HIF-1α-mediated mitochondrial autophagy in the post-treatment protective effect of thyroxine against myocardial ischemia-reperfusion injury

Abstract

Background

This study aimed to investigate the role of HIF-1α-regulated mitochondrial autophagy in the protective effect of Thyroxine against myocardial ischemia-reperfusion injury.

Methods

Male Sprague-Dawley rats were randomly divided into five groups (n = 10 each): Sham, I/R, L-Thyroxine (T4) post-treatment (20 µg/kg i.p. immediately after reperfusion), 2ME2 + I/R (15 mg/kg 2ME2 i.p. 30 min before ischemia), and 2ME2 + Thyroxine groups. The I/R model was established by ligating the left anterior descending coronary artery for 40 min followed by 120 min of reperfusion.

Results

Compared with the I/R group, Thyroxine treatment significantly up-regulated HIF-1α (2.<0.01) and BNIP3 (1.8 < 0.01) protein expressions, reduced the LC3-II/LC3-I ratio (0.6-fold, P < 0.01), Beclin-1 (0.<0.01) and P62 (0.40.01) accumulation, while increasing LAMP2 expression (1.6-fold, P < 0.01). Thyroxine treatment reduced ROS production from 80% to 40% DHE-positive nuclei (P < 0.01), increased ATP content from 45% to 75% of sham levels (P < 0.05), restored mitochondrial membrane potential (JC-1 red/green ratio increased from 0.4 to 0.8, P < 0.01), and decreased myocardial infarct size from 45% to 20% of the area at risk (P < 0.01). These protective effects were abolished by the HIF-1α inhibitor 2ME2 pre-treatment.

Conclusion

Thyroxine reduced the area of myocardial infarction and promoted mitochondrial autophagy by activating the HIF-1α/BNIP3 signaling pathway.

Introduction

The first choice for patients with acute myocardial infarction is reperfusion therapy after myocardial ischemia. Through thrombolytic therapy, percutaneous coronary intervention, and surgical coronary artery bypass grafting, providing the ischemic myocardium with timely successful reperfusion is the most effective treatment plan to limit the infarct size, improve heart function, and clinical prognosis [1]. Unfortunately, when the ischemic myocardium undergoes reperfusion, it will inevitably lead to unnecessary heart damage: myocardial ischemia reperfusion injury. The occurrence of myocardial I/R injury may offset some of the beneficial effects of restoring blood flow and is the main cause of adverse cardiac events [2].

The key to reducing myocardial I/R damage is to find out the factors that mediate I/RI. At the myocardial I/R injury site, myocardial cell swelling, myofibrillar contracture, myositis, and phosphate particles appear in mitochondria. Therefore, it is believed that the harmful factors that mediate myocardial reperfusion injury include mitochondrial dysfunction, oxidative stress, intracellular Ca²⁺ overload, inflammation and apoptosis [3]. Briefly, restoration of physiological pH and Ca²⁺ overload during myocardial perfusion may lead to the destruction of mitochondrial membrane potential and depletion of ATP. The opening of mitochondrial permeability transition pores may lead to the massive production of active oxygen. In addition to inducing cell apoptosis, ROS may directly cause cell DNA damage. The ROS accumulated during I/R injury can also stimulate the secretion of pro-inflammatory cytokines and chemokines [4].

The hypoxia-inducible factor-1α signaling pathway is the “center” of the healthy myocardium in resisting I/R injury [5]. Under hypoxic conditions, the PI3K-Akt pathway, the NO-cGMP-PKG pathway, and the adenosine pathway protect against organ hypoxic damage through upstream HIF-1α regulation [6]. In the case of hypoxia, HIF-1α can form a heterodimer with HIF-1β to generate active HIF-1 and combine with hypoxia response elements to regulate downstream target genes such as vascular endothelial growth factor (VEGF), heme oxygenase-1 (HO-1), and BNIP3, which is conducive to the survival of cells under hypoxic conditions. Among them, BNIP3 is a member of the BH3-only subfamily of the Bcl-2 family. It is an outer mitochondrial membrane protein and a target gene of HIF-1. It can be transcriptionally activated by HIF-1 under hypoxia. The degree of phosphorylation of BNIP3 directly determines whether it promotes apoptosis or autophagy downstream of mitochondrial fusion and fission. BNIP3 plays an important role in the HIF-1- mediated hypoxia response pathway.

The phosphorylation of BNIP3 at serines 17 and 24 can directly promote its binding with Bcl-xL, and directly recruit LC3-II to promote the initiation of mitochondrial autophagic flux. Studies have also found that under hypoxic conditions, HIF-1α is activated and promotes mitochondrial autophagy by regulating the expression of BNIP3 [7]. However, it is rarely reported whether HIF-1/BNIP3-mediated mitochondrial autophagy plays a role in combating myocardial ischemia-reperfusion injury.

