Abstract Due to the cardiotoxicity of doxorubicin (DOX), its clinical application is limited. Lipid peroxidation caused by excessive ferrous iron is believed to be a key molecular mechanism of DOX-induced cardiomyopathy (DIC). Dexrazoxane (DXZ), an iron chelator, is the only drug approved by the FDA for reducing DIC, but it has many side effects and cannot be used as a preventive drug in clinical practice. Single-nucleus RNA sequencing (snRNA-seq) analysis identified myocardial and epithelial cells that are susceptible to DOX-induced ferroptosis. The glutathione peroxidase 4 (GPX4) activator selenomethione (SeMet) significantly reduced polyunsaturated fatty acids (PUFAs) and oxidized lipid levels in vitro. Consistently, SeMet significantly decreased DOX-induced lipid peroxidation in H9C2 cells and mortality in C57BL/6 mice compared to DXZ, ferrostatin-1, and normal saline. SeMet can effectively reduce serum markers of cardiac injury in C57BL/6 mice and breast cancer patients. Depletion of the GPX4 gene in C57BL/6 mice resulted in an increase in polyunsaturated fatty acid (PUFA) levels and eliminated the protective effect of SeMet against DIC. Notably, SeMet exerted antitumor effects on breast cancer models with DOX while providing cardiac protection for the same animal without detectable toxicities. These findings suggest that pharmacological activation of GPX4 is a valuable and promising strategy for preventing the cardiotoxicity of doxorubicin. Keywords: DOX, Cardiotoxicity, GPX4, scRNA-seq, Selenium Abbreviations DOX doxorubicin DIC DOX-induced cardiomyopathy DXZ dexrazoxane scRNA-seq single-cell RNA sequencing snRNA-seq single-nucleus RNA sequencing DC dendritic cells SMC smooth muscle cells UFAs unsaturated fatty acids PUFAs polyunsaturated fatty acids PE phosphatidylethanolamine PI phosphatidylinositol PC phosphatidylcholine AA arachidonic acid GSH glutathione GPX4 glutathione peroxidase 4 SeMet selenomethione Se selenium NS normal saline DCM dilated cardiomyopathy FDA Food and Drug Administration UMAP uniform manifold approximation and projection DEGs differentially expressed genes CM cardiomyocytes ECG electrocardiogram GPX4hetko heterozygous GPX4-knockout LVEF left ventricular ejection fraction LVFS left ventricular fraction shortening HR heart rate VERP ventricular effective refractory period VF ventricular fibrillation AF atrial fibrillation ICP‒MS inductively coupled plasma‒mass spectrometry Nrf2 nuclear factor erythroid 2-related factor 2 1. Introduction Doxorubicin (DOX) is a foundation of chemotherapy regimens for some malignant tumors, including breast cancer, lymphoma, and soft tissue sarcoma [[69]1]. However, cardiotoxicity caused by DOX is one of the most serious adverse effects and has dose-dependent and cumulative characteristics, which greatly limits its clinical application. The incidence of DIC is as high as 17% and is mainly characterized as dilated cardiomyopathy, which is progressive and irreversible and can lead to heart failure in severe cases [[70]2]. Cardiovascular disease is one of the most common complications among cancer survivors and a major cause of death, especially in the long-term follow-up of child cancer survivors [[71]3,[72]4]. Dexrazoxane (DXZ) is the only drug approved by the US Food and Drug Administration (FDA) to reduce the cardiotoxicity of DOX. However, it still has some shortcomings: it can aggravate the bone marrow suppression caused by chemotherapy drugs, affect the antitumor effect of DOX, and may lead to secondary malignant tumors after long-term use [[73]5,[74]6]. Therefore, there is an urgent need to explore new strategies to prevent DIC in clinical practice. DOX-induced excessive accumulation of lipid peroxides in mitochondria is believed to play a critical role in the progression of DIC [[75]7,[76]8]. Thus, the pharmacological activation of GPX4 to suppress lipid peroxidation by selenium compounds is potentially valuable for preventing DIC. 2. Results 2.1. Single-cell characterization of cardiac cells in DIC To elucidate the mechanism of DOX-induced myocardial damage and screen new drugs with few side effects to prevent DIC, we conducted single-nucleus RNA sequencing (snRNA-seq) on heart samples from DOX-treated mice (DOX 20 mg/kg, n = 3) and untreated mice (n = 3) ([77]Fig. 1a). Using unbiased clustering analysis, we identified and visualized 32 clusters, which included twelve different cell types ([78]Fig. 1b, Extended Data [79]Fig. 1a–d). Five nonimmune cardiac cell populations were identified by examining the expression of known lineage markers in cardiomyocytes (Ryr2, Trdn, Myh7, Myh6, Ttn), fibroblasts (Pdgfra, Ckap4, Col1a1), endothelial cells (Pecam1, Vwf, Cdh5, Cd93, Ldb2, Tie1), adipocytes (Plin1, Pparg, Adam12), smooth muscle cells (Acta2, Tagln, Myh11), epithelial cells (Wt1, Tbx18, Krt18, Msln), and pericytes (Pdprb) ([80]Fig. 1c). Most studies on DOX-related cardiotoxicity are based on cardiomyocytes. Using unsupervised dimensionality reduction and clustering, we identified four cardiomyocyte subclusters that exhibited different gene expression profiles ([81]Fig. 1c). To further study the characteristics of cardiomyocytes after chemotherapy, we