A Hybrid Model for Safety Pharmacology on an Automated Patch Clamp Platform: Using Dynamic Clamp to Join iPSC-Derived Cardiomyocytes and Simulations of Ik1 Ion Channels in Real-Time

Abstract

An important aspect of the Comprehensive In Vitro Proarrhythmia Assay (CiPA) proposal is the use of human stem cell-derived cardiomyocytes and the confirmation of their predictive power in drug safety assays. The benefits of this cell source are clear; drugs can be tested in vitro on human cardiomyocytes, with patient-specific genotypes if needed, and differentiation efficiencies are generally excellent, resulting in a virtually limitless supply of cardiomyocytes. There are, however, several challenges that will have to be surmounted before successful establishment of hSC-CMs as an all-round predictive model for drug safety assays. An important factor is the relative electrophysiological immaturity of hSC-CMs, which limits arrhythmic responses to unsafe drugs that are pro-arrhythmic in humans. Potentially, immaturity may be improved functionally by creation of hybrid models, in which the dynamic clamp technique joins simulations of lacking cardiac ion channels (e.g., I_K1) with hSC-CMs in real-time during patch clamp experiments. This approach has been used successfully in manual patch clamp experiments, but throughput is low. In this study, we combined dynamic clamp with automated patch clamp of iPSC-CMs in current clamp mode, and demonstrate that I_K1 conductance can be added to iPSC-CMs on an automated patch clamp platform, resulting in an improved electrophysiological maturity.

Keywords: automated patch clamp electrophysiology; cardiomyocyte; dynamic clamp; inward rectifying potassium ion channels; safety pharmacology; stem cell.

Figure 1

Block diagram showing connections between Patchliner, patch clamp amplifier and dynamic clamp system. For a more detailed depiction of the dynamic clamp system we refer to Supplementary Figure 1.

Figure 2

Typical recordings from hiPSC Cellartis Cardiomyocytes recorded on the Patchliner. (A) Na⁺ currents in response to increasing voltage steps. (B) Corresponding current-voltage plot for an average of 7 cells. A Boltzmann fit revealed a V_half of activation of −46 mV. (C) Ca²⁺ currents in response to increasing voltage steps. (D) Corresponding current-voltage plot for an average of 18 cells. A Boltzmann fit revealed a V_half of activation of −5.8 mV. (E) Raw traces of Ca²⁺ current in control conditions (black) and after inhibition by increasing concentrations of nifedipine (blue). (F) The concentration response curve (normalized to maximum block) for nifedipine for an average of 5 cells. The average concentration response curve was fitted with a standard Hill-equation which revealed an IC₅₀ = 252 ± 186 nM (n = 5).

Figure 3

I_K1 recorded in Cor.4U cells on the Patchliner. (A) Example of a cell with I_K1, shown are responses to a voltage step protocol to −120 mV for 1,200 ms from a holding potential of −40 mV in control conditions (black) and in the presence of 10 μM BaCl₂ (red). (B) Average current-voltage relationship of the Ba²⁺-sensitive current for an average of 7 cells. (C) Current traces of an example cell which does not express I_K1 in control (black) and with 10 μM BaCl₂ (red). (D) Corresponding current-voltage plot of an average of 3 cells showing no Ba²⁺-sensitive current.

Figure 4

Dynamic clamp used to simulate I_K1 conductance on action potentials (APs) of Cellartis Cardiomyocytes. The simulated I_K1 conductance could replace injected current to achieve a native resting membrane potential (RMP) of approximately −94 mV. The cells repolarized faster and the AP duration decreased with increasing I_K1 conductance. Note: in order to keep a RMP of −94 mV in the absence of simulated I_K1 conductance, −180 pA of holding current was injected. This was removed upon addition of I_K1 and RMP remains at −94 mV.

Figure 5

Effects of adding simulated I_K1 on AP parameters. (A) Adding I_K1 prolongs the APD₉₀ of action potentials recorded from Cellartis Cardiomyocytes (n = 4) compared to constant current injection. With increasing I_K1 conductance, the prolongation of the action potential becomes smaller, consistent with the role of I_K1 in the final repolarization of the cardiac action potential. (B) Upstroke velocity of the action potentials after addition of simulated I_K1 is high, as with constant current injections, and decreases slightly with increasing I_K1 conductance.

Figure 6

Action potentials recorded simultaneously after adding simulated I_K1. (A) Action potentials from 3 cells recorded in parallel on a Patchliner Quattro. (B) Simulated I_K1 was recorded for each of the 3 cells and is shown. Note the brief increase in I_K1 during the upstroke of the action potential, where I_K1 channels respond with increased current to the depolarization induced by the pacing stimulus, which is in line with the slight decrease observed in upstroke velocity with increasing I_K1 conductance.

Figure 7

(A) The calcium channel agonist BayK-8644 increased AP duration while simulated I_K1 was added (blue trace). Conversely, the calcium channel antagonist nifedipine decreased AP duration (red trace) as compared with control (black trace). In this experiment, I_K1 was injected at 400 pS/pF and RMP was −94 mV. (B) Average responses from 6 cells showing significantly increased APD₉₀ after exposure to 1 μM BayK-8644 (^*p < 0.05) and decreasing APD₉₀ after exposure to 30 μM nifedipine.