Protection of dystrophic muscle cells using Idebenone correlates with the interplay between calcium, oxidative stress and inflammation

Abstract

There is strong cross‐talk between abnormal intracellular calcium concentration, high levels of reactive oxygen species (ROS) and an exacerbated inflammatory process in the dystrophic muscles of mdx mice, the experimental model of Duchenne muscular dystrophy (DMD). In this study, we investigated effects of Idebenone, a potent anti‐oxidant, on oxidative stress markers, the anti‐oxidant defence system, intracellular calcium concentrations and the inflammatory process in primary dystrophic muscle cells from mdx mice. Dystrophic muscle cells were treated with Idebenone (0.05 μM) for 24 h. The untreated mdx muscle cells were used as controls. The MTT assay showed that Idebenone did not have a cytotoxic effect on the dystrophic muscle cells. The Idebenone treatment was able to reduce the levels of oxidative stress markers, such as H₂O₂ and 4‐HNE, as well as decreasing intracellular calcium influx in the dystrophic muscle cells. Regarding Idebenone effects on the anti‐oxidant defence system, an up‐regulation of catalase levels, glutathione reductase (GR), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity was observed in the dystrophic muscle cells. In addition, the Idebenone treatment was also associated with reduction in inflammatory molecules, such as nuclear factor kappa‐B (NF‐κB) and tumour necrosis factor (TNF) in mdx muscle cells. These outcomes supported the use of Idebenone as a protective agent against oxidative stress and related signalling mechanisms involved in dystrophinopathies, such as DMD.

1. INTRODUCTION

Duchenne muscular dystrophy (DMD) is a progressive muscle‐wasting disease, which affects about 1 in 6000 live male births. ¹ , ² Most DMD patients become wheelchair dependent by the age of 10–12, require assisted ventilation by the age of 20 and usually die between the ages of 20–40 of heart and/or respiratory system failure. ¹ , ³

Duchenne muscular dystrophy is caused by the mutation or deletion of the dystrophin protein, leading to membrane muscle fibre damage with consequent changes in intracellular calcium and muscle fibre necrosis. ⁴ Associated with the high concentration of intracellular calcium in the dystrophic muscle fibres, the oxidative stress and the exacerbated inflammatory process are recognized as key pathogenic events in DMD. ⁵ , ⁶ , ⁷ Previous studies showed a strong cross‐talk between high levels of reactive oxygen species (ROS); nuclear factor kappa‐B (NF‐κB), a transcription factor that regulates the expression of pro‐inflammatory cytokines; and intracellular calcium concentration in the dystrophic muscles of mdx mice, the experimental model of DMD. ⁸ , ⁹

Nowadays, glucocorticoid steroids are the main therapy used by dystrophic patients, showing efficacy in muscle strength and respiratory function. However, the side effect profile restricts its long‐term use. ¹⁰ Searching for new drugs for DMD treatment, it was reported that Idebenone significantly reduced the loss of respiratory function in 8‐ to 18‐year‐old DMD patients who were not using concomitant glucocorticoids. ¹¹ Idebenone, which is a short‐chain benzoquinone structurally related to coenzyme Q10, is a potent anti‐oxidant and electron carrier, whose anti‐oxidant properties were reflected in inhibition of oxidative stress markers. ¹¹ , ¹² , ¹³ In addition, a study with mdx mice showed that Idebenone significantly improved voluntary wheel running performance, reduced cardiac inflammation and fibrosis, as well as enhancing cardiac function. The researchers suggested that the beneficial effects of Idebenone reported on the dystrophic cardiac muscle are related to its anti‐oxidant activity. ¹⁴

There are few experimental and clinical studies that have demonstrated the potential effects of Idebenone in the dystrophic disease, and these effects are not completely understood. Thus, in this study, we evaluated the Idebenone effect on oxidative stress markers, on the anti‐oxidant defence system, on intracellular calcium concentration and on the inflammatory process in the primary dystrophic muscle cell cultures from mdx mice.

