Identification and expression analysis of kcnh2 genes in the zebrafish Ivone Un San Leong a , Jonathan R. Skinner b , Andrew N. Shelling c , Donald R. Love a,d, * a School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland Mail Centre, Auckland 1142, New Zealand b Greenlane Paediatric and Congenital Cardiac Service, Starship Children’s Hospital, Grafton Auckland, Private Bag 92024, New Zealand c Department of Obstetrics and Gynaecology, University of Auckland, Private Bag 92019, Auckland Mail Centre, Auckland 1142, New Zealand d LabPLUS, Auckland City Hospital, PO Box 110031, Auckland Mail Centre, Auckland 1142, New Zealand article info Article history: Received 19 April 2010 Available online 11 May 2010 Keywords: Zebrafish KCNH2 HERG Zerg Gene targeting abstract Long QT syndrome is a disorder that is characterised by a prolonged QT-interval and can lead to fatal car- diac arrhythmias. Many animal models have been created to study congenital long QT syndrome. Of these, zebrafish models have involved targeting two different KCNH2 gene (long QT syndrome 2) ortho- logues, termed zerg-2 and zerg-3, with differing cardiac phenotypes. In order to clarify this situation, this study uses a bioinformatic approach to search the current zebrafish genome sequence (Zv7 and Zv8 builds) to investigate and locate all likely zebrafish orthologues of the human KCNH2 gene. Quantitative real-time RT-PCR was also used to determine the temporal and spatial gene expression profile of the zeb- rafish orthologues. The data support the conclusion that zerg-2 and zerg-3 are apparent orthologues of different human genes encoding potassium ion channels, but that their functions have switched com- pared to the respective human proteins. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction Long QT syndrome (LQTS) is a group of disorders characterised by a prolonged QT-interval that may lead to fatal cardiac arrhyth- mias, and can be characterised as congenital or drug-acquired. Twelve genes have been associated with congenital LQTS, which encode for either ion channel proteins (LQT 1, 2, 3, 5, 6, 7, 8 and 10) involved with the generation of cardiac action potential, or scaffolding proteins (LQT 4, 9, 11 and 12) that are responsible for anchoring ion channels to the cell membrane. In drug-acquired LQTS, the KCNH2 protein (mutations in which are implicated in LQT 2) is the major ion channel that is blocked by QT-prolonging drugs [1]. Recently, the zebrafish has emerged as a modelling spe- cies for LQTS. This species has a cardiac action potential which resembles that in the human more closely than other animal mod- els, and when exposed to known QT-prolonging drugs exhibits bra- dycardia (slowing of the heart rate) [2]. Mutants of the zebrafish orthologue of the human KCNH2 gene have been identified that ex- hibit bradycardia [2], slight to severe arrhythmia, including atrial fibrillation [3], and atrioventricular block [4], all of which can occur in human LQT syndrome or with KCNH2 gene mutations. Just as mutants in the human KCNH2 gene can cause long or short QT syn- drome (SQT), the zebrafish reggae mutant (in the zebrafish kcnh2 gene) causes electrophysiological changes analogous to human short QT syndrome [5]. There still remains, however, uncertainty as to which gene in the zebrafish is the true orthologue of the human KCNH2 gene. The KCNH2 gene encodes the HERG protein, which is the a-sub- unit of the rapid delayed rectifier current (I Kr ). HERG is responsible for the repolarisation phase of cardiac action potential in ventricu- lar myocytes. However, the kcnh2 gene transcript targeted by Milan et al. [2] (zerg-2 gene; located on zebrafish chromosome 2) differs from that targeted by Langheinrich et al. [3] and Arnaout et al. [4] (zerg-3 gene; located on zebrafish chromosome 3). In the case of zerg-3, conserved primers were used to amplify a small region of the zebrafish genome followed by 5 0 and 3 0 rapid ampli- fication of cDNA ends (RACE) to obtain the full length sequence [3]. Whole mount RNA in situ hybridisation showed that the zerg-3 mRNA is localised in the heart of 4 day old zebrafish larvae [3] and a recent biophysical study of the zerg-3 protein showed that it displayed properties similar to the HERG protein [6]. The methods used to identify the zerg-2 gene are unclear. There is no accession number of the target gene [2]; however, a BLAST search using the antisense morpholino (MO) sequence described by Milan et al. [2] located the zerg-2 gene to chromosome 2 (ENS- DARG00000029881). The MO knockdown of the zerg-2 gene in zeb- rafish caused bradycardia in a dose-dependent manner [2], suggesting that zerg-2 plays a role in cardiac action potential [6]. The existence of two possible zebrafish orthologues of the KCNH2 gene may be due to the whole genome duplication event that arose prior to the teleost radiation [7]. However, it remains 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.04.157 * Corresponding author. Address: Diagnostic Genetics, LabPLUS, Auckland City Hospital, PO Box 110031, Auckland Mail Centre, Auckland 1142, New Zealand. Fax: +64 9 3074939. E-mail addresses: donaldl@adhb.govt.nz, d.love@auckland.ac.nz (D.R. Love). Biochemical and Biophysical Research Communications 396 (2010) 817–824 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc