Mitochondria-targeted antioxidant (MitoQ) and non-targeted antioxidant (Idebenone) mitigate mitochondrial dysfunction in corneal endothelial cells

Abstract

Purpose:

To investigate the effectiveness of mitochondrial-targeted antioxidant Mitoquinone (MitoQ) and non-targeted antioxidant Idebenone (Idb) in alleviating mitochondrial dysfunction in corneal endothelial cells (CEnCs).

Methods:

In vitro experiments were carried out using immortalized normal human corneal endothelial cells (HCEnC-21T; SVN1-67F) and Fuchs endothelial corneal dystrophy (FECD) cells (SVF5-54F; SVF3-76M). Cells were pre-treated with MitoQ or Idebenone (Idb) and then exposed to menadione (MN) with simultaneous antioxidant treatment. Mitochondrial parameters were evaluated through ATP viability assays, JC-1 staining for membrane potential (MMP), and Tom-20 antibody staining for fragmentation, with analysis performed using ImageJ software. HCEnC-21T cells were additionally exposed to UVA (25 J/cm²) to assess drug effects under physiological stress. Mitochondrial fragmentation in FECD specimens was analyzed pre- and post-treatment with the drugs. Statistical analysis was conducted using one-/two-way ANOVA with post-hoc Tukey’s test.

Results:

MitoQ and Idb enhanced cell viability and MMP in both normal and FECD cells under MN-induced stress. Idb reduced MN-induced mitochondrial fragmentation by 32% more than MitoQ in HCEnC-21T cells and by 13% more in SVF5-54F cells. Under UVA stress, Idb and MitoQ improved mitochondrial function by 31% and 25%, respectively, with MitoQ increasing mitochondrial function by 42% in FECD specimens.

Conclusion:

Differential responses in mitochondrial dysfunction across cell lines highlight disease heterogeneity. MitoQ and Idb protected CEnCs from oxidative stress and improved mitochondrial bioenergetics, suggesting that mitochondrial-targeted antioxidants could be considered for mitochondrial dysfunction in CEnCs.

Introduction

Human corneal endothelial cells (CEnCs) are post-mitotic cells, arrested in the G1 phase of the cell cycle,^1,2 that have limited regenerative potential. Multiple factors (environmental or genetic) could lead to increased intracellular reactive oxygen species (ROS) production and oxidative stress, resulting in accumulation of mitochondrial damage and dysfunction in CEnCs.

This supports the proposition of a vicious cycle of oxidant-antioxidant imbalance in the pathogenesis of corneal endothelial disease such as Fuchs endothelial corneal dystrophy (FECD).³ To date, no pharmacological treatment exists, and as the corneal endothelial dysfunction advances, corneal transplantation remains the only treatment option.

Mitochondria that are directly exposed to the ROS generate electron transport chain (ETC) leak and lack histone protection, thus making mitochondrial DNA (mtDNA) susceptible to oxidative damage. Mitochondrial dysfunction is a hallmark of aging resulting from oxidative stress, which plays a critical role in the pathogenesis of FECD and leads to cumulative mitochondrial damage.^4–6 CEnCs contain a high density of mitochondria that generate adenosine triphosphate (ATP) for energy supply to multiple Na+/K+-ATPase and other pumps essential for the function of CEnCs.^7–11 Prior studies have investigated the role of mitochondria in the degenerative process of FECD and observed an accumulation of 8-hydroxy-2-deoxyguanosine (8-OHdG) a marker of oxidized DNA lesions, primarily in mtDNA of CEnCs clustered in rosettes. This finding indicates that mtDNA is a specific target of oxidative stress in FECD.^6,12–14 Importantly, the colocalization of oxidative DNA damage, mostly to mitochondria, and apoptosis was found to be specific to FECD as similar findings were not detected in the CEnCs from pseudophakic bullous keratopathy (PBK) patients.⁶

Antioxidants, such as exogeneous ubiquinones can prevent oxidative damage and redox signaling and could alleviate mitochondrial stress. These molecules are based on the predominant human form of endogenous ubiquinone, coenzyme Q10 (CoQ10), which is synthesized in the mitochondrial inner membrane.¹⁵ With mitochondrial dysfunction, mitochondrial enhancement by the short-chain analog of CoQ10, Idebenone (Idb), could decrease oxidative damage and improve mitochondrial bioenergetics. Consequently, a mitochondria-targeted ubiquinone, Mitoquinone (MitoQ), that accumulates within mitochondria was also developed to mitigate mitochondrial dysfunction.¹⁶

