A Study Of The Role Of ATP6AP2 In The Remodeling Induced By Pressure Overload And The Underlying Mechanism

BackgroundHeart failure is a group of syndromes with clinical manifestations caused by various cardiac structural and functional diseases,resulting in impaired cardiac filling and/or ejection function,cardiac output unable to meet the metabolic needs of the body tissue,resulting in pulmonary circulation or systemic circulation congestion,and insufficiency of blood perfusion in organs and tissues.In the past 20 years,drug treatment and instruments for the heart failure treatment significantly improved the symptoms in patients with heart failure and delay the progress of heart failure,but the overall prognosis is still relatively poor,mortality rates is about 50%within five years.To identify the mechanism and potential therapeutic targets for effective interventions that are likely to slow the heart failure disease progression.The pathogenesis of heart failure involves oxidative stress,mitochondrial dysfunction,inflammation,activation of neuro-humoral endocrine system and cardiac remodeling.Among which inflammation plays a key role in cardiac remodeling.Serum inflammatory cytokines are significantly elevated in all types of heart failure and are significantly associated with adverse clinical outcomes.NLRP3 is an intracellular inflammatory signaling platform,a key multiprotein signaling platform that tightly controls the inflammatory response and coordinates host defense by regulating the cleavage and activation of Caspasel,resulting in the cleavage of IL1β,IL-18 precursor into mature fragments.Previous studies have shown that activation of NLRP3 inflammasome promotes the pathogenesis and cardiac remodeling of various cardiovascular diseases such as arteriosclerosis,hypertension and diabetes.Autophagy is an evolutionary highly conserved intracellular biological process that plays an important protective role by removing senescence,dysfunctional organelles,damaged or misfolded proteins,degrading them in lysosomes and converting them into substances that can be reused by the body,as well as destroying pathogens that invade cells.It’s critical for maintaining cellular homeostasis.Abnormal activation of inflammasome is associated with intense inflammation,leading to the development of inflammatory disease.Therefore,inflammasome must be activated at the right intensity to prevent infection and avoid tissue damage.A large number of studies have shown that the deletion of autophagy related proteins can cause abnormal activation of inflammasome,leading to severe tissue damage.In contrast,autophagy inducers improved symptoms of inflammasome-related diseases.ATP6AP2/PRR widely distributed in the body for multiple organizations,kidney,heart,brain,eye,placenta and the immune system,it involved in many important physiological processes,such as the cell cycle,autophagy,acid-base balance,energy metabolism,embryonic development,T cells,water balance,regulation of blood pressure,cardiac remodeling and maintain podocyte structure and so on.PRR has also been reported to be involved in diabetic fibrosis,hypertension,diabetic microangiopaplasia,acute kidney injury,cardiovascular disease and other pathological conditions,but some relevant conclusions are inconsistent.At present,several studies have shown that the loss of ATP6AP2 function under physiological conditions leads to the accumulation of a large number of multiple vesicle fusion structures around the nuclear,partially digested or undigested cellular components such as mitochondria can be seen.Increased expression of autophagy related proteins,such as LC3B,SQSTM1/P62,and amino acid starvation response genes,such as Asna,Atf4,Gadd 153,were associated with other serious phenotypes,such as severe heart failure,cardiac fibrosis,and fatal.These results suggest that ATP6AP2-regulated autophagy plays an important role in cell homeostasis.Structurally,ATP6AP2,as a key component of vacuolar-ATPase,plays an important role in lysosomal degradation and autophagy.In conclusion,the loss of Atp6ap2 leads to severe intracellular homeostasis disruption and cell death.At present,our understanding of Atp6ap2 is not very clear,large number of research are still needed.At present,there is no relevant study on the role of ATP6AP2 in stress-induced heart failure.Clinical diseases such as hypertension and aortic stenosis,ultimatedly progressed to heart failure.Inflammation plays important role in the process.We hypothesized that ATP6AP2 may play a key regulatory role in stress-induced heart failure by mediating autophagy and NLRP3.Therefore,we raised the following scientific questions:What’s does ATP6AP2 response in stress-induced cardiac remodeling and progression of heart failure?How