2. MATERIALS AND METHODS

2.1. Animals

Male and female mdx (C57BL/10‐Dmdmdx/PasUnib) mice and their normal counterparts, from the same genetic background, C57BL/10 (C57BL/10ScCr/PasUnib) mice, were maintained on a regimen of 12 h dark, 12 h light cycles at room temperature (21°C), receiving ad libitum access to food and water. All experiments described here were carried out based on the guidelines of the Brazilian College for Animal Experimentation (COBEA) and approved by the Ethics Committee of our institution (Process n° 3588‐1).

2.2. Cell culture and treatment

Primary skeletal muscle cell culture was performed using the hind limb muscles of both male and female mdx and C57BL/10 mice (n‐3–6 animals per culture; 28 days old). The hind limb muscles of the mice were dissected, and the muscles were rapidly isolated in Dulbecco's phosphate‐buffered saline (DPBS) containing 1% glucose (v/v) and 1% penicillin (v/v) to prepare primary skeletal muscle cell culture. ¹⁵ The muscle tissue was crushed for 10 min using scissors and the suspension was enzymatically digested with collagenase and trypsin solutions at 37°C. The skeletal muscle‐derived cells (5 × 10⁴ cells/cm²) were plated in Matrigel‐coated dishes (Matrigel at 9 mg/ml was diluted in DMEM to a working concentration of 0.9 mg/ml). The skeletal muscle‐derived cells were cultured in a proliferation and growth medium containing Dulbecco's Modified Eagle Medium (DMEM) with glucose (5.5 mM), L‐glutamine (2 mM), foetal bovine serum (10% v/v), horse serum (10% v/v) and penicillin/streptomycin (1% v/v) for 2 days. Myotube differentiation was induced by the addition of a fusion medium (FM) made up of DMEM with glucose (5.5 mM), Lglutamine (2 mM) and horse serum (10% v/v). The culture was maintained in a 5% Carbon Dioxide (CO2) incubator at 37°C.

Undifferentiated muscle cells were observed on the first day of culture; primary muscle cell fusion was triggered 2 days after plating and the maturation process on day 3. Morphological maturation with organized sarcomeric structures was achieved by day 6. Idebenone was added on the 6th day of culture. For the Idebenone (IDB) treatment, IDB powder (Sigma Aldrich; #15659) was dissolved in absolute ethyl alcohol to obtain a 10 mM stock solution. Further, the dilution was made in the incubation medium and the muscle cells were treated with 0.05 μM Idebenone for 24 h. Fresh solutions were prepared for each experiment.

The differentiated muscle cells with contractile properties were used in all experiments and all the measurements were performed in triplicate, in three independent cultures, on the 7th day. Skeletal muscle cell cultures were divided into four experimental groups: Ctrl (skeletal muscle cells from C57BL/10 mice that did not receive any treatment), CtrlId (skeletal muscle cells from C57BL/10 mice treated with Idebenone), mdx (skeletal muscle cells from mdx mice that did not receive any treatment) and mdxId (skeletal muscle cells from mdx mice treated with Idebenone). The Ctrl and CtrlId experimental groups were used for certain focused experiments.

2.2.1. Cell cytotoxicity and viability by MTT assay

For the quantification of mitochondrial metabolism and activity of the respiratory chain of cells, a tetrazolium [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] (MTT; Sigma) assay was used. In this experiment, the primary muscle cells were washed in phosphate buffered saline (PBS) once and MTT solution (5 mg/ml, tetrazolium salt) was added and incubated for 4 h at 37°C. After the incubation period, the whole medium was discarded and MTT crystals were dissolved with isopropanol acid and the absorbance was measured in a spectrophotometer (Synergy H1, Hybrid Reader, Biotek Instruments, Winooski, VT, USA) at 570 nm with a 655 nm reference wave length to quantify the amount of formazan product, which reflects the number of cells in culture. Wells that did not contain cells were used as a zero point of absorbance.

