Nuclear accumulation of MLKL induces necroptosis in cardiomyocytes: potential implication in Doxorubicin-induced cardiotoxicity

Abstract

Abstract Background The treatment with doxorubicin, a powerful chemotherapeutic agent, has been shown to be associated with an increased risk of lethal heart failure. Although various types of cell death pathway such as apoptosis and ferroptosis have been shown to be involved in the development of doxorubicin-induced cardiotoxicity, DIC, the involvement of necroptosis, a novel programmed necrosis induced by translocation of activated mixed lineage kinase domain-like protein, MLKL, to plasma membrane, remains unclear. Purpose The aim of this study was to determine whether necroptosis is involved in the development of DIC. Methods and results DIC was induced in C57BL/6J mice by intraperitoneal injection of doxorubicin at a dose of 10 mg/kg 3 times for a week. Eight days after the commencement of injection, echocardiographic analyses showed that left ventricular ejection fraction assessed by echocardiography was significantly lower in the doxorubicin-treated mice than in the vehicle-treated mice (44.0±13.7 vs. 70.5±3.7%), indicating the development of DIC. Immunoblot analysis showed that MLKL protein level was higher by 1.6 fold in the doxorubicin-treated mice than in the vehicle-treated mice. Interestingly, immunohistochemical analysis showed that signals of phospho-Ser345-MLKL, an activated form of MLKL, was found in the nuclei in addition to cytosol and intercalated discs of cardiomyocytes in the doxorubicin-treated mice. To get novel insight into significance of nuclear MLKL accumulation, a leucine-rich nuclear export signal (NES) spanning amino acids 280–284 of rat MLKL was identified by site-directed mutation analyses, and H9c2 cells, cultured rat cardiomyoblasts, were transfected with expression constructs for nucleus-directed MLKL (FLAG-mtNES-MLKL) or its wild type (FLAG-WT-MLKL). Percentage of FLAG-positive cells stained with Zombie Red, a fluorescent dye that is non-permeant to live cells, was higher in FLAG-mtNES-MLKL-transfected cells than in FLAG-WT-MLKL-transfected cells (80.0±3.5% vs. 6.3±1.3%, p<0.05), whereas percentage of cells immunostained with cleaved caspase-3 to FLAG-positive cells was similar in the two groups. The effect of the MLKL mutant on necroptosis was attenuated by treatment with GppNHp, an inhibitor of Ran-mediated nuclear protein import. Conclusion Nuclear accumulation of MLKL induces necroptosis in cardiomyocytes, which may contribute to progression of DIC. Funding Acknowledgement Type of funding sources: None.