Long QT 7 syndrome (LQT7, also known as Andersen-Tawil syndrome) is a rare autosomal-dominant disorder that causes cardiac arrhythmias, periodic paralysis, and dysmorphic features. Mutations in the human KCNJ2 gene, which encodes for the subunit of the potassium inwardly-rectifying channel ( ), have been associated with the disorder. The majority of mutations are considered to be dominant-negative as mutant proteins interact to limit the function of wild type KCNJ2 proteins. Several LQT7 syndrome mouse models have been created that vary in the physiological similarity to the human disease. To complement the LQT7 mouse models, we investigated the usefulness of the zebrafish as an alternative model via a transient approach. Initial bioinformatic analysis identified the zebrafish orthologue of the human KCNJ2 gene, together with a spatial expression profile that was similar to that of human. The expression of a kcnj2-12 transcript carrying an in-frame deletion of critical amino acids identified in human studies resulted in embryos that exhibited defects in muscle development, thereby affecting movement, a decrease in jaw size, pupil-pupil distance, and signs of scoliosis. These defects correspond to some phenotypes expressed by human LQT7 patients. 1. Introduction Long QT 7 syndrome (LQT7, also known as Andersen-Tawil syndrome) is a rare autosomal-dominant disorder that causes periodic paralysis, ventricular arrhythmias with QT-prolongation, and dysmorphic features, which may not be present in all affected individuals. The dysmorphic features include scoliosis (curvature of the spine), clinodactyly (permanent lateral or medial curve of a finger or toe), wide-set eyes, low set or slanted ears, small jaw, and broad forehead [1]. Mutations in the human KCNJ2 gene, which encodes the potassium inwardly rectifying channel ( ) subunit, have been associated with the disorder. The KCNJ2 gene is expressed in the heart, brain, lung, skeletal muscle, kidney, and the eyes [2–4]. In the heart, the channel is involved in the resting phase of the cardiac action potential (AP) cycle. The KCNJ2 protein consists of two transmembrane domains encompassing a selective pore region. There are currently 38 mutations reported in the Inherited Arrhythmias Database (http://www.fsm.it/cardmoc/), and 19 of these mutations have dominant-negative effects. Many of these mutations have been characterised by electrophysiological measurements of transfected CHO cells. This in vitro system is widely used, but for the purposes of functional studies, in vivo modelling is the approach-of-choice.
References
[1]
R. Tawil, L. J. Ptacek, S. G. Pavlakis et al., “Andersen's syndrome: potassium-sensitive periodic paralysis, ventricular ectopy, and dysmorphic features,” Annals of Neurology, vol. 35, no. 3, pp. 326–330, 1994.
[2]
A. Llobet, X. Gasull, J. Palés, E. Martí, and A. Gual, “Identification of kir2.1 channel activity in cultured trabecular meshwork cells,” Investigative Ophthalmology and Visual Science, vol. 42, no. 10, pp. 2371–2379, 2001.
[3]
K. F. Raab-Graham, C. M. Radeke, and C. A. Vandenberg, “Molecular cloning and expression of a human heart inward rectifier potassium channel,” NeuroReport, vol. 5, no. 18, pp. 2501–2505, 1994.
[4]
J. B. Redell and B. L. Tempel, “Multiple promoter elements interact to control the transcription of the potassium channel gene, KCNJ2,” Journal of Biological Chemistry, vol. 273, no. 35, pp. 22807–22818, 1998.
[5]
J. J. Zaritsky, D. M. Eckman, G. C. Wellman, M. T. Nelson, and T. L. Schwarz, “Targeted disruption of kir2.1 and kir2.2 genes reveals the essential role of the inwardly rectifying k+ current in k+-mediated vasodilation,” Circulation Research, vol. 87, no. 2, pp. 160–166, 2000.
[6]
J. J. Zaritsky, J. B. Redell, B. L. Tempel, and T. L. Schwarz, “The consequences of disrupting cardiac in wardly rectifying k+ current ( ) as revealed by the targeted deletion of the murine kir2.1 and kir2.2 genes,” Journal of Physiology, vol. 533, no. 3, pp. 697–710, 2001.
[7]
J. Li, M. McLerie, and A. N. Lopatin, “Transgenic upregulation of in the mouse heart leads to multiple abnormalities of cardiac excitability,” American Journal of Physiology: Heart and Circulatory Physiology, vol. 287, no. 6, pp. H2790–H2802, 2004.
[8]
C. W. Lu, J. H. Lin, Y. S. Rajawat et al., “Functional and clinical characterization of a mutation in KCNJ2 associated with Andersen-Tawil syndrome,” Journal of Medical Genetics, vol. 43, no. 8, pp. 653–659, 2006.
[9]
M. McLerie and A. Lopatin, “Dominant-negative suppression of in the mouse heart leads to altered cardiac excitability,” Journal of Molecular and Cellular Cardiology, vol. 35, no. 4, pp. 367–378, 2003.
