AT1-AA Induces Cardiac Insufficiency In Rats Via Up-regulating Autophagy

Background:Cardiovascular disease is one of the most common diseases in China and even in the world.Heart failure is the most common cause of death.Apoptosis of cardiomyocytes can gradually decrease cardiac function and eventually lead to heart failure.Therefore,inhibiting the apoptosis of cardiomyocytes is an important way to delay the occurrence of cardiac insufficiency and treat heart failure.Studies have found that high titers of AT1-AA were detected in the serum of patients with heart failure,suggesting that AT1-AA may be involved in the process of heart failure.However,whether this antibody can induce apoptosis of cardiomyocytes and cause cardiac dysfunction in the overall situation and its specific mechanism remains unclear.Autophagy is generally considered to be a normal protective mechanism of cells,which is necessary to maintain cell homeostasis.Under physiological conditions,it protects against certain diseases by dissolving some organelles,but excessive and uncontrolled autophagy can cause cell apoptosis.Some studies have shown that angiotensin II（Ang II）can induce autophagy.So does AT1-AA,which has the resemble effect of Ang II,can affect autophagy in cardiomyocytes?It has been found that endoplasmic reticulum stress is closely related to autophagy and the change of the latter can affect the former.So does AT1-AA induce endoplasmic reticulum stress?Whether or not endoplasmic reticulum stress plays a role between autophagy and apoptosis,and what role it plays are still unclear.Therefore,in this study,it was assumed that AT1-AA,through binding with AT1receptor,could up-regulate autophagy to induce apoptosis of rat cardiomyocytes,causing cardiac dysfunction and participating in the process of heart failure.Objective:To establish a rat model that actively immunized with AT1-AA,to observe the effects of AT1-AA on cardiomyocyte autophagy,endoplasmic reticulum stress,and apoptosis,and to explore the underlying mechanism of cardiac dysfunction,so as to provide intervention methods and therapeutic strategies for the clinical treatment of heart failure.Methods:1.Identification of rat genotypes by PCR.The AT1a receptor gene was knocked out in SD rats（heterozygous）,and after breeding,the male rats aged 8 weeks were obtained.The genotypes of the rats were identified by PCR.2.Establishment of AT1R-ECⅡpeptide actively immunized rat model.After gene identification,SD rats were divided into Vehicle control group,AT1-AA immunized group,and AT1-AA+3MA group.AT1A receptor knockout rats（homozygous）were divided into AT1a R^-/-+AT1-AA group（n=6 for each group）.The latter three groups were firstly treated with active immunization with AT1R-ECⅡpeptides dissolved in Na₂CO₃ solution after well mixed with freund’s complete adjuvant;two weeks later,enhancing immunization was given,which only changed complete freund’s adjuvant to incomplete adjuvant as compared with active immunization,and this was performed once every 2 weeks,for a total of 12 weeks.For immunization of the Vehicle control group,only the antigen solution was replaced by the same amount of Na₂CO₃ solution.In AT1-AA+3MA group,3-MA was injected intraperitoneally at 8 weeks after immunization,once every other day,for 4weeks.3.The serum titer of AT1-AA in AT1-AA group was determined by ELISA.Blood samples were collected intravenously and serum samples were kept before each immunization in AT1-AA group.4.The changes of heart function and structure in each group were detected by ultrasonography.5.HE staining was used to observe the changes of cardiac structure.6.TUNEL staining was used to detect the apoptosis of heart tissue cells.7.Apoptosis,ER stress,and autophagy-related protein expressions were detected by Western blot.Results:1.Identification of rat genotypes by PCR.According to PCR identification results,they were divided into SD rats（homozygous negative）,AT1a receptor knockout rats（homozygous positive）,and AT1a receptor knockout rats（heterozygous）.2.The successful establishment of AT1-AA positive rat model.ELISA results showed that the serum