Department of Cardiology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, 310009, Hangzhou, China.
Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health, The First Affiliated Hospital, Zhejiang University School of Medicine, 310003, Hangzhou, China; Institute of Translational Medicine, Zhejiang University, 310029, Hangzhou, China.
EBioMedicine. 2023 Sep;95:104741. doi: 10.1016/j.ebiom.2023.104741. Epub 2023 Aug 4.
Brugada syndrome (BrS) is a cardiac channelopathy that can result in sudden cardiac death (SCD). SCN5A is the most frequent gene linked to BrS, but the genotype-phenotype correlations are not completely matched. Clinical phenotypes of a particular SCN5A variant may range from asymptomatic to SCD. Here, we used comparison of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) derived from a SCN5A mutation-positive (D356Y) BrS family with severely affected proband, asymptomatic mutation carriers (AMCs) and healthy controls to investigate this variation.
26 iPSC lines were generated from skin fibroblasts using nonintegrated Sendai virus. The generated iPSCs were differentiated into cardiomyocytes using a monolayer-based differentiation protocol.
D356Y iPSC-CMs exhibited increased beat interval variability, slower depolarization, cardiac arrhythmias, defects of Na channel function and irregular Ca signaling, when compared to controls. Importantly, the phenotype severity observed in AMC iPSC-CMs was milder than that of proband iPSC-CMs, an observation exacerbated by flecainide. Interestingly, the iPSC-CMs of the proband exhibited markedly decreased Ca currents in comparison with control and AMC iPSC-CMs. CRISPR/Cas9-mediated genome editing to correct D356Y in proband iPSC-CMs effectively rescued the arrhythmic phenotype and restored Na and Ca currents. Moreover, drug screening using established BrS iPSC-CM models demonstrated that quinidine and sotalol possessed antiarrhythmic effects in an individual-dependent manner. Clinically, venous and oral administration of calcium partially reduced the malignant arrhythmic events of the proband in mid-term follow-up.
Patient-specific and genome-edited iPSC-CMs can recapitulate the varying phenotypic severity of BrS. Our findings suggest that preservation of the Ca currents might be a compensatory mechanism to resist arrhythmogenesis in BrS AMCs.
National Key R&D Program of China (2017YFA0103700), National Natural Science Foundation of China (81922006, 81870175), Natural Science Foundation of Zhejiang Province (LD21H020001, LR15H020001), National Natural Science Foundation of China (81970269), Key Research and Development Program of Zhejiang Province (2019C03022) and Natural Science Foundation of Zhejiang Province (LY16H020002).
Brugada 综合征（BrS）是一种心脏通道病，可导致心脏性猝死（SCD）。SCN5A 是与 BrS 关联最密切的基因，但基因型-表型相关性并不完全匹配。特定 SCN5A 变体的临床表型范围从无症状到 SCD。在这里，我们使用源自 SCN5A 突变阳性（D356Y）BrS 家族中具有严重影响的先证者、无症状突变携带者（AMC）和健康对照的诱导多能干细胞衍生的心肌细胞（iPSC-CMs）比较来研究这种变化。
使用非整合性 Sendai 病毒从皮肤成纤维细胞中生成了 26 个 iPSC 系。使用基于单层的分化方案将生成的 iPSCs 分化为心肌细胞。
与对照相比，D356Y iPSC-CMs 表现出更长的心动间隔变异性、更慢的去极化、心律失常、钠通道功能缺陷和不规则的 Ca 信号传导。重要的是，在 AMC iPSC-CMs 中观察到的表型严重程度比先证者 iPSC-CMs 轻，在 flecainide 的作用下这种观察结果更加恶化。有趣的是，与对照和 AMC iPSC-CMs 相比，先证者的 iPSC-CMs 中的 Ca 电流明显减少。使用 CRISPR/Cas9 介导的基因组编辑纠正先证者 iPSC-CMs 中的 D356Y 有效地挽救了心律失常表型，并恢复了 Na 和 Ca 电流。此外，使用已建立的 BrS iPSC-CM 模型进行药物筛选表明，quinidine 和 sotalol 以个体依赖的方式具有抗心律失常作用。临床上，静脉和口服给予钙部分减少了先证者在中期随访中的恶性心律失常事件。
患者特异性和基因组编辑的 iPSC-CMs 可以再现 BrS 表型严重程度的变化。我们的研究结果表明，Ca 电流的保存可能是 BrS AMC 抵抗心律失常发生的代偿机制。
国家重点研发计划（2017YFA0103700）、国家自然科学基金（81922006、81870175）、浙江省自然科学基金（LD21H020001、LR15H020001）、国家自然科学基金（81970269）、浙江省重点研发计划（2019C03022）和浙江省自然科学基金（LY16H020002）。