Thyroid hormones have a central role in cardiovascular homeostasis. In the myocardium, these hormones stimulate both diastolic myocardial relaxation and systolic myocardial contraction, exert a pro-angiogenic effect, and play an important role in extracellular matrix maintenance [8]. T3 enhances the expression of HIF-1α, limiting the opening of mitochondrial permeability transition pores and thereby protecting cardiomyocyte from reperfusion injury [9]. However, relatively few studies have investigated thyroxine’s effects on cardiovascular aspects, particularly in the context of myocardial ischemia-reperfusion injury. Therefore, our research aims to explore whether thyroxine exerts cardioprotective effects against myocardial ischemia-reperfusion injury through the HIF-1α/BNIP3-mediated mitophagy pathway, and whether these effects can be blocked by the HIF-1α inhibitor 2ME2.

Methods

Animals and ethical approval

Male Sprague-Dawley rats (280–350 g, 8–10 weeks old) were obtained from the Laboratory Animal Center. All animals were housed in a temperature-controlled environment (22 ± 2 °C) with a 12-hour light/dark cycle and had free access to standard rat chow and water. All experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. The animals were acclimatized for at least one week before the experimental procedures.

I/R model establishment and grouping

The model of ischemia-reperfusion injury was simulated by ligating the anterior descending coronary artery in rats. Rats were anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneally) and mechanically ventilated after endotracheal intubation. The chest was opened via a left thoracotomy at the fourth intercostal space to expose the heart. The left anterior descending coronary artery (LAD) was identified, and a 6 − 0 silk suture was placed around the LAD approximately 2 mm from its origin. Regional ischemia was induced by tightening the suture, and successful occlusion was confirmed by the appearance of pallor in the left ventricular wall and ST-segment elevation on the electrocardiogram. After 40 min of ischemia, the suture was released to allow reperfusion, which was confirmed by visible hyperemia in the previously ischemic region. The chest was then closed in layers, and the animals were allowed to recover. Body temperature was maintained at 37 °C throughout the procedure using a heating pad.

Intraoperative physiological parameters were continuously monitored throughout all surgical procedures. Cardiac rhythm and heart rate were monitored via lead II electrocardiography (ECG). Mechanical ventilation was maintained at a rate of 60–70 breaths per minute with a tidal volume of 8–10 mL/kg using a rodent small animal ventilator (e.g., RoVent Jr., Kent Scientific). Core body temperature was monitored via a rectal probe and maintained within 36.5–37.5 °C using a homeothermic blanket system. Mean arterial blood pressure was intermittently measured via tail-cuff plethysmography. Animals exhibiting sustained ventricular arrhythmias, failure of ECG to demonstrate ST-segment elevation upon LAD occlusion, or body temperature deviating by more than ± 0.5 °C from 37 °C were excluded from the study. Of the total animals prepared, [X] were excluded based on these criteria.

SD rats were randomly divided into the following 5 groups (n = 10 per group):

(1) Sham operation (Sham) group: The rats’ chests were opened without ligation, and perfusion was performed for 180 min. All surgical procedures were identical to those of the other groups except that the suture around the LAD was not tightened.

(2) Ischemia-reperfusion (I/R) group: The rats were stabilized for 20 min, subjected to LAD ligation, and then subjected to reperfusion for 120 min. Vehicle solution (normal saline containing 1% DMSO) was administered intraperitoneally immediately after reperfusion.

(3) Thyroxine post-treatment group: The rats were stabilized for 20 min, subjected to LAD for 40 min, injected with 20 µg/kg L-Thyroxine (levothyroxine sodium, T4) intraperitoneally immediately after reperfusion, and then subjected to reperfusion for 120 min. The 120-minute reperfusion duration was chosen because previous studies have shown that autophagic activity, as indicated by LC3-II accumulation, peaks around 2 h after reperfusion, making this time point optimal for detecting both autophagy and ATP changes [10]. We specifically used L-thyroxine (T4, levothyroxine sodium) rather than T3 (triiodothyronine) because T4 represents the primary circulating form of thyroid hormone and has a longer half-life, making it more clinically relevant for therapeutic applications. The dose of 20 µg/kg was selected based on previous studies demonstrating cardioprotective effects in rat I/R models without inducing thyrotoxicosis or significant hemodynamic disturbances [11, 12].

(4) 2ME2 + I/R group: 15 mg/kg 2-ME2 (2-methoxyestradiol, a specific HIF-1α inhibitor) was injected intraperitoneally 30 min before LAD ligation, followed by 40 min of ischemia and 120 min of reperfusion. The dose of 2-ME2 was based on previous studies showing effective HIF-1α inhibition [13].

(5) 2ME2 + Thyroxine group: 15 mg/kg 2-ME2 was injected into the abdominal cavity 30 min before LAD ligation, and 20 µg/kg L-thyroxine (levothyroxine sodium, T4) was injected intraperitoneally immediately after reperfusion for 120 min.

Drug preparation and storage

L-Thyroxine (levothyroxine sodium, T4; Sigma-Aldrich, Cat# T2376) was dissolved in a minimal volume of 0.1 mol/L sodium hydroxide (NaOH) and then diluted to the desired working concentration in sterile 0.9% saline (pH adjusted to 7.2–7.4). The final solution was freshly prepared on the day of experiment and kept at 4 °C until use; unused portions were discarded. 2-Methoxyestradiol (2ME2; Sigma-Aldrich, Cat# M6383) was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution at 50 mg/mL and stored at − 20 °C protected from light. On the day of the experiment, the stock solution was diluted in 0.9% saline containing 1% DMSO to achieve the working concentration of 15 mg/kg for intraperitoneal injection. The vehicle control (0.9% saline with 1% DMSO) was administered to Sham and I/R group animals at equivalent volumes.

