Epicardial adipose tissue-derived kynurenic acid induces atrial lymphatic dysfunction in atrial fibrillation

Background The cardiac lymphatic system maintains myocardial fluid and immune cell homeostasis by draining excess fluid and pro-inflammatory immune cells. We have recently reported that an epicardial adipose tissue (EAT)-derived factor induces inflammation and fibrosis in the atrial myocardium, leading to atrial fibrillation (AFib). However, the impact of specific EAT-derived metabolites on cardiac lymphatic endothelial cells (LECs) remains unknown. Purpose To identify and validate key EAT-derived metabolites that mediate cardiac lymphatic dysfunction, a potential contributor to AFib. Methods Human EAT samples were collected from 16 patients with or without AFib undergoing cardiovascular surgery. Subjects were categorized into the No-AFib group (n=8, 72 ± 9.7 years) and the AFib group (n=8, 74 ± 8.1 years). Stromal vascular fractions (SVF) were extracted from the EAT and cultured to confluence. Following differentiation into adipocytes, the cell culture media (No-AFib or AFib EAT-conditioned media) was collected and analyzed by LC-TOF/MS. Subsequently, kynurenic acid was administered to human LECs. Results LC-TOF/MS analysis revealed significantly higher levels of kynurenic acid in AFib EAT-conditioned media compared to No-AFib EAT-conditioned media (p=0.0015). To elucidate the effect of kynurenic acid on LECs, we assessed endothelial barrier function using FITC-dextran permeability assays and evaluated tube-forming ability. Kynurenic acid-treated LECs exhibited significantly increased permeability, indicating impaired barrier function, and a marked reduction in tube formation (p=0.0038). Furthermore, RT-PCR analysis demonstrated that kynurenic acid-treated LECs showed a significant increase in the mRNA expression of the mesenchymal marker TAGLN (p=0.0019), whereas the expression of lymphatic endothelial markers LYVE1 (p<0.0001) and PROX1 (p<0.0001) was significantly decreased. Additionally, kynurenic acid treatment markedly reduced the mRNA levels of mitochondrially encoded genes, including CYTB (p=0.0075), COX1 (p=0.0040), COX3 (p=0.0002), ND4 (p=0.0124), ATP6 (p<0.0001), and ATP8 (p=0.0002). Metabolomic analysis using GC/MS revealed that kynurenic acid treatment led to decreased intracellular levels of branched-chain amino acids (valine; p<0.0001, leucine; p=0.0002, and isoleucine; p<0.0001) and TCA cycle metabolites (citrate; p<0.0001, cis-aconitate; p=0.0002, isocitrate; p<0.0001, succinate; p=0.0016, fumarate; p=0.0018, and malate; p=0.0015) in LECs. These data suggest that kynurenic acid induces endothelial-to-mesenchymal transition (EndMT) and disrupts energy metabolism in LECs. Conclusion Our study demonstrates that EAT-secreted kynurenic acid impairs lymphatic function via the induction of EndMT and mitochondrial energy dysfunction. Targeting EAT-derived atrial lymphatic dysfunction may offer a novel therapeutic approach for atrial fibrillation.