Nucleophosmin 1 mediates cell death through the cuproptosis pathway in cobalt chloride-treated H9C2 cells

Myocardial ischemia remains a leading cause of mortality worldwide, yet the molecular mechanisms underlying ischemia-induced cardiomyocyte death are incompletely understood. Here, we investigated whether nucleophosmin 1 (NPM1) regulates cuproptosis, a newly identified form of copper-dependent cell death, in the setting of ischemic injury. Using a CoCl₂-induced H9C2 cardiomyocyte ischemia model, we found that CoCl₂ triggered hallmark features of cuproptosis, including loss of Fe-S cluster proteins (FDX1, DLAT, and DLST), reduced lipoylation of DLAT and DLST, and enhanced DLAT oligomerization, accompanied by impaired cell viability, all of which were reversed by the cuproptosis inhibitor TTM. ES-Cu, a copper ionophore, similarly induced dose-dependent cuproptosis and cytotoxicity. Protein-protein interaction analysis suggested associations between NPM1 and key regulators of cuproptosis. Functionally, siRNA-mediated NPM1 knockdown attenuated CoCl₂-induced cuproptotic alterations and rescued cell viability, whereas NPM1 overexpression exacerbated these effects. Collectively, these loss- and gain-of-function studies establish NPM1 as a positive regulator of cuproptosis in myocardial ischemia. Our findings uncover a previously unrecognized role for NPM1 in mediating ischemic cardiomyocyte death via the cuproptosis pathway and highlight the NPM1-cuproptosis axis as a potential therapeutic target for ischemic heart disease.