The Role And Mechanism Of Up-regulation Of SNX7 Expression In β₁-AA-induced Decrease Of Cardiomyocyte Autophagy

Heart failure is the final stage of the development of various cardiovascular diseases.According to epidemiological data,there are 64.3 million people suffering from heart failure worldwide,which has become an important social and public problem endangering human health.Finding the key target of precise treatment of heart failure has become an important topic of concern to many scholars in the field of cardiovascular research.Clinical data show that autoantibodies（β1-adrenergic receptor autoantibodies,β₁-AA）can be detected in the serum of about 40%-60% patients with heart failure.β₁-AA can mediate the occurrence and development of heart failure by activating β₁-AR.The removal of β₁-AA from patients’ blood by immunoadsorption technology can obviously improve patients’ cardiac function,suggesting that β₁-AA is closely related to heart failure.Our previous experiments found that myocardial autophagy inhibition is an important reason for β₁-AA-induced myocardial cell death and even heart failure.However,the mechanism of autophagy inhibition in cardiomyocytes caused by β₁-AA is not clear.Therefore,in the first part of this study,the myocardial tissue samples of β₁-AA positive mice after active immunization for 4 weeks were analyzed by quantitative protein proteomics,and then the differentially expressed proteins in the histological results were intersected with the proteins in the autophagy database,and only Sorting Nexin 7（SNX7）with up-regulated expression was obtained.So,does β₁-AA induce myocardial autophagy inhibition by up-regulating SNX7 expression? Immunofluorescence staining and Western blot experiments confirmed that β₁-AA could lead to the up-regulation of SNX7 protein expression in mouse myocardial tissue and H9c2 cardiomyocytes,and further downregulation by si RNA could significantly improve the autophagy inhibition of cardiomyocytes induced by β₁-AA,suggesting that the up-regulation of SNX7 expression did participate in the autophagy inhibition of cardiomyocytes induced by β₁-AA.Subsequently,the molecular mechanism of up-regulation of SNX7 expression involved in β₁-AA inhibiting autophagy of cardiomyocytes will be further explored.SNX7 has PX domain and BAR domain which are closely related to autophagy.At present,only one document reports that it can form a complex with SNX4 to participate in the extension and closure of autophagy membrane.However,the experimental results of this study show that β₁-AA does not affect the combination of SNX4 and SNX7 in cardiomyocytes,suggesting that the up-regulation of SNX7 expression induced by β₁-AA does not affect autophagy by combining with SXN4.So,how does SNX7 play a role in the inhibition of myocardial autophagy induced by β₁-AA?In the second part of this study,the IP solution of SNX7 was prepared for mass spectrometry detection and the results were analyzed to explore the interaction protein and signal network of SNX7 in myocardial cells.Through GO and KEGG enrichment analysis of autophagy-related differential proteins,it was found that death-related protein kinase 3（DAPK3）,vacuole membrane protein 1（VMP1）and cathepsin D（CTSD）may participate in β₁-AA-induced myocardial autophagy inhibition through interaction with SNX7,among which DAPK3’ s Further Co-IP experiments also confirmed that β₁-AA could induce the increase of the binding between SNX7 and DAPK3.In recent years,DAPK3 is regarded as a new type of autophagy positive regulator.We found that the autophagy level of cardiomyocytes decreased after knocking down DAPK3 with si RNA,but the increase of expression of DAPK3 induced by β₁-AA could not promote autophagy,suggesting that β₁-AA inhibited the autophagy promotion of DAP3.So,does β₁-AA inhibit the autophagy promoted by DAPK3 by up-regulating SNX7? In this study,β₁-AA treatment was given on the basis of knocking down the expression of SNX7 in cardiomyocytes.It was found that β₁-AA could not inhibit the autophagy promoting effect of DAPK3.It is suggested that β₁-AA inhibits DAPK3 from promoting autophagy by increasing the interaction between SNX7 and DAPK3 in cardiomyocytes.To sum up,this study draws the following conclusions: β₁-AA can increase its binding with DAPK3 by up-regulating the expression of SNX7 protein in myocardial cells,but DAPK3 can not promote autophagy,which leads to autophagy inhibition.This study innovatively confirmed the new mechanism of SNX7 affecting autophagy,which not only expanded the first class members of SNXs protein family as autophagy regulators,but also provided new targets for cardiovascular diseases related to autophagy disorder.Part 1: Up-regulation of SNX7 protein expression participates in β₁-AA-induced decrease of myocardial autophagyObjective:To investigate the role of the expression changes of SNX7 protein in β₁-AA-induced decreased myocardial autophagy.Methods:（1）synthesizing the extracellular second loop（β₁-AR-ECII）antigen peptide of β adrenergic receptor type 1.（2）Establish an animal model of active immunization with autoantibody against β1 adrenoceptor（β₁-AA）: Randomly divide 6-8-week-old male SD rats into a control group and a primary immunization group,with 6 rats in each group.（3）streptavidin-enzyme-linked immunosorbent assay（SA-ELISA）: The content of β₁-AA in serum of primary immunized mice was detected by double antibody sandwich method.