Electron transfer flavoprotein subunit beta suppresses hypoxia/reoxygenation-induced mitochondrial dysfunction and apoptosis in cardiomyocytes

Abstract

Objective

This study aimed to investigate the role of the electron transfer flavoprotein subunit beta in myocardial ischemia–reperfusion injury.

Methods

An in vitro ischemia–reperfusion model was established in H9c2 cardiomyocytes using hypoxia/reoxygenation. Myocardial injury was assessed by measuring the levels of creatine kinase-MB, cardiac troponin I, cardiac troponin T, and lactate dehydrogenase. Apoptosis was evaluated via terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and Annexin V-PE/7-AAD flow cytometry. Mitochondrial morphology was observed via transmission electron microscopy, while mitochondrial function was assessed by measuring reactive oxygen species and membrane potential. Oxidative stress markers and apoptosis-related proteins were also assessed.

Results

Hypoxia/reoxygenation treatment significantly increased the levels of myocardial injury markers and downregulated electron transfer flavoprotein subunit beta expression. The model group exhibited enhanced apoptosis, impaired mitochondrial structure, elevated reactive oxygen species levels, reduced mitochondrial membrane potential, and increased oxidative stress. Overexpression of electron transfer flavoprotein subunit beta effectively reversed these changes: it reduced the injury markers, decreased apoptosis, improved mitochondrial morphology and function, attenuated oxidative stress, and modulated the expression of apoptosis-related proteins.

Conclusions

Electron transfer flavoprotein subunit beta protects against myocardial ischemia–reperfusion injury by ameliorating mitochondrial dysfunction, reducing oxidative stress, and inhibiting apoptosis, identifying it as a potential therapeutic target.

Keywords

Myocardial ischemia–reperfusion injury electron transfer flavoprotein subunit beta apoptosis mitochondrial function H9c2 cardiomyocytes

Introduction

Myocardial ischemia–reperfusion (I/R) injury is a common clinical pathophysiological process that often occurs following reperfusion therapy in patients with acute myocardial infarction (AMI). Paradoxically, the restoration of blood flow can exacerbate myocardial damage, leading to adverse cardiac remodeling, aggravated arrhythmias, and impaired cardiac function.¹ As a major cause of perioperative adverse cardiac events and mortality, severe myocardial I/R injury may trigger life-threatening ventricular arrhythmias and sudden cardiac death, significantly worsening treatment outcomes and prognosis in patients with AMI.² Although considerable progress has been made in understanding the complex mechanisms underlying myocardial I/R injury—a central focus and significant challenge in cardiovascular medicine—effective preventive and therapeutic strategies remain elusive. Current clinical interventions and preventive measures, such as pharmacological treatments, ischemic preconditioning, and physical interventions, offer limited clinical benefits for comprehensive myocardial protection.³ Therefore, further research into the underlying mechanisms of myocardial I/R injury is warranted for developing effective therapies and improving the management of patients with AMI.

In recent years, extensive research has confirmed that myocardial I/R injury involves multiple pathological mechanisms, primarily including cardiomyocyte death, oxidative stress, and inflammation.^4–6 Among these, cardiomyocyte death—an irreversible process—can directly lead to severe complications such as heart failure and pathological cardiac remodeling. Therefore, effective inhibition of cardiomyocyte death is crucial for preserving cardiac function following I/R.⁷ Apoptosis, a major form of programmed cell death, plays a significant role in myocardial injury.⁸ Molecular evidence indicates that I/R-induced apoptosis is positively correlated with the severity of cardiomyocyte damage.⁹ In this context, mitochondria—central to cardiomyocyte energy metabolism—play a central role. Specifically, oxidative stress triggered by mitochondrial-derived reactive oxygen species (ROS) exacerbates cardiomyocyte injury by activating the mitochondrial-dependent apoptotic pathway. This process involves the collapse of the mitochondrial membrane potential (MMP), activation of caspase cascades, and coordinated dysregulation of oxidative stress pathways.^10–12 Thus, investigating the molecular mechanisms of genes involved in regulating cardiomyocyte apoptosis during myocardial I/R injury holds important clinical value for mitigating cardiac damage.

