormal;2. β₁-AA can induce the death of cardiomyocytes and decrease of heart function.Part 2 β₁-AA disrupts myocardial autophagy rhythmObjective:Autophagy maintains the normal structure and function of the heart by wrapping,degrading,and reusing some damaged,denatured or aging proteins and organelles.Our previous research results indicate that β₁-AA can inhibit myocardial autophagy and promote myocardial cell death by continuously activating β₁-AR.However,other studies have reported that myocardial autophagy function is inhibited to play a protective role in cardiac function.It can be seen that autophagy itself is not the only mechanism that affects the death of cardiomyocytes.Studies have found that there is a circadian rhythm in myocardial autophagy,and this change in circadian rhythm may also be an important cause of myocardial cell death.However,there is currently no research to show whether β₁-AA inhibits myocardial autophagy while changing its circadian rhythm.Therefore,this part of the experiment will explore the effect of β₁-AA on myocardial autophagy rhythm.Methods:1.Enrichment analysis through KEGG:Perform KEGG enrichment analysis through the use of R language;2.SA-ELISA:Please refer to the first part for details;3.Affinity chromatography:Please refer to the first part for details;4.Real time-PCR:Detect the rhythmic expression of autophagy marker LC3m RNA in myocardial tissue or myocardial cells;5.Western blot:Detect the rhythmic expression of autophagy marker LC3 protein in myocardial tissue and myocardial cells.Results:1.KEGG enrichment results show that the abnormal expression of adrenergic receptor signaling pathway in the left ventricle of patients with cardiac insufficiency is related to the circadian entrainment signaling pathway;2.Real-time PCR and Western blot results show that β₁-AA can cause the destruction of myocardial tissue autophagy rhythm in the LD environment,which is more significant in ZT12;3.Real-time PCR and Western blot results show that β₁-AA can cause the destruction of myocardial tissue autophagy rhythm in the DD environment,which is more significant in CT8 and CT12;4.Real-time PCR and Western blot results show that β₁-AA can cause the destruction of cardiomyocyte autophagy rhythm,which is more significant in CT12.Conclusion:1.Biological entrainment signal pathway is abnormal in the left ventricle of patients with cardiac insufficiency;2.β₁-AA disturbs the autophagy rhythm of myocardial tissue;3.β₁-AA disturbs the autophagy rhythm of H9c2 cardiomyocytes.Part 3 β₁-AA induces cardiomyocyte death by disturbing the rhythm of myocardial autophagyObjective:In order to explore the role of disordered autophagy rhythm in inducing the death of cardiomyocytes.In this part of the experiment,the survival rate of cardiomyocytes whose autophagy rhythm was disrupted by the Per2 gene of lentivirus was tested;and the survival rate of cardiomyocytes whose autophagy rhythm was restored by antigen neutralizing antibody was tested.Methods:1.Real time-PCR:Detect the rhythmic expression of per2 m RNA in myocardial tissue or myocardial cells;Detect the inhibition efficiency of lentivirus on per2m RNA.Detect the rhythmic expression of LC3 m RNA in myocardial cells;2.Western blot:Detect the rhythmic expression of Per2 protein in myocardial tissue and myocardial cells;Detect the inhibition efficiency of lentivirus on Per2 protein.Detect the rhythmic expression of LC3 protein in myocardial cells;3.Lentivirus infection:Use 1/2 small volume method for lentivirus infection;4.ABC-ELISA:Use ELISA kit to detect the content of c AMP in the supernatant of H9c2 cardiomyocytes;5.CCK8 method:The survival rate of each group of cells is tested.Results:1.Real-time PCR and Western blot results show that β₁-AA induces circadian rhythm disorder of the circadian rhythm gene Per2 in myocardial tissue;2.Real-time PCR and Western blot results show that β₁-AA destroys the circadian rhythm of the cardiomyocyte circadian clock gene Per2;3.Laser scanning confocal microscope observation showed that when MOI=30,LV-sh PER2 had the highest infection efficiency,so MOI=30 was selected for subsequent experiments;Real-time PCR and Western blot results show that LV-sh PER2 can significantly inhibit cardiomyocytes Per2 expression;4.Real-time PCR and Western blot results show that LV-sh PER2 can destroy the autophagy rhythm of cardiomyocytes after infecting cardiomyocytes;5.CCK8 results show that the survival rate of cardiomyocytes is significantly reduced after the autophagy rhythm is disrupted;6.ABC-ELISA test results show that antigen-neutralizing antibodies can inhibit the activation of c AMP in the downstream pathway of β₁-AA;7.Real-time PCR and Western blot results show that the destructive effect of β₁-AA on the autophagy rhythm at CT12 is reversed by the antigen neutralizing antibody;8.The results of CCK8 showed that the survival rate of cardiomyocytes after the autophagy rhythm was restored.Conclusion:Autophagy rhythm disorder is an important reason for β₁-AA to induce cardiomyocyte death.