（4）Immunoaffinity chromatography: β₁-AA in serum of β₁-AA positive mice was purified for in vitro experiments.（5）protein omics: Protein extraction,enzyme digestion,TMT labeling,high performance liquid chromatography classification,liquid chromatography-mass spectrometry tandem analysis,database search,biological information analysis,etc.were carried out on the myocardial tissue samples of β₁-AA positive mice that had been actively immunized for 4 weeks.（6）Analysis of life information: Cross the differentially expressed proteins obtained by mass spectrometry with 887 proteins derived from autophagy-related protein database to screen out autophagy-related proteins.（7）CCK8 method: to detect the change of myocardial cell survival rate with or without β₁-AA treatment,and whether there is any effect on myocardial cell survival rate after knocking down SNX7.（8）Western blot: detect the expression of SNX7 protein and autophagy marker proteins LC3 II and P62 in myocardial tissue and myocytes of mice after β₁-AA treatment,and whether knocking down SNX7 has any effect on the expression of autophagy marker proteins LC3 II and P62.（9）Immunofluorescence: Using the principle of antigen-antibody specific binding,the target protein was labeled with fluorescent antibodies of different colors,and the localization and quantification of SNX7 protein in myocardial cells under the action of β₁-AA were observed more intuitively by confocal microscope.（10）Small interfering RNA and cell transfection: Knock down myocardial cell SNX7 with SNX7 small interfering RNA,and detect the interference efficiency of small interfering RNA on the m RNA and protein level of SNX7 by Real time-PCR and Western blot,and select the sequence with the highest interference efficiency for subsequent experiments.（11）lentivirus construction and cell infection: SNX7 knock-down stable cell line was constructed,and the survival rate and autophagy level of myocardial cells were detected with or without β₁-AA treatment.Results:（1）β₁-AA can inhibit autophagy in myocardial tissue of mice.（2）β₁-AA induced the decrease of autophagy level in cardiomyocytes.（3）The results of protein omics and letter analysis showed that the expression of autophagyrelated protein SNX7 in myocardium of mice immunized actively increased.（4）β₁-AA can induce the up-regulation of SNX7 protein expression in myocardial tissue of mice.（5）β₁-AA can induce the up-regulation of SNX7 protein expression in cardiomyocytes.（6）Knocking down the expression of SNX7 can improve the decrease of autophagy induced by β₁-AA in cardiomyocytes.（7）Knocking down the expression of SNX7 can reverse the decline of myocardial cell survival rate induced by β₁-AA.Conclusion:β₁-AA can induce the decrease of myocardial autophagy by up-regulating the expression of SNX7 protein.Part 2: The up-regulation of SNX7 expression induced by β₁-AA inhibits autophagy of cardiomyocytes by interacting with DAPK3Objective:The protein binding to SNX7 in myocardial cells was significantly changed under the action of β₁-AA by mass spectrometry and signal analysis.Methods:（1）SA-ELISA,affinity chromatography and Western blot: as shown in the first part.（2）Co-IP: incubating the antibody with Protein A/G Magnetic Beads,magnetically separating,collecting the beads,adding cell lysate containing protease inhibitor,fully suspending and incubating,washing off the nonspecific binding,and eluting the eluate to verify other proteins interacting with SNX7 protein by Western blot.（3）Mass spectrometry analysis（bioinformatics analysis of Shotgun protein Group）: The IP solution of SNX7 was obtained by immune coprecipitation experiment,and the data were collected by protein extraction,peptide digestion and LC-MS/MS（liquid chromatography-tandem mass spectrometry）.Based on the detected protein abundance difference,the relevant databases were searched,and combined with bioinformatics analysis methods,the signal pathways that differentially expressed proteins mainly participated in and the interaction networks between proteins were analyzed.（4）Bio-information analysis: GO and KEGG enrichment of differential protein.（5）Small interfering RNA and cell transfection: The interference efficiency of three synthesized small interfering RNA sequences of DAPK3 was tested,and the most effective knock-down sequence was selected for subsequent experiments.Results:（1）β₁-AA did not affect the expression of SNX4 and the combination of SNX4 and SNX7 in cardiomyocytes.（2）The results of mass spectrometry showed that β₁-AA could significantly change the binding of 326 proteins to SNX7.（3）There are 10 autophagy-related proteins in cardiomyocytes that change their binding to SNX7 under the action of β₁-AA.（4）The results of GO and KEGG show that DAPK3 may participate in β₁-AA-induced myocardial autophagy inhibition through its interaction with SNX7.（5）β₁-AA can induce the increase of DAPK3 protein expression in myocardial tissue and cardiomyocytes of mice.（6）β₁-AA can increase the binding between SNX7 and DAPK3 protein.（7）Up-regulation of DAPK3 expression induced by β₁-AA can not promote autophagy of cardiomyocytes.（8）Up-regulation of SNX7 protein expression induced by β₁-AA inhibits the autophagypromoting effect of DAPK3.Conclusion:β₁-AA inhibits DAPK3 from promoting autophagy by promoting the binding between SNX7 and DAPK3.