Grhl3 Downregulation Facilitates ECM Adaptation for Fibroblast to iCM Commitment

BACKGROUND: Direct cardiac reprogramming offers a promising therapeutic strategy for heart regeneration by converting endogenous fibroblasts to functional induced cardiomyocytes (iCMs) that integrate into the myocardium to restore heart structure and function. While ECM (extracellular matrix) plays critical roles in cardiac disease and repair, the dynamic changes and transcriptional regulation underlying ECM remodeling during reprogramming remain poorly understood. METHODS: We investigated ECM dynamics during iCM reprogramming using integrated transcriptomic, proteomic, and epigenetic analyses, focusing on cell type-specific ECM components. A loss-of-function screen was used to identify critical ECM components and regulators, including Itga8 (integrin alpha-8) and Grhl3 (grainyhead-like protein 3 homolog), respectively, as reprogramming barriers. Mechanistic studies integrated RNA sequencing, mass spectrometry, and Cleavage Under Targets and Tagmentation to define Grhl3-dependent regulation. Functional outcomes were evaluated in vitro using decellularized ECM and in vivo using a myocardial infarction model with genetic lineage tracing. RESULTS: Cardiac reprogramming induced dynamic ECM remodeling, with significant changes in collagen, fibrillar proteins, and integrins. Itga8 was identified as a pivotal ECM component that restricts iCM conversion via the TGF-β (transforming growth factor-β)/SMAD (small mothers against decapentaplegic) pathway. Grhl3 emerged as a key transcriptional regulator for ECM components, including Itga8. ECM derived from Grhl3-deficient fibroblasts enhanced iCM induction, while Grhl3 depletion also reduced fibroblast activation and increased cellular plasticity. These effects synergized with TF (transcription factor)-mediated reprogramming to improve iCM efficiency, structural organization, and functional maturation. In vivo, removing Grhl3 enhanced fibroblast-to-cardiomyocyte conversion, reduced scar formation, and improved cardiac function after myocardial infarction. CONCLUSIONS: Our findings establish ECM adaptation as a critical determinant of cardiac reprogramming and identify Grhl3 as a promising therapeutic target to advance myocardial repair strategies.