Uncoupling Protein 2 Alleviates Myocardial Ischemia/Reperfusion Injury by Inhibiting Cardiomyocyte Ferroptosis Open Access

Ischemic heart disease (IHD) is a significant cause of morbidity and mortality worldwide [1]. Timely restoration of blood flow is the most logical and standardized approach for saving cardiomyocytes in patients with IHD; paradoxically, ischemia/reperfusion (I/R) induces additional cardiomyocyte death and increases the infarct size, which is referred to as myocardial ischemia/reperfusion injury (MI/RI) [2]. The development of MI/RI strongly limits the clinical therapeutic effects on IHD. Therefore, methods to alleviate MI/RI and improve the clinical treatment of IHD are urgently needed.

Complex network systems, such as oxidant stress, inflammation, and intracellular calcium overload [3‒5], are involved in the pathological mechanisms of MI/RI. In addition, mitochondrial dysfunction is a pivotal factor in MI/RI; during MI/RI, mitochondria are considered the main source of reactive oxygen species (ROS), and their structure and function undergo alterations following injury [6‒8]. Thus, preventing mitochondrial dysfunction in myocardial I/R is an important therapeutic strategy for cardioprotection. Uncoupling protein 2 (UCP2) is a proton transporter located in the inner mitochondrial membrane that modulates mitochondrial biogenesis and function. UCP2 is a significant member of the uncoupling protein family that is predominantly expressed in organs such as the heart and kidney. By uncoupling mitochondrial oxidative phosphorylation, UCP2 reduces the transmembrane potential of hydrogen ions and suppresses the production of ROS to ameliorate intracellular oxidative stress, thereby protecting tissues from injury [9]. Increasing evidence has confirmed that UCP2 has a protective effect on the heart. UCP2 is upregulated after myocardial I/R and can protect the heart from I/R injury by inducing mitochondrial autophagy [10]. Overexpression of UCP2 in cardiomyocytes inhibits the oxidative stress-induced mitochondrial death pathway [11]. Additionally, UCP2 can protect H9c2 cardiomyocytes from hypoxia/reoxygenation injury [12]. In contrast, shRNA-mediated Ucp2 silencing increases lipopolysaccharide-induced cardiomyocyte injury and oxidative stress [13]. However, the exact role of UCP2 in MI/RI and the mechanism are still not fully understood.

Various forms of cell death, such as apoptosis, necrosis, and autophagy, are involved in MI/RI [14‒16]. Although various types of cell death have been investigated for their ability to mitigate adverse effects, their use in ameliorating clinical I/R injury has not been satisfactory, and there have not been significant improvements in fatality rates [17]. There is a critical need to further explore the underlying mechanism of MI/RI and identify new targets for preventing this condition. With further research, ferroptosis was confirmed to be an important factor in MI/RI, and inhibiting ferroptosis can alleviate MI/RI [18]. Ferroptosis, which is a novel form of nonapoptotic programmed cell death characterized by lipid peroxidation that depends on ROS production and iron overload, was first proposed by Dixon et al. [19] in 2012. The main morphological features of ferroptosis include mitochondrial shrinkage, thickening of the mitochondrial membrane, decreased or absent cristae, and rupture of the outer mitochondrial membrane [20]. Interestingly, ferroptosis is characterized by the accumulation of ROS and mitochondrial dysfunction, and UCP2 inhibits ROS generation and protects mitochondrial function. Thus, we examined whether UCP2 exerted an antiferroptotic effect to protect the myocardium from MI/RI. The purpose of this study was to verify the cardioprotective effect of UCP2 against MI/RI, evaluate the effect of UCP2 on ferroptosis, investigate whether UCP2 inhibits ferroptosis to alleviate MI/RI by using the ferroptosis inhibitor ferrostatin-1 (Fer-1) and the inducer erastin (Era), and determine whether UCP2 inhibits ferroptosis in I/R-induced mouse hearts by inhibiting the p53/TfR1 pathway to prevent MI/RI.

