Acyl-CoA thioesterase 1 prevents cardiomyocytes from Doxorubicin-induced ferroptosis via shaping the lipid composition

Doxorubicin (DOX) causes cardiotoxicity involving multiple regulated cell death pathways (apoptosis, necroptosis, pyroptosis) and is linked to disrupted iron metabolism and excess ROS. Classical antioxidants have largely failed to prevent DIC, suggesting other mechanisms. Ferroptosis, an iron-dependent lipid peroxidation-driven cell death, is implicated in DIC, with prior work indicating Nrf2/Hmox1-mediated iron dysregulation promotes ferroptosis. Lipid metabolic enzymes such as ACSL4 and LPCAT3 modulate ferroptosis sensitivity by promoting PUFA incorporation into phospholipids. Acyl-CoA thioesterase 1 (Acot1) hydrolyzes fatty acyl-CoAs to free fatty acids and CoA-SH, acting biochemically opposite to ACSL4. Given cardiomyocytes’ reliance on fatty acid metabolism and vulnerability to lipid peroxidation, the study hypothesizes that Acot1 protects against DOX-induced ferroptosis by shaping lipid composition.

Prior studies attribute DIC to iron overload and ROS injury, but antioxidant strategies have been inadequate; mitochondrial-targeted redox modulation (e.g., Mito-Q) showed benefits via signaling rather than ROS scavenging. Ferroptosis was defined as iron-dependent, non-apoptotic death marked by lipid peroxide accumulation, with ω-6 PUFA-PE oxidation as a key step. Enzymes such as ACSL4 (PUFA-CoA synthesis) and LPCAT3 (PUFA incorporation into phospholipids) heighten ferroptosis susceptibility. In DIC, Nrf2/Hmox1 activation can disrupt iron homeostasis to increase ferroptosis. Other ferroptosis regulators include GPX4 and FSP1 (suppressors). The role of other PUFA metabolism-related enzymes, including Acot1, in DIC-associated ferroptosis had not been clarified.

In vivo DOX model: Male C57BL/6 mice (7–8 weeks) received intraperitoneal DOX (15 mg/kg Day 1 and 10 mg/kg Day 8; cumulative 25 mg/kg) and were assessed on Day 15. Echocardiography (Vevo770, 30-MHz) evaluated EF, FS, LVIDs/d. Fer-1 (1 mg/kg i.p., every other day from Day 0) or DMSO vehicle was administered in selected cohorts. Survival analyses were performed. Hearts were collected for histology (H&E, Sirius Red), transmission electron microscopy (standard fixation, osmium tetroxide, epoxy embedding, sectioning, uranyl/lead staining), and biochemical assays. RNA-seq: Heart RNA from DOX vs control mice was sequenced (BGISEQ-500). Reads mapped with Bowtie2 to GRCm38.P6; quantification with RSEM. Low-count genes filtered (FPKM < 0.5829). Differential expression by limma (≥1.44-fold change, P<0.05). KEGG enrichment via clusterProfiler; GSEA with GSKB gene sets. Visualization by ggplot2/ggpubr/pheatmap. PPI network constructed. Validation by RT-qPCR and western blot for selected targets. Biochemical assays: Cardiac MDA measured (normalized to protein via BCA). Serum cTnI assessed (supplementary). GSH/GSSG measured in cells using commercial kits. Cell culture: HL-1 cardiomyocytes in supplemented Claycomb medium. DOX (2 µM for 24 h or 6 h) and RSL-3 (5 µM; ranges tested) used to induce cytotoxicity/ferroptosis. Cell viability measured by CCK-8. Lipid peroxidation assessed by C11-BODIPY 581/591 via flow cytometry and confocal microscopy. Genetic manipulation: Acot1 knockdown via siRNAs (100 nM; si-Acot1 #1/#2) and overexpression via pcDNA3.1(+)-Acot1 (200 ng/well). Protein and mRNA levels verified by western blot (anti-ACOT1) and RT-qPCR (primers provided). Transgenic mice and lipidomics: aMHC-Acot1 transgenic mice (cardiomyocyte-specific Acot1) previously generated were used for free fatty acid profiling by GC-MS in heart tissue. Total fatty acids extracted, methyl esterified, analyzed by GC-MS (Agilent 7890A/5975C, DB-WAX column, SIM mode). QC samples monitored instrument performance. Concentrations determined via MSD ChemStation. Fatty acid sensitization experiments: HL-1 cells incubated overnight with varying concentrations of stearic acid (C18:0), arachidonic acid (C20:4n6), and docosahexaenoic acid (C22:6n3), followed by DOX exposure±Fer-1 or Acot1 overexpression to assess effects on DIC. Statistics: Data presented as means with individual data points. Two-tailed unpaired Student’s t-test for two-group comparisons; one-way ANOVA with Tamhane’s T2 post-hoc for ≥3 groups. Survival by log-rank test. No blinding; no a priori sample size calculation; normality and variance homogeneity assessed.

