Rapid communication Effect of experimental hypercholesterolaemia on K + channel α-subunit mRNA levels in rabbit hearts Angelika Varga a, ⁎ , Péter Bagossi b , József Tözsér b , Barna Peitl a , Zoltán Szilvássy a a Department of Pharmacology and Pharmacotherapy, Medical and Health Science Center, University of Debrecen, Hungary b Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Hungary Received 11 December 2006; received in revised form 28 January 2007; accepted 1 February 2007 Available online 16 February 2007 Abstract We investigated the effect of dietary cholesterol on gene transcription of delayed rectifier (I Kr — ERG1 and I Ks — KvLQT1) and transient outward (I to,fast — Kv4.2 and Kv4.3) potassium channel subunits in rabbit hearts using real-time quantitative reverse transcription-polymerase chain reaction (RT- PCR). While the level of Kv4.3 mRNA did not change, both Kv4.2 and ERG1 mRNAs were downregulated, whereas the level of KvLQT1 was increased in hypercholesterolaemic rabbits, indicating that hypercholesterolaemia altered ventricular K + channel α-subunit gene transcription. © 2007 Elsevier B.V. All rights reserved. Keywords: K + channel; mRNA expression; Hypercholesterolaemia Hypercholesterolaemia is often associated with abnormal electrical activities in the myocardium characterized by longer QT interval in the ECG, afterdepolarizations and ventricular arrhythmias (Adamantidis et al., 1992; Liu et al., 2003). Nevertheless, no data are available whether the expression of K + channel α-subunits implicated in the development of arrhythmias, as summarized previously by Tristani-Firouzi et al. (2001), changes in hypercholesterolaemia. Therefore, the purpose of the present study was to evaluate the effects of diet- induced hypercholesterolaemia on gene transcription of these repolarizing outward currents in rabbit ventricles. Adult, male New Zealand white rabbits were maintained on 1.5% cholesterol-enriched diet (n = 4) over 8 weeks preceding the experiments as described previously (Szilvassy et al., 2001), yielding an increase in serum cholesterol level from 1.7 ± 0.4 to 24.1 ± 2.9 mmol/l. Control animals were given standard lab chow (n = 4). All experiments were approved by the ethics committee of the Medical and Health Science Center of our University. Total RNA was extracted from left ventricular muscle samples (2–3/rabbit) using the RNeasy Fibrous Tissue Mini Kit (Qiagen) according to the supplier's instructions. DNase-treated total RNA preparations (2 μg) were reverse transcribed by using random primers (Invitrogen) and Super- Script™ II reverse transcription kit (Invitrogen) according to the manufacturer's recommendations, followed by RT-PCR using ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Primer pairs and TaqMan probes for RT-PCR were designed by Primer Express software (Applied Biosys- tems) based on published rabbit mRNA sequences (http://www. ncbi.nlm.nih.gov/GenBank) for Kv4.2 (GenBank accession no. AF493547), for Kv4.3 (AF085170), for KvLQT1 (AJ291316), for ERG1 (U87513) and for 18S rRNA (X06778). The oligonucleotide sequences are available at http://biochemistry. med.unideb.hu/SBBG/OligoDB.htlm. PCR reaction was carried out in a 10 μl volume containing final concentrations of 1× Taq buffer (Fermentas), 0.1 mM dNTP mix (Fermentas), 2.5 mM MgCl 2 (Fermentas), 1 × ROX reference Dye (Invitrogen), 500 nM forward and reverse primer each (Bio-Science), 100 nM TaqMan probe (Bio-Science), 1 μl cDNA and 0.25 units of Taq DNA polymerase (Fermentas). Thermal cycling conditions for amplification were 1 min at 94 °C, followed by 40 cycles of 15 sec at 94 °C, 60 sec at 60 °C (for ERG1 and Kv4.2), at 58 °C (for KvLQT1) or at 55 °C (for Kv4.3), respectively. In present RT-PCR studies left ventricular mRNA expres- sions of ERG1, KvLQT1, Kv4.2, Kv4.3 were quantified from European Journal of Pharmacology 562 (2007) 130 – 131 www.elsevier.com/locate/ejphar ⁎ Corresponding author. Postal address: Department of Pharmacology and Pharmacotherapy, University of Debrecen, H-4032 Debrecen, Nagyerdei krt 98, Hungary. Tel./fax: +36 52 427 899. E-mail address: angelika.varga@king.pharmacol.dote.hu (A. Varga). 0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.02.003