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. 2023 Aug 14;28(6):773–785. doi: 10.1007/s12192-023-01369-2

LED therapy plus idebenone treatment targeting calcium and mitochondrial signaling pathways in dystrophic muscle cells

Heloina Nathalliê Mariano da Silva 1, Daniela Sayuri Mizobuti 1, Valéria Andrade Pereira 1, Guilherme Luiz da Rocha 1, Marcos Vinícius da Cruz 1,2, André Gustavo de Oliveira 1,2, Leonardo Reis Silveira 1,2, Elaine Minatel 1,✉
PMCID: PMC10746663  PMID: 37578579

Abstract

Intracellular calcium dysregulation, oxidative stress, and mitochondrial dysfunction are some of the main pathway contributors towards disease progression in Duchenne muscular dystrophy (DMD). This study is aimed at investigating the effects of light emitting diode therapy (LEDT) and idebenone antioxidant treatment, applied alone or together in dystrophic primary muscle cells from mdx mice, the experimental model of DMD. Mdx primary muscle cells were submitted to LEDT and idebenone treatment and evaluated for cytotoxic effects and calcium and mitochondrial signaling pathways. LEDT and idebenone treatment showed no cytotoxic effects on the dystrophic muscle cells. Regarding the calcium pathways, after LEDT and idebenone treatment, a significant reduction in intracellular calcium content, calpain-1, calsequestrin, and sarcolipin levels, was observed. In addition, a significant reduction in oxidative stress level markers, such as H2O2, and 4-HNE levels, was observed. Regarding mitochondrial signaling pathways, a significant increase in oxidative capacity (by OCR and OXPHOS levels) was observed. In addition, the PGC-1α, SIRT-1, and PPARδ levels were significantly higher in the LEDT plus idebenone treated-dystrophic muscle cells. Together, the findings suggest that LEDT and idebenone treatment, alone or in conjunction, can modulate the calcium and mitochondrial signaling pathways, such as SLN, SERCA 1, and PGC-1α, contributing towards the improvement of the dystrophic phenotype in mdx muscle cells. In addition, data from the LEDT plus idebenone treatment showed slightly better results than those of each separate treatment in terms of SLN, OXPHOS, and SIRT-1.

Keywords: Dystrophic muscle cells, Photobiomodulation, Calcium signaling, Oxidative stress, Mitochondrial parameters

Introduction

Duchenne muscular dystrophy (DMD) is a genetic X-linked disease characterized by mutations of dystrophin protein, which leads to progressive muscle degeneration with a consequent loss of ambulation in adolescence and to death, at approximately 30 years of age, usually due to cardio-respiratory failure (Emery 2002; Landfeldt et al. 2018). There is currently no cure for DMD, and glucocorticoids are the main drug treatment used by dystrophic patients (Gloss et al. 2016). However, glucocorticoid continued use causes several adverse effects (Moxley et al. 2010), making it necessary to find new therapeutic approaches.

In addition to the primary genetic defect of DMD, the absence of dystrophin protein, several studies suggest that abnormal intracellular calcium concentration [Ca2+]i, reactive oxygen species (ROS), and mitochondrial dysfunction play a crucial role in the pathophysiology of this disease (Moore et al. 2020; González-Jamett et al. 2022).

Previous studies suggest that the sustained elevation of [Ca2+]i underlies muscle pathology and dysfunction in DMD. Research showed that the increase of [Ca2+]i enhances the expression and activity of calpains, the Ca2+-dependent proteases in dystrophic muscles (Spencer et al. 1995; Hussain et al. 2000; Sundaram et al. 2006; Voit et al. 2017). Furthermore, it was also observed that increased [Ca2+]i influences mitochondrial Ca2+ uptake, which in turn leads to altered metabolism and increased ROS production (Mareedu et al. 2021).

Mitochondria are the main source of ROS in dystrophic muscles (Moore et al. 2020). A recent study showed abnormal mitochondrial structure and reduced mitochondrial function in the dystrophic muscle of mdx mice (the experimental model of DMD), prior to the onset of muscle dystrophic fiber damage, suggesting an early mitochondrial role in the pathophysiology of DMD (Moore et al. 2020).

