Proteasomal Degradation of TRAF2 Mediates Mitochondrial Dysfunction in Doxorubicin-Cardiomyopathy.
Proteasomal Degradation of TRAF2 Mediates Mitochondrial Dysfunction in Doxorubicin-Cardiomyopathy.
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DOI：
10.1161/circulationaha.121.058411
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发表时间：
2022-09-20
期刊：
影响因子：
37.8
通讯作者：
Kirshenbaum, Lorrie A.
中科院分区：
文献类型：
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作者：
Dhingra, Rimpy;Rabinovich-Nikitin, Inna;Rothman, Sonny;Guberman, Matthew;Gang, Hongying;Margulets, Victoria;Jassal, Davinder S.;Alagarsamy, Keshav N.;Dhingra, Sanjiv;Ripoll, Carla Valenzuela;Billia, Filio;Diwan, Abhinav;Javaheri, Ali;Kirshenbaum, Lorrie A.
关键词：
细胞因子如TNFα与多柔比星（DOX）相关的心功能障碍和毒性有关。虽然TNFα可以引起不同的细胞反应，包括存活或死亡，但心脏中这些不同结果的潜在机制仍然是神秘的。E3泛素连接酶TRAF 2为K63连接的RIPK 1多聚泛素化提供了一个关键的信号平台，这对TNFα激活NF-κB和存活至关重要。TNFα-TRAF 2-NF-κB信号的改变是否是DOX心脏毒性作用的基础，目前仍知之甚少。探讨TRAF 2信号通路在阿霉素心脏毒性发病机制中的作用。使用体内（在AAV 9-GFP和AAV 9-TRAF 2小鼠（C57/BL 6 J）的心肌细胞限制性表达中每周4次注射DOX（5 mg/kg/周））和体外方法（大鼠、小鼠和人iPSC衍生的心肌细胞）的组合，我们监测TNFα水平、LDH、心脏超微结构和功能、线粒体生物能量学和心肌细胞活力。与溶剂处理的小鼠相反，在DOX处理的小鼠心脏中观察到超微结构缺陷，包括细胞质肿胀、线粒体扰动和TNFα水平升高。在研究TNFα在DOX心脏毒性中的作用时，我们发现在没有DOX的情况下，NF-κB很容易被TNFα激活。然而，TNFα介导的NF-κB活化在DOX处理的心肌细胞中受损。这与RIPK 1的K63连接的多聚泛素化的丧失相一致，这归因于TRAF 2的蛋白酶体降解。此外，TRAF 2蛋白丰度在用DOX治疗的癌症患者的心脏中显著降低。受损的TRAF 2信号转导导致Bnip 3的激活和线粒体扰动，包括生物能学破坏、膜电位损失和渗透性转换孔开放。我们进一步确定，泛素化和去泛素化酶c-IAP 1和USP 19的相互作用分别调节DOX处理的心肌细胞中TRAF 2的蛋白酶体降解。重要的是，c-IAP 1的E3连接酶突变体（c-IAP 1 H588 A）或USP 19功能的获得阻止了TRAF 2的蛋白酶体降解和DOX诱导的细胞死亡。此外，野生型TRAF 2而不是RING指突变体，其在RIPK 1的K63连接的多聚泛素化中有缺陷，恢复了NF-κB信号传导并抑制了DOX诱导的心脏细胞死亡。最后，心肌细胞限制性表达TRAF 2（AAV 9-TRAF 2）在体内保护免受DOX诱导的线粒体缺陷和心脏功能障碍。我们的研究结果揭示了一种新的信号传导轴，它在功能上将DOX的心脏毒性作用与TRAF 2的蛋白酶体降解联系起来。DOX破坏关键TRAF 2存活途径，使心肌细胞对TNFα和Bnip 3介导的坏死性细胞死亡敏感。
Cytokines such as TNFα have been implicated in cardiac dysfunction and toxicity associated with doxorubicin (DOX). While TNFα can elicit different cellular responses including survival or death, the mechanisms underlying these divergent outcomes in the heart remains cryptic. The E3 ubiquitin ligase TRAF2 provides a critical signaling platform for K63 - linked polyubiquitination of RIPK1, crucial for NF-κB activation by TNFα and survival. Whether alterations in TNFα-TRAF2-NF-κB signaling underlie the cardiotoxic effects of DOX, remains poorly understood. To investigate TRAF2 signaling in the pathogenesis of DOX cardiotoxicity. Using a combination of in vivo (4 weekly injections of DOX (5mg/kg/week) in cardiac-myocyte restricted expression of AAV9-GFP and AAV9-TRAF2 mice (C57/BL6J), and in vitro approaches (rat, mouse and human iPSCs derived cardiac myocytes), we monitored TNFα levels, LDH, cardiac ultrastructure and function, mitochondrial bioenergetics and cardiac cell viability. In contrast to vehicle treated mice, ultrastructural defects including cytoplasmic swelling, mitochondrial perturbations, and elevated TNFα levels were observed in the hearts of mice treated with DOX. While investigating the involvement of TNFα in DOX cardiotoxicity, we discovered that in the absence of DOX, NF-κB was readily activated by TNFα. However, TNFα -mediated NF-κB activation was impaired in cardiac myocytes treated with DOX. This coincided with loss of K63- linked poly-ubiquitination of RIPK1, attributed to the proteasomal degradation of TRAF2. Further, TRAF2 protein abundance was markedly reduced in hearts of cancer patients treated with DOX. Impaired TRAF2 signaling resulted in the activation of Bnip3 and mitochondrial perturbations, including disrupted bioenergetics, loss of membrane potential and permeability transition pore opening. We further established that the reciprocal actions of the ubiquitinating and de-ubiquitinating enzymes c-IAP1 and USP19 respectively regulated the proteasomal degradation of TRAF2 in DOX treated cardiac myocytes. Importantly, an E3 ligase mutant of c-IAP1(c-IAP1 H588A) or gain of function of USP19, prevented proteasomal degradation of TRAF2 and DOX -induced cell death. Further, wild type TRAF2 but not a RING finger mutant defective for K63 linked polyubiquitination of RIPK1, restored NF-κB signaling and suppressed DOX-induced cardiac cell death. Finally, cardiomyocyte-restricted expression of TRAF2 (AAV9-TRAF2) in vivo protected against mitochondrial defects and cardiac dysfunction induced by DOX. Our findings reveal a novel signaling axis that functionally connects the cardiotoxic effects of DOX to proteasomal degradation of TRAF2. Disruption of the critical TRAF2 survival pathway by DOX, sensitizes cardiac myocytes to TNFα and Bnip3 mediated necrotic cell death.