Specific pathogen-free healthy male C57BL/6 mice (8 weeks old, 20–25 g) were obtained from the Laboratory Animal Center of Nantong University (Nantong, China). The Animal Ethics Committee of Nantong University approved all animal studies, which were consistent with the Guide for the Care and Use of Laboratory Animals. The animal model mice were raised in a standard laboratory environment that consisted of a 12-hour light/dark cycle, with a constant temperature of 25 °C. The mice had free access to animal feed and tap water. Mice were randomized into Sham+sesame oil (as a control for E${}_2$), Sham+E${}_2$, transverse aortic constriction (TAC)+sesame oil, and TAC+E${}_2$ groups (n = 6/group). After inducing anesthesia with 4% isoflurane, anesthetization was maintained via the inhalation of 1.5% isoflurane through a mask. After cutting open the sternum, a chest expander was used to open the chest so that the thymus could be separated, exposing the aortic arch and its branches. A 5-0 suture was passed through the soft tissue under the aortic arch using an L-shaped needle prepared in-house, after which a 27G probe was placed on the needle and ligated to narrow the vessel. In the sham group, the suture was placed through the soft tissue under the aortic arch without ligation, while all other steps were the same as in the experimental group. E${}_2$ was subcutaneously injected for 6 consecutive weeks beginning 2 days after the procedure, using the same dose as in prior studies [14] (dissolving E${}_2$ (20 µg/kg bw/day) dissolved in sesame oil (1 mL/kg), utilizing ethanol (10 µg/µL) as an adjuvant) or an equivalent volume of sesame oil.

Information on the materials used in this study can be found in the Supplementary Material.

Measurements of the left ventricular end-diastolic diameter (LVIDd), left ventricular end-systolic diameter (LVIDs), left ventricular end-diastolic interventricular septum thickness (IVSd), and other indices were made along the long axis and short axis sections of the left ventricle. M-mode ultrasound measurements were processed to calculate indicators that reflect left ventricular systolic function, including left ventricular ejection fraction (LVEF) and fractional shortening (FS). Cardiac functional parameters were measured based on the average values from 3–5 cardiac cycles.

Cardiac tissues were fixed for 24 h using 4% paraformaldehyde, dehydrated with an ethanol gradient (75%, 85%, 95%, 95%, 100%, 100%), treated with xylene, immersed for 3 h in pre-dissolved paraffin, and placed in an embedding box and frozen at –20 °C. A microtome was then used to cut 5 µm sections that were stored at room temperature. For analysis, these sections were immersed twice in xylene (10 min each), rehydrated with an ethanol gradient (100%, 100%, 95%, 85%, and 75%; 5 min each), washed for 5 min with phosphate buffered saline (PBS), stained with hematoxylin, washed for 5 min with distilled water, and immersed in 1% HCl for 30 min for differentiation. Then, samples were incubated for 30 min in 0.1% ammonia in water, rinsed, immersed for 5 min in eosin staining solution, dehydrated using 90%, 95%, and 100% ethanol for 20 s each, incubated twice for 10 min in xylene, sealed using glue, allowed to air dry, and analyzed via microscopy.

Place the sections in the following order in xylene I for 20 minutes, xylene II for 20 minutes, absolute ethanol I for 10 minutes, absolute ethanol II for 10 minutes, 95% alcohol for 5 minutes, 90% alcohol for 5 minutes, 80% alcohol for 5 minutes, 70% alcohol for 5 minutes, and then wash with distilled water. Stain the cell nuclei with hematoxylin: immerse the sections in Weigert’s hematoxylin stain from the Masson staining kit for 5 minutes, wash with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, wash with tap water, and rinse with running water for several minutes to return blue. Stain with eosin and phloxine for 5–10 minutes using the Masson staining kit, and quickly wash with distilled water. Treat with phosphomolybdic acid: immerse the sections in the phosphomolybdic acid solution from the Masson staining kit for about 3–5 minutes. Do not wash with water, and directly stain with Fast Blue solution from the Masson staining kit for 5 minutes. Differentiate: treat with 1% acetic acid for 1 minute. Dehydrate and clear the sections by immersing them in 95% alcohol I for 5 minutes, 95% alcohol II for 5 minutes, absolute ethanol I for 5 minutes, and absolute ethanol II for 5 minutes, and then in xylene I and xylene II for 5 minutes. Remove the sections from xylene and allow them to air-dry slightly. Mount the sections with neutral resin and cover them with a cover slip. Examine the sections under a microscope and capture and analyze the images.

