A transcriptional enhancer regulates cardiac maturation [scRNA-seq]. A transcriptional enhancer regulates cardiac maturation [scRNA-seq]

Cardiomyocyte maturation is crucial for generating adult cardiomyocytes and the application of human pluripotent stem cell derived-cardiomyocytes (hPSC-CMs). However, regulation at the cis-regulatory element level, and its role in heart disease remain unclear. Alpha-actinin 2 (ACTN2) levels increase during CM maturation. Here, we investigate a clinically relevant, conserved ACTN2 enhancer’s effects on CM maturation using hPSC and mouse models. Heterozygous ACTN2 enhancer deletion led to abnormal CM morphology, reduced function, and mitochondrial respiration. Transcriptomic analyses in vitro and in vivo showed disrupted CM maturation and upregulated anabolic mammalian target for rapamycin (mTOR) signaling promoting senescence and hindering maturation. As confirmation, ACTN2 enhancer deletion induced heat shock protein 90A expression, a chaperone mediating mTOR activation. Conversely, targeting the ACTN2 enhancer via enhancer CRISPR activation (enCRISPRa) promoted hPSC-CM maturation. Our studies reveal the transcriptional enhancer’s role in cardiac maturation and disease, offering insights into potentially fine-tuning gene expression to modulate cardiomyocyte physiology. Overall design: To investigate the role of a conserved ACTN2 enhancer in vitro, we used CRISPR/Cas9 and deleted this enhancer genomic region in human pluripotent stem cells (hPSCs) (H9). We then differentiated the cells to cardiomyoaytes (hPSC-CMs) and perfomed single cell RNA-Seq at differentiated day 20 CMs.

心肌细胞成熟对于获取成体心肌细胞以及人多能干细胞衍生心肌细胞（human pluripotent stem cell derived-cardiomyocytes，hPSC-CMs）的应用均至关重要。然而，顺式调控元件层面的调控机制及其在心脏疾病中的作用仍未明确。α-辅肌动蛋白2（Alpha-actinin 2，ACTN2）的表达水平在心肌细胞成熟过程中显著升高。本研究借助人多能干细胞与小鼠模型，探究一个具有临床相关性的保守ACTN2增强子对心肌细胞成熟的调控效应。杂合子ACTN2增强子缺失会导致心肌细胞形态异常、功能受损以及线粒体呼吸功能障碍。体外与体内转录组分析结果显示，心肌细胞成熟过程被扰乱，且合成代谢型雷帕霉素靶蛋白（mammalian target for rapamycin，mTOR）信号通路出现上调，该通路可促进细胞衰老并阻碍心肌细胞成熟。为验证上述结论，我们发现ACTN2增强子缺失会诱导热休克蛋白90A的表达，而该分子伴侣可介导mTOR的激活。反之，通过增强子CRISPR激活（enhancer CRISPR activation，enCRISPRa）靶向调控ACTN2增强子，可有效促进hPSC-CMs的成熟。本研究揭示了转录增强子在心脏成熟与疾病发生中的关键作用，为通过精准调控基因表达以调节心肌细胞生理功能提供了全新的研究思路。总体实验设计：为在体外探究保守ACTN2增强子的功能，我们利用CRISPR/Cas9技术在人多能干细胞（H9细胞系）中删除该增强子的基因组区域，随后将修饰后的细胞诱导分化为心肌细胞（hPSC-CMs），并在细胞分化第20天的心肌细胞中开展单细胞RNA测序。