compared their transcriptional profiles and pathway enrichment between the two groups. Here, we found that after DOX treatment, hypertrophic cardiomyopathy, dilated cardiomyopathy, adrenergic signaling in cardiomyocytes, cardiac muscle contraction, arrhythmogenic right ventricular cardiomyopathy, and ferroptosis pathways were significantly enriched ([82]Fig. 2a, Extended Data [83]Fig. 1e). To determine which populations were most susceptible to ferroptosis, pathway enrichment of DEGs was conducted in four cardiomyocyte (CM) subclusters. We found that ferroptosis pathways were significantly enriched in the CM1 and CM3 clusters (Extended Data [84]Fig. 3a). We further examined the expression levels of ferroptosis-related genes that were previously reported [[85]9]. We confirmed that after DOX treatment, ferroptosis-related genes such as Gclm (glutamate-cysteine ligase modifier subunit), Fth1 (ferritin heavy chain 1), Hmox1 (heme oxygenase-1), Slc39a14 (solute carrier family 39 member 14), Ftl1 (ferritin light polypeptide 1), Nqo1(NAD(P)H quinone oxidoreductase 1), Cp (ceruloplasmin), Slc39a8 (solute carrier family 39 member 8), Map1lc3b (microtubule-associated protein 1 light chain 3 beta), and Ncoa4 (nuclear receptor coactivator 4) were significantly upregulated in cardiomyocytes and epithelial cells ([86]Fig. 2b–g, Extended Data [87]Fig. 3a and b, [88]Extended Data Tables 1–4). To further address the alterations in immune cell types, seven subclusters were identified based on their genetic markers, including four myeloid cell populations (Wt1, Fcgr1), one neutrophil population (S100a8, S1009a), one NK/T-cell population (Cd3d, Cd3e), and one B-cell population (Ms4a1, Cd79a). Among them, myeloid cells were further divided into subgroups, including one monocyte population (Itgal), two dendritic cell populations (H2-Eb1, H2-Ab1), four macrophage populations (Adgre1), and one proliferating cell population (Top2a, Mki67) (Extended Data [89]Fig. 2). The infiltration and activation of neutrophils contribute to DIC [[90]10], and pharmacological inhibition of neutrophils significantly prevents the cardiotoxic effects of DOX [[91]11]. The acute inflammatory response to DOX is associated with the apoptosis of monocytes and macrophages [[92]12,[93]13]. DOX treatment disrupts the cardiac M1/M2 macrophage balance and suppresses M2 macrophage differentiation [[94]14], which plays a critical role in cardiac injury and repair. M2 macrophage transplantation alleviated DOX-induced cardiac apoptosis and remodeling [[95]15,[96]16]. More studies are needed to illuminate the relationship between cardiac immune cells and DIC in the future. Taken together, our snRNA-seq analysis identified cardiomyocytes and epithelial cells as the main specific cell types that contribute to DOX-induced ferroptosis. Fig. 1. [97]Fig. 1 [98]Open in a new tab Single-nucleus profiling of cardiac cells during DOX treatment. (a) Schematic diagram showing the single nucleus RNA-sequencing (snRNA-seq) experimental design and analytical workflow. (b) Heatmap of the expression of cell type-specific markers in the no-immune cell atlas (left) and uniform manifold approximation and projection (UMAP) plots of cardiac no-immune cells, colored by clusters (center) and treatment (right). (c) UMAP plots of cardiac cells from mice treated with DOX (20 mg/kg) or saline. Fig. 2. [99]Fig. 2 [100]Open in a new tab Ferroptosis is associated with DOX-induced cardiotoxicity. (a) Dot plots showing the top 20 biological terms for the DEG processes of cardiomyocytes by KEGG pathway enrichment analysis. Red arrows indicate ferroptosis-related pathways. (b–g) Violin plots showing the expression of ferroptosis-related genes in cardiomyocytes after DOX (20 mg/kg) treatment. (h) Kaplan‒Meier survival curves of mice pretreated with saline (control), SeMet, l-selenocystine, Se-methylselenocysteine, sodium selenite, and Se-rich cardamine enshiensis (0.75 mg/kg, by gavage, weekly) before DOX (20 mg/kg) administration and after the start of DOX (0.375 mg/kg, by gavage, weekly). (i) Kaplan‒Meier survival curves of mice pretreated with saline (control), Fer-1 (a ferroptosis inhibitor, 1 mg/kg), DXZ (dexrazoxane, an iron chelator, 50 mg/kg), or SeMet (a GPX4 activator), followed by DOX (15 mg/kg, i.p.) on day 0 (n = 10 mice per group). (j) Serum cTnT activity in mice on day 2 after DOX injection. DOX (20 mg/kg DOX, i.p), n = 10 mice for each group. (k) Cell viability of H9C2 cells treated with various concentrations of DOX (0 μM, 0.001 μM, 0.05 μM, 0.5 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 25 μM) or DOX + SeMet for 24 h. (l–m) C11-BODIPY 581/591 staining showing lipid peroxidation levels in H9C2 cells treated with vehicle, DOX (2 μM, 6 h), or DOX (2 μM, 6 h) + SeMet (0.1 μM). Scale bar, 20 μm. A quantitative image analysis of fluorescence median intensity was performed with ImageJ software. (n–q) Levels of selected PUFAs with the indicated treatment (n = 4). Groups were compared using one-way ANOVA or Student’s t-test. Data show the mean ± s.d. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). (For interpretation of the references to color in this figure legend, the