Background:Cytokines such as tumor necrosis factor-α (TNFα) have been implicated in cardiac dysfunction and toxicity associated with doxorubicin (DOX). Although TNFα can elicit different cellular responses, including survival or death, the mechanisms underlying these divergent outcomes in the heart remain cryptic. The E3 ubiquitin ligase TRAF2 (TNF receptor associated factor 2) provides a critical signaling platform for K63-linked polyubiquitination of RIPK1 (receptor interacting protein 1), crucial for nuclear factor-κB (NF-κB) activation by TNFα and survival. Here, we investigate alterations in TNFα–TRAF2–NF-κB signaling in the pathogenesis of DOX cardiotoxicity.Methods:Using a combination of in vivo (4 weekly injections of DOX 5 mg·kg⁻¹·wk⁻¹) in C57/BL6J mice and in vitro approaches (rat, mouse, and human inducible pluripotent stem cell–derived cardiac myocytes), we monitored TNFα levels, lactate dehydrogenase, cardiac ultrastructure and function, mitochondrial bioenergetics, and cardiac cell viability.Results: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 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 polyubiquitination of RIPK1 from the proteasomal degradation of TRAF2. Furthermore, TRAF2 protein abundance was markedly reduced in hearts of patients with cancer treated with DOX. We further established that the reciprocal actions of the ubiquitinating and deubiquitinating enzymes cellular inhibitors of apoptosis 1 and USP19 (ubiquitin-specific peptidase 19), respectively, regulated the proteasomal degradation of TRAF2 in DOX-treated cardiac myocytes. An E3-ligase mutant of cellular inhibitors of apoptosis 1 (H588A) or gain of function of USP19 prevented proteasomal degradation of TRAF2 and DOX-induced cell death. Furthermore, 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. Last, cardiomyocyte-restricted expression of TRAF2 (cardiac troponin T–adeno-associated virus 9–TRAF2) in vivo protected against mitochondrial defects and cardiac dysfunction induced by DOX.Conclusions: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-mediated necrotic cell death and DOX cardiotoxicity.

中文翻译：

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TRAF2 的蛋白酶体降解介导多柔比星心肌病中的线粒体功能障碍

背景：肿瘤坏死因子-α (TNFα) 等细胞因子与阿霉素 (DOX) 相关的心脏功能障碍和毒性有关。尽管 TNFα 可以引发不同的细胞反应，包括生存或死亡，但心脏中这些不同结果的潜在机制仍然神秘。E3 泛素连接酶 TRAF2（TNF 受体相关因子 2）为 RIPK1（受体相互作用蛋白 1）的 K63 连接多泛素化提供了关键的信号传导平台，对于 TNFα 激活核因子 -κB (NF-κB) 和生存至关重要。在这里，我们研究了 DOX 心脏毒性发病机制中 TNFα–TRAF2–NF-κB 信号传导的变化。方法：在 C57/BL6J 中使用体内组合（每周 4 次注射 DOX 5 mg·kg -1 · ^wk -1 ^）在小鼠和体外方法（大鼠、小鼠和人类诱导多能干细胞衍生的心肌细胞）中，我们监测了 TNFα 水平、乳酸脱氢酶、心脏超微结构和功能、线粒体生物能学和心肌细胞活力。结果：与载体相反在接受 DOX 治疗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 与 DOX 心脏毒性的关系时，我们发现 NF-κB 很容易被 TNFα 激活。然而，用 DOX 处理的心肌细胞中 TNFα 介导的 NF-κB 激活受到损害。这与 TRAF2 蛋白酶体降解导致 RIPK1 的 K63 连接多泛素化丧失同时发生。此外，接受 DOX 治疗的癌症患者心脏中 TRAF2 蛋白丰度显着降低。我们进一步确定，泛素化和去泛素化酶细胞凋亡抑制剂 1 和 USP19（泛素特异性肽酶 19）的相互作用分别调节 DOX 处理的心肌细胞中 TRAF2 的蛋白酶体降解。细胞凋亡抑制剂 1 (H588A) 的 E3 连接酶突变体或 USP19 的功能获得可阻止 TRAF2 的蛋白酶体降解和 DOX 诱导的细胞死亡。此外，野生型TRAF2（而非RIPK1的K63连接多聚泛素化缺陷的RING指突变体）恢复了NF-κB信号传导并抑制了DOX诱导的心肌细胞死亡。最后，TRAF2（心肌肌钙蛋白 T-腺相关病毒 9-TRAF2）在体内的心肌细胞限制性表达可防止 DOX 引起的线粒体缺陷和心脏功能障碍。 结论：我们的研究结果揭示了一个新的信号轴，该信号轴在功能上连接了 DOX 的心脏毒性作用DOX 对 TRAF2 的蛋白酶体降解。DOX 对关键 TRAF2 生存途径的破坏使心肌细胞对 TNFα 介导的坏死细胞死亡和 DOX 心脏毒性敏感。