In this study, we investigated whether the mitochondria-targeted antioxidant MitoQ or the non-targeted antioxidant Idb enhance mitochondrial bioenergetics and alleviate mitochondrial dysfunction of CEnCs following oxidative stress.¹⁶

Methods

Human tissue

This study was conducted according to the tenets of the Declaration of Helsinki and approved by the Massachusetts Eye and Ear Institutional Review Board. FECD patient specimen (n=9) were isolated during endothelial keratoplasty (EK) at Price Vision Group (Indianapolis, IN, USA) and immediately placed in storage medium (Optisol-GS; Bausch & Lomb) at 4°C and shipped on ice. Normal human cadaveric donor tissues (n=3) were directly obtained from Eversight eye bank (Ann Arbor, MI, USA).

Cell culture

Normal human corneal endothelial cell lines generated from cadaveric donor corneal endothelial tissue (SVN1-67F, from a 67 year old female with 15/16 TCF4 CTG repeats and HCEnC-21T, from a 21 year old male with 30/32 TCF4 CTG repeats) and FECD cell lines generated from post-keratoplasty patient tissues (SVF5-54F from a 54 year old female with 11/73 monoallelic CTG repeats in TCF4 and SVF3-76M from a 76 year male without CTG repeat expansion in TCF4) were immortalized using the simian virus 40 (SV40) T antigen cell immortalization kit (Alstem Cell Advancements, Richmond, CA) as per the methodology described previously.¹⁷ HCEnC-21T cells were immortalized by retrovirus transfection containing pBABE-puro-hTERT.¹⁸

Cells were cultured for 24 hours in Chen’s medium¹⁹ containing low serum basal medium (OptiMEM-I; cat# 51985091; Life Technologies), 8% fetal bovine serum (cat# 10082147; Thermo Fisher Scientific), 5 ng/mL epidermal growth factor (cat# 01-101; EMD Millipore, Billerica, MA), 100 mg/mL No-Worries^™ bovine pituitary extract (cat# 500-102-100; Gemini Bio-products), 200 mg/L calcium chloride dihydrate (cat# C7902; Sigma-Aldrich, St. Louis, MO), 0.08% chondroitin sulfate (cat# C9819; Sigma-Aldrich), 50 mg/mL gentamicin (cat# 15750078; Thermo Fisher Scientific), and 1:100 diluted antibiotic/antimycotic solution (cat# A5955; Sigma-Aldrich). The HCEnCs were passaged after dislodging the cells with 0.05% trypsin (cat# 25300120; Thermo Fisher Scientific) for 5 minutes at 37°C.

Determination of cell viability by quantifying adenosine triphosphate (ATP)

The four cell lines, HCEnC-21T, SVN1-67F, SV5-54F, and SVF3-76M, were pre-treated in Chen’s medium with the desired concentration of Mitoquinone mesylate (MitoQ, cat# HY-100116A, Medchem Express) (0.01 μm, 0.05 μM) or Idebenone (Idb, cat# HY-N0303, Medchem Express) (2.5 μM, 5 μM) for two hours and then co-treated with the desired concentration of Idb or MitoQ with menadinone (MN) (Sigma-Aldrich) (25 μM, 50 μM) in Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies) with no serum in a 96-well plate.²⁰ Cell numbers were measured using an automatic cell counter (Countess; Thermo Fisher Scientific) using trypan blue stain to account for the dead cells. Phase-contrast microscopy (Leica DM IL LED) was employed to visualize cell morphology. Cell viability was determined by the CellTiter-Glo 2.0 Assay (Promega, Madison, WI) according to the manufacturer’s protocol. The assay provides a homogeneous method to determine the number of viable cells in culture by quantifying the amount of adenosine triphosphate (ATP) present, which indicates the presence of metabolically active cells. The luminescence was determined by a luminometer (Turner Biosystems, Sunnyvale, CA).