2.3. Fluo‐4 assay for intracellular calcium content analyses

This assay used calcium‐sensitive Fluo‐4 dye (Molecular Probes, Oregon, USA) to measure intracellular calcium concentrations [Ca²⁺]i. Cells were incubated with the Fluo‐4 AM dye for 60 min at room temperature at a final concentration of 1 mmol/L (plus 0.005% Pluronic F‐127; Invitrogen, Oregon, USA). ¹⁶ The intensities of fluo‐4 fluorescence were monitored on a fluorescent inverted microscope (Nikon, Eclipse TS100/TS100F) for qualitative analyses. Quantitative measurements were performed using a spectrophotometer (Synergy H1, Hybrid Reader, Biotek Instruments, Winooski, VT, USA) at 494 and 516 nm excitation and emission wavelengths respectively.

2.4. Determination of Hydrogen Peroxide (H₂O₂ ) production

Amplex Red assay kit (Molecular Probes, Life Technologies, California, EUA) was used to determine H₂O₂ levels according to the manufacturer's instructions. The Amplex UltraRed reagent (50 μM) and Horseradish Peroxidase (HRP; 0.1 U/ml) were added for 60 min. The absorbance was determined at 530‐nm (excitation) and 590‐nm wavelength (emission). Measurements of ROS were previously calibrated using exogenous 10 μM H₂O₂ (positive control). All measurements were performed in phenol red‐free culture medium (1 ml), pH 7.4, at 37°C.

2.5. Western blotting

Cell cultures were washed thrice with PBS, before the addition of the lysis buffer (Tris–HCl, 100 mM, pH 7.5; Ethylenediaminetetraacetic acid (EDTA) 10 mM, pH 8.0; sodium pyrophosphate, 10 mM; sodium fluoride, 0.1 mM; sodium orthovanadate, 10 mM; phenylmethylsulfonyl fluoride (PMSF), 2 mM and aprotinin, 10 μg/ml). The cell extracts were sonicated for 30 s at 4°C. Cell debris was removed by centrifugation at 11,000 g for 20 min at 4°C and the cleared lysate was subjected to sodium dodecyl‐sulphate polyacrylamide gel electrophoresis (SDS‐PAGE). An aliquot from the supernatant was used to determine the total protein content by the Bradford method. Thirty μg of total protein homogenate was loaded on 6%–15% SDS‐polyacrylamide gels. The proteins, following electrophoresis, were transferred on to nitrocellulose membranes using an iBlot Gel Transfer Device (Bio‐Rad Laboratories, Hercules, California). All membranes were blocked for 2 h at room temperature with 5% skim milk/Tris–HCl buffer saline‐Tween buffer (TBST; 10 mM Tris–HCl, pH 8, 150 mM NaCl, and 0.05% Tween 20). Primary antibodies used were 4‐Hydroxynonenal (4‐HNE, Santa Cruz Biotechnology, SC‐202019), anti‐catalase (Sigma‐Aldrich C0979), Nuclear factor kappa B (NF‐κB; Santa Cruz Biotechnology, SC‐372), Tumour necrosis factor alpha (TNF‐α; Cell‐Signaling #3707) and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology, SC‐25558) were incubated overnight at 4°C with gentle shaking. Then, membranes were incubated for 2 h at room temperature with peroxidase‐conjugated secondary purified goat, mouse or rabbit IgG antibodies (KPL) respectively. Membranes were washed 3 × for 10 min with TBST after both incubations. All membranes were examined using the SuperSignal West Pico Chemiluminescent Substrate kit (Pierce Biotechnology, Rockford, Illinois). To control protein loading, Western blot transfer and nonspecific changes in protein levels, the blots were stripped and reprobed for glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). Band intensities were quantified using ImageJ 1.38X (National Institutes of Health, Bethesda, Maryland) software. Figure S1 represent three independent cultures in sequence for representative bands analysed (4‐HNE‐protein adducts; GAPDH; catalase; TNF and NF‐κB).

2.6. Anti‐oxidant defence analysis

2.6.1. Glutathione (GSH) content

Total GSH content was determined by Ellman's reaction using 5′5′‐dithio‐bis‐2‐nitrobenzoic acid (DTNB). The intensity of the yellow colour was read at 412 nm. The results were expressed as nmol per mg of protein.

2.6.2. Superoxide dismutase (SOD) activity

Superoxide dismutase activity was analysed by the reduction of nitroblue tetrazolium using a Xanthine–Xanthine oxidase system, that is, superoxide generation. The results were expressed as SOD units per mg of protein.

