Mechanistic insights into the role of mitochondrial iron in doxorubicin-induced cardiomyopathy using human induced pluripotent stem cells

使用人类诱导多能干细胞深入了解线粒体铁在阿霉素诱导的心肌病中的作用

• 批准号: 9916208
• 负责人: Hossein Ardehali
• 金额: $ 39.5万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-15 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9916208
• 关键词: Address; Animal Disease Models; Animal Model; Antineoplastic Agents; Apoptosis; Biogenesis; CRISPR/Cas technology; Cardiac Myocytes; Cardiomyopathies; Cardiotoxicity; Cell Death; Cell Survival; Cells; Chelating Agents; Clinical; Collaborations; Cultured Cells; Data; Defect; Disease; Doxorubicin; Exposure to; Generations; Genes; Goals; Grant; Heart; Homeostasis; Human; Impairment; In Vitro; Iron; Iron Chelating Agents; Knockout Mice; Lysosomes; Measurement; Mediating; Messenger RNA; Mitochondria; Mus; Pathogenesis; Patients; Permeability; Phenotype; Play; Predisposition; Process; Protein Export Pathway; Proteins; Research Personnel; Respiration; Role; Sampling; Testing; Toxic effect; Transgenic Mice; experience; human disease; improved; induced pluripotent stem cell; insight; mouse model; protective effect; side effect; tissue culture; transcriptome sequencing

The clinical use of doxorubicin (DOX, and anticancer drug) is limited due to its cardiotoxic side effects. The mechanism of DOX-induced cardiotoxicity (DIC) is not totally understood. We recently showed that DOX causes mitochondrial (as opposed to total cellular) iron accumulation, and that a reduction in mitochondrial iron leads to protection against DIC. However, these studies were predominantly performed in vitro and in animal models, and it is not clear whether higher mitochondrial iron contributes to DIC susceptibility in patients. To extend these findings beyond animal models, we have established several lines of human induced pluripotent stem cell- derived cardiomyocytes (hiPSC-CM) from patients who either experienced DIC (DIC-CM) or did not have cardiotoxicity (noDIC-CM) after exposure to DOX, and showed that they recapitulate the patients’ phenotype in vitro, and that they are susceptible to a special form of iron-mediated cell death called “ferroptosis”. Using RNA- seq, we have also identified a number of mitochondrial iron genes to be altered in DIC samples after DOX treatment. Finally, we have demonstrated that hiPSC-CM from DIC patients display elevated mitochondrial iron after DOX exposure. Our central hypothesis is that mitochondrial iron plays a key role in the susceptibility to DIC in humans, and that a reduction in mitochondrial iron (either by iron chelators or by modulation of genes involved in mitochondrial iron homeostasis) alter cardiomyocyte susceptibility to DOX. We also hypothesize that DOX clearance from subcellular compartments is regulated by mitochondrial iron through mitophagy, and that this process is impaired in DIC. In Aim 1, we will determine whether DIC-CM are more susceptible to DOX toxicity due to mitochondrial iron accumulation, and that a reduction in mitochondrial iron improves cell viability in these cells. We will modulate mitochondrial iron in DIC-CM and noDIC-CM using mitochondrial permeable iron chelators (including new chelators identified in our lab), followed by exposure to DOX and assessment of ferroptosis and cell viability. In Aim 2, we will determine whether modulation of mitochondrial iron-related genes identified in our RNA-seq analysis alters susceptibility to DOX. Our studies demonstrated that mRNA levels of ABCB7 and ABCB8 (proteins that mediate mitochondrial iron export) and MFRN2 (a mitochondrial iron importer) are altered with DOX treatment. We will delete ABCB7 or ABCB8 in hiPSC from noDIC (which would result in an increase in mitochondrial iron) and MFRN2 in hiPSC from DIC (resulting in a reduction in mitochondrial iron) using CRISPR-Cas9, followed by generation of hiPSC-CM, exposure to DOX, and assessment of cell viability. Finally, in Aim 3, we will determine whether mitochondrial iron regulates DOX clearance and that inherent defects in DOX clearance promotes susceptibility to DIC. We will assess DOX clearance, mitochondrial dynamics and mitophagy in noDIC-CM and DIC-CM after DOX and in the presence and absence of iron chelators, and with modulation of mitochondrial iron genes. We will also assess DOX clearance and mitophagy in the hearts of Abcb8 knockout and transgenic mouse models after DOX.

阿霉素（DOX，抗癌药物）的临床使用由于其心脏毒性副作用而受到限制。这 DOX 引起的心脏毒性 (DIC) 的机制尚不完全清楚。我们最近表明 DOX 会导致 线粒体（而不是总细胞）铁积累，线粒体铁的减少会导致 预防 DIC。然而，这些研究主要是在体外和动物模型中进行的， 目前尚不清楚较高的线粒体铁是否会导致患者 DIC 易感性。为了扩展这些 除了动物模型之外的发现，我们已经建立了几种人类诱导多能干细胞系- 来自经历过 DIC (DIC-CM) 或未经历过 DIC 的患者的衍生心肌细胞 (hiPSC-CM) 暴露于 DOX 后的心脏毒性（noDIC-CM），并表明它们概括了患者的表型 体外，并且它们容易受到一种特殊形式的铁介导的细胞死亡的影响，称为“铁死亡”。使用RNA- seq，我们还发现了 DOX 后 DIC 样品中许多线粒体铁基因发生改变 治疗。最后，我们证明来自 DIC 患者的 hiPSC-CM 线粒体铁含量升高 DOX 暴露后。我们的中心假设是线粒体铁在易感性中起着关键作用 人类 DIC 的发生，以及线粒体铁的减少（通过铁螯合剂或通过调节） 参与线粒体铁稳态的基因）改变心肌细胞对 DOX 的敏感性。我们也 假设 DOX 从亚细胞区室的清除受到线粒体铁的调节 通过线粒体自噬，并且这个过程在 DIC 中受到损害。在目标 1 中，我们将确定 DIC-CM 是否 由于线粒体铁积累，更容易受到 DOX 毒性的影响，并且 线粒体铁可以提高这些细胞的活力。我们将调节 DIC-CM 中的线粒体铁 noDIC-CM 使用线粒体可渗透性铁螯合剂（包括我们实验室鉴定的新螯合剂），然后 通过暴露于 DOX 并评估铁死亡和细胞活力。在目标 2 中，我们将确定是否 RNA-seq 分析中发现的线粒体铁相关基因的调节会改变对 DOX 的敏感性。 我们的研究表明，ABCB7 和 ABCB8（介导线粒体铁的蛋白质）的 mRNA 水平 DOX 处理会改变 MFRN2（线粒体铁输入蛋白）和 MFRN2（线粒体铁输入蛋白）。我们将删除 ABCB7 或 来自 noDIC 的 hiPSC 中的 ABCB8（这将导致线粒体铁增加）和来自 noDIC 的 hiPSC 中的 MFRN2 使用 CRISPR-Cas9 进行 DIC（导致线粒体铁减少），然后生成 hiPSC-CM， 暴露于 DOX，并评估细胞活力。最后，在目标 3 中，我们将确定线粒体是否 铁调节 DOX 清除，而 DOX 清除的固有缺陷会增加 DIC 的易感性。我们 将评估 DOX 后和 in 后 noDIC-CM 和 DIC-CM 中的 DOX 清除率、线粒体动力学和线粒体自噬 铁螯合剂的存在和缺乏，以及线粒体铁基因的调节。我们还将评估 DOX 后 Abcb8 敲除和转基因小鼠模型心脏中的 DOX 清除和线粒体自噬。