Hand-held suckling mice (1–2 days old), scrub the chest and abdomen with 75% alcohol, then use an ophthalmic scissor to cut open the sternum and extrude the heart, put it into a PBS dish and wash it repeatedly, then transfer it into a small glass bottle and fully cut it into a pulpy shape. The cut heart was transferred into a blue-covered wide-mouth bottle containing 10 mL collagenase, gently blow the tissue fragments, and place them into a constant temperature oscillator at 37 °C for 5 minutes. The cell suspension after oscillation was transferred to a 15 mL centrifuge tube, centrifuged at 1000 rpm for 5 minutes, and the rest of the heart tissue was added with 10 mL collagenase, gently blown, and then placed into a constant temperature oscillator at 37 °C for 5 minutes. After centrifugation, 2 mL of complete culture was added to the cells, and the cells were blown evenly and left standing. The suspension after centrifugation was digested again, and the cycle was repeated for 7 times in total to make the tissue completely digested. All the cells collected above were collected and transferred into a new centrifuge tube, and then centrifuged again, and the supernatant was discarded. Then add an appropriate amount of complete medium to the centrifuge tube and gently shake to re-suspend the cells. After filtration with a cell sieve, transfer the cells to a Petri dish. Add Brdu (5-bromo-2-deoxyuridine) to the medium and place it in an incubator for 1.5 hours at a differential speed. The cells that are adherent to the wall are fibroblasts, and the cells that are not adherent to the wall are cardiomyocytes. Take the supernatant and inoculate it in a Petri dish. Change to serum-free medium after 48 hours, and starve for 24 hours before drug addition and transfection.

All cell lines were validated by short tandem repeat (STR) profiling and tested negative for mycoplasma. Retrieve the cryovial labeled with H9C2 cells and thaw it rapidly in a 37 °C water bath. Cells were all cultured in a humidified incubator at 37 $\mathrm{℃}$ and 5% CO${}_2$. Using a pipette, carefully mix 2 mL of cell suspension containing 10% fetal bovine serum (FBS) from fully grown to frozen vial cells by gentle pipetting. Centrifuge at 1000 rpm for 5 minutes, disinfect the centrifuge tube with alcohol, and discard the supernatant in a laminar flow hood. Allow the cell sediment to be suspended in complete culture medium for 5 minutes. Transfer the cell mixture to a culture dish using a sterile pipette and place it in a 37 °C incubator for growth and incubation. Replace the culture medium after 24 hours of incubation and continue cultivation. Upon reaching 80%–90% confluence, perform a subculture at a ratio of 1:3: Discard old culture medium, wash three times with PBS buffer solution, add 1.5 mL of digestive solution (0.25% trypsin), then incubate cells at 37 °C for five minutes under microscopic observation. When cells assume round granular shape due to digestion termination, gently resuspend them using a pipette; subsequently centrifuge at 1000 rpm for five minutes, discard supernatant, add fresh complete culture medium to suspend cells before transferring them into a culture dish containing approximately four milliliters of complete culture medium.

The cells were inoculated into 96-well plates at a concentration of 5 $\times$10${}^3$ cells/well and cultured for 24 hours at 37 ℃, 95% air and 5% CO${}_2$ environment. The cells were treated with different concentrations (1, 5, 10, 25, 50, 100, 150, 200 µm) of E${}_2$, and 6 groups of parallel culture groups were set up for each concentration. After continued culture for 24 h, the cells were incubated with CCK-8 (10 µL/well) for 2 hours, and the spectrophotometric quantification was performed at 450 nm. Similarly, after 24 hours of cell culture, E${}_2$ was added in increasing concentrations (1, 5, 10, 25, 50, 100, 150, 200 µm), and 6 groups of parallel culture groups were set up for each concentration. After continued culture for 24 hours, the cell survival rate was determined by using a rapid and sensitive CCK-8 detection kit. Optical density (OD) value, which directly reflects the survival status and vitality of cells. The survival rate of cardiomyocytes in the control well was set as 100%, and the percentage of other wells was calculated accordingly.

