1 INTRODUCTION AND BACKGROUND

Superparamagnetic iron oxide nanoparticles (SPION) have been widely used in the diagnosis and treatment for cardiovascular diseases.1-6 Correspondingly, the myocardial tissue safety of SPION is becoming a bottleneck to seriously restrict its clinical translation. In recent years, in vitro and in vivo experiments have confirmed that SPION‐induced oxidative stress of normal myocardium in mice, leading to myocardial cell injury, apoptosis or necrosis.7-9 More alarmingly, SPION applied to ischemic myocardium could accumulate in the target sites for a long time with high concentration,5, 6, 10 thereby probably further aggravating oxidative stress injury and cardiomyocytes death.11, 12

So far, however, the specific molecular mechanism of cardiotoxicity of SPION remains unclear. Previous studies have reported that SPION‐induced apoptosis of murine macrophage (J774) cells 13 and necrosis of human endothelial cells.14 SPION can selectively induce autophagy‐mediated cell death of human cancer cells (A549).15 After SPION pre‐treatment, H9C2 cardiomyocytes were exposed to acrolein or H₂O₂, leading to reactive oxygen species (ROS) dependent cell necrosis.7 Our in vitro experiment showed that SPION significantly increased oxidative stress damage to overactivate autophagy and endoplasmic reticulum stress, eventually resulting in cardiomyocyte apoptosis.12 Furthermore, SPION could elicit IL‐1βrelease and pyroptosis in macrophages, especially with the octapod and plate morphology.16 Notably, it has been recently reported that sorafenib or cisplatin assembled into nano‐devices containing SPION, which are phagocytized by tumour cells and degraded into free divalent iron to accelerate Fenton reaction, leading to the lipid peroxidation burst to promote ferroptosis of tumour cells.17, 18

Taken together, SPION can induce apoptosis, necrosis, autophagy, pyroptosis or ferroptosis in vitro and in vivo studies. The discrepancy may be attributed to distinct cell types and experiments design. It has already been well documented that the toxicity of SPION is mainly due to its degradation and release of free iron to catalyse Fenton reaction, leading to oxidative stress by a large number of ROS generation.19, 20 Then, what is the downstream molecular mechanism of SPION mediated cardiotoxicity?

Ferroptosis is a novel form of regulated cell death characterized by the iron‐dependent accumulation of lipid peroxides to lethal levels, which is morphologically, biochemically, and genetically distinct from apoptosis, necroptosis and autophagy.21 Recent studies found that ferroptosis is not only an important pathological mechanism in the case of circulating iron overload of hemochromatosis,22 but also a key molecular mechanism of cellular iron overload in doxorubicin (DOX) induced cardiomyopathy.23 DOX induced mitochondria iron overload by down‐regulating ABCB8,24 a mitochondrial protein that facilitates iron export, to elicit lipid peroxidation and mitochondria dysfunction, eventually causing cardiomyocytes ferroptosis.23 Mice that were subjected to 30 minutes of myocardial ischemia followed by 24 hours of reperfusion had significantly higher levels of cardiac non‐heme iron, cardiac ferritin H, ferritin L and Ptgs2 mRNA. Both ferroptosis inhibitor Ferrostatin‐1 (Fer‐1) and iron chelator Dexrazoxane (DXZ) pre‐treatment significantly reduced I/R‐induced cardiac remodelling and fibrosis, indicating that ischemia‐reperfusion could also induce cardiomyocytes iron overload to cause ferroptosis and subsequent left ventricular remodelling.23 Myocardial haemorrhage is a frequent complication after successful myocardial reperfusion,25, 26 which is associated with residual myocardial iron in post‐myocardial infarction (MI) patients received reperfusion therapy.27 It is reasonable to infer that this iron accumulation has a potential to generate excessive ROS and trigger pathological events such as ferroptosis. A previous study also confirmed that ferroptosis is a significant type of cell death in cardiomyocytes; moreover, mechanistic target of rapamycin (mTOR) was found to play an important role in protecting cardiomyocytes against excess iron and ferroptosis by regulating ROS production.28 In addition, glutathione peroxidase 4 (GPX4), which protects cells from ferroptosis, was down‐regulated in the early and middle stages of MI mouse model, suggesting that ferroptosis during MI was in part due to a reduction in GPX4 protein.29

