473 将人类诱导多能干细胞衍生的心肌细胞(iPSC-CMs)应用于 Ankyrin-2 p.R990Q 变异诱导的室性心律失常建模和个性化医疗

IF 2.1 Q3 MEDICINE, RESEARCH & EXPERIMENTAL Journal of Clinical and Translational Science Pub Date : 2024-04-03 DOI:10.1017/cts.2024.401
Jyotsna Joshi, Neill Schwieterman, Nate Smole, Shuliang Guo, Xiaoping Wan, Angelina Ramirez-Navarro, Cemantha Lane, Isabelle Deschenes, Thomas Hund, Loren Wold, Sakima Smith
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METHODS/STUDY POPULATION: We previously published a case of a 36-year-old woman with an ankyrin-2 p.R990Q (ANK2) variant, presented with severe ventricular arrhythmias and sudden cardiac arrest, caused by a novel mutation in the ankyrin-B gene (c.2969G>A) that disrupts the interaction of ankyrin-B/βII spectrin. To model the condition, we will use human induced pluripotent stem cell (DF 19-9-7T, WiCell)-derived ventricular cardiomyocytes (iPSC-CMs) having ANK2 variant, engineered using CRISPR/Cas9 method (Synthego Corp.). We will validate the differentiation of iPSCs into ventricular lineage and characterize the ANK2 ventricular phenotype. Next, we will express light-gated cation channel Channelrhodopsin (ChR2) in the ANK2 iPSC-CMs and investigate the potential role of optogenetics in treating such severe arrhythmias. RESULTS/ANTICIPATED RESULTS: Immunostaining shows 87.339% of iPSC-CMs, treated with All-trans retinoic acid (RA) (1 uM) on days 7 and 12 [RA 7,12], and 23.84% of those, treated on days 3 and 5 [RA 3,5], expressed MLC-2V (p<0.001). Calcium reuptake (τ) is 0.5914 s in RA 7, 12 while 0.2247s in RA 3, 5 (p<0.001). APD90 and APD50 of RA 7, 12 are 2- and 5-fold higher than RA 3, 5, showing distinct ventricular and atrial phenotypes. Protein expression of βII-spectrin and ankyrin-2 and their co-localizations were reduced in the ANK2 phenotype compared to the healthy phenotype. We found prolongation of Ca2+ waves and τ with blue light on iPSC-CMs, expressing ChR2. We anticipate that such prolongation of calcium transients would prevent aberrant calcium spikes, rescue Ca2+/calpain-induced βII-spectrin loss and provide electrical stability. DISCUSSION/SIGNIFICANCE: Animal models cannot accurately recapitulate human cardiac electrophysiology. 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引用次数: 0