Negative control siRNA (si-NC) and si-CoQ were synthesized by GenePharma (Suzhou, China). Use Lipo3000 transfection reagent to transfect cells according to the manufacturer’s scheme. Add an appropriate amount of Lipo3000 transfection reagent to a certain amount of Opti-Minimal essential medium (Opti-MEM) medium and mix it thoroughly; then use a certain amount of serum-free DMEM and an appropriate amount of si-NC or si-CoQ transfection reagent to mix it thoroughly; shake and mix the two dilutions that have been configured at a ratio of 1:1, and place them at room temperature for 20 min. Add the above mixture to the well plate containing cells one by one with a pipette gun, and then place it in the cell incubator for incubation. After 6 hours, replace it with fresh culture, and then culture for 24–48 hours.

Total RNA was extracted from the above-mentioned mouse heart tissues and cells. The reverse transcription reaction system was prepared by using the reverse transcription kit (G3337), gently mixed and centrifuged, and the reverse transcription program was set to complete the reverse transcription on the PCR instrument. Take 0.1 mL PCR reaction plate, prepare the following reaction system, and prepare 3 tubes for each reverse transcription product. After the sample was added, the PCR sealing plate membrane was used to complete the sealing with the sealing plate instrument, and the microwell plate centrifuge was used to complete the centrifugation. Then the amplification was completed on the fluorescence quantitative PCR instrument.

Total protein was extracted from mouse heart tissues or H9C2 cells. The bicinchoninic acid (BCA) protein detection kit (ThermoFisher, MI, USA) was used to measure the protein content. According to the measured protein concentration, 5$\times$Loading Buffer was added in proportion, and the protein was cooked at 100 °C for 10 minutes after vortex oscillation and mixing. An equal amount of protein is loaded onto a sodium dodecyl sulfate (SDS)-polyacrylamide gel. The gel was prepared by using Omni-Easy one-step PAGE gel rapid preparation kit (PG213, EpiZyme, China), and then prepare the electrophoresis solution, confirming that the positive and negative electrodes are connected correctly before electrophoresis. Set the program as follows: 80 V constant voltage for 40 minutes, then switch to 120 V until the bromophenol blue band reaches the bottom of the glass plate. The primary and secondary antibodies were incubated after being blocked by rapid blocking solution for 20 min. After washing the membrane with Tris-Borate-Sodium Tween-20 (TBST), the membrane was put into a chemiluminescence imaging system and a small amount of configured electrochemiluminescence (ECL) luminescence solution was added to the membrane to display the strip, and the image analysis software (Image J, Bethesda, MD, USA) was used for quantitative analysis. The following antibodies were used: DHODH Antibody, E9X8R, CellSignaling, USA; CoQ Antibody, A15193, Abclonal, China; GPX4 Antibody, HY-P80450, MCE, China; SLC7A11 Antibody, D2M7A, CellSignaling, USA; FSP1 Antibody, A21808, Abclonal, China; GAPDH Antibody, A19056, Abclonal, China; PCBP1 Antibody, A19276, Abclonal, China; TFR Antibody, A5865, Abclonal, China; Ferritin Light Chain Antibody, A11241, Abclonal, China; IgG H&L Antibody, AB150081, Abcam, USA.

Cell samples were homogenized or lysed using PBS or lysis buffer, and the supernatant was collected by centrifugation. Thiobarbituric acid (TBA) storage solution and MDA detection working solution were prepared and dissolved by heating. For the measurement, add the blank control, standard, or sample to the centrifuge tube, then add the MDA detection working solution. Mix well and heat for 15 minutes. Cool to room temperature, centrifuge, and collect the supernatant. Transfer the supernatant to a 96-well plate and measure the absorbance at 532 nm using a microplate reader. Calculate the molar concentration of MDA based on the standard curve.

