Utilizing Human-Induced Pluripotent Stem Cells to Study Cardiac Electroporation Pulsed-Field Ablation.

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.

Optogenetic modulation of cardiac action potential properties may prevent arrhythmogenesis in short and long QT syndromes.

Abnormal action potential (AP) properties, as occurs in long or short QT syndromes (LQTS and SQTS, respectively), can cause life-threatening arrhythmias. Optogenetics strategies, utilizing light-sensitive proteins, have emerged as experimental platforms for cardiac pacing, resynchronization, and defibrillation. We tested the hypothesis that similar optogenetic tools can modulate the cardiomyocyte's AP properties, as a potentially novel antiarrhythmic strategy. Healthy control and LQTS/SQTS patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were transduced to express the light-sensitive cationic channel channelrhodopsin-2 (ChR2) or the anionic-selective opsin, ACR2. Detailed patch-clamp, confocal-microscopy, and optical mapping studies evaluated the ability of spatiotemporally defined optogenetic protocols to modulate AP properties and prevent arrhythmogenesis in the hiPSC-CMs cell/tissue models. Depending on illumination timing, light-induced ChR2 activation induced robust prolongation or mild shortening of AP duration (APD), while ACR2 activation allowed effective APD shortening. Fine-tuning these approaches allowed for the normalization of pathological AP properties and suppression of arrhythmogenicity in the LQTS/SQTS hiPSC-CM cellular models. We next established a SQTS-hiPSC-CMs-based tissue model of reentrant-arrhythmias using optogenetic cross-field stimulation. An APD-modulating optogenetic protocol was then designed to dynamically prolong APD of the propagating wavefront, completely preventing arrhythmogenesis in this model. This work highlights the potential of optogenetics in studying repolarization abnormalities and in developing novel antiarrhythmic therapies.