目标：长QT综合征7型（Andersen-Tawil综合征，ATS)，这是由KCNJ2基因突变引起的，常导致室性心律失常,周期性瘫痪和骨骼畸形。的发展,心肌细胞（CMs）分化和电生理成熟的变化促进了长QT综合征7型（LQT7）的病理生理。我们旨在特异性再现ATS疾病表型并研究其致病机制。
结果：我们建立了来源于人诱导多能干细胞（hiPSCs）的心脏细胞模型，而特异性再现ATS症状的人心肌细胞模型的建立为探索这种疾病的机制或潜在药物提供了可靠的平台。突变组心肌细胞自发搏动率显著低于修复CRISPR组,动作电位持续时间延长，内向整流钾离子通道的Kir2.1电流降低，这与ATS患者的临床症状一致。只有ZNF528，一种与致病性相关的染色质可及的TF，从心脏中胚层前体细胞阶段（第4天）开始连续调节，并继续以低水平表示，通过WGCNA方法鉴定，并在突变组中使用ATAC-seq数据进行验证。随后，通过单样本基因组富集分析来评估富集在晚期成熟CMs的转录组和蛋白质组中的钾相关通路的整体调控，这表明有7条通路被下调（均p<0.05）。其中，涉及包含突变基因KCNJ2的三种途径（GO：0008076，GO：1990573和GO：0030007），这些途径与钾离子通过内向整流钾通道进入细胞以发挥其作用的整个过程有关被抑制。其他四种途径与钾跨膜途径和钠：钾交换ATP酶的调节有关（p<0.05）。将ZNF528小干扰(si)-RNA应用于CRISPR组的hiPSC来源的心肌细胞,以探索影响疾病表型的钾离子电流和生长发育相关靶蛋白水平的变化。通过相关性和交叉分析验证了与致病性相关的三种一致下调蛋白（KCNJ2，CTTN和ATP1B1）。
结论：这项研究揭示了与ATS心肌细胞电生理和发育致病性相关的TFs和靶蛋白，获得不依赖于基因编辑的潜在治疗候选开发的新靶点。
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.
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