In search of alternative treatments for DMD, previous studies have pointed to the use of photobiomodulation (PBM) as a potential therapy (Silva et al. 2015; Macedo et al. 2020). PBM has presented several benefits in dystrophic muscles, such as the mitigation of inflammatory response and oxidative stress (Silva et al. 2015; Macedo et al. 2020). The effects of PBM result in the absorption of the light through the mitochondria, leading to an increase in membrane potential, the electron transport, and the oxygen consumption (Karu 2008; Freitas and Hamblin 2016). In this study, seeking to increase the therapeutic efficiency of PBM, we associated the PBM therapy (PBMT) with idebenone, a potent antioxidant and electron carrier. Idebenone is a synthetic short-chain benzoquinone, which improves mitochondrial function, restores ATP production, and reduces ROS (Buyse et al. 2015). In addition, recently, our research group showed that idebenone protected the dystrophic muscle cells by reducing the [Ca2+]I, oxidative stress, and inflammatory process (Valduga et al. 2023).

Thus, in the present study, we evaluated the LED therapy plus idebenone treatment effects in dystrophic muscle cells from mdx mice, focusing on the calcium and mitochondrial signaling pathways.

Materials and methods

Mdx primary muscle cells

The development of primary skeletal muscle cell cultures from mdx (C57BL/10-Dmdmdx/PasUnib) mice was based on protocol previously described (Rando and Blau 1994; Mizobuti et al. 2019). All the experiments were performed under an approved protocol of the Committee on the Ethics of Animal Experiments of UNICAMP (#5603-1/2020) and were in accordance with the Brazilian College for Animal Experimentation guidelines.

Experimental design (see Fig. 1)

Fig. 1.

Fig. 1

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Timescale of LEDT and idebenone treatment in mdx muscle cells

LEDT

LEDT at 850 nm was applied on mdx primary muscle cells, using a ThorLabs Mounted High-Power equipment, and the parameters used (Table 1) are based on previous study (Rocha et al. 2022). The LEDT was applied to muscle cells at a perpendicular angle, by a single application, and irradiation was performed by one point at the center of each culture well. The cells were irradiated inside a laminar flow in a dark room without radiation. Mdx muscle cells not irradiated were used as control.

Table 1.

Specifications of LEDT parameters

Device information
  Manufacturer ThorLabs Mounted High-Power equipment
Irradiation parameters 96 weels 6 weels
  Output power (mW) 110 38
  Spot size (cm2) 0.32 9.5
  Power density (w/cm2) 343 4
  Energy density (J/cm2) 1.5 0.05
  Time per point (s) 4.5 13.1
  Wavelength (nm) 850
  Beam shape Circular
  Operating mode Continuous
  Number and frequency of treatment sessions 1
  Application technique Applied directed to the cells through the bottom of each well
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Idebenone treatment

The first experiment tried to establish the best dose of idebenone for mdx muscle cell proliferation by MTT assay. Mdx muscle cells received different doses of idebenone (0.5, 0.25, 0.12, 0.06, and 0.03 μM) diluted in 0.5% carboxymethylcellulose sodium salt (CMC, Fluka, Buchs, Switzerland) in water and were evaluated after 24 and 48 h of treatment. Subsequently, the mdx muscle cells received only two doses (0.06 and 0.03 μM; selected based on the results of MTT assay and IC50) and were evaluated after 24 and 48 h of treatment for cell viability (Neutral Red assay), [Ca2+]i, mitochondrial superoxide (O2•−), and H2O2 production analyses. Lastly, the other analyses were performed in mdx muscle cells treated with idebenone, after 48 h of treatment, at the chosen dose (0.06 μM). For all experiments, the untreated mdx muscle cells were used as controls.