In accordance with the standard process, the cells were collected and cleavage, and then the BCA protein analysis kit was used for the accurate determination of protein concentration. At the same time, the ferrous ion colorimetric test kit was used for the scientific analysis of ferrous ion content: the probe was loaded with ferrous ions in the cells, and the optical density value (OD value) of the generated substance at the strong absorption peak of 593 nm wavelength was determined, and the corresponding calculation was performed, so as to determine the content of ferrous ions (Fe${}^{2+}$) in the cells.

Fresh cell model was measured according to the standard procedure. The cells were washed twice with PBS and centrifuged. The supernatant was discarded, and protein removal reagent was added, followed by full eddy current oscillation. The samples were rapidly frozen-thawed twice with liquid nitrogen and 37 °C water bath, and then placed in a 4 °C refrigerator or ice bath for 5 minutes, and then centrifuged for 10 minutes at 4 °C. The supernatant was used for the determination of GSH. The absorbance was measured at 405 nm with an Enzyme-Linked Tmmunosorbent Assay (ELISA) kit, and compared with the standard curve.

Place the cells in a PBS solution containing 3% to 4% formaldehyde at room temperature for 10 to 30 minutes to fix them. Then, suck out the fixative solution and wash the cells with PBS 2 to 3 times. To quench excess formaldehyde, place the cells in a PBS solution containing 10 mM ethanolamine (or 0.1M glycine) for 5 minutes. Next, add a PBS solution containing 0.1% Triton X-100 to the fixed cells, and incubate them for 3–5 minutes to enhance their permeability. Then, wash the cells with PBS 2–3 times again. Add DAPI solution to the cell culture medium and incubate the cells at 37 °C for about 5 minutes. Wash the cells with PBS 2–3 times again. Then, add a sealing agent to preserve the fluorescence (if using a cover slip, seal the cells with a cover slip). Finally, observe the cells under the excitation/emission wavelength of 493/517 nm.

If the data follows a normal distribution, ANOVA (Analysis of Variance) can be used, which is a method to compare the differences in means between two or more groups assuming that the data comes from a normal distribution. If the data does not follow a normal distribution, Kruskal-Wallis test should be chosen. This is a non-parametric method that does not depend on the distribution assumption of the data and is mainly used to compare whether the median of three or more independent samples is equal. Cell culture and animal experiments were independently performed at least three times. Data are reported as means $\pm$ SEM, and were analyzed using GraphPad Prism 9.3.0. (Dotmatics, Boston, MA, USA) and differences between groups were compared via Student’s t-tests or one-way ANOVAs with Bonferroni post hoc tests. p 0.05 was selected as the threshold of significance.

C57BL/6 mice were initially subjected to TAC surgery in order to generate a murine model of HF (Fig. 1A), after which animals in the experimental group were injected subcutaneously with E${}_2$. At 8 weeks following the TAC procedure, echocardiographic analyses revealed that the LVEF and left ventricular FS in the TAC group were significantly reduced as compared to the TAC group compared with the sham group (Fig. 1A). After 8 weeks of TAC surgery, we used Western blot analysis to detect the expression level of DHODH, and found that the expression level of DHODH was decreased after TAC surgery, while the expression level of DHODH was up-regulated by E${}_2$ administration (Fig. 1B,C). The partial restoration of cardiac function was observed in E${}_2$-treated mice (Fig. 1D–F). Mice in the TAC group also presented with characteristic features of HF including a significant increase in heart volume, heart weight to body weight ratio, and heart weight to tibial length ratio (Fig. 1G–I). Masson staining analyses of the fibrotic area in cardiac tissue samples from these mice revealed that TAC model mice treated with E${}_2$ exhibited a reduction in the extent of cardiac fibrosis as compared to the TAC group (Fig. 1H). Masson and WGA staining approaches additionally revealed significant increases in the cross-sectional cardiomyocyte area in the TAC group, with E2 treatment having significantly reversed these changes (Fig. 1J,K). These data suggest that E${}_2$ can have a beneficial effect in TAC model mice, partially alleviating cardiac hypertrophy and fibrosis. The mRNA levels of HF and myocardial fibrosis-associated markers including ANP, BNP, $\beta$-MHC, Acta1, CTGF, and COL1a1 were also significantly increased following TAC surgery, while their upregulation was suppressed in the cardiac tissue of TAC model mice treated using E${}_2$ (Fig. 1L).

