Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and primary human cardiomyocytes are used for in vitro cardiac safety testing. hiPSC-CMs have been associated with a vast heterogeneity regarding single-cell morphology, beating behavior and action potential duration, prompting a systematic analysis of single-cell characteristics. Previously published hiPSC-CM studies revealed action potentials with nodal-, atrial- or ventricular-like morphology, although ion channel expression of singular hiPSC-CMs is not fully understood. Other studies used single-cell RNA-sequencing, however, these studies did not extensively focus on expression patterns of cardiac ion channels or failed to detect ion channel transcripts. Thus, the current study used a single-cell patch-clamp-RT-qPCR approach to get insights into single-cell electrophysiology (capacitance, action potential duration at 90% of repolarization, upstroke velocity, spontaneous beat rate, and sodium-driven fast inward current) and ion channel expression (HCN4, CACNA1G, CACNA1D, KCNA5, KCNJ4, SCN5A, KCNJ2, CACNA1D, and KCNH2), the combination of both within individual cells, and their correlations in single cardiomyocytes. We used commercially available hiPSC-CMs (iCell cardiomyocytes, atrial and ventricular Pluricytes) and primary human adult atrial and ventricular cardiomyocytes. Recordings of electrophysiological parameters revealed differences between the cell groups and variation within the hiPSC-CMs groups as well as within primary ventricular cardiomyocytes. Expression analysis on mRNA level showed no-clear-cut discrimination between primary cardiac subtypes and revealed both similarities and differences between all cell groups. Higher expression of atrial-associated ion channels in primary atrial cardiomyocytes and atrial Pluricytes compared to their ventricular counterpart indicates a successful chamber-specific hiPSC differentiation. Interpretation of correlations between the single-cell parameters was challenging, as the total data set is complex, particularly for parameters depending on multiple processes, like the spontaneous beat rate. Yet, for example, expression of SCN5A correlated well with the fast inward current amplitude for all three hiPSC-CM groups. To further enhance our understanding of the physiology and composition of the investigated hiPSC-CMs, we compared beating and non-beating cells and assessed distributions of single-cell data. Investigating the single-cell phenotypes of hiPSC-CMs revealed a combination of attributes which may be interpreted as a mixture of traits of different adult cardiac cell types: (i) nodal-related pacemaking attributes are spontaneous generation of action potentials and high HCN4 expression; and (ii) non-nodal attributes: cells have a prominent INa-driven fast inward current, a fast upstroke velocity and a high expression of SCN5A. In conclusion, the combination of nodal- and non-nodal attributes in single hiPSC-CMs may hamper the interpretation of drug effects on complex electrophysiological parameters like beat rate and action potential duration. However, the proven expression of specific ion channels enables the evaluation of drug effects on ionic currents in a more realistic environment than in recombinant systems.

中文翻译：

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单个人（原代和诱导多能干细胞衍生）心肌细胞的离子通道表达和电生理

人类诱导的多能干细胞衍生的心肌细胞（hiPSC-CM）和原代人类心肌细胞用于体外心脏安全性测试。在单细胞形态，跳动行为和动作电位持续时间方面，hiPSC-CM与巨大的异质性相关联，从而促使对单细胞特征进行系统的分析。先前发表的hiPSC-CM研究揭示了结节，心房或心室样形态的动作电位，尽管尚未完全了解单个hiPSC-CM的离子通道表达。其他研究使用单细胞RNA测序，但是，这些研究并未广泛关注于心脏离子通道的表达模式或未能检测到离子通道转录本。因此，当前的研究使用单细胞膜片钳RT-qPCR方法深入了解单细胞电生理学（电容，复极化90％时的动作电位持续时间，上冲速度，自发搏动率和钠驱动的快速内向电流）和离子通道表达（HCN4，CACNA1G，CACNA1D，KCNA5，KCNJ4，SCN5A，KCNJ2，CACNA1D和KCNH2），两者在单个细胞内的组合以及它们在单个心肌细胞中的相关性。我们使用了市售的hiPSC-CMs（iCell心肌细胞，心房和心室肥大细胞）和原代人成年心房和心室心肌细胞。电生理参数的记录揭示了细胞组之间的差异以及hiPSC-CMs组以及原发性心室心肌细胞内的变化。对mRNA水平的表达分析表明，原发性心脏亚型之间没有清晰的区分，并且揭示了所有细胞组之间的相似性和差异性。与它们的心室对应物相比，原发性心房心肌细胞和心房多发性心房相关离子通道的更高表达表明成功的腔室特异性hiPSC分化。解释单细胞参数之间的相关性具有挑战性，因为总数据集非常复杂，尤其是对于取决于多个过程的参数，例如自发搏动率。然而，例如，对于所有三个hiPSC-CM组，SCN5A的表达与快速向内的电流幅度密切相关。为了进一步增进我们对所研究的hiPSC-CM的生理和组成的了解，我们比较了跳动和无跳动的细胞，并评估了单细胞数据的分布。研究hiPSC-CM的单细胞表型揭示了属性的组合，这些属性可以解释为不同成年心脏细胞类型的特征的混合：（i）与结节相关的起搏属性是动作电位的自发产生和HCN4高表达；（ii）非节点属性：细胞具有明显的INa驱动的快速内向电流，快速的上冲速度和SCN5A的高表达。总之，单个hiPSC-CM中节点属性和非节点属性的组合可能会妨碍药物对复杂电生理参数（如心跳率和动作电位持续时间）的影响的解释。然而，