HERG block in HEK cells incorporated in real time into computer models of in silico cardiac repolarization using dynamic clamp

Abstract

Using dynamic clamp in Synthetic Cell Mode, we input experimentally-obtained HERG current, in real time, into in-silico cardiomyocytes. In-silico models were either generated by the dynamic clamp system or by interfacing with NEURON software (www.neuron.yale.edu/neuron/). The in-silico action potential (AP) voltage generated in real time was applied to HEK cells expressing the cloned HERG current. The experimental HERG current was then input into the in-silico cardiomyocytes. The objective was to examine the experimental effect of HERG block by dofetilide on AP morphology in the in-silico cardiomyocytes. HERG currents were recorded from a stable HERG_A: HEK cell line in the whole cell ruptured patch configuration (200B amplifier, Molecular Devices). Using dynamic clamp (Cybercyte DC-1, Cytocybernetics), experimental HERG currents were incorporated into either an in-silico ventricular cardiomyocyte consisting of electronically-expressed I_Na, I_K1, I_KS, I_{Ca_L,} and I_to or an in-silico NEURON atrial cardiomyocyte (Courtemanche et al., 1998; Jacobson, 1998). HERG current magnitude was adjusted so AP duration (APD) ranged from 300 to 500 ms (ventricular) and 200–300 ms (atrial) for the in-silico cardiomyocytes. HERG current block with dofetilide (10–100 nM) on AP morphology was examined. Dofetilide blocked HERG current with an IC₅₀ of 22.5 ± 0.9 nM and a Hill coefficient of 1.03 ± 0.05 (n = 6). For the in-silico ventricular cardiomyocytes, dofetilide prolonged the AP (APD90 baseline: 442 ± 21 ms, 100 nM: 974 ± 104 ms, p < 0.01, n = 6). For the in-silico atrial cardiomyocytes, the NEURON model I_Kr was replaced with HERG current. Dofetilide block of HERG current (30 nM dofetilide: 62 ± 8 %, n = 6) increased the APD90 (baseline: 263 ± 13 ms, 30 nM dofetilide: 347 ± 15 ms, p < 0.001, n = 6). Using dynamic clamp in Synthetic Cell Mode, experimentally-expressed ion channel currents can be incorporated in real time into in-silico cardiomyocytes, and the effects of drugs targeting the expressed ion channel on AP morphology/behavior assessed. Dynamic Clamp Synthetic Cell Mode can readily be implemented on automated patch clamp systems, allowing for HT screening of drugs targeting cardiac ion channels with AP morphology as the readout. These studies were supported, in part, by NIH NS011613, 1R43NS125749 and 5R44MH119842.