2.6.3. Glutathione peroxidase (GPx) activity

Glutathione peroxidase activity was quantified by following the decrease in absorbance at 365 nm induced by 0.25 mM H₂O₂ in the presence of reduced glutathione (10 mM), NADPH, (4 mM) and 1 U enzymatic activity of GR. Results were expressed as nmol per min per mg of protein.

2.6.4. Glutathione reductase (GR) activity

Glutathione reductase activity was measured following the decrease in absorbance at 340 nm induced by oxidized glutathione in the presence of NADPH in phosphate buffer, pH 7.8. Absorbance changes were read between 1 and 10 min. Results were expressed as nmol per min per mg of protein.

2.7. Statistical analysis

All the data analysis is presented as mean ± standard deviation (SD). Comparisons of multiple groups were analysed by one‐way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test. The unpaired T test was used to compare two groups. A 5% (p ≤ .05) significance level was used. The GraphPad Prims5 software package (GraphPad Software, CA, USA) was used.

3. RESULTS

After 24 h of Idebenone treatment, no change in the morphologic features of control (C57BL/10) and dystrophic (mdx) muscle cells was observed (Figure 1A,B,D,E). The control and dystrophic muscle cells treated with Idebenone or untreated demonstrated a similar morphology expressed by thick and branching myotubes (Figure 1A,B,D,E). It was also verified by MTT assay that the Idebenone treatment does not modify either the control or the dystrophic muscle cell viability (Figure 1C,F).

FIGURE 1.

Morphology of (A) untreated C57BL/10 muscle cells (Ctrl), (B) C57BL/10 muscle cells treated with Idebenone (CtrlId), (D) untreated mdx muscle cells (mdx) and (E) mdx muscle cells treated with Idebenone (mdxId). Scale bar 100 μm. Cell viability was assessed by measurement of MTT assay in the muscle cells from the Ctrl and CtrlId (C) and mdx and mdxId (F) groups. All the biological experiments were performed in triplicate and data expressed as mean ± SD.

Regarding intracellular calcium concentration, the dystrophic muscle cells treated with Idebenone showed a reduction in intracellular calcium (by 8.45%; p ≤ .0081) compared to untreated dystrophic muscle cells (Figure 2A–C).

FIGURE 2.

Intracellular calcium [Ca²⁺] concentrations, assessed by the measurement of calcium‐sensitive dye Fluo‐4 (green), in (A) untreated mdx muscle cells (mdx) and (B) mdx muscle cells treated with Idebenone (mdxId). Scale bar 100 μm. In (C), graph showing fluorescence intensity of [Ca²⁺] in mdx and mdxId groups. All the biological experiments were performed in triplicate and data expressed as mean ± SD. ^## p < .001 versus mdx.

To test whether the Idebenone treatment affected oxidative stress in dystrophic muscle cells, the 4‐hydroxynonenal (4‐HNE)‐protein adduct levels, H₂O₂ production and anti‐oxidant defence system were analysed.

Bands of 4‐HNE‐protein adducts from 25 to 130 kDa were detected in control and dystrophic muscle cells (Figure 3A). The 4‐HNE‐protein adduct levels significantly increased in the mdx group (by 12.2; p ≤ .05) compared to the Ctrl group (Figure 3A,B). In the dystrophic muscle cells, Idebenone treatment promoted a significant reduction in 4‐HNE‐protein adduct levels (by 13.6%; p ≤ .006) compared to the mdx group (Figure 3A,B). In addition, the Idebenone‐treated dystrophic muscle cells also showed a significant reduction in H₂O₂ production (by 22.25%; p ≤ .0001) when compared to the untreated dystrophic muscle cells (Figure 3C).

FIGURE 3.

In (A) the immunoblot analysis shows several bands of 4‐HNE‐protein adducts, ranging from 25 to 130 kDa in untreated C57BL/10 muscle cells (Ctrl), untreated mdx muscle cells (mdx) and mdx muscle cells treated with Idebenone (mdxId). (B) Graphs show 4‐HNE levels in the Ctrl, mdx and mdxId groups. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was used as a loading control. The relative value of the band intensity was quantified and normalized by the corresponding control. In (C), graphs show H₂O₂ production in mdx and mdxId groups. In (D), there is an immunoblot analysis of catalase and in (E) graphs show protein level in Ctrl, mdx and mdxId groups. All the biological experiments were performed in triplicate and data expressed as mean ± SD. *p < 0.5 versus Ctrl; ^## p < .001 versus mdx.

