TBX20 Consolidates Cardiac Reprogramming Factors to Enhance Contractility and Energy Metabolism in Human Induced Cardiomyocytes [CUT&RUN]

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. Epigenomic profiling of H3K4me1, H3K27ac and H3K27me3 in human induced cardiomyocytes by CUT&RUN sequencing. Biological duplicates were applied for each group.