Even though signalling pathways of ferroptosis in cardiovascular diseases is not yet well characterized, it has been confirmed that ischemia‐reperfusion (I/R) could induce mitochondrial iron overload in cardiomyocytes rather than the increase of iron content in cytoplasm.30 In this study, mice treated with 2,2′‐bipyridyl (BPD), which has high membrane permeability and thus is able to access mitochondria, had demonstrated protective effects on I/R myocardium, while deferoxamine (DFO) failed to protect mice against I/R damage due to poor penetrance into mitochondria. Notably, overexpression of ABCB8 in cardiomyocytes in mice reduces mitochondrial iron and protects against I/R damage,30 suggesting that ABCB8 might play an important role in maintaining iron homeostasis in myocardial mitochondria and regulating ferroptosis after I/R injury. Thus, it is not difficult to speculate that SPION could aggravate mitochondrial iron load in I/R myocardium. SPION applied in ischemic myocardium could be directly degraded by cardiomyocytes,12 leading to severe mitochondrial iron overload.

We detected prominently mitochondrial lipid peroxidation (malondialdehyde, MDA), mitochondrial membrane potential (MMP) loss and ATP depletion at 24 hours and 4 weeks after SPION injected into the peri‐infarcted zones of myocardial ischemia‐reperfusion rats compared with the control group (all P < .01). We found that iron content of mitochondria was significantly higher than that in the control group (P < .001), and the distorted mitochondria were observed by transmission electron microscopy in the SPION group, suggesting that SPION have the potential to destroy mitochondrial structure and function by inducing mitochondria iron overload (data not published). Mitochondria are the major site of iron metabolism and ROS production, thereby cardiomyocytes iron accumulation is especially prone to induce mitochondria iron overload to trigger mitochondrial oxidative damage. Based on the above results, we speculate that SPION might further promote ferroptosis to aggravate left ventricular remodelling and cardiac deterioration by inducing severe mitochondria iron overload to promote lipid peroxidation burst.

中文翻译：

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超顺磁性氧化铁纳米颗粒促进缺血心肌细胞的铁死亡。

1 引言和背景

超顺磁性氧化铁纳米粒子（SPION）已广泛用于心血管疾病的诊断和治疗。1-6相应地，SPION的心肌组织安全性正成为严重制约其临床转化的瓶颈。近年来，体外和体内实验证实，SPION诱导小鼠正常心肌氧化应激，导致心肌细胞损伤、凋亡或坏死。7-9更令人担忧的是，SPION 应用于缺血心肌可在靶部位长时间高浓度蓄积，5, 6, 10从而可能进一步加重氧化应激损伤和心肌细胞死亡。11、12

然而，迄今为止，SPION心脏毒性的具体分子机制仍不清楚。以前的研究报道了 SPION 诱导的小鼠巨噬细胞 (J774) 细胞凋亡13和人内皮细胞坏死。14 SPION 可以选择性地诱导自噬介导的人类癌细胞死亡 (A549)。15在 SPION 预处理后，H9C2 心肌细胞暴露于丙烯醛或 H ₂ O ₂中，导致活性氧 (ROS) 依赖性细胞坏死。7我们的体外实验表明，SPION 显着增加了氧化应激损伤以过度激活自噬和内质网应激，最终导致心肌细胞凋亡。12此外，SPION 可以在巨噬细胞中引发 IL-1β 释放和焦亡，尤其是在八足和平板形态中。16值得注意的是，最近有报道称，索拉非尼或顺铂组装成含有 SPION 的纳米器件，它们被肿瘤细胞吞噬并降解为游离二价铁以加速 Fenton 反应，导致脂质过氧化爆发，促进肿瘤细胞的铁死亡。17、18

