Arrhythmic risk biomarkers for the assessment of drug cardiotoxicity: from experiments to computer simulations

In this paper, we illustrate how advanced computational modelling and simulation can be used to investigate drug-induced effects on cardiac electrophysiology and on specific biomarkers of pro-arrhythmic risk. To do so, we first perform a thorough literature review of proposed arrhythmic risk biomarkers from the ionic to the electrocardiogram levels. The review highlights the variety of proposed biomarkers, the complexity of the mechanisms of drug-induced pro-arrhythmia and the existence of significant animal species differences in drug-induced effects on cardiac electrophysiology. Predicting drug-induced pro-arrhythmic risk solely using experiments is challenging both preclinically and clinically, as attested by the rise in the cost of releasing new compounds to the market. Computational modelling and simulation has significantly contributed to the understanding of cardiac electrophysiology and arrhythmias over the last 40 years. In the second part of this paper, we illustrate how state-of-the-art open source computational modelling and simulation tools can be used to simulate multi-scale effects of drug-induced ion channel block in ventricular electrophysiology at the cellular, tissue and whole ventricular levels for different animal species. We believe that the use of computational modelling and simulation in combination with experimental techniques could be a powerful tool for the assessment of drug safety pharmacology

Inhibitory effects of oxidants on n-type K+ channels in T lymphocytes and Xenopus oocytes (Szabo è corresponding author)

Reactive oxygen species (ROS) appear to be involved in Fas-induced programmed cell death. We have previously demonstrated a tyrosine-kinase-dependent inhibition of the n-type K+ channels (Kn) by Fas stimulation. Thus, the effect of hydrogen peroxide (H2O2) on the function of Kn was examined using the patch-clamp technique. Incubation of Jurkat human T lymphocytes with 100 microM H2O2 resulted in a 46 +/- 5% inhibition of the macroscopic whole-cell current. Experiments performed at the single-channel level using the cell-attached configuration revealed that the probability of the channel being open diminished upon incubation in H2O2. The effect was not dependent on src-like kinases, since H2O2 did not trigger tyrosine phosphorylation of the Kn channel protein and herbimycin A did not prevent channel inhibition. Kv1.3 channels underly the Kn of T lymphocytes and were expressed in Xenopus oocytes and subjected to electrophysiological analysis by the two-electrode voltage-clamp technique. Application of 1 mM H2O2 and 500 microM t-BOOH (tert, butylhydroperoxide) resulted in a marked inhibition of the K+ current within 20 min. Both the membrane-permeable thiol-group oxidizing agent DTNP [2,2'-dithiobis-(5-nitropyridine)] and the membrane-impermeable DTNB [5,5'-Dithiobis-(2-nitrobenzoic acid)] (50 microM) inhibited Kv1.3 channels, suggesting that extracellular domains of Kv1.3 are affected. These results point to a direct modulation of Kn by various oxidative agents

Specific blockade of slowly activating IsK channels by chromanols - impact on the role of IsK channels in epithelia

AbstractChromanols, which were recently shown to inhibit cAMP-mediated Cl− secretion in colon crypts via a blockade of a cAMP-activated K+ conductance, were analyzed for their effects on distinct cloned K+ channels expressed in Xenopus oocytes. The lead chromanol 293B specifically inhibited IsK channels with an IC50 of 7 μmol/l without affecting the delayed rectifier Kv1.1 or the inward rectifier Kir2.1. Moreover, several other chromanols displayed the same rank order of potency for IsK inhibition as demonstrated in colon crypts. Finally, we tested the effects of the previously described IsK blocker azimilide on cAMP mediated Cl− secretion in rat colon crypts. Similar to 293B azimilide inhibited the forskolin induced Cl− secretion. These data suggest that IsK protein induced K+ conductances are the targets for the chromanol 293B and its analogues, and azimilide

Inhibition of hEAG1 and hERG1 potassium channels by clofilium and its tertiary analogue LY97241

1. We investigated the inhibition of hEAG1 potassium channels, expressed in mammalian cells and Xenopus oocytes, by several blockers that have previously been reported to be blockers of hERG1 channels. 2. In the whole-cell mode of mammalian cells, LY97241 was shown to be a potent inhibitor of both hEAG1 and hERG1 channels (IC(50) of 4.9 and 2.2 nM, respectively). Clofilium, E4031, and haloperidol apparently inhibited hEAG1 channels with lower potency than hERG1 channels, but they cannot be considered hERG1-specific. 3. The block of hEAG1 channels by LY97241 and clofilium was time-, use-, and voltage-dependent, best explained by an open-channel block mechanism. 4. Both drugs apparently bind from the intracellular side of the membrane at (a) specific site(s) within the central cavity of the channel pore. They can be trapped by closure of the activation gate. 5. In inside-out patches from Xenopus oocytes, hEAG1 block by clofilium was stronger than by LY97241 (IC(50) of 0.8 and 1.9 nM, respectively). In addition, hEAG1 block by clofilium was much faster than by LY97241 although there was no difference in the voltage dependence of the on-rate of block. 6. Physico-chemical differences of clofilium and the weak base LY97241 determine the access of the drugs to the binding site and thereby the influence of the recording mode on the apparent block potencies. This phenomenon must be considered when assessing the inhibitory action of drugs on ion channels

