A Better Understanding of Atrial-like and Ventricular-like Action Potentials in Stem Cell-Derived Cardiomyocytes: The Underestimated Role of the L-Type Ca2+ Current

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) tend to show a mixed population of action potential (AP) types, including atrial-like (A-like) and ventricular-like (V-like) APs. In the present study, we investigated the membrane currents underlying these two AP types in hESC-CMs. These were generated using standard (Std) and retinoic acid (RA)-based differentiation protocols. Patch clamp methodology was used to correlate AP morphology with major cardiac ion currents by applying alternating current and voltage clamp protocols to each cell, and to measure L-type Ca²⁺ current (I_Ca,L) and Na⁺-Ca²⁺ exchange current (I_NCX) in detail, whereas Ca²⁺ transients were measured ratiometrically using Indo-1. A- and V-like APs were found in both Std and RA-treated hESC-CMs and the AP plateau amplitude (AP_plat), as a measure of fast phase-1 repolarization, appeared the best AP criterion to separate these two AP types. Traditional voltage clamp experiments revealed a significantly smaller I_Ca,L density in RA-treated hESC-CMs, as well as larger densities of the transient outward and delayed rectifier K⁺ currents (I_to1 and I_K, respectively), without changes in the inward rectifier K⁺ current (I_K1). The AP_plat showed strong and moderate correlations with the densities of I_Ca,L and I_K, respectively, in the absence of a clear-cut correlation with the density of I_to1. Using pre-recorded, typical A- and V-like APs, AP clamp demonstrated that the I_Ca,L-mediated Ca²⁺ influx during the V-like AP in Std hESC-CMs is 3.15 times larger than the influx during the A-like AP in RA-treated hESC-CMs. Ca²⁺ transients of A-like hESC-CMs have a lower diastolic and systolic level, as well as a lower amplitude, than those of Std hESC-CMs, while their duration is shorter due to enhanced SERCA activity. In conclusion, I_Ca,L is an important determinant of the differently shaped A- and V-like APs in hESC-CMs. Furthermore, the Ca²⁺ homeostasis differs between A- and V-like hESC-CMs due to the smaller I_Ca,L and enhanced SERCA activity during A-like APs, resulting in a strongly reduced Ca²⁺ influx, which will cause a substantial reduction in I_NCX, further contributing to the shorter A-like APs.