Dual-Target Cardiac Regeneration: Controllable Nfyb/Nr3c1 Cointervention Spurs Cardiomyocyte Cytokinesis After Myocardial Infarction

BACKGROUND: Myocardial infarction can cause a massive loss of functional cardiomyocytes, yet effective strategies to stimulate cardiac regeneration remain lacking. A key barrier to adult cardiomyocyte proliferation appears to be cytokinesis inhibition. This study aimed to determine whether combining proliferation stimulators with the removal of cytokinesis-inhibitory constraints could unlock regenerative potential after myocardial infarction. METHODS: Transcriptomic profile from 5 regeneration models, including Aurkb -tdTomato cytokinesis reporter mice and Myh6 -MerCreMer;mosaic analysis with double markers (MADM), and cross-species comparative analysis were used to explore the regulatory networks for cardiomyocyte proliferation. MADM mice were used to evaluate cardiac regeneration by quantifying after cytokinesis new cardiomyocytes and clonal clusters. RESULTS: Integrative multimodel analysis revealed a dual regulatory control system for cardiomyocyte proliferation, along with key associated genes and transcriptional regulators. Nfyb was identified as an activator and Nr3c1 as a repressor of cardiomyocyte proliferation. Nfyb overexpression enhanced cardiomyocyte proliferation and post–myocardial infarction cardiac repair through propelling cell-cycle gene transcription. Nr3c1 inhibition promoted cardiomyocyte proliferation, improved cardiac function, and reduced infarct size. A spatiotemporally controlled adeno-associated virus 9 system combining drug-inducible Nfyb overexpression and CRISPR/enOsCas12f1–mediated Nr3c1 deletion efficiently induces cardiomyocyte proliferation. Clonal analysis using MADM mice showed that the dual intervention synergistically increased new cardiomyocyte formation (28% clustered, >28-fold versus control). The enhanced regenerative effect of the dual intervention was demonstrated in post–myocardial infarction mice and human engineered heart tissues. CONCLUSIONS: This work documents a dual-control paradigm of cardiomyocyte proliferation and establishes Nfyb / Nr3c1 cointervention as a putative new therapy to induce and control cardiac regeneration after injury.