In terms of the Idebenone effects on the anti‐oxidant defence system, an increase in catalase levels was observed in the mdxId group compared to the Ctrl and mdx groups (by 30.7%; p ≤ .05 and 54.6%; p ≤ .001 respectively; Figure 3D). In addition, the Idebenone‐treated dystrophic muscle cells also showed a significant increase in GR, GPx and SOD activity (by 99%, 109% and 27% respectively) when compared to the untreated dystrophic muscle cells (Table 1). No significant difference in the GSH content was found between the mdxID and mdx groups (Table 1).

TABLE 1.

SOD, GPx and GR activity and GSH content in dystrophic muscle cells.

Group	SOD (OD/mg protein)	GPx (nmol/min/mg protein)	GR (nmol/min/mg protein)	GSH (nmol/mg protein)
mdx	12.27 ± 0.69	4.84 ± 0.91	4.24 ± 0.69	45.89 ± 8.21
mdxId	15.63 ± 1.42##	10.14 ± 2.32####	8.76 ± 2.47####	43.87 ± 5.18

Note: Analysis of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase activity (GR) and glutathione (GSH) content in untreated mdx muscle cells (mdx) and mdx muscle cells treated with Idebenone (mdxId). All the experiments were performed in triplicate and data were expressed as mean ± SD. ^## p < .001 versus mdx; ^#### p < .00001 versus mdx.

Regarding the Idebenone effect on the inflammatory process in dystrophic muscle cells, a significant increase in TNF (by 14.8%; p ≤ .0001) and NF‐κB (by 24.9%; p ≤ .05) levels was observed in the mdx group compared with the Ctrl group (Figure 4A–C). The Idebenone‐treated dystrophic muscle cells showed a significant reduction in TNF (by 24.8%; p ≤ .05) and NF‐κB (by 40.6%; p ≤ .001) levels when compared to the untreated dystrophic muscle cells (Figure 4A–C).

FIGURE 4.

In (A), there is the immunoblot analysis of TNF and NF‐κB in untreated C57BL/10 muscle cells (Ctrl), untreated mdx muscle cells (mdx) and mdx muscle cells treated with Idebenone (mdxId). The graphs show TNF (B) and NF‐κB (C) levels in the Ctrl, mdx and mdxId groups. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was used as a loading control. The relative value of the band intensity was quantified and normalized by the corresponding control. All the biological experiments were performed in triplicate and data expressed as mean ± SD. *p < .05 versus Ctrl; ***p < .0001 versus Ctrl; ^# p < .05 versus mdx; ^### p < .0001 versus mdx.

4. DISCUSSION

We observed the therapeutic potential of Idebenone in the current study in dystrophic muscle cells by simultaneously exerting both anti‐oxidant and anti‐inflammatory activities, while concomitantly regulating the intracellular calcium concentration.

Muscular dystrophy is characterized by increased membrane permeability to calcium (Ca²⁺), resulting in greater Ca²⁺ influx across the sarcolemma. ¹⁷ , ¹⁸ , ¹⁹ Sarcolemmal Ca²⁺ influxes are decisive early events in DMD pathophysiology, preceding the start of histological damage and an inflammatory process in the dystrophic muscles. ²⁰ , ²¹ Previous studies showed a mutual interplay of high intracellular Ca²⁺ concentration and ROS signalling in the dystrophic primary muscle cells from mdx mice. ⁸ , ⁹ In addition, it was also reported that the calcium channels are sensitive to oxidants, probably due to the direct redox alteration of cysteine residues, or by the redox modification of regulatory proteins involved in the channel function. ²² , ²³

