the resulting ERG1 current can be modified. We therefore hypothesized that the variation in ‘peak potential’ of the IKr transient observed in native cardiac cells [10] can be explained by differences in macroscopic channel kinetics caused by a variation in the relative abundance of ERG1 isoforms. As the functional consequences of regional differences in the relative abundance of ERG1 isoforms are largely unknown we explored this as a potential mechanism underlying native heterogeneity of action potential characteristics. We find that the characteristics of native IKr transients can be reproduced in heterologous expression systems by differential expression of hERG1a and hERG1b isoforms. Furthermore, we describe how differential expression of hERG1a and hERG1b may in turn modulate both action potential duration (APD) and restitution properties. Methods DNA Constructs cDNAs encoding hERG1a (acc. number NM_000238) and hERG1b (acc. number NM_172057) were inserted into either pcDNA3 (for mammalian expression) or into pXOOM [13] (for expression in X. laevis oocytes) plasmids. The hERG1b clone was obtained from A. Arcangeli (Universita` degli Studi di Firenze, Italy). Expression in HEK293 Cells HEK293 cells were maintained in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and 5% CO2 at 37uC. At 50–60% confluency, cells were transiently transfected with a total of 2 mg of cDNA using Lipofectamine (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. For co-transfection, 1 mg of each cDNA was used. 0.3 mg of eGFP in pcDNA3 was co-transfected to identify successfully transfected cells. 48 hours after transfection, the cells were trypsinized and transferred to cover slips for experiments. Expression in X. laevis Oocytes cRNA for injection was prepared from the linearized DNA constructs using the T7 m-Message Machine kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. RNA concentrations were quantified using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and RNA quality was checked by gel electrophoresis. X. laevis oocytes were either purchased from Ecocyte Bioscience (Castrop-Rauxel, Germany) or prepared in-house. In the latter case X. laevis surgery and oocyte treatment were performed according to the guidelines of the Danish National Committee for Animal Studies. For co-expression of hERG1a and hERG1b cRNAs were mixed in different molar ratios (20%, 40%, 60% and 80% hERG1b) before injection. After injection the oocytes were kept in Kulori solution (in mM: 87 NaCl, 4 KCl, 1 MgCl2, 1 CaCl2, 5 HEPES, pH 7.4) at 19uC for 2–3 days before experiments were performed. Electrophysiological Procedures Measurements on HEK293 cells were performed in the whole- cell patch clamp configuration using an EPC-9 patch clamp amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany). The glass pipettes for the recording electrodes had a tip resistance of 1.5–2.5 MOhm when filled with intracellular solution. The series resistance recorded in the whole-cell configuration was 2–8 MOhm and was compensated (80%). The extracellular solution Role of Cardiac hERG1 Isoforms contained (in mM): NaCl 140, KCl 4, CaCl2 2, MgCl2 1 and HEPES 10, pH 7.4. The intracellular solution used in the pipettes contained (in mM): KCl 110, EGTA 10, CaCl2 5.17, MgCl2 1.42, K2ATP 4, HEPES 10, pH 7.2. Measurements on X. laevis oocytes were performed with the two- electrode voltage-clamp technique using a Dagan CA-1B amplifier (Dagan Corporation, Minneapolis, MN, USA). The recordings were performed under continuous superfusion with Kulori solution. The glass pipettes for the recording electrodes were filled with 2 M KCl and had tip resistances of 0.5–2.5 MOhm. All recordings were performed at room temperature and data was acquired using the Pulse software (HEKA Elektronik, Lambrecht/Pfalz, Germany). The time constant of activation at 0 mV was determined using the standard envelope of tails protocol, measuring the peak tail current at either 260 mV or 2100 mV. A single-exponential function was fitted to the normalized peak tail currents to obtain tact. The time constants of channel deactivation (tslow and tfast) were obtained by fitting a double-exponential function to tail current traces measured at 260 mV following a voltage step to +20 mV for 1000 ms to achieve maximal activation. Similarly, the time constant of recovery from inactivation (trec) at 260 mV was determined from the initial rising phase of the tail currents recorded with this protocol. The extrapolated fit of the deactivation process was subtracted from the initial rising phase of the tail currents. The difference between the extrapolated values and the recorded current values represents the time course of recovery from inactivation. The time constant of this process was estimated by fitting a single-exponential function to the resulting curve. Please see the Results section and figure legends for details on the voltage ramp protocols. For all protocols, the holding potential was 280 mV. Computational Modeling All simulations were performed using the COR program [14]. The built-in CVODE integrator was used with a time step of 0.01 ms. All models were encoded in CellML 1.0 (http://www. cellml.org/). We adopted the Markov model formulation of hERG1 as described by Fink et al. [15]. The model includes three closed states (C1-C3), an open state (O) and an inactivated state (I). The topology of the model is shown in figure 1. The model has been shown to reproduce data recorded both at room temperature and at physiological temperature reasonably well. The hERG1 Markov simulations were performed at 296 K with 4 mM extracellular potassium. In our simulations we modified the transition rate of activation a3 (transition from the closed state C3 to the open state O), the transition rate of deactivation b3 (transition from the open state O to the closed state C3) and the transition rate of recovery from inactivation b4 (transition from the inactivated state I to the open state O). For single cell action potential (AP) simulations we used the ten Tusscher model (TT) of the human ventricular epicardial cell [16]. Figure 1. Topology of the hERG1 Markov model. The hERG1 Markov model developed by Fink et al. [15], consists of three closed states (C1-C3), an open state (O) and an inactivated state (I). Allowed transitions between states are indicated with arrows. The transition rates are indicated above or below the corresponding transitions. doi:10.1371/journal.pone.0009021.g001 PLoS ONE | www.plosone.org 2 February 2010 | Volume 5 | Issue 2 | e9021