TBX20 Consolidates Cardiac Reprogramming Factors to Enhance Contractility and Energy Metabolism in Human Induced Cardiomyocytes [ATAC-Seq]

Background: Direct cardiac reprogramming of fibroblasts into cardiomyocytes has emerged as one of the promising strategies to remuscularize the injured myocardium. Yet, it is still insufficient to generate functional induced cardiomyocytes (iCMs) from human fibroblasts using conventional reprogramming cocktails, such as our previously published combination consisting of MEF2C, GATA4, TBX5 and microRNA miR-133 (MGT133). Results: To discover potential missing factors for human direct reprogramming, we performed transcriptomic comparison between human iCMs and functional cardiomyocytes (CMs). We identified T-box transcription factor TBX20 as the top CM gene that is unable to be activated by MGT133. TBX20 is required for normal heart development and cardiac function in adult CMs but its role on cardiac reprogramming remains undefined. Here, we found that transduction of MGT133+TBX20 in human cardiac fibroblasts resulted in enhanced reprogramming featured with significantly activated contractility gene programs and signatures more similar to ventricular CMs. Human iCMs produced with MGT133+TBX20 more frequently demonstrated beating and calcium oscillation in co-culture with pluripotent stem cell derived CMs. More mitochondria and higher mitochondrial respiration were also detected in iCMs after TBX20 overexpression. Mechanistically, comprehensive transcriptomic, chromatin occupancy and epigenomic integration revealed that TBX20 localized to the cis-regulatory enhancers of under-expressed cardiac genes, such as MYBPC3, MYH7 and MYL4, to activate gene expression via strengthening the occupancy and co-occupancy of transcription factors. Furthermore, we identified TBX20-regulated enhancers and confirmed the synergistic effect of MGT and TBX20 on enhancer activation. Conclusions: TBX20 promotes cardiac cell fate conversion via direct activating cardiac enhancers. Human iCMs generated with TBX20 showed enhanced cardiac function in terms of contractility and mitochondrial respiration. Examination of genome-wide accessibility of induced cardiomyocytes derived from human fibroblasts. Biological triplicate were used for statistical analysis.

研究背景：将成纤维细胞直接重编程为心肌细胞，已成为受损心肌再肌化的极具前景的策略之一。然而，使用传统重编程组合（如我们此前发表的由MEF2C、GATA4、TBX5以及微小RNA miR-133组成的MGT133组合），从人类成纤维细胞中获取具有功能的诱导型心肌细胞（induced cardiomyocytes，iCMs）仍存在不足。

研究结果：为挖掘人类直接重编程中可能缺失的因子，我们对人类诱导型心肌细胞（induced cardiomyocytes，iCMs）与功能性心肌细胞（cardiomyocytes，CMs）开展了转录组比较分析。我们鉴定出T-box转录因子TBX20是MGT133无法激活的优势心肌细胞基因。TBX20对正常心脏发育及成年心肌细胞的心脏功能至关重要，但其在心脏重编程中的作用仍未明确。本研究发现，在人类心脏成纤维细胞中转导MGT133+TBX20组合，可增强重编程效果，具体表现为收缩相关基因程序显著激活，且分子特征更接近心室心肌细胞。与多能干细胞来源的心肌细胞共培养时，MGT133+TBX20诱导产生的人类iCMs更易出现搏动及钙振荡现象。TBX20过表达后的iCMs中，线粒体数量更多，线粒体呼吸水平也更高。从机制层面来看，整合多组学转录组、染色质占据及表观基因组数据后发现，TBX20可靶向结合表达量低下的心肌基因（如MYBPC3、MYH7及MYL4）的顺式调控增强子，通过增强转录因子的占据及共占据情况来激活基因表达。此外，我们还鉴定出TBX20调控的增强子，并证实了MGT与TBX20在增强子激活中的协同效应。

研究结论：TBX20通过直接激活心肌增强子促进心脏细胞命运转化。采用TBX20诱导产生的人类iCMs，其收缩功能及线粒体呼吸水平均得到显著提升。本数据集包含人类成纤维细胞来源的诱导型心肌细胞的全基因组染色质开放程度检测数据。实验采用3次生物学重复进行统计学分析。