Maintenance of Fibroblast Identity During Cardiac Reprogramming and Heart Repair [ATAC-seq]

Direct cardiac reprogramming (CR) of fibroblasts into induced cardiomyocytes represents a promising therapeutic avenue to treat heart failure as it would use the injury by-product and transform fibroblasts into new cardiac muscle thereby achieving the unmet medical need of cardiac regeneration. However, low reprogramming efficiency limits its clinical potential. Our lab identified four transcription factors playing a conserved role as barriers to CR. Knock-down of these barriers increase CR five-fold compared to MGT (MEF2C; GATA4; TBX5) overexpression alone. Exploration of direct downstream targets of these barriers identified proteoglycan sulfation as potential pathway regulating cell fate stability. Proteoglycan sulfation, specifically Carbohydrate Sulfotransferase 7 (CHST7), was identified as a conserved barrier to CR. Knockdown of Chst7 in mouse and human fibroblasts significantly enhances CR, increasing expression of cardiac-specific markers and promoting sarcomeric organization. Functional screening of proteoglycan core proteins identifies CD44 as a conserved barrier to reprogramming. Additionally, Chst7 and Cd44 functionally interact to mediate cell fate stability. RNA sequencing and transcription factor analysis identify Junb as the main TF mediating Chst7-regulated transcription during CR. Mechanistic studies of Chst7-regulated pathway genes demonstrate that Chst7 knockdown reduces Pip4k2c expression, establishing a novel regulatory axis between Chst7/Cd44/Junb and Pip4k2c. In vivo, shRNA-mediated Pip4k2c knockdown significantly enhances MGT-mediated CR, increasing cardiac muscle content and reducing fibrosis after myocardial infarction. These findings establish proteoglycan sulfation as a key regulator of cell fate stability, offering new opportunities to improve CR therapies. Thus, proteoglycan sulfation-associated genes might represent an innovative target space to enhance CR for therapeutic purposes.