Stable expression of human Nav1.5 for high-throughput cardiac safety assessment

Background: Cardiac safety of new drugs is essential for public health. Nav1.5 is the cardiac sodium channel responsible for action potentials in cardiomyocytes. Objective: For high-throughput cardiotoxicity assays in the development of antiarrhythmic drugs, we have established an electrophysiologically validated stable HEK293 cell line expressing human Nav1.5 (hNav1.5). To validate the cell line, we examined the effects of lidocaine, an antiarrhythmic agent, and compared its effects using conventional and automated patch-clamp systems. Results: We isolated three stable cell lines originating from a single clonal cell and selected one stable cell line that produced a minimum 5 nA of hNav1.5 currents without any change in biophysical properties compared to the current from the transiently expressed hNav1.5. We further compared the pharmacological effects of lidocaine on this cell line using the conventional patch-clamp and automated patch-clamp systems. Lidocaine blocked hNav1.5 currents in a concentration- and voltage-dependent manner. The IC₅₀ values at holding potentials of − 90 mV, near the resting membrane potential of cardiomyocytes, and − 120 mV were 18.4 ± 2.6 μM and 775.6 ± 37.1 μM, respectively. In the automated patch-clamp system, the IC₅₀ values at holding potentials of − 90 mV and − 120 mV were 17.9 ± 2.0 μM and 578.7 ± 74.3 μM, respectively, indicating no difference between the systems. In both systems, lidocaine caused significant shifts toward hyperpolarization in the steady-state inactivation curves by ~ 20 mV and induced slower recovery from inactivation and stronger use-dependent inhibition. Conclusion: The new HEK293 cell line stably expressing hNav1.5 channels produced a current that could be tested using both conventional and automated patch-clamp systems with similar results. This current would be strong enough to evaluate cardiac safety, thus allowing the use of the automated patch-clamp system for drug screening and functional kinetic studies to reveal the mechanism of drug action.