Determination of mitochondrial membrane potential (ΔΨm)

Mitochondrial membrane potential was determined as per the manufacturer’s protocol available in the MitoProbeTM JC-1 Assay Kit (Cat #M34152, Thermo Fisher Scientific). Cells were cultured in a 96-well plate. Subsequently, the cells underwent stress with MN followed by co-treatment with MitoQ or Idb and incubated with a 2 μM MitoProbe JC-1 assay kit for a duration of 30 minutes at room temperature. Using a Synergy H1 microplate reader, the measurements of red fluorescence (excitation 550 nm/emission 600 nm) and green fluorescence (excitation 485 nm/emission 535 nm) were obtained. The ratio of red to green fluorescence was then compared to untreated controls or stressors in order to ascertain the percentage of Mitochondrial Membrane Potential (MMP) positivity.

Immunostaining and microscopy

For assessment of mitochondrial fragmentation, the two most promising and robust cell lines, HCEnC-21T (normal) and SVF5-54F (FECD), were seeded in Chen’s medium at a density of 0.22 × 10⁶ cells/well in a 12-well glass-bottom plate pre-coated with FNC Coating Mix (0407, AthenaES). After 24 hours, the cells were pre-treated with 0.05 μM MitoQ or 5 μM Idb for three hours and then co-treated with MitoQ or Idb supplemented with 25 μM MN for SVF5-54F cells and 50 μM MN for HCEnC-21T cells in OptiMEM-I (no serum) for 24 hours at 37°C with 5% CO₂. In addition, HCEnC-21T cells were exposed to UVA light (25 J/cm²) and recovered in OptiMEM-I for 6 hours at 37°C with 5% CO2 with or without MitoQ and Idb to investigate the effect of the drugs on physiological stress-induced mitochondrial fragmentation.

After treatment, cells were washed with 1 mL 1x PBS for 10 minutes, fixed with 4% paraformaldehyde (PFA) in 1x PBS (pH 7.4) for 10 min, and permeabilized using 0.1% Triton-X-100 diluted in 1x PBS for 30 min at room temperature (RT), followed by 2 washes with 1x PBS. Next, cells were blocked in 2% bovine serum albumin (BSA) in 1x PBS for 1 hour at RT and incubated in TOM20 primary antibody (FL-147, rabbit polyclonal IgG) (sc-11415, Santa Cruz Biotechnology: 1:200 in 1x PBS) overnight at 4°C with gentle shaking. The following day, the plate was kept at RT for 30 min before the cells were washed twice with 1x PBS for 10 min each. For the secondary antibody incubation, cells were incubated in donkey anti-Rabbit IgG (Alexa Fluor^™ 594) (A-21207, Invitrogen: 1:400 in 1x PBS) with a tinfoil cover for 1 hour with gentle shaking. Cells were then washed twice with 1x PBS for 5 min each at RT and incubated with 1–2 drops of DAPI (1:1000, # D9542, Sigma Aldrich). Round coverslips were placed in each well and cells were imaged under TRITC/DAPI channels at 63X with immersion oil using a Leica DMi8 inverted fluorescent microscope.

Drug treatment of ex-vivo Specimen

After receiving the FECD specimen, the tissues were incubated for 6h in OptiMEM-I media with or without MitoQ (0.05 μM) / Idb (5 μM) at 37°C, 5% CO₂. The samples were then washed with PBS and fixed in 4% PFA overnight at 4°C. The specimens were stained with TOM20 antibody using immunostaining protocol, as described above and the mitochondrial fragments were imaged after flat mounting on a glass slide and counted using ImageJ analysis, as described in the later paragraph.

Quantification of Mitochondrial Morphology

Images of cells fixed and stained for TOM20 and DAPI were acquired for each condition using 63X oil objective on a Leica DMI8 microscope. The acquired image was opened using Image J FIJI and brightness contrast was adjusted to visualize the immunofluorescent signal clearly for all cells. The image was processed by adapting the pre-process, threshold, and denoising steps from the mitochondrial analysis pipeline described by Chaudhry et al²¹. The processing parameters (described in the supplementary material) were optimized by comparing the final thresholded image to the original image as a quality control check for object identification and segmentation. Using the FIJI freehand tool, an in-focus mitochondrion of a single cell from the processed image was selected carefully avoiding overlap with neighboring mitochondria. The Yeast Mitomap plugin (http://www.gurdon.cam.ac.uk/stafflinks/downloadspublic/imaging-plugins) was applied to open only the selected mitochondrion in a new window. Next, the 3D objects counter plugin was applied to obtain a log of total number of objects and the number of object voxels in the selected mitochondrion. Mitochondrial fragmentation was analyzed by calculating the mitochondrial fragmentation count (MFC) using the formula adapted from the study by Rehman et al as shown below.²²

