BACKGROUND:Germline HRAS gain-of-function pathogenic variants cause Costello syndrome (CS). During early childhood, 50% of patients develop multifocal atrial tachycardia, a treatment-resistant tachyarrhythmia of unknown pathogenesis. This study investigated how overactive HRAS activity triggers arrhythmogenesis in atrial-like cardiomyocytes (ACMs) derived from human-induced pluripotent stem cells bearing CS-associated HRAS variants.METHODS:HRAS Gly12 mutations were introduced into a human-induced pluripotent stem cells-ACM reporter line. Human-induced pluripotent stem cells were generated from patients with CS exhibiting tachyarrhythmia. Calcium transients and action potentials were assessed in induced pluripotent stem cell-derived ACMs. Automated patch clamping assessed funny currents. HCN inhibitors targeted pacemaker-like activity in mutant ACMs. Transcriptomic data were analyzed via differential gene expression and gene ontology. Immunoblotting evaluated protein expression associated with calcium handling and pacemaker-nodal expression.RESULTS:ACMs harboring HRAS variants displayed higher beating rates compared with healthy controls. The hyperpolarization activated cyclic nucleotide gated potassium channel inhibitor ivabradine and the Na_v1.5 blocker flecainide significantly decreased beating rates in mutant ACMs, whereas voltage-gated calcium channel 1.2 blocker verapamil attenuated their irregularity. Electrophysiological assessment revealed an increased number of pacemaker-like cells with elevated funny current densities among mutant ACMs. Mutant ACMs demonstrated elevated gene expression (ie, ISL1, TBX3, TBX18) related to intracellular calcium homeostasis, heart rate, RAS signaling, and induction of pacemaker-nodal-like transcriptional programming. Immunoblotting confirmed increased protein levels for genes of interest and suppressed MAPK (mitogen-activated protein kinase) activity in mutant ACMs.CONCLUSIONS:CS-associated gain-of-function HRAS^G12 mutations in induced pluripotent stem cells-derived ACMs trigger transcriptional changes associated with enhanced automaticity and arrhythmic activity consistent with multifocal atrial tachycardia. This is the first human-induced pluripotent stem cell model establishing the mechanistic basis for multifocal atrial tachycardia in CS.

中文翻译：

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多灶性房性心动过速的 HRAS 突变心肌细胞模型

背景：种系HRAS功能获得性致病变异导致 Costello 综合征 (CS)。在儿童早期，50% 的患者会出现多灶性房性心动过速，这是一种发病机制不明的难治性快速心律失常。本研究调查了过度活跃的 HRAS 活性如何触发心房样心肌细胞 (ACM) 中的心律失常，这些心肌细胞源自携带 CS 相关HRAS变异的人诱导多能干细胞。方法：将HRAS Gly12 突变引入人诱导多能干细胞 -ACM 报告基因中线。人类诱导的多能干细胞是从表现出快速心律失常的 CS 患者中产生的。在诱导多能干细胞衍生的 ACM 中评估钙瞬变和动作电位。自动膜片钳评估有趣的电流。 HCN 抑制剂针对突变 ACM 中的起搏器样活性。通过差异基因表达和基因本体分析转录组数据。免疫印迹评估了与钙处理和起搏器节点表达相关的蛋白质表达。结果：与健康对照相比，含有HRAS变异的 ACM 显示出更高的搏动率。超极化激活的环核苷酸门控钾通道抑制剂伊伐布雷定和 Na _v 1.5 阻滞剂氟卡尼显着降低了突变型 ACM 的搏动率，而电压门控钙通道 1.2 阻滞剂维拉帕米则减弱了其不规则性。电生理评估显示，突变 ACM 中起搏器样细胞数量增加，且有趣电流密度升高。突变的 ACM 表现出与细胞内钙稳态、心率、RAS 信号传导和起搏器节点样转录编程诱导相关的基因表达升高（即ISL1、TBX3、TBX18 ）。免疫印迹证实突变 ACM 中感兴趣基因的蛋白质水平增加，并抑制 MAPK（丝裂原激活蛋白激酶）活性。结论：诱导多能干细胞衍生的 ACM 中与CS 相关的功能获得性HRAS ^G12突变触发与自主性和心律失常活性增强，与多灶性房性心动过速一致。这是第一个人类诱导的多能干细胞模型，为 CS 中多灶性房性心动过速奠定了机制基础。