Blocking Nuclear Receptor Nr4a3 Unlocks the Senescence Barrier to Promote Direct Cardiac Reprogramming

Direct reprogramming of scar-forming fibroblasts into induced cardiomyocytes (iCMs) offers a regenerative strategy to repair injured myocardium. However, reprogramming efficiency remains low in fibroblasts from adult and aged hearts, and the molecular barriers underlying this resistance remain poorly understood. Here, we used transcriptomic and epigenetic profiling to uncover cellular senescence as a key obstacle limiting fibroblast plasticity and cardiogenic conversion. Fibroblasts from post-neonatal stages exhibited impaired activation of cardiac gene programs and persistent expression of fibrotic and inflammatory signatures. A loss of function screen identified Nr4a3 as a key repressor of cardiac reprogramming, particularly in senescent and aged cardiac fibroblasts (CFs). Nr4a3 overexpression promoted senescence and suppressed iCM reprogramming, whereas Nr4a3 knockdown significantly enhanced iCM induction from both murine and human senescent CFs. Mechanistically, Nr4a3 depletion remodeled the chromatin landscape by shifting it from a fibrotic and inflammatory state to a regenerative cardiac program. We further identified Cxcl14, a senescence-associated secretory phenotype (SASP) factor upregulated by Nr4a3, as a key downstream effector. Blocking Cxcl14 restored reprogramming in otherwise refractory fibroblasts. In vivo, Nr4a3 knockdown enhanced reprogramming-based improvement of heart function following myocardial infarction. These findings demonstrated that cellular senescence is a major barrier to direct cardiac reprogramming and identified Nr4a3 as a central regulator of this block. Targeting Nr4a3 and its downstream effectors may represent a promising therapeutic avenue to enhance cardiac regeneration in aged individuals following injury. Overall design: To determine the molecular basis by which knockdown Nr4a3 enhances iCM reprogramming, we conducted RNA-seq using shNr4a3-CFs and shNT-CFs collected at day 7 of treatment.