Mitochondrial protection role of oxidoreductase-like domain containing 1 in myocardial cells under hypoxia

Abstract

Although mitochondria and related proteins are essential for mitochondrial preservation, the functions of some of these proteins remain unknown. The novel protein oxidoreductase-like domain containing 1 (OXLD1/C17orf90, UniProtKB Q5BKU9) have attracted our attention because of its correlation with mitochondria. This study revealed a decrease in OXLD1 levels in cardiomyocytes cultured in 1% oxygen for 24 hours. Suppressing OXLD1 increases mitochondrial injury under both normoxic and hypoxic conditions. This is evidenced by decreased mitochondrial membrane potential and increased reactive oxygen species production. Meanwhile, suppressing OXLD1 decreased mitochondrial oxidative phosphorylation. Overexpression of OXLD1 decreased mitochondrial injury under normoxia and hypoxia, as indicated by an increase in the mitochondrial membrane potential and a decrease in reactive oxygen species production. Moreover, overexpression of OXLD1 enhanced mitochondrial oxidative phosphorylation. Additionally, we found that OXLD1 regulates mitochondrial oxidative phosphorylation by affecting mitochondrial complexes I and V. OXLD1 plays a crucial role in protecting cardiomyocytes by improving mitochondrial function under low-oxygen conditions. OXLD1 achieves this protection through interactions with mitochondrial complexes I and V. Therefore, OXLD1 may serve as a new and important regulator of mitochondrial function.

Introduction

Mitochondria, the powerhouse of the cell, rely on a complex network of more than 1200 proteins to execute essential cellular processes.1 When mitochondrial function is compromised, a wide array of metabolic diseases can occur, highlighting the critical importance of these organelles in maintaining cellular health.2 Disruption of proteins associated with mitochondria can have profound effects on cellular function.3 Despite the extensive research on mitochondrial proteins, the specific roles of certain proteins remain largely undetermined.4 Among these proteins, we have become particularly interested in a novel protein known as oxidoreductase-like domain containing 1 (OXLD1/C17orf90, UniProtKB Q5BKU9) due to its unique properties and potential biological significance. OXLD1 is situated within the terminal region of chromosome 17 at 17q25.3. Although the functional role of the OXLD1 protein has not yet been fully elucidated, some studies have indicated that genes located in this terminal region may serve as assembly factors for mitochondrial complex I, playing crucial roles in various mitochondrial processes, including the operation of the respiratory chain. Additionally, this region has been identified as a novel locus associated with cardiac susceptibility.5,6 Moreover, emerging research has suggested a potential link between OXLD1 and the critical mitochondrial regulatory protein C7orf55, further emphasizing the need for further investigations into OXLD1 functions.7

Given the established connection between mitochondrial dysfunction and various heart diseases, coupled with the positioning of OXLD1 within a cardiac susceptibility locus, we hypothesized that OXLD1 might serve as a key regulator of mitochondrial function. This study aimed to test the hypothesis that OXLD1 protects mitochondrial function under hypoxia and to investigate its regulatory mechanisms.

Methods

Plasmid construction and RNA interference

The OXLD1 overexpression (OE) plasmid was constructed by cloning the subclone OXLD1 [Rattus norvegicus (Norway rat)] (NCBI accession No. NM_001305985.1) into the plenti-GII-CMV-CBH-GFP-2A-Puro (Amp) vector at the EcoRI and XbaI sites. The OXLD1 overexpression (OE) plasmid and siRNA_{OXLD1 186, 348, and 558} were provided by Qianzhao Xinye Biology Science and Technology Co., Ltd., Beijing, China. siRNA_{OXLD1 186, 348, and 558} consisted of the following sequences: forward: 5’-GCU CAC UGA GAG CUU UCU UTT-3’ and reverse: 5’-AAG AAA GCU CUC AGU GAG ATT-3’; forward: 5’-CCA AGU AGG UAC AGA GGU UTT-3’ and reverse: 5’-AAC CUC UGU ACC UAC UUG GTT-3’; forward: 5’-GCA CGU GAC UGA UGA GAA CTT-3’ and reverse: 5’-GUU CUC AUC AGU CAC GUG CTT-3’.

Groups of experiments

The study timeline is shown in Figure 1.

Figure 1.

Groups of experiments.

