【作者】 李蕾；

【导师】 董波；

【作者基本信息】 山东大学 ， 心血管内科学， 2022， 博士

【摘要】 研究背景心力衰竭是由各种心脏结构和功能性疾病导致心脏充盈和/或射血功能受损,心脏排血量不能满足机体组织代谢需要,引起肺循环或者体循环淤血,器官组织血液灌注不足为临床表现的一组综合征。在过去的20年里,心力衰竭的药物治疗和器械治疗显著改善了低射血分数心力衰竭患者的症状并延缓了心衰进展,但这些患者的总体预后仍然相对较差,5年内死亡率接近50%,因此,新途径的识别和对潜在治疗靶点的有效干预可能减缓心衰疾病的进展。心衰的发病机制涉及氧化应激、线粒体功能障碍、炎症、神经体液内分泌系统激活、心脏重构等多个方面,其中炎症在心脏重构中发挥着关键的作用。不管何种类型的心衰,其血清中炎症细胞因子均明显升高,且与不良的临床结局存在明显的相关性。NLRP3是细胞内的炎症信号平台,它是一种关键的多蛋白信号传导平台,通过调控caspase1的裂解和活化,严格控制炎症反应并协调宿主防御,导致IL-1β,IL-18前体裂解为成熟片段。已有的研究表明NLRP3炎性小体的活化促进动脉硬化、高血压、糖尿病等多种心血管疾病的发病机制及心脏重塑。自噬是进化上高度保守的细胞内生物过程,通过清除衰老、功能异常的细胞器、损伤或错误折叠蛋白质,在溶酶体内将其降解,转化为被机体可重新利用的物质,还通过破坏侵袭细胞的病原体发挥保护作用。对于维持细胞稳态至关重要。炎症小体的异常激活与强烈的炎症相关,导致炎症性疾病的发展。因此,炎症小体必须在适当的强度下激活,以防止感染和避免组织损伤。大量研究表明,自噬相关蛋白的敲除会引起炎症小体的异常激活,导致严重的组织损伤。相反,自噬诱导剂可改善炎症小体相关疾病的症状。ATP6AP2/PRR广泛分布在体内多个组织,肾脏,心脏,脑,眼,胎盘和免疫系统,它参与多个重要的生理过程,比如细胞周期,自噬,酸碱平衡,能量代谢,胚胎发展,T细胞稳态,水钠平衡,血压调控,心脏重塑和维持足细胞结构等。也有报道PRR参与糖尿病纤维化,高血压,糖尿病微血管病变,急性肾损伤,心血管疾病等多种病理情况,但是部分结论存在较多不一致。目前多个研究表明生理情况下ATP6AP2/PRR功能缺失后导致胞浆内细胞核周围积累了大量的、多个囊泡融合性结构,包含部分消化或未消化的细胞成分如线粒体,并伴随着自噬相关蛋白如LC3B,SQSTM1/P62及对氨基酸饥饿反应的基因表达增加如Asna、Atf4、Gadd153等,并伴随着其他严重表型如严重心衰、心脏纤维化并伴有致死性。说明ATP6AP2/PRR调节的自噬对于细胞稳态有重要作用。结构上ATP6AP2/PRR作为vacuolar-ATPase的关键组成部分,在溶酶体降解和自噬中发挥了重要作用。综上由于Atp6ap2/PRR的缺失导致严重的胞内稳态破坏及细胞死亡,目前对ATP6AP2/PRR的理解并不很深入,仍需要大量的探索。目前ATP6AP2/PRR在压力负荷应激诱导的心衰中并无相关研究。由于高血压、瓣膜钙化病变如主动脉瓣狭窄等压力负荷增加的临床疾病最终导致心衰,炎症在心衰的发病及进展发挥着重要作用。我们推测ATP6AP2/PRR可以通过介导自噬,调控NLRP3,在压力负荷诱导的心衰中发挥关键的调控作用。因此我们提出以下科学问题:在压力负荷诱导的心脏重构、心衰进展中,ATP6AP2/PRR的表达如何改变?自噬及NLRP3在压力负荷诱导的心脏重构中是如何变化的?ATP6AP2是否与NLRP3存在调控关系?ATP6AP2通过哪种机制调控NLRP3在心衰的进展中发挥作用。研究目的1.构建压力负荷诱导的心衰模型,观察ATP6AP2/PRR在TAC小鼠心肌组织中的表达情况。2.构建ATP6AP2/PRR过表达基因鼠,构建靶向ATP6AP2/PRR的腺病毒敲减片段并体内注射,观察ATP6AP2/PRR对压力负荷应激诱导的心脏重构及心功能的影响。3.探讨ATP6AP2/PRR对压力负荷应激诱导的心脏重构及心功能影响的机制。研究方法1.构建野生小鼠TAC模型,模拟压力负荷诱导的心衰模型(1)用C57BL/6J小鼠构建TAC模型,在sham组及TAC术后不同时间点5day,7day,14day,28day,56day检测重塑和自噬、NLRP3的变化。