Functional analysis of KCNH2 gene mutations of type 2 long QT syndrome in larval zebrafish using microscopy and electrocardiography
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 human KCNH2 orthologue, kcnh2a-knockdown. A morpholino (MO) targeting kcnh2a was 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 of KCNH2 mutations.
KeywordsLong QT syndrome KCNH2 In vivo cardiac assay Zebrafish Electrocardiography
The authors gratefully acknowledge Dr. Attila Sik and Dr. Ferenc Muller (University of Birmingham) for technical advice and helpful discussion concerning larval ECG measurement in Zebrafish, and thank Takako Obayashi for technical assistance. The work was supported by grants from the Ministry of Health, Labor and Welfare of Japan for Clinical Research on Intractable Diseases (H26-040 and H24-033 to K.H.), grant-in-aid for Scientific Research from Japan Society for the Promotion of Science (26460670 and 15KK0302 to K.H.), Takeda Science Foundation to K.H.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
- 8.Liu L, Hayashi K, Kaneda T, Ino H, Fujino N, Uchiyama K, Konno T, Tsuda T, Kawashiri MA, Ueda K, Higashikata T, Shuai W, Kupershmidt S, Higashida H, Yamagishi M (2013) A novel mutation in the transmembrane nonpore region of the KCNH2 gene causes severe clinical manifestations of long QT syndrome. Heart Rhythm 10:61–67CrossRefGoogle Scholar
- 10.Berghmans S, Butler P, Goldsmith P, Waldron G, Gardner I, Golder Z, Richards FM, Kimber G, Roach A, Alderton W, Fleming A (2008) Zebrafish based assays for the assessment of cardiac, visual and gut function—potential safety screens for early drug discovery. J Pharmacol Toxicol Methods 58:59–68CrossRefGoogle Scholar
- 15.Hodatsu A, Konno T, Hayashi K, Funada A, Fujita T, Nagata Y, Fujino N, Kawashiri MA, Yamagishi M (2014) Compound heterozygosity deteriorates phenotypes of hypertrophic cardiomyopathy with founder MYBPC3 mutation: evidence from patients and zebrafish models. Am J Physiol Heart Circ Physiol 307:H1594–H1604CrossRefGoogle Scholar