H9c2 cells were transfected with OXLD1 overexpression vector and OXLD1 siRNA. Control group cells were cultured in DMEM with 20% O₂, while hypoxia group cells were cultured with 1% O₂. The normoxia group H9c2 cells were cultured in DMEM with 20% O₂ and 5% CO₂ for 24 hours. H9c2 cells from all groups were harvested for subsequent experiments. Created with Microsoft Office PowerPoint (2019). DEME: Dulbecco’s modified Eagle medium; Hypoxia: cells were grown under 1% O₂ and 5% CO₂ for 24 hours; KD: knockdown; NC: negative control; Normoxia: cells grew under 20% O₂ and 5% CO₂; OE: overexpression; OXLD1: oxidoreductase like domain containing 1.

The H9c2 (RRID:CVCL_0286) rat cardiomyoblast cell line was obtained from Qianzhao Xinye Biology Science and Technology Co., Ltd. (Beijing, China). H9c2 cells were recently authenticated by short tandem repeat profiling and were confirmed to be free of mycoplasma contamination. H9c2 cells were transfected with the OXLD1 overexpression plasmid and siRNA_{OXLD1 186, 348, and 558}. The normoxia group cells were grown in complete medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA, 10100147), 1% penicillin‒streptomycin, and high-sugar Dulbecco’s modified Eagle medium (Invitrogen, 11965092) under 20% O₂ and 5% CO₂ at 37 °C, while hypoxia group cells were cultured with 1% O₂.

Detection of OXLD1 by reverse transcription-quantitative polymerase chain reaction

Total RNA was extracted from cells using the RNAqueous® Total RNA Isolation Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA; AM1912).8 Reverse transcription-quantitative polymerase chain reaction (PCR) was conducted using the PrimeScript^TM RT reagent kit (Takara Biomedical Technology Co., Ltd., Osaka, Japan, RR047A)9 and GoTaq qPCR master mix (Promega Corporation, Madison, WI, USA, A6001).10 The CFX96 touch real-time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA; C1000 Touch) was employed for detection. β-Actin was used as a reference gene. The primer sequences used for PCR were as follows: OXLD1 forward: 5′-GCT TGG AGG AGT CAT GGT CA-3′, reverse: 5′-TTC TGC GGC TAT CAG TGG TT-3′; β-actin forward: 5′-AGG CAT CCT GAC CCT GAA GTA C-3′, and reverse: 5′-GAG GCA TAC AGG GAC AAC ACA G-3′.

Detection of OXLD1 by western blotting

The cells were lysed with radio immunoprecipitation assay lysis buffer containing phenylmethanesulfonyl fluoride (Coolaber, Beijing, China, SL1020) for protein extraction. The proteins were separated by 10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (Bio-Rad Laboratories, Inc., 5671034) and transferred to polyvinylidene fluoride transfer membranes (Thermo Fisher Scientific Inc., 88520). Following blocking, we incubated the polyvinylidene fluoride membranes overnight with primary antibodies at 4°C. Then, the polyvinylidene fluoride membranes were incubated with the secondary antibody goat anti-rabbit IgG (1:3000, Abcam, Cambridge, USA, Cat# ab6721, RRID:AB_955447) at room temperature for 2 hours. We obtained the images using a General Electric Company Imager 600 series detector (Boston, MA, USA) with chemiluminescence detection reagents from Applygen Technologies, Inc. (Beijing, China). Primary antibodies against OXLD1 (rabbit, 1:1000, Abcam, ab188306) and β-actin (mouse, 1:1000, Abcam, Cat# ab8226, RRID:AB_306371) were used. The images were quantified by scanning densitometry with ImageJ software (ImageJ 1.8.0, National Institutes of Health, Bethesda, MD, USA). β-Actin served as the loading control. The results from each experimental group are expressed as the relative integrated intensity compared with that of the control group measured at the same time.

Colocalization of OXLD1 with mitochondria

In this study, the subcellular localization of the OXLD1 protein was predicted using the GeneCards database (https://www.genecards.org/).11

The cells were fixed in 4% paraformaldehyde at room temperature for 30 minutes. After blocking, the cells were incubated overnight with an anti-OXLD1 antibody (rabbit, 1:500) at 4°C. Next, the cells were incubated at room temperature for 2 hours with secondary antibody (goat anti-rabbit IgG H&L-Alexa Fluor® 488, 1:200, Abcam, Cat# ab150077, RRID:AB_2630356). After washing, the cells were incubated with anti-translocase of the outer membrane 20 (TOM20) antibody (rabbit, CoraLite® 594-conjugated, 1:200, Proteintech Group, Inc., Chicago, IL, USA, CL594-11802, RRID:AB_2919775) at room temperature for 2 hours, followed by nuclear staining. Nuclei were stained with 4′,6-diamidino-2-phenylindole (Beyotime, Shanghai, China, C1002), which emits blue fluorescence. The subcellular localization of OXLD1 was detected by laser confocal microscopy (Leica, Heidelberg, Germany, STELLARIS 8).

