Introducing Simulated IK1 into Human iPSC-Cardiomyocytes using Dynamic Clamp on an Automated Patch Clamp System

Abstract

Dynamic clamp is a powerful tool involving injection of real-time simulated currents into patch-clamped cells, and has been employed in conventional patch-clamp to inject IK1 current into human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). hiPSC-CMs are attractive cell types because of their unlimited availability and human origin. However, IK1 is expressed at low levels in those cells, hence they display a more depolarized membrane potential than adult cardiomyocytes (CM). Introducing simulated IK1 into hiPSC-CMs improves resting potential and makes them a viable alternative to the scarcely available adult human CMs. Another limitation of hiPSC-CMs is that the ratio of seal resistance (Rseal) to membrane resistance (Rm) is small due to smaller capacitance and typically slightly lower Rseal in automated patch clamp (APC). Thus, membrane potential dissipation is more pronounced compared to patch-clamp recordings of adult CMs. To correct for that, we implemented a Rseal compensation mechanism to inject current compensating for the potential dissipation through Rseal. In this study, we combined dynamic clamp with APC to demonstrate IK1 conductance can be added to hiPSC-CMs, and applying automatic Rseal compensation simultaneously. Our results show virtual IK1 can be successfully injected into hiPSC-CMs in up to 4 cells simultaneously and Rseal is correctly compensated avoiding overcompensation. Our approach results in more stable resting membrane potentials and improved action potential (AP) shape. Increased IK1 resulted in AP shortening and acceleration of the upstroke. We measured native, but small, Ba2+-sensitive IK1 in voltage-clamp mode in approximately 50% of these cells. Adding a Ca2+ channel activator (BayK 8644), or blocker (Nifedipine) caused an increase and decrease of the AP duration, respectively. In conclusion, combining dynamic clamp and Rseal compensation with APC resulted in an enhanced, medium-throughput platform for safety pharmacology.