总之，在体外和体内研究中，SPION 可以诱导细胞凋亡、坏死、自噬、细胞焦亡或铁死亡。差异可能归因于不同的细胞类型和实验设计。SPION的毒性主要是由于其降解和释放游离铁催化Fenton反应，大量产生ROS导致氧化应激。19, 20那么，SPION介导心脏毒性的下游分子机制是什么？

铁死亡是一种新的受调控的细胞死亡形式，其特征在于铁依赖性过氧化脂质积累至致死水平，其在形态、生化和遗传上与细胞凋亡、坏死性凋亡和自噬不同。21最近的研究发现，铁死亡不仅是血色素沉着症循环铁超负荷情况下的重要病理机制，22也是阿霉素 (DOX) 诱导的心肌病中细胞铁超负荷的关键分子机制。23 DOX 通过下调 ABCB8（一种促进铁输出的线粒体蛋白24 ）诱导线粒体铁过载，从而引发脂质过氧化和线粒体功能障碍，最终导致心肌细胞铁死亡。23心肌缺血 30 分钟后再灌注 24 小时的小鼠心脏非血红素铁、心脏铁蛋白 H、铁蛋白 L 和Ptgs2 mRNA 水平显着升高。ferroptosis 抑制剂 Ferrostatin-1 (Fer-1) 和铁螯合剂 Dexrazoxane (DXZ) 预处理均显着降低了 I/R 诱导的心脏重塑和纤维化，表明缺血再灌注也可诱导心肌细胞铁过载导致铁死亡和随后的左心室重构。23心肌出血是心肌再灌注成功后的常见并发症，25, 26这与接受再灌注治疗的心肌梗死 (MI) 患者的残余心肌铁有关。27可以合理地推断，这种铁积累有可能产生过多的 ROS 并引发诸如铁死亡等病理事件。先前的一项研究也证实，铁死亡是心肌细胞中一种重要的细胞死亡类型。此外，发现雷帕霉素的机械靶点（mTOR）通过调节活性氧的产生在保护心肌细胞免受过量铁和铁死亡方面发挥重要作用。28此外，保护细胞免于铁死亡的谷胱甘肽过氧化物酶 4 (GPX4) 在 MI 小鼠模型的早期和中期下调，这表明 MI 期间的铁死亡部分是由于 GPX4 蛋白的减少。29

尽管心血管疾病中铁死亡的信号通路尚未得到很好的表征，但已证实缺血再灌注 (I/R) 可诱导心肌细胞中的线粒体铁超负荷，而不是细胞质中铁含量的增加。30在这项研究中，2,2'-联吡啶 (BPD) 处理的小鼠具有高膜通透性，因此能够进入线粒体，显示出对 I/R 心肌的保护作用，而去铁胺 (DFO) 未能保护小鼠抗由于对线粒体的渗透性差导致的 I/R 损伤。值得注意的是，ABCB8 在小鼠心肌细胞中的过度表达会降低线粒体铁并防止 I/R 损伤，30提示 ABCB8 可能在维持心肌线粒体铁稳态和调节 I/R 损伤后铁死亡中起重要作用。因此，不难推测 SPION 会加重 I/R 心肌中的线粒体铁负荷。应用于缺血心肌的 SPION 可被心肌细胞直接降解，12导致严重的线粒体铁过载。

与对照组相比，我们在 SPION 注射到心肌缺血再灌注大鼠的围梗死区后 24 小时和 4 周检测到显着的线粒体脂质过氧化（丙二醛，MDA）、线粒体膜电位（MMP）损失和 ATP 耗竭（所有P  < .01)。我们发现线粒体的铁含量显着高于对照组（P < .001)，并且在 SPION 组中通过透射电子显微镜观察到了扭曲的线粒体，表明 SPION 有可能通过诱导线粒体铁过载来破坏线粒体的结构和功能（数据未发表）。线粒体是铁代谢和产生活性氧的主要场所，因此心肌细胞铁积累特别容易诱发线粒体铁过载，从而引发线粒体氧化损伤。基于上述结果，我们推测SPION可能通过诱导严重的线粒体铁超负荷促进脂质过氧化爆发，进一步促进铁死亡，从而加剧左心室重构和心脏恶化。