The electron transfer flavoprotein (ETF) is a mitochondrial protein containing FAD, composed of two subunits: α and β (ETFα and ETFβ, respectively). ETFB encodes the ETFβ subunit, which participates in mitochondrial fatty acid and amino acid metabolism by transferring electrons between flavoprotein dehydrogenases.¹³ A study has shown that ETFB is associated with prognosis in patients with acute myeloid leukemia (AML).¹⁴ Functionally, silencing ETFB in AML cells induces mitochondrial stress, promotes differentiation and apoptosis, and increases sensitivity to venetoclax.¹⁵ In cardiomyocytes, mitochondrial connexin 43 can interact with ETFB and apoptosis-inducing factor (AIF) to form a multiprotein complex that influences mitochondrial respiration and ROS signaling.¹⁶ Furthermore, transcriptome analysis revealed that following coronary microembolism in a rat model, the left ventricular tissue exhibited downregulation of ETFB expression compared with control groups.¹⁷ However, the role and underlying mechanisms of ETFB in myocardial I/R injury remain unexplored.

Thus, in this study, hypoxia/reoxygenation (H/R) was applied to cardiomyocytes to simulate myocardial I/R injury, and the role and underlying mechanism of ETFB in H/R-induced cellular damage were investigated.

Materials and methods

Cell culture and establishment of the H/R model

H9c2 cardiomyocytes (RRID:CVCL_0286) were purchased from the Wuhan Procell Life Science & Technology Co., Ltd. (China; Cat# CL-0089) and routinely cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, USA; Cat# 12100046) supplemented with 10% fetal bovine serum (FBS; Gibco, USA; Cat# 10099-141) at 37°C in a humidified incubator with 5% CO₂. Cells between passages 4 and 6 were used in all experiments. To establish an in vitro model of myocardial I/R injury, cells were subjected to hypoxia (95% N₂ and 5% CO₂) for 6 h. Following hypoxia, the medium was replaced with fresh medium, and the cells were then subjected to reoxygenation for 12 h under normoxic conditions (95% air and 5% CO₂). Control cells were maintained under normal culture conditions (37°C, 5% CO₂) throughout the experiment.

Cell transfection

H9c2 cells were transfected with either an ETFB overexpression plasmid (pcDNA3.1-ETFB) or an empty pcDNA3.1 vector (serving as a negative control) at a dose of 5 pg DNA per cell using Lipofectamine 3000 (Invitrogen, USA; Cat# L3000015). Prior to transfection, cells were serum-starved for 4 h. After 48 h of transfection, the cells were harvested for subsequent experiments. Transfection efficiency was validated by detecting the overexpression of ETFB protein using western blot (WB) analysis.

MTT assay

H9c2 cells from each group were transferred to a 96-well plate at a density of 2 × 10³ cells/well. According to the instructions of the MTT Cell Proliferation and Cytotoxicity Assay Kit (Beyotime, China; Cat# C0009S), 10 µL of MTT solution was added to each well, followed by continued incubation in the cell culture incubator for 4 h. Subsequently, 100 µL of formazan dissolving solution was added to each well and mixed thoroughly, after which the plate was further incubated. The incubation proceeded until complete dissolution of formazan was observed under a standard optical microscope. Finally, absorbance was measured at 570 nm, and cell viability was calculated.

Detection of creatine kinase-MB (CK-MB), cardiac troponin I (CTnI), cardiac troponin T (CTnT), lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione disulphide (GSSG) levels

The CK-MB, CTnI, and CTnT levels in cells were measured and quantified using enzyme-linked immunosorbent assay kits purchased from Wuhan Cloud-Clone Corp. (China; Cat# SEA479Ra, SEA478Ra, and SED232Ra), following the manufacturer’s instructions. The activities of LDH and SOD as well as the concentrations of MDA and oxidized glutathione GSSG were measured using commercial assay kits from NanJing JianCheng Bioengineering Institute (China; Cat# A020-2-2, A001-3-1, A003-1-1, and A061-2-1) following the manufacturer’s protocols.

TUNEL staining

Apoptosis was assessed using the One Step terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) Apoptosis Assay Kit (Beyotime, China, Cat# C1090). Briefly, cells were fixed with 4% formaldehyde and permeabilized with 0.5% Triton X-100. After washing with phosphate-buffered saline, the cells were incubated with the TdT incubation buffer at 37°C for 1 h. Subsequently, the nuclei were counterstained with DAPI in the dark at room temperature for 5 min. Fluorescent signals were visualized and captured using a fluorescence microscope (Olympus Corporation, Japan; DP73). The proportion of TUNEL-positive cells was quantified using ImageJ software (National Institutes of Health, USA; version 1.52).