Detection of the real-time ATP rate

After serial addition of mitochondrial inhibitors (oligomycin and rotenone/antimycin A), Seahorse Analyzers (Agilent, Santa Clara, CA, USA) directly measure the real-time extracellular acidification rate and oxygen consumption rate of cells, indicators of the two major energy-producing pathways: glycolysis and oxidative phosphorylation. After analysis, the cells in each well were counted, and the results were normalized to a value per 5000 cells. The calculation of the mitochondrial and glycolytic ATP production rates provides a real-time measurement of the cellular ATP production rates and a quantitative phenotype of cellular energy poise.12

Detection of cell mito-stress

The cell mito-stress test directly measures the oxygen consumption rate to assess key parameters of mitochondrial function. The compounds (oligomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, and a mixture of rotenone/antimycin A) were serially injected to measure ATP production, maximal respiration, and nonmitochondrial respiration, respectively. These parameters, along with basal respiration, are used to calculate proton leakage and spare respiratory capacity.13 After analysis, we counted the cells in each well and normalized the results to a value per 5000 cells.

Detection of the glycolytic rate

The glycolytic rate assay is a dependable method for measuring glycolysis in cells. The compounds Rot/AA and 2-deoxy-D-glucose are injected serially to measure the glycolytic proton efflux rate of the cells.14 After analysis, we counted the cells in each well and normalized the results to a value per 5000 cells.

Detection of the mitochondrial membrane potential by laser confocal microscopy

The fluorescent dye tetramethylrhodamine ethyl ester (TMRE) was utilized to detect the mitochondrial membrane potential (MMP).15 The cells were collected and detected via laser confocal microscopy. The integrated density/area of MMP production was measured by ImageJ software.

Detection of the reactive oxygen species content by flow cytometry

A fluorometric intracellular ROS kit (Sigma‒Aldrich Corporation, St. Louis, MO, USA, MAK145) was used to measure the ROS content in H9c2 cells. The cells were collected and detected by flow cytometry (SONY, Tokyo, Japan, SH800).16,17

Detection of mitochondrial complex activity

Complex I activity (Abcam, Cat# ab109721) was measured, and Complex II to V activities (Cayman, Cat# 700950, 700990, and 701000) were measured according to the manufacturer’s instructions.18,19,20 Complex activities were calculated by dividing the changes in absorbance over time in the linear range of the readings.

Statistical analysis

Statistical analysis was conducted using GraphPad Prism 7.0.0 (GraphPad Software, Boston, MA, USA; www.graphpad.com). Significant differences between two groups were evaluated using Student’s t-test. The 95% confidence interval (CI) for the parameters in our analysis was estimated, and a two-sided P < 0.05 (the threshold) was defined as statistically significant. Continuous data variables are summarized as the means and standard deviations (SD). To compare continuous data variables among multiple groups, we performed one-way analysis of variance followed by Tukey’s post hoc test.

Results

OXLD1 expression is suppressed under hypoxia in H9c2 cells

We observed that OXLD1 expression levels were reduced in cardiomyocytes exposed to hypoxia. To further investigate the reduction in OXLD1 expression, we conducted a thorough examination of cardiomyocytes subjected to hypoxia. We quantified OXLD1 expression at the mRNA and protein levels using PCR and Western blot analyses, as shown in Figure 2A and B. Our results showed a significant decrease in OXLD1 expression in the hypoxia group compared with the control group (n = 3, P < 0.01), emphasizing the effect of hypoxic stress on this regulatory protein. In addition to measuring expression levels, we also sought to determine the subcellular localization of OXLD1. Predictions from GeneCards suggested that OXLD1 is primarily localized in the mitochondria and the cytosol (Figure 2C). To confirm this localization, we assessed the enrichment of OXLD1 within mitochondrial compartments. The co-localization of OXLD1 with mitochondria was determined using immunofluorescence staining, employing TOMM20 as a mitochondrial marker alongside OXLD1 staining. Our observations revealed co-localization of OXLD1 protein with mitochondria, as depicted in Figure 2D. Considering the experimental evidence that demonstrates a reduction in OXLD1 expression under hypoxic conditions, along with its notable enrichment within the mitochondrial population, we propose that OXLD1 is potentially associated with mitochondrial respiratory function.