do autophagy and NLRP3 change in stress-induced cardiac remodeling?Is there a regulatory relationship between ATP6AP2 and NLRP3?By what mechanism does ATP6AP2 regulate NLRP3 in the progression of heart failure?Objective1.The pressure overload induced hypertrophy and heart failure-TAC model(transverse aortic constriction)was constructed.2.The genetic mice with ATP6AP2 overexpression was constructed.The knockdown of ATP6AP2 was achieved by adenovirus as a vector.The virus was injected in vivo to observe the effects of ATP6AP2/PRR on cardiac remodeling and cardiac function induced by stress stress.3.To explore the mechanism of the effects of ATP6AP2 on cardiac remodeling and cardiac function induced by stress.Method1.TAC model was established in wild mice to simulate the stress-induced heart failure model(1)construction of TAC in C57BL/6J mice model,the different time points in the sham group and post TAC at day 5,day7,day14,day28,day56.It covers from compensatory hypertrophy to decompensated heart failure.(2)Using C57BL/6J as background,ATP6AP2/PRR knock-in mice(CAS9-PRR-KI mice)and ATP6AP2/PRR Floxed mice were constructed using the Crisper-Cas9 technique and hybridized with α-MHC-CRE mice.Cardiomyocyte specific ATP6AP2/PRR overexpression mice were constructed.Through genotypic identification,homozygous mice(Tg)of 8-12 weeks and littermate control wild mice were selected to construct TAC model.(3)Adenovirus as a vector targeting ATP6AP2/PRR knockout virus(Ad-shR-ATP6AP2)was constructed,i.e.,the control empty vector virus Ad-sh-Scr was injected into wild mice(109PFU/mouse),TAC model was established,and the same dose of virus was injected again 2 weeks later.2.Animal experimental drug intervention and groupingEarly autophagic flow changes of TAC were detected:Sham,Sham+BafA1,TAC-5day,TAC-5day+BafA1;MCC950 animal response test:Ad-ShR-SCr-TAC,Ad-ShR-Scr-TAC+MCC950,Ad-ShR-ATP6AP2-TAC,Ad-ShR-ATP6AP2-TAC+MCC9503.General detection indexMouse body weight,heart weight,lung weight,liver weight,tibia length,etc4.Cardiac ultrasoundIn the wild mouse model,cardiac ultrasound was performed at different time points after TAC to detect left ventricular systolic and diastolic functions,and ventricular inner diameter and ventricular wall thickness were detected to determine the compensatory and decompensated stages.The TAC model constructed by gene overexpression mice and control group,and the TAC model constructed after transfection of Ad-ShR-ATP6AP2 and Ad-ShR-SCR adenovirus in control group were used to detect cardiac function related parameters at 4 weeks.5.Histopathological examination(1)Immunohistochemical related protein expression:collagen 1(Col1),collagen 3(Col3),ATP6AP2,NLRP3,Caspase-1,IL-1β,IL-18.(2)Myocardial tissue fibrosis was detected by Masson(3)Immunohistofluorescence detection:co-location of SQSTM1/P62,NLRP3 and ATP6AP2,detection of SQSTM1/P62 and LAMP2 protein(4)HE test:cross-sectional area of myocardial cells on the cross-section of myocardial tissue was detected6.ELISA detectionThe expression levels of IL-1β and TNF-α in serum were detected7.Western Blot assayTissues and cells of ATP6AP2/PRR,NLRP3,Caspase1,LC3A/B,P62/SQSTM1,Atg58.The PCR detectionATP6AP2/PRR,Myh6,Myh7,IL-1β,IL-18 were detected in myocardial tissue and primary myocardial cells9.Apoptosis was detected by TUNELTUNEL assay was used to detect the changes of myocardial apoptosis during TAC induced cardiac remodeling,TAC mice after knockout virus injection,and MCC950 inhibitor treated model group.10.Primary myocardial cells were extracted and stimulated with phenylephrine to construct an in vitro cell model of stress stressPrimary cardiomyocytes were extracted from the hearts of Suckling rats at 1-3 days by differential adhesion method,and the purity was identified and related experiments were started 3 days later.Phenyleterine(PE,100μM)was given to stimulate ATP6AP2 and NLRP3 at different time gradients.Western blot was used to detect the expression changes of ATP6AP2 and NLRP3,providing a basis for the subsequent ATP6AP2 intervention time point.11.Effects of ATP6AP2 on autophagy flow detected by mCHerry-GFP-LC3B autophagy dual fluorescence virusThe groups are as follows:ShR-Scr,ShR-Scr+BafA1,ShR-ATP6AP2,ShR-ATP6AP2+BafA1The primary cardiomyocytes were pretreated with lysosomal inhibitor BafA1 and transfected with Ad-ShR-ATP6AP2 virus and mCherry-GFP-LC3B autophagy dual fluorescence virus.The ratio and change of autophagosome(yellow speck)and autophagolysosome(red speck)were calculated to detect the effect of ATP6AP2 on autophagy flow.12.Lysosomal probe LYS-tracker was used to detect the influence of ATP6AP2 on lysosomal PHThe groups are as follows:ShR-Scr,ShR-Scr+BafA1,ShR-ATP6AP2,ShR-ATP6AP2+BafA1Primary cardiomyocytes were pretreated