$$\mathrm{M}\mathrm{F}\mathrm{C}=\frac{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{o}\mathrm{b}\mathrm{j}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{s}\mathrm{\times }1000}{\mathrm{S}\mathrm{u}\mathrm{m}\mathrm{o}\mathrm{f}\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{o}\mathrm{b}\mathrm{j}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{v}\mathrm{o}\mathrm{x}\mathrm{e}\mathrm{l}\mathrm{s}}$$

MFC was calculated for 25 cells per condition and the experiment was repeated three times to obtain MFC values for a total of 75 cells per condition per cell line or at least 10 cells per normal donor tissue or FECD specimen.

Statistics

Statistical analysis was conducted using SPSS and GraphPad Prism. Data are expressed as the mean ± standard error (SE). The normality of the distribution of results was estimated using the Shapiro–Wilk test. Student’s T-test, one or two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was used to compare measured parameters. Values of p< 0.05 were considered statistically significant.

Results

Mitochondrial fragmentation in FECD specimens and dose-response effects of antioxidants MitoQ and Idb on cell lines

To examine the mitochondrial morphology in FECD specimens compared to normal human cadaveric donor tissues, we stained the tissues with TOM20. Normal tissues exhibited a filamentous mitochondrial network with intact tubular structure (Fig. 1A). In contrast, FECD specimens displayed fragmented mitochondria (Fig. 1B).

Figure 1.

Mitochondrial morphology and dose response for drug screening. (A) normal human cadaveric donor corneal endothelial cells showing tubular and intact mitochondrial network and (B) FECD cells showing mitochondrial dysfunction in the form of fragmentation. Chemical structures of (C) Mitoquinone (MitoQ) and (D) Idebenone (Idb). (E) Dose–response analysis using cellular viability of (E) MitoQ and (F) Idb-treated HCEn-21T, SVN1-67F, SVF3-76M and SVF5-54 cells using the Cell-Titer Glo assay. The data are expressed as mean ± SEM. Red=mitochondria (TOM20), Blue=nucleus (DAPI). White arrow indicates guttae. Scale bar = 20 μm. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

The chemical structures of Mitoquinone (MitoQ), a coenzyme Q derivative with a quinone moiety linked to a lipophilic triphenylphosphonium (TPP) cation via a 10-carbon alkyl chain (Fig. 1C), and Idebenone (Idb), a 1,4-benzoquinone with methoxy groups at positions 2 and 3, a methyl group at position 5, and a 10-hydroxydecyl group at position 6 (Fig. 1D) are shown.

For the dose-response analysis, we assessed the effects of MitoQ (0.05–25 μM) and Idb (2.5–50 μM) on cell viability in HCEnC-21T, SVN1-67F, SVF3-76M, and SVF5-54F cells using the Cell-Titer Glo assay to measure ATP production. MitoQ caused a significant decrease in cell viability at concentrations starting from 0.5 μM in HCEnC-21T cells (Fig. 1E). Idb led to significant reductions in cell viability starting at 10 μM in HCEnC-21T, SVN1-67F, and SVF5-54F cells (Fig. 1F). Based on these results, we selected the concentrations of 0.05 μM for MitoQ and 5 μM for Idb for subsequent assays in all four cell lines.

MitoQ and Idb rescue cell viability of immortalized normal and FECD cells after menadione treatment.

The pro-oxidant MN has been previously established as an in vitro oxidative stress model to investigate the disease pathogenesis in CEnCs²³. Therefore, we investigated whether the mitochondrial-targeted antioxidant MitoQ or non-targeted Idb enhance mitochondrial bioenergetics of normal and FECD CEnCs protecting from mitochondrial dysfunction.

The cells were pre-treated with MitoQ (0.05 μM) or Idb (5 μM) followed by co-treatment with 25 μM MN for SVN1-67F, SVF3-76M, and SVF5-54F cells and 50 μM MN for HCEnC-21T cells, since 25 μM MN did not show the desired phenotypic change in HCEnC-21T cells (Supplementary Fig. 1).

