Differential Expression of ERG1a and ERG1b Reproduces Properties of IKr Transients We hypothesized that the observed variation in ‘peak potential’ of the native IKr transient [10] could be explained by variation in kinetic rates as a result of differential expression of ERG1 isoforms. Indeed, by varying the relative abundance of hERG1a and hERG1b we were able to manipulate the ‘peak potential’ over a wide range of potentials. We were able to do this in both mammalian cells and in X. laevis oocytes. In oocytes, where we could control the exact ratio of the two isoforms the ‘peak potential’ showed a very good correlation to the relative abundance of the isoforms. The differences in the absolute values of ‘peak potentials’ and peak amplitudes between ERG1 channels expressed in mammalian cells and in oocytes may be attributed to differences in the expression systems. Compared to hERG1a channels, increased rates of both activation, deactivation and recovery from inactivation have been described for heteromeric hERG1a/b channels [8,9]. We were able to show that the relative changes in the kinetics of deactivation and recovery from inactivation is a linear function of the relative hERG1b abundance in X. laevis oocytes. Interestingly, a similar relationship was not obtained for the process of activation. The reason for the non-linear relationship between activation kinetics and isoform abundance is beyond the scope of this study. However, it is worth noting that when expressed in CHO cells hERG1a/b channels display deactivation and recovery kinetics intermediate of those of hERG1a and hERG1b homomeric channels while activation kinetics of hERG1a/b are similar to hERG1b [8]. hERG1 channels are believed to be tetramers. Possibly, the number of long 1a-type N- terminals in the channel complex may play a crucial role in the activation process. Future studies focusing on molecular determi- nants of gating characteristics of heteromeric channels may explain these findings. Nevertheless, the systematic changes in kinetics of deactivation and recovery as a function of hERG1b abundance explain the variation in ‘peak potential’. The absolute variation in the ‘peak potential’ observed by Gintant [10] in canine cardiomyocytes (20–30 mV) is within the range observed in our experiments (,40 mV in HEK293 cells and ,30 mV in X. laevis oocytes). Although there were differences in the experimental conditions, the data suggest a large variation in the relative abundance of ERG1a and ERG1b in native cardiomyocytes. However, our results show that the ‘peak potential’ of hERG1 channels in general occurs at more depolarized potentials as compared to the data from canine cardiomyocytes [10]. This difference can not be fully explained by differences in the temperature at which the data was obtained. It may, however, partly be attributed to species-dependent differ- ences in IKr kinetics (see [18] for review). IKr is known to deactivate slower in canine than in human cardiomyocytes [18]. Thus, the ‘peak potential’ is expected to occur at more depolarized potentials for human ERG1 channels. Interestingly, Hua et al. [19] have shown that over-expression of hERG1a by adenoviral transfection in isolated canine cardiomyocytes shifted the ‘peak potential’ towards more depolarized potentials as compared to control. Additionally, differences in post-translational modifications of hERG1 channels and/or interactions with auxiliary proteins may play a role [18]. When we normalized the recorded currents to a value proportional to the number of expressed channels, we observed only little variation in the peak current density over the whole range of isoform ratios in both expression systems. Similarly, Gintant [10] reported only small variations in the absolute IKr peak density. Although speculative, taken together these data Role of Cardiac hERG1 Isoforms may indicate that the absolute number of ERG1 channels in the membrane of cardiomyocytes is relatively constant from cell to cell but that the relative abundance of ERG1a and ERG1b varies. We have based our study on a comparison of canine IKr [10] and heterologously expressed hERG1a and hERG1b channels. Although our results qualitatively show that differential expression of hERG1a and hERG1b may explain the observed variation in canine IKr, it remains to be shown to which extend post- translational modifications and/or interactions with auxiliary proteins influence the properties of native IKr. Also, it is unknown whether a similar variation is present in the human heart. Given the relatively high abundance of hERG1b in the human heart [5,8] it seems plausible to suggest that at least some degree of variation is present. Interestingly, the existence of separate promoter regions for hERG1a and hERG1b has been described [20] providing a potential mechanism for such differences. Therefore, differences in the relative abundance of hERG1a and hERG1b may underlie regional heterogeneity of IKr kinetics. Future studies looking into these issues are needed to fully establish the importance of ERG1b in the human heart. To this end, our results suggest that it is the relative (to ERG1a) rather than the absolute abundance of ERG1b that determines the properties of IKr. Functional Implications of ERG1b in the Heart To address the possible functional implications of differential expression of hERG1a and hERG1b in the heart we conducted a series of computer simulations. First, we used a previously published hERG1 Markov model [15] to reproduce the experimental data. In our simulations we changed the kinetic rates of the Markov model by factors corresponding to the relative changes in kinetics observed in the in vitro experiments. Qualitatively, the model reproduced the experimental data well, especially considering that the intrinsic kinetics of the model were somewhat different than the experimental observations (single vs. double-exponential kinetics of deactivation). Second, the Markov model was introduced into the TT model of the ventricular cardiomyocyte. The results of the ventricular cardiomyocyte simulations showed that kinetic changes in IKr corresponding to increases in the relative abundance of hERG1b resulted not only in gradually shorter steady-state APD but also in flatter restitution curves. Mechanistically, inspection of the changes in channel state occupancy revealed that the shortening of APD was mainly due to the increase in recovery rate. Similarly, the decrease in restitution slope was mainly a result of the increase in deactivation rate. The existence of a transmural APD gradient has been well established and has been attributed to differences in IK density [21–23]. Our results show that differences in IKr kinetics, especially differences in kinetics of recovery from inactivation, may also contribute to APD dispersion. It is therefore interesting to note that in the study by Szabo et al. [11] the shortest APDs were recorded in the epicardial cells where also the deactivation kinetics of IKr were fastest. Assuming that faster deactivation rate is due to greater relative abundance of ERG1b, IKr in these cells would be expected also to display a faster rate of recovery from inactivation. In our simulations such differences are predicted to shorten APD, suggesting that differences in IKr kinetics may indeed contribute to the APD dispersion observed in vivo. APD restitution describes the relationship between APD and DI. When the slope of the restitution curve is steep, APD is very sensitive to even small changes in DI and as a result APD alternans may develop. Accordingly, steep restitution slopes has been associated with an increased risk of ventricular fibrillation PLoS ONE | www.plosone.org 7 February 2010 | Volume 5 | Issue 2 | e9021