Figure 2.

The level of OXLD1 expression in H9c2 cells under hypoxia.

(A) The expression level of the OXLD1 gene in H9c2 cells was assessed using qRT‒PCR. n = 3 in each group. β-Actin served as the internal control reference gene. (B) The expression level of the OXLD1 protein in H9c2 cells was measured by Western blot. n = 3 in each group. β-Actin was used as a reference protein. (C) Prediction of OXLD1 subcellular localization in the GeneCards database. The varying shades of green indicate confidence levels from 1 to 5. (D) Co-localization of OXLD1 with mitochondria. The cells were stained with TOM20, which exhibited red fluorescence, and OXLD1, which exhibited green fluorescence, simultaneously. The orange particles indicated fusions between mitochondria and OXLD1. Scale bar: 50 μm, 10 μm (magnified image). Data are presented as the mean ± SD. **P < 0.01, vs. control group (two-tailed Student’s t-test). Control group: H9c2 cells were cultured in 20% O₂; DAPI: 4′,6-diamidino-2-phenylindole; hypoxia group: H9c2 cells were cultured in 1% O₂ for 12 hours; OXLD1: oxidoreductase like domain containing 1; qRT‒PCR: reverse transcription-quantitative polymerase chain reaction; TOM20: translocase of the outer membrane 20.

OXLD1 enhances the respiratory function of H9c2 cells

To elucidate the role of OXLD1, we evaluated respiratory function in OXLD1-OE and OXLD1-KD H9c2 cells under hypoxic conditions. H9c2 cells were transfected with the OXLD1-OE vector or specific small interfering RNAs (siRNAs) (containing siRNA_{OXLD1 186, 348, and 558}) targeting OXLD1 for 48 hours. We chose siRNA_{OXLD1 558}, due to its superior knockdown efficiency, to generate OXLD1-KD cells for further experiments. We successfully generated both OXLD1-OE and OXLD1-KD H9c2 cells (Figure 3). Firstly, we assessed the levels of OXLD1 mRNA expression in both the OXLD1-OE and OXLD1-KD groups under hypoxia. Compared with that in the control group, the expression of OXLD1 mRNA increased in the OXLD1-OE group under hypoxic conditions (Figure 4A). Secondly, we measured total ATP production rates in OXLD1-OE and OXLD1-KD cells under both normoxic and hypoxic conditions, utilizing the Agilent Seahorse real-time ATP rate test. The results indicated a significant increase in ATP production rates in the OXLD1-OE group, while the OXLD1-KD group presented a marked decrease compared with the control group under both conditions (Figure 4B). Thirdly, we evaluated oxidative phosphorylation (OXPHOS) in OXLD1-OE and OXLD1-KD cells under normoxia and hypoxia using the Agilent Seahorse cell mito-stress test. Lastly, we measured glycolysis using the Agilent Seahorse glycolytic rate assay. Our findings showed that OXPHOS was significantly increased in the OXLD1-OE group but significantly decreased in the OXLD1-KD group under both normoxic and hypoxic conditions (Figure 4C). Notably, there were no significant changes in glycolysis between the OXLD1-OE and OXLD1-KD groups (Figure 4D). These data suggest that OXLD1 plays a crucial role in enhancing mitochondrial respiratory function in cardiomyocytes under hypoxic conditions.

Figure 3.

Development of cell models for OXLD1 overexpression and knockdown.

(A) OXLD1 mRNA expression levels in both the control and OXLD1-OE groups were measured using RT-qPCR. β-Actin served as the internal control reference gene. H9c2 cells were transfected with the OXLD1 overexpression vector containing GFP (green fluorescence) after 48 hours. Scale bars: 50 μm. (B) OXLD1 mRNA expression levels in the control and OXLD1-KD groups (transfected with siRNA_{OXLD1 186, 348, and 558}) were detected by RT-qPCR. siRNA_{OXLD1 558} was best knockdown effect to construct OXLD1-KD cells. β-Actin served as the internal control reference gene. (C) The protein expression levels of OXLD1 in the control, OXLD1-OE, and OXLD1-KD groups. β-Actin served as the internal control reference protein. n = 3 in each group. Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, vs. control group (Student’s t-test (A) or one-way analysis of variance followed by Tukey’s post hoc test (B, C)). KD: Knockdown; OE: overexpression; OXLD1: oxidoreductase like domain containing 1; qRT‒PCR: reverse transcription-quantitative polymerase chain reaction.

