Nuclear receptor coactivator 4 -mediated ferritinophagy is involved in LPS-induced ferroptosis in cardiomyocytes

Abstract

Objective To investigate whether ferritin autophagy mediated by nuclear receptor coactivator 4 (NCOA4) is involved in lipopolysaccharide (LPS)-induced ferroptosis in septic cardiomyocytes. Methods Different doses of LPS (5, 10 and 20 μg/mL) were adopted to induce rat cardiomyocytes H9c2 to form an in vitro model of cardiomyocyte injury, and normal H9c2 cells were used as control group. The cell viability, Fe2+ level, releases of myocardial enzymes and oxidative stress level were detected by corresponding reagent kits, and the change in mitochondrial membrane potential was measured with fluorescent probe. Immunofluorescence assay was performed to determine the expression and localization of NCOA4 and ferritin in the cardiomyocytes. Moreover, small interfering RNA (siRNA) was used to reduce NCOA4 expression in H9c2 cells, and the levels of ferroptosis regulatory proteins as well as autophagy proteins were detected with Western blotting. Results After stimulation with different doses of LPS, the viability of H9c2 cells was decreased significantly (P < 0.05), the concentration of Fe2+ was increased (P < 0.05), the levels of oxidative stress products such as malondialdehyde (MDA) and reactive oxygen species (ROS) were both elevated (P < 0.05), and the mitochondrial membrane potential was declined (P < 0.05). Increased expression of NCOA4-Ferritin colocalization was observed under fluoroscopy (P < 0.05). Western blotting showed that the expression levels of ferroptosis regulatory protein NCOA4 and autophagy protein LC3-Ⅱwere up-regulated (P < 0.05), while those of ferritin, SLC7A11 and GPX4 were down-regulated (P < 0.05). NCOA4 knockdown resulted in significantly enhanced expression of ferritin, SLC7A11 and GPx4 (P < 0.05), whereas reduced expression of LC3-Ⅱ (P < 0.05). Conclusion NCOA44-mediated ferritin autophagy is involved in the occurrence of ferroptosis in septic cardiomyocytes.

Keywords

• ferritinophagy
• ferroptosis
• sepsis
• cardiomyocyte injury