Verkerk Arie O, Veerman Christiaan C, Hoekstra Maaike, Devalla Harsha D, Wilders Ronald
Department of Medical Biology, Amsterdam Cardiovascular Sciences, Amsterdam University Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.
Department of Experimental Cardiology, Heart Center, Amsterdam Cardiovascular Sciences, Amsterdam University Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.
Cells. 2025 Aug 8;14(16):1226. doi: 10.3390/cells14161226.
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 Ca current (I) and Na-Ca exchange current (I) in detail, whereas Ca 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), 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 density in RA-treated hESC-CMs, as well as larger densities of the transient outward and delayed rectifier K currents (I and I, respectively), without changes in the inward rectifier K current (I). The AP showed strong and moderate correlations with the densities of I and I, respectively, in the absence of a clear-cut correlation with the density of I. Using pre-recorded, typical A- and V-like APs, AP clamp demonstrated that the I-mediated Ca 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. Ca 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 is an important determinant of the differently shaped A- and V-like APs in hESC-CMs. Furthermore, the Ca homeostasis differs between A- and V-like hESC-CMs due to the smaller I and enhanced SERCA activity during A-like APs, resulting in a strongly reduced Ca influx, which will cause a substantial reduction in I, further contributing to the shorter A-like APs.
人胚胎干细胞衍生的心肌细胞（hESC-CMs）往往表现出混合的动作电位（AP）类型群体，包括心房样（A样）和心室样（V样）动作电位。在本研究中，我们调查了hESC-CMs中这两种动作电位类型背后的膜电流。这些细胞是使用基于标准（Std）和视黄酸（RA）的分化方案生成的。采用膜片钳技术，通过对每个细胞应用交流和电压钳方案，将动作电位形态与主要心脏离子电流相关联，并详细测量L型钙电流（I）和钠钙交换电流（I），而使用Indo-1以比率法测量钙瞬变。在Std和RA处理的hESC-CMs中均发现了A样和V样动作电位，作为快速1期复极化的指标，动作电位平台期幅度（AP）似乎是区分这两种动作电位类型的最佳动作电位标准。传统的电压钳实验显示，RA处理的hESC-CMs中I密度显著较小，而瞬时外向和延迟整流钾电流（分别为I和I）的密度较大，内向整流钾电流（I）无变化。在与I密度无明确相关性的情况下，动作电位分别与I和I密度呈强相关性和中等相关性。使用预先记录的典型A样和V样动作电位，动作电位钳实验表明，Std hESC-CMs中V样动作电位期间I介导的钙内流比RA处理的hESC-CMs中A样动作电位期间的钙内流大3.15倍。与Std hESC-CMs相比，A样hESC-CMs的钙瞬变具有较低的舒张期和收缩期水平以及较低的幅度，而由于SERCA活性增强，其持续时间较短。总之，I是hESC-CMs中形状不同的A样和V样动作电位的重要决定因素。此外，A样和V样hESC-CMs之间的钙稳态不同，这是由于A样动作电位期间I较小且SERCA活性增强，导致钙内流大幅减少，这将导致I大幅降低，进一步导致A样动作电位持续时间缩短。
