Department of Cardiovascular Surgery of the First Affiliated Hospital & Institute for Cardiovascular Science, Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Protection, Medical College, Soochow University, Suzhou 215000, China.
Institute of Microcirculation & Department of Pathophysiology of Basic Medical College, Hebei North University, Zhangjiakou 075000, China.
Cardiovasc Res. 2022 Feb 21;118(3):859-871. doi: 10.1093/cvr/cvab154.
Congenital heart disease (CHD) frequently occurs in newborns due to abnormal formation of the heart or major blood vessels. Mutations in the GATA4 gene, which encodes GATA binding protein 4, are responsible for atrial septal defect (ASD), a common CHD. This study aims to gain insights into the molecular mechanisms of CHD using human-induced pluripotent stem cells (iPSCs) from a family cohort with ASD.
Patient-specific iPSCs possess the same genetic information as the donor and can differentiate into various cell types from all three germ layers in vitro, thus presenting a promising approach for disease modelling and molecular mechanism research. Here, we generated a patient-specific iPSC line (iPSC-G4T280M) from a family cohort carrying a hereditary ASD mutation in GATA4 gene (T280M), as well as a human embryonic stem cell line (ESC-G4T280M) carrying the isogenic T280M mutation using the CRISPR/Cas9 genome editing method. The GATA4-mutant iPSCs and ESCs were then differentiated into cardiomyocytes (CMs) to model GATA4 mutation-associated ASD. We observed an obvious defect in cell proliferation in cardiomyocytes derived from both GATA4T280M-mutant iPSCs (iPSC-G4T280M-CMs) and ESCs (ESC-G4T280M-CMs), while the impaired proliferation ability of iPSC-G4T280M-CMs could be restored by gene correction. Integrated analysis of RNA-Seq and ChIP-Seq data indicated that FGF16 is a direct target of wild-type GATA4. However, the T280M mutation obstructed GATA4 occupancy at the FGF16 promoter region, leading to impaired activation of FGF16 transcription. Overexpression of FGF16 in GATA4-mutant cardiomyocytes rescued the cell proliferation defect. The direct relationship between GATA4T280M and ASD was demonstrated in a human iPSC model for the first time.
In summary, our study revealed the molecular mechanism of the GATA4T280M mutation in ASD. Understanding the roles of the GATA4-FGF16 axis in iPSC-CMs will shed light on heart development and provide novel insights for the treatment of ASD and other CHD disorders.
由于心脏或主要血管的异常形成，新生儿常发生先天性心脏病（CHD）。编码 GATA 结合蛋白 4 的 GATA4 基因突变是房间隔缺损（ASD）的原因，ASD 是一种常见的 CHD。本研究旨在利用携带 ASD 的家系队列中的人诱导多能干细胞（iPSC）深入了解 CHD 的分子机制。
患者特异性 iPSC 具有与供体相同的遗传信息，并且可以在体外分化为来自所有三个胚层的各种细胞类型，因此为疾病建模和分子机制研究提供了一种很有前途的方法。在这里，我们使用 CRISPR/Cas9 基因组编辑方法，从携带 GATA4 基因突变（T280M）的家系队列中生成了一个患者特异性 iPSC 系（iPSC-G4T280M），以及一个携带同源 T280M 突变的人胚胎干细胞系（ESC-G4T280M）。然后，将 GATA4 突变型 iPSC 和 ESC 分化为心肌细胞（CM），以模拟 GATA4 突变相关的 ASD。我们观察到源自 GATA4T280M 突变的 iPSC（iPSC-G4T280M-CM）和 ESC（ESC-G4T280M-CM）的心肌细胞增殖明显受损，而 iPSC-G4T280M-CM 的增殖能力受损可以通过基因纠正来恢复。RNA-Seq 和 ChIP-Seq 数据的综合分析表明，FGF16 是野生型 GATA4 的直接靶标。然而，T280M 突变阻止了 GATA4 在 FGF16 启动子区域的占据，导致 FGF16 转录的激活受损。在 GATA4 突变型心肌细胞中过表达 FGF16 可挽救细胞增殖缺陷。首次在人类 iPSC 模型中证明了 GATA4T280M 与 ASD 的直接关系。
总之，本研究揭示了 ASD 中 GATA4T280M 突变的分子机制。了解 GATA4-FGF16 轴在 iPSC-CM 中的作用将为心脏发育提供新的认识，并为 ASD 和其他 CHD 疾病的治疗提供新的思路。