Wild-type (WT) and Ucp2^−/− C57BL/6 mice (male, 6–8 weeks old) were purchased from Chengdu Dossy Experimental Animals Co., Ltd., and Jiangsu GemPharmatech Co., Ltd., respectively. The mice were kept in a specific-pathogen-free room for 1 week of acclimatization and were provided free access to food and water. All experiments were performed with the approval of the Laboratory Animal Welfare and Ethics Committee of the General Hospital of the Western Theater Command (Chengdu, Sichuan, China). The approval number was 2022EC1-005. The care and treatment of the animals were conducted in strict accordance with the Regulations on the Management of Experimental Animals.

The MI/RI model was generated as previously described [18, 21, 22]. The animals were fasted for 12 h before the experiments and given free access to water. The mice were placed in the supine position after being anesthetized by an intraperitoneal injection of 2% pentobarbital sodium (45 mg/kg). Subsequently, a 16-channel physiological recorder (PL3516, PowerLab) was used to monitor the II lead electrocardiogram (ECG). Then, the mice were orally intubated and mechanically ventilated on a small animal ventilator (DW-3000, Zhenghua). Core body temperature was maintained at 37°C. Then, a left thoracotomy was performed between the 3rd and 4th ribs to expose the heart, and the left anterior descending coronary artery was reversibly ligated for 30 min to induce ischemia. Cyanosis in the left ventricular wall and ST-segment elevation on the ECG indicated successful ischemia. After 30 min of regional ischemia, myocardial reperfusion was performed for 2 h by releasing the slipknot. The ischemic area turned red, and the elevated ST-segment retreated by more than half, indicating successful reperfusion. Mice in the sham group underwent the same surgical procedure without the ligature.

WT and Ucp2^−/− mice were randomly divided into four groups: the sham, I/R, I/R + Fer-1, and I/R + Era groups. To determine the relationship between UCP2 and ferroptosis, Fer-1 (0.8 mg/kg) [18, 23] (HY-100579, MCE, USA) and Era (50 mg/kg) (HY-15763, MCE, USA) were injected intraperitoneally 1 h before surgery in the I/R + Fer-1 and I/R + Era groups, respectively. Fer-1 and Era were dissolved in vehicle (DMSO:normal saline = 1:9, v/v). The sham and I/R groups were treated with an equal volume of vehicle (a mixture of DMSO and normal saline).

ECG was performed to evaluate cardiac function as previously described [24]. Briefly, after 2 h of reperfusion, the left ventricular end-diastolic diameter and left ventricular end-systolic diameter were examined in the two-dimensional long-axis view by B-ultrasound and M-ultrasound using a small animal high-frequency ultrasound system (Vevo3100LT, FUJI). Vevo LAB 5.5.1 (FUJI) data analysis software was used to calculate the left ventricular ejection fraction and left ventricular fraction shortening, which are indices of LV systolic function.

The cardioprotective effect of UCP2 was determined by triphenylttrazolium chloride (TTC) staining. The mice were sacrificed after a 2-h reperfusion period. After being washed in PBS, the heart was frozen at −20°C for 20 min. The heart was immediately cut into 2 mm-thick short-axis sections from the apex toward the base and incubated with 2% TTC (Solarbio, G3005) for 20 min at 37°C in the dark [25]. Then, the slices were observed and photographed. The noninfarcted areas were stained red with TTC, and the infarcted areas appeared grayish white after TTC staining. Image-Pro Plus 6.0 (Media Cybernetics, Inc., Sliver Spring, MD, USA) was used to quantify the infarct area and the total area of the heart slice, and the following calculation was performed: infarct rate (%) = infarct area/total area *100% [26, 27].

Hematoxylin-eosin staining [25, 28] was performed to observe the morphological changes in cardiac tissues. Briefly, after 2 h of reperfusion, myocardial tissues were collected, washed twice in PBS, fixed with 4% paraformaldehyde for 24 h at 4°C, and embedded in paraffin. Then, the tissues were cut into 4-μm-thick sections. After being dewaxed and hydrated, the slices were stained with hematoxylin and eosin (Biosharp, BL700B). Pathological changes in myocardial tissue were observed and assessed under an optical microscope (Leica, DM3000).