Cell viability and proliferation

MTT analysis

This colorimetric analysis was performed for quantification of cell metabolic activity. The protocol of this assay was previously described by Macedo et al. (2015) and Mizobuti et al. (2019). Briefy, after the MTT incubation phase, the crystals were dissolved with isopropanol acid, and the amount of formazan product was measured by a spectrophotometer (Synergy H1, Hybrid Reader, Biotek Instruments, Winooski, VT, USA) at 570 nm with a 655-nm reference wavelength. In addition, the IC50 was calculated based on the MTT data.

Neutral Red

This colorimetric analysis was performed to quantify membrane permeability and lysosomal cell activity in response to different doses of idebenone (0.06 and 0.03 μM). The protocol of this assay was previously described by Borenfreund and Puerner (1985). Briefy, primary muscle cells were washed in PBS once, and NR medium was added (250 μl), and the cells were incubated (37 °C) for 3 h. After this period, the NR medium was removed, the muscle cells were washed in PBS, and desorption solution was added. A shaker was used to extract the NR from the cells, and the absorbance was measured in a spectrophotometer (Synergy H1, Hybrid Reader, Biotek Instruments, Winooski, VT, USA) at 540 nm.

Intracellular calcium content

[Ca2+]i analysis was performed by Fluo-4 dye (Molecular Probes, Oregon, USA). The protocol was previously described (Macedo et al. 2015; Mizobuti et al. 2019). Briefly, after incubation with Fluo-4 AM, the calcium-sensitive dye intensities were observed on a fluorescent inverted microscope (Nikon, Eclipse TS100/TS100F) for qualitative data. Quantitative measurements were investigated using a spectrophotometer (Synergy H1, Hybrid Reader, Biotek Instruments, Winooski, VT, USA) at excitation and emission wavelengths of 494 and 516 nm, respectively.

H2O2 production

H2O2 levels were determined by Amplex® Red assay kit (Molecular Probes, Life Technologies, California, EUA) according to the manufacturer’s instructions. The Amplex UltraRed reagent (50 μM) and HRP (0.1 U/ml) were added for 60 min. The absorbance was determined at 530- (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.

Mitochondrial superoxide (O2•−) production

The mitochondrial superoxide (O2•−) production was determined by fluorescent dye MitoSOXTM Red (M36008, ThermoFisher) according to the manufacturer’s instructions. Briefly, the dystrophic primary muscle cells were incubated with MitoSOXTM Red for 15 min at 37 °C. MitoSOXTM is selectively accumulated in the mitochondria, and it emits red fluorescence when oxidized by the superoxide anion. The intensities of MitoSOXTM fluorescence were monitored on a fluorescent inverted microscope (Nikon, Eclipse TS100/TS100F) for qualitative analyses. MitoSOXTM Red was excited at 514 nm with the fluorescent images been collected at 570–600 nm. Quantitative measurements were performed using a spectrophotometer (Synergy H1, Hybrid Reader, Biotek Instruments, Winooski, VT, USA) at 510-nm excitation and 580-nm emission wavelengths.

Oxygen consumption

We evaluated oxygen consumption rates using an O2k-FluoRespirometer and used the DatLab software package (OROBOROS, Innsbruck, Austria) for data acquisition and analysis. After treatment, we trypsinized dystrophic muscle cells, centrifuged them (1250 RPM), and incubated them with 2 ml of air-saturated respiration medium in an Oxygraph-2k (O2k, OROBOROS Instruments). We used the following drugs in the assay: 1 μM oligomycin (Oligo), 2 μM carbonyl cyanate m-chlorophenyl hydrazone (CCCP), and 1 μM antimycin (Ant). We determined the basal oxygen consumption rate (OCR) by subtracting OCR pre-oligomycin from OCR post-antimycin; ATP-linked OCR was calculated by subtracting OCR post-oligomycin from OCR pre-oligomycin; proton leak was obtained by subtracting OCR post-oligomycin from OCR post-antimycin; maximal OCR was determined by subtracting OCR post-CCCP from OCR post-antimycin; reserve capacity was determined by subtracting OCR post-CCCP from OCR pre-oligomycin; and non-mitochondrial was the value of OCR post-antimycin.

