Rapamycin ameliorates doxorubicin-induced cardiomyopathy through suppression of RIPK1-independent necroptosis

Abstract

Abstract Background Although several strategies to mitigate doxorubicin-induced cardiomyopathy (DIC), such as early induction of neurohormonal regulators, have been attempted, there is no currently available therapy focusing on molecular mechanisms of DIC. Necroptosis is a novel type of regulated necrosis and has been implicated in the mechanisms of various diseases including heart failure. Activation of MLKL by its phosphorylation, leading to loss of plasma membrane integrity, is a hallmark of necroptosis, and RIPK1 activation is an upstream event in a canonical necroptotic pathway, whereas RIPK1-independent MLKL activation has been reported. There are no available necroptosis inhibitors in the clinical setting, but our experimental studies have shown that mTOR inhibitors suppress necroptosis in cardiomyocytes. Purpose This study aimed to determine the involvement of necroptosis and the effect of rapamycin in DIC. Methods and results C57BL/6N mice received intraperitoneal injection of doxorubicin (10 mg/kg, three times a week) and its vehicle to induce DIC. Rapamycin and necrostatin-1, a RIPK1 inhibitor, were intraperitoneally administered at 0.25 mg/kg and 6 mg/kg one hour before doxorubicin administration, respectively. Eight days after the commencement of doxorubicin injection, cardiac MRI analyses showed that left ventricular ejection fraction (LVEF) was significantly lower in the doxorubicin-treated mice than in the vehicle-treated mice (45.5±5.1% vs. 59.6±2.2%, p<0.05), indicating the development of DIC. The treatment of rapamycin significantly improved LVEF in DIC mice (65.4±4.2%, P<0.05). Kaplan-Meier survival curve analyses during a follow-up period of 12 days showed that the survival rate was significantly higher in the rapamycin-treated DIC mice than in vehicle-treated DIC mice (40% vs. 0%, p<0.001 by the log-rank test), but it was comparable in the necrostatin-1-treated DIC mice and the vehicle-treated DIC mice (both 0%). Quantitative immunohistochemical analyses showed that the level of phospho-Ser345-MLKL, an activated form of MLKL, was significantly higher by 1.5-fold in the doxorubicin-treated mice than in the vehicle-treated mice, which was reversed by the treatment with rapamycin, suggesting that rapamycin improves DIC through suppression of necroptosis. In the second series of experiments, the effect of doxorubicin on necroptosis was analyzed. H9c2 cardiomyoblasts received doxorubicin (5 µmol/L) or its vehicle for 24 h, and cell death was determined by LDH release (as % of total). LDH release was higher in the doxorubicin-treated cells than in the vehicle (45.5±6.7% vs. 8.4±2.2%), and doxorubicin-induced increase in LDH release was significantly attenuated by siRNA-mediated knockdown of MLKL (36.5±3.9%) but not by pretreatment with necrostatin-1 (50 nmol/L, 49.9±3.2%). Conclusion RIPK1-independent necroptosis is involved in the pathogenesis of DIC, and rapamycin prevents the development of DIC through the reduction in MLKL activity.