Annexin V-PE/7-AAD apoptosis detection

Apoptosis in H9c2 cells was also assessed using the Annexin V-PE/7-AAD Apoptosis Detection Kit (Meilunbio, China; Cat# MA0429) according to the manufacturer’s instructions. After staining with Annexin V-PE and 7-AAD in the dark (15 min, room temperature), cells were analyzed via flow cytometry (Agilent Technologies, USA; NovoCyte), and the resulting data were processed to quantify apoptosis.

ROS detection and JC-1 assay

Intracellular ROS levels were evaluated using the fluorescent probe DCFH-DA (Beyotime, China; Cat# S0035S). After washing, cells were incubated with 10 μM DCFH-DA at 37°C for 30 min, followed by fluorescence imaging to quantify ROS production. MMP was assessed using the JC-1 probe (Beyotime, China; Cat# C2006). Cells were treated with the JC-1 reagent at 37°C for 20 min. Following washing, fluorescence was measured immediately using a fluorescence microscope (Olympus Corporation, Japan; DP73). The relative intensity of ROS and the red/green fluorescence ratio from the JC-1 assay were quantified using ImageJ software (National Institutes of Health, USA; version 1.52).

WB analysis

Following specific treatments, H9c2 cells were harvested and lysed with RIPA buffer (Beyotime, China; Cat# P0013K) for total protein extraction. Protein quantification was performed using a BCA assay kit (Beyotime, China; Cat# P0398S), after which the samples were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The separated proteins were then electrotransferred onto a membrane at 200 mA for 90 min. The membrane was subsequently blocked with 5% (w/v) skim milk for 2 h at room temperature. Following blocking and washing with Tris-buffered saline with Tween 20 (TBST), the membrane was incubated overnight at 4°C with the following primary antibodies: ETFB (Proteintech, USA; Cat# 17925-1-AP; 1:1000), Bax (Proteintech, USA; Cat# 50599-2-Ig; 1:50000), Bcl2 (Proteintech, USA; Cat# 26593-1-AP; 1:1000), cleaved caspase-3 (Proteintech, USA; Cat# 68773-1-Ig; 1:10000), cleaved caspase-6 (Proteintech, USA; Cat# 10198-1-AP; 1:1000), and β-actin (Proteintech, USA; Cat# 66009-1-Ig; 1:20000). After removing the primary antibodies and washing with TBST, the membrane was incubated with the corresponding secondary antibodies: horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Proteintech, USA; Cat# SA00001-1; 1:10000) or HRP-conjugated goat anti-rabbit IgG (Proteintech, USA; Cat# RGAR001; 1:10000) at room temperature for 1 h. Finally, after three additional TBST washes (5 min each), protein bands were visualized using a chemiluminescence imaging system (Shanghai Tanon Science & Technology Co., Ltd., China; Tanon 5200) and quantified via grayscale analysis using ImageJ software (National Institutes of Health, USA; version 1.52).

Transmission electron microscopy (TEM)

The collected cells were fixed in 2.5% glutaraldehyde for 4 h, rinsed thoroughly, and then post-fixed in 1% osmium tetroxide for 1–2 h. After dehydration through a graded ethanol series, the H9c2 cells were embedded, sectioned into 50–70 nm ultrathin slices, and stained with uranyl acetate and lead citrate. The prepared samples were examined under a transmission electron microscope (Thermo Fisher Scientific, USA; Talos) to observe ultrastructural alterations.

Statistical analysis

Statistical analysis was performed using GraphPad Prism software (GraphPad Software, USA; version 10.2). Data were presented as the mean ± standard deviation (SD) from three independent biological replicates. Comparisons between two groups were made using an unpaired two-tailed Student’s t-test. Differences among three or more groups were analyzed using one-way analysis of variance, with post-hoc comparisons performed using Tukey’s test. A p-value <0.05 was considered statistically significant.