ATP level measurement

Tissue samples were collected at the end of 120 min of reperfusion for analysis.Following the kit instructions (ATP Bioluminescence Assay Kit, Beyotime Biotechnology, China), ATP levels were measured with a firefly luciferase-based ATP assay kit. Briefly, left ventricular tissue samples (50 mg) were rapidly homogenized in ice-cold lysis buffer provided in the kit. The homogenate was centrifuged at 12,000 × g for 5 min at 4 °C, and the supernatant was collected. ATP content was determined by mixing 100 µL of supernatant with 100 µL of ATP detection working solution in a white 96-well plate. Luminescence was immediately measured using a microplate reader (SpectraMax M5, Molecular Devices). ATP concentration was calculated from a standard curve and normalized to the protein content determined by the BCA assay. Results were expressed as nmol ATP/mg protein.

Measurement of mitochondrial membrane potential

Tissue samples were collected at the end of 120 min of reperfusion for analysis.According to the kit instructions (JC-1 Mitochondrial Membrane Potential Assay Kit, Beyotime Biotechnology), the mitochondrial membrane potential was measured with the fluorescent probe JC-1. Fresh myocardial tissue was minced and incubated in collagenase II solution to obtain single cardiomyocytes. Cells were incubated with JC-1 staining solution (5 µg/mL) for 20 min at 37 °C in the dark. After washing twice with JC-1 staining buffer, cells were analyzed by flow cytometry (BD FACSCalibur). JC-1 exists as monomers at low membrane potentials and emits green fluorescence (525 nm), while at high membrane potentials, it forms aggregates that emit red fluorescence (590 nm). The ratio of red to green fluorescence intensity was used as an indicator of mitochondrial membrane potential. Data from at least 10,000 cells per sample were collected and analyzed using FlowJo software.

Measurement of mitochondrial ROS production

Tissue samples were collected at the end of 120 min reperfusion for analysis.The fluorescent probe DCFH-DA (2’,7’-dichlorofluorescin diacetate) and DHE (dihydroethidium) method were used to detect the expression of ROS in myocardial tissue. For DCFH-DA staining, fresh tissue Sect.  (10 μm) were incubated with 10 µM DCFH-DA in PBS for 30 min at 37 °C in the dark. For DHE staining, frozen sections were incubated with 5 µM DHE for 30 min at 37 °C. Nuclei were counterstained with DAPI (4’,6-diamidino-2-phenylindole). Images were captured using a confocal laser scanning microscope (Zeiss LSM 880) with consistent settings across all samples. Fluorescence intensity was quantified using ImageJ software by measuring the mean fluorescence intensity in at least 5 random fields per section. Fields were selected by a systematic grid-based random sampling method with predefined stage coordinates, and image acquisition was performed by an investigator blinded to the experimental group assignments. Confocal settings (laser power, photomultiplier gain, and pinhole diameter) were fixed and kept identical across all samples within each experiment. The percentage of DHE-positive nuclei was calculated by counting DHE-positive cells among total DAPI-positive cells.

Measurement of myocardial infarction area

Tissue samples were collected at the end of 120 min of reperfusion for analysis.The area of myocardial infarction was measured by 2,3,5-triphenyl-2 H-tetrazolium chloride (TTC) staining. After 120 min of reperfusion, rats were re-anesthetized without re-occluding the LAD. Evans blue dye (2%, 2 mL) was injected via the femoral vein, and the original LAD ligature site was identified to distinguish the area at risk from the non-ischemic zone. Evans blue dye (2%, 2 mL) was injected via the femoral vein to delineate the area at risk (AAR). Hearts were rapidly excised, frozen at -20 °C for 20 min, and cut into 2-mm-thick transverse slices. The slices were incubated in 1% TTC solution at 37 °C for 15 min and then fixed in 4% paraformaldehyde overnight. The viable myocardium stained red, while the infarcted tissue appeared pale. Digital images of both sides of each slice were captured, and the areas of infarction, AAR, and left ventricle were measured using ImageJ software. Infarct size was expressed as a percentage of the AAR.

Observation of autophagosomes under electron microscope

Tissue samples were collected at the end of 120 minutes’ reperfusion for analysis.Myocardial ultrastructure and autophagosomes were observed under transmission electron microscope. Small pieces of left ventricular tissue (1 mm³) were rapidly fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 2 h at 4 °C, followed by post-fixation in 1% osmium tetroxide for 1 h. After dehydration through a graded ethanol series and propylene oxide, tissues were embedded in epoxy resin. Ultrathin Sects.  (70–80 nm) were cut using an ultramicrotome (Leica EM UC7), stained with uranyl acetate and lead citrate, and examined under a transmission electron microscope (JEOL JEM-1400Plus) at 80 kV. Images were captured at various magnifications to assess mitochondrial morphology, autophagosomes, and mitophagosomes. Quantitative analysis included counting the number of autophagosomes per 100 μm² and assessing mitochondrial damage using a scoring system (0 = normal, 1 = mild swelling, 2 = moderate swelling with cristae disruption, 3 = severe damage with membrane rupture). All TEM image acquisition and quantitative scoring were performed by two investigators blinded to the group assignments independently. The final scores were averaged between the two raters.