Figure 4.

OXLD1 enhances mitochondrial oxidative phosphorylation in H9c2 cells both in normoxia and hypoxia.

(A) qRT-PCR was used to measure OXLD1 mRNA expression levels in the OXLD1-OE and OXLD1-KD groups under normoxia and hypoxia. β-Actin served as the internal control reference gene. n = 3 in each group. (B) Total ATP production rates were measured using the Agilent Seahorse analyzer under both normoxic and hypoxic conditions. The ECAR and OCR were measured under basal conditions. By obtaining these data under basal conditions and after serial addition of Rotenone/antimycin A, total cellular ATP production rates could be measured in real-time. n = 5 in each group. (C) Mitochondrial respiration was measured, including ATP production, maximal respiration, proton leak, respiratory reserve capacity, and basal respiration under normoxia or hypoxia conditions. The compounds (oligomycin, FCCP, and a mix of Rote/AA) were serially injected to measure mitochondrial respiration. n = 5 in each group. (D) Glycolysis was measured, including basal glycolysis and compensatory glycolysis, under normoxia or hypoxia conditions. Glycolysis was measured in the presence of Rote/AA and 2-DG at indicated time points. n = 5 in each group. The results were normalized to a value per 5000 cells for standardization. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, vs. normoxia; ^#P < 0.05, ^##P < 0.01, vs. hypoxia (one-way analysis of variance followed by Tukey’s post hoc test). 2-DG: 2-Deoxy-d-glucose; ECAR: extracellular acidification rate; FCCP: carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone; Hypoxia: cells were grown under 1% O₂ and 5% CO₂ for 24 hours; KD: knockdown; Normoxia: cells were grown under 20% O₂ and 5% CO₂; OCR: oxygen consumption rate; OE: overexpression; OXLD1: oxidoreductase like domain containing 1; Rote/AA: rotenone/antimycin A.

OXLD1 protects the mitochondrial membrane potential in H9c2 cells under hypoxia

We measured the MMP to determine whether OXLD1 preserves mitochondrial function. Confocal microscopy results showed that the MMP in the OXLD1-OE group was higher than that in the control group. This phenomenon was observed under both normoxic and hypoxic conditions. In contrast, the MMP in the OXLD1-KD group was lower than that in the control group under both normoxia and hypoxia (Figure 5). These findings suggest that OXLD1 helps prevent the decrease in the MMP caused by hypoxia.

Figure 5.

The function of OXLD1 on mitochondrial membrane potential in H9c2 cells under normoxia and hypoxia.

(A) MMP fluorescence staining was stained by TMRE fluorescent dye (red). Nuclei were stained with DAPI, which emits blue fluorescence. (B) MMP fluorescence value. n = 3 in each group. Scale bar: 50 μm, 10 μm (magnified image). Data are presented as the mean ± SD. **P < 0.01, vs. normoxia; ^##P < 0.01, vs. hypoxia (one-way analysis of variance followed by Tukey’s post hoc test). DAPI: 4’,6-Diamidino-2-phenylindole; Hypoxia: cells were grown under 1% O₂ and 5% CO₂ for 24 hours; KD: knockdown; MMP: mitochondrial membrane potential; Normoxia: cells were grown under 20% O₂ and 5% CO₂; OE: overexpression; OXLD1: oxidoreductase like domain containing 1; TMRE: tetramethylrhodamine ethyl ester.

OXLD1 reduces reactive oxygen species production in H9c2 cells under hypoxia

We measured mitochondrial ROS levels to investigate the role of OXLD1 in this process. We used a fluorescent probe to specifically label the ROS. Flow cytometry was then employed to quantify the levels of ROS. As shown in Figure 6, ROS production was significantly lower in the OXLD1-OE group than in the control group under normoxic and hypoxic conditions (P < 0.01). We observed that the ROS levels in the OXLD1-KD group were significantly higher than those in the control group under normoxia and hypoxia (P < 0.01). Overall, these data suggest that OXLD1 plays a crucial protective role by reducing ROS production during hypoxia, thereby contributing to mitochondrial function and cellular homeostasis.

Figure 6.

