Maturation of hiPSC-derived cardiomyocytes in tri-cellular cardiac microtissues promotes adult alternative splicing of SCN5A revealing effects of mutations in cardiac disease

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used to examine in vitro the effect of mutations in the cardiac sodium channel gene SCN5A, associated with cardiac arrhythmias. Postnatally SCN5A undergoes a fetal-to-adult isoform switch, but hiPSC-CMs in conventional 2-dimensional cultures are fetal-like. This impedes evaluation of mutations in the adult isoform. Here, we derived hiPSC-CMs from a patient carrying compound mutations in the adult SCN5A exon 6B and in exon 4 and generated isogenic corrected lines. In hiPSC-CM 2-dimensional culture, exon 6B mutation did not affect single-cell electrophysiology because of its limited expression. CRISPR/Cas9-mediated excision of the fetal exon 6A with did not promote adult SCN5A expression, rather it impaired the splicing. By maturing hiPSC-CMs in three-dimensional tri-cell type cardiac microtissues, SCN5A underwent isoform switch and revealed the functional effect of exon 6B mutation. Upregulation of the splicing factor MBNL1 in hiPSC-CMs either by culture in microtissues or by overexpression was sufficient to promote exon 6B inclusion. Our results support the ability to study developmentally regulated cardiac genes and postnatal cardiac arrhythmias using hiPSC cardiac cells. Overall design: We derived hiPSC-CMs from a patient carrying compound mutations in the adult SCN5A exon 6B and in exon 4 and generated isogenic corrected lines we sequenced.

人类诱导多能干细胞衍生心肌细胞（human induced pluripotent stem cell-derived cardiomyocytes, hiPSC-CMs）被用于体外探究与心律失常相关的心脏钠通道基因SCN5A突变的效应。出生后，SCN5A会发生胎儿型至成人型剪接异构体的转换，但常规二维培养体系中的hiPSC-CMs仍维持胎儿样表型，这极大阻碍了针对成人型异构体突变的功能评估。本研究从一名携带成人型SCN5A外显子6B与外显子4复合突变的患者样本中诱导获得hiPSC-CMs，并构建了同基因校正细胞系。在二维培养的hiPSC-CMs中，由于外显子6B的表达水平极低，其突变并未对单细胞电生理特性造成显著影响。通过CRISPR/Cas9介导切除胎儿型外显子6A，不仅未能促进成人型SCN5A的表达，反而损害了其剪接过程。将hiPSC-CMs置于三维三细胞型心脏微组织中进行成熟培养后，SCN5A发生了剪接异构体转换，从而明确了外显子6B突变的功能效应。无论是通过微组织培养体系，还是通过过表达手段，上调hiPSC-CMs中的剪接因子MBNL1均可有效促进外显子6B的剪接包含。本研究结果证实，利用hiPSC来源的心脏细胞可有效研究发育调控型心脏基因以及出生后心律失常相关机制。实验整体设计：本研究从一名携带成人型SCN5A外显子6B与外显子4复合突变的患者样本中诱导获得hiPSC-CMs，并构建同基因校正细胞系进行测序。