In HCEnC-21T cells, menadione (MN) reduced cell viability to 32%. MitoQ provided a 32% rescue, while Idebenone (Idb) achieved a 53% rescue, with Idb offering a significantly greater 21% improvement over MitoQ (Fig. 2A). In SVN1-67F cells, MN decreased cell viability to 13%. Both MitoQ and Idb restored cell viability by 44%, demonstrating comparable efficacy in mitigating MN-induced damage (Fig. 2B). In SVF3-76M cells, MN reduced cell viability to 8%. MitoQ rescued 48% of the viability, whereas Idb rescued 66%, with Idb providing a significantly greater 23% improvement compared to MitoQ (Fig. 2C). For SVF5-54F cells, MN decreased cell viability to 29%. MitoQ rescued 25%, while Idb restored 42% of the viability, with Idb showing a significantly greater 17% improvement over MitoQ (Fig. 2D).

Figure 2.

Mitochondria-targeted antioxidant MitoQ and non-mitochondria-targeted antioxidant Idb rescue menadione (MN)-induced loss of cell viability in normal cell lines, (A) HCEnC-21T and (B) SVN1-67F, and FECD cell lines, (C) SVF3-76M and (D) SVF5-54F as measured by % ATP with Cell Titer Glo assay. Cell Viability (% of ATP) was measured after pre-treatment with MitoQ or Idb and co-treatment with 50 μM MN in HCEnC-21T and 25 μM MN in SVN1-67F, SVF3-76M, SVF5-54F cells. The data are expressed as mean ± SEM. **p<0.01, ****p<0.0001.

MitoQ and Idb rescue mitochondrial membrane potential in normal and FECD cell lines after menadione treatment.

Mitochondrial depolarization and ATP loss are key indicators of mitochondrial dysfunction.^6,24 We evaluated the impact of MN on mitochondrial membrane potential (MMP) in normal and FECD cell lines and tested the efficacy of MitoQ and Idb. In HCEnC-21T cells, MN reduced MMP to 23% of the control, with MitoQ and Idb restoring it by 35% and 31%, respectively (Fig. 3A). In SVN1-67F cells, MN decreased MMP to 50%, with MitoQ and Idb providing 18% and 9% rescue, respectively (Fig. 3B). For the FECD cells SVF3-76M, MN lowered MMP to 54%, with MitoQ and Idb restoring it to 19% and 4%, respectively (Fig. 3C). In FECD SVF5-54F cells, MN reduced MMP to 41%, with MitoQ and Idb rescuing it to 21% and 16%, respectively (Fig. 3D). Interestingly, neither antioxidant significantly outperformed the other in rescuing MMP across the four cell lines, indicating equivalent efficacy in restoring mitochondrial membrane potential.

Figure 3.

MN-induced loss of mitochondrial membrane potential (MMP) was rescued after treatment with MitoQ or Idb in normal and FECD cell lines. (A) HCEnC-21T cells showed MMP rescue with both MitoQ and Idb after 50 μM MN stress, (B) SVN1-67F and (C) SVF3-76M cells showed MMP rescue with MitoQ alone, and (D) SVF5-54F cells showed MMP rescue with both MitoQ and Idb after 25 μM MN stress. Data are expressed as mean ± SEM. *p<0.05, **p<0.01, ****p<0.0001.

MitoQ and Idb rescue menadione-induced mitochondrial fragmentation in HCEnC-21T and SVF5-54F cells.

Based on previous experiments, we focused on mitochondrial fragmentation in HCEnC-21T (normal) and SVF5-54F (FECD) cells, where MN stress and drug effects were most pronounced.

At baseline, SVF5-54F cells exhibited 28% greater mitochondrial fragmentation (MFC = 7.6) compared to HCEnC-21T cells (MFC = 5.9), suggesting that the FECD cells have less efficient mitochondria at baseline (see Fig. 4A, 4B).

Figure 4.

MitoQ and Idb rescue menadione (MN)-induced mitochondrial fragmentation in normal and FECD cells. (A) Baseline comparison of mitochondrial morphology using TOM20 staining and (B) mitochondrial fragmentation count (MFC) between normal (HCEnC-21T) and FECD (SVF5-54F) cells. (C) comparison of mitochondrial morphology and MFC analysis between no treatment (control), menadione (MN), and menadione supplemented with either MitoQ (MN+MitoQ) or Idb (MN+Idb) in (C, D) HCEnC-21T and (E, F) SVF5-54F cells. The data are expressed as mean ± SEM of n=3 with a minimum of 25 cells analyzed per treatment per n. Red=mitochondria (TOM20), Blue=nucleus (DAPI), white=skeletonized mitochondria as observed after ImageJ 3D objects cell counter analysis. Scale bar = 20 μm. **p<0.01, ***p<0.001, ****p<0.0001.