[10]
A. Dodd, P. M. Curtis, L. C. Williams, and D. R. Love, “Zebrafish: bridging the gap between development and disease,” Human Molecular Genetics, vol. 9, no. 16, pp. 2443–2449, 2000.
[11]
K. Dooley and L. I. Zon, “Zebrafish: a model system for the study of human disease,” Current Opinion in Genetics and Development, vol. 10, no. 3, pp. 252–256, 2000.
[12]
F. Brette, G. Luxan, C. Cros, H. Dixey, C. Wilson, and H. A. Shiels, “Characterization of isolated ventricular myocytes from adult Zebrafish (Danio rerio),” Biochemical and Biophysical Research Communications, vol. 374, no. 1, pp. 143–146, 2008.
[13]
N. Hu, D. Sedmera, H. J. Yost, and E. B. Clark, “Structure and function of the developing Zebrafish heart,” The Anatomical Record, vol. 260, no. 2, pp. 148–157, 2000.
[14]
D. J. Milan, I. L. Jones, P. T. Ellinor, and C. A. MacRae, “In vivo recording of adult Zebrafish electrocardiogram and assessment of drug-induced QT prolongation,” American Journal of Physiology: Heart and Circulatory Physiology, vol. 291, no. 1, pp. H269–H273, 2006.
[15]
P. Nemtsas, E. Wettwer, T. Christ, G. Weidinger, and U. Ravens, “Adult Zebrafish heart as a model for human heart? an electrophysiological study,” Journal of Molecular and Cellular Cardiology, vol. 48, no. 1, pp. 161–171, 2010.
[16]
D. Sedmera, M. Reckova, A. DeAlmeida et al., “Functional and morphological evidence for a ventricular conduction system in Zebrafish and xenopus hearts,” American Journal of Physiology: Heart and Circulatory Physiology, vol. 284, no. 4, pp. H1152–H1160, 2003.
[17]
R. Arnaout, T. Ferrer, J. Huisken et al., “Zebrafish model for human long QT syndrome,” Proceedings of the National Academy of Sciences of the United States of America, vol. 104, no. 27, pp. 11316–11321, 2007.
[18]
U. Langheinrich, G. Vacun, and T. Wagner, “Zebrafish embryos express an orthologue of HERG and are sensitive toward a range of QT-prolonging drugs inducing severe arrhythmia,” Toxicology and Applied Pharmacology, vol. 193, no. 3, pp. 370–382, 2003.
[19]
B. Meder, E. P. Scholz, D. Hassel et al., “Reconstitution of defective protein trafficking rescues long-QT syndrome in Zebrafish,” Biochemical and Biophysical Research Communications, vol. 408, no. 2, pp. 218–224, 2011.
[20]
S. W. Mittelstadt, C. L. Hemenway, M. P. Craig, and J. R. Hove, “Evaluation of Zebrafish embryos as a model for assessing inhibition of hERG,” Journal of Pharmacological and Toxicological Methods, vol. 57, no. 2, pp. 100–105, 2008.
[21]
D. S. Peal, R. W. Mills, S. N. Lynch et al., “Novel chemical suppressors of long QT syndrome identified by an in vivo functional screen,” Circulation, vol. 123, no. 1, pp. 23–30, 2011.
[22]
D. P. Wall, H. B. Fraser, and A. E. Hirsh, “Detecting putative orthologs,” Bioinformatics, vol. 19, no. 13, pp. 1710–1711, 2003.
[23]
K. Tamura, J. Dudley, M. Nei, and S. Kumar, “MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0,” Molecular Biology and Evolution, vol. 24, no. 8, pp. 1596–1599, 2007.
[24]
R. Tang, A. Dodd, D. Lai, W. C. McNabb, and D. R. Love, “Validation of Zebrafish (Danio rerio) reference genes for quantitative real-time RT-PCR normalization,” Acta Biochimica et Biophysica Sinica, vol. 39, no. 5, pp. 384–390, 2007.
[25]
I. U. S. Leong, J. R. Skinner, A. N. Shelling, and D. R. Love, “Identification and expression analysis of kcnh2 genes in the Zebrafish,” Biochemical and Biophysical Research Communications, vol. 396, no. 4, pp. 817–824, 2010.
[26]
C.-C. Lan, R. Tang, I. U. S. Leong, and D. R. Love, “Quantitative real-time RT-PCR (qRT-PCR) of Zebrafish transcripts: optimization of RNA extraction, quality control considerations, and data analysis,” Cold Spring Harbor Protocols, vol. 4, prot 5314, no. 10, 2009.
[27]
A. St？hlberg, P. ？man, B. Ridell, P. Mostad, and M. Kubista, “Quantitative real-time PCR method for detection of b-lymphocyte monoclonality by comparison of κ and λ immunoglobulin light chain expression,” Clinical Chemistry, vol. 49, no. 1, pp. 51–59, 2003.
