MITOL/MARCH5 is an E3 ubiquitin ligase that plays a crucial role in the control of mitochondrial quality and function. However, the significance of MITOL in cardiomyocytes under physiological and pathological conditions remains unclear.

First, to determine the significance of MITOL in unstressed hearts, we assessed the cellular changes with the reduction of MITOL expression by siRNA in neonatal rat primary ventricular cardiomyocytes (NRVMs). MITOL knockdown in NRVMs induced cell death via ferroptosis, a newly defined non-apoptotic programmed cell death, even under no stress conditions. This phenomenon was observed only in NRVMs, not in other cell types. MITOL knockdown markedly reduced mitochondria-localized GPX4, a key enzyme associated with ferroptosis, promoting accumulation of lipid peroxides in mitochondria. In contrast, the activation of GPX4 in MITOL knockdown cells suppressed lipid peroxidation and cell death. MITOL knockdown reduced the glutathione/oxidized glutathione (GSH/GSSG) ratio that regulated GPX4 expression. Indeed, the administration of GSH or N-acetylcysteine improved the expression of GPX4 and viability in MITOL-knockdown NRVMs. MITOL-knockdown increased the expression of the glutathione-degrading enzyme, ChaC glutathione-specific γ-glutamylcyclotransferase 1 (Chac1). The knockdown of Chac1 restored the GSH/GSSG ratio, GPX4 expression, and viability in MITOL-knockdown NRVMs.

Further, in cultured cardiomyocytes stressed with DOX, both MITOL and GPX4 were reduced, whereas forced-expression of MITOL suppressed DOX-induced ferroptosis by maintaining GPX4 content. Additionally, MITOL knockdown worsened vulnerability to DOX, which was almost completely rescued by treatment with ferrostatin-1, a ferroptosis inhibitor. In vivo, cardiac-specific depletion of MITOL did not produce obvious abnormality, but enhanced susceptibility to DOX toxicity. Finally, administration of ferrostatin-1 suppressed exacerbation of DOX-induced myocardial damage in MITOL-knockout hearts.

The present study demonstrates that MITOL determines the cell fate of cardiomyocytes via the ferroptosis process and plays a key role in regulating vulnerability to DOX treatment.

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MITOL/MARCH5 是一种 E3 泛素连接酶，在控制线粒体质量和功能中起关键作用。然而，在生理和病理条件下，MITOL 在心肌细胞中的意义仍不清楚。

首先，为了确定 MITOL 在无压力心脏中的重要性，我们评估了新生大鼠原发性心室心肌细胞 (NRVM) 中 siRNA 降低 MITOL 表达的细胞变化。NRVM 中的 MITOL 敲低通过铁死亡诱导细胞死亡，这是一种新定义的非凋亡程序性细胞死亡，即使在没有压力条件下也是如此。这种现象仅在 NRVM 中观察到，而在其他细胞类型中则没有。MITOL 敲低显着降低了线粒体定位的 GPX4，这是一种与铁死亡相关的关键酶，促进脂质过氧化物在线粒体中的积累。相比之下，MITOL 敲低细胞中 GPX4 的激活抑制了脂质过氧化和细胞死亡。MITOL 敲低降低了调节 GPX4 表达的谷胱甘肽/氧化谷胱甘肽 (GSH/GSSG) 比率。事实上，GSH 的管理或N-乙酰半胱氨酸改善了 MITOL 敲低 NRVM 中 GPX4 的表达和活力。MITOL 敲低增加了谷胱甘肽降解酶 ChaC 谷胱甘肽特异性 γ-谷氨酰环转移酶 1 (Chac1) 的表达。Chac1 的敲低恢复了 MITOL 敲低 NRVM 中的 GSH/GSSG 比率、GPX4 表达和活力。

此外，在用 DOX 应激的培养心肌细胞中，MITOL 和 GPX4 均减少，而 MITOL 的强制表达通过维持 GPX4 含量来抑制 DOX 诱导的铁死亡。此外，MITOL 敲低使 DOX 的易感性恶化，这种情况几乎完全通过使用 ferrostatin-1（一种铁死亡抑制剂）治疗得以挽救。在体内，MITOL 的心脏特异性消耗没有产生明显的异常，但增强了对 DOX 毒性的敏感性。最后，ferrostatin-1 的施用抑制了 MITOL 敲除心脏中 DOX 诱导的心肌损伤的恶化。

本研究表明，MITOL 通过铁死亡过程决定心肌细胞的细胞命运，并在调节对 DOX 治疗的易感性中起关键作用。

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