Abstract Body: Introduction: Direct cardiac reprogramming emerges as a promising strategy for in situ conversion of fibroblasts into induced cardiomyocyte-like cells (iCMs). While the conversion of mouse fibroblasts into functional, beating iCMs has been achieved efficiently, the reprogramming of human iCMs remains challenging, particularly their maturation with existing transcription factor combinations. Given the pivotal role of epigenetic regulation in this process, our study sought to leverage heart-specific epigenetic factors alongside cardiac-enriched transcription factors to enhance cardiac maturation, thereby yielding more reliable human iCMs.
Result: Through analyzing the proteomic data across various human tissues, we found that while many epigenetic factors are broadly expressed, SMYD1 is uniquely heart-specific. To determine its involvement in reprogramming, we overexpressed SMYD1 and evaluated the reprogramming efficiency and iCM quality via flow cytometry, immunofluorescence staining, and transcriptome analysis. Notably, we observed an increase in iCMs expressing sarcomere markers, such as α-myosin heavy chain and α-actinin, as well as the ventricular specific myosin-light-chain 2v in SMYD1-treated group. Gene set enrichment analysis of RNA-sequencing data further indicated that SMYD1-induced iCMs exhibit ventricular gene signatures. Additionally, we discovered moderate activation of SMYD1 in mature iCMs generated with MEF2C, GATA4, TBX5, and TBX20. TBX20 is the key factor that leads to the co-binding of MEF2C, TBX5, and TBX20 on the SMYD1 promoter region and increases active histone marks, H3K27ac and H3K4me1. Rescue assay and transcriptomic comparison confirmed SMYD1 as the primary downstream target of TBX20 in promoting iCM functional maturation. Mechanistically, SMYD1 directly interacts with MEF2C and GATA4 to modify histone marks on cardiac regulatory regions.
Conclusion: Our study provides comprehensive phenotypic and molecular evidence of SMYD1’s role as a cardiac-specific regulator during direct cardiac reprogramming. By interacting with key reprogramming factors and rewriting histone marks, SMYD1 significantly advances the maturation of human iCMs towards a ventricular subtype identity.