ERG1介导Integrinβ1维持大鼠心脏发育信号稳态的研究

The fate of cardiomyocyte differentiation is regulated precisely by specific transcription factors. It is not entirely clear how the complex signaling pathways involved are mediated by the cell membrane proteins. In particular, it is still unknown that whether the ion channel, as a functional cardiomyocyte membrane protein, involves in the regulation of cardiac fate. Our previous study found that knockout of Kcnh2 could lead to abnormal heart development and down-expression of genes related to heart development in rat. We also found ERG1 could interact with Integrin β1 to form a complex during the embryonic stem cell differentiation. After loss-of-function of ERG1, the level of β-catenin, which locates in the downstream of Integrinβ1, was significantly reduced. Accordingly, we speculate that ERG1 interacts with Integrinβ1, which mediates PI3K / FAK / AKT signaling axis to regulate cardiomyocyte differentiation, during the early stage of heart development. Therefore, we will explore the mechanism of ERG1/ Integrinβ1 in regulation of early cardiac development using molecular biology and other methods in Kcnh2-knockout rat model and the cardiomyocyte differentiation model of rat embryonic stem cells in vitro. We hope this study could expand our understanding of regulation of heart fate by ion channel protein.

心肌细胞分化的命运受到特异转录因子精确的时空动态调控，涉及的复杂信号通路如何由细胞膜蛋白介导并不完全清楚，特别是离子通道作为心肌细胞功能膜蛋白是否参与调控心脏命运走向知之甚少。我们前期研究发现，敲除编码ERG1的Kcnh2基因导致大鼠心脏发育异常，心脏发育相关基因被抑制；进一步发现在胚胎干细胞分化时，ERG1可与Integrinβ1形成复合体，当ERG1功能缺失后，Integrinβ1下游的β-catenin水平显著降低。据此推测：在心脏发育早期，ERG1与Integrinβ1相互作用，介导了下游PI3K/FAK/AKT信号轴，从而维持着心肌细胞的正常分化。为此，我们将利用Kcnh2敲除大鼠以及大鼠胚胎干细胞定向心肌细胞分化模型，结合分子、细胞和生化等手段，阐明ERG1/Integrinβ1维系心脏发育调控的新机制，从而拓展对离子通道蛋白调控心脏命运的认识。

在建立了rESC定向心肌细胞体外高效分化系统的基础上，研究ERG1调控rESC定向心肌细胞分化过程的详细分子机制。首先使用同源重组的方法建立了Kcnh2敲除大鼠模型并分离建立了野生型（wild type，WT）、Kcnh2+/-、Kcnh2-/-这三种基因型的rESC。利用rESC定向心肌细胞体外高效分化系统我们精确地分析了ERG1在rESC定向心肌细胞分化过程不同阶段的具体作用，解析了其对心肌细胞分化调控的分子机制。通过一系列实验研究，发现了以下结果：1. ERG1对rESC定向心肌细胞分化以及细胞凋亡的调控存在一定的剂量依赖性，既有一半的Kcnh2被敲除的Kcnh2+/- rESC定向心肌细胞分化的能力没有发生异常改变并且也没有细胞凋亡产生。而当Kcnh2水平继续降低， Kcnh2纯合敲除的rESC定向心肌细胞分化出现了显著抑制且rEB在分化中出现了细胞凋亡。2. ERG1在rESC定向心肌细胞分化早期可以与Integrinβ1、FAK、PI3K p85形成复合体。3. ERG1能够通过FAK/PI3K/AKT/GSK3β/β-catenin信号轴调控rESC定向心肌细胞分化。4. ERG1能够通过FAK/PI3K/AKT/IKKβ/NF-κB信号轴抑制rESC定向心肌细胞分化过程中凋亡发生。5. PTEN抑制剂SF1670可以通过激活AKT拯救Kcnh2-/- rESC定向心肌细胞的分化并且抑制分化过程中凋亡的发生。最终我们得出以下结论：在rESC定向心肌细胞分化过程中ERG1可以与Integrinβ1、PI3K、FAK形成复合体，通过该复合体进而影响下游AKT/GSK3β/β-catenin以及AKT/IKKβ/NF-κB这两条信号轴从而调控rESC定向心肌细胞的分化以及抑制分化过程中凋亡的发生。