Circulation. 2026 Sep 08.
Jingdong Wu,
Chunyan Yang,
Donghui Wu,
Shaoqi Zhu,
Yiran Liu,
Yang Yang,
Ze Xu,
Yitong Xu,
Xiaochun Yang,
Lichao Mao,
Jinxuan Chen,
Huan Wang,
Dongyu Zhao,
Shiqiang Wang,
Chao Tang,
Bin Zhou,
Yang Zhao.
BACKGROUND: Direct reprogramming of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds promise as a therapeutic strategy for heart regeneration. After myocardial infarction (MI), resident quiescent CFs (QCFs) activate and differentiate into myofibroblasts (MFs) in the infarcted region that drive pathological cardiac fibrosis. Converting these injury-activated MFs into iCMs could simultaneously alleviate fibrosis and replenish lost cardiomyocytes. However, whether the marked heterogeneity of CFs in the infarcted heart, and in particular the activation of QCFs into MFs, creates an intrinsic barrier that limits the reprogramming of these injury-activated MFs remains unknown.
METHODS: To define the molecular basis of this heterogeneity, we purified PDGFRα+ CFs and used Postn lineage tracing to distinguish injury-activated MFs from quiescent CFs, enabling matched comparison of their reprogramming competence and single-cell RNA sequencing (scRNA-seq) profiling of post-MI CF subpopulations. A targeted in vitro shRNA screen was conducted against 9 MF-enriched TFs as candidate molecular barriers for cardiac reprogramming. The lead candidate was validated in mouse MFs, human iPSC-derived MFs, primary human MFs, and in vivo using dual-recombinase-mediated lineage tracing. Mechanistic insights were gained through integrated bulk RNA-seq, scRNA-seq and Cleavage Under Targets and Tagmentation (CUT&Tag), together with functional assays including DNA-binding-deficient and domain-swap MEOX1 mutants.
RESULTS: We identified the upregulated transcription factor MEOX1, a known fibrosis determinant downstream of the key post-MI cytokines transforming growth factor-beta 1 and interleukin-1 beta, as the principal molecular barrier responsible for the profound reprogramming resistance of MFs. MEOX1 knockdown markedly enhanced reprogramming efficiency in both mouse and human MFs and enabled GATA4-free reprogramming combinations. This inhibition depended on the transcriptional activation activity of MEOX1, as disrupting its DNA-binding domain or fusing it to a repressor domain rescued reprogramming. Integrated scRNA-seq and CUT&Tag analyses revealed that MEOX1 binds and stabilizes a fibrotic, MF-defining transcriptional program that antagonizes the cardiogenic program while also modulating the inflammatory response; its knockdown disrupted this fibrotic network to favor iCM fate acquisition. Using stringent dual-recombinase lineage tracing, we demonstrated that MEOX1 knockdown enables highly efficient in vivo MF-to-iCM conversion, leading to significant reductions in cardiac fibrosis and substantial improvement in cardiac function after MI.
CONCLUSIONS: Our study identifies pathological MEOX1 upregulation as a key mechanism underlying the reprogramming resistance of post-MI mouse MFs and activated human MFs. Overcoming this barrier achieves unprecedented, lineage-confirmed in vivo reprogramming efficiency, thereby addressing a significant obstacle for the clinical translation of in situ reprogramming therapies.
Keywords: cellular reprogramming; fibrosis; myocardial infarction; myofibroblasts; regeneration