Western blotting

The Western blotting protocol was previously described (Mizobuti et al. 2019; Rocha et al. 2022). Briefly, the Bradford method was used to determine the total protein content in cell extracts. Thirty micrograms of total protein homogenate was loaded on 6–15% SDS-polyacrylamide gels. After electrophoresis, the proteins were transferred onto nitrocellulose membranes, and the membranes were incubated with primary antibodies: 4-HNE (Bio-Rad AHP1251), Calpain 1 (Santa Cruz Biotechnology sc-7530), calsequestrin (Affinity BioReagents VIIID12), sarcolispin (Merckmilipore ABT 13), serca 2a (Cell-Sinaling 4388S), serca 1a (Cell-Sinaling D54G12), Oxphos (Abcam STN-19467), PGC-1α (Calbiochem 4C1.3), PPARδ (Invitrogen PA1-823A), SIRT-1 (Cell-Sinaling C14H4), and β-actin (Sigma-Aldrich A1978). Next, the membranes were incubated with the peroxidase-conjugated secondary antibodies: anti-rabbit (Promega Corporation W4011), anti-mouse (Promega Corporation W4021), and anti-goat (KPL14-13-06). To control protein loading, Western blot transfer, and nonspecific changes in protein levels, the blots were stripped and re-probed for β-actin. The ImageJ software was used to determine the band intensities.

Statistical analysis

All experiments were repeated independently at least three times. IC50 values of compounds were analyzed using GraphPad Prims 8 software package (GraphPad Software, CA, USA) using non-linear regression curve fitting with the normalized response. Statistical differences were analyzed using one-way ANOVA followed by Tukey test or Student’s t-test using GraphPad Prism 8. Significant differences were defined as P < 0.05. All results are expressed as mean ± standard deviation (SD).

Results

Cell proliferation and viability

To assess the dose-dependent toxicities of idebenone on dystrophic muscle cells, we used the MTT and Neutral Red assays.

A significant reduction in cell proliferation in treated mdx muscle cells was observed in the MTT assay (Fig. 2A, B), when compared to untreated mdx muscle cells after 24-h treatment (93.0% for Ide 0.5 μM; 76.0% for Ide 0.25 μM; 42.4% for Ide 0.12 μM; 21.3% for Ide 0.06 μM; 93.0% for Ide 0.5 μM + LEDT; 81.6% for Ide 0.25 μM + LEDT; and 43.6% for Ide 0.12 μM + LEDT) and after 48-h treatment (93.7% for Ide 0.5 μM; 56.6% for Ide 0.25 μM; 21.5% for Ide 0.12 μM; 91.9% for Ide 0.5 μM + LEDT; and 67.0% for Ide 0.25 μM + LEDT).

Fig. 2.

Fig. 2

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MTT assay in untreated mdx muscle cells (mdxC); mdx muscle cells treated with carboxymethylcellulose sodium salt (Vehicle); mdx muscle cells treated with different doses of idebenone; mdx muscle cells treated with LEDT (LEDT); and mdx muscle cells treated with LEDT and different doses of idebenone, after 24 h (A) and 48 h (B). The half maximal inhibitory concentrations (IC50) in mdx muscle cells treated with different doses of idebenone and mdx muscle cells treated with LEDT and different doses of idebenone, after 24 h (C) and 48 h (D). Neutral Red assay in mdxC; vehicle; mdx muscle cells treated with 0.06 μM and 0.03μM of idebenone, LEDT, and LEDT and 0.06μM and 0.03μM of idebenone, after 24 h (E) and 48 h (F). All data are expressed by mean ± SD, and the experiments were carried out in triplicate. *P < 0.05 versus mdxC; **P < 0.01 versus mdxC; ***P < 0.001 versus mdxC; ****P < 0.00001 versus mdxC; °P < 0.05 versus vehicle; °°°°P < 0.00001 versus vehicle; φP < 0.05 versus Ide 0.03 μM

The half maximal inhibitory concentrations (IC50) (Fig. 2C, D) at 24 h were 0.15 μM for idebenone and idebenone plus LEDT and at 48 h were 0.21 μM for idebenone and 0.20 μM idebenone plus LEDT.