Under our experimental conditions, we observed that Idebenone, a potent antioxidant, decreased the intracellular Ca²⁺ concentration in mdx muscle cells, which was accompanied by a reduction in the 4‐HNE levels and H₂O₂ production. 4‐HNE is one of the quantitatively most important products of lipid peroxidation ²⁴ and its levels are abnormally increased in the dystrophic muscle cells. ⁸ , ⁹ , ²⁵ , ²⁶ , ²⁷ In addition, the reduction of H₂O₂ production is a relevant outcome, because H₂O₂ in the presence of iron may lead to the formation of highly reactive hydroxyl radicals that can exacerbate the lipid peroxidation process. ²⁶ , ²⁸ In agreement with our results, in vivo and in vitro studies indicated that Idebenone inhibits lipid peroxidation and regulated the redox balance by scavenging free radicals. ²⁹ , ³⁰ , ³¹ , ³² , ³³

Previous work suggests that the anti‐oxidant capacity of Idebenone is also likely to be linked to the consequence of an increased expression of a range of endogenous anti‐oxidants such as SOD, catalase and GPx, ³² , ³⁴ , ³⁵ , ³⁶ , ³⁷ which are some of the major anti‐oxidant enzymes directly involved in ROS neutralization. In this study, we also observed elevated catalase levels and an increase in SOD, GR and GPX activity in the dystrophic muscle cells treated with Idebenone. These results suggest that the potential effect of Idebenone on dystrophic muscle cells may also be related to its ability to strengthen the anti‐oxidant defence machinery.

The therapeutic effect of idebenone has also been linked to its anti‐inflammatory activity in some diseases. ³² , ³⁵ , ³⁸ , ³⁹ , ⁴⁰ , ⁴¹ This is an interesting aspect of Idebenone, since muscle degeneration in DMD is exacerbated by endogenous inflammatory response, with a key role being played by NF‐κB and other related factors such as TNF. ⁴² , ⁴³ , ⁴⁴ , ⁴⁵ Therefore, the suppression of pro‐inflammatory cytokines is thought to represent an essential part of the therapeutic approach against DMD. Evidence of a new insight into anti‐inflammatory Idebenone effects was put forward as a result of both in vitro and in vivo studies, which involved the suppression of interleukins 1 and 6 (IL‐1; IL‐6) and TNF. ⁴¹ In addition, this study suggests that the effects of Idebenone may be mediated by the inhibition of the NF‐κB pathway. ⁴¹ In agreement with this previous study, our results showed a substantial suppression of the NF‐κB and TNF in Idebenone‐treated dystrophic muscle cells. In addition, a previous study also observed that Idebenone treatment morphologically reduced cardiac inflammation in mdx mice. ¹⁴

Regarding Idebenone clinical trials, previous work reported that treatment with Idebenone, for 12 months, can slow the loss of pulmonary function in adolescent patients who have reached the pulmonary function decline stage. ⁴⁶ In addition, research with DMD patients treated with Idebenone for a long‐term (up to 6 years) added data to the previously reported body of evidence, demonstrating that Idebenone holds a disease‐modifying therapeutic potential to preserve respiratory function. ⁴⁷ Based on these studies, we speculate that the anti‐oxidant and anti‐inflammatory activity of Idebenone, observed under our experimental conditions, can justify in part the beneficial effects of Idebenone on the respiratory function reported in dystrophic patients.

As DMD involves multiple dystrophic events, the pharmacological targeting of individual factors appears to have limited use to provide effective and therapeutic results. The present study highlights that Idebenone appeared to target not just one, but several factors relevant to DMD which included oxidative stress, calcium concentration and inflammatory markers. To summarize, these outcomes supported the use of Idebenone as a protective agent against the oxidative stress and related signalling mechanisms involved in the dystrophinopathies, such as DMD.

AUTHOR CONTRIBUTIONS

The authors contributed substantially to conception and design, acquisition of data, analysis and interpretation of data. All authors participated in drafting the article, revised it critically for important intellectual content and gave final approval of the version to be submitted.

CONFLICT OF INTEREST

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supporting information

Figure S1.

Valduga AH, Mizobuti DS, Moraes FdSR, et al. Protection of dystrophic muscle cells using Idebenone correlates with the interplay between calcium, oxidative stress and inflammation. Int J Exp Path. 2023;104:4‐12. doi: 10.1111/iep.12463