The role of OXLD1 in regulating reactive oxygen species production in H9c2 cells under normoxic and hypoxic conditions.

(A, B) The ROS content in OXLD1-OE cell groups was measured under normoxic and hypoxic conditions using flow cytometry. (C, D) The ROS content in OXLD1-KD cell groups was measured under normoxic and hypoxic conditions using flow cytometry. n = 3 in each group. Data are presented as the mean ± SD. **P < 0.01 (one-way analysis of variance followed by Tukey’s post hoc test). Hypoxia: Cells were cultured in 1% O₂ and 5% CO₂ for 24 hours; KD: knockdown; Normoxia: cells were cultured in 20% O₂ and 5% CO₂; OE: overexpression; OXLD1: oxidoreductase like domain containing 1.

OXLD1 enhances mitochondrial complex activity in H9c2 cells

We explored the impact of OXLD1 on the enzymatic activities of mitochondrial complexes I–V. Our findings showed that, in the OXLD1-OE group, the activities of mitochondrial complexes I and V were enhanced under both normoxic and hypoxic conditions compared with those in the control group. In the OXLD1-KD group, we noted a decrease in the activities of mitochondrial complexes I and V under both normoxic and hypoxic conditions (Figure 7A and D). Our analysis revealed no changes in the activities of mitochondrial complexes II, III, and IV across all experimental conditions (Figure 7B and C). This specificity indicates that OXLD1 primarily modulates mitochondrial respiratory efficiency through its effects on complexes I and V. Overall, our results demonstrate that OXLD1 positively affects mitochondrial respiratory capacity mainly through its influence on complexes I and V.

Figure 7.

OXLD1 enhances the activities of mitochondrial complexes I and V in H9c2 cells.

(A) The activities of mitochondrial complexes I in H9c2 cells are measured in mOD per minute per milligram of total protein. (B) The activities of mitochondrial complexes II and III in H9c2 cells are measured in mOD per minute per milligram of total protein. (C) The activities of mitochondrial complexes IV in H9c2 cells are measured in mOD per minute per milligram of total protein. (D) The activities of mitochondrial complexes V in H9c2 cells are measured in mOD per minute per milligram of total protein. n = 3 in each group. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, vs. normoxia; ^#P < 0.05, ^##P < 0.01, vs. to hypoxia (one-way analysis of variance followed by Tukey’s post hoc test). Hypoxia: cells were cultured in 1% O₂ and 5% CO₂ for 24 hours; KD: knockdown; mOD: milli-optical density; Normoxia: cells were cultured in 20% O₂ and 5% CO₂; OE: overexpression; OXLD1: oxidoreductase like domain containing 1.

Discussion

The terminal segment of chromosome 17, specifically at the locus 17q25.3, has been identified as a novel genetic region that is associated with cardiac dysfunction, as noted in various studies.6,21 However, the precise function of the gene OXLD1, which is located in the terminal region of 17q25.3, remains largely unclear and warrants further investigation. The heart, a vital organ in the human body, plays an essential role in facilitating blood circulation and ensuring the distribution of oxygen throughout the organism, which is crucial for maintaining overall health. Furthermore, the heart itself has an intrinsic requirement for oxygen to sustain effective contractility, which is necessary for its proper function.22 Our observations revealed that the expression level of OXLD1 in H9c2 cardiac cells was significantly decreased under hypoxic conditions, indicating a potential link between oxygen availability and the expression of this gene. Previous research has highlighted that there are notable alterations in the respiratory function of cardiomyocytes that are associated with cardiac dysfunction, suggesting a complex interplay between these factors.23,24 To delve deeper into the relationship between OXLD1 and cardiac dysfunction, we conducted an assessment of total ATP production rates in both OXLD1-OE and OXLD1-KD H9c2 cells under hypoxic conditions, aiming to elucidate the role of OXLD1 in the context of cardiac health and disease.

The overexpression of OXLD1 has the potential to significantly elevate the rate of ATP production, a critical process for cellular energy. Total ATP is generated through two primary metabolic pathways, mitochondrial OXPHOS and glycolysis, both of which are essential for ATP synthesis in mammalian cells. This raises an important question: is the impact of OXLD1 expression on the ATP production rate primarily attributable to OXPHOS, or does it also involve glycolysis? To address this issue, we conducted a thorough evaluation of both the OXPHOS and glycolysis pathways. The results of our investigation indicated that OXLD1 enhances the respiratory function of cardiomyocytes predominantly through the OXPHOS pathway rather than through glycolysis. Mitochondria, often referred to as the powerhouse of the cell, serve as the central hub for aerobic respiration.25,26 Over 90% of ATP is generated by OXPHOS occurring within the mitochondria. The OXPHOS system plays a crucial role in the conversion of cellular energy, and any impairment in this system can lead to various mitochondrial disorders, highlighting its importance in maintaining cellular health.