涵盖从代偿性肥厚阶段,至失代偿性心衰阶段。(2)以C57BL/6J为背景,采用CRISPER-Cas9技术构建ATP6AP2/PRR基因敲入小鼠(Cas9-PRR-KI 小鼠)和 ATP6AP2/PRR 的 floxed 小鼠,并与 α-MHC-Cre 小鼠杂交,及构建出心肌细胞特异性的ATP6AP2/PRR过表达小鼠。通过基因型鉴定,挑选8-12周大小的纯合子小鼠(Tg)和同窝对照野生鼠构建TAC模型。(3)构建腺病毒为载体的靶向ATP6AP2/PRR的敲减病毒(Ad-shR-ATP6AP2)即和对照空载体病毒Ad-shR-Scr,注射到野生小鼠体内(109PFU/只),构建TAC模型,并于2周后补充注射一次相同剂量的病毒。2.动物实验药物干预处理及分组检测 TAC 早期自噬流改变:sham,sham+BafA1,TAC-5day,TAC-5day+BafA1;MCC950 动物回复实验:Ad-shR-Scr-TAC,Ad-shR-Scr-TAC+MCC950,Ad-shR-ATP6AP2-TAC,Ad-shR-ATP6AP2-TAC+MCC9503.一般检测指标小鼠体重、心脏重量、肺重量、肝脏重量、胫骨长度等4.心脏超声野生小鼠模型,在TAC后不同时间点行心脏超声检测左室收缩和舒张功能,并检测心室内径、室壁厚度等,确定代偿、失代偿性阶段。基因过表达小鼠及对照组构建的TAC模型、转染Ad-shR-ATP6AP2及对照组Ad-shR-Scr腺病毒后构建的TAC模型于4周时检测心功能相关参数。5.组织病理学检测(1)免疫组化相关蛋白表达:胶原蛋白1(Co11)、胶原蛋白3(Col3),ATP6AP2/PRR,NLRP3,caspase-1,IL-1β、IL-18.(2)Masson检测心肌组织纤维化改变(3)免疫组织荧光检测:SQSTM1/P62、NLRP3、ATP6AP2/PRR的组织荧光共定位,SQSTM1/P62,LAMP2蛋白免疫荧光检测(4)HE检测:心肌组织切面上心肌细胞横截面积检测6.ELISA 检测:血清中IL-1 β、TNF-α的表达水平检测7.western blot 检测:组织和细胞中的 ATP6AP2/PRR,NLRP3,Caspase1,LC3A/B,SQSTM1/P62,Atg58.PCR检测心肌组织和原代心肌细胞中检测ATP6AP2/PRR,Myh6,Myh7,IL-1β,IL-189.TUNEL检测凋亡用TUNEL法检测TAC诱导的心脏重塑过程中、敲减病毒注射后的TAC鼠、MCC950抑制剂处理的模型组中心肌细胞凋亡的变化。10.原代心肌细胞提取并给予苯肾上腺素(PE)刺激构建压力负荷应激的体外细胞模型用差速贴壁法从1-3天的大鼠乳鼠心脏中提取原代心肌细胞,鉴定纯度,于3天后开始相关实验。先给予苯肾上腺素(PE,100 μ M)刺激不同时间梯度,用western检测ATP6AP2/PRR、NLRP3的表达变化,为后续ATP6AP2的干预时间点提供依据。11.mCherry-GFP-LC3B自噬双荧光病毒检测ATP6AP2对于自噬流的影响分组如下:shR-Scr,shR-Scr+BafA1,shR-ATP6AP2,shR-ATP6AP2+BafA1原代心肌细胞用溶酶体抑制剂BafA1预处理后,转染Ad-shR-ATP6AP2病毒和mCherry-GFP-LC3B自噬双荧光病毒,荧光显微镜下观察。通过统计自噬小体(黄色斑点)和自噬溶酶体(红色斑点)比例及变化,检测ATP6AP2对自噬流的影响。12.溶酶体探针Lyso-Tracker检测ATP6AP2对溶酶体PH的影响分组如下:shR-Scr,shR-Scr+BafA1,shR-ATP6AP2,shR-ATP6AP2+BafA1原代心肌细胞用溶酶体抑制剂BafA1预处理原代心肌细胞后,转染Ad-shR-ATP6AP2病毒,溶酶体探针Lyso-Tracker处理后荧光显微镜下观察并统计荧光强度。13.透射电镜观察胞内线粒体自噬形态改变原代心肌细胞转染过表达、敲减病毒及对照病毒,相应的用PE刺激不同时间,在透射电镜下观察不同时间的PE及ATP6AP2对线粒体自噬的影响。