Through the analysis of Neutral Red assay (Fig. 2E, F), a significant increase in the viability of treated mdx muscle cells was observed when compared to untreated mdx muscle cells after 24-h treatment (18.8% for Ide 0.06 μM; 15.3% for Ide 0.03 μM; 30.6% for LEDT; 22.6% for Ide 0.06 μM + LEDT; and 23.2% for Ide 0.03 μM + LEDT) and after 48-h treatment (23.6% for LEDT; 14.8% for Ide 0.06 μM + LEDT; and 22.7% for Ide 0.03 μM + LEDT).

Intracellular calcium content

Twenty-four hours after applying the treatments (idebenone, LEDT and/or idebenone plus LEDT), no significant difference in [Ca2+]i was observed between the experimental groups (Fig. 3A, B). On the other hand, 48 h after treatments, the treated-mdx muscle cells showed a significant reduction of [Ca2+]i (13.0% for Ide 0.06 μM; 9.1% for LEDT; 11.6% for Ide 0.06 μM + LEDT; and 11.0% for Ide 0.03 μM + LEDT) compared to the untreated mdx muscle cells (Fig. 3C, D).

Fig. 3.

Fig. 3

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Intracellular calcium concentrations [Ca2+]i in untreated mdx muscle cells (mdxC); mdx muscle cells treated with 0.06 μM and 0.03 μM of idebenone; mdx muscle cells treated with LEDT; and mdx muscle cells treated with LEDT and 0.06 μM and 0.03 μM of idebenone, after 24 h (A) and 48 h (C). Scale bar: 100 μm, × 20. Graph showing fluorescence intensity of [Ca2+] in experimental groups, after 24 h (B) and 48 h (D) of LEDT and idebenone treatment. All data are expressed by mean ± SD, and the experiments were carried out in triplicate. *P < 0.05 versus mdxC; **P < 0.01 versus mdxC; ***P < 0.001 versus mdxC; #P < 0.05 versus Ide 0.06 μM

Oxidative stress

Twenty-four hours after applying the treatments (idebenone, LEDT and/or idebenone plus LEDT), the treated-mdx muscle cells showed a significant reduction of O2•− production (16.6% for Ide 0.06 μM; 15.6% for Ide 0.03 μM; 12.1% for LEDT; 13.6% for Ide 0.06 μM + LEDT; and 12.0% for Ide 0.03 μM + LEDT) compared to the untreated mdx muscle cells (Fig. 4A, B). Similar results were also observed after 48 h of treatments (14.7% for Ide 0.06 μM; 15.1% for Ide 0.03 μM; 19.3% for LEDT; 15.3% for Ide 0.06 μM + LEDT; and 12.8% for Ide 0.03 μM + LEDT) compared to the untreated mdx muscle cells (Fig. 4D, E).

Fig. 4.

Fig. 4

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MitoSOXTM fluorescence (red) images in untreated mdx muscle cells (mdxC); mdx muscle cells treated with 0.06 μM and 0.03 μM of idebenone; mdx muscle cells treated with LEDT; and mdx muscle cells treated with LEDT and 0.06 μM and 0.03 μM of idebenone, after 24 h (A) and 48 h (D). Scale bar 100 μm, × 20. Graphs showing MitoSOXTM fluorescence intensity in experimental groups, after 24 h (B) and 48 h (E) of LEDT and idebenone treatments. The graphs show H2O2 production in experimental groups, after 24 h (C) and 48 h (F) of LEDT and idebenone treatments. Western blotting analysis of 4-HNE protein adducts (G) in mdxC; mdx muscle cells treated with 0.06 μM of idebenone; LEDT; and mdx muscle cells treated with LEDT and 0.06 μM of idebenone. The graphs (H) show 4-HNE protein adducts levels in experimental groups. β-Actin was used as a loading control. The relative value of the band intensity was quantified and normalized by the corresponding control. All data are expressed by mean ± SD, and the experiments were carried out in triplicate. *P < 0.05 versus mdxC; **P < 0.01 versus mdxC; ***P < 0.001 versus mdxC; ****P < 0.00001 versus mdxC; ##P < 0.01 versus Ide 0.06 μM; ####P < 0.00001 versus Ide 0.06 μM; φφφP < 0.001 versus Ide 0.03 μM; ††††P < 0.00001 versus LEDT; δδδδP < 0.00001 versus Ide 0.06 μM+LEDT

