【作者】 李文静；

【导师】 夏豪；

【作者基本信息】 武汉大学 ， 临床医学 内科学， 2021， 博士

【摘要】 研究背景:心肌肥厚是心脏血流动力学在应激状态下产生的缓慢但较有效的代偿功能,在代偿初期,心肌细胞体积增大,蛋白质合成增多,心脏重量增加,以此来减轻内外界刺激对心脏造成的损伤,但如果刺激不消除,在持续一段时间后,则会导致心肌肥厚的失代偿,表现为心肌细胞凋亡或坏死,心肌组织胶原生成增加,室壁顺应性及应变力下降,造成心功能不全,最终发展为心力衰竭。而心力衰竭是大多数心血管疾病的终末阶段,其发病率、病死率和再住院率一直较高。因此研究影响心肌肥厚的发生发展因素并对其进行干预对于降低心力衰竭的发病率具有重要意义。自噬是一种溶酶体降解途径,能够清除未折叠蛋白、受损的DNA及细胞器,并将其降解为新的代谢物,发挥其对生存、分化、发育和体内平衡的作用。其中,Atg7作为调控自噬的关键因子,能够与Atg10或Atg3共同作用,促进LC3I向LC3II的转化,激活自噬。越来越多的研究表明,自噬与心肌肥厚的关系密切,但作为自噬的关键调节因子,Atg7与压力负荷诱导的心肌肥厚的关系及机制仍不明确。研究目的:探究Atg7在压力负荷诱导的心肌肥厚中的作用并探索其具体作用机制研究方案:第一部分人体心衰标本来源于心脏移植中的终末期心衰患者,对照心脏来自因意外如车祸丧生的捐赠者;动物实验则利用8周龄,体重在25-27g的C57BL/6背景小鼠,通过对其进行假手术和主动脉缩窄术构建心肌肥厚模型,并于术后不同时间取材检测;细胞实验通过提取1-3天的大鼠乳鼠心肌细胞并对其进行PE刺激诱导心肌细胞肥大,在这三种模型中,我们分别通过Western Blot和RT-PCR检测了对照组与实验组中Atg7的蛋白和mRNA的表达水平。第二部分动物实验:选取8周龄,体重25-27g,经过基因鉴定及四环素诱导的Atg7-CTG小鼠及同窝出生的非Atg7转基因小鼠(即WT小鼠),通过主动脉缩窄术构建心肌肥厚模型,根据手术方式和小鼠类型将其分为4组,分别是Sham+WT组、Sham+Atg7-CTG组、AB+WT组和AB+Atg7-CTG组,手术4W后进行超声心动图和血流动力学检测LVEF、FS、LVEDd、LVEDs、dp/dt max等指标,通过病理染色检测心肌细胞横截面积变化及心肌纤维化的情况,通过RT-PCR来检测不同刺激对心肌肥厚(ANP、BNP、β-MHC)和心肌纤维化(Collagen I、Collagen III、CTGF)相关基因的影响。细胞实验:SD背景的大鼠互相交配繁殖得到1-3d的乳鼠,然后利用乳鼠进行心肌细胞的提取,并用PE刺激模拟心肌肥厚模型,心肌细胞过表达Atg7基因则通过转染腺病毒来实现,细胞实验分为4组,分别是:GFP+PBS、Ad-Atg7+PBS、PE和PE+Ad-Atg7组,在PE刺激24h后通过α-actinin染色观察PE刺激及Atg7过表达对心肌细胞的影响,通过提取蛋白和RT-PCR定量分析肥厚相关指标(ANP、BNP和β-MHC)的变化。第三部分应用高通量二代测序技术对AB术后4W的WT和Atg7-CTG两组小鼠心脏组织进行转录组测序,并通过对差异表达基因GO富集和KEGG富集分析来确定Atg7对心肌肥厚的作用机制。第四部分动物实验中构建p53-CKO+Atg7-CTG双基因小鼠并将其分为4组:p53-CK O+Sham、p53-CKO+Atg7-CTG+Sham、p53-CKO+AB和p53-CKO+Atg7-CTG+A B组,通过心功能检测、病理染色、通路蛋白检测和RT-PCR定量实验来验证A tg7对心肌肥厚的作用机制。细胞实验利用Adshp53,对原代大鼠心肌细胞进行刺激,将细胞实验分为4组,分别是Adshp53+PBS、Adshp53+Ad-Atg7+PBS、dsh P53+PE和Adsh P53+Ad-Atg7+PE组,PE刺激24h后进行α-actinin的染色及心肌肥厚指标ANP、B NP和β-MHC的定量分析。研究结果:第一部分Western Blot结果显示,心力衰竭患者心肌组织中,Atg7的表达显著增加,且mRNA的转录水平也显著升高;与Sham组相比,AB术后Atg7的表达显著增加,且术后8W比4W的表达更高;细胞实验中,PE刺激的心肌细胞中Atg7的表达显著高于PBS组。第二部分超声结果显示,AB术后,小鼠的心功能显著下降,表现为LVEF和FS显著降低,LVEDd和LVEDs则显著增加,与WT小鼠AB术后相比,心脏特异性过表达Atg7加重心功能的恶化;取材结果分析可得,与Sham组相比,AB术后HW/BW和HW/TL均显著增加,与WT+AB组相比,Atg7-CTG+AB组HW/BW和HW/TL显著增加。HE染色结果显示,AB术后,心肌细胞横截面积显著增加,PSR染色结果显示,心肌血管周围和间质胶原沉积增加,RT-PCR检测结果显示ANP、BNP和β-MHC的转录水平明显增加,Collagen I、Collagen III、CTGF的表达均显著增加,表明心肌肥厚和纤维化均显著增加,心脏特异性过表达Atg7后心肌肥厚和心肌纤维化更加明显。细胞α-actinin染色和RNA定量结果显示,心肌细胞加入PE培养24h后,心肌细胞面积显著增加,且ANP、BNP和β-MHC的mRNA水平显著升高,Ad-Atg7和PE共同刺激心肌细胞后,其心肌细胞面积和肥厚指标表达水平与PE单独刺激相比更高。第三部分对WT组和Atg7-CTG两组的高通量二代测序结果显示,在筛选条件为|Log1.2Fold Chage|>1,P-value<0.05的情况下,两组间的表达有差异的基因共2550个,把WT作为对照组,其中表达增加的基因有1319个,表达降低的基因有1231个,GO富集可得差异基因富集的GO term有3059个,KEGG富集得到的pathway有114个。第四部分同第一部分结果一致,与Sham组相比,AB术后心功能下降,心肌肥厚和心肌纤维化增加,但与p53-CKO小鼠AB术后心功能进行比较,可发现Atg7过表达及p53-CKO的双基因小鼠与之并无差异。研究结论:1.人、小鼠和大鼠心肌细胞中均有Atg7的表达,且与对照组相比,心肌肥厚模型中Atg7的表达显著增加。2.主动脉缩窄术可致心功能降低、心肌肥厚和心肌纤维增加,心脏特异性过表达Atg7可加重心功能降低和增加心肌肥厚和心肌纤维化;与单独PE刺激心肌细胞相比,腺病毒Atg7和PE共同刺激心肌细胞可使心肌细胞肥大更加显著。3.WT和Atg7-CTG小鼠的差异表达基因有2550个,GO富集和KEGG分析显示过表达Atg7可以激活p53信号通路,增加细胞凋亡和线粒体分裂,抑制线粒体自噬。4.敲除小鼠心肌细胞和沉默原代大鼠心肌细胞中p53的表达可以改善过表达Atg7对心肌肥厚的影响。更多还原

【Abstract】 Background:Cardiac hypertrophy is an adaptive compensatory response of cardiac hemodynamics in response to various physiological or pathological stimuli.In the early stage of compensation,the volume of myocardial cells,protein synthesis and heart weight increases,which reduce the damage caused by various stimulus.However,if the stimulation is not eliminated,it will lead to decompensation of cardiac hypertrophy after a period of time,which is characterized by cardiomycyte apoptosis or necrosis,cardiac fibrosis,dicreased ventricular wall compliance and strain force,resulting in heart failure.Heart