Automated patch-clamp of human induced pluripotent stem cell (hiPSC) derived atrial cardiomyocytes as a promising technique to determine the electrophysiological impact of genetic risk variants

Abstract Introduction Several studies have associated atrial fibrillation (AF) with abnormalities in cardiac ion channels in native cardiomyocytes with patch-clamp techniques. However, this complex low-throughput technique is not well suited for studying common intronic or intergenic genetic risk variants. On the other hand, cardiomyocytes derived from isogenic hiPSC-lines have emerged as an attractive alternative to study genetic disorders in cardiovascular disease. Purpose Therefore, this study aimed to develop protocols to characterize major ion currents in hiPSC-derived atrial cardiomyocytes (hiPSC-aCMs) using high-throughput automated patch-clamp technology in order to set the basis for studying the impact of common genetic variants associated with AF expected to affect ion channels. Methods hiPSCs were differentiated into hiPSC-aCMs using retinoic acid and matured for 5 weeks. Subsequently, myocytes were separated from other cell types using Magnetic Activated Cell Sorting (MACS) and replated at low density (600K single aCMs/well). After 2 weeks, myocytes were dissociated using Collagenase B and Accumax and the cell suspension was transferred to a Nanion PatchLiner for automated whole-cell patch-clamp recordings of sodium current (INa), apamin-sensitive SK current, HCN current (If), L-type calcium current (ICaL) and transient outward currents (Ito) in isolated hiPSC-aCMs. Results The current-voltage relationship of INa reached a peak amplitude of -141±29.41 pA/pF (n=11) at -40 mV with 14 mM Nao. A voltage ramp protocol was used to record the current-voltage relationship before and after exposure to 100 nM apamin in order to obtain apamin-sensitive SK current, which was proportional to the membrane potential above -30 mV. The If density was -11.10±2.79 pA/pF in control, and 1 mM Ivabradine reduced it to -6.39±2.31 pA/pF (n=11, p=0.0099). The Ito density at + 50 mV was 8.00±1.53 pA/pF, and 2mM 4-aminopyridine reduced it to 4.72±0.76 pA/pF (p=0.0059, n=6). The peak ICaL density recorded at 0 mV was -5.91±1.18 pA/pF in control conditions. It increased to -8.62±1.25 pA/pF after exposing myocytes to the ICaL agonist BAY-K8644 (1 mM), and subsequent exposure to 1 mM of the selective inhibitor nifedipine reduced the ICa density to -3.43±0.56 pA/pF (n=9, p=0.0011). Conclusion The automated patch-clamp technique can be used to record current-voltage relationships and current densities in hiPSC-derived atrial myocytes that compare to recordings in native atrial myocytes in similar experimental conditions, opening the possibility of assessing the functional electrophysiological impact of AF risk SNPs affecting ion channels using isogenic hiPSC lines.