An in silico–in vitro pipeline for drug cardiotoxicity screening identifies ionic pro‐arrhythmia mechanisms

FIGURE 1. An in silico–in vitro pipeline to determine drug cardiotoxicity risk and mechanism. Step 1, the Kernik–Clancy model with experimental artefacts is used to develop a voltage clamp (VC) protocol that is specifically designed to isolate currents. Step 2, spontaneous, IK1 dynamic clamp and paced action potentials (AP), and optimized VC data are acquired from a patient‐derived induced pluripotent stem cell‐derived cardiomyocyte (iPSC‐CM) before and after drug application. Step 3, the change in IK1 dynamic clamp and paced AP data from pre‐ to post‐drug application is used to identify AP prolongation, a surrogate marker of cardiotoxicity. Step 4, changes in VC data are used to determine the ion channels targeted by a drug. Step 5, after identifying the ion channel targeted by a drug, a dose–response curve is developed for each of these ion channels using expression line cells.

FIGURE 2. A genetic algorithm (GA) optimized a voltage clamp (VC) protocol to isolate individual current contributions in an in silico induced pluripotent stem cell‐derived cardiomyocyte (iPSC‐CM) model. (a) The GA was used to develop individual VC protocols for seven of the most predominant ionic currents in cardiac cells. (b) These seven protocols were systematically shortened and joined together by a −80‐mV holding step to produce an optimized VC protocol that isolates each of the seven currents.

FIGURE 3. Synthetic maturation of induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs) with dynamically clamped IK1 ensures consistent action potentials (APs) under 1‐Hz pacing conditions. (a) Spontaneous behaviour from a subpopulation of six cells (each with a different coloured trace) that indicates the level of heterogeneity and inconsistent presence of APs in the cell population. (b) IK1 dynamic clamp applied to the same cells makes them appear more mature when paced at 1 Hz. (c and d) Histograms for the IK1 dynamically clamped and paced AP resting membrane potential (−74.2 ± 2.8 mV) and action potential duration at 90% repolarization (142.0 ± 48.3 ms) for the 40 cells in this study

FIGURE 4. Drug treatment resulted in action potential (AP) morphology changes consistent with the effects caused by drug block of the expected currents. Four cells with dynamically clamped IK1 and paced at 1 Hz before (black) and after (red) treatment with cisapride (a), verapamil (b), quinidine (c) and quinine (d). (e) Percent change in APD20 and (f) APD90 for drugs treated with cisapride ( C), verapamil (V), quinidine (Qd), quinine (Qn) or a DMSO control solution (Ctrl). No drugs caused a significant change in APD20 compared to control cells. Quinidine and quinine showed significant prolongation when compared to control cells. *P< 0.05, significantly different as indicated.

FIGURE 5. Optimized voltage clamp (VC) protocol correctly identifies IKr as a target of cisapride, quinidine and quinine. (a) Representative cell shows the effect of quinine on the current response during the segment of the VC protocol designed to isolate IKr. The black trace is pre‐drug, and the red trace is post‐drug. (b) The current trace during the segment meant to isolate IKr (shaded grey in panel a). (c) Cells treated with cisapride (C), quinidine (Qd) and quinine (Qn) showed a decrease in total current during the VC segment designed to isolate IKr. At the concentrations used in this experiment, cisapride, quinidine and quinine should block 95%, 89% and 72% of IKr, respectively. *P< 0.05, significantly different as indicated. (d–f) Functional t tests show a significant difference in the average change in current during the IKr‐isolating segment when comparing cells treated with DMSO to cells treated with cisapride (d), quinidine (e) and quinine (f). Verapamil was excluded because there was no significant difference during this segment of the protocol.

FIGURE 6. Voltage clamp (VC) protocol identifies funny current as a target of quinine. (a) Representative cell treated with quinine shows a reduction in inward current when the cell is clamped to a hyperpolarized potential of −120 mV, before 6000 ms. After 6000 ms, outward current is reduced as If reverses in direction before inactivating. (b) The current trace during and around the segment meant to isolate If (shaded grey in panel a). (c) Functional t test shows a significant difference between quinine‐treated cells and control cells throughout the period of the protocol when funny current would be active (c, top). The average difference between quinine‐treated and control cells is between 0.8 and 1.2 pA/pF throughout this period (c, middle). The Kernik–Clancy induced pluripotent stem cell‐derived cardiomyocyte (iPSC‐CM) model funny current becomes active during the period when quinine‐treated cells show a significant change in current and turns off just before the P value increases above 0.05 (c, bottom). (d) Cells treated with quinine (Qn) show a decrease in total current during the VC segment designed to isolate If. *P< 0.05, significantly different as indicated.

FIGURE 7. Dose–response curve shows quinine block of funny current in HEK‐293 cells stably expressing HCN1. (a) IV curve with averages and errors calculated from six HEK‐HCN1 cells. (b) Representative HEK‐HCN1 current–voltage traces. (c) Dose–response curve fit to pharmacology data from six HEK‐HCN1 cells. (d) Traces generated from a representative cell by clamping at −20 mV for 3000 ms, and then stepping to −110 mV for 3500 ms at all tested concentrations