Zhong Rujia, Zhang Feng, Yang Zhen, Li Yingrui, Xu Qiang, Lan Huan, Cyganek Lukas, El-Battrawy Ibrahim, Zhou Xiaobo, Akin Ibrahim, Borggrefe Martin
First Department of Medicine (Department of Cardiology, Angiology, Haemostaseology and Medical Intensive Care), Medical Faculty Mannheim, University Medical Centre Mannheim (UMM), University of Heidelberg, Theodor-Kutzer-Ufer 1-3, Mannheim 68167, Germany.
Key Laboratory of Medical Electrophysiology of Ministry of Education and Medical Electrophysiological Key Laboratory of Sichuan Province, Institute of Cardiovascular Research, Southwest Medical University, Luzhou 646000, Sichuan, China.
Europace. 2022 Dec 9;24(12):2028-2036. doi: 10.1093/europace/euac091.
A loss-of-function mutation in L-type calcium (Ca2+) channel subunit gene CACNB2 has been reported to cause short QT syndrome subtype 5 (SQT5). However, the mechanism underlying the loss-of-function of the Ca2+ channel has not been clarified. In the present study, we aim to explore the DNA methylation mechanism of L-type Ca2+ channel downregulation in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) of SQT5.
The hiPSC-CMs were generated from a healthy donor and a SQT5 patient carrying the CACNB2 variant c.1439C > T/p.S480L. The variant was genetically corrected using ribonucleoprotein-based CRISPR/Cas9 technique to obtain an isogenic control cell line. The action potential (AP) and Ca2+ current were measured by patch clamp. Protein expression levels were determined by western blotting. Dot blotting and bisulfite sequence were performed for epigenetic study. Our results showed that AP durations at 10% repolarization (APD10) and 50% repolarization (APD50) were significantly shortened in SQT5 cells and both the expression level of the β-subunit and channel current of L-type Ca2+ channel were reduced. Besides, an increased level of whole-genome DNA methylation and DNA methylation of CpG island in the promoter region of CACNB2 gene was detected. Overexpression of demethylation enzyme could rescue the decreased expression of CACNB2 and the L-type Ca2+ current.
In SQT5 hiPSC-CMs carrying the CACNB2-S480L variant, the decreased L-type Ca2+ current resulting from decreased CACNB2 protein expression was caused by enhanced methylation in the promoter region of the CACNB2 gene and upregulation of DNA methyltransferases might be one of the mechanisms.
据报道，L型钙（Ca2+）通道亚基基因CACNB2的功能丧失突变可导致短QT综合征5型（SQT5）。然而，Ca2+通道功能丧失的潜在机制尚未阐明。在本研究中，我们旨在探讨SQT5患者的人诱导多能干细胞衍生心肌细胞（hiPSC-CMs）中L型Ca2+通道下调的DNA甲基化机制。
hiPSC-CMs由一名健康供体和一名携带CACNB2变体c.1439C>T/p.S480L的SQT5患者产生。使用基于核糖核蛋白的CRISPR/Cas9技术对该变体进行基因校正，以获得同基因对照细胞系。通过膜片钳测量动作电位（AP）和Ca2+电流。通过蛋白质免疫印迹法测定蛋白质表达水平。进行斑点印迹和亚硫酸氢盐测序以进行表观遗传学研究。我们的结果表明，SQT5细胞中10%复极化时的动作电位时程（APD10）和50%复极化时的动作电位时程（APD50）显著缩短，并且L型Ca2+通道的β亚基表达水平和通道电流均降低。此外，检测到CACNB2基因启动子区域的全基因组DNA甲基化水平和CpG岛的DNA甲基化增加。去甲基化酶的过表达可以挽救CACNB2表达的降低和L型Ca2+电流。
在携带CACNB2-S480L变体的SQT5 hiPSC-CMs中，CACNB2蛋白表达降低导致的L型Ca2+电流降低是由CACNB2基因启动子区域甲基化增强引起的，DNA甲基转移酶上调可能是其中一种机制。
