Overexpression of MFN2 alleviates sorafenib-induced cardiomyocyte necroptosis via the MAM-CaMKIIδ pathway in vitro and in vivo.
Overexpression of MFN2 alleviates sorafenib-induced cardiomyocyte necroptosis via the MAM-CaMKIIδ pathway in vitro and in vivo.
复制标题
MFN2 的过度表达通过体外和体内 MAM-CaMKIIδ 途径减轻索拉非尼诱导的心肌细胞坏死性凋亡
作者：
Song Z;Song H;Liu D;Yan B;Wang D;Zhang Y;Zhao X;Tian X;Yan C;Han Y
背景：肿瘤治疗的持续成功依赖于与治疗相关的不良事件的缓解，特别是心血管毒性。因此，有必要了解抗肿瘤治疗过程中药物毒性的基本机制。本研究的目的是阐明索拉非尼(SOR)诱导心肌细胞损伤的潜在机制。方法：原代培养小鼠心肌细胞，用SOR和其他各种处理方法处理。用流式细胞仪、Western blotting和CCK8检测心肌细胞坏死性下垂。用Rhod-2探针共聚焦成像检测线粒体钙摄取。用透射电子显微镜和共聚焦显微镜观察线粒体和线粒体相关内质网(ER)膜的形态变化。通过心脏灌流检测Mfn2过表达在体内的心脏特异性作用。结果：我们报道了线粒体钙超载，随后钙调素依赖的蛋白激酶II增量(CaMKIIδ)的增加和RIP3/MLKL级联激活，参与了SOR诱导的心脏坏死性下垂。MAM的过度形成和内质网与线粒体的紧密接触是SOR诱导的钙超载的重要机制。SOR以浓度依赖的方式介导Mfn2下调。此外，我们还发现，心肌细胞中丝裂原蛋白-2(Mfn2)水平的降低增加了SOR介导的MAM生物合成，并增加了线粒体-MAM的连接。SOR诱导哺乳动物雷帕霉素靶标(MTOR)失活，随后转录因子EB(TFEB)激活和核转位，导致有丝分裂和Mfn2降解。在体内模型中，接受SOR治疗的小鼠出现心功能障碍、自噬激活和坏死性下垂；我们的研究发现，Mfn2的全局和心脏特异性过表达抑制了心功能障碍，并通过抑制MAM-CaMKIIδ-RIP3/MLKL途径诱导心肌细胞坏死性下垂。结论：索拉非尼在体内外通过Mfn2-MAM-钙-CaMKIIδ通路介导心肌细胞坏死性下垂。Mfn2的过表达可以挽救SOR诱导的心肌细胞坏死性下垂，而不干扰其抗肿瘤作用。
Background: The continued success of oncological therapeutics is dependent on the mitigation of treatment-related adverse events, particularly cardiovascular toxicities. As such, there is an important need to understand the basic mechanisms of drug toxicities in the process of antitumor therapy. Our aim in this study was to elucidate the underlying mechanisms of sorafenib (sor)-induced cardiomyocyte damage. Methods: Primary mouse cardiomyocytes were prepared and treated with sor and various other treatments. Cardiomyocyte necroptosis was detected by flow cytometry, western blotting, and CCK8 assays. Mitochondrial Ca2+ uptake was detected by the Rhod-2 probe using confocal imaging. Morphological changes in mitochondria and mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) were imaged using transmission electron microscopy (TEM) and confocal microscopy. Cardiac perfusion was performed to detect cardiac specific role of MFN2 overexpression in vivo. Results: We reported that mitochondrial Ca2+ overload, the subsequent increase in calmodulin-dependent protein kinase II delta (CaMKIIδ) and RIP3/MLKL cascade activation, contributed to sor-induced cardiac necroptosis. Excess MAM formation and close ER-mitochondria contact were key pathogenesis of sor-induced Ca2+ overload. Sor mediated MFN2 downregulation in a concentration-dependent manner. Furthermore, we found that reduced mitofusin-2 (MFN2) level augmented sor-mediated elevated MAM biogenesis and increased mitochondria-MAM tethering in cardiomyocytes. Sor-induced Mammalian Target of Rapamycin (mTOR) inactivation, followed by the activation and nuclear translocation of Transcription Factor EB (TFEB), contributed to mitophagy and MFN2 degradation. In an in vivo model, mice subjected to sor administration developed cardiac dysfunction, autophagy activation and necroptosis; our investigation found that global and cardiac-specific overexpression of MFN2 repressed cardiac dysfunction, and sor-induced cardiomyocyte necroptosis via repressing the MAM-CaMKIIδ-RIP3/MLKL pathway. Conclusion: Sorafenib mediated cardiomyocyte necroptosis through the MFN2-MAM-Ca2+-CaMKIIδ pathway in vitro and in vivo. The overexpression of MFN2 could rescue sor-induced cardiomyocyte necroptosis without disturbing the anti-tumor effects.
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