Department of Clinical Nutrition & Health Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Department of Cardiology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100730, China.
J Transl Med. 2024 Mar 25;22(1):307. doi: 10.1186/s12967-024-05125-7.
Long QT syndrome type 7 (Andersen-Tawil syndrome, ATS), which is caused by KCNJ2 gene mutation, often leads to ventricular arrhythmia, periodic paralysis and skeletal malformations. The development, differentiation and electrophysiological maturation of cardiomyocytes (CMs) changes promote the pathophysiology of Long QT syndrome type 7(LQT7). We aimed to specifically reproduce the ATS disease phenotype and study the pathogenic mechanism.
We established a cardiac cell model derived from human induced pluripotent stem cells (hiPSCs) to the phenotypes and electrophysiological function, and the establishment of a human myocardial cell model that specifically reproduces the symptoms of ATS provides a reliable platform for exploring the mechanism of this disease or potential drugs. The spontaneous pulsation rate of myocardial cells in the mutation group was significantly lower than that in the repair CRISPR group, the action potential duration was prolonged, and the Kir2.1 current of the inward rectifier potassium ion channel was decreased, which is consistent with the clinical symptoms of ATS patients. Only ZNF528, a chromatin-accessible TF related to pathogenicity, was continuously regulated beginning from the cardiac mesodermal precursor cell stage (day 4), and continued to be expressed at low levels, which was identified by WGCNA method and verified with ATAC-seq data in the mutation group. Subsequently, it indicated that seven pathways were downregulated (all p < 0.05) by used single sample Gene Set Enrichment Analysis to evaluate the overall regulation of potassium-related pathways enriched in the transcriptome and proteome of late mature CMs. Among them, the three pathways (GO: 0008076, GO: 1990573 and GO: 0030007) containing the mutated gene KCNJ2 is involved that are related to the whole process by which a potassium ion enters the cell via the inward rectifier potassium channel to exert its effect were inhibited. The other four pathways are related to regulation of the potassium transmembrane pathway and sodium:potassium exchange ATPase (p < 0.05). ZNF528 small interfering (si)-RNA was applied to hiPSC-derived cardiomyocytes for CRISPR group to explore changes in potassium ion currents and growth and development related target protein levels that affect disease phenotype. Three consistently downregulated proteins (KCNJ2, CTTN and ATP1B1) associated with pathogenicity were verificated through correlation and intersection analysis.
This study uncovers TFs and target proteins related to electrophysiology and developmental pathogenicity in ATS myocardial cells, obtaining novel targets for potential therapeutic candidate development that does not rely on gene editing.
长 QT 综合征 7 型（Andersen-Tawil 综合征，ATS）由 KCNJ2 基因突变引起，常导致室性心律失常、周期性瘫痪和骨骼畸形。心肌细胞（CMs）的发育、分化和电生理成熟变化促进了长 QT 综合征 7 型（LQT7）的病理生理学。我们旨在专门复制 ATS 疾病表型并研究致病机制。
我们建立了一种源自人类诱导多能干细胞（hiPSC）的心脏细胞模型，以表型和电生理功能为特征，并建立了一种专门复制 ATS 症状的人类心肌细胞模型，为探索该疾病的机制或潜在药物提供了可靠的平台。突变组心肌细胞的自发搏动率明显低于修复 CRISPR 组，动作电位持续时间延长，内向整流钾离子通道的 Kir2.1 电流减少，与 ATS 患者的临床症状一致。只有与致病性相关的染色质可及性 TF（转录因子）ZNF528 从心脏中胚层前体细胞阶段（第 4 天）开始持续被调控，并在突变组中通过 ATAC-seq 数据进行验证，表达水平持续较低。随后，通过单样本基因集富集分析（GSVA）评估富含成熟晚期 CMs 转录组和蛋白质组的钾相关途径的整体调控，发现七个途径被下调（均 p<0.05）。其中，涉及突变基因 KCNJ2 的三个途径（GO：0008076、GO：1990573 和 GO：0030007）被抑制，这些途径涉及钾离子通过内向整流钾通道进入细胞以发挥作用的整个过程。其他四个途径与钾跨膜途径和钠：钾交换 ATP 酶（p<0.05）的调节有关。为了探索影响疾病表型的钾离子电流和与生长发育相关的靶蛋白水平的变化，我们将 ZNF528 小干扰（si）-RNA 应用于 hiPSC 衍生的心肌细胞的 CRISPR 组。通过相关性和交集分析验证了与致病性相关的三个一致下调的蛋白质（KCNJ2、CTTN 和 ATP1B1）。
本研究揭示了 ATS 心肌细胞中与电生理和发育致病性相关的 TFs 和靶蛋白，为不依赖于基因编辑的潜在治疗候选物开发提供了新的靶点。