failure is the end stage of most cardiovascular diseases,and its incidence rate,mortality rate and rehospitalization rate have been higher.Therefore,studying the factors that influence the development of cardiac hypertrophy is of great significance in reducing the incidence rate of heart failure.Autophagy is a lysosome degradation pathway.It is a process that removes unfolded proteins,damaged DNA and organelles,which are degraded into new metabolities.Therefore,autophagy plays an improtant role in survival,differentiation,development and homeostasis of body.As a key factor in regulating autophagy,Atg7 interacts with atg10 or Atg3 to complete the transformation process of LC3 I to lc3 II.More studies have shown that autophagy is closely related to cardiac hypertrophy,but the relationship and mechanism between Atg7 and pressure-overload induced cardiac hypertrophy are still unclear.Objective:Exploring the effect of Atg7 on pressure-overload induced cardiac hypertrophy and its mechanisms.Methods:Part one:Left ventricular tissues of failing human hearts were obtained from patients with end-stage heart failure during heart transplantation.Normal heart specimens were procured from donors who died in accidents such as car accidents.In animal experiments,8-week-old C57 BL / 6 background mice weighing 25-27 g were used to establish cardiac hypertrophy models with sham operation and aortic banding.The samples were collected at different time after operation.In cell experiment,1～3-day-old neonatal rat cardiomyocytes were collected and cultured.In these three models,the protein and mRNA expression levels of Atg7 were detected by Western Blot and RT-PCR.Part two:Experimental mice contained WT and Atg7-CTG mice.WT mice was born in the litter with Atg7-CTG mice but didn’t overexprssed Atg7.The two kinds of mice which growed up to 8-week-old,weighing 25-2g,and was oral tetracycline.After that,they underwent Aortic Banding to establish cardiac hypertrophy model.According to the operation mode and mouse type,they were divided into four groups,namely WT+sham group,Atg7-CTG +Sham group,AB + WT group and AB +Atg7-CTG group.The changes of cross-sectional area of cardiomyocytes and cardiac fibrosis were detected by pathological staining.The effects of different stimulation on genes of cardiac hypertrophy(ANP,BNP,β-MHC)and fibrosis(collagen I,collagen III,CTGF)were determined by quantitative analysis of mRNA level.Cell experiment: 1～3-day-old neonatal rat cardiomyocytes were collected and stimulated with PE to induce cardiomyocyte hypertrophy in vitro.The overexpression of Atg7 gene in cardiomyocytes was achieved by adenovirus transfection.Cell experiment was divided into four groups: GFP +PBS group,Ad-Atg7+PBS group,PE group and PE + Ad-Atg7 group.After PE stimulation for 24 hours,the effects of PE stimulation and Atg7 virus on cardiomyocytes were observed by α-actinin staining.The changes of ANP,BNP and β-MHC were analyzed by RT-PCR.Part three:The WT and Atg7-CTG groups that underwent AB for four weeks were sequenced by high-throughput second-generation sequencing technology,and the mechanism of Atg7 on cardiac hypertrophy was determined by analyzing the differentially expressed genes.Part four:In animal experiments,we constructed p53-CKO + Atg7-CTG double gene mice and divided them into four groups: p53-CKO+Sham,p53-CKO + Atg7-CTG+Sham,p53-CKO + AB and p53-CKO + Atg7-CTG + AB groups.The mechanism of Atg7 on cardiac hypertrophy was verified by cardiac function detection,pathological staining and RT-PCR.The cell experiment was divided into four groups: Adshp53+PBS,Adshp53 +Ad-Atg7+PBS,Adshp53+ PE and Adshp53 + ad Atg7 + PE groups.