Fig. 1.

Estradiol (E${}_{\mathrm{2}}$) treatment promotes Dihydroorotate dehydrogenase (DHODH) upregulation in murine cardiomyocytes and alleviates transverse aortic constriction (TAC)-induced cardiac hypertrophy and fibrosis. (A) Construction of a mouse model of heart failure and a timeline of drug treatments. (B,C) Representative Western blot and quantitative analysis showing DHODH expression in cardiomyocytes. (D–F) Left ventricular M-mode echocardiographic images and parameters, including ejection fraction (EF) and fractional shortening (FS) of the left ventricle short axis in mice. (G) Overall cardiac morphology and hematoxylin-eosin (H&E)-stained sections (40- and 200-fold) were used to assess cardiomyocyte morphology in mice. Scale bar: 1000 µm, 50 µm. (H,I) Bar graphs showing quantitative data on heart weight/body weight (HW/BW) and heart weight/tibial length (HW/TL). (J) Detection of the degree of cardiomyocyte fibrosis and corresponding quantification results by Masson staining. Scale bar: 50 µm. (K) Wheat germ agglutinin (WGA) staining and cell surface area quantification showing cardiomyocyte cross-sectional area (CSA). Scale bar: 20 µm. (L) Q-PCR detection of mRNA expression levels of cardiomyocyte fibrosis markers ANP, BNP, $\beta$-MHC, Acta1, CTGF, and COL1a1. Data are shown as mean $\pm$ SEM. ****p 0.0001, ***p 0.0005, **p 0.005, *p 0.05, n = 3 per group.

As the above results support the successful establishment of the murine TAC model system, analyses of iron levels in murine cardiomyocytes were next conducted. A significant increase in these iron levels was observed in mice from the TAC group, whereas iron concentrations in mice from the TAC+E${}_2$ group were reduced as compared to mice from the TAC group (Fig. 2A). Significantly elevated MDA levels were observed in the cardiac tissue of mice from the TAC group as compared to the control group, while these levels were restored to baseline following treatment with E${}_2$. Conversely, TAC mice presented with significant reductions in cardiac GSH levels, while this change was reversed in animals that had undergone E${}_2$ treatment (Fig. 2B,C).

Fig. 2.

E${}_{\mathrm{2}}$ suppresses cardiomyocyte ferroptosis in TAC model mice. (A–C) Measurement of iron (A), Malondialdehyde (MDA) (B), and Glutathione (GSH) (C) levels in mouse cardiomyocytes using commercial kits. (D,E) Representative Western blot and quantitative analysis showing the expression of phospholipid hydroperoxidase (GPX4), solute carrier family 7 member 11 (SLC7A11), ferroptosis suppressor protein 1 (FSP1), coenzyme Q (CoQ), and Poly(rC) Binding Protein 1 (PCBP1) in cardiomyocytes. Data are shown as mean $\pm$ SEM. ****p 0.0001, ***p 0.0005, **p 0.005, *p 0.05, n = 3 per group.

Western blotting was next used to analyze the expression of ferroptosis-associated factors in tissue samples from these mice including DHODH, GPX4, SLC7A11, FSP1, CoQ and PCBP1, with the goal of further confirming the anti-ferroptotic effects of E${}_2$in vivo. These analyses revealed that relative to the TAC group, GPX4, SLC7A11, FSP1, and CoQ were upregulated, whereas the opposite was true for the ferroptosis marker PCBP1. Treatment with E${}_2$ was sufficient to partially reverse these effects (Fig. 2D,E). These data thus underscore the value of further studies focused on the mechanisms underlying these phenotypic outcomes.