AT1-AA content in AT1-AA group was increased compared with that in Vehicle group in the second week of immunization（P<0.05）.After immunization,the serum AT1-AA content showed an time-dependent upward trend,reached the peak after 8 weeks of immunization,and remained at a high level until the end of the experiment（P<0.01）.3.AT1-AA could cause cardiac function decline and cardiac structure change in rats.Compared with the Vehicle group,LVIDs and LVESV of AT1-AA immunized rats were significantly increased（P<0.05）,while LVEF and LVFS were significantly decreased（P<0.05）after 12 weeks of immunization.HE staining showed that,compared with Vehicle control group,after12 weeks of immunization,the organization of myocardial cells in AT1-AA immunized group were disordered,with significantly different nuclear sizes,vacuolation of cytoplasm,blurred horizontal stripes,distortion and widening of intercellular spaces,and disordered direction and rupture of myocardial fibers.4.AT1-AA could increase the expression of apoptosis in heart tissue cells.After 12 weeks of immunization,the brown-yellow nuclei in TUNEL staining were increased and the apoptosis index was increased in immunized group compared with that of the Vehicle group（P<0.05）.Western blot showed an increase in cleaved caspase-3/caspase-3 protein levels in heart tissue after 12 weeks of immunization in AT1-AA group compared with taht of the Vehicle control group（P<0.05）.5.AT1-AA could up-regulate autophagy expression in heart tissue.Western blot results showed that compared with the Vehicle group,aftter 12weeks,LC3Ⅱ/LC3Ⅰand Beclin1 protein expressions in heart tissues of AT1-AA group were increased（P<0.05）,but the p62 protein levels were decreased（P<0.05）.6.AT1-AA could activate endoplasmic reticulum stress in heart tissue.Western blot results of endoplasmic reticulum stress showed that compared with Vehicle control group,the levels of GRP78,CHOP,and Caspase-12 proteins in heart tissue were increased in AT1-AA immunized group after 12 weeks of immunization（P<0.05）.7.The autophagy inhibitor 3-MA could alleviate the increased levels of autophagy and apoptosis in rat heart tissue induced by AT1-AA and improve cardiac dysfunction.After intraperitoneal injection of 3-MA for 4 weeks,the results showed that compared with the AT1-AA immunized group,LC3Ⅱ/LC3Ⅰ,Beclin1,Cleaved caspase 3/caspase 3,CHOP,GRP78 and Caspase-12 protein levels of the AT1-AA+3MA group were decreased（P<0.05）,while p62 protein level was increased（P<0.05）.The brown-yellow granules in heart tissue were less,and the apoptosis index of myocardial cells was decreased（P<0.05）.The LVIDs and LVESV were decreased,while LVEF and LVFS were significantly increased.The disordered arrangement of myocardial cells was reduced,the vacuolation of cytoplasm was reduced,the horizontal striations were clear,the degree of intercellular distortion and widening was reduced,the disordered direction and breakage of myocardial fibers were alleviated,and the lysis of myocardial cells was reduced.8.AT1a receptor knockout could reduce the levels of autophagy and apoptosis induced by AT1-AA in heart tissue and improve cardiac dysfunction.After 12 weeks of active immunization,it was found that compared with the AT1-AA immunization group,the expression of LC3Ⅱ/LC3Ⅰ,Beclin1,cleaved caspase-3/caspase-3,CHOP,GRP78 and Caspase-12 protein levels in AT1a R^-/-+AT1-AA group were decreased（P<0.05）,while p62 protein levels were elevated（P<0.05）.The brown-yellow granules in heart tissue were less,and the apoptosis index of myocardial cells was decreased（P<0.05）.The LVIDs and LVESV were decreased（P<0.05）,while LVEF and LVFS were significantly increased（P<0.05）.The disordered arrangement of myocardial cells was reduced,the horizontal striations were clear,the degree of intercellular distortion and widening were reduced,and the disordered direction and the degree of rupture of myocardial fibers was were decreased.Conclusion:AT1-AA acts on AT1a receptor and induces apoptosis of myocardial tissue in rats by upregulating autophagy and then activating endoplasmic reticulum stress,thus leads to cardiac dysfunction.