EPI64C在左室逆重构中的作用及机制研究

Aortic valve replacement induces spontaneous reverse remodeling of cardiac hypertrophy and functional recovery of left ventricle in aortic stenosis patient. However, cardiac recovery is always far from satisfactory in late-stage patient due to insufficient reverse remodeling. A deeper insight into the molecular mechanism of reverse remodeling will aid the therapeutic effect of aortic valve replacement in these patient. We have established a cardiac reverse remodeling animal model, in which a debanding surgery was performed to unload the pressure overload created by transverse aortic constriction. We thus successfully mimicked the reverse remodeling after aortic valve replacement. Then, high-throughput expression profiling through RNA-sequencing was performed to determine the gene expression profile when reverse remodeling was initiated. Surprisingly, we found that expression of EPI64C was sharply downregulated in pressure overload-stressed myocardium, but acutely increased upon unloading. Furthermore, we found that loss of EPI64C attenuated reverse remodeling upon unloading in mice. These results indicate that EPI64C serves as a key factor in reverse remodeling. Our current study will further determine the role of EPI64C in reverse remodeling by means of EPI64C overexpression. Moreover, both the direct and indirect molecular mechanisms downstream of EPI64C will be dissected by combined analysis of interaction proteomics and gene expression profiling. At last, an EPI64C-based gene therapy will be performed to explore the translational potential. Our current study thus provide a new therapeutic target for cardiac remodeling.

主动脉瓣狭窄患者在瓣膜置换术后，原肥大的左室会自发出现形态复原和功能恢复，该现象被称为左室逆重构，然而晚期换瓣患者往往因逆重构不显著而疗效不佳。为促进换瓣术后心脏结构及功能恢复，需深入发掘机体内在复原机制并加以利用。申请人前期建立了小鼠主动脉弓缩窄后缩窄松解模型，模拟换瓣术后左室逆重构。为寻找左室逆重构关键始动机制，本课题前期通过高通量基因表达谱测序的方法，发现EPI64C基因表达在左室重构过程中急剧下调，而在左室逆重构过程中迅速上调。进一步研究发现逆重构启动前EPI64C基因敲除显著抑制了左室逆重构，表明EPI64C是左室逆重构的关键调控因子。本课题拟从EPI64C功能增强的角度进一步探明其在左室逆重构中的作用，并综合运用互作蛋白质谱和基因表达谱测序等方法，解析EPI64C调控逆重构的直接和间接机制。最后，探索基于EPI64C的基因治疗在增强左室逆重构中的应用，为治疗左室重构提供新思路。

心肌肥厚（Cardiac hypertrophy），是由于心脏在长期压力负荷过重的情况下，收缩力加强，导致心肌细胞肥大。当心脏长期处于高负荷状态下，左室舒张末期容积扩张，并出现间质纤维化和心肌细胞凋亡等病理改变，即左室重构。病理性心肌肥厚和左室重构是心力衰竭、心律失常和心源性猝死发生发展最重要的独立危险因素，然而目前已有的药物往往只能部分缓解症状而不能治愈。因此，深入了解心肌肥厚和左室重构的细胞及分子机制，进而为研发治疗心肌肥厚的药物提供新思路和新靶点是心胸血管外科领域迫切需要解决的问题。GTP酶激活蛋白（EPI64C）是一种对Calcineurin与Ras信号通路双重抑制的蛋白,相关研究证实其在T细胞增殖及活化中起着重要的负调控作用。本项目从分子、细胞以及动物水平系统的阐明了EPI64C对心肌肥厚下左室重构的调控作用及分子机制。首先我们建立了稳定的小鼠左室逆重构模型，发现模型动物中主动脉弓缩窄术（TAC）组的心脏左室射血分数（EF）、心重体重比（HW/BW）、左室收缩末期内径（LVEDd）等心脏重构指标相比假手术组（Sham）显著升高等，而主动脉弓缩窄松解术（DB）组的以上指标则与假手术组无显著差异。我们将以上三组小鼠左心室样品进行三代测序，测序结果发现，同一个基因可存在多个可变剪切体，且其表达趋势可完全相反。通过三代测序结果与参考基因组对比，我们还发现了大量之前未被测出的中长链转录本。其次，我们构建了EPI64C敲低和过表达小鼠，从动物体内实验验证了我们的猜想。同时，我们构建了EPI64C敲低和过表达的慢病毒质粒，分别感染大鼠原代心肌细胞，结果显示，EPI64C敲低显著促进了TAC诱导下的心肌肥大状态，而EPI64C过表达则显著抑制心肌细胞的肥大情况。通过萤光素酶报告基因实验和免疫共沉淀（IP）富集与EPI64C相互作用的蛋白发现，钙调神经磷酸酶在EPI64C调控心肌细胞肥大过程中发挥重要作用。本项目的研究成果基本阐述了EPI64C调控心肌肥厚的作用机制，建立了由EPI64C与钙调神经磷酸酶的相互联系，为治疗心肌肥厚的后续研究提供了新思路和新的药物靶点。

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