Gonzalez David M, Dariolli Rafael, Moyett Julia, Song Stephanie, Shewale Bhavana, Bliley Jacqueline, Clarke Daniel, Ma'ayan Avi, Rentschler Stacey, Feinberg Adam, Sobie Eric, Dubois Nicole C
Department of Cell, Developmental, and Regenerative Biology, Mount Sinai, New York, NY 10029, USA.
Mindich Child Health and Development Institute, Mount Sinai, New York, NY 10029, USA.
bioRxiv. 2024 Sep 28:2024.09.22.614353. doi: 10.1101/2024.09.22.614353.
Cardiac Purkinje fibers form the most distal part of the ventricular conduction system. They coordinate contraction and play a key role in ventricular arrhythmias. While many cardiac cell types can be generated from human pluripotent stem cells, methods to generate Purkinje fiber cells remain limited, hampering our understanding of Purkinje fiber biology and conduction system defects. To identify signaling pathways involved in Purkinje fiber formation, we analyzed single cell data from murine embryonic hearts and compared Purkinje fiber cells to trabecular cardiomyocytes. This identified several genes, processes, and signaling pathways putatively involved in cardiac conduction, including Notch signaling. We next tested whether Notch activation could convert human pluripotent stem cell-derived cardiomyocytes to Purkinje fiber cells. Following Notch activation, cardiomyocytes adopted an elongated morphology and displayed altered electrophysiological properties including increases in conduction velocity, spike slope, and action potential duration, all characteristic features of Purkinje fiber cells. RNA-sequencing demonstrated that Notch-activated cardiomyocytes undergo a sequential transcriptome shift, which included upregulation of key Purkinje fiber marker genes involved in fast conduction such as and downregulation of genes involved in contractile maturation. Correspondingly, we demonstrate that Notch-induced cardiomyocytes have decreased contractile force in bioengineered tissues compared to control cardiomyocytes. We next modified existing models of human pluripotent stem cell-derived cardiomyocytes using our transcriptomic data and modeled the effect of several anti-arrhythmogenic drugs on action potential and calcium transient waveforms. Our models predicted that Purkinje fiber cells respond more strongly to dofetilide and amiodarone, while cardiomyocytes are more sensitive to treatment with nifedipine. We validated these findings , demonstrating that our new cell-specific model can be utilized to better understand human Purkinje fiber physiology and its relevance to disease.
心脏浦肯野纤维形成心室传导系统的最远端部分。它们协调收缩并在室性心律失常中起关键作用。虽然许多心脏细胞类型可以从人类多能干细胞中产生，但产生浦肯野纤维细胞的方法仍然有限，这阻碍了我们对浦肯野纤维生物学和传导系统缺陷的理解。为了确定参与浦肯野纤维形成的信号通路，我们分析了来自小鼠胚胎心脏的单细胞数据，并将浦肯野纤维细胞与小梁心肌细胞进行了比较。这确定了几个可能参与心脏传导的基因、过程和信号通路，包括Notch信号通路。接下来，我们测试了Notch激活是否可以将人类多能干细胞衍生的心肌细胞转化为浦肯野纤维细胞。Notch激活后，心肌细胞呈现出细长的形态，并表现出改变的电生理特性，包括传导速度、动作电位上升斜率和动作电位持续时间增加，这些都是浦肯野纤维细胞的特征性特征。RNA测序表明，Notch激活的心肌细胞经历了连续的转录组转变，其中包括参与快速传导的关键浦肯野纤维标记基因的上调，如 ，以及参与收缩成熟的基因的下调。相应地，我们证明，与对照心肌细胞相比，Notch诱导的心肌细胞在生物工程组织中的收缩力降低。接下来，我们利用我们的转录组数据修改了现有的人类多能干细胞衍生心肌细胞模型，并模拟了几种抗心律失常药物对动作电位和钙瞬变波形的影响。我们的模型预测，浦肯野纤维细胞对多非利特和胺碘酮的反应更强，而心肌细胞对硝苯地平治疗更敏感。我们验证了这些发现 ，证明我们新的细胞特异性 模型可用于更好地理解人类浦肯野纤维生理学及其与疾病的相关性。