- DOX induces cardiotoxicity in mice: increased mortality; reduced body/heart weight and heart/tibia index; significant systolic dysfunction (decreased EF and FS; increased LVIDs) versus controls. - RNA-seq identified significant downregulation of the biosynthesis of unsaturated fatty acids pathway in DOX hearts (including Acot1, Acot2, Acot3, Scd2, Scd4). GSEA showed negative enrichment of the same pathway with Acot1 as a leading-edge gene. Western blot confirmed decreased Acot1 protein. - Despite ferroptosis involvement, canonical ferroptosis/iron pathways were not enriched in RNA-seq; expression of GPX4, ACSL4, and FSP1 showed no significant differences across groups. - Ferroptosis contributes to DIC: Fer-1 treatment reduced mortality and preserved cardiac function in DOX-treated mice; improved histology (less edema/fibrosis) and mitochondrial ultrastructure (less crista loss and OMM rupture). Fer-1 lowered Ptgs2 mRNA and cardiac MDA levels elevated by DOX. - In HL-1 cells, DOX and RSL-3 decreased viability dose-dependently; Fer-1 (10 µM) rescued viability. Both agents reduced GSH/total glutathione and increased lipid peroxidation (C11-BODIPY), all mitigated by Fer-1. DOX increased Ptgs2 mRNA, blunted by Fer-1. - DOX decreased Acot1 mRNA/protein in HL-1 cells in a dose-dependent manner; Fer-1 partially prevented Acot1 downregulation. - Acot1 knockdown (si-Acot1) sensitized HL-1 cells to DOX: higher Ptgs2 mRNA, more lipid peroxidation, reduced GSH/total GSH, decreased viability; Fer-1 partially rescued. - Acot1 overexpression reduced DOX-induced Ptgs2, increased GSH/total GSH, decreased lipid peroxidation, and improved viability; Fer-1 provided additional protection. - aMHC-Acot1 transgenic hearts showed altered FFA composition: significantly higher C22:6n3 (DHA) and C18:0 (stearic acid); no significant changes in C18:2n6 (linoleic), C16:0 (palmitic), or C20:4n6 (arachidonic acid). - Exogenous DHA sensitized HL-1 cells to DOX-induced cytotoxicity in a concentration-dependent manner; AA also enhanced DIC. Fer-1 mitigated, and Acot1 overexpression desensitized cells to DHA/AA-enhanced DIC.

The study demonstrates that ferroptosis is a key contributor to DOX-induced cardiotoxicity and that Acot1 protects cardiomyocytes against ferroptotic damage. Fer-1 preserved cardiac function and mitochondrial integrity in vivo and reduced lipid peroxidation signatures, linking ferroptosis to DIC pathogenesis. Although RNA-seq did not enrich classical ferroptosis pathways or show changes in GPX4/ACSL4/FSP1, it revealed suppression of the biosynthesis of unsaturated fatty acids pathway, prominently featuring Acot1 downregulation. Functional experiments corroborated that decreasing Acot1 enhances susceptibility to lipid peroxidation and DOX cytotoxicity, while augmenting Acot1 reduces lipid ROS and cell death. The protective mechanism is consistent with Acot1’s enzymatic role counteracting ACSL4, thereby shaping cellular lipid pools and membrane composition that dictate ferroptosis sensitivity. Altered cardiac FFA profiles in Acot1 transgenic hearts and modulation of DOX toxicity by exogenous DHA/AA support a lipid composition-based mechanism. The data also suggest cross-talk between ferroptosis and apoptosis, as Fer-1 partially reversed apoptotic markers in DOX-treated hearts. Overall, Acot1 emerges as a modulator of lipid metabolism that desensitizes cardiomyocytes to ferroptosis in DIC.

Acot1 is downregulated in DOX-induced cardiotoxicity, and restoring Acot1 activity attenuates ferroptosis, lipid peroxidation, and cell death in cardiomyocytes. Ferroptosis inhibition with Fer-1 improves survival and cardiac function in DOX-treated mice, underscoring ferroptosis as a therapeutic target in DIC. Acot1 likely confers protection by reshaping lipid composition, thereby reducing susceptibility to lipid peroxidation-driven ferroptosis. Future studies should: (1) validate cardioprotective effects of Acot1 modulation in vivo during DOX challenge; (2) delineate how Acot1 alters membrane phospholipid PUFA incorporation relative to ACSL4/LPCAT3 pathways; (3) explore roles of other lipid enzymes (e.g., SCD family) in DIC; and (4) examine interactions between ferroptosis and other death pathways to optimize combinatorial cardioprotective strategies.

- Ferroptosis inhibition (Fer-1) only partially rescued DOX cardiotoxicity, indicating additional mechanisms contribute to DIC. - Key ferroptosis regulators (GPX4, ACSL4, FSP1) were not significantly altered at the expression level, and ferroptosis-specific pathways were not enriched by RNA-seq, potentially reflecting timing/tissue heterogeneity. - In vivo Acot1 overexpression was assessed for baseline cardiac FFA composition but not directly tested for protection against DOX challenge in mice within this study. - HL-1 cardiomyocytes are a cell line model and may not fully recapitulate primary cardiomyocyte biology. - No blinding and no a priori sample size calculations were used, which may introduce bias or limit power/generalizability.