AimsThe level of inhibition of the human Ether-à-go-go-related gene (hERG) channel is one of the earliest preclinical markers used to predict the risk of a compound causing Torsade-de-Pointes (TdP) arrhythmias. While avoiding the use of drugs with maximum therapeutic concentrations within 30-fold of their hERG inhibitory concentration 50% (IC50) values has been suggested, there are drugs that are exceptions to this rule: hERG inhibitors that do not cause TdP, and drugs that can cause TdP but are not strong hERG inhibitors. In this study, we investigate whether a simulated evaluation of multi-channel effects could be used to improve this early prediction of TdP risk.Methods and resultsWe collected multiple ion channel data (hERG, Na, l-type Ca) on 31 drugs associated with varied risks of TdP. To integrate the information on multi-channel block, we have performed simulations with a variety of mathematical models of cardiac cells (for rabbit, dog, and human ventricular myocyte models). Drug action is modelled using IC50 values, and therapeutic drug concentrations to calculate the proportion of blocked channels and the channel conductances are modified accordingly. Various pacing protocols are simulated, and classification analysis is performed to evaluate the predictive power of the models for TdP risk. We find that simulation of action potential duration prolongation, at therapeutic concentrations, provides improved prediction of the TdP risk associated with a compound, above that provided by existing markers.ConclusionThe suggested calculations improve the reliability of early cardiac safety assessments, beyond those based solely on a hERG block effect.

The rapidly activating delayed rectifier potassium current, IKr, was studied in guinea‐pig ventricular myocytes in the presence of thiopentone, which blocks the more slowly activating component of the delayed rectifier potassium current, IKs, and using whole cell perforated patch clamp or switched voltage clamp with sharp electrodes to minimise intracellular dialysis. Activation of protein kinase A (PKA) by isoprenaline or forskolin caused an increase in IKr tail currents. Following a 300 ms depolarising step to +20 mV, mean tail current amplitude was increased 47 ± 12% by isoprenaline, and 73 ± 13% by forskolin. No increase in IKr was observed when IKr was studied using whole cell ruptured patch clamp and there was no change in the reversal potential of IKr in the presence of isoprenaline. The rectification of the current sensitive to E4031, a selective IKr blocker, was markedly reduced in the presence of isoprenaline and the region of negative slope was absent. This is consistent with a reduction in the inactivation of IKr and was supported by the finding that IKr, in the presence of isoprenaline, was somewhat less sensitive to block. E4031 (5 μm) blocked only 81 ± 5% of IKr in the presence of isoprenaline compared to 100 ± 0% in control. The forskolin‐ and isoprenaline‐induced increases in IKr were inhibited by staurosporine and by the selective protein kinase C (PKC) inhibitor bisindolylmaleimide I. Direct activation of PKC by phorbol dibutyrate increased IKr tail currents by 24 ± 5%. Both the isoprenaline‐ and forskolin‐induced increases in IKr were inhibited when calcium entry was reduced by block of ICa with nifedipine or when myocytes were pre‐incubated in BAPTA‐AM. The selective PKA inhibitor KT5720 prevented the isoprenaline‐induced increase in IKr only when the increase in ICa was also suppressed. These data show a novel mechanism of regulation of IKr by PKC and this kinase was activated by β‐adrenoceptor stimulation. IKr seems to be enhanced through a reduction in the C‐type inactivation which underlies the rectification of the channel and such a mechanism may occur in other channels with this type of inactivation.

Delayed rectifier potassium current (IK) was investigated in guinea‐pig isolated ventricular myocytes under voltage‐clamp conditions (‘switched’ single electrode clamp), using selective blockers and/or different activation protocols to separate its rapid (IKr) and slow (IKs) components. The class III antiarrhythmic compound E4031 (5 microM) was used to block IKr and the anaesthetic drugs propofol (100 microM) or thiopentone (100 microM) to block IKs. In all experiments L‐type calcium currents were blocked with nifedipine (2 microM). Complementary effects of E4031 and the anaesthetic drugs on the components of IK were observed. The E4031‐sensitive current (IKr) resembled the current remaining in the presence of the anaesthetics and, likewise, the anaesthetic‐sensitive current (IKs) resembled the current remaining in the presence of E4031. Under the conditions of these experiments, the relative contribution of the two components to total IK tail current was found to be approximately equal after a 400 ms depolarization to +40 mV. For example, IKr was 58 +/− 10% of total IK tail current when measured as the E4031‐sensitive current, 41 +/− 6% as the propofol‐insensitive current and 43 +/− 7% as the thiopentone‐insensitive current. In the presence of both E4031 and propofol or thiopentone the IK tail current deactivating at ‐40 mV was completely eliminated, leaving a residual current during the pulse which reversed at ‐46 +/− 1 mV. To avoid complication of the ‘envelope of tails’ test with this residual current, the tail:pulse ratio was calculated for the anaesthetic‐sensitive component and this was constant, consistent with block of a single component of IK. Forskolin (1 microM) enhanced the current most consistent with IKs. Propofol (300 microM) caused a 64 +/− 3% increase in action potential duration in the presence of both E4031 (5 microM) and nifedipine (2 microM), consistent with an important role for IKs in the repolarization of the action potential in the guinea‐pig heart. The observations therefore provide further support for separate components of IK with different characteristics in the guinea‐pig heart; it appears that E4031 and propofol or thiopentone are useful complementary tools for their separation.

The aim of this study was to investigate possible regulation of the hyperpolarization-activated current (I(f)) by cytosolic calcium in guinea-pig sino-atrial (SA) node cells. Isolated SA node cells were superfused with physiological saline solution (36 degrees C) and the perforated patch voltage-clamp technique used to record I(f) activated by hyperpolarizing voltage steps. A 10-min loading of SA node cells with the calcium chelator BAPTA (using 10 microM BAPTA-AM) significantly reduced the amplitude of I(f) at all potentials studied (69+/-8% at -80 mV, n=6). BAPTA loading also shifted the voltage of half-activation (V(h)) of the conductance from -83+/-2 mV in control to -93+/-2 mV in BAPTA (n=6) without significantly altering the slope of activation. The calmodulin antagonists W-7 (10 microM), calmidazolium (25 microM) and ophiobolin A (20 microM) caused similar reductions in I(f) amplitude (73+/-4, 86+/-9 and 59+/-6% at -80 mV, n=6, 5 and 4, respectively) and shifts in V(h) (11+/-3, 14+/-3 and 8+/-2 mV). In cells pre-treated with W-7, exposure to BAPTA caused no further reduction in current amplitude (n=6). I(f) current amplitude was unaffected by the calmodulin dependent kinase (CaMKII) inhibitor KN-93 (1 microM) although this CaMKII inhibition did reduce L-type calcium by 48+/-19% at 0 mV (n=3). These results are consistent with a role for calcium and calmodulin in the regulation of I(f), via a mechanism that is independent of CaMKII. Alterations in intracellular calcium during the cardiac cycle may be involved in fine tuning the voltage-dependent properties of I(f) and may thus determine its relative contribution to pacemaking in the SA node.

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