In untreated HCEnC-21T controls, mitochondria displayed a network of interconnected, elongated filaments, indicating a healthy state (Fig. 4C). MN (50 μM) induced fragmentation and disrupted the filamentous network. Co-treatment with MN and MitoQ (0.05 μM) resulted in a mixture of long filaments and fragmented areas. Similarly, MN and Idb (5 μM) also showed mixed morphology but with less pronounced fragmentation compared to MitoQ. Quantitatively, MN increased mitochondrial fragmentation by 65% in HCEnC-21T cells (MFC = 9.7), whereas MitoQ rescued 28% of this fragmentation (MFC = 8.1). Idb rescued 60% of MN-induced fragmentation (MFC = 6.2), providing a 32% greater rescue compared to MitoQ (Fig. 4D).

In SVF5-54F untreated controls, mitochondria exhibited long, interconnected filaments (Fig. 4E). MN stress caused fragmentation and varied cluster sizes. MitoQ treatment resulted in long filaments with minimal fragmentation, while Idb similarly maintained filamentous structures but with slightly less fragmentation. MN increased mitochondrial fragmentation by 40% in SVF5-54F cells (MFC = 10.5). MitoQ rescued 19% of this fragmentation (MFC = 9.2), whereas Idb achieved a 32% rescue (MFC = 8.1), providing a 13% greater rescue compared to MitoQ (Fig. 4F). Overall, Idb showed significantly better protection against MN-induced mitochondrial fragmentation than MitoQ, especially in HCEnC-21T cells.

MitoQ and Idb rescues UVA-induced mitochondrial fragmentation in HCEnC-21T cells.

To evaluate the effects of MitoQ and Idb under physiological stress, we exposed HCEnC-21T cells to UVA light and assessed mitochondrial morphology with and without drug treatment (Fig. 5A). In untreated controls, mitochondria were interconnected and filamentous, indicating a healthy state. UVA exposure caused mitochondrial fragmentation and disrupted the filamentous network similar to MN. Co-treatment with MitoQ and Idb resulted in a mix of long filaments and fragmented areas, with no notable differences between the two drugs (Fig. 5A).

Figure 5.

MitoQ and Idb rescues UVA-induced mitochondrial fragmentation in normal HCEnC-21T cells. (A) Left panel shows images acquired at 63X, middle panel shows an inset of a single cell while right panel shows its skeletonized image after ImageJ analysis. (C) mitochondrial fragmentation count (MFC) between no treatment (control), UVA-stress, UVA-stress supplemented with either MitoQ (UVA+MitoQ) or Idb (UVA+Idb). The data are expressed as mean ± SEM of n=3 with a minimum of 25 cells analyzed per treatment per n. Red=mitochondria (TOM20), Blue=nucleus (DAPI), white=skeletonized mitochondria as observed after ImageJ 3D objects cell counter analysis. Scale bar = 20 μm. **p<0.01, ***p<0.001, ****p<0.0001.

UVA exposure increased mitochondrial fragmentation to 69% in HCEnC-21T cells (MFC = 13.1) compared to untreated cells (MFC = 7.8). MitoQ treatment rescued fragmentation to 25.2% (MFC = 9.8), while Idb treatment achieved a 30.6% rescue (MFC = 9.1). Both drugs significantly improved mitochondrial morphology under UVA stress, demonstrating their protective effects (Fig. 5B).

