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

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 Vhalf 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 Vhalf 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. IK1 recorded in Cor.4U cells on the Patchliner. (A) Example of a cell with IK1, 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 IK1 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 IK1 conductance on action potentials (APs) of Cellartis Cardiomyocytes. The simulated IK1 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 IK1 conductance. Note: in order to keep a RMP of −94 mV in the absence of simulated IK1 conductance, −180 pA of holding current was injected. This was removed upon addition of IK1 and RMP remains at −94 mV.

Figure 5. Effects of adding simulated IK1 on AP parameters. (A) Adding IK1 prolongs the APD90 of action potentials recorded from Cellartis Cardiomyocytes (n = 4) compared to constant current injection. With increasing IK1 conductance, the prolongation of the action potential becomes smaller, consistent with the role of IK1 in the final repolarization of the cardiac action potential. (B) Upstroke velocity of the action potentials after addition of simulated IK1 is high, as with constant current injections, and decreases slightly with increasing IK1 conductance.

Figure 6. Action potentials recorded simultaneously after adding simulated IK1. (A) Action potentials from 3 cells recorded in parallel on a Patchliner Quattro. (B) Simulated IK1 was recorded for each of the 3 cells and is shown. Note the brief increase in IK1 during the upstroke of the action potential, where IK1 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 IK1 conductance.

Figure 7. (A) The calcium channel agonist BayK-8644 increased AP duration while simulated IK1 was added (blue trace). Conversely, the calcium channel antagonist nifedipine decreased AP duration (red trace) as compared with control (black trace). In this experiment, IK1 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.