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

Doxorubicin (DOX), a widely used chemotherapeutic agent, causes cardiotoxicity (DIC) through various mechanisms including apoptosis, necroptosis, and pyroptosis. While iron overload and reactive oxygen species (ROS) are implicated, traditional antioxidant therapies have proven ineffective. Recent research points to ferroptosis, an iron-dependent form of cell death driven by lipid peroxidation, as a significant contributor to DIC. This process is largely driven by the oxidation of ω-6 polyunsaturated fatty acids (PUFAs) within phosphatidylethanolamines (PEs). Genes like acyl-CoA synthetase long-chain family member 4 (Acsl4) and lysophosphatidylcholine acyltransferase 3 (Lpcat3) influence this process. While ferroptosis's role in cancer is well-studied, its significance in DIC is less understood. Acyl-CoA thioesterase 1 (Acot1), an enzyme crucial in fatty acid metabolism, catalyzes the conversion of fatty acyl-CoAs to free fatty acids and CoA-SH. Cardiomyocytes, with their high energy demands and reliance on fatty acid metabolism, may be particularly susceptible to ferroptosis due to mitochondrial lipid peroxidation. Previous research has demonstrated Acot1's ability to reduce oxidative stress and protect cardiac function in various disease models. Given Acot1's enzymatic function being opposite that of Acsl4, it was hypothesized that Acot1 could potentially inhibit ferroptosis in DIC. This study aimed to investigate the role of Acot1 in ferroptosis and its potential as a therapeutic target for DIC.

The literature extensively documents the cardiotoxicity of doxorubicin, implicating various forms of regulated cell death. Studies have shown the involvement of apoptosis, necroptosis, and pyroptosis in doxorubicin-induced cardiomyocyte damage. The role of disrupted iron metabolism and ROS generation is established, with iron dysregulation leading to increased ROS production and damage to cellular components. However, the limitations of antioxidant therapies have highlighted the complexity of the process. Recent research introduced ferroptosis as a novel mechanism contributing to doxorubicin cardiotoxicity. This study builds upon this emerging understanding, focusing on the potential role of Acot1 in modulating ferroptosis and influencing the overall outcome of doxorubicin-induced cardiac damage.

The study employed both in vivo and in vitro models. An in vivo sub-acute DIC model was established in C57BL/6 mice using intraperitoneal injections of doxorubicin. Cardiac function was assessed using transthoracic echocardiography, and RNA sequencing was performed on murine heart tissue to identify differentially expressed genes. In vitro studies utilized HL-1 cardiomyocytes. Acot1 expression was manipulated using siRNA-mediated knockdown and plasmid-mediated overexpression. Cell viability assays (CCK-8), measurements of intracellular glutathione, and lipid peroxidation assessment (C11-BODIPY 581/591) were used to evaluate ferroptosis and cellular damage. Transmission electron microscopy was used to analyze ultrastructural changes in cardiomyocytes. Gas chromatography-mass spectrometry (GC-MS) was used to analyze free fatty acid composition in the hearts of aMHC-Acot1 transgenic mice and their littermates. Statistical analysis included two-tailed unpaired Student's t-tests and one-way ANOVA with post-hoc tests.

Doxorubicin treatment in mice resulted in significant cardiac dysfunction, reduced survival rate, and morphological changes consistent with cardiac injury. RNA sequencing revealed downregulation of the biosynthesis of unsaturated fatty acid pathway and Acot1 as a leading-edge core gene in the doxorubicin-treated murine heart. Fer-1, a ferroptosis inhibitor, partially rescued cardiac dysfunction and improved survival in DOX-treated mice. In HL-1 cardiomyocytes, doxorubicin induced ferroptosis-like cell death, characterized by decreased glutathione levels, increased lipid peroxidation, and elevated Ptgs2 mRNA expression. Acot1 knockdown sensitized cardiomyocytes to doxorubicin-induced cell death, while Acot1 overexpression provided protection. GC-MS analysis of aMHC-Acot1 transgenic mice revealed altered free fatty acid composition in their hearts, particularly increased levels of docosahexaenoic acid (DHA) and stearic acid. In vitro studies showed that DHA increased cardiomyocyte sensitivity to doxorubicin, but this was counteracted by Acot1 overexpression, suggesting a protective effect of Acot1 related to lipid composition.

The study provides compelling evidence supporting the involvement of ferroptosis in doxorubicin-induced cardiotoxicity. The observed downregulation of Acot1 in doxorubicin-treated hearts and its correlation with increased ferroptosis vulnerability highlight its potential protective role. The findings suggest that Acot1's enzymatic function in shaping the lipid composition of cardiomyocyte membranes is crucial in determining their susceptibility to ferroptosis. The observation that Acot1 overexpression mitigates doxorubicin-induced damage, even in the presence of high levels of DHA, points towards Acot1's ability to regulate cellular lipid homeostasis beyond simply DHA concentrations. While ferroptosis inhibition offers partial protection, the incomplete rescue underscores the multifaceted nature of doxorubicin-induced cardiotoxicity, with other cell death mechanisms likely playing a role.

This study demonstrates a crucial role for Acot1 in protecting cardiomyocytes against doxorubicin-induced ferroptosis. Acot1's influence on lipid composition appears to be a key mechanism behind its cardioprotective effects. These findings suggest Acot1 as a potential therapeutic target for preventing doxorubicin-induced cardiotoxicity. Further research should focus on the precise mechanisms through which Acot1 modulates lipid metabolism and ferroptosis, as well as investigating the potential synergistic effects of Acot1 modulation combined with other cardioprotective strategies.

The study primarily focused on Acot1's role in ferroptosis, and other contributing factors to doxorubicin-induced cardiotoxicity warrant further investigation. While the transgenic mouse model provides valuable in vivo evidence, the complexity of cardiac function necessitates further studies to fully understand the long-term effects of Acot1 modulation in vivo. Additionally, the study mainly used a single cell line which limits generalizability across different cardiomyocyte types.