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Mitochondria-dependent ferroptosis plays a pivotal role in doxorubicin cardiotoxicity
Tomonori Tadokoro, … , Hirotaka Imai, Hiroyuki Tsutsui
Tomonori Tadokoro, … , Hirotaka Imai, Hiroyuki Tsutsui
Published May 7, 2020
Citation Information: JCI Insight. 2020;5(9):e132747. https://doi.org/10.1172/jci.insight.132747.
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Research Article Cardiology

Mitochondria-dependent ferroptosis plays a pivotal role in doxorubicin cardiotoxicity

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Abstract

Doxorubicin (DOX), a chemotherapeutic agent, induces a cardiotoxicity referred to as doxorubicin-induced cardiomyopathy (DIC). This cardiotoxicity often limits chemotherapy for malignancies and is associated with poor prognosis. However, the molecular mechanism underlying this cardiotoxicity is yet to be fully elucidated. Here, we show that DOX downregulated glutathione peroxidase 4 (GPx4) and induced excessive lipid peroxidation through DOX-Fe2+ complex in mitochondria, leading to mitochondria-dependent ferroptosis; we also show that mitochondria-dependent ferroptosis is a major cause of DOX cardiotoxicity. In DIC mice, the left ventricular ejection fraction was significantly impaired, and fibrosis and TUNEL+ cells were induced at day 14. Additionally, GPx4, an endogenous regulator of ferroptosis, was downregulated, accompanied by the accumulation of lipid peroxides, especially in mitochondria. These cardiac impairments were ameliorated in GPx4 Tg mice and exacerbated in GPx4 heterodeletion mice. In cultured cardiomyocytes, GPx4 overexpression or iron chelation targeting Fe2+ in mitochondria prevented DOX-induced ferroptosis, demonstrating that DOX triggered ferroptosis in mitochondria. Furthermore, concomitant inhibition of ferroptosis and apoptosis with ferrostatin-1 and zVAD-FMK fully prevented DOX-induced cardiomyocyte death. Our findings suggest that mitochondria-dependent ferroptosis plays a key role in progression of DIC and that ferroptosis is the major form of regulated cell death in DOX cardiotoxicity.

Authors

Tomonori Tadokoro, Masataka Ikeda, Tomomi Ide, Hiroko Deguchi, Soichiro Ikeda, Kosuke Okabe, Akihito Ishikita, Shouji Matsushima, Tomoko Koumura, Ken-ichi Yamada, Hirotaka Imai, Hiroyuki Tsutsui

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Figure 4

Ferroptosis is involved in DOX-induced cell death in cultured cardiomyocytes.

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Ferroptosis is involved in DOX-induced cell death in cultured cardiomyoc...
(A) Total (left) and mitochondrial Gpx4 (right) expression in the cultured cardiomyocytes treated with vehicle or doxorubicin (DOX) was quantified by real-time PCR (n = 3 and 5, respectively). (B) Western blot of GPx4 in lysates from the cultured cardiomyocytes (n = 6). (C) MDA in the cultured cardiomyocyte was measured using the TBARs assay (left; n = 3). MDA in the cytosolic fraction of the cultured cardiomyocytes (middle; n = 8 and 7, each). MDA in the mitochondrial fraction of the cultured cardiomyocytes (right; n = 4). (D) Representative fluorescence imaging of mitochondrial LPs using MitoPeDPP in cultured cardiomyocytes under control conditions, in the presence of DOX (2 μM, 30 hours) or Fer-1 (50 μM, 30 hours) (green, left panels). Mitochondria were counterstained with MitoTracker Red (red, central panels). Scale bars: 10 μm. (E) Mitochondrial lipid peroxidation was measured using MitoPeDPP (n = 12–18). (F) MDA in the cultured cardiomyocyte, treated with DOX or Fer-1, was measured using the TBARs assay (n = 4, each). (G) Cell viability was assessed 30 hours after treatment with DOX or Fer-1 (n = 6). Data are shown as the mean ± SEM. Statistical significance was determined using 2-tailed Student’s t test (A–C) or 1-way ANOVA with a post hoc Tukey’s HSD test (E–G) *P < 0.05, **P < 0.01.

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