The inhibitory effect of the antipsychotic drug haloperidol on HERG potassium channels expressed in Xenopus oocytes

1. The antipsychotic drug haloperidol can induce a marked QT prolongation and polymorphic ventricular arrhythmias. In this study, we expressed several cloned cardiac K(+) channels, including the human ether-a-go-go related gene (HERG) channels, in Xenopus oocytes and tested them for their haloperidol sensitivity. 2. Haloperidol had only little effects on the delayed rectifier channels Kv1.1, Kv1.2, Kv1.5 and I(sK), the A-type channel Kv1.4 and the inward rectifier channel Kir2.1 (inhibition <6% at 3 μM haloperidol). 3. In contrast, haloperidol blocked HERG channels potently with an IC(50) value of approximately 1 μM. Reduced haloperidol, the primary metabolite of haloperidol, produced a block with an IC(50) value of 2.6 μM. 4. Haloperidol block was use- and voltage-dependent, suggesting that it binds preferentially to either open or inactivated HERG channels. As haloperidol increased the degree and rate of HERG inactivation, binding to inactivated HERG channels is suggested. 5. The channel mutant HERG S631A has been shown to exhibit greatly reduced C-type inactivation which occurs only at potentials greater than 0 mV. Haloperidol block of HERG S631A at 0 mV was four fold weaker than for HERG wild-type channels. Haloperidol affinity for HERG S631A was increased four fold at +40 mV compared to 0 mV. 6. In summary, the data suggest that HERG channel blockade is involved in the arrhythmogenic side effects of haloperidol. The mechanism of haloperidol block involves binding to inactivated HERG channels

Heterogeneous expression of the delayed-rectifier K+ currents iK,r and iK,s in rabbit sinoatrial node cells

The electrical activity of sinoatrial node cells is heterogeneous. To understand the reasons for this, the density of the delayed-rectifier K+ current and its two components, iK,r and iK,s, as a function of the size (as measured by cell capacitance) of rabbit sinoatrial node cells was investigated using the whole-cell voltage-clamp technique at 35 °C.iK,r and iK,s were isolated using E-4031 and 293B. Features of the E-4031-sensitive and 293B-insensitive currents corresponded well to those of iK,r, while features of the E-4031-insensitive and 293B-sensitive currents corresponded well to those of iK,s.The densities of the outward current under control conditions and the drug-sensitive and -insensitive currents were significantly (P < 0.01) correlated with cell capacitance, with current densities being greater in larger cells.The effects of partial blockade of iK,r by 0.1 μm E-4031 on spontaneous action potentials were greater in smaller cells.It is concluded that there are cell size-dependent differences in the density of the iK,r and iK,s components, and these may be involved in the heterogeneity of the electrical activity of single sinoatrial node cells as well as that of the intact sinoatrial node

The role of the I(sK) protein in the specific pharmacological properties of the I(Ks) channel complex

I(Ks) channels are composed of I(sK) and KvLQT1 subunits and underly the slowly activating, voltage-dependent I(Ks) conductance in heart. Although it appears clear that the I(sK) protein affects both the biophysical properties and regulation of I(Ks) channels, its role in channel pharmacology is unclear. In the present study we demonstrate that KvLQT1 homopolymeric K(+) channels are inhibited by the I(Ks) blockers 293B, azimilide and 17-β-oestradiol. However, I(Ks) channels induced by the coexpression of I(sK) and KvLQT1 subunits have a 6–100 fold higher affinity for these blockers. Moreover, the I(Ks) activators mefenamic acid and DIDS had little effect on KvLQT1 homopolymeric channels, although they dramatically enhanced steady-state currents through heteropolymeric I(Ks) channels by arresting them in an open state. In summary, the I(sK) protein modulates the effects of both blockers and activators of I(Ks) channels. This finding is important for the action and specificity of these drugs as I(sK) protein expression in heart and other tissues is regulated during development and by hormones

Blockade of HERG channels by the class III antiarrhythmic azimilide: mode of action

1. The class III antiarrhythmic azimilide has previously been shown to inhibit I(Ks) and I(Kr) in guinea-pig cardiac myocytes and I(Ks) (minK) channels expressed in Xenopus oocytes. Because HERG channels underly the conductance I(Kr) in human heart, the effects of azimilide on HERG channels expressed in Xenopus oocytes were the focus of the present study. 2. In contrast to other well characterized HERG channel blockers, azimilide blockade was reverse use-dependent, i.e., the relative block and apparent affinity of azimilide decreased with an increase in channel activation frequency. Azimilide blocked HERG channels at 0.1 and 1 Hz with IC(50) s of 1.4 μM and 5.2  μM respectively. 3. In an envelope of tail test, HERG channel blockade increased with increasing channel activation, indicating binding of azimilide to open channels. 4. Azimilide blockade of HERG channels expressed in Xenopus oocytes and I(Kr) in mouse AT-1 cells was decreased under conditions of high [K(+)](e), whereas block of slowly activating I(Ks) channels was not affected by changes in [K(+)](e). 5. In summary, azimilide is a blocker of cardiac delayed rectifier channels, I(Ks) and HERG. Because of the distinct effects of stimulation frequency and [K(+)](e) on azimilide block of I(Kr) and I(Ks) channels, we conclude that the relative contribution of block of each of these cardiac delayed rectifier channels depends on heart frequency. [K(+)](e) and regulatory status of the respective channels