Protein electrophoresis

Tissue samples were collected at the end of 120 minutes’ reperfusion for analysis.Left ventricular tissues of each group were collected to detect the expression levels of HIF-1α, BNIP3, LC3B, Beclin-1, P62, and LAMP2 by Western blot method. Tissue samples were homogenized in RIPA lysis buffer containing protease and phosphatase inhibitors. Protein concentration was determined using the BCA method. Equal amounts of protein (30 µg) were separated by SDS-PAGE on 10–15% gels and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk for 1 h and incubated overnight at 4 °C with primary antibodies: HIF-1α (1:1000, Cell Signaling Technology, Cat# 14179), BNIP3 (1:1000, Cell Signaling Technology, Cat# 44060), LC3B (1:1000, Cell Signaling Technology, Cat# 3868), Beclin-1 (1:1000, Cell Signaling Technology, Cat# 3495), P62/SQSTM1 (1:1000, Cell Signaling Technology, Cat# 5114]), LAMP2 (1:1000, Abcam, Cat# ab25631, GAPDH (1:5000, Cell Signaling Technology, Cat# 5174). After washing, membranes were incubated with HRP-conjugated secondary antibodies for 1 h. Protein bands were visualized using enhanced chemiluminescence and quantified by densitometry using ImageJ software. All Western blot experiments were performed with at least three independent biological replicates (n = 3), and representative blots are shown in the figures. Each biological replicate was run in technical duplicate, and the mean value from the two technical replicates was used for downstream statistical analysis. A total of three independent biological replicates (n = 3 animals per group) were included in all densitometric analyses. Densitometric quantification was performed on all replicates, and data are presented as mean ± SD.

Statistical analysis

All data are expressed as mean ± standard deviation (mean ± SD). Prior to statistical analysis, data were tested for normality using the Shapiro-Wilk test and for homogeneity of variance using the Brown-Forsythe test to ensure appropriateness of parametric tests. For data meeting these assumptions, one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was used for multiple group comparisons. For comparisons between two groups, Student’s t-test was applied. In cases where data did not meet normality or equal variance assumptions, the Kruskal-Wallis test followed by Dunn’s multiple comparison test was used. Statistical analyses were performed using GraphPad Prism 8.0 software. A P value < 0.05 was considered statistically significant. All experiments were performed with appropriate biological replicates as indicated in each figure legend, and the investigator performing the analysis was blinded to the experimental groups to minimize bias.

Results

ATP level determination

The changes in ATP levels were observed in each group. Compared with the Sham group, the ATP content in the I/R group was < 0.01), indicating severe mitochondrial dysfunction following ischemia-reperfusion injury. Treatment with Thyroxine significantly increased ATP levels compared to the I/R group < 0.05), suggesting improved mitochondrial function. Pre-treatment with 2ME2 alone did not significantly affect ATP levels compared to the I/R group. However, the beneficial effect of Thyroxine on ATP content was completely abolished by 2ME2 pre-treatment, as there was no statistically significant difference in ATP content between the 2ME2 + Thyroxine group and the I/R group (Fig. 1).

Fig. 1.

The effect of Thyroxine on ATP levels. ATP content was measured in left ventricular tissue from different experimental groups. The Sham group showed normal ATP levels, while the I/R group exhibited significantly decreased ATP content. Thyroxine treatment partially restored ATP levels, but this effect was abolished by pre-treatment with the HIF-1α inhibitor 2ME2. Data are expressed as mean ± SD (n = 10). *P < 0.05, **P < 0.01 vs. Sham group; ^#P < 0.05, ^##P < 0.01 vs. Thyroxine group; NS, not significant

Measurement of mitochondrial membrane potential

The mitochondrial membrane potential of each group was measured by the JC-1 kit. Compared with the Sham group, the mitochondrial membrane potential of the I/R group was significantly lower (P < 0.01), as indicated by a decreased red/green fluorescence ratio. This suggests severe mitochondrial depolarization following ischemia-reperfusion. Compared with the I/R group, the mitochondrial membrane potential of the Thyroxine group increased significantly (< 0.01), indicating preservation of mitochondrial integrity. The 2ME2 group showed no significant improvement compared to the I/R. Importantly, pre-treatment with 2ME2 completely blocked the protective effect of Thyroxine on mitochondrial membrane potential, as the 2ME2 + Thyroxine group showed similar values to the I/R group (Fig. 2).

Fig. 2.

The effect of Thyroxine on mitochondrial membrane potential. Mitochondrial membrane potential was assessed using JC-1 staining and expressed as the ratio of red to green fluorescence. I/R injury caused significant mitochondrial depolarization compared to the Sham group. Thyroxine treatment preserved mitochondrial membrane potential, while 2ME2 pre-treatment prevented this protective effect. Data are expressed as mean ± SD (n = 10). *P < 0.05, **P < 0.01 vs. Sham group; ^#P < 0.05, ^##P < 0.01 vs. Thyroxine group; NS, not significant

Measurement of mitochondrial ROS production

Compared with the Sham group, through semi-quantitative and qualitative analysis using DHE staining, the mitochondrial ROS production rate of the I/R group was significantly increased (P < 0.01), with approximately 80% of nuclei showing positive DHE staining. However, the production of mitochondrial ROS in the Thyroxine group was significantly reduced compared to the I/R group (< 0.01), with only about 40% DHE-positive nuclei. There was no statistically significant difference in ROS content between the 2ME2, 2ME2 + Thyroxine and I/R groups, indicating that HIF-1α inhibition prevented the antioxidant effects of Thyroxine (Fig. 3).

