Doxorubicin-induced myocardial cytotoxicity is associated with the downregulation of the Klotho-Nrf2 signaling pathway

Introduction: Doxorubicin (DOX), a widely used chemotherapeutic agent, induces dose-dependent cardiotoxicity through mechanisms involving oxidative stress and inflammatory cell death. This study investigates the role of the Klotho-Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) signaling axis in mitigating DOX-induced pyroptosis in cardiomyocytes. Methods: AC16 cells were treated with DOX to induce pyroptosis. Klotho was overexpressed or knocked down via plasmids and small interfering RNA (siRNA). Cell viability, Lactate dehydrogenase (LDH) release, Reactive oxygen species (ROS)/ Malondialdehyde (MDA)/ Glutathione (GSH) levels, and pyroptosis markers were measured. Methylation-specific polymerase chain reaction (PCR) and methylation-sensitive high-resolution melting (MS-HRM) analyzed Klotho promoter methylation and DNA methyltransferase (DNMT) binding, while quantitative real-time polymerase chain reaction (qRT-PCR) and luciferase assays assessed gene expression and Nrf2 activity. DNA methyltransferase 3a (DNMT3a) and DNMT3b were silenced with specific small interfering RNAs (siRNAs) to explore their epigenetic roles. Results: We demonstrate that DOX triggers pyroptosis via caspase-1 activation, Gasdermin D N-terminal fragment (GSDMD-N), Interleukin-1β (IL-1β) and Interleukin-18 (IL-18) secretion, accompanied by an increase in ROS and depletion of GSH and antioxidant enzymes. Pyroptosis was ROS-dependent, as pretreatment with N-Acetyl-L-cysteine (NAC) restored redox balance and suppressed cell death. Mechanistically, DOX downregulated Klotho expression via promoter Cytosine-phosphate-Guanine (CpG) hypermethylation mediated by DNA methyltransferases DNMT3a and DNMT3b, with DNMT3b playing a dominant role. Klotho suppression exacerbated pyroptosis, while its overexpression reducing Apoptosis-Associated Speck-Like Protein Containing a CARD (ASC) speck formation. Furthermore, Klotho activated the Nrf2 pathway, restoring nuclear Nrf2 levels and upregulating antioxidant genes Heme Oxygenase-1 (HO-1) and NAD(P)H Quinone Oxidoreductase 1 (NQO1). Critically, the Nrf2 inhibitor ML385 reversed Klotho’s protective effects, confirming Nrf2’s necessity. Silencing of DNMT3a/3b attenuated DOX-induced Klotho promoter hypermethylation, restored Klotho expression, and inhibited pyroptosis, with DNMT3b siRNA exerting a more pronounced protective effect. Conclusion: These findings reveal that DOX induces myocardial cytotoxicity by epigenetically silencing Klotho via DNMT3a/3b-mediated promoter hypermethylation, thereby disrupting the Klotho-Nrf2 antioxidant axis and unleashing inflammasome-driven pyroptosis, with DNMT3b serving as the key epigenetic regulator in this process.