Exercise-Induced Epoxy-Eicosanoids Promote Cardiomyocyte Proliferation and Post-Infarction Repair via NR4A1

ABSTRACT BACKGROUND Myocardial infarction (MI) causes irreversible cardiomyocyte loss that exceeds the adult heart’s regenerative capacity, leading to adverse remodeling and heart failure. While exercise provides cardiovascular protection, the mechanisms underlying exercise-induced cardiac repair remain unclear. METHODS Plasma levels of free fatty acids and polyunsaturated fatty acid metabolites were quantified by liquid chromatography–tandem mass spectrometry (LC-MS/MS). RNA sequencing was performed to identify differentially expressed genes and enriched pathways. NR4A1 was knocked down in mice via tail vein injection of AAV9 carrying shRNA targeting NR4A1 (AAV9-shNR4A1). The role of 17,18-EEQ was evaluated by continuous subcutaneous infusion via osmotic minipump; the involvement of the cAMP–PKA–CREB axis was assessed using specific pharmacological inhibitor. Molecular analyses included ELISA, immunofluorescence staining, QPCR, and western blotting. Additionally, clinical samples were collected from 36 endurance-trained athletes and 30 sedentary individuals (age 18-22, male) for comparative analysis. RESULTS Here we demonstrate that preventive aerobic training promotes post-MI cardiomyocyte proliferation and confers cardioprotection by reducing fibrosis area and improving cardiac function. Targeted lipidomics revealed that exercise elevates circulating epoxyeicosatetraenoic acids (EEQs), particularly 17,18-EEQ; osmotic pump delivery of 17,18-EEQ mimicked the protective effects of exercise, reducing infarct burden and enhancing proliferation markers (Ki67, pH3, Aurora B). Transcriptomic analysis identified NR4A1 as the most prominent up-regulated mRNA under exercise induction; AAV9-mediated NR4A1 knockdown abolished exercise-mediated cardioprotection and proliferative benefits, establishing NR4A1 as essential for exercise-driven repair. Phosphoproteomic profiling revealed that 17,18-EEQ activates the PKA–CREB pathway to induce NR4A1 expression; pharmacological inhibition of adenylyl cyclase, PKA, or CREB phosphorylation reversed EEQ-mediated cardioprotection and proliferation, confirming pathway necessity. Dietary supplementation with eicosapentaenoic acid (EPA), the metabolic precursor of 17,18-EEQ, amplified exercise-induced cardioprotection by enhancing EEQ biosynthesis. CONCLUSION Our study identifies 17,18-EEQ as an exercise-derived exerkine that promotes cardiomyocyte proliferation and post-MI repair through a PKA–CREB–NR4A1 signaling axis, advancing our understanding of exercise cardioprotection and myocardial regeneration mechanisms while providing novel therapeutic targets for cardiac intervention. What IS New? Aerobic exercise enhances post-myocardial infarction cardiac repair by upregulating the cardio-protective lipid mediator 17,18-EEQ and its downstream effector NR4A1. 17,18-EEQ promotes cardiomyocyte proliferation and cardiac repair through activation of the cAMP–PKA–NR4A1 signaling axis. Combining aerobic exercise with omega-3 fatty acid supplementation synergistically improves cardiac remodeling and functional recovery after ischemic injury. What Are the Clinical Implications? Targeting endogenous cardiomyocyte proliferation represents a promising therapeutic strategy for cardiac regeneration after ischemic injury. Dietary supplementation with omega-3 fatty acids may be a clinically feasible approach to amplify exercise-induced cardioprotection and improve outcomes after myocardial infarction.