摘要

目的/目标:细胞骨架蛋白α²II谱林与心肌细胞中的肌动蛋白和ankyrin-2相互作用,对协调心肌细胞中的离子通道和膜蛋白至关重要。我们的目标是了解光谱蛋白功能障碍导致严重室性心律失常的分子机制,并探索治疗此类病症的新型疗法。方法/研究对象:我们以前发表过一例 36 岁女性患者的病例,她患有ankin-2 p.R990Q (ANK2)变异,表现为严重室性心律失常和心脏骤停,其原因是ankin-B 基因(c.2969G>A)的新型突变破坏了ankin-B/βII spectrin 的相互作用。为了模拟这种情况,我们将使用通过 CRISPR/Cas9 方法(Synthego 公司)设计的具有 ANK2 变异的人类诱导多能干细胞(DF 19-9-7T,WiCell)衍生的心室心肌细胞(iPSC-CMs)。我们将验证 iPSCs 向心室系的分化,并鉴定 ANK2 心室表型。接下来,我们将在 ANK2 iPSC-CMs 中表达光门控阳离子通道 Channelrhodopsin (ChR2),并研究光遗传学在治疗此类严重心律失常中的潜在作用。结果/预期结果:免疫染色显示,在第 7 天和第 12 天[RA 7,12]用全反式维甲酸(RA)(1 uM)处理的 iPSC-CMs 中,有 87.339% 表达了 MLC-2V(p<0.001);在第 3 天和第 5 天[RA 3,5]用全反式维甲酸(RA)(1 uM)处理的 iPSC-CMs 中,有 23.84% 表达了 MLC-2V(p<0.001)。钙再摄取(τ)在 RA 7、12 中为 0.5914 秒,而在 RA 3、5 中为 0.2247 秒(p<0.001)。RA 7、12 的 APD90 和 APD50 分别比 RA 3、5 高 2 倍和 5 倍,显示出不同的心室和心房表型。与健康表型相比,ANK2 表型中βII-pectrin和ankyrin-2的蛋白表达及其共定位减少。我们发现,在表达 ChR2 的 iPSC-CMs 上,蓝光会延长 Ca2+ 波和τ。我们预计,这种钙离子瞬态的延长将防止钙离子尖峰的异常出现,挽救 Ca2+/calpain 诱导的 βII-spectrin 损失,并提供电稳定性。讨论/意义:动物模型无法准确再现人类心脏电生理学。拟议的基于人类 iPSC-CM 的 ANK2 模型能更好地揭示严重室性心律失常的机理。此外,拟议中的光遗传学心脏起搏器有可能提供安全、有针对性和无痛的心脏起搏器来控制心律失常。
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473 Application of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) for modeling of Ankyrin-2 p.R990Q variant-induced ventricular arrhythmia and personalized medicine
OBJECTIVES/GOALS: The cytoskeletal protein α²II spectrin interacts with actin and ankyrin-2 in cardiomyocytes which is essential to orchestrate ion channels and membrane proteins in the cardiac dyad. Our goal is to understand molecular mechanism causing severe ventricular arrhythmias due to spectrin dysfunction and explore novel therapies to treat such conditions. METHODS/STUDY POPULATION: We previously published a case of a 36-year-old woman with an ankyrin-2 p.R990Q (ANK2) variant, presented with severe ventricular arrhythmias and sudden cardiac arrest, caused by a novel mutation in the ankyrin-B gene (c.2969G>A) that disrupts the interaction of ankyrin-B/βII spectrin. To model the condition, we will use human induced pluripotent stem cell (DF 19-9-7T, WiCell)-derived ventricular cardiomyocytes (iPSC-CMs) having ANK2 variant, engineered using CRISPR/Cas9 method (Synthego Corp.). We will validate the differentiation of iPSCs into ventricular lineage and characterize the ANK2 ventricular phenotype. Next, we will express light-gated cation channel Channelrhodopsin (ChR2) in the ANK2 iPSC-CMs and investigate the potential role of optogenetics in treating such severe arrhythmias. RESULTS/ANTICIPATED RESULTS: Immunostaining shows 87.339% of iPSC-CMs, treated with All-trans retinoic acid (RA) (1 uM) on days 7 and 12 [RA 7,12], and 23.84% of those, treated on days 3 and 5 [RA 3,5], expressed MLC-2V (p<0.001). Calcium reuptake (τ) is 0.5914 s in RA 7, 12 while 0.2247s in RA 3, 5 (p<0.001). APD90 and APD50 of RA 7, 12 are 2- and 5-fold higher than RA 3, 5, showing distinct ventricular and atrial phenotypes. Protein expression of βII-spectrin and ankyrin-2 and their co-localizations were reduced in the ANK2 phenotype compared to the healthy phenotype. We found prolongation of Ca2+ waves and τ with blue light on iPSC-CMs, expressing ChR2. We anticipate that such prolongation of calcium transients would prevent aberrant calcium spikes, rescue Ca2+/calpain-induced βII-spectrin loss and provide electrical stability. DISCUSSION/SIGNIFICANCE: Animal models cannot accurately recapitulate human cardiac electrophysiology. The proposed human iPSC-CM-based ANK2 model would provide better mechanistic insights of severe ventricular arrhythmias. Also, the proposed optogenetic cardioversion has the potential to provide safe, targeted and painless cardioversion to manage arrhythmias.
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来源期刊
Journal of Clinical and Translational Science
Journal of Clinical and Translational Science MEDICINE, RESEARCH & EXPERIMENTAL-
CiteScore
2.80
自引率
26.90%
发文量
437
审稿时长
18 weeks
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