To probe the mechanistic role of DHODH in cardiomyocytes, a series of in vitro experiments was next conducted. Initially, H9C2 cardiomyocytes were treated for 24 h with a range of E${}_2$ concentrations (1–200 µm), and a CCK-8 kit was used to quantify cell survival. No toxic effects of E${}_2$ treatment were observed within this dose range. In light of prior studies [14], E${}_2$ was selected for use at a 10 µm concentration in further experiments (Fig. 3A,B). A PE dose of 50 µm was also selected based upon prior research [23].

Fig. 3.

DHODH upregulation protects against phenylephrine (PE)-induced cardiomyocytes with failure. (A,B) Effects of different concentrations of E${}_2$ on the optical density values and cell viability of H9C2 cells. (C,D) Representative images of ghost pen peptide fluorescence staining and cell surface area quantification of primary cardiomyocytes. Scale bar: 50 µm. (E–G) Determination of Fe${}^{2+}$ (E), MDA (F) and GSH (G) levels in primary mouse cardiomyocytes using commercial kits. (H,I) Western blotting and quantitative analysis showing the expression levels of DHODH, Ferritin light chain, transferrin receptor (TFR) and PCBP1 in H9C2 cells. Data are shown as mean $\pm$ SEM. ****p 0.0001, ***p 0.0005, *p 0.05, compared with PE group; ns, not significant. n = 3 per group, and the cell survival rate in control group was set at 100%.

The impact of E${}_2$ on the morphological changes associated with PE-induced cardiomyocyte hypertrophy was next assessed via a ghost pen cyclic peptide fluorescence staining approach. Primary cardiomyocytes that had not undergone any treatment were fusiform and exhibited a uniform distribution, whereas following PE stimulation there was a significant increase in cell volume and greater morphological irregularity. This suggests that PE can induce cardiomyocyte failure. Relative to PE-treated primary cardiomyocytes, those cells that underwent E${}_2$ treatment exhibited more normal morphological characteristics and a significant reduction in volume (Fig. 3C,D). E${}_2$ treatment can thus significantly inhibit PE-induced primary cardiomyocyte failure.

Further analyses of Fe${}^{2+}$ levels and the lipid peroxidation markers GSH and MDA revealed significant increases in Fe${}^{2+}$ and MDA levels in cardiomyocytes treated with PE, whereas there was a significant decrease in GSH levels. E${}_2$ significantly reversed these observed changes in PE-treated hypertrophic cardiomyocytes (Fig. 3E–G).

Subsequently, a ferroptosis-specific inhibitor Ferrostatin-1 (Fer-1) was added as a positive control. Analysis of protein levels of DHODH, Ferritin light chain and transferrin receptor (TFR), which are downregulated in ferroptosis, and ferroptosis marker PCBP1 further showed that these three proteins were significantly upregulated in PE-induced primary cardiomyocytes with failure, but returned to baseline levels after E${}_2$ treatment, which was consistent with the results of the Fer-1 group. In summary, these experiments demonstrated the ability of E${}_2$ to resist ferroptosis (Fig. 3H,I).

To further interrogate the effects that DHODH has on mitochondria, transmission electron microscopy was employed to assess mitochondrial changes in the cardiomyocytes of TAC model mice. These analyses revealed significantly reduced mitochondria in TAC model mice, with the remaining mitochondria exhibiting disorderly arrangement, outer membrane rupture, and the partial or total loss of cristae. In contrast, TAC+E${}_2$ treatment was associated with regularly arranged mitochondria with more cristae and substantially reduced mitochondrial outer membrane rupture (Fig. 4A).

Fig. 4.

