Development of a perforated patch clamp assay for studying IPSC-derived cardiomyocytes

Ion channel profiling of drug candidates using heterologous cells is a robust de-risking strategy. However, this approach often neglects the intricate interactions of intracellular components, leading to a growing interest in more physiologically relevant models. The quality of iPSC-derived cardiomyocytes has steadily improved, making these cells widely used in drug discovery due to their close resemblance to native cardiomyocytes. Patch clamp recordings, particularly action potential investigations, are the preferred method for evaluating the electrophysiological properties of cardiomyocytes. In automated patch clamp (APC) recordings, the whole-cell (WC) configuration is typically employed, but this can hinder action potential measurements as cytoplasmic components are ‘washed out,’ altering channel activities and disrupting Ca2+ buffering systems. Consequently, recorded action potentials are very short, especially during early repolarization. This effect is further exacerbated when fluoride is used as a seal enhancer, as Ca2+ and F- readily precipitate as CaF2. In this study, we developed an assay utilizing perforated patch clamp on a high-throughput automated patch-clamp (APC) platform to record action potentials from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using nystatin as a pore-forming agent. The application of the intracellular voltage-gated sodium channel (VGSC) blocker QX314 via internal solution exchange resulted in complete inhibition of VGSC currents in cells recorded in WC configuration, but had minimal effect on cells recorded in the perforated configuration, confirming that cells did not spontaneously transition to the WC configuration. Action potentials were recorded from cells matured between 8 and 21 days in vitro. Depending on the maturation time, action potential duration at 30 % repolarization (APD30) values were between 3.5 and 1.6 times longer in perforated patch clamp recordings compared to cells recorded in the WC configuration. Voltage clamp recordings revealed that this change in APD was attributable to larger voltage-gated calcium channel (VGCC) currents. These findings These findings underscore the advantage of using perforated patch clamp, as it allows for more accurate electrophysiological measurements by preserving the cell's native intracellular environment.