Regarding H2O2 production, the treated-mdx muscle cells analyzed 24 h after all the treatments showed a significant reduction in its production (12.6% for Ide 0.06 μM; 12.9% for Ide 0.03 μM; 10.9% for LEDT; 18.0% for Ide 0.06 μM + LEDT; and 14.3% for Ide 0.03 μM + LEDT) compared to the untreated mdx muscle cells (Fig. 4C). However, 48 h after the treatments, only mdx muscle cells treated with Ide 0.06 μM, LEDT, and Ide 0.06 μM + LEDT showed a significant reduction in H2O2 production (by 16.0%, 8.9%, and 13.0%, respectively) compared to the untreated mdx muscle cells (Fig. 4F).

Regarding the lipidic peroxidation marker, the mdx muscle cells treated with Ide 0.06 μM, LEDT, and Ide 0.06μM + LEDT (analyzed 48 h after treatments) showed a significant reduction in 4-HNE protein adduct levels (by 10.0%, 11.0%, and 13%, respectively) compared to the untreated mdx muscle cells (Fig. 4G, H).

Calcium pathways

Regarding calpain-1, the mdx muscle cells treated with Ide 0.06 μM, LEDT, and Ide 0.06 μM + LEDT showed a significant reduction in its levels (by 57.7%, 46.2%, and 64.7%, respectively) compared to the untreated mdx muscle cells (Fig. 5A, B).

Fig. 5.

Fig. 5

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A Western blotting analysis of calpain-1, calsequestrin, sarcolipin, serca-1a, serca-2a in untreated mdx muscle cells (mdxC); mdx muscle cells treated with 0.06 μM of idebenone; mdx muscle cells treated with LEDT; and mdx muscle cells treated with LEDT and 0.06 μM of idebenone. B The graph shows the protein levels. β-Actin was used as an internal control. The relative value of the band intensity was quantified and normalized by the corresponding control. All data are expressed by mean ± SD, and the experiments were carried out in triplicate. *P < 0.05 versus mdxC; **P < 0.01 versus mdxC; ***P < 0.001 versus mdxC; ****P < 0.00001 versus mdxC; ##P < 0.01 versus Ide 0.06 μM; ††P < 0.05 versus LEDT

The calsequestrin levels were significantly reduced in the mdx muscle cells treated with LEDT and Ide 0.06 μM + LEDT (by 83.0% and 80.9%%, respectively) compared to the untreated mdx muscle cells (Fig. 5A, B).

In relation to sarcolispin and serca 2a levels, only the mdx muscle cells treated with Ide 0.06μM + LEDT showed a significant reduction in their levels (by 49.2% and 30.0%, respectively) compared to the untreated mdx muscle cells (Fig. 5A, B).

The serca 1a levels were significantly increased in the mdx muscle cells treated with Ide 0.06 μM, LEDT, and Ide 0.06 μM + LEDT (by 227.4%, 201.9%, and 205.8%, respectively) compared to the untreated mdx muscle cells (Fig. 5A, B).

Mitochondrial pathways

The OCR was evaluated, and it was found that the mdx muscle cells treated with Ide 0.06 μM increased the basal, ATP-linked, and maximal capacity (by 98.8%, 100.0%, and 55.2%, respectively) compared to the mdx untreated muscle cells (Fig. 6A). In addition, the basal, ATP-linked, maximal and spare capacity were increased in the mdx muscle cells treated with LEDT (by 107.8%, 201.5%, 138.7%, and 511.7%, respectively) and/or Ide 0.06 μM + LEDT (by 51.8%, 85.8%, 96.9%, and 198.6%, respectively) compared to the untreated mdx muscle cells (Fig. 6A).