Results

ETFB expression is downregulated in the myocardial I/R injury cell model

To investigate the role of ETFB in myocardial I/R, we first established a myocardial I/R injury cell model through H/R. The MTT assay indicated that the H/R treatment group showed significantly reduced viability of H9c2 cells compared with the control (Con) group (Figure 1(a)). Following H/R treatment, markers of myocardial injury—CK-MB, CTnI, CTnT, and LDH—were detected. The results revealed that all four markers were significantly elevated in the model group compared with that in the Con group (Figure 1(b)–(e)), confirming successful model establishment. Further WB analysis revealed that ETFB was downregulated in cells after H/R, suggesting a potential protective role of ETFB against myocardial I/R injury (Figure 1(f)).

Figure 1.

Hypoxia/reoxygenation (H/R) treatment induces myocardial injury and electron transfer flavoprotein subunit beta (ETFB) downregulation in H9c2 cells. (a) Analysis of cell viability in the control (Con) and H/R model (Model) groups. (b–e) Analysis of myocardial injury markers (creatine kinase–myocardial band, cardiac troponin I, cardiac troponin T, and lactate dehydrogenase) in the Con and Model groups, respectively. (f) Protein expression levels of ETFB, Bcl-2-associated X protein, B-cell lymphoma 2, cysteine-aspartic protease-3, and cleaved cysteine-aspartic protease-6 were detected by western blot after H/R treatment. Data are presented as mean ± SD; each experiment was repeated independently three times (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs. Con.

Apoptosis and mitochondrial damage were observed in the myocardial I/R injury cell model

To further investigate how ETFB influences cardiomyocyte damage during myocardial I/R, we first examined whether the cellular injury in the H/R cell model was associated with apoptosis and mitochondrial damage. TUNEL assay indicated that H/R treatment induced apoptosis in H9c2 cells (Figure 2(a)). This finding was further confirmed via Annexin V-PE/7-AAD staining and flow cytometry, which revealed a higher apoptosis rate in the Model group than in the Con group (Figure 2(b)). Concurrently, WB analysis revealed marked alterations in the expression of key apoptosis-related proteins—Bax, Bcl-2, cleaved caspase-3, and cleaved caspase-6—in H/R-treated cells. Specifically, compared with the Con group, the Model group exhibited decreased Bcl-2 expression and increased levels of Bax, cleaved caspase-3, and cleaved caspase-6 (Figure 1(f)). TEM revealed impaired mitochondrial structure in the Model group, characterized by swelling and blurred cristae (Figure 2(c)). The levels of key oxidative stress indicators—SOD, MDA, and GSSG—were further measured and compared. A significant decrease in SOD activity, along with significant increases in MDA and GSSG levels, was observed in the Model group compared with that in the Con group (Figure 2(d)–(f)).

Figure 2.

Hypoxia/reoxygenation (H/R) treatment induces apoptosis, mitochondrial damage, and oxidative stress in H9c2 cells. (a) Apoptosis was detected via terminal deoxynucleotidyl transferase dUTP nick-end labeling assay (with Cyanine 3-labeled dUTP) in the control (Con) and H/R model (Model) groups. Nuclei were counterstained with DAPI. Scale bar, 50 µm (corresponding to a 200× magnification). (b) The apoptosis rate was quantitatively analyzed via flow cytometry with Annexin V-PE/7-AAD staining. (c) Mitochondrial ultrastructure was observed via transmission electron microscopy (TEM) in the Con and Model groups. Red arrows indicate swollen mitochondria. Green arrows point to blurred cristae. Scale bar, 500 nm. (d–f) The levels of oxidative stress markers, including superoxide dismutase (d), malondialdehyde (e), and glutathione disulfide (f), were measured in the Con and Model groups. All quantitative data (panels a–b, d–f) are presented as mean ± SD from three independent experiments (n = 3). **p < 0.01, ***p < 0.001 vs. Con.

ETFB overexpression mitigates H/R-induced injury and suppresses apoptosis in H9c2 cardiomyocytes

