Novel Calmodulin Variant p.E46K Associated With Severe Catecholaminergic Polymorphic Ventricular Tachycardia Produces Robust Arrhythmogenicity in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.
Novel Calmodulin Variant p.E46K Associated With Severe Catecholaminergic Polymorphic Ventricular Tachycardia Produces Robust Arrhythmogenicity in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.
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与严重儿茶酚胺能多形性室性心动过速相关的新型钙调蛋白变体 p.E46K 在人诱导多能干细胞来源的心肌细胞中产生强烈的心律失常性。
DOI：
10.1161/circep.122.011387
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Oh
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文献类型：
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作者：
Gao J;Makiyama T;Yamamoto Y;Kobayashi T;Aoki H;Maurissen TL;Wuriyanghai Y;Kashiwa A;Imamura T;Aizawa T;Huang H;Kohjitani H;Nishikawa M;Chonabayashi K;Fukuyama M;Manabe H;Nakau K;Wada T;Kato K;Toyoda F;Yoshida Y;Makita N;Woltjen K;Oh
背景CaM（钙调蛋白）是一种普遍表达的多功能Ca 2+传感蛋白，可调节多种蛋白质。最近，在恶性遗传性心律失常患者中发现了钙调素错义变异体，如长QT综合征和儿茶酚胺能多形性室性心动过速（CPVT）。然而，钙调素相关的CPVT在人类心肌细胞的确切机制仍不清楚。在这项研究中，我们试图调查一种新的变体使用人类诱导多能干细胞（iPSC）模型和生化assays.MethodsWe产生iPSC从CPVT bearingCALM2p.E46K的患者引起的CPVT的致瘤机制。作为比较，我们使用了2个对照系，包括一个同基因系，和另一个来自携带CALM2p.N98S的长QT综合征患者的iPSC系（也在CPVT中报道）。使用iPSC-心肌细胞研究电生理特性。我们进一步研究了RyR 2（ryanodine受体2）和Ca 2+亲和力的钙调素使用重组protein.ResultsWe确定了一种新的从头杂合变异，CALM2p.E46K，在2个无关的患者与CPVT伴随神经发育障碍。E46 K心肌细胞表现出更频繁的异常电兴奋和Ca 2+波比其他线与增加Ca 2+泄漏从肌浆网通过RyR 2。此外，[3 H]ryanodine结合试验表明，E46 K-CaM促进RyR 2的功能，特别是通过激活在低[Ca 2 +]水平。实时荧光定量分析结果表明，E46 K-CaM与RyR 2的结合能力是野生型CaM的10倍，这可能是突变型CaM的优势作用。此外，E46 K-CaM不影响CaM-Ca 2+结合或L型钙通道功能。最后，抗心律失常药物，纳多洛尔和氟卡尼，抑制异常的Ca 2+波在E46 K-myocardialcytos.ConclusionsWe，第一次，建立了一个钙调素相关的CPVT iPSC-CM模型，概括了严重的致心律失常的功能，导致E46 K-CaM占主导地位的结合和促进RyR 2。此外，基于iPSC的药物测试结果将有助于精准医学。
BackgroundCaM (calmodulin) is a ubiquitously expressed, multifunctional Ca2+sensor protein that regulates numerous proteins. Recently, CaM missense variants have been identified in patients with malignant inherited arrhythmias, such as long QT syndrome and catecholaminergic polymorphic ventricular tachycardia (CPVT). However, the exact mechanism of CaM-related CPVT in human cardiomyocytes remains unclear. In this study, we sought to investigate the arrhythmogenic mechanism of CPVT caused by a novel variant using human induced pluripotent stem cell (iPSC) models and biochemical assays.MethodsWe generated iPSCs from a patient with CPVT bearingCALM2p.E46K. As comparisons, we used 2 control lines including an isogenic line, and another iPSC line from a patient with long QT syndrome bearingCALM2p.N98S (also reported in CPVT). Electrophysiological properties were investigated using iPSC-cardiomyocytes. We further examined the RyR2 (ryanodine receptor 2) and Ca2+affinities of CaM using recombinant proteins.ResultsWe identified a novel de novo heterozygous variant,CALM2p.E46K, in 2 unrelated patients with CPVT accompanied by neurodevelopmental disorders. The E46K-cardiomyocytes exhibited more frequent abnormal electrical excitations and Ca2+waves than the other lines in association with increased Ca2+leakage from the sarcoplasmic reticulum via RyR2. Furthermore, the [3H]ryanodine binding assay revealed that E46K-CaM facilitated RyR2 function especially by activating at low [Ca2+] levels. The real-time CaM-RyR2 binding analysis demonstrated that E46K-CaM had a 10-fold increased RyR2 binding affinity compared with wild-type CaM which may account for the dominant effect of the mutant CaM. Additionally, the E46K-CaM did not affect CaM-Ca2+binding or L-type calcium channel function. Finally, antiarrhythmic agents, nadolol and flecainide, suppressed abnormal Ca2+waves in E46K-cardiomyocytes.ConclusionsWe, for the first time, established a CaM-related CPVT iPSC-CM model which recapitulated severe arrhythmogenic features resulting from E46K-CaM dominantly binding and facilitating RyR2. In addition, the findings in iPSC-based drug testing will contribute to precision medicine.