BACKGROUND:Electroporation is a promising nonthermal ablation method for cardiac arrhythmia treatment. Although initial clinical studies found electroporation pulsed-field ablation (PFA) both safe and efficacious, there are significant knowledge gaps concerning the mechanistic nature and electrophysiological consequences of cardiomyocyte electroporation, contributed by the paucity of suitable human in vitro models. Here, we aimed to establish and characterize a functional in vitro model based on human-induced pluripotent stem cells (hiPSCs)-derived cardiac tissue, and to study the fundamentals of cardiac PFA.METHODS:hiPSC-derived cardiomyocytes were seeded as circular cell sheets and subjected to different PFA protocols. Detailed optical mapping, cellular, and molecular characterizations were performed to study PFA mechanisms and electrophysiological outcomes.RESULTS:PFA generated electrically silenced lesions within the hiPSC-derived cardiac circular cell sheets, resulting in areas of conduction block. Both reversible and irreversible electroporation components were identified. Significant electroporation reversibility was documented within 5 to 15-minutes post-PFA. Irreversibly electroporated regions persisted at 24-hours post-PFA. Per single pulse, high-frequency PFA was less efficacious than standard (monophasic) PFA, whereas increasing pulse-number augmented lesion size and diminished reversible electroporation. PFA augmentation could also be achieved by increasing extracellular Ca²⁺ levels. Flow-cytometry experiments revealed that regulated cell death played an important role following PFA. Assessing for PFA antiarrhythmic properties, sustainable lines of conduction block could be generated using PFA, which could either terminate or isolate arrhythmic activity in the hiPSC-derived cardiac circular cell sheets.CONCLUSIONS:Cardiac electroporation may be studied using hiPSC-derived cardiac tissue, providing novel insights into PFA temporal and electrophysiological characteristics, facilitating electroporation protocol optimization, screening for potential PFA-sensitizers, and investigating the mechanistic nature of PFA antiarrhythmic properties.

中文翻译：

---

利用人类诱导多能干细胞研究心脏电穿孔脉冲场消融

背景：电穿孔是一种很有前景的治疗心律失常的非热消融方法。尽管最初的临床研究发现电穿孔脉冲场消融（PFA）既安全又有效，但由于缺乏合适的人体体外模型，关于心肌细胞电穿孔的机械性质和电生理学后果仍存在显着的知识差距。在这里，我们的目的是建立和表征基于人诱导多能干细胞（hiPSC）来源的心脏组织的功能性体外模型，并研究心脏 PFA 的基本原理。方法：将 hiPSC 来源的心肌细胞接种为圆形细胞片并接受不同的 PFA 协议。进行详细的光学测绘、细胞和分子表征，以研究 PFA 机制和电生理结果。结果：PFA 在 hiPSC 衍生的心脏圆形细胞片内产生电沉默损伤，导致传导阻滞区域。鉴定了可逆和不可逆电穿孔成分。 PFA 后 5 至 15 分钟内记录了显着的电穿孔可逆性。 PFA 后 24 小时仍存在不可逆的电穿孔区域。对于每个单脉冲，高频 PFA 的效果低于标准（单相）PFA，而增加脉冲数会增大病变大小并减弱可逆电穿孔。 PFA 增强也可以通过增加细胞外 Ca ²⁺水平来实现。流式细胞术实验表明，PFA 后调节的细胞死亡发挥着重要作用。评估 PFA 的抗心律失常特性，可以使用 PFA 产生可持续的传导阻滞线，从而可以终止或隔离 hiPSC 衍生的心脏圆形细胞片中的心律失常活动。结论：可以使用 hiPSC 衍生的心脏组织来研究心脏电穿孔，提供对 PFA 时间和电生理学特征的新见解，促进电穿孔方案优化，筛选潜在的 PFA 敏化剂，并研究 PFA 抗心律失常特性的机制性质。