Fig. 6.

Fig. 6

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A OCR values were measured in untreated mdx muscle cells (mdxC), mdx muscle cells treated with 0.06 μM of idebenone, mdx muscle cells treated with LEDT, and mdx muscle cells treated with LEDT and 0.06 μM of idebenone. The measurements were taken for basal, ATP-linked, leak, maximal, and spare capacity. Non-mitochondrial OCR was subtracted from all data before being used for these analyses. B Western blotting analysis of OXPHOS and D of PGC-1α, PPARδ, and SIRT-1in untreated mdx muscle cells (mdxC); mdx muscle cells treated with 0.06 μM of idebenone; mdx muscle cells treated with LEDT; and mdx muscle cells treated with LEDT and 0.06 μM of idebenone. C, E The graph shows the protein levels. β-Actin was used as an internal control. The relative value of the band intensity was quantified and normalized by the corresponding control. All data are expressed by mean ± SD, and the experiments were carried out in triplicate. *P < 0.05 versus mdxC; **P < 0.01 versus mdxC; ***P < 0.001 versus mdxC; ****P < 0.00001 versus mdxC; #P < 0.05 versus Ide 0.06 μM; ##P < 0.01 versus Ide 0.06 μM; †P < 0.05 versus LEDT; ††P < 0.01 versus LEDT; ††††P < 0.00001 versus LEDT

The mdx muscle cells treated with Ide 0.06 μM, LEDT, and Ide 0.06 μM + LEDT presented a significant increase in the OXPHOS levels in complex V (by 125.0%, 94.1%, and 182.3%, respectively) compared to the untreated mdx muscle cells (Fig. 6B, C). In addition, the mdx muscle cells treated with Ide 0.06μM + LEDT, also showed a significant increase in complex II and I (by 39.1% and 55.4%, respectively) compared to the untreated mdx muscle cells (Fig. 6B, C).

Regarding PGC-1α, the mdx muscle cells treated with Ide 0.06 μM, LEDT, and Ide 0.06μM + LEDT presented a significant increase in its levels (by 27.0%, 32.8%, and 27.0%, respectively) compared to the untreated mdx muscle cells (Fig. 6D, E). Similar results were also observed in PPARδ where the mdx muscle cells treated with Ide 0.06μM, LEDT, and Ide 0.06μM + LEDT presented a significant increase in its levels (by 66.6%, 33.3%, and 38.8%, respectively) compared to the untreated mdx muscle cells (Fig. 6D, E).

On the other hand, only the mdx muscle cells treated with Ide 0.06 μM + LEDT showed a significant increase in SIRT-1 levels (31.8%), compared to the untreated mdx muscle cells (Fig. 6D, E).

Discussion

Here, we demonstrated beneficial effects in calcium and mitochondrial signaling pathways in dystrophic muscle cells subjected to LEDT and idebenone treatment applied separately and/or together.

Although the mechanisms involved in DMD are complex and multifactorial, impaired calcium handling and calcium overload are key contributors to disease onset and progression (Mareedu et al. 2021). Increased intracellular calcium in the context of dystrophin deficiency is a main mediator in myocyte death and fibrotic development (Shirokova et al. 2013). Under our experimental conditions, we observed that LEDT and idebenone treatment decreased the [Ca2+]i in mdx muscle cells. Concomitant with the decrease in [Ca2+]i, a significant finding of this study was the decrease in sarcolipin (SLN) levels. In agreement with our results, a previous study found improvement in the cytosolic Ca2+ homeostasis in mdx mice lacking SLN (Tanihata et al. 2018).