with lysosomal inhibitor BafA1 and transfected with Ad-Shr-ATP6AP2 virus.Lysosomal probe Lyso-Tracker was used to observe the fluorescence intensity under a fluorescence microscope.13.Morphological changes of intracellular mitochondrial autophagy were observed by transmission electron microscopyThe effect of PE and ATP6AP2 at different time on mitochondrial autophagy was observed under transmission electron microscope.14.Survival of cardiomyocytes detected by SYTO/SYTOX fluorescent probeGroup 1 is as follows:ShR-Scr,ShR-Scr+PE,ShR-ATP6AP2,ShR-ATP6AP2+PE;The effect of ATP6AP2 on PE induced cardiomyocyte death was investigated.Primary cardiomyocytes were transfected with AD-SR-ATP6AP2 virus and control virus after PE stimulation for at least 24h,and then treated with SYTO/SYTOX fluorescence probe,observed and counted under the fluorescence microscope.Group 2 is as follows:ShR-Scr+PE,ShR-Scr+PE+MCC950,ShR-ATP6AP2+PE,SRATP6AP2+PE+MCC950;To determine whether the effect of ATP6AP2 on myocardial cell death is related to NLRP3.MCC950 pretreated primary cardiomyocytes,transfected with ADSR-ATP6AP2 virus and control virus,PE was stimulated for at least 24h.SYTO/SYTOX fluorescence probe was used to observe and count cardiomyocytes under the fluorescence microscope.15.Total ROS and mitochondrial ROS were detected by DCFDA or MitoSoxThe groups are as follows:ShR-Scr,ShR-Scr+PE,ShR-ATP6AP2,ShR-ATP6AP2+PE;The effect of ATP6AP2 on PE-induced ROS was detected.Primary cardiomyocytes were transfected with Ad-ShR-ATP6AP2 virus with or without PE treatment.The fluorescence intensity was observed under a fluorescence microscope according to the instructions of the DCFDA or MitoSox method.16.Mitochondrial membrane potential changes were detected by TMREThe subgroups were as follows:ShR-Scr,ShR-Scr+PE,ShR-Scr+PE+Mito-tempo,ShRATP6AP2,ShR-ATP6AP2+PE,ShR-ATP6AP2+PE+Mito-tempo.To investigate the effect of ATP6AP2 on PE-induced mitochondrial membrane potential and whether mitochondrial ROS mediated this damage.17.Statistical methodsAll data were tested and statistically analyzed by SPSS28.X±SEM were used for counting data in accordance with normal distribution.Student T test was used for 2 groups of counting data consistent with normal distribution,and one-way ANOVA was used for 3 or more groups.Kruskal-wallis test and Dunn test were used for those who did not conform to normal distribution or had uneven variance.P<0.05 was considered statistically significant.Result(1)After TAC,the cardiac function of mice developed from compensatory hypertrophy to decompensated heart failure(8W),which was transitional at 4-6 weeks.Autophagy shifts from early compensatory activation to late inhibition.However,the expression of NLRP3-related protein gradually increased,and significantly increased at 8W.(2)ATP6AP2 expression began to increase on the 5th day after TAC operation,and it was statistically significant.Gradually increased,and still significantly increased at 8W.(3)Compared with Ad-ShR-Scr-TAC,the expression of NLRP3,Caspase1 and IL-18 in ad-ShR-ATP6AP2-TAC group was increased,and the concentration of IL-1β and TNF-α in serum was increased.(4)Compared with Ad-ShR-Scr-TAC,autophagy was blocked in Ad-ShR-ATP6AP2-TAC group,and LAMP2,SQSTM1/P62 were significantly increased.(5)Compared with Ad-ShR-Scr-TAC,Ad-ShR-ATP6AP2-TAC group showed increased fibrosis,increased myocardial hypertrophy,enlarged heart cavity,and significantly decreased cardiac systolic function.(6)Compared with Ad-ShR-Scr-TAC,mice in Ad-ShR-ATP6AP2-TAC group had more obvious cardiac hypertrophy,increased heart weight and increased myocardial cell crosssectional area.(7)Compared with WT-TAC,the expression of hypertrophic gene(Myh6/Myh7),systolic function,cardiac myocyte cross-sectional area and NLRP3 were not significantly different in tg-TAC group,but the expression of SQSTM1/P62 was significantly reduced.(8)The inhibitor oF NLRP3,MCC950,restored cardiac remodeling,reduced expression fibrosis,improved cardiac function,reduced cardiac hypertrophy,decreased expression of NLRP3 and Caspase1,and reduced apoptosis in the Ad-ShR-ATP6AP2-TAC group.Conclusion(1)The expression of ATP6AP2/PRR was significantly increased in stress-induced cardiac remodeling.(2)ATP6AP2/PRR regulates the late stages of autophagy in stress-induced cardiac hypertrophy and heart failure models.(3)ATP6AP2/PRR can reduce the activation and injury of NLRP3 and improve the cardiac remodeling induced by pressure load by promoting the patency of autophagy flow.(4)ATP6AP2/PRR plays a role in the late regulation of autophagy by affecting lysosomal acidification function.(5)ATP6AP2/PRR alleviates ROS and accumulation of dysfunctional mitochondria and NLRP3 activation by promoting patency of mitochondrial autophagy.