NFATc4乙酰化修饰对心肌肥大的影响及其机制研究

Prolong hypertrophy ultimately progresses to heart failure, there are still many questions on the pathogenesis of cardiac hypertrophy. Cardiac transcription factors have attracted considerable attention for their important role in the onset of cardiac hypertrophy, and NFATc4 is one of the most important factors. We found that acetylation NFATc4 was upregulated in cardiomyocyte hypertrophy and abdominal aortic coarctation (AAC) model, and cardiac hypertrophy was improved by downregulation of acetylation NFATc4. It also reminds us that NFATc4 may be acetylated in cell processes for its special structure and posttranslation modification features. So we hypothesis that acetylation regulated the activity of NFATc4 by many mechanisms, which cardiac hypertrophy was affected by the changed activity of NFATc4. We aimed to explore the involvement of NFATc4 acetylation in cardiomyocyte hypertrophy and the underlying mechanisms. We will focus on effects on NFATc4 activation, DNA binding activity, transcription activity and the interaction with other posttranslation modification. The acetylated amino acid residues were analyzed by affinity chromatography and LC-MS/MS. The potential of acetylation NFATc4 in intervene of cardiac hypertrophy was evaluated in cardiomyocyte hypertrophy and AAC model. The project will explain the molecular basis of NFATc4 activity regulation and its cellular compartmentalization, and set the light on posttranslation modification in cardiac hypertrophy, which provide a new strategy for the treatment of cardiac hypertrophy and heart failure.

心肌肥厚持续发展是导致心力衰竭的重要原因，而心肌肥厚发生的病理机制仍存在许多疑问。转录因子在心肌肥厚发展中的作用受到关注，NFATc4是介导心肌肥厚的重要转录因子。我们前期在体内外心肌肥大模型中发现，NFATc4乙酰化水平升高，降低NFATc4乙酰化水平改善心肌肥大。NFATc4的功能和结构特点以及自身修饰状态也提示，NFATc4可能发生乙酰化修饰。在此基础上提出假说：乙酰化修饰通过多种机制调节NFATc4活性，影响心肌肥大。本课题拟在体内外心肌肥大模型中探索乙酰化修饰影响NFATc4活性的机制，包括NFATc4激活、DNA结合活性、转录活性以及其他翻译后修饰改变等，采用免疫亲和层析和LC-MS/MS检测乙酰化位点，探讨不同位点乙酰化修饰对NFATc4功能的重要性。本研究将阐明NFATc4功能调节和亚细胞定位的结构基础，发现心肌肥厚发展的新机制，可能为心肌肥厚治疗药物的研发提供新靶点。

心肌肥厚持续发展是导致心力衰竭的重要原因，而心肌肥厚发生的病理机制仍存在许多疑问。转录因子在心肌肥厚发展中的作用受到关注，包括磷酸化、乙酰化、泛素化等在内的多种翻译后修饰被认为在多种因子的功能调节中起重要作用，并且不同的翻译后修饰之间可能相互影响，在心肌肥大进程中起重要作用。NFATc4是介导心肌肥厚的重要转录因子，高度磷酸化的NFATc4被钙调磷酸酶去磷酸化激活后结合到ANF、BNP的启动子上，促进肥大基因的表达。项目探索了心肌肥大过程中NFATc4的变化以及这些变化影响心肌肥大机制，并对NFATc4乙酰化区域进行探索。结果发现，心肌肥大进程中，除了磷酸化水平显著降低外，NFATc4乙酰化修饰显著升高。NFATc4乙酰化水平的升高与心肌肥大过程中去乙酰化酶SIRT1酶活性水平下降有关。通过增强和减弱去乙酰化酶SIRT1的方法，我们发现乙酰化修饰升高能够促进NFATc4与钙调磷酸酶结合而减少与14-3-3胞浆蛋白结合，促进了NFATc4去磷酸化激活，同时NFATc4的DNA活性和转录活性增加，加剧了心肌肥大。相反，当SIRT1酶活性增加使NFATc4乙酰化水平下降后，NFATc4磷酸化激活被抑制，与DNA的结合活性和转录活性降低，心肌肥大得到缓解。进一步的研究还发现，NFATc4关键的去乙酰化位点可能位于DNA结合域。项目具有两大科学意义。新机制：NFATc4翻译后修饰改变尤其是乙酰化修饰调节后而使其功能发生改变，进而影响心肌肥大，项目不仅揭示NFATc4的自身修饰状态与功能活性之间的关系，也揭示了NFATc4乙酰化水平的变化与心肌肥大的关系，这是对心肌肥大发生机制的补充。新靶点：从基因水平进行调节是改善心肌肥大的关键。NFATc4直接与肥大基因启动子结合，促进肥大基因的表达，我们从NFATc4的关键乙酰化位点入手，探索NFATc4与肥大基因结合与肥大基因表达间的关系，设计特异性的抑制剂，为心肌肥大治疗提供新的特异性靶点。