The ETC consists of multimeric enzymes known as complexes I to V, which are essential for transferring electrons from nicotinamide adenine dinucleotide (NADH) and reduced flavin adenine dinucleotide (FADH₂) to molecular oxygen (O₂) to support cellular respiration. Complex V harnesses the proton motive force that is generated during the electron transfer process to synthesize ATP, the energy currency of the cell.27 In our comprehensive study assessing the impact of OXLD1 on the enzymatic activities of mitochondrial complexes, we observed that both complex I and complex V exhibited significant alterations, indicating a profound influence of OXLD1 on mitochondrial function. Complex I, in particular, serves as a critical regulatory hub for mitochondrial respiratory function, acting as the primary site for the production of ROS and playing a pivotal role in apoptosis.28 Disorders related to mitochondrial oxidative phosphorylation are among the most common metabolic disorders in clinical practice. Notably, among all OXPHOS deficiencies, deficiency of respiratory chain complex I stands out as the most frequently observed. A previous study suggests that a gene on chromosome 17q25.3 acts as an assembly factor for complex I and is involved in various mitochondrial functions, including those of the respiratory chain.29 Despite this knowledge, no research has yet been conducted to elucidate the specific role of OXLD1 as a complex I assembly factor within mitochondrial processes. Our data demonstrated that OXLD1 significantly enhanced the activities of both ETC complex I and complex V, leading to an increase in electron acceptor activity. As a result of these OXLD1 enhancements, ATP production from the OXPHOS pathway significantly increased.

In addition to its primary role in producing ATP, the OXPHOS process is responsible for generating approximately 90% of ROS, which are byproducts of cellular respiration. Among the various complexes involved in this process, complex I is recognized as the principal contributor to ROS production. Our study revealed that OXLD1 plays a regulatory role in both complex I and the overall OXPHOS pathway. During our developmental investigations, we discovered that OXLD1 has the ability to reduce ROS production, particularly under hypoxic conditions, where oxygen levels are significantly low. The accumulation of excessive mitochondrial ROS is often regarded as an early indicator of mitochondrial dysfunction.30,31 Generally, ROS are perceived as toxic byproducts resulting from aerobic metabolism, and they are primarily responsible for causing damage to macromolecules within the cell. Interestingly, a lack of oxygen can trigger a process known as reductive carboxylation, which has been shown to lead to an increase in ROS production.32 Therefore, OXLD1 is essential for preventing the excessive formation of ROS during hypoxic episodes. The mitochondrial ETC is crucial because it generates an electrochemical gradient through a series of intricate redox reactions. This electrochemical gradient not only drives the synthesis of ATP but also contributes to the generation of MMP. MMP serves as a critical parameter for assessing mitochondrial function and health.33 Our findings indicate that OXLD1 can alleviate the decline in MMP that is typically induced by hypoxia. OXLD1 plays a vital role in preserving both the structure and function of mitochondria.

Our research also has some limitations. First, we focused exclusively on how OXLD1 protects cardiomyocytes. This protection occurs through a reduction in ROS levels, enhancement of MMP, and improvement in mitochondrial respiration in a cell model. Therefore, to confirm these effects in animal models, we will pursue additional research next. This area of research will be our next focus. Second, OXLD1 is a novel regulator of OXPHOS and represents a promising research avenue. However, its mechanisms influencing complexes I and V require further investigation.

In summary, our comprehensive data suggest that OXLD1 plays a crucial role in protecting cardiomyocytes by effectively decreasing the level of ROS, enhancing the expression of MMP, and promoting increased mitochondrial respiration. The beneficial effects of OXLD1 on mitochondrial function are mediated specifically through its interaction with mitochondrial complexes I and V. Consequently, OXLD1 has emerged as a novel and significant regulator of mitochondrial function, highlighting its potential importance in cardiac health and disease management.

Funding Statement

Funding: This study was supported by the National Natural Science Foundation of China (Nos. 82001994 and 82000311).

Data availability statement:

Data will be made available on request.