MitoQ rescued mitochondrial fragmentation in FECD specimen

We further examined the impact of drugs on corneal specimens from FECD patients (n=9; age 67±10.2 yrs; 7F:2M; storage duration 2±1 days) by treating them with MitoQ and Idb, and compared the results with normal tissues (n=3; age 70±6.2 years; 2 females:1 male; 12±3 storage days). Notably, normal tissues displayed interconnected and filamentous mitochondria alongside some mitochondrial fragments (Fig. 6A). In contrast, FECD specimens exhibited increased fragmentation and reduced filamentous networks, which was improved by MitoQ treatment (Fig. 6B). Quantitatively, baseline mitochondrial fragmentation was higher in FECD specimens (MFC = 12.8) compared to normal corneal donor tissues (MFC = 6.8). Treatment with MitoQ significantly reduced mitochondrial fragmentation by 41.6% (MFC = 7.5). Although Idb reduced fragmentation by 15.6% (MFC = 10.8), this change was not statistically significant (Fig. 6C). Overall, MitoQ demonstrated a significant reduction in mitochondrial fragmentation in FECD cells, indicating its potential as a promising candidate for further investigation.

Figure 6.

MitoQ rescues mitochondrial fragmentation in FECD specimen. Immunofluorescence staining with TOM20 shows the mitochondrial morphology of (A) normal tissues and (B) FECD cells derived from the patient specimen untreated and treated with MitoQ or Idb. Left panel shows images acquired at 63X, middle panel shows an inset of a single cell while right panel shows its skeletonized image obtained through ImageJ. (C) Comparison of mitochondrial morphology and MFC analysis between normal tissues, untreated FECD specimen, MitoQ and Idb treated FECD cells. The data are expressed as mean ± SEM of n=3 with a minimum of 25 cells analyzed per treatment per n. Red=mitochondria (TOM20), Blue=nucleus (DAPI), white=skeletonized mitochondria as observed after ImageJ 3D objects cell counter analysis. Scale bar = 20 μm. **p<0.01, ***p<0.001, ****p<0.0001.

Discussion

Oxidative stress plays a pivotal role in the pathogenesis of FECD and leads to increased apoptosis of mitotically incompetent CEnCs. Mitochondrial dysfunction is a key aspect of this oxidative stress-induced pathology, as these organelles are crucial for maintaining cellular energy balance and integrity.⁶ The balance of mitochondrial function is disrupted in FECD, as also seen in many neurodegenerative disorders, as a part of aging process.^25,26 This further results in decreased ATP production, loss of MMP, heightened generation of ROS, and aberrant mitochondrial dynamics including fragmentation.^17,27 These abnormalities stem from the vulnerability of mitochondrial DNA (mtDNA) to oxidative damage, compounded by the direct exposure of mitochondria to ROS generated during the ETC and the absence of protective histones.^4–6 The greater accumulation of acquired mtDNA and nuclear DNA (nDNA) damage in FECD compared to normal aging CEnCs provided the first evidence that lifelong accumulation of DNA damage has detrimental effects on mitochondrial bioenergetics leading to mitochondrial dysfunction and apoptosis.²³ To protect mitochondria from dysfunction and to keep them in a state of homeostasis is a complex interplay between mitochondrial biogenesis and degradation.^28,29

Skeie et al. demonstrated that ubiquinol, an electron rich form of coenzyme Q, could enhance mitochondrial function of CEnCs.³⁰ Ubiquinol plays a crucial role in the mitochondrial ETC and ATP biosynthesis, acting as an antioxidant localized to cell and mitochondrial membranes thus mitigating ROS. In this context, we further explored the potential of two antioxidants, the mitochondrial-targeted ubiquinone, MitoQ, and the non-targeted antioxidant Idb, in preserving mitochondrial function.

In the present study, we observed mitochondrial fragmentation concurrent with disruption of the tubular and dynamic network in FECD specimen, however the normal cadaveric donor tissues exhibited an intact tubular mitochondrial network. Similar disruption of mitochondria was observed in MN-induced mitochondrial dysfunction model. To mitigate this response, we used lower doses of the drugs that showed cytoprotective properties (0.05 μM MitoQ and 5 μM Idb) compared to higher doses which were found to be toxic. Such phenomenon has previously been observed with quinones, as they are capable of covalent and redox modifications of biomolecules. Notably, these low doses of moderately electrophilic quinones, such as Idb and MitoQ, are generally associated with cytoprotection through the activation of the Keap1/Nrf2 pathway, a pivotal regulator of the phase II antioxidant response, by inducing the transcription of enzymes involved in antioxidant and anti-inflammatory responses.³¹

