Department of Cardiovascular Medicine (T.K., M.S., S.K., T.T., H.I., S. Okuno, S.Ogawa, Y.K., J.-K.L., S. Hikoso, Y.S.).
Department of Medical Therapeutics for Heart Failure (S. Higo).
Circ Genom Precis Med. 2022 Oct;15(5):e003522. doi: 10.1161/CIRCGEN.121.003522. Epub 2022 Jul 12.
The Δ160E mutation in , which encodes troponin T, is a rare pathogenic variant identified in patients with hypertrophic cardiomyopathy and is associated with poor prognosis. Thus, a convenient human model recapitulating the pathological phenotype caused by Δ160E is required for therapeutic development.
We identified a heterozygous in-frame deletion mutation (c.478_480del, p.Δ160E) in in a patient with familial hypertrophic cardiomyopathy showing progressive left ventricular systolic dysfunction, leading to advanced heart failure. To investigate the pathological phenotype caused by Δ160E, we generated a set of isogenic induced pluripotent stem cells carrying the heterozygous Δ160E, homozygously corrected or homozygously introduced Δ160E using genome editing and differentiated them into cardiomyocytes (Hetero-Δ160E-, wild type-, and Homo-Δ160E-induced pluripotent stem cells [iPSC]-derived cardiomyocytes [iPSC-CMs]).
Hetero-Δ160E-iPSC-CMs exhibited prolonged calcium decay, relaxation impairment, and hypertrophy compared to wild type-iPSC-CMs. Notably, these phenotypes were further exacerbated in Homo-Δ160E-iPSC-CMs. Overexpression of R-GECO-fused Δ160E mutant troponin T prolonged decay time and time to peak of the myofilament-localized calcium transient in iPSC-CMs, indicating that sarcomeric calcium retention with Δ160E may affect intracellular calcium concentration. High-content imaging analysis detected remarkable nuclear translocation of NFATc1, especially in Homo-Δ160E-iPSC-CMs, indicating that the Δ160E mutation promotes hypertrophic signaling pathway in a dose-dependent manner. Increased phosphorylation of CaMKIIδ (calcium/calmodulin-dependent protein kinase IIδ) and phospholamban at Thr17 was observed in Homo- and Hetero-Δ160E-iPSC-CMs. Epigallocatechin-3-gallate, a calcium desensitizing compound, shortened prolonged calcium decay and relaxation duration in Δ160E-iPSC-CMs.
Isogenic iPSC-CMs recapitulate the prolonged calcium decay, relaxation impairment, and subsequent calcium-regulated signaling pathways caused by the Δ160E mutation and can serve as a human model for therapeutic development to prevent hypertrophic cardiomyopathy pathology.
肌钙蛋白 T 编码基因 中的 Δ160E 突变是一种罕见的致病性变异，已在肥厚型心肌病患者中被发现，与预后不良相关。因此，需要一种方便的人类模型来重现由 Δ160E 引起的病理表型，以进行治疗开发。
我们在一名家族性肥厚型心肌病患者中发现了一种异质框内缺失突变（c.478_480del，p.Δ160E），该患者表现为进行性左心室收缩功能障碍，导致晚期心力衰竭。为了研究 Δ160E 引起的病理表型，我们使用基因组编辑生成了一组携带杂合性 Δ160E、纯合性校正或纯合性引入 Δ160E 的同源诱导多能干细胞，并将其分化为心肌细胞（杂合性 Δ160E-、野生型和纯合性 Δ160E-诱导多能干细胞 [iPSC]-衍生的心肌细胞 [iPSC-CMs]）。
与野生型 iPSC-CMs 相比，杂合性 Δ160E-iPSC-CMs 表现出钙衰减延长、松弛受损和肥大。值得注意的是，这些表型在纯合性 Δ160E-iPSC-CMs 中进一步加重。过表达融合了 R-GECO 的 Δ160E 突变肌钙蛋白 T 延长了 iPSC-CMs 中肌球蛋白定位钙瞬变的衰减时间和峰值时间，表明 Δ160E 可能影响肌节内钙浓度的钙保留。高内涵成像分析检测到 NFATc1 的显著核易位，特别是在纯合性 Δ160E-iPSC-CMs 中，表明 Δ160E 突变以剂量依赖的方式促进了肥大信号通路。在 Homo- 和 Hetero-Δ160E-iPSC-CMs 中观察到钙调蛋白依赖性蛋白激酶 IIδ（CaMKIIδ）和肌浆网磷蛋白 at Thr17 的磷酸化增加。表没食子儿茶素没食子酸酯（一种钙脱敏化合物）缩短了 Δ160E-iPSC-CMs 中钙衰减延长和松弛持续时间。
同基因 iPSC-CMs 重现了由 Δ160E 突变引起的钙衰减延长、松弛受损和随后的钙调节信号通路，可作为治疗开发的人类模型，以预防肥厚型心肌病的病理。