Fig. 3.

The effect of Thyroxine on mitochondrial ROS production. Representative confocal microscopy images showing DHE (red) and DAPI (blue) staining in myocardial tissue sections. Scale bar = 50 μm. All images were acquired with identical laser power (X%), detector gain (X), and pinhole settings (1 Airy unit). Fields were selected using a systematic random grid approach by an observer blinded to group identity. The bar graph shows quantification of DHE-positive nuclei as a percentage of total nuclei. I/R injury induced significant ROS production, which was attenuated by Thyroxine treatment. Pre-treatment with 2ME2 blocked the antioxidant effect of Thyroxine. Data are expressed as mean ± SD (n = 10). *P < 0.05, **P < 0.01 vs. Sham group; ^#P < 0.05, ^##P < 0.01 vs. Thyroxine group; NS, not significant

Measurement of myocardial infarction area

Through TTC staining, the Sham group showed no evidence of infarction, with all myocardial tissue staining red. The I/R group showed extensive infarction, with approximately 45% of the area at risk showing pale, infarcted tissue. Compared with the I/R group, the area of myocardial infarction in the Thyroxine group was significantly reduced to approximately 20% of the AAR (< 0.01). However, among the 2ME2, 2ME2 + Thyroxine, and I/R groups, there was no significant difference in the myocardial infarction area, with all showing approximately 40–45% infarction of the AAR. This demonstrates that HIF-1α inhibition completely abolished the cardioprotective effect of Thyroxine (Fig. 4).

Fig. 4.

The effect of Thyroxine on the myocardial infarction area. Representative images of TTC-stained heart slices from each experimental group. Viable myocardium appears red, while infarcted tissue appears pale. The Sham group showed no infarction, while the I/R group exhibited extensive infarction. Thyroxine treatment significantly reduced infarct size, but this cardioprotective effect was abolished by 2ME2 pre-treatment. Scale bar = 5 mm. Data are expressed as mean ± SD (n = 10 per group). *P < 0.05, **P < 0.01 vs. I/R group; ^#P < 0.05, ^##P < 0.01 vs. Thyroxine group; NS, not significant

Observation of mitochondrial ultrastructure and autophagosomes under electron microscope

Under the electron microscope, the Sham group showed normal myocardial ultrastructure with intact myofilaments, well-organized mitochondria with clear cristae, and no evidence of autophagosomes. In contrast, myofilaments in the I/R group was found to be dissolved, mitochondria were swollen and arranged disorderly with disrupted cristae, interstitial exudation increased, and accumulated autophagosomes were seen, indicating impaired autophagy flux. The mitochondria in the Thyroxine group were basically intact and neatly arranged, but they showed still slight swelling and some autophagy. Notably, the autophagosomes were significantly reduced compared to the I/R group, suggesting enhanced autophagy flux and clearance of damaged organelles. After the application of the HIF-1α selective inhibitor 2ME2, it was found that the mitochondrial structure damage was similar among the 2ME2, 2ME2 + Thyroxine and I/R groups, with extensive mitochondrial swelling, cristae disruption, and accumulation of autophagosomes (Fig. 5).

Fig. 5.

The effect of Thyroxine on the mitochondria ultrastructure and autophagosomes. Representative transmission electron microscopy images showing myocardial ultrastructure. In the Sham group, mitochondria displayed normal morphology with intact cristae and well-organized myofibrils. The I/R group showed damaged mitochondria with disrupted cristae (black arrows) and vacuolization. Thyroxine treatment preserved mitochondrial structure with clearer cristae and reduced cellular damage. The 2ME2 and 2ME2 + Thyroxine groups showed mitochondrial swelling, cristae disruption (black arrows), and varying degrees of myofibril disorganization similar to the I/R group. Scale bar = 1 μm

Protein electrophoresis

The protein levels of each group were observed by Western blot analysis. Compared with the I/R group, the expression of HIF-1α and BNIP3 proteins increased significantly in the Thyroxine group (P < 0.01), while LAMP2 expression also < 0.01), indicating enhanced autophagy flux. Interestingly, the expressions of LC3B-II, P62, and Beclin-1 was significantly reduced in the Thyroxine group compared to the I/R group (P < 0.01), suggesting active autophagy with efficient clearance of autophagosomes. Compared with the Thyroxine group, myocardial HIF-1α and BNIP3 expression decreased significantly in the 2ME2 and 2ME2 + Thyroxine groups (P < 0.01), confirming effective HIF-1α inhibition by 2ME2. Additionally, LAMP2 expression was reduced while the of LC3B-II, P62, and Beclin-1 increased in these groups, indicating impaired autophagy flux similar to the I/R group (Fig. 6).

Fig. 6.

The effect of Thyroxine on various proteins. Western blot analysis of HIF-1α (120 kDa), BNIP3 (30 kDa), Beclin-1 (60 kDa), LC3B-II/LC3B-I (14–16 kDa), p62 (62 kDa), LAMP2 (114 kDa), and GAPDH (37 kDa) in left ventricular tissue. A: Sham, B: I/R, C: Thyroxine, D: 2ME2, E: 2ME2 + Thyroxine. Thyroxine treatment increased HIF-1α, BNIP3, and LAMP2 expression while decreasing LC3B-II, Beclin-1, and p62 accumulation. These effects were reversed by 2ME2 pre-treatment. GAPDH was used as a loading control. Data are expressed as mean ± SD (n = 3 biological replicates)

Discussion

The present study explored the mechanisms involved in reducing ischemic myocardial injury through HIF-1α-BNIP3-mediated mitophagy. Myocardial ischemia is caused by insufficient myocardial blood flow, which can lead to necrosis, autophagy, and apoptotic cell death, accompanied by fibrosis, inflammatory cell infiltration, and contractile dysfunction. Mitochondria play an important role in the survival and death of cardiomyocytes. Mitochondria are rich and dynamic organelles that provides ATP for cells. They participate in different intracellular processes, especially during cell damage. Mitochondria are the continuous source of superoxide anions and reactive oxygen species [14].

Basically, the ATP production in the adult mammalian heart depends on mitochondrial oxidative phosphorylation. Abnormalities in the function and structure of mitochondrial are found in cardiovascular diseases, such as hypertrophic and enlarged dilated cardiomyopathy, cardiac conduction defects, and alcoholic and ischemic cardiomyopathy. In most cases, ischemic heart disease with congestive heart failure and myocardial infarction is the main cause of death. During the process of ischemia/reperfusion, the decrease of respiratory oxygen supply and oxidative phosphorylation leads to a rapid decrease in ATP, and stimulates the production of anaerobic ATP, while increasing glycolysis and lactic acid production [15]. As reperfusion progresses, mitochondria and cardiomyocytes are further damaged due to the production of mitochondrial reactive oxygen species. Therefore, mitochondrial dysfunction may be one of the main mechanisms of cardiomyopathy and ischemia/reperfusion injury (I/RI) [16].

Prolonged ischemia leads to the death of cardiomyocytes because of insufficient oxygen supply. However, it has been demonstrated that when the oxygen supply is supplemented, the cells around the infarct zone are at risk of further damage during reperfusion. Interestingly, in both cases, the formation of reactive oxygen species is the determinant of the ultimate damage, including increased fibrosis, angiogenesis, and vascular remodeling. Ischemia leads to hypoxia in myocardial cells, causing decreased function of mitochondrial respiratory chain enzymes. Superoxide dismutase, hydrogen peroxide, peroxynitrite and hydroxyl free radicals are formed. Specifically, I/R damage impairs the function of mitochondrial complexes I and III and stimulates NADH to produce ROS [17]. The reduction of Complex I function is related to mitochondrial cardiolipin damage, which accelerates the leakage of electrons and stimulates the vicious cycle of free radical generation. In order to break this vicious cycle, the key is to quickly remove damaged mitochondria.

Autophagy is the only mechanism of mitochondrial degradation and is called mitochondrial autophagy or mitophagy. Sufficient mitochondrial autophagy is necessary to maintain the survival of cardiomyocytes. Mitophagy breaks down the proteins and membranes of damaged mitochondria into amino acids, which enter the tricarboxylic acid (TCA) cycle to generate ATP. Glucose deprivation significantly reduces cell ATP content, which is related to the up-regulation of cardiomyocyte autophagy [18, 19]. Mitophagy refers to the degradation of mitochondria through autophagy. Bcl-2-related proteins such as BNIP3 and BNIP3L/Nix are another group of regulators that participate in mitochondrial autophagy. These atypical BH3-only proteins are well-known activators of cell death. BNIP3 activates the mitochondrial outer membrane Bax/BAK to open the mitochondrial permeability transition pore [20]. According to reports, a common mechanism of BNIP3-mediated mitochondrial autophagy is by opening the mitochondrial permeability transition pore (MPTP) to depolarize the mitochondrial membrane potential, thereby transmitting a “discard me” signal. This signal allows the autophagosome marker LC3-II to bind to the LIR domain of BNIP3, and encapsulate the mitochondria in the autophagosome, thereby selectively removing the mitochondria through mitophagy [21].

BNIP3 is not only a mitochondrial autophagy receptor closely related to hypoxia, but also an important downstream target gene of HIF-1α, and is considered to be an important signal molecule for hypoxia-induced mitochondrial autophagy [22]. Previous studies have shown that HIF-1α-BNIP3-mediated mitophagy plays a protective role through inhibition of apoptosis and of ROS production in various ischemia-reperfusion injury models [23, 24]. Our experiment also found that the expression of BNIP3 protein in the I/R group increased with the increase of HIF-1α, but that in the inhibitor group decreased with the inhibition of HIF-1α. In addition, under the electron microscope, myofilament dissolution, mitochondrial swelling, disordered arrangement and increased interstitial exudation in were observed in the I/R group. Autophagosomes were obviously accumulated, indicating that hypoxia-induced mitophagy is related to the HIF-1/BNIP3 pathway.

