Peroxiredoxin Ⅲ safeguards cardiac function against doxorubicin by regulating mitochondrial quality control via H2O2 detoxification

Doxorubicin (DOX) is a widely used anticancer drug associated with severe cardiotoxicity primarily driven by mitochondrial dysfunction and reactive oxygen species (ROS) production. Hydrogen peroxide (H 2 O 2 ), a major mitochondrial ROS, significantly contributes to cardiotoxicity, yet its precise mechanistic role in DOX-induced cardiotoxicity remains incompletely understood. Here, we investigated the critical role of mitochondrial H 2 O 2 in DOX-induced cardiotoxicity using rat cardiomyocyte H9c2 cells with stable knockdown or reconstitution of mitochondrial antioxidant enzyme peroxiredoxin Ⅲ (PrxⅢ), which specifically regulates mitochondrial H 2 O 2 levels. Our results demonstrated that severe mitochondrial H 2 O 2 accumulation (>10-fold compared to control) in PrxⅢ-depleted cells exacerbated mitochondrial oxidative stress, evidenced by increased cardiolipin oxidation and mitochondrial membrane potential dissipation. Furthermore, excessive mitochondrial H 2 O 2 impaired mitochondrial fusion by reducing fusion-related protein expression, disrupted autophagic flux via lysosomal dysfunction, and significantly attenuated mitophagy, ultimately leading to enhanced apoptosis. Conversely, moderate mitochondrial H 2 O 2 levels (5- to 8-fold increase compared to control) observed in PrxⅢ-expressing cells promoted mitochondrial elongation, enhanced mitophagy, and preserved autophagic flux, suggesting a protective adaptation against DOX-induced oxidative stress. In vivo experiments using PrxⅢ knockout mice confirmed that loss of PrxⅢ aggravated DOX-induced cardiac dysfunction. Bioinformatic analysis of independent public transcriptome datasets and targeted qPCR validation in PrxⅢ-deficient cardiac tissues further confirmed that the mitochondrial quality control pathways identified in vitro are robustly dysregulated in vivo . Additionally, PrxⅢ deficiency markedly increased mitochondrial structural damage without significantly affecting cardiac fibrosis or hypertrophy. Notably, mitigating the mitochondrial H 2 O 2 burden and protecting the mitochondrial inner membrane using the mitochondria-targeted antioxidant peptide SS-31 successfully rescued this exacerbated cardiac dysfunction. In conclusion, our findings establish mitochondrial H 2 O 2 as a pivotal determinant in DOX-induced mitochondrial dysfunction and cardiotoxicity, highlighting PrxⅢ as a promising therapeutic target for mitigating oxidative cardiac injury. In the presence of PrxⅢ, DOX-induced moderate mitochondrial H 2 O 2 activates adaptive mitochondrial quality control responses, including mitochondrial fusion, enhanced mitophagy, functional autophagic flux, and mitochondrial biogenesis, thereby preserving cardiomyocyte homeostasis. In PrxⅢ-deficient cardiomyocytes, excessive mitochondrial H 2 O 2 accumulation causes cardiolipin oxidation, impairs mitochondrial fusion, attenuates mitophagy, blocks autophagic flux through lysosomal dysfunction, and impairs mitochondrial biogenesis, collectively leading to mitochondrial quality control collapse, enhanced apoptosis, and severe cardiotoxicity. • Doxorubicin increases mitochondrial H 2 O 2 (mtH 2 O 2 ) to cause cardiomyocyte toxicity. • Peroxiredoxin III prevents oxidative damage by doxorubicin in cardiomyocytes. • Moderate mtH 2 O 2 promotes adaptive MQC; excessive mtH 2 O 2 causes MQC collapse. • Excessive mtH 2 O 2 blocks autophagic flux and mitophagy via lysosomal dysfunction. • SS-31 rescues DOX-induced cardiac dysfunction in PrxⅢ-deficient mice in vivo.