转录因子锌指蛋白91(ZFP91)在肥厚性心肌病发病过程中的作用及其机制研究

Hypertrophic cardiomyopathy (HCM) is a common heart disease and some studies have shown that HCM is associated with gene mutation of some transcriptional factors. Zinc finger protein 91（ZFP91）belongs to the zinc finger family. ZFP91 protein contains the classical zinc finger domain, C2H2 that was found in numerous nucleic acid-binding proteins. ZFP91 may regulate the expression of many downstream target genes. We deleted ZFP91 specifically in cardiomyocytes in mice and found that ZFP91 loss can lead to HCM and accelerate its development under the disease stress. However, the mechanism remains unclear. It is reported that phospholamban (PLN) promoter had zinc protein-binding sites. In addition, previous results indicated that ZFP91 may regulate PLN protein expression. Thus, we hypothesized that cardiac-specific ZFP91 loss can lead to increase of PLN expression, resulting in HCM development. Consequently, we will first over-express ZFP91 in heart of wild type mice and observe whether cardiac hypertrophy is inhibited in mice after transverse aortic constriction and myocardial infarcton. Second, we will study whether PLN promoter is directly bound by ZFP91 by constructing luciferase reporter gene vector and ChIP analysis. Finally, we will interfere PLN expression in heart of mice and observe whether cardiac hypertrophy is reversed. From this study, we hope to identify a new method to treat human HCM.

肥厚性心肌病（HCM）是常见的心脏病，研究表明其可能与一些转录因子的基因突变相关。锌指蛋白91（ZFP91）属于锌指蛋白家族，其含有与核酸结合的经典结构域C2H2，可能参与众多基因的表达调控。我们在小鼠心肌细胞中特异剔除ZFP91基因，发现ZFP91缺失能引发HCM并在疾病应激下加速其发展,但这个作用机制尚不清楚。文献报道受磷蛋白（PLN）的启动子有锌指类蛋白结合位点，前期结果也提示ZFP91可能会调控PLN蛋白的表达，因此我们假设心肌特异ZFP91缺失可致PLN表达增多而促进HCM发生。于是我们首先在野生小鼠心肌过表达ZFP91，观察主动脉弓缩窄和心肌梗死术后小鼠心肌肥厚是否得到抑制；再通过构建荧光素酶报告基因载体和ChIP检测分析ZFP91是否能直接结合PLN启动子；最后干扰心肌特异ZFP91缺失小鼠PLN的表达，观察心肌肥厚是否逆转。通过该研究,我们希望找到治疗HCM的新途径。

背景：Zfp91的功能主要是在体外研究，而目前尚无体内研究。这些研究表明，Zfp91可能是调节人类反应各个方面的重要基因。一旦发生变异，就可能诱发多种疾病。然而，目前还没有关于Zfp91对心脏内稳态的转录调控的研究。.目的：观察小鼠心肌细胞Zfp91基因在压力超负荷下对心肌内稳态的作用及其相关机制。.方法：24只雄性小鼠均分为4组：Zfp91F/F和Zfp91F/F；αMHC-Cre假手术组以及Zfp91F/F和Zfp91F/F；αMHC-Cre TAC手术组。小鼠TAC术后腹腔注射N6-环戊基腺苷（CPA）（2mg/kg/d）治疗6周。测量心脏和体重。超声心动图评价心功能。分别用PCR、HE染色、Masson染色和WGA染色检测心肌细胞的基因型、心肌大小、胶原沉积和心肌细胞面积。分别用RT-PCR和western blot分析zfp91、Adora1和GAPDH的mRNA和蛋白表达。采用双荧光素酶报告法和ChIP-qPCR法研究zfp91与腺苷酸A1受体（adora1）启动子的相互作用。.结果：与对照组相比，Zfp91缺失小鼠Zfp91 mRNA和蛋白水平显著降低（P<0.01）。Zfp91基因敲除可导致TAC术后病理性心脏重塑和心功能不全加重（P<0.05或P<0.01）。基因芯片、RT-PCR和western blot分析显示TAC小鼠心脏Adora1基因和蛋白表达显著下调（P<0.01）。双荧光素酶报告和ChIP-qPCR分析表明zfp91可以通过其结合位点作用于adora1启动子。最后，Adora1激动剂可挽救TAC术后zfp91丢失小鼠的心功能不全和不良的心脏重构表型（P<0.05或P<0.01）。.结论：转录因子zfp91直接调节心脏Adora1的表达，在压力超负荷状态下主要维持心脏的稳态，为心肌肥厚的治疗提供了新的途径。