These findings align with and are further contextualized by recent mechanistic advances in the field. Alanova et al. (2024) demonstrated in a genetic mouse model that partial Hif1a deficiency significantly impaired the mitophagy response induced by chronic hypoxia and abolished the associated reduction in infarct size, providing direct genetic evidence that HIF-1α is an indispensable upstream driver of cardioprotective mitophagy [25]. This is consistent with our pharmacological approach using 2ME2 to inhibit HIF-1α and reversal of thyroxine-conferred protection. At the post-translational level, a recent Nature Communications study demonstrated that BNIP3 protein stability is actively regulated by the deubiquitinase USP20, which protects BNIP3 from proteasomal degradation at K131, thereby sustaining mitophagy flux during I/R injury [26]. This raises the intriguing possibility that thyroxine may not only upregulate BNIP3 transcription via HIF-1α but could also influence BNIP3 protein stability, a hypothesis warranting future investigation. Furthermore, a comprehensive review on mitophagy in ischemic heart disease highlighted that the dynamics of BNIP3-mediated mitophagy are highly context-dependent, with moderate activation being cardioprotective and excessive activation potentially contributing to cardiomyocyte loss [27]. This dual-role model reinforces the clinical relevance of the dosing strategy employed in our study and underscores the importance of future dose-response experiments to define the therapeutic window of thyroxine post-treatment.

The role of thyroid hormones in cardioprotection is increasingly being recognized. Thyroid hormones modulate cardiac mitochondrial function, and dysfunction of thyroid axis impairs myocardial bioenergetic status [28]. T3 pretreatment may confer protection against subsequent IRI by inducing pharmacological preconditioning in cardiomyocytes, mainly by enhancing heat-shock protein 27 (HSP27) and heat-shock protein 70 (HSP70) expression and decreasing the activation of proapoptotic p38MAPK [29, 30]. Our study demonstrates that thyroxine post-treatment significantly reduces myocardial infarct size and improves mitochondrial function through activation of the HIF-1α/BNIP3 pathway.

The HIF-1α inhibitor 2-methoxyestradiol (2ME2) is a natural metabolite of estrogen that inhibits HIF-1α at the post-transcriptional level [31]. 2ME2 has been shown to block HIF-1α nuclear accumulation and HIF-1α transcriptional activity by disrupting microtubules and inhibiting HIF-1α protein translation [32, 33]. In our study, 2ME2 effectively blocked the protective effects of thyroxine, confirming that the cardioprotective mechanism is HIF-1α-dependent. It is important to acknowledge that 2ME2, while widely used as an HIF-1α inhibitor, may have off-target effects including disruption of microtubule dynamics and potential modulation of estrogen receptor signaling [34]. However, at the concentration used in our study (15 mg/kg), previous research has demonstrated that the predominant mechanism is HIF-1α inhibition through prevention of its nuclear accumulation and suppression of transcriptional activity. The fact that 2ME2 completely abolished the upregulation of both HIF-1α and its downstream target BNIP3, while reversing the protective effects of thyroxine, strongly suggests that the observed effects are primarily mediated through the HIF-1α pathway inhibition. Nevertheless, future studies employing alternative HIF-1α inhibitors (such as YC-1 or digoxin) or genetic approaches (HIF-1α siRNA or conditional knockout models) would provide additional validation of the HIF-1α-dependent mechanism. Additionally, direct measurement of microtubule integrity and estrogen receptor activation in our model would help rule out significant contributions from off-target effects. To more rigorously establish causal evidence for the HIF-1α/BNIP3 mitophagy axis, future studies should employ genetic approaches including cardiomyocyte-specific conditional HIF-1α deletion (e.g., HIF-1α^flox/flox × αMHC-Cre transgenic mice) or adeno-associated virus (AAV9)-mediated cardiac-specific HIF-1α or BNIP3 knockdown. These strategies would allow definitive attribution of the observed cardioprotective effects to HIF-1α-dependent signaling, independent of 2ME2’s pharmacological profile. Additionally, rescue experiments using BNIP3 overexpression in the context of HIF-1α inhibition could further substantiate the hierarchical relationship within this pathway.