As described previously [29], Masson’s trichrome staining (Solarbio, G1340) was performed to determine the fibrotic area of the I/R-injured heart after 4 weeks of reperfusion. In brief, the heart was immediately removed and washed twice in precooled PBS. Then, the heart was fixed in 4% paraformaldehyde for 24 h at 4°C. Next, the heart tissue was prepared as conventional paraffin slides. After deparaffinization and rehydration, the paraffin slides were stained with Weigert’s iron hematoxylin solution for 5 min, followed by incubation with acid ethanol differentiation solution for 10 s. Then, the sections were stained with Masson bluing solution for 5 min and washed with water. Subsequently, the sections were stained with Ponceau for 5 min and washed with weak staining solution for 1 min, phosphorus or molybdenum staining solution for 1 min, and weak staining solution for 1 min. Finally, the sections were counterstained with aniline blue for 1 min and washed with weak staining solution for 1 min, dehydrated with 95% ethanol and anhydrous ethanol, cleared with xylene, and sealed with neutral resin. The collagen fibers appeared blue, and the viable myocardium was shown in red. Imaging of the heart sections were collected with a slide scanner (Olympus, VS200). The fibrotic areas were quantified using ImageJ software, version 1.53t (National Institutes of Health, Bethesda, MD, USA).

Sirius red (G-Clone, RS1240) staining was performed to measure the fibrotic area of the I/R-injured heart after 4 weeks of reperfusion. In brief, the heart was immediately removed and washed twice in precooled PBS. Then, the heart was fixed in 4% paraformaldehyde for 24 h at 4°C. Next, the heart tissue was prepared as conventional paraffin slides. After deparaffinization and rehydration, the paraffin slides were stained with Weigert’s iron hematoxylin solution for 10 min, incubated with acid ethanol differentiation solution for 5 s, and washed with water for 5 min. Then, the sections were stained with Sirius red for 1 h and washed with water. Finally, the sections were dehydrated, cleared, and sealed. The collagen fibers appeared red, and the muscle fibers appeared yellow. Imaging of the heart sections was performed with a slide scanner (Olympus, VS200). The fibrotic areas were quantified using ImageJ software.

A lactate dehydrogenase (LDH) activity assay kit (Solarbio, BC0685) and a creatine kinase (CK) activity assay kit (Solarbio, BC1145) were used to measure the activity of LDH and CK, respectively. In brief, after reperfusion, blood samples were collected and centrifuged for 10 min at 3,500 rpm to obtain serum. LDH and CK activity was analyzed by Multiskan Spectrum (Multiskan GO, Thermo) according to the manufacturer’s protocol.

The total iron and Fe²⁺ levels in heart tissues were assessed with tissue iron (Solarbio, BC4355) and Fe²⁺ (Solarbio, BC5415) assay kits, respectively. Briefly, after 2 h of reperfusion, the myocardial tissue was removed and homogenized. After centrifugation, the supernatant was collected and analyzed by Multiskan Spectrum (Multiskan GO, Thermo) according to the manufacturer’s protocol.

Myocardial tissues were homogenized with precooled lysis buffer, and the supernatant was collected for analysis. Lipid peroxide (LPO) (Nanjing Jiangcheng Institute of Biological Engineering, A106-1), malondialdehyde (MDA) (Solarbio, BC0025), superoxide dismutase (SOD) (Solarbio, BC0175), and glutathione (GSH) (Solarbio, BC1175) assay kits were used according to the kit instructions.

After 2 h of reperfusion, myocardial tissues were collected and washed twice in precooled PBS. Frozen sections (8 μm) were removed and washed twice in PBS as previously described [30]. The sections were incubated in dihydroethidium (Beyotime, S0063) (diluted with PBS to 10 μm) at 37°C for 60 min. A fluorescence microscope (Olympus, IX-81) was used to observe and collect the images. The average fluorescence intensity was calculated by Image-Pro Plus 6.0 to determine the ROS concentration.

Western blotting was performed to analyze protein expression as previously described [31]. In brief, myocardial tissue was homogenized with ice-cold RIPA lysis buffer containing protease inhibitors. Total protein was extracted and quantified using a BCA Protein Assay Kit (KGP902, KeyGEN, China). Samples containing ∼30 μg of protein were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, USA). Subsequently, the membranes were incubated with primary antibodies overnight at 4°C. After the membranes were incubated with secondary antibodies for 2 h at room temperature, the signals were measured by a chemical luminescence imaging system (Azure, A300). Densitometric quantification was carried out with ImageJ software. GAPDH was used as a loading control to calculate the relative expression levels of the target proteins.

