Inhibition of NETs prevents doxorubicin-induced cardiotoxicity by attenuating IL-18-IFN-γ-Cx43 axis induced cardiac conduction abnormalities.

Doxorubicin-induced cardiotoxicity (DIC) is one of the most severe side effects of doxorubicin, yet the underlying mechanisms remain incompletely understood. Our results showed that Neutrophil extracellular traps (NETs) accumulated in plasma and cardiac tissue after doxorubicin treatment. The inhibition of NETs formation by Pad4 gene ablation significantly attenuated doxorubicin-induced arrhythmia, prolonged survival time and reduced the levels of Troponin T (cTnT) and creatine kinase MB (CK-MB) in mice. In addition, reductions in left ventricular fractional shortening and ejection fraction induced by doxorubicin were more severe in WT mice than in Pad4 mice. Immunostaining and qPCR analyses revealed that NETs activated macrophages to release pro-inflammatory cytokines such as IL-18, IL-1β, and TNF-α. IL-18, in turn, activated T cells to produce IFN-γ, which, along with TNF-α, downregulated the expression of Cx43, thereby inducing cardiac conduction abnormalities. We identify that IL-18-IFN-γ-Cx43-induced cardiac conduction abnormalities triggered by neutrophil extracellular traps is the key molecular and cellular determinants of DIC. Furthermore, targeting NETs formation using ozone therapy significantly alleviated DIC. This study highlights the critical role of NETs in the development of DIC and proposes ozone therapy as a potential therapeutic strategy for treating DIC.