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. 2024 Jun 19;14(6):730.
doi: 10.3390/biom14060730.

Exogenous Iron Induces Mitochondrial Lipid Peroxidation, Lipofuscin Accumulation, and Ferroptosis in H9c2 Cardiomyocytes

Konstantin G Lyamzaev  1   2 , He Huan  1 , Alisa A Panteleeva  1 , Ruben A Simonyan  1 , Armine V Avetisyan  1 , Boris V Chernyak  1
Affiliations

Exogenous Iron Induces Mitochondrial Lipid Peroxidation, Lipofuscin Accumulation, and Ferroptosis in H9c2 Cardiomyocytes

Konstantin G Lyamzaev et al. Biomolecules. .

Abstract

Lipid peroxidation plays an important role in various pathologies and aging, at least partially mediated by ferroptosis. The role of mitochondrial lipid peroxidation during ferroptosis remains poorly understood. We show that supplementation of exogenous iron in the form of ferric ammonium citrate at submillimolar doses induces production of reactive oxygen species (ROS) and lipid peroxidation in mitochondria that precede ferroptosis in H9c2 cardiomyocytes. The mitochondria-targeted antioxidant SkQ1 and the redox mediator methylene blue, which inhibits the production of ROS in complex I of the mitochondrial electron transport chain, prevent both mitochondrial lipid peroxidation and ferroptosis. SkQ1 and methylene blue also prevented accumulation of lipofuscin observed after 24 h incubation of cardiomyocytes with ferric ammonium citrate. Using isolated cardiac mitochondria as an in vitro ferroptosis model, it was shown that rotenone (complex I inhibitor) in the presence of ferrous iron stimulates lipid peroxidation and lipofuscin accumulation. Our data indicate that ROS generated in complex I stimulate mitochondrial lipid peroxidation, lipofuscin accumulation, and ferroptosis induced by exogenous iron.

Keywords: ferroptosis; lipid peroxidation; lipofuscin; mitochondria; mitochondria-targeted antioxidants.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Ferric ammonium citrate induces ferroptosis in H9c2 cardiomyocytes. (a) Cells were incubated with FAC for 48 h. Cell viability was measured using the CellTiterBlue reagent. A total of 50 nM SkQ1 (red points) was added simultaneously with FAC. (b) Cells were incubated with 0.6 mM FAC alone and in combination with 50 nM SkQ1 or 0.1 mM Ferr-1 for 48 h, stained with 50 μg/mL propidium iodide (red) and 8 μM Hoechst 33,258 (blue) for 30 min, and analyzed using Olympus IX83. Bar 50 μm. (c) Cells were incubated with 0.6 mM FAC alone and in combination with 10 μM zVADfmk (zVAD), 0.1 mM Ferr-1, 50 nM SkQ1, 50 nM C12TPP, and 250 nM MB for 48 h. (d) Cells were incubated with 0.6 mM FAC alone and in combination with 50 nM SkQ1, 50 nM C12TPP, 0.1 mM Ferr-1, 0.2 mM Trolox, and 250 nM MB for 24 h. Then, cells were stained with 2 μM C11-BODIPY581/591 for 30 min and analyzed using flow cytometry. Ratio of green/red fluorescence was measured. Mean values are presented. (e) Cells were incubated with 0.6 mM FAC alone and in combination with 1 mM BSO for 48 h. A total of 50 nM SkQ1, 50 nM C12TPP, 0.1 mM Ferr-1, 0.2 mM Trolox, and 250 nM MB were added simultaneously with FAC and BSO. p < 0.05 (#)—the significance of the difference between control and FAC or FAC+BSO sample. p < 0.05 (*)—the significance of the difference between samples treated with FAC or FAC+BSO and other samples.
Figure 2
Figure 2
Ferrous ammonium citrate induces oxidative stress, ROS production, and lipid peroxidation in mitochondria. Cells were incubated with 0.6 mM FAC alone and in combination with 20 nM or 50 nM SkQ1, 50 nM C12TPP, 250 nM MB, or 0.2 mM Trolox for 24 h and analyzed using flow cytometry. (a) Cells were stained with 1 µM MitoSOX for 30 min. Mean values of fluorescence are presented. (b) Cells were stained with 1.8 µM CM-H2DCFDA for 30 min. Mean values of fluorescence are presented. (c) Cells were stained with 100 nM MitoCLox for 1 h. Ratio of green/red fluorescence was measured and analyzed using gating procedure. p < 0.05 (#)—the significance of the difference between control and FAC sample. p < 0.05 (*)—the significance of the difference between samples treated with FAC and other samples.
Figure 3
Figure 3
Ferrous ammonium citrate induces accumulation of lipofuscin-like material in H9c2 cells. Cells were incubated with 0.6 mM FAC alone and in combination with 20 nM or 50 nM SkQ1, 50 nM C12TPP, 250 nM MB, or 0.2 mM Trolox for 24 h and analyzed using flow cytometry without staining. Representative histograms (a) and mean values of fluorescence (b) are presented. p < 0.05 (#)—the significance of the difference between control and FAC sample. p < 0.05 (*)—the significance of the difference between samples treated with FAC and other samples.
Figure 4
Figure 4
Mitochondrial lipid peroxidation (a,b) and accumulation of lipofuscin-like material (c,d) induced by Fe2+ in isolated rat heart mitochondria. (a,b) Mitochondria were incubated with 50 nM MitoCLox and various concentrations of FeSO4 in the presence of glutamate (5 mM), malate (5 mM), rotenone (rot, 10 μM), or in the presence of succinate (succ, 5 mM) and antimycine A (Ant, 1 μM). A total of 300 nM SkQ1, 300 nM C12TPP, or 1 μM MB were added simultaneously with 50 μM FeSO4. Where indicated, rotenone was omitted or succinate and antimycine A were added. In the probe “no Fe”, 0.1 mM EGTA was added. Fluorescent spectra were analyzed after subtraction of light scattering. Kinetics of MitoCLox oxidation calculated as a ratio of fluorescence at 521 nm and 596 nm are shown. (c,d) Mitochondria were incubated with 50 μM FeSO4 in the presence of glutamate (5 mM), malate (5 mM), rotenone (10 μM), or in the presence of succinate (succ, 5 mM) and antimycine A (1 μM, Ant) for 40 min or 24 h at 37 °C and destructed with 0.5% SDS. A total of 300 nM SkQ1 or 300 nM C12TPP were added simultaneously with FeSO4. In the probe “no Fe”, 0.1 mM EGTA was added in the presence of glutamate (5 mM), malate (5 mM), and rotenone (10 μM). Fluorescent spectra at 360 nm excitation were analyzed. The representative spectra obtained by subtracting the 40 min spectra from the corresponding 24 h spectra (c) and fluorescence at 430 nM from these spectra (d) are shown. p < 0.05 (*)—the significance of the difference between samples treated with Rot+Fe and other samples.
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