The antibodies used for Western blotting were anti-UCP2 (CST, 89326), anti-GPX4 (Abcam, ab125066), anti-FTH1 (Abcam, ab183781), anti-ACSL4 (Abcam, ab155282), anti-TF (Abcam, ab82411), anti-p53 (Abcam, ab241566), anti-TfR1 (Abcam, ab269513), GAPDH (Bioss, bs-0755R), goat anti-rabbit (Bioss, bs-0295G-HRP, China), goat anti-mouse (Bioss, bs-0296G-HRP, China), and goat anti-rat (Bioss, bs-0293G-HRP, China).

Immunofluorescence analysis was performed to evaluate the protein levels of GPX4 and ACSL4, as previously described [32]. Briefly, cardiac tissues were fixed in 4% paraformaldehyde for 24 h, after which paraffin blocks were prepared. Then, the tissues were cut into 4-μm-thick sections. After being dewaxed, hydrated, and subjected to antigen retrieval, the slices were incubated in 5% BSA for 30 min at room temperature. Then, the sections were incubated with primary antibodies overnight at 4°C. After the sections were rewarmed, they were incubated with the fluorescently labeled secondary antibody for 2 h at room temperature. After being rinsed and stained with DAPI (Biosharp, BL739B), the slices were observed, and images were captured with a fluorescence microscope (Olympus, IX-81). The average fluorescence intensity was evaluated by Image-Pro Plus 6.0 to determine the protein levels of GPX4 and ACSL4.

Total RNA was extracted with TRIzol reagent (Thermo, 155596-026) according to the general experimental protocol for RNA isolation. Complementary DNA was synthesized using a reverse transcription kit (Takara, RR047A). Real-time quantitative PCR (RT-qPCR) was performed by using TB Green dye (Takara, RR820A) and an RT-qPCR instrument (CFX96, Bio-Rad). GAPDH served as an internal quantitative control. The relative mRNA expression of each gene was calculated by the 2^−△△Ct method [31, 33]. The RT-qPCR primer sequences are listed in Table 1 [21, 22].

A one-step mouse gene identification kit (Beyotime, D7284S) was used for PCR according to the manufacturer’s instructions. The sequences of the primers used were as follows: forward: 5′-ATG​AGA​TGT​TCC​TCG​TGT​CTC​G-3′; reverse: 5′-AGG​TGA​GGT​GGG​AAG​TAA​ATC​G-3′; forward: 5′-AGG​TGA​AGA​GTT​GAG​CAG​CTA​CTG-3′; and reverse: 5′-CAG​ATG​TGA​AAG​AGC​CAT​GAA​GC-3′.

First, 2% agarose gel was added. After the gel was completely dissolved in a microwave oven, it was cooled to 60°C in a water bath, after which a 1/20,000 volume of ethidium bromide (Solarbio, E1020) was added. Then, the mixture was gently shaken and poured into the mold, the comb was inserted, and the gel was cooled at room temperature for 30 min, after which the comb was vertically removed. The gel was placed into the electrophoresis tank, and the samples were added. After 30 min of electrophoresis, the gel was removed. The gel was observed and photographed using a gel imaging system (GelDoc XR+, Bio-Rad) [34].

GraphPad Prism 8.0.2 software (GraphPad Software, USA) and SPSS 25.0 (IBM Corp., USA) were used for statistical analysis. The data are shown as the mean ± standard deviation. Analysis of variance followed by Student’s t test was used to determine the significance of differences between two groups, and one-way ANOVA and Tukey’s post hoc test were used to determine the significance of differences among multiple groups. p < 0.05 was considered to indicate statistical significance.

To determine the role of UCP2 in MI/RI, we performed the procedures shown in Figure 1a. Successful I/R was verified by the presence of an ST segment on the ECG. As shown in Figure 1b, after ligation of the left anterior descending coronary artery in WT and Ucp2^−/− mice, ST-segment elevation was detected and was accompanied by visible cyanosis of the left ventricular wall. Following reperfusion, the ST segment exhibited a decrease exceeding 50%, while the left ventricular ischemic area exhibited conspicuous reddening. These findings suggest that the MI/RI model was successfully established. In subsequent experiments, we used Western blotting and RT-qPCR to evaluate the expression of UCP2 in the myocardial tissue of WT mice following myocardial I/R. As shown in Figure 1c, d, the protein and gene expression levels of UCP2 were notably increased compared to those in the sham group (p < 0.01).

