The model was obtained from the CellML repository (http:// www.cellml.org/models/tentusscher_panfilov_2006_version04) on the 11th of April 2009 (Note: after the model was down- loaded the CellML website has been restructured and the model can now be found at http://models.cellml.org/exposure/ de5058f16f829f91a1e4e5990a10ed71). This version of the model has reached curation level 2 indicating that it qualitatively reproduces the results published in the original paper (see http://www.cellml.org/repository-info/info for details on the curation procedure). We exchanged the original Hodgkin-Huxley formulation of IKr for the hERG1 Markov model described above [15]. In the modified model (TTF) we adjusted GKr,max by a factor of 0.3 to 0.046 nS/pF. Otherwise, no changes were made to the downloaded model. To ensure steady-state conditions the ventricular cell model was paced at a basic cycle length (BCL) of 1000 ms for 500 beats prior to any modification of hERG1 kinetics. After the kinetic modifications the models were paced for another 100 beats (BCL 1000 ms) to ensure that a new steady- state was reached. APD restitution was determined using the standard S1-S2 protocol. After reaching steady-state (BCL 1000 ms) a premature stimulus (S2) was introduced at progres- sively shorter diastolic intervals (DI). The APD restitution curve was obtained by plotting APD against the preceding DI. A single- exponential function was fitted to each restitution curve and the slope was calculated by differentiation of the fitted curve. Data Analysis Data analysis was performed with Igor Pro (Wavemetrics, Lake Oswego, OR, USA), GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA) and R (http://www.R-project.org) software. Statistical comparisons were performed using ANOVA followed by Tukey’s procedure or an appropriate set of custom- defined contrasts. A P-value of less than 0.05 was considered statistically significant. Unless otherwise stated, data are repre- sented as mean 6 SEM. The electrophysiological experiments were not designed to compare the absolute current levels between hERG1a and hERG1b channels. In order to compare data independently of differences in transfection efficiency and/or translation/trafficking processes, we normalized the current levels to a value (N) proportional to the number of expressed channels as described by Sale et al. [9]. Briefly, we subjected cells/oocytes to a depolarizing voltage step to +40/+20 mV for 1000 ms to achieve maximal activation of hERG1 channels followed by a step to 260 mV for 3 s to record channel deactivation. A double- exponential function was fitted to the current decay at 260 mV. By extrapolation back to the beginning of the 260 mV voltage step we obtained an estimate of N. This normalization is valid under the assumption that the single channel conductances are identical. It has been shown, that for hERG1a single channel conductance does not depend on the length of the N-terminus [17]. As the only difference between hERG1a and hERG1b is the N-termini the assumption seems justified. Results Differential Expression of hERG1a and hERG1b Modulates the ‘Peak Potential’ In order to address whether differential expression of hERG1a and hERG1b was able to modulate the ‘peak potential’, i.e. the potential where the current peaks during a repolarizing voltage ramp, we transfected HEK293 cells with either hERG1a, hERG1b or both channel subunits. The cells were clamped at +40 mV for 200 ms to activate the channels and then repolarized Role of Cardiac hERG1 Isoforms to 280 mV using a ramp with a slope of 20.12 mV/ms. Representative currents recorded during the ramp are shown in figure 2A. Currents were normalized to the maximum peak current for comparison. The data show that expression of hERG1b was able to shift the ‘peak potential’ significantly towards more depolarized potentials. Summary data are shown in figure 2B. To compare peak amplitudes independently of expression levels data was normalized to a value, N, proportional to the number of expressed channels (see Methods for details). For hERG1a channels the ‘peak potential’ was 239.661.7 mV, for hERG1a/b channels it was shifted to 219.362.9 mV and for hERG1b channels it was 1.961.9 mV (P,0.05 for all compar- isons). The peak amplitudes, normalized to N, were 0.3960.01 Figure 2. The relative abundance of hERG1a and hERG1b determines the ‘peak potential’ in mammalian cells. A, Representative recordings of hERG1a, hERG1b and hERG1a/b channels expressed in HEK293 cells are shown. Currents were recorded using the protocol depicted in the inset. Only currents recorded during the ramp are shown. For comparison, currents were normalized to the maximum peak current during the ramp and plotted against the command voltage. The dotted lines indicate the potential where the currents peaked (‘peak potential’) and are also used to indicate the expressed channels. B, Summary data of the variation in ‘peak potential’. For comparison, the peak amplitude was normalized to an estimate of the number of channels (N) and plotted as a function of the ‘peak potential’. doi:10.1371/journal.pone.0009021.g002 PLoS ONE | www.plosone.org 3 February 2010 | Volume 5 | Issue 2 | e9021