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: 10577780
• 负责人: Hossein Ardehali
• 金额: $ 40万
• 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 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10577780
• 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; Cytoplasm; Data; Defect; Disease; Doxorubicin; Exposure to; Generations; Genes; Goals; Grant; Heart; Homeostasis; Human; Impairment; In Vitro; Iron; Iron Chelation; Knock-out; 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; induced pluripotent stem cell derived cardiomyocytes; 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和抗癌药）的临床用途由于心脏毒性副作用而受到限制。这 DOX诱导的心脏毒性（DIC）的机制尚不完全了解。我们最近表明Dox原因 线粒体（与总细胞）的铁积累相反，线粒体铁的降低导致 防止DIC。然而，这些研究主要是在体外和动物模型中进行的 尚不清楚较高的线粒体铁是否有助于患者的DIC敏感性。扩展这些 除了动物模型之外的发现，我们已经建立了几条人类诱导的多能干细胞 - 来自经历DIC（DIC-CM）或没有的患者的衍生心肌细胞（HIPSC-CM） 暴露于DOX后的心脏毒性（Nodic-CM），并表明他们概括了患者的表型 体外，它们容易受到一种特殊形式的铁介导的细胞死亡，称为“铁毒性”。使用RNA- SEQ，我们还确定了许多线粒体铁基因在DOX之后在DIC样品中改变 治疗。最后，我们已经证明了来自DIC患者的HIPSC-CM表现出升高的线粒体铁 DOX暴露后。我们的中心假设是线粒体铁在易感性中起关键作用 在人类中进行DIC，并减少线粒体铁（通过铁螯合剂或通过调节 线粒体铁稳态涉及的基因）改变了心肌细胞对DOX的敏感性。我们也是 假设来自亚细胞室的DOX清除受线粒体铁的调节 通过线粒体，这一过程在DIC中受到损害。在AIM 1中，我们将确定DIC-CM是否 由于线粒体的积累而更容易受到DOX毒性的影响，并且减少了 线粒体铁可以改善这些细胞中的细胞活力。我们将在DIC-CM中调节线粒体铁和 使用线粒体可渗透铁螯合剂（包括在我们实验室中确定的新螯合剂），随后是Nodic-CM，随后 通过暴露于DOX并评估铁凋亡和细胞活力。在AIM 2中，我们将确定是否 在我们的RNA-seq分析中鉴定的线粒体铁相关基因的调节改变了对DOX的敏感性。 我们的研究表明，ABCB7和ABCB8的mRNA水平（介导线粒体铁的蛋白 DOX处理改变了出口）和MFRN2（线粒体铁的进口商）。我们将删除ABCB7或 来自NoDIC的HIPSC中的ABCB8（这将导致线粒体铁的增加）和MFRN2在HIPSC中的MFRN2 DIC（导致线粒体铁的降低）使用CRISPR-CAS9，然后产生HIPSC-CM， 暴露于DOX，并评估细胞活力。最后，在AIM 3中，我们将确定线粒体是否 铁调节DOX清除率，而遗传DOX清除中的缺陷促进了对DIC的敏感性。我们 将评估DOX和DOX和DIC-CM中的DOX清除率，线粒体动力学和线粒体。 铁螯合剂的存在和不存在，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的心脏中的DOX清除和线粒体。