Given the cardioprotective effects of UCP2 on the heart and its ability to protect against MI/RI [10‒12, 35], we conducted experiments using WT and Ucp2^−/− mice to confirm the cardioprotective effect of UCP2 on the MI/RI model. First, we confirmed the knockout of Ucp2 in Ucp2^−/− mice (Fig. 1e, f). The results showed that, compared to the I/R group of WT mice, Ucp2^−/− mice exhibited more severe cardiac remodeling and functional impairment following myocardial I/R, as evidenced by notable reductions in left ventricular ejection fraction (p < 0.01) and left ventricular fraction shortening (p < 0.05) (Fig. 2a, c, d; Table 2), as well as significant increases in LDH (p < 0.05) and CK (p < 0.01) activity (Fig. 2b, e) and in the myocardial infarct size (p < 0.05) (Fig. 3a, b). Additionally, the I/R group of WT mice exhibited disrupted cardiac structure, myocardial cell swelling and necrosis, irregular arrangement of myocardial fibers, and localized infiltration of inflammatory cells. Knocking out Ucp2 resulted in more pronounced damage in the I/R group of Ucp2^−/− mice compared to the I/R group of WT mice (Fig. 3c). Moreover, after Ucp2 ablation, the I/R group of Ucp2^−/− mice exhibited a notable increase in myocardial fibrosis compared to the I/R group of WT mice (p < 0.05) (Fig. 3d–g). These findings indicate that UCP2 plays a cardioprotective role in MI/RI.

Ferroptosis is characterized by iron overload, ROS accumulation, and lipid peroxidation. More ferroptosis was observed in the myocardium in the I/R group of Ucp2^−/− mice, and iron levels (total iron, Fe²⁺, FTH1, and TF) (p < 0.05) (Fig. 4a, b, g–i, l, m) and lipid peroxide levels (LPO, MDA, ACSL4, and ROS) (p < 0.05) (Fig. 4c, d, g, j, n, Fig. 5a–d) in the myocardial tissue of the I/R group of Ucp2^−/− mice were significantly higher than those in the I/R group of WT mice. Moreover, antioxidant levels (SOD, GSH, and GPX4) (p < 0.05) (Fig. 4e–g, k, o, Fig. 5e, f) were dramatically lower than those in the I/R group of WT mice.

To explore whether the cardioprotective effect of UCP2 involved the regulation of ferroptosis, we used the ferroptosis inhibitor Fer-1 and the inducer Era and evaluated various indicators of ferroptosis. After the administration of Fer-1, iron levels (total iron, Fe²⁺, FTH1, and TF) (p < 0.05) (Fig. 4 a, b, g–i, l, m) and lipid peroxides (LPO, MDA, ACSL4, and ROS) (p < 0.05) (Fig. 4c, d, g, j, n, Fig. 5a–d) in the myocardial tissues of the I/R + Fer-1 group of WT mice were markedly reduced, and these changes were accompanied by a significant increase in antioxidant levels (SOD, GSH, and GPX4) (p < 0.05) (Fig. 4e–g, k, o, Fig.5e, f). However, iron and lipid peroxide levels in the I/R + Fer-1 group of Ucp2^−/− mice were higher than those in the I/R + Fer-1 group of WT mice. Following treatment with Era, iron and lipid peroxide levels were dramatically increased in the I/R + Era group of WT mice, and there was a noteworthy decrease in antioxidant levels. However, iron and lipid peroxide levels in the I/R + Era group of Ucp2^−/− mice were markedly higher than those in the I/R + Era group of the WT mice, and antioxidant levels were markedly lower than those in the I/R + Era group of the WT mice. In conclusion, these findings suggest that UCP2 inhibits ferroptosis.

