Blocking nuclear receptor Nr4a3 unlocks the senescence barrier to promote direct cardiac reprogramming

Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) offers a regenerative strategy for heart repair, but efficiency declines in adult and aged cells. Transcriptomic and epigenetic profiling identified cellular senescence as a major barrier limiting cardiac fibroblast (CF) plasticity and cardiogenic conversion. Postneonatal fibroblasts exhibited impaired activation of cardiac gene programs and persistent expression of fibrotic and inflammatory signatures. A loss-of-function screen identified Nr4a3 as a central repressor. Nr4a3 overexpression promoted senescence and suppressed iCM induction, whereas knockdown enhanced reprogramming in murine and human senescent CFs. Mechanistically, Nr4a3 depletion remodeled the chromatin landscape from a fibrotic and inflammatory state to a regenerative cardiac program. Blocking downstream Cxcl14 restored reprogramming in refractory fibroblasts. In vivo, Nr4a3 knockdown improved heart function following myocardial infarction. These findings established cellular senescence as a major barrier to cardiac reprogramming and identified Nr4a3 and its effectors as potential targets to enhance heart regeneration.