14.SYTO/SYTOX荧光探针检测心肌细胞存活分组 1如下:shR-Scr,shR-Scr+PE,shR-ATP6AP2,shR-ATP6AP2+PE;检测ATP6AP2对PE诱导的心肌细胞死亡的影响。原代心肌细胞用转染Ad-shR-ATP6AP2病毒及对照病毒后PE刺激至少24h,SYTO/SYTOX荧光探针处理后荧光显微镜下观察并计数。分组 2 如下:shR-Scr+PE,shR-Scr+PE+MCC950,shR-ATP6AP2+PE,shR-ATP6AP2+PE+MCC950;检测ATP6AP2对心肌细胞死亡的影响是否与NLRP3有关。MCC950预处理原代心肌细胞,转染Ad-shR-ATP6AP2病毒及对照病毒后PE刺激至少24h,SYTO/SYTOX荧光探针处理后荧光显微镜下观察并计数。15.DCFDA或者MitoSox检测总ROS和线粒体ROS分组如下:shR-Scr,shR-Scr+PE,shR-ATP6AP2,shR-ATP6AP2+PE;检测 ATP6AP2对PE诱导的ROS的影响。原代心肌细胞转染Ad-shR-ATP6AP2病毒,加或者不加PE处理,参照DCFDA或者MitoSox法说明书,并用荧光显微镜下观察并统计荧光强度。16.TMRE检测线粒体膜电位改变分组如下:shR-Scr,shR-Scr+PE,shR-Scr+PE+Mito-Tempo,shR-ATP6AP2,shR-ATP6AP2+PE,shR-ATP6AP2+PE+Mito-Tempo。检测 ATP6AP2 对 PE 诱导线粒体膜电位的影响,及是否通过线粒体ROS介导了这种损伤。17.统计学方法所有数据用SPSS28进行检验和统计分析。计数资料符合正态分布的用均数±标准误(X±SEM)。2组计数资料符合正态分布者用studentt检验,3组或以上这用one-way ANOVA。对于不符合正态分布者或者方差不齐者用Kruskal-Wallis检验及Dunn检验。P<0.05认为有统计学差异。实验结果(1)TAC术后小鼠心功能由代偿性肥厚阶段,最终发展为失代偿性心衰阶段(8w),在4-6周为过度阶段。自噬由早期的代偿性活化,转为后期的抑制。而NLRP3相关蛋白的表达逐渐升高,在8w时明显增加。(2)TAC术后第5day时ATP6AP2的表达即开始升高,差异有统计学意义。逐渐升高,8w时仍明显升高。(3)与 Ad-shR-Scr-TAC 相比,Ad-shR-ATP6AP2-TAC 组小鼠组织中 NLRP3,caspase1、IL-18的表达增加,血清IL-1 β、TNF-α的浓度升高。(4)与 Ad-shR-Scr-TAC 相比,Ad-shR-ATP6AP2-TAC 组小鼠自噬被阻断,LAMP2,SQSTM1/P62 明显增加。(5)与Ad-shR-Scr-TAC相比,Ad-shR-ATP6AP2-TAC组小鼠纤维化加重,心肌肥厚增加,心腔扩大,心脏收缩功能明显降低。(6)与Ad-shR-Scr-TAC相比,Ad-shR-ATP6AP2-TAC组小鼠的心肌肥厚较明显,心脏重量增加,心肌细胞横截面积增加。(7)与WT-TAC相比,Tg-TAC组小鼠中肥厚基因(Myh6/Myh7)表达、收缩功能、心肌细胞横截面积、NLRP3表达差异不明显,但是明显减轻了 SQSTM1/P62的表达。(8)NLRP3的抑制剂MCC950回复了 Ad-shR-ATP6AP2-TAC组小鼠的心脏重构,表现力纤维化减轻,心功能改善,心肌肥厚减轻,NLRP3及caspase1表达减少,凋亡减轻。结论(1)ATP6AP2/PRR在压力负荷诱导的心脏重塑中表达明显增加。(2)ATP6AP2/PRR在压力负荷诱导的心肌肥厚和心衰模型中,调控自噬的晚期阶段。(3)ATP6AP2/PRR可以通过促进自噬流的通畅,减少NLRP3活化及损伤,改善压力负荷诱导的心脏重构。(4)ATP6AP2/PRR通过影响溶酶体酸化功能在自噬的晚期调控中发挥作用。(5)ATP6AP2/PRR通过促进线粒体自噬的通畅,减轻ROS和减少功能异常的线粒体积累,减轻NLRP3活化。更多还原

【Abstract】 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.更多还原