【作者】 周倩；

【导师】 何西淼；

【作者基本信息】 华中科技大学 ， 基础医学， 2024， 博士

【摘要】 背景与目的:扩张型心肌病(dilated cardiomyopathy,DCM)的特征为心室扩大和心脏收缩功能下降。由于DCM在早期往往没有明显的临床症状,大多数患者最终会发展到心力衰竭阶段。因此,对DCM患者进行早期诊断和干预,可以有效改善患者的生活质量。本研究旨在探索DCM的潜在生物标志物,并揭示其在DCM中的作用机制,以期为DCM的诊断和治疗提供新的思路和方法。方法:本研究通过分析公共数据库中健康个体和DCM患者左心室组织的转录组测序(RNA sequencing,RNA-seq)数据,揭示DCM的基因表达谱变化,并确定与DCM相关的生物标志物。本研究共收集22例健康对照和18例DCM患者的血清样本,运用酶联免疫吸附试验测定血清中生物标志物的水平,并评估其诊断效能。构建Frzb敲除(knockout,KO)小鼠,并在其10-12周龄时建立阿霉素(doxorubicin,Dox)诱导的DCM模型。通过对野生型(wild-type,WT)和Frzb KO小鼠的心脏进行心功能评估和组织病理学检测,揭示Frzb在Dox诱导的DCM模型中的作用。进一步,运用单细胞核转录组测序(single-nucleus RNA sequencing,snRNA-seq)、RNA-seq和全基因组亚硫酸氢盐测序(whole genome bisulfite sequencing,WGBS)技术,以及体外细胞干预实验深入研究FRZB在DCM中的作用机制。结果:(1)RNA-seq数据分析结果表明,细胞外基质相关基因与DCM之间存在紧密联系,其中FRZB在DCM患者的心脏组织中表达显著升高。(2)FRZB在DCM患者的血清中水平也显著升高;与常见的DCM诊断标志物,如BNP、NT-proBNP和sST2相比,FRZB展现出更优异的诊断性能;血清中FRZB水平与左心室直径大小呈正相关性,与左心室射血分数呈负相关性。(3)Frzb在Dox诱导的DCM模型小鼠心脏组织中的mRNA和蛋白水平均显著升高。生理盐水处理小鼠和Dox诱导小鼠心脏组织的snRNA-seq结果表明,Frzb在Dox诱导小鼠的心肌细胞中表达显著升高,与此同时,Ppargc1a的表达显著下降,氧化磷酸化(oxidative phosphorylation,OXPHOS)途径相关基因的表达显著下降。在Dox诱导的人源AC16心肌细胞损伤模型中,FRZB的mRNA和蛋白水平也显著升高,与此同时,PPARGC1A的mRNA和蛋白水平显著下降,以及OXPHOS途径相关基因的表达水平下降。(4)动物实验结果表明,Frzb KO小鼠的心功能相较于WT小鼠明显增强,且心脏萎缩程度、心肌纤维化程度、心肌细胞萎缩程度和心肌细胞凋亡均明显降低。WT和Frzb KO小鼠的心脏组织的snRNA-seq结果表明,一群心肌细胞特异性存在于Frzb KO小鼠中,并且该群细胞中Ppargc1a表达水平显著升高,OXPHOS途径相关基因表达水平也显著升高。降低AC16细胞中FRZB的表达水平后,PPARGC1A的表达水平显著升高,OXPHOS能力增强。(5)综合分析汉族人群的健康个体和DCM患者的WGBS和RNA-seq数据,揭示FRZB基因体区域的高甲基化水平可能促进其在DCM中表达升高。结论:FRZB能够作为DCM潜在诊断标志物,且具有良好的诊断效能。FRZB通过抑制心肌细胞中PPARGC1A的表达,从而抑制线粒体OXPHOS途径,最终导致DCM的发生和发展。在DCM中,FRZB的表达水平与其DNA甲基化水平密切相关。更多还原

【Abstract】 Background and Objectives:Dilated cardiomyopathy(DCM)is characterized by ventricular enlargement and decreased cardiac contractile function.Since DCM often presents without obvious clinical symptoms in its early stages,most patients eventually progress to heart failure.Therefore,early diagnosis and intervention for DCM patients can effectively improve their quality of life.This study aims to explore potential biomarkers for DCM and elucidate the mechanisms of the disease,with the goal of providing new insights and approaches for the diagnosis and treatment of DCM.Methods:This study utilized the RNA sequencing(RNA-seq)data from left ventricular tissues of healthy individuals and DCM patients available in public databases to identify biomarkers associated with DCM.A total of 22 healthy controls and 18 DCM patients’serum samples were collected for this study.The levels of biomarkers in the serum were measured using enzyme-linked immunosorbent assay and their diagnostic efficacy was evaluated.Frzb knockout(KO)mice were constructed,and a doxorubicin(Dox)-induced DCM model was established in these mice at 10-12 weeks of age.Cardiac function assessment and histopathological examination of the hearts of wild-type(WT)and Frzb KO mice were performed to reveal the role of Frzb in the Dox-induced DCM model.Furthermore,single-nucleus RNA sequencing(snRNA-seq),RNA-seq,and whole genome bisulfite sequencing(WGBS)techniques,along with in vitro cell intervention experiments,were used to investigate the mechanism of FRZB in DCM.Results:(1)RNA-seq data analysis revealed a close association between extracellular matrix-related genes and DCM,with FRZB expression significantly elevated in the cardiac tissues of DCM patients.(2)The level of FRZB was also significantly elevated in the serum of DCM patients.Compared to common DCM diagnostic markers such as BNP,NT-proBNP,and sST2,FRZB demonstrated superior diagnostic performance.The serum level of FRZB was positively correlated with left ventricular diameter and negatively correlated with left ventricular ejection fraction.(3)In the hearts of Dox-induced DCM model mice,the mRNA and protein levels of Frzb were significantly elevated.snRNA-seq results from saline-treated and Dox-induced mice indicated that Frzb expression was significantly increased in the cardiomyocytes of Dox-induced mice,while the expression of Ppargc1a was significantly decreased,along with a marked reduction in the expression of genes related to the oxidative phosphorylation(OXPHOS)pathway.In the Dox-induced AC16 cardiomyocyte injury model,the mRNA and protein levels of FRZB were significantly elevated,whereas the mRNA and protein levels of PPARGC1A were significantly decreased,as was the expression of genes associated with the OXPHOS pathway.(4)Animal experiment results showed that the cardiac function of Frzb KO mice was significantly improved compared to WT mice.Additionally,the extent of cardiac atrophy,myocardial fibrosis,myocardial cell atrophy,and cardiomyocyte apoptosis were all significantly reduced in Frzb KO mice.snRNA-seq results of cardiac tissues from WT and Frzb KO mice revealed a specific group of cardiomyocytes present in Frzb KO mice,with significantly elevated expression levels of Ppargc1a and genes related to the oxidative phosphorylation(OXPHOS)pathway.Reducing FRZB expression in AC16 cells resulted in a significant increase in PPARGC1A expression and enhanced OXPHOS capacity.(5)Comprehensive analysis of WGBS and RNA-seq data from healthy individuals and DCM patients in the Han population revealed that high methylation levels in the FRZB gene body may promote its expression in DCM.Conclusion:FRZB may serve as a potential diagnostic biomarker for DCM for its good diagnostic performance.FRZB inhibits the expression of PPARGC1A in cardiomyocytes,suppressing the mitochondrial OXPHOS pathway,and ultimately leading to the occurrence and progression of DCM.The expression level of FRZB in DCM is closely related to its DNA methylation level.更多还原