In subsequent experiments, cardiac structure and function were analyzed and revealed that after the administration of Fer-1, there were slight reductions in LDH and CK activities (p < 0.05) (Fig. 2d, e), myocardial infarct size (p < 0.05) (Fig. 3a, b), pathological myocardial injury (Fig. 3c), and myocardial fibrosis (p < 0.05) (Fig. 3d–g) in the I/R + Fer-1 group of WT mice. However, these indices were dramatically higher in the myocardial tissues in the I/R + Fer-1 group of Ucp2^−/− mice than in the I/R + Fer-1 group of WT mice. In contrast, after the administration of Era, LDH and CK activities, myocardial infarct size, myocardial pathological injury, and myocardial fibrosis in I/R + Era group of WT mice were significantly increased, while these indexes in the myocardium in the I/R + Era group of Ucp2^−/− mice were significantly higher than those in the I/R + Era group of WT mice. These findings indicate that UCP2 plays an antiferroptotic role and can alleviate MI/RI.

Research has shown that the p53/TfR1 pathway plays an important role in ferroptosis [36]. Therefore, to preliminarily determine the underlying molecular mechanisms involved, we assessed the expression of p53 and TfR1. The results revealed that following I/R, both WT and Ucp2^−/− mice exhibited significantly upregulated protein and mRNA expression of p53 and TfR1 in comparison to those in the corresponding sham groups (p < 0.01). However, the administration of Fer-1 led to a marked decrease in the levels of p53 and TfR1 in the I/R + Fer-1 group of WT mice. The levels of p53 and TfR1 in the I/R + Fer-1 group of Ucp2^−/− mice were dramatically higher than those in the I/R + Fer-1 group of WT mice (p < 0.05). Conversely, Era markedly increased the levels of p53 and TfR1 in the I/R + Era group of WT mice. However, the levels of p53 and TfR1 in the myocardial tissue of the I/R + Era group of Ucp2^−/− mice were markedly higher than those in the I/R + Era group of WT mice (p < 0.05) (Fig. 6a–e).

In the present study, we performed a series of in vivo experiments and first confirmed the upregulation of UCP2 in the myocardium after I/R and demonstrated that UCP2 significantly protected the myocardium from I/R injury, ultimately leading to improvements in cardiac function. Previous studies have shown that UCP2 can inhibit the generation of ROS, reduce oxidative stress, suppress cell apoptosis, and ameliorate mitochondrial dysfunction, thereby exerting a protective effect on the heart [10, 11, 37, 38]. Our results extend these findings. Knockout of Ucp2 resulted in increased ferroptosis. By intervening with ferroptosis inhibitors and inducers, we found that UCP2 could mitigate MI/RI by inhibiting ferroptosis to exert a cardioprotective effect. Furthermore, through preliminary mechanistic exploration, we found that UCP2 could inhibit ferroptosis by suppressing the p53/TfR1 pathway. These findings help to elucidate the molecular mechanisms of MI/RI and provide new insights on alleviating MI/RI and improving the prognosis of patients with IHD.

The discovery of ferroptosis in MI/RI has provided a new direction for the prevention and treatment of MI/RI. Some recent studies have focused on targeting ferroptosis to alleviate MI/RI through the use of ferroptosis inhibitors, such as Fer-1, deferoxamine, and liproxstatin-1; however, there are certain problems with these drugs, such as some being metabolically unstable and others requiring careful determination of their selectivity and adaptability in vivo [19, 39, 40]. Additionally, other scholars have found that some natural active substances can inhibit ferroptosis to reduce MI/RI, but their clinical application is limited by their low solubility, short half-life, and unstable chemical properties [41, 42]. Therefore, it is necessary to explore the potential molecular mechanisms and new therapeutic targets for MI/RI. Numerous studies have shown that UCP2 has a protective effect on the heart, but the specific mechanism has still not been fully elucidated. Considering that the characteristics of ferroptosis include the accumulation of ROS and mitochondrial dysfunction and that UCP2 can inhibit ROS production and protect mitochondrial function, we hypothesized that UCP2 could mitigate MI/RI by inhibiting ferroptosis. To investigate this hypothesis, we used Fer-1, which is a ferroptosis inhibitor, to inhibit ferroptosis and alleviate I/R injury and found that knockout of Ucp2 could reverse this effect. Similarly, when ferroptosis was induced using the ferroptosis inducer Era to exacerbate myocardial injury, knocking out UCP2 further exacerbated this effect. Taken together, these results suggest that UCP2 protects the myocardium from myocardial I/R damage by inhibiting ferroptosis.

