组蛋白甲基转移酶SETDB1在心脏重构中的作用及分子机制研究

Cardiac remodeling is the pathologic basis of several heart diseases, and also one of the most important risk factor of heart failure. However, the regulatory mechanisms of cardiac remodeling have not yet been fully elucidated. Previous studies indicated that protein methylation closely related to cardiac remodeling, while the role and mechanisms of SETDB1, a kind of histone methyltransferase, on cardiac remodeling remains unknown. Therefore, in our present proposal, we will investigate this scientific issue through four parts of experiments. Firstly, we will detect the expression pattern of SETDB1 during cardiac remodeling and unravel the upstream regulatory mechanism responsible for upregulated SETDB1 expression. Secondly, primarily cultured neonatal rat cardiomyocytes (NRCMs) and fibroblasts were infected with lenti-shRNA, lenti-shSETDB1, lenti-GFP or lenti-SETDB1 lentivirus to knockdown or overexpression of SETDB1, respectively. Then, these cells were treated with Ang II or TGFβ to induce cellular disease model to investigate the role of SETDB1 on NRCMs and fibroblasts. Thirdly, in order to ascertain the function of SETDB1 on cardiac remodeling, the cardiac-specific SETDB1 knockout or overexpression mice were subjected to transverse aortic constriction (TAC) surgery to induce cardiac remodeling. Finally, the methylation of H3K9 and non-histone protein were detected in SETDB1 knockdown or overexpression cells or mice to clarify the mechanism of SETDB1 on cardiac remodeling. This proposal tries to provide therapeutic target or direction for clinicians to remedy cardiac remodeling.

心脏重构是多种心脏疾病的病理基础，也是导致心力衰竭的重要危险因素，但其调节机制尚未完全明确。研究提示，蛋白质的甲基化修饰与心脏重构有密切联系，而组蛋白甲基转移酶SETDB1对心脏重构的影响及其分子机制尚无相关报道。因此，本项目拟通过四部分研究内容阐明SETDB1对心脏重构的影响及其机制。首先拟检测SETDB1在心脏重构过程中的表达模式及其上游调控机制；其次，通过在原代大鼠心肌细胞和成纤维细胞中过表达或沉默SETDB1，并建立细胞模型，研究SETDB1对体外培养的心肌细胞和成纤维细胞的影响；再次，通过构建心脏特异性SETDB1基因敲除或过表达小鼠，并采用主动脉缩窄术建立心脏重构模型，明确SETDB1对小鼠心脏重构的影响；最后，通过检测心脏重构过程中SETDB1对组蛋白H3K9以及非组蛋白的甲基化阐明其影响心脏重构的分子机制。以期为临床干预心脏重构提供新的靶点和方向。

心脏重构（Cardiac remodeling，CR）是多数心脏疾病晚期发展为心力衰竭的重要病理机制，极大加速了疾病进程，限制了心血管疾病患者的治疗预后。因此，深入了解心力衰竭背后心脏重构独特的细胞及分子机制，进而为研发治疗心力衰竭的药物提供新思路和新靶点，是心胸外科领域亟待解决的关键问题。组蛋白甲基化修饰作为重要的表观遗传学修饰之一在心血管领域中的作用被广泛认可，本项目从分子、细胞以及在体动物水平系统的阐释了组蛋白甲基转移酶SETDB1在心脏重构中的表达模式和调控作用。首先，本项目组收集了临床扩心病患者左心室前壁心肌组织，H&E和PSR染色可见病变组织中心肌细胞排列紊乱、心肌纤维断裂和纤维化显著；进一步的免疫荧光染色及Western blot显示病变心肌组织中SETDB1表达水平相较正常组织显著上调。进一步地，本项目组构建了心肌特异性SETDB1转基因和敲除小鼠，并通过TAC手术建立了心肌肥厚小鼠模型，术后小鼠心脏超声结果显示，TAC手术后小鼠LVIDs、LVIDd上升而EF%和FS%减低提示小鼠心功能损害严重，SETDB1转基因小鼠进一步加重了以上心功能指标的偏移，而敲除SETDB1则有效改善了TAC手术介导的心功能下降；解剖取材后可见，TAC手术使得小鼠心脏、肺脏明显增大，心重/体重（HW/BW）、肺重/体重（LW/BW）增加即发生了严重的心肌肥厚和心源性肺水肿，而SETDB1转基因小鼠HW/BW、LW/BW上升更为显著，相反SETDB1敲除后心脏和肺脏病理改变得到明显逆转；组织病理学角度来看，小鼠心肌病理切片H&E染色提示与单纯手术组相比，SETDB1转基因小鼠在TAC术后心肌结构紊乱和心肌纤维断裂程度更剧，WGA细胞膜染色以及定量结果显示其平均单个细胞面积显著大于单纯手术组，而敲除SETDB1则明显改善了负荷下的心肌病理结构变化，逆转了心肌细胞病理性肥大。其次，体外研究中，我们构建了SETDB1过表达/敲低质粒并导入大鼠原代心肌细胞，α-actinin免疫荧光染色显示SETDB1在细胞层面促进了AngII诱导的心肌细胞肥厚，而敲低SETDB1则有效逆转了此过程。本项目的研究成果基本阐述了SETDB1在心脏重构中的表达模式和调控作用，提出了心脏重构与表观遗传学修饰的重要联系，为今后的研究提供了新的思路，并有望在心力衰竭疾病预防和治疗中实现转化应用。

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