DHODH protects against PE-induced ferroptosis in cardiomyocytes via enhancing mitochondrial function. (A) Representative image of mitochondrial ultrastructure morphology in mouse cardiomyocytes. Scale bar: 5 µm, 2 µm, 500 nm. (B) Representative JC-1 green/red fluorescence image. Scale bar: 50 µm. (C,D) Detection of intracellular reactive oxygen species (ROS) levels in H9C2 cells using DCFH-DA fluorescence probe. Scale bar: 100 µm. (E) Detection of ATP concentration in H9C2 cells using mouse adenosine triphosphate (ATP) ELISA kit. Data are shown as mean $\pm$ SEM. ****p 0.0001, ***p 0.0005, n = 3 per group.

The impact of E${}_2$ on mitochondrial membrane potential in H9C2 cardiomyocytes that had been treated with PE was next evaluated via JC-1 fluorescent staining. JC-1 polymerizes within the mitochondrial matrix under normal conditions and emits red fluorescence, whereas it remains in a monomeric state in cells with damaged mitochondria owing to a drop in mitochondrial membrane potential. Cardiomyocytes in the control and E${}_2$ treatment groups exhibited a strong red fluorescent signal, whereas PE treatment was associated with a significant increase in green fluorescence. The simultaneous treatment of cells with PE and E${}_2$ significantly reduced this induction of green fluorescence. E${}_2$ treatment can thus protect against the loss of mitochondrial membrane potential (Fig. 4B).

To explore the in vitro inhibition of ferroptotic cell death by E${}_2$, 2${}^{\prime }$,7${}^{\prime }$-Dichlorodihydrofluorescein diacetate (DCFH-DA) was employed to detect ROS levels in H9C2 cells. These analyses indicate that relative to the control or E${}_2$ treatment groups, ROS levels were significantly elevated following PE treatment for 48 h, while E${}_2$ was sufficient to reverse this PE-induced ROS production (Fig. 4C,D).

Mitochondrial function was analyzed by detecting ATP concentrations in H9C2 cells via ELISA. A significant decline in ATP levels was detected in PE-treated H9C2 cells, while treatment with both PE and E${}_2$ led to an increase in ATP concentrations (Fig. 4E). This suggests that DHODH upregulation can have reparative effects on damaged mitochondria in cardiomyocytes.

To confirm the functional role that CoQ plays as a regulator of the anti-ferroptotic effects of DHODH, the knockdown of CoQ in H9C2 cells was performed prior to PE treatment, with Western blotting being used to confirm successful CoQ knockdown following si-CoQ transfection. Cells treated with PE also exhibited lower levels of ferroptosis-related proteins including DHODH, Ferritin light chain, and TFR as compared to control levels, with the expression of these proteins being significantly elevated in the PE+E${}_2$ group relative to the PE-treated group. However, CoQ knockdown was sufficient to disrupt the upregulation of these genes (Fig. 5A,B). CoQ knockdown in the PE+E${}_2$+si-CoQ treatment group also prevented E${}_2$-mediated reductions in ROS levels (Fig. 5C,D). When an ELISA approach was employed to detect ATP levels in H9C2 cells, PE treatment was found to significantly reduce ATP concentrations. PE+E${}_2$ treatment was associated with higher ATP concentrations, while the silencing of CoQ reversed this increase (Fig. 5E).

Fig. 5.

DHODH regulates CoQ to exert cardioprotective effects and prevent ferroptotic induction. (A,B) Western blotting and quantitative analysis of DHODH, CoQ, Ferritin light chain and TFR expression in H9C2 cells. (C,D) Determination of ROS levels in H9C2 cells using DCFH-DA fluorescent probe. Scale bar: 100 μm. (E) Detection of ATP concentration in H9C2 cells using mouse adenosine triphosphate (ATP) ELISA kit. (F–H) Determination of Fe${}^{2+}$, MDA and GSH levels in H9C2 cells using commercial kits; Data shown as mean $\pm$ SEM; ns, not significant. ****p 0.0001, ***p 0.0005, n = 3 per group.

Fe${}^{2+}$ concentrations, as well as levels of MDA and GSH, were also analyzed to explore the link between CoQ and ferroptosis, revealing that the knockdown of CoQ was sufficient to attenuate the anti-ferroptotic effects associated with DHODH upregulation (Fig. 5F–H).