To further investigate the role of ETFB in myocardial I/R injury, we established empty vector control (NC) and ETFB-overexpressing (ETFB-OE) groups in H9c2 cells via transfection. A simulated I/R injury cell model was then induced using H/R to systematically evaluate the effect of ETFB on cardiomyocyte injury and apoptosis. The results showed that the Model+NC group exhibited outcomes consistent with the Model group, confirming the successful establishment of the model and indicating no therapeutic effect from the plasmid vector alone (Figure 3). The MTT assay indicated that cell viability in the Model+ETFB-OE group was significantly higher than that in the Model+NC group (Figure 3(a)). Importantly, in the Model+ETFB-OE group, the levels of myocardial injury markers (CK-MB, CTnI, CTnT, and LDH) were significantly reduced, with values intermediate between the Con and Model groups (Figure 3(b)–(e)), suggesting that ETFB overexpression effectively alleviates cardiomyocyte injury. At the molecular level, WB analysis revealed that the apoptotic pathway was activated in the Model group, as evidenced by elevated expression of pro-apoptotic proteins (cleaved Caspase-3, cleaved Caspase-6, and Bax) and decreased expression of the anti-apoptotic protein Bcl-2. In contrast, ETFB overexpression reversed these alterations, effectively suppressing the activation of the apoptotic pathway (Figure 3(f)). Further results from TUNEL staining and flow cytometry revealed that the apoptotic rate was significantly higher in both the Model and Model+NC groups than in the Con group; in contrast, ETFB overexpression (Model+ETFB-OE) significantly reduced the proportion of apoptotic cells (Figure 4(a)–(d)). This provides direct evidence at the cellular level that ETFB overexpression effectively inhibits model-induced apoptosis. In summary, ETFB overexpression mitigates H/R-induced cardiomyocyte injury and inhibits apoptosis by modulating key proteins in the apoptotic pathway.

Figure 3.

Electron transfer flavoprotein subunit beta (ETFB) overexpression mitigates hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury and inhibits apoptosis by modulating key proteins in the apoptotic pathway. (a) Analysis of cell viability in the control (Con), H/R model (Model), empty vector control (Model+NC), and ETFB-overexpressing (Model+ETFB-OE) groups. (b–e) Analysis of myocardial injury markers (creatine kinase–myocardial band, cardiac troponin I, cardiac troponin T, and lactate dehydrogenase) in the Con, Model, Model+NC, and Model+ETFB-OE groups, respectively. (f) Protein expression and quantitative analysis of ETFB, Bcl-2-associated X protein, B-cell lymphoma 2, cleaved cysteine-aspartic protease-3, and cleaved cysteine-aspartic protease-6 in the Con, Model, Model+NC, and Model+ETFB-OE groups, as determined by western blot. Data are presented as mean ± SD; each experiment was repeated independently three times (n = 3). **p < 0.01, ***p < 0.001 vs. Con. ns represents not significant vs. Model. ^##p < 0.01, ^###p < 0.001 vs. Model+NC.

Figure 4.

Electron transfer flavoprotein subunit beta (ETFB) overexpression suppresses apoptosis and ameliorates mitochondrial morphology in hypoxia/reoxygenation (H/R)-induced H9c2 cells. (a–b) Apoptosis was detected via terminal deoxynucleotidyl transferase dUTP nick-end labeling assay (with Cyanine 3-labeled dUTP) in the control (Con), H/R model (Model), empty vector control (Model+NC), and ETFB-overexpressing (Model+ETFB-OE) groups. Nuclei were counterstained with DAPI. Scale bar, 50 µm (corresponding to a 200× magnification). (c–d) The apoptosis rate was quantitatively analyzed via flow cytometry with Annexin V-PE/7-AAD staining in the Con, Model, Model+NC, and Model+ETFB-OE groups. (e) Mitochondrial ultrastructure was observed via transmission electron microscopy (TEM) in the Con, Model, Model+NC, and Model+ETFB-OE groups. Red arrows indicate swollen mitochondria. Green arrows indicate blurred cristae. Scale bar, 500 nm. All quantitative data (panels b and d) are presented as mean ± SD from three independent experiments (n = 3). ***p < 0.001 vs. Con. ns represents not significant vs. Model. ^##p < 0.01, ^###p < 0.001 vs. Model+NC.

ETFB overexpression ameliorates mitochondrial function and alleviates oxidative stress in H/R-induced H9c2 cells