The assessment of autophagy flux in our study revealed an interesting pattern. While LC3-II, Beclin-1, and p62 levels were elevated in the I/R group, indicating autophagosome accumulation, these markers were reduced in the thyroxine-treated group despite increased HIF-1α and BNIP3 expression. This apparent paradox can be explained by enhanced autophagy flux rather than reduced autophagy initiation. The increased LAMP2 expression in the thyroxine group supports this interpretation, as LAMP2 is essential for autophagosome-lysosomal fusion [35]. The accumulation of p62 and LC3-II in untreated I/R injury suggests impaired autophagy flux, while their reduction with thyroxine treatment indicates efficient clearance of autophagosomes through enhanced lysosomal degradation [36, 37]. It should be noted, however, that the current study did not employ lysosomal flux inhibitors (e.g., chloroquine or bafilomycin A1) to directly quantify autophagic flux. Without comparative experiments using these agents, the possibility that reduced LC3-II and p62 levels reflect decreased autophagy initiation rather than enhanced completion cannot be entirely excluded. Future studies should utilize tandem fluorescence reporters (mRFP-GFP-LC3) and lysosomal inhibitor co-treatment paradigms to definitively characterize autophagic flux dynamics in this model. Additionally, the present study did not evaluate in vivo cardiac functional parameters, including left ventricular ejection fraction (LVEF), fractional shortening, end-diastolic and end-systolic volumes, or pressure-volume loop indices (e.g., maximal rate of pressure rise, +dP/dt_max). These measurements are essential to determine whether thyroxine-mediated mitochondrial protection translates into genuine improvement of global cardiac contractile function. Furthermore, long-term outcomes including adverse ventricular remodeling, myocardial fibrosis, and cardiac hypertrophy were not assessed. Future studies should include serial echocardiographic evaluation and invasive hemodynamic monitoring at multiple time points following I/R injury to provide a comprehensive assessment of thyroxine’s cardioprotective effects in vivo.

Our findings hold important implications for the clinical management of myocardial ischemia-reperfusion injury. The post-reperfusion administration strategy used in this study is particularly clinically translatable, as it can be applied immediately after reperfusion therapies such as percutaneous coronary intervention (PCI) or thrombolysis—avoiding the impracticality of pre-treatment in acute myocardial infarction settings. This timing aligns with existing clinical workflows, representing a feasible therapeutic window that could be readily integrated into standard care. However, key considerations must be addressed before clinical application. Notably, safety remains a critical focus: while our single 20 µg/kg thyroxine dose showed no adverse effects, high doses may pose risks including arrhythmogenicity or increased myocardial oxygen consumption. Previous clinical studies have demonstrated the safety of short-term thyroid hormone administration in cardiac patients under close monitoring, but dosing optimization specifically for acute I/R injury remains necessary. Equally important is patient stratification: thyroxine may offer particular benefit to patients with subclinical hypothyroidism or low-T3 syndrome—conditions common in critically ill populations—while being contraindicated for those with pre-existing hyperthyroidism or atrial fibrillation. Future phase I/II trials will be essential to define optimal timing, dosage, and treatment duration in human cohorts. Beyond dosing and patient selection, combination therapy merits exploration: thyroxine’s HIF-1α-mediated protection could synergize with ischemic conditioning or other cardioprotective agents targeting distinct pathways, potentially amplifying therapeutic efficacy. Several important limitations of the present study should be acknowledged. First, only a single dose of thyroxine (20 µg/kg) and a single post-reperfusion administration time point were evaluated. A systematic dose-response experiment and assessment of multiple therapeutic time windows (e.g., 0, 30, 60 min post-reperfusion) are needed to define the optimal therapeutic range and intervention window. Second, real-time hemodynamic parameters (heart rate, mean arterial pressure, cardiac output), myocardial oxygen consumption, and serum thyroid hormone levels (T3, T4, TSH) were not monitored following thyroxine administration, which precludes a full safety and pharmacodynamic characterization. Future preclinical studies should incorporate these measurements to bridge the gap toward clinical application. Moving forward, clinical research should prioritize validating thyroxine’s safety and efficacy in ST-elevation myocardial infarction patients undergoing primary PCI, with rigorous monitoring of infarct size, cardiac function, and adverse events to establish its role in standard clinical practice. Furthermore, all experiments in this study were conducted in young, healthy, male Sprague-Dawley rats (8–10 weeks, 280–350 g), which represent a simplified model that does not fully reflect the clinical profile of patients suffering acute myocardial infarction. The majority of such patients are elderly and present with multiple cardiovascular risk factors including diabetes mellitus, hyperlipidemia, and hypertension, all of which are known to alter mitophagic capacity and HIF-1α signaling. Future studies should therefore validate the cardioprotective effects of thyroxine in aged rat models, streptozotocin-induced type 2 diabetic models, high-fat-diet-fed hyperlipidemic models, and female cohorts to strengthen the translational applicability of these findings.

Conclusion

In conclusion, thyroxine promotes mitochondrial autophagy by activating the HIF-1α/BNIP3 signaling pathway, clears damaged mitochondria in time, reduces ROS production, stabilizes mitochondrial function, and ultimately reduces myocardial infarction area. This effect is blocked by the HIF-1α inhibitor 2ME2. These findings provide new insights into the cardioprotective mechanisms of thyroid hormones and suggest potential therapeutic strategies for myocardial ischemia-reperfusion injury.

Author contributions

Hongbo Yu is responsible for the guarantor of the integrity of the entire study, study concepts & design, literature research, data acquisition & analysis, manuscript preparation editing and review; Jingsheng Feng is responsible for the guarantor of the integrity of the entire study, definition of intellectual content, clinical studies, experimental studies, data analysis, statistical analysis; Kepeng Wang is responsible for the definition of intellectual content, literature research, clinical studies, manuscript review; Xuehui Zhang is responsible for the guarantor of the integrity of the entire study, study concepts, definition of intellectual content. All authors read and approved the final manuscript.

Funding

The authors have not received any funding support.

Data availability

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The research was conducted in accordance with the Declaration of Helsinki and with the Guide for Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Clinical trial number

Not applicable.