Indoxyl Sulfate Induces Ferroptosis via the AhR/ACSL4 Axis to Mediate Uremic Cardiomyopathy

Background: Uremic cardiomyopathy is a leading cause of cardiovascular death in chronic kidney disease (CKD), yet the cell death mechanisms linking uremic toxins to myocardial injury remain unclear. Indoxyl sulfate (IS), a protein-bound uremic toxin and endogenous aryl hydrocarbon receptor (AhR) ligand, has been implicated in cardiotoxicity; however, whether it induces cardiomyocyte ferroptosis through a defined signaling pathway remains to be investigated. This study aimed to determine whether IS induces cardiomyocyte ferroptosis via an AhR/Acyl-CoA synthetase long-chain family member 4 (ACSL4) signaling axis and to evaluate its contribution to uremic cardiomyopathy. Methods: A 5/6 nephrectomy CKD mouse model was used to characterize myocardial ferroptosis in the uremic setting. H9c2 cardiomyocytes were treated with IS, with or without the AhR antagonist CH-223191, ferrostatin-1, or ACSL4-targeting siRNA, to dissect the upstream signaling mechanism. An IS injection model combined with AAV9-mediated cardiac-targeted ACSL4 knockdown was employed to evaluate ACSL4's role in vivo. Results: CKD mice exhibited elevated serum IS alongside myocardial ferroptosis signatures and cardiac remodeling (all p < 0.001). IS promoted AhR nuclear translocation and ACSL4 upregulation in cardiomyocytes (p < 0.001), both of which were attenuated by CH-223191 (p < 0.001). Bioinformatic analysis identified multiple xenobiotic response element motifs in the ACSL4 promoter. ACSL4 silencing or ferrostatin-1 attenuated IS-induced lipid peroxidation and rescued cell viability in vitro (all p < 0.001). Cardiac-targeted ACSL4 knockdown alleviated IS-induced hypertrophy, fibrosis, and myocardial ferroptosis in vivo without altering systemic IS levels (all p < 0.001). Conclusion: Using complementary CKD and direct IS exposure models, we demonstrate that IS exposure is sufficient to induce cardiomyocyte ferroptosis in association with AhR-dependent upregulation of ACSL4, and that cardiac-targeted ACSL4 knockdown attenuates IS-induced cardiac injury in vivo. The AhR/ACSL4/ferroptosis axis represents a potential pathogenic mechanism in uremic cardiomyopathy and a candidate therapeutic target.