Department of Cardiology, Zhongshan Hospital, Fudan University, Research Unit of Cardiovascular Techniques and Devices, Chinese Academy of Medical Sciences, Shanghai, China; Shanghai Institute of Cardiovascular Diseases, Shanghai, China; National Clinical Research Center for Interventional Medicine & Shanghai Clinical Research Center for Interventional Medicine (19MC1910300), Shanghai, China; Department of Cardiology and Laboratory of Heart Valve Disease, West China Hospital, Sichuan University, Chengdu, Sichuan, China.
Department of Cardiology, Zhongshan Hospital, Fudan University, Research Unit of Cardiovascular Techniques and Devices, Chinese Academy of Medical Sciences, Shanghai, China; Shanghai Institute of Cardiovascular Diseases, Shanghai, China; National Clinical Research Center for Interventional Medicine & Shanghai Clinical Research Center for Interventional Medicine (19MC1910300), Shanghai, China.
Cell Signal. 2024 Jan;113:110937. doi: 10.1016/j.cellsig.2023.110937. Epub 2023 Oct 21.
Doxorubicin (DOX), an anthracycline drug widely used in antitumor therapies, has dose-dependent toxicity that can cause cardiomyocyte apoptosis and oxidative stress, thus limiting its clinical application. OTUB1 (ovarian tumor associated proteinase B1) is an OTU superfamily deubiquitinase that effectively regulates cell proliferation, inflammatory responses, apoptosis, and oxidative stress by specifically removing K48- and K63-linked ubiquitination; however, its role in DOX-induced cardiotoxicity remains unknown.
A DOX-induced subacute cardiotoxicity mouse model was established by intraperitoneal injection, and cardiac injury was assessed by echocardiography, serum cardiac markers, and histopathological staining. Western blotting, qRT-PCR, and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) immunohistochemistry were used to analyze cell apoptosis, tissue oxidative stress was assessed by superoxide dismutase (SOD) activity, malondialdehyde (MDA), and glutathione peroxidase (GSH-PX) activity. Cell counting kit-8 (CCK-8) assay, TUNEL staining, Western blotting, qRT-PCR, and reactive oxygen species (ROS) flow cytometry were applied on isolated neonatal mice cardiomyocytes to assess apoptosis and oxidative stress. Differentially expressed genes were analyzed using RNA sequencing and clustering analyses. c-MYC inhibitor 10,058-F4 and siRNA targeting c-Myc were used to investigate the roles of c-MYC in OTUB1's regulations of DOX-induced cardiotoxicity. Immunoprecipitation and Western blotting were performed to reveal the deubiquitinating effects of OTUB1 on c-MYC expression.
We found that global Otub1-knockdown in vivo alleviated the subacute DOX treatment-induced cardiac dysfunction, fibrosis, and cardiomyocyte atrophy. Mechanistically, unbiased RNA sequencing and molecular biology experiments revealed that cardiomyocyte apoptosis, inflammation, and oxidative stress in DOX-induced cardiotoxicity were significantly compromised in the Otub1-knockdown group. Further in vitro studies have shown that c-MYC, a critical regulator of apoptosis, is indispensable in OTUB1's regulations of DOX-induced cardiotoxicity. Deubiquitinating effects of OTUB1 on K48- and K63-linked ubiquitination of c-MYC protein are essential for promoting cardiomyocyte apoptosis and oxidative responses.
OTUB1-c-MYC inhibition protected cardiomyocytes against DOX-induced apoptosis and oxidative stress, suggesting that OTUB1 is a potential translational therapeutic target for preventing DOX-induced cardiotoxicity.
多柔比星（DOX）是一种广泛用于抗肿瘤治疗的蒽环类药物，具有剂量依赖性毒性，可导致心肌细胞凋亡和氧化应激，从而限制了其临床应用。OTUB1（卵巢肿瘤相关蛋白酶 B1）是一种 OTU 超家族去泛素化酶，可通过特异性去除 K48-和 K63-连接的泛素化，有效调节细胞增殖、炎症反应、凋亡和氧化应激；然而，其在 DOX 诱导的心脏毒性中的作用尚不清楚。
通过腹腔注射建立 DOX 诱导的亚急性心脏毒性小鼠模型，并通过超声心动图、血清心脏标志物和组织病理学染色评估心脏损伤。Western blot、qRT-PCR 和末端脱氧核苷酸转移酶 dUTP 缺口末端标记（TUNEL）免疫组织化学用于分析细胞凋亡，超氧化物歧化酶（SOD）活性、丙二醛（MDA）和谷胱甘肽过氧化物酶（GSH-PX）活性评估组织氧化应激。细胞计数试剂盒-8（CCK-8）测定、TUNEL 染色、Western blot、qRT-PCR 和活性氧（ROS）流式细胞术用于评估分离的新生小鼠心肌细胞的凋亡和氧化应激。使用 RNA 测序和聚类分析分析差异表达基因。使用 c-MYC 抑制剂 10,058-F4 和针对 c-MYC 的 siRNA 研究 OTUB1 在 DOX 诱导的心脏毒性中的调节作用。免疫沉淀和 Western blot 用于揭示 OTUB1 对 c-MYC 表达的去泛素化作用。
我们发现体内敲低全局 Otub1 可减轻亚急性 DOX 处理诱导的心脏功能障碍、纤维化和心肌细胞萎缩。在机制上，无偏 RNA 测序和分子生物学实验表明，在 Otub1 敲低组中，DOX 诱导的心脏毒性中的心肌细胞凋亡、炎症和氧化应激显著受损。进一步的体外研究表明，凋亡的关键调节因子 c-MYC 在 OTUB1 调节 DOX 诱导的心脏毒性中不可或缺。OTUB1 对 c-MYC 蛋白 K48-和 K63-连接泛素化的去泛素化作用对于促进心肌细胞凋亡和氧化反应至关重要。
OTUB1-c-MYC 抑制可保护心肌细胞免受 DOX 诱导的凋亡和氧化应激，表明 OTUB1 是预防 DOX 诱导的心脏毒性的潜在转化治疗靶点。
