Functional analysis of KCNH2 gene mutations of type 2 long QT syndrome in larval zebrafish using microscopy and electrocardiography
Functional analysis of KCNH2 gene mutations of type 2 long QT syndrome in larval zebrafish using microscopy and electrocardiography
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DOI：
10.1007/s00380-018-1231-4
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发表时间：
2018-07
影响因子：
1.5
通讯作者：
Yoshihiro Tanaka;K. Hayashi;N. Fujino;T. Konno;H. Tada;C. Nakanishi;A. Hodatsu;T. Tsuda;Y. Nagata;R. Teramoto;S. Yoshida;A. Nomura;M. Kawashiri;M. Yamagishi
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文献类型：
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作者：
Yoshihiro Tanaka;K. Hayashi;N. Fujino;T. Konno;H. Tada;C. Nakanishi;A. Hodatsu;T. Tsuda;Y. Nagata;R. Teramoto;S. Yoshida;A. Nomura;M. Kawashiri;M. Yamagishi
异源表达系统在钾电压门控通道亚家族H成员2（KCNH 2）基因突变的表征中起着至关重要的作用，例如与长QT综合征2型（LQT 2）相关的E637 K。使用斑马鱼的体内测定提供了用于测试心脏病的遗传变异的手段;然而，从体内系统中可获得关于E637 K突变的作用的有限信息，并且它们的效用尚未在LQT 2的背景下被充分利用。我们试图评估E637 K突变通道恢复正常复极的能力，在斑马鱼幼鱼与人类KCNH 2的同源物，kcnh 2a敲低。将靶向KCNH 2a的吗啉代（MO）单独或与野生型（WT）或E637 K KCNH 2cRNA一起注射到1-2细胞期的斑马鱼胚胎中。在受精后72小时，使用光学显微镜筛选心脏复极表型，并通过单导联心电图（ECG）分析测量QT间期。在MO单独组中，17%的斑马鱼具有正常表型;在注射WT KCNH 2 cRNA的斑马鱼中，该比率增加至60%，在注射E637 K的斑马鱼中增加至35%。斑马鱼幼鱼的ECG显示，与对照组相比，MO单独组的QTc显著延长。同时注射WT KCNH 2 cRNA可缩短QTc间期，而E637 K则无此作用。我们认为，这种在体内的心脏分析，使用显微镜和心电图在幼斑马鱼提供了一个可靠的方法来区分KCNH 2突变的风险。
Heterologous expression systems play a vital role in the characterization of potassium voltage-gated channel subfamily H member 2 (KCNH2) gene mutations, such as E637K which is associated with long QT syndrome type 2 (LQT2). In vivo assays using zebrafish provide a means for testing genetic variants of cardiac disease; however, limited information on the role of the E637K mutation is available from in vivo systems and their utility has yet to be fully exploited in the context of LQT2. We sought to evaluate the ability of the E637K mutant channel to restore normal repolarization in larval zebrafish with a humanKCNH2orthologue,kcnh2a-knockdown. A morpholino (MO) targetingkcnh2awas injected alone or with wild type (WT) or E637K KCNH2 cRNA into zebrafish embryos at the 1–2 cell stage. Cardiac repolarization phenotypes were screened using light microscopy and the QT interval was measured by single lead electrocardiograph (ECG) analysis at 72-h post-fertilization. In the MO alone group, 17% of zebrafish had a normal phenotype; this rate increased to 60% in the WT KCNH2 cRNA injected zebrafish and to 35% in the E637K injected zebrafish. The ECG of larval zebrafish revealed that QTc was significantly prolonged in the MO alone group compared to the control group. Co-injection of WT KCNH2 cRNA shortened the QTc interval, however, that of the E637K did not. We suggest that this in vivo cardiac assay using microscopy and ECG in larval zebrafish offers a reliable approach for risk discrimination ofKCNH2mutations.