To further elucidate the protective mechanism of ETFB, its effects on mitochondrial structure and function as well as oxidative stress levels were evaluated. TEM revealed normal mitochondrial morphology in the Con group, whereas the Model and Model+NC groups exhibited obvious mitochondrial swelling and disruption of cristae structure (Figure 4(e)). In contrast, ETFB overexpression (Model+ETFB-OE) ameliorated these morphological abnormalities, resulting in more regular mitochondrial structure, indicating that ETFB overexpression effectively mitigates H/R-induced mitochondrial damage (Figure 4(e)). Consistent with the morphological impairment, the Model group showed a significant increase in mitochondrial ROS levels and a decrease in MMP levels, indicating severe mitochondrial dysfunction (Figure 5(a)–(d)). However, ETFB overexpression effectively suppressed excessive ROS generation and attenuated the H/R-induced loss of MMP (Figure 5(a)–(d)), demonstrating its role in maintaining mitochondrial functional homeostasis. In terms of oxidative stress, ETFB overexpression reversed the H/R-induced changes by significantly enhancing SOD activity and reducing MDA and GSSG levels (Figure 5(e)–(g)). These findings suggest that the protective role of ETFB in cardiomyocytes is closely associated with the preservation of mitochondrial integrity and the reduction of oxidative stress.

Figure 5.

Electron transfer flavoprotein subunit beta (ETFB) overexpression alleviates oxidative stress in hypoxia/reoxygenation (H/R)-induced H9c2 cells. (a–b) Mitochondrial membrane potential was detected using the JC-1 probe in cells from the following groups: control (Con), H/R model (Model), empty vector control (Model+NC), and ETFB-overexpressing (Model+ETFB-OE). Nuclei were counterstained with DAPI. Scale bar, 50 µm (corresponding to a 200× magnification). (c–d) Intracellular reactive oxygen species levels were assessed using the DCFH-DA probe in the Con, Model, Model+NC, and Model+ETFB-OE groups. Nuclei were counterstained with DAPI. Scale bar, 50 µm (corresponding to a 200× magnification). (e–g) The levels of oxidative stress markers, including superoxide dismutase (e), malondialdehyde (f), and glutathione disulfide (g), were measured in the Con, Model, Model+NC, and Model+ETFB-OE groups. Data are presented as mean ± SD; each experiment was repeated independently three times (n = 3). ***p < 0.001 vs. Con. ns represents not significant vs. Model. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. Model+NC.

Discussion

This study provides the first systematic evidence for the crucial protective role of ETFB in myocardial I/R injury. Our principal findings were as follows: 1. ETFB expression was downregulated in the H/R-induced cardiomyocyte injury model; 2. The downregulation of ETFB is closely associated with enhanced cardiomyocyte apoptosis, impaired mitochondrial structure and function, and elevated oxidative stress; and 3. Overexpression of ETFB attenuates H/R-induced cardiomyocyte injury by suppressing apoptosis, restoring mitochondrial function, and alleviating oxidative stress. Our results not only underscore the importance of ETFB in myocardial protection but also offer novel experimental insights into its underlying mechanism.

WB revealed that ETFB expression was significantly downregulated following H/R treatment. This finding is consistent with the observations by Jiang et al.,¹⁷ who reported decreased ETFB expression in the left ventricular tissue of rats with coronary microembolization, thereby corroborating this phenomenon across different experimental models. Following H/R induction, the levels of myocardial injury markers were significantly elevated in H9c2 cells. This confirms the successful establishment of the myocardial I/R injury cell model, which is consistent with previous reports that myocardial I/R injury elevates these markers.^18,19 Importantly, ETFB overexpression suppressed the H/R-induced elevation of these markers, demonstrating its protective role in I/R injury.

Oxidative stress plays a critical role in the progression of myocardial I/R injury.²⁰ Given that ETFB is the beta-subunit of the electron transfer flavoprotein, we hypothesized that it mitigates cardiomyocyte damage by modulating mitochondrial function and alleviating oxidative stress. To test this hypothesis, we investigated the role of ETFB in the regulation of oxidative stress. Intracellular ROS levels are well-established hallmarks of oxidative stress.^21–23 Key indicators of oxidative stress status—including SOD, MDA, and GSSG—are integral to the maintenance of intracellular redox homeostasis.^24,25 In this study, we found that ETFB overexpression significantly modulated the SOD, MDA, GSSG, and ROS levels, effectively attenuating H/R-induced oxidative stress in H9c2 cells. These results suggest that ETFB plays an important role in preserving cellular redox balance under H/R conditions.

