Abstract

Ferroptosis is a major cause of cardiotoxicity induced by doxorubicin (DOX). Previous studies have shown that hydrogen sulfide (H 2 S) inhibits ferroptosis in cardiomyocytes and myoblasts, but the underlying mechanism has not been fully elucidated. In this study, we investigated the role of H 2 S in protecting against DOX-induced cardiotoxicity both in vivo and in vitro, and elucidated the potential mechanisms involved. We found that DOX downregulated the expression of glutathione peroxidase 4 (GPX4) and NFS1, and upregulated the expression of acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) expression level, resulting in increased lipid peroxidation and ferroptosis. Additionally, DOX inhibited MFN2 expression and increased DRP1 and FIS1 expression, leading to abnormal mitochondrial structure and function. In contrast, exogenous H 2 S inhibited DOX-induced ferroptosis by restoring GPX4 and NFS1 expression, and reducing lipid peroxidation in H9C2 cells. This effect was similar to that of the ferroptosis antagonist ferrostatin-1 (Fer-1) in protecting against DOX-induced cardiotoxicity. We further demonstrated that the protective effect of H 2 S was mediated by the key mitochondrial membrane protein optic atrophy 3 (OPA3), which was downregulated by DOX and restored by exogenous H 2 S. Overexpression of OPA3 alleviated DOX-induced mitochondrial dysfunction and ferroptosis both in vivo and in vitro. Mechanistically, NFS1 has an inhibitory effect on ferroptosis, and NFS1 deficiency increases the susceptibility of cardiomyocytes to ferroptosis. OPA3 is involved in the regulation of ferroptosis by interacting with NFS1. Post-translationally, DOX promoted OPA3 ubiquitination , while exogenous H 2 S antagonized OPA3 ubiquitination by promoting OPA3 s-sulfhydration. In summary, our findings suggested that H 2 S protects against DOX-induced cardiotoxicity by inhibiting ferroptosis via targeting the OPA3–NFS1 axis. This provides a potential therapeut