α-actinin staining and quantitative analysis of ANP,BNP and β-MHC were performed 24 hours after PE stimulation.Results:Part one:Western Blot results showed that the expression of Atg7 was significantly increased in heart failure patients,and the mRNA level was also significantly increased.Compared with sham group,the expression of Atg7 was significantly increased after AB,and the expression was higher at 8W than 4W;In cell experiment,the expression of Atg7 in PE stimulated cardiomyocytes was significantly higher than that in PBS group.Part two:The results of echocardiogram showed that the cardiac function of mice after AB operation was significantly decreased,which showed that LVEF and FS were significantly decreased,while LVEDd and LVEDs were significantly increased.Compared with WT mice after AB operation,the cardiac specific overexpression of Atg7 deteriorated pressure-overload induced cardiac function dysfunction.Compared with sham group,the HW /BW and HW/ TL after AB operation were significantly increased,and the cardiac specific overexpression Atg7 made these ratios more higher after AB.The results of HE and PSR staining showed that the cross-sectional area of cardiomyocytes increased significantly after AB operation,and the cardiac fibrosis increased significantly.The results of mRNA detection showed that the transcription levels of ANP,BNP and β-MHC increased significantly,and Collagen I,Collagen III,CTGF also increased significantly.Cardiac hypertrophy and fibrosis were more obvious after Atg7 overexpression.The results of α-actinin staining and mRNA quantification showed that the area of cardiomyocytes was significantly increased after PE stimulation for 24 h,and the mRNA levels of ANP,BNP and β-MHC were significantly increased.After Ad-Atg7 and PE stimulation,the area of cardiomyocytes was higher than those stimulated by PE alone.Part three:The results of high-throughput second-generation sequencing showed that 2550 genes were differentially expressed between WT group and Atg7-CTG group under the condition of | log1.2foldchage |>1 and p-value < 0.05.WT group was used as control group.331 genes were up-regulated and 415 genes were down regulated.In addition,there were 3059 GO terms and 114 pathways enriched by GO and KEGG enrichment analysis.Part four:Compared with sham group,cardiac function of AB group decreased,cardiac hypertrophy and fibrosis increased.But compared with p53-CKO mice,there was no difference in Atg7-CTG+p53-CKO group.Conclusions:1.The expression of Atg7 in different cardiac hypertrophy models was significantly higher than that in the control group.2.AB can lead to deterioration of cardiac function,increase of cardiac hypertrophy and fibrosis.Cardiac specific overexpression Atg7 can further deteriorate cardiac function and increase cardiac hypertrophy and fibrosis.Compared with PE stimulated cardiomyocytes,Ad-Atg7 and PE stimulated cardiomyocytes can make cardiomyocyte hypertrophy more significantly.3.There were 2550 differentially expressed genes between WT and Atg7 CTG mice.GO and KEGG enrichment analysis showed that the mechanism of Atg7 on cardiac hypertrophy may be related to p53 signaling pathway.4.Cardiomyocyte-specific knockou p53 can improve the effect of Atg7 overexpression on cardiac hypertrophy.更多还原