Limitations of Ferroptosis Inhibitors on the Doxorubicin-Induced Cardiotoxicity

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  Figure 1. The application of ferroptosis inhibitors does not mitigate toxicity in H9c2 cells following treatment with doxorubicin. (A) Relative cell viability of H9c2 cells as determined by EZ-Cytox assay. H9c2 were pre-treated with ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1) at concentrations of 2 and 0.2 μM, respectively, for 24 h. Additionally, doxorubicin was treated at concentrations of 0.5 and 1 μM for 24 h. **** p < 0.001. (B) Effect of Fer1, Lip-1, and/or doxorubicin treatment on apoptosis in H9c2 cells. Apoptosis was quantified by flow cytometry, measuring the percentage of annexin V-positive H9c2 cells. The fluorescence intensity of annexin V is plotted on the x-axis, and Zombie NIR is plotted on the y-axis. The percentage of apoptotic cells was investigated by using annexin V/Zombie NIR staining. Each bar is represented as the mean ± standard deviation. **** p < 0.001. (C) Relative cell viability of H9c2 cells following co-treatment with Fer-1, Lip-1, doxorubicin, and RSL3. H9c2 cells were pre-treated with Fer-1 and Lip-1 at concentrations of 2 and 0.2 μM, respectively, for 24 h. Additionally, doxorubicin and RSL3 were used at a concentration of 0.5 μM each for 24 h. (A–C),…

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  Figure 2. Ferroptosis inhibitors do not alleviate doxorubicin-induced toxicity in induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs). (A) Schematic representation of the iPSC-CMs differentiation. The validated hiPSCs were cultured in the indicated media during the differentiation period and finally differentiated to mature hiPSC-CMs through cardiac mesoderm, cardiac progenitors, and cardiomyocytes. (B) To validate iPSC-CMs, cells were stained with the indicated antibodies recognizing cardiac markers (Troponin T2, NK2 Homeobox 5), and immunofluorescence was analyzed; Scale bar = 100 µm. (C) Troponin T2-positive cells were assessed using flow cytometry. (D) Relative cell viability of iPSC-CMs as determined by EZ-Cytox assay. iPSC-CMs were pre-treated with ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1) at concentrations of 2 and 0.2 μM, respectively, for 24 h. Additionally, doxorubicin was treated at concentrations of 5 μM for 24 h. (E) Flow cytometric quantification of apoptosis in iPSC-CMs treated with Fer-1, Lip-1, and/or doxorubicin. Fer-1 or Lip-1 was applied to iPSC-CMs, and the cells were exposed to 5 µM doxorubicin for 24 h Apoptosis was quantified by flow cytometry,…

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  Figure 3. Electrophysiological alterations of induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) induced by doxorubicin treatment are not ameliorated by ferroptosis inhibitors. (A) Schematic diagram of drug and doxorubicin treatment experiments. (B) Plate schematic for seeding iPSC-CMs to perform a multi-electrode assay. (C) An activity map of the total active electrode count is shown. The red and blue colors indicate higher and lower spike amplitudes for iPSC-CMs, respectively. Representative images are shown from three independent experiments (left panel). The percentage of total active electrodes and the percentage changes in spike amplitude compared to the baseline were measured using a multi-electrode array (MEA) assay (right two panels). (D) Field potential trace data recorded with the MEA, exhibiting mean spontaneous beating traces, are shown. Representative trace images recorded with the MEA display field potential traces in iPSC-CMs treated with ferroptosis inhibitors with or without DOX (left six panels). The graph indicates beat period and spike amplitude measured across the treatment groups (right panel). (E) The graph displays field potential duration corrected…

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  Figure 4. Ferroptosis inhibitors fail to rescue cardiac dysfunction. (A) Brief schedule of in vivo experiment. C57BL/6J mice were treated with ferrostatin-1 (Fer-1) or liproxstatin-1 (Lip-1) 24 h before doxorubicin injection and were dosed every 24 h. A single dose of doxorubicin was administered intraperitoneally once on day 1. Experimental groups: Vehicle, DOX (20 mg/kg, i.p.), DOX + Fer-1 (2 mg/kg, i.p.), and DOX + Lip-1 (10 mg/kg, i.p.) (B) The survival graph of mice from different groups was shown by Kaplan–Meier survival analysis. (C) Three days after intraperitoneal injection of doxorubicin, the mouse cardiac function was analyzed with echocardiography. The echocardiography data showed decreased fractional shortening and ejection fraction in mice treated with doxorubicin and Fer-1 or Lip-1. Survival rate, n = 5 per group; ECG, n = 5 per group. (D) Bar graph representation of the heart weight to body weight ratio of mice. (E) 3,3’-Diaminobenzidine immunohistochemical staining for 4-hydroxynonenal (4-HNE) in the mouse heart tissue; scale bars, 200 µm (left panel). Quantification of 4-HNE-specific 3,3’-Diaminobenzidine staining (right panel). (F) Bar graph representation of the…

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  Figure 5. Ferroptosis inhibitors do not alleviate doxorubicin-induced arrhythmic changes in mice. (A) Electrocardiogram (ECG) traces recorded on day 1 after DOX administration with or without ferroptosis inhibitors, showing various arrhythmic events, including tachyarrhythmia, frequent premature ventricular contractions, irregular rhythm, and cardiac arrest. Red arrows indicate representative arrhythmic events, including premature ventricular contractions and irregular R−R intervals. (B) ECG traces recorded on day 21, demonstrating the progression of arrhythmic changes, including atrioventricular (AV) block and bradycardia with AV block, indicated by red arrows. n = 4 per group. Data are presented as mean ± S.D., where n represents the number of individual mice per group. Statistical significance was determined by one-way ANOVA followed by Sidak’s multiple comparison test.