目的:人诱导多能干细胞-心肌细胞(hiPSC-CMs)被广泛用于研究离子通道中心律失常相关的突变。其中,心脏钠通道SCN5A在出生前后经历胎儿到成人的同工型转换.传统的hiPSC-CM培养,它们的表型是胎儿的,到目前为止,还无法捕获成年基因亚型中的突变。这里,我们研究了三维心脏微组织中的三细胞串扰是否促进了hiPSC-CM的出生后SCN5A成熟。
结果:我们得出了在成年SCN5A外显子6B和外显子4中携带复合突变的患者hiPSC-CM。与等基因对照相比,单层患者hiPSC-CM的电生理特性未被外显子6B突变改变,因为它不表达;此外,CRISPR/Cas9介导的胎儿外显子6A切除不促进成人SCN5A表达。然而,当hiPSC-CM在三维心脏微组织中成熟时,SCN5A经历了同工型转换,并揭示了位于外显子6B的突变的功能后果。剪接因子肌盲样蛋白1(MBNL1)的上调导致SCN5A在微组织中的出生后成熟,因为其在hiPSC-CM中的过度表达足以促进外显子6B的包含,而敲除MBNL1未能促进同工型转换。
结论:我们的研究表明(i)三细胞心脏微组织促进hiPSC-CM中的出生后SCN5A同工型转换(ii)这些组织中的MBNL1驱动SCN5A的成人剪接(iii)该模型可用于检查由于外显子6B突变引起的出生后心律失常。
UNASSIGNED:心脏钠通道对于传导心脏中的电脉冲至关重要。出生后的选择性剪接调节导致相应基因的胎儿或成人外显子相互排斥,SCN5A.通常,未成熟的hiPSC-CM在研究位于成年外显子的突变的影响方面不足。我们在这里描述了创新的三细胞三维心脏微组织培养物通过上调MBNL1促进hiPSC-CM的成熟,从而揭示了位于SCN5A成人外显子中的致病性遗传变异的作用。这些结果有助于推进hiPSC-CM在研究成人心脏病和开发个性化医学应用中的使用。
Human-induced pluripotent stem cell-cardiomyocytes (hiPSC-CMs) are widely used to study arrhythmia-associated mutations in ion channels. Among these, the cardiac sodium channel SCN5A undergoes foetal-to-adult isoform switching around birth. Conventional hiPSC-CM cultures, which are phenotypically foetal, have thus far been unable to capture mutations in adult gene isoforms. Here, we investigated whether tri-cellular cross-talk in a three-dimensional (3D) cardiac microtissue (MT) promoted post-natal SCN5A maturation in hiPSC-CMs.
We derived patient hiPSC-CMs carrying compound mutations in the adult SCN5A exon 6B and exon 4. Electrophysiological properties of patient hiPSC-CMs in monolayer were not altered by the exon 6B mutation compared with isogenic controls since it is not expressed; further, CRISPR/Cas9-mediated excision of the foetal exon 6A did not promote adult SCN5A expression. However, when hiPSC-CMs were matured in 3D cardiac MTs, SCN5A underwent isoform switch and the functional consequences of the mutation located in exon 6B were revealed. Up-regulation of the splicing factor muscleblind-like protein 1 (MBNL1) drove SCN5A post-natal maturation in microtissues since its overexpression in hiPSC-CMs was sufficient to promote exon 6B inclusion, whilst knocking-out MBNL1 failed to foster isoform switch.
Our study shows that (i) the tri-cellular cardiac microtissues promote post-natal SCN5A isoform switch in hiPSC-CMs, (ii) adult splicing of SCN5A is driven by MBNL1 in these tissues, and (iii) this model can be used for examining post-natal cardiac arrhythmias due to mutations in the exon 6B.
The cardiac sodium channel is essential for conducting the electrical impulse in the heart. Postnatal alternative splicing regulation causes mutual exclusive inclusion of fetal or adult exons of the corresponding gene, SCN5A. Typically, immature hiPSCCMs fall short in studying the effect of mutations located in the adult exon. We describe here that an innovative tri-cellular three-dimensional cardiac microtissue culture promotes hiPSC-CMs maturation through upregulation of MBNL1, thus revealing the effect of a pathogenic genetic variant located in the SCN5A adult exon. These results help advancing the use of hiPSC-CMs in studying adult heart disease and for developing personalized medicine applications.