Although the triggers of injury may differ, mitochondrial dysfunction and cell death represent common and critical pathological mechanisms underlying cellular damage in numerous cardiovascular diseases.²⁶ Oxidative stress and mitochondrial dysfunction can form a vicious cycle.²⁷ Specifically, ROS can trigger further massive ROS production by mitochondria and increase the permeability of the mitochondrial permeability transition pore after I/R.²⁸ This leads to an imbalance in mitochondrial osmotic pressure, resulting in mitochondrial swelling, cristae disruption, and outer membrane rupture. Such structural damage to mitochondria, in turn, further stimulates ROS production.²⁹ Therefore, maintaining mitochondrial structural integrity is considered crucial for mitigating I/R injury.³⁰ Our results demonstrate that ETFB overexpression enhances MMP and improves mitochondrial structure, suggesting that ETFB may exert its protective effects by interrupting this vicious cycle. This finding not only establishes ETFB as a potential therapeutic target but also further corroborates that maintaining mitochondrial homeostasis is an effective protective strategy for alleviating cardiovascular injury.^31–33

The fate of injured cells—whether they undergo necrosis or apoptosis—is largely determined by the interplay between ROS production and mitochondrial dysfunction.^34,35 Mitochondrial dysfunction, in particular, is a critical event in the initiation of apoptosis.^36,37 Key mitochondrial events, such as MMP depolarization, cristae disruption, and outer membrane rupture, facilitate the release of apoptogenic proteins into the cytosol, thereby initiating the apoptotic cascade.³⁸ In this study, we found that ETFB overexpression significantly reduced the apoptosis rate in H9c2 cells subjected to H/R. At the molecular level, WB analysis revealed that ETFB downregulated the expression of pro-apoptotic proteins, including cleaved caspase-3, cleaved caspase-6, and Bax, while upregulating the expression of the anti-apoptotic protein Bcl-2. These findings suggest that ETFB may exert its anti-apoptotic effect by modulating the critical Bcl-2/Bax balance, thereby inhibiting the activation of the caspase cascade.^39,40 Notably, the imbalance of mitochondrial homeostasis in myocardial I/R injury is regulated by multiple aspects. For instance, the nuclear receptor NR4A1 has been shown to promote apoptosis-mediated myocardial I/R injury by inducing excessive mitochondrial fission and inhibiting FUNDC1-mediated autophagy, thereby disrupting mitochondrial quality control.⁴¹ In contrast, our study demonstrates that ETFB preserves mitochondrial functional integrity and inhibits apoptosis in myocardial I/R injury. Collectively, these findings highlight that ETFB and NR4A1 influence apoptosis-mediated I/R injury progression by maintaining mitochondrial functional integrity and regulating its structural renewal, respectively. This suggests that simultaneous intervention targeting multiple pathways may offer greater myocardial protection than single-target strategies.

Thus, this study confirms that ETFB serves as a protective factor in myocardial I/R injury, and its mechanism involves the amelioration of mitochondrial function, thereby reducing oxidative stress and inhibiting apoptosis. These findings provide new insights into the pathophysiological mechanisms of myocardial I/R injury and identify ETFB as a potential target for future therapeutic interventions. However, this study has several limitations that should be acknowledged. First, all experiments were conducted in cell lines, and further validation in animal models is required. Second, the specific molecular mechanisms by which ETFB influences mitochondrial function warrant further investigation. Notably, future research could assess the distribution levels of mitochondrial integrity markers (such as HSP60 and cytochrome c) in the cytoplasm and mitochondria, which would help more precisely elucidate the role of ETFB in maintaining mitochondrial structural integrity and function. Additionally, future studies could explore the long-term effects of ETFB overexpression on cardiomyocytes under normoxic steady-state conditions. Finally, the upstream regulatory mechanisms controlling ETFB expression remain to be elucidated. Addressing these limitations in future work will facilitate a more comprehensive understanding of ETFB’s role in myocardial protection and promote its translation into clinical applications.

Conclusion

This study systematically demonstrates that ETFB protects cardiomyocytes from I/R injury by improving mitochondrial function, alleviating oxidative stress, and inhibiting apoptosis. These findings confirm the crucial role of ETFB in myocardial protection and establish a foundation for developing new therapeutic strategies aimed at maintaining mitochondrial homeostasis against myocardial I/R injury.

Footnotes

Acknowledgements

Not applicable.

Author contributions

Jing Liu conceptualized the study, performed experiments, analyzed the data, and wrote the manuscript.

Data availability statement

The data underlying this article will be shared upon reasonable request to the corresponding author.

Declaration for conflicting interests

The author declares no conflicting interests.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

ORCID iD

Jing Liu

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