[28]
J. Hellemans, G. Mortier, A. De Paepe, F. Speleman, and J. Vandesompele, “qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data,” Genome Biology, vol. 8, no. 2, article R19, 2007.
[29]
C. Thisse and B. Thisse, “High-resolution in situ hybridization to whole-mount Zebrafish embryos,” Nature Protocols, vol. 3, no. 1, pp. 59–69, 2008.
[30]
M. B. Walker and C. B. Kimmel, “A two-color acid-free cartilage and bone stain for Zebrafish larvae,” Biotechnic and Histochemistry, vol. 82, no. 1, pp. 23–28, 2007.
[31]
M. D. Abramoff, P. J. Magalhaes, and S. J. Ram, “Image processing with IMageJ,” Biophotonics International, vol. 1, pp. 36–42, 2004.
[32]
H. Hibino, A. Inanobe, K. Furutani, S. Murakami, I. Findlay, and Y. Kurachi, “Inwardly rectifying potassium channels: their structure, function, and physiological roles,” Physiological Reviews, vol. 90, no. 1, pp. 291–366, 2010.
[33]
W. B. Barbazuk, I. Korf, C. Kadavi et al., “The syntenic relationship of the Zebrafish and human genomes,” Genome Research, vol. 10, no. 9, pp. 1351–1358, 2000.
[34]
C. B. Kimmel, W. W. Ballard, S. R. Kimmel, B. Ullmann, and T. F. Schilling, “Stages of embryonic development of the Zebrafish,” Developmental Dynamics, vol. 203, no. 3, pp. 253–310, 1995.
[35]
M. Tristani-Firouzi, J. L. Jensen, M. R. Donaldson et al., “Functional and clinical characterization of KCNJ2 mutations associated with LQT7 (Andersen syndrome),” Journal of Clinical Investigation, vol. 110, no. 3, pp. 381–388, 2002.
[36]
J. Burrone, M. O'Byrne, and V. N. Murthy, “Multiple forms of synaptic plasticity triggered by selective suppression of activity in individual neurons,” Nature, vol. 420, no. 6914, pp. 414–418, 2002.
[37]
J. Y. Hua, M. C. Smear, H. Baier, and S. J. Smith, “Regulation of axon growth in vivo by activity-based competition,” Nature, vol. 434, no. 7036, pp. 1022–1026, 2005.
[38]
T. Yoshida, S. Uchida, and M. Mishina, “Regulation of synaptic vesicle accumulation and axon terminal remodeling during synapse formation by distinct Ca2+ signaling,” Journal of Neurochemistry, vol. 111, no. 1, pp. 160–170, 2009.
[39]
N. M. Plaster, R. Tawil, M. Tristani-Firouzi et al., “Mutations in kir2.1 cause the developmental and episodic electrical phenotypes of Andersen's syndrome,” Cell, vol. 105, no. 4, pp. 511–519, 2001.
[40]
K. A. McKeown, G. B. Downes, and L. D. Hutson, “Modular laboratory exercises to analyze the development of Zebrafish motor behavior,” Zebrafish, vol. 6, no. 2, pp. 179–185, 2009.
[41]
Y. Naganawa and H. Hirata, “Developmental transition of touch response from slow muscle-mediated coilings to fast muscle-mediated burst swimming in Zebrafish,” Developmental Biology, vol. 355, no. 2, pp. 194–204, 2011.
[42]
M. J. Airhart, D. H. Lee, T. D. Wilson, B. E. Miller, M. N. Miller, and R. G. Skalko, “Movement disorders and neurochemical changes in Zebrafish larvae after bath exposure to fluoxetine (PROZAC),” Neurotoxicology and Teratology, vol. 29, no. 6, pp. 652–664, 2007.
[43]
M. Dong, Y. F. Fu, T. T. Du et al., “Heritable and lineage-specific gene knockdown in Zebrafish embryo,” PLoS One, vol. 4, no. 7, Article ID e6125, 2009.
[44]
T. F. Schilling and C. B. Kimmel, “Musculoskeletal patterning in the pharyngeal segments of the Zebrafish embryo,” Development, vol. 124, no. 15, pp. 2945–2960, 1997.
[45]
M. L. Suster, H. Kikuta, A. Urasaki, K. Asakawa, and K. Kawakami, “Transgenesis in Zebrafish with the Tol2 transposon system,” Methods in Molecular Biology, vol. 561, pp. 41–63, 2009.
[46]
Y. Doyon, J. M. McCammon, J. C. Miller et al., “Heritable targeted gene disruption in Zebrafish using designed zinc-finger nucleases,” Nature Biotechnology, vol. 26, no. 6, pp. 702–708, 2008.