Furthermore, our study demonstrated that pre-treatment with MitoQ and Idb effectively preserved cell viability in all the studied cell lines following exposure to MN. Moreover, MitoQ exhibited the ability to rescue MMP across all cell lines tested, whereas Idb was effective in restoring MMP in one normal and one FECD cell line indicating a differential efficacy that may be contingent upon the specific cellular phenotype. Thus, we observed that prophylactic treatment with MitoQ and Idb can protect mitochondrial bioenergetics of normal and FECD cells by rescuing ATP depletion and MMP depolarization, induced by oxidative stress. However, the differential response could be influenced by individual cellular characteristics.³²

Previous studies have shown that the depletion of ATP and the alteration in MMP triggers the fragmentation of mitochondria.^33,34 Prior research has also associated heightened mitochondrial DNA damage³⁵, loss of MMP, and decreased ATP production with mitochondrial fragmentation, highlighting the role of dystrophy-induced mitochondrial dysfunction.²³ We observed that FECD cell lines had inherently higher baseline mitochondrial fragmentation compared to normal CEnCs before exposure to oxidative stress. Similarly, ex vivo FECD patient specimens showed significantly greater mitochondrial fragmentation compared to normal donor tissue, indicating that FECD cells possess dysfunctional mitochondria.

In a recent study concluded by our group, Bannon et al. showed that MitoQ treatment significantly improved cellular morphology and health of HCEnC-21T cells exposed to UVA or UVA combined with cigarette smoke condensate (CSC). MitoQ enhanced cell viability, reduced mitochondrial fragmentation and ROS levels, and maintained mitochondrial membrane potential (MMP). It also decreased cytochrome C release, thus reducing apoptosis, and lowered oxygen consumption rate (OCR). These results suggest that MitoQ could be a promising strategy for addressing CEnC bioenergetic dysfunction.³⁶ In the current study, we observed a similar trend in MitoQ and Idb when HCEnC-21T cells were exposed to UVA indicating the effectiveness of these drugs on physiological stress.

Herein, we thus provide initial evidence that the antioxidants MitoQ and Idb successfully rescued mitochondrial fragmentation, likely attributed to their antioxidant properties^31,37,38 and their ability to modulate mitochondrial electron flow.^39–42 Idb exhibited higher efficacy in preserving mitochondrial morphology and mitigate mitochondrial fragmentation against MN-induced oxidative stress compared to MitoQ in both normal and FECD cell lines. However, it did not show any difference in mitigating mitochondrial fragmentation in UVA-induced mitochondrial dysfunction compared to MitoQ. Idb is distributed through extracellular and intracellular compartments, with a small proportion accumulating in the mitochondria, thus being a non-mitochondrial targeted antioxidant.⁴³ On the contrary, MitoQ (CoQ10) can directly affect the mitochondria by targeting mitochondrial ETC preventing cell apoptosis and death. While Idb effectively improved two parameters across various models, MitoQ proved to be more effective amongst all the parameters in addition to mitigating mitochondrial fragmentation in ex vivo FECD specimens. This suggests that a mitochondrial-targeted therapy, as opposed to a non-targeted one, may be a better option for addressing mitochondrial dysfunction in CEnCs.

The current study acknowledges several limitations - a) FECD is a heterogeneously complex disorder with varied individual responses, therefore our findings may not fully capture the range of responses across all FECD cases; b) neither quinones fully restored MMP, as achieving complete rescue is challenging due to the dynamic nature of mitochondrial bioenergetics in CEnCs. The study primarily aimed to assess potential therapeutic benefits rather than complete restoration; and c) while both quinones improved some parameters, more extensive functional assays are needed to fully assess their therapeutic potential. Additionally, the study did not explore different dosages or treatment regimens, which should be examined in future research to optimize effectiveness if intended for in vivo use. The varied responses of MitoQ and Idb across cell lines indicate the need for personalized treatment approaches. Moreover, the impact of these quinones on mitochondrial electron transport chain alterations was not detailed and requires further investigation. Finally, as the study is based on in vitro models, and only highlights the fragmentation parameter in ex vivo tissues, further validation with in vivo animal models is necessary to confirm the therapeutic efficacy of MitoQ and Idb for corneal endothelial disorder.

Overall, while the study provides preliminary evidence supporting the potential of Idb and MitoQ for mitigating mitochondrial dysfunction, additional research is essential to confirm these findings and optimize treatment strategies.

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Supplementary Material

supp figure

Supp. Figure 1. Dose response analysis of of 25 μM and 50 μM menadione on HCEnC-21T cells.