Intraperitoneal injection of Fer-1 (1 mg/kg) 24 h and 2 h before MI/RI in mice significantly reduced the extent of myocardial infarction and prevented increases in LDH and creatine kinase-MB levels. In addition, intraperitoneal injection of Fer-1 every 2 days after surgery could significantly reduce I/R-induced cardiac remodeling and fibrosis [18]. Another study showed that intraperitoneal injection of Fer-1 (1 mg/kg) the day before and every day after MI/RI markedly reduced myocardial infarction size 24 h later, inhibited cardiac dysfunction, and alleviated myocardial fibrosis within 7 days [23]. The results of the present study showed that intraperitoneal injection of Fer-1 (0.8 mg/kg) 1 h before surgery could significantly reduce the myocardial infarction size, ameliorate myocardial pathological injury, inhibit the increases in LDH and CK activities, and significantly reduce I/R-induced myocardial fibrosis 4 weeks after reperfusion.

As a crucial tumor suppressor, p53 not only regulates processes such as the cell cycle and apoptosis but also controls ferroptosis [43, 44]. Studies have shown that in colon cancer cells, UCP2 exerts its antiapoptotic effect by reducing the production of ROS and inhibiting the phosphorylation of proteins within the trans-activated domain of p53 [45]. Furthermore, under hypoxic conditions, p53 can translocate to mitochondria, leading to the loss of mitochondrial membrane potential, the generation of ROS, the release of cytochrome c, and the activation of caspases, ultimately triggering ferroptosis [46]. However, the specific mechanisms that influence the mitochondrial translocation of p53 have not been identified. Research indicates that mitochondrial translocation of p53 is influenced by mitochondrial uncoupling. Notably, knocking out UCP2 in JB6 cells treated with the tumor promoter TPA significantly enhanced the mitochondrial translocation of p53 [47]. Further investigations are needed to determine whether UCP2 can inhibit ferroptosis to alleviate MI/RI by inhibiting the phosphorylation or mitochondrial translocation of p53. Furthermore, recent studies have demonstrated that spermidine/spermine N1-acetyltransferase 1 (SAT1) is a transcriptional target of p53 in cells such as human lung cancer H1299 cells, human melanoma A375 cells, and human breast cancer MCF7 cells. ROS-induced cell death in cells overexpressing SAT1 was inhibited by Fer-1 but not by the apoptosis inhibitor Z-VAD-FMK, the necrosis inhibitor necrostatin-1, or the autophagy inhibitor 3-methyladenine. Moreover, arachidonate 15-lipoxygenase (ALOX15) was identified as a downstream molecule of p53-induced SAT1 expression during ferroptosis [48]. Treatment of lung cancer A549 cells and mouse embryonic fibroblasts with the ferroptosis inducer Era can induce the accumulation of ROS, followed by the activation of p53. Activated p53 can in turn regulate and induce ROS production and increase the sensitivity of cells to ferroptosis. This is because activated p53 can inhibit the expression of the specific light chain subunit (SLC711A) of system Xc− and hinder the uptake of cystine, leading to a reduction in GSH synthesis and resulting in a decrease in GPX4 activity and cellular antioxidant capacity, thereby inducing ferroptosis [49, 50]. Additionally, this study revealed that UCP2 could inhibit the expression of p53. Therefore, UCP2 may ameliorate MI/RI by inhibiting the p53/SAT1/ALOX15 metabolic pathway and thereby inhibiting iron death; however, further investigation is needed. These findings require further validation in vitro.

All animal treatments were approved by the Laboratory Animal Welfare and Ethics Committee of General Hospital of Western Theater Command (approval number 2022EC1-005).

The authors declare no conflicts of interest.

This work was supported by the National Nature Science Foundation of Sichuan Province (No. 2022NSFSC0738).

P.-T.Z. and Y.-L.Z. contributed equally to this work. P.-T.Z., Y.-L.Z., X.C., and L.-F.Z. made significant contributions to the study conception and design. P.-T.Z., Y. -L.Z., K.-W. X., Y.-C.L., J.H., and Q.-Z.Y. contributed to the completion of the experiment and data acquisition, analysis, and interpretation. P.-T.Z. contributed to the writing of the manuscript, and X.C. and L.-F.Z. made strict revisions to the manuscript. All the authors have approved the final version of the manuscript.

The data that support the findings of this study are not publicly available due to privacy reasons but are available from the corresponding author upon reasonable request.