Sex-biased metabolic reprogramming is associated with AP-1–mediated cardiomyocyte remodeling in pediatric Tetralogy of Fallot

Background/Introduction Sex differences in congenital heart disease (CHD) outcomes are increasingly recognized, yet their myocardial basis remains unclear. Tetralogy of Fallot (TOF) induces chronic hypoxic stress in the right ventricular outflow tract (RVOT). Although surgical repair markedly improves survival in cyanotic CHD, myocardial structural and functional deterioration often develops before intervention and continues to affect postoperative recovery. Experimental and human transcriptomic studies suggest that female myocardium exhibits enhanced fatty acid oxidation and oxidative phosphorylation under physiological conditions. Hypoxia induces a metabolic shift towards increased glycolytic dependence in cardiomyocytes, raising the possibility that a pre-existing fatty acid preference may influence this adaptive response. Whether this sex-biased metabolic program influences stress-responsive cardiomyocyte remodeling in pediatric TOF remains unknown. Purpose To define sex-specific cardiomyocyte transcriptional states in pediatric TOF and to investigate the role of metabolic modulation under hypoxic stress. Methods RVOT tissue from pediatric TOF patients was analyzed by single-nucleus RNA sequencing. Neonatal rat cardiomyocytes (NRCMs) were subjected to hypoxia with pharmacological activation of fatty acid oxidation for mechanistic studies. Results Single-nucleus RNA sequencing identified five cardiomyocyte subpopulations. Among them, CM3 displayed high NPPA and NPPB expression and was enriched for hypoxia response and developmental remodeling pathways, consistent with a stress-activated phenotype. Notably, 86.8% of CM3 nuclei were derived from female patients, indicating a marked sex bias. Trajectory analysis positioned CM3 at the terminal stage of disease progression. Transcription factor analysis revealed selective activation of the Activator protein-1 (AP-1) network, including JUN, JUNB, and FOS, with downstream targets enriched in hypoxia and cardiac functional regulation pathways. Immunofluorescence confirmed increased p-c-Jun activation in female RVOT myocardium. In neonatal rat cardiomyocytes, activation of fatty acid oxidation using a peroxisome proliferator-activated receptor-α agonist under hypoxia (1% O2, 48 h) significantly increased p-c-Jun levels compared with normoxia or hypoxia alone, findings corroborated by immunofluorescence. Conclusion(s) Sex-biased metabolic reprogramming is associated with the emergence of a distinct AP-1–activated terminal cardiomyocyte state in pediatric TOF. Functional data support that enhanced fatty acid oxidation under hypoxic conditions amplifies AP-1 signaling, linking intrinsic metabolic bias to stress-responsive myocardial remodeling. These findings highlight AP-1 as a potential mediator of sex-dependent cardiac adaptation in CHD.