SLN is an important regulator of the SERCA pump, which is expressed exclusively in the striated muscle of all mammals (Vangheluwe et al. 2005). A previous study provides evidence that SLN upregulation is a molecular basis for SERCA dysfunction in both DMD skeletal and cardiac muscles (Voit et al. 2017). In addition, SLN knockout mice display an increase in the SERCA function (Tupling et al. 2011; Bombardier et al. 2013). Enhancing the SERCA expression or activity appears to be a promising strategy to treat the dystrophic skeletal muscle, since SERCA accounts for ≥ 70% of Ca2+ removal from the cytosol (Periasamy and Kalyanasundaram 2007; Tupling 2009). It is important to note that in the present study, we also showed an increase in SERCA 1 levels, which is the primary isoform express in the fast-twitch skeletal muscle (Wu and Lytton 1993). However, we also found a reduction in the SERCA 2 levels. In the skeletal muscle, there are some inhibitory proteins that alter the SERCA action, such as SLN (as mentioned before) and phospholamban (PLN) (Gamu et al. 2020). In vivo studies have consistently found that PLN preferentially binds with SERCA 2, which is highly expressed in the cardiac and slow-twitch muscle (Fujii et al. 1988; Briggs et al. 1992; Babu et al. 2007; Fajardo et al. 2013). On the other hand, SLN has been associated to SERCA 1 (Maclennan et al. 1973). Thus, these previous studies can collaborate with our findings about SERCA and SLN.

Still regarding the calcium signaling pathways, we found a reduction in the levels of calpain-1 and calsequestrin after LEDT and idebenone treatment. It is already well established that the increase in [Ca2+]i could activate calpains, thereby promoting muscle wasting through increased proteolysis in dystrophic muscles (Bodensteiner and Engel 1978; Spencer et al. 1995). In addition, it was reported that isolated mdx muscle fibers exhibit heightened calpain-mediated proteolysis when compared with normal muscle fibers (Gailly et al. 2007). Thus, targeting a reduction in calpain levels is a promising approach in DMD treatment. On the other hand, calsequestrin (the most abundant calcium-binding protein) showed higher levels in the spared muscles of mdx mice (Pertille et al. 2010). However, in our experimental conditions, we observed a reduction in calsequestrin levels, suggesting that the decrease of [Ca2+]i, by other pathways, after LEDT and idebenone treatment, reduced the need for its upregulation.

Another potential target related to impaired Ca2+ homeostasis in dystrophic muscles is the mitochondria (Robert et al. 2001). Reduced mitochondrial respiration and increased calcium deposits were reported in the fast-twitch muscle fibers of mdx mice (Gaglianone et al. 2019). In addition, mitochondrial Ca2+ overload can cause mitochondrial ROS production contributing to oxidative stress in dystrophic muscles (Kyrychenko et al. 2015). In the present study, using LEDT and idebenone treatment, we observed improved oxidative capacity in dystrophic muscle cells, by OCR and OXPHOS analyzes. Concomitantly, we also found reduced levels of ROS and lipid peroxidation after treatment. A main mechanism of the beneficial effect of LEDT relies on the improvement of the mitochondrial function by the direct stimulation of the electron transfer chain (Salechpour et al. 2018b). Several studies showed that PBMT can improve mitochondrial functions by increasing ATP and the mitochondrial membrane potential and by decreasing ROS production (Salechpour et al. 2017, 2018a, 2018b). Regarding idebenone, its ability to modulate the activity of the mitochondrial respiratory chain complexes, as well as its capacity to protect membranes against lipid peroxidation, is well known (Muscoli et al. 2002).

It is also important to highlight that the reduction in SLN levels correlates with the interplay between mitochondrial function and oxidative stress. Supporting what was said before, the reduction of SLN restored the complex proteins (OXPHOS) and their activities, as well as reducing the protein carbonyl content and lipid peroxidation, thus indicating reduced oxidative stress in the dystrophic muscles (Balakrishnan et al. 2022). Interestingly, in the present study, we also observed a reduction in SLN levels in the dystrophic muscle cells treated, suggesting that this signaling pathway may also be one of the mechanisms by which the LEDT and idebenone treatment showed antioxidant effects and improved mitochondrial function in the dystrophic muscle.

The signaling pathway between the silent mating type information regulation 2 homolog 1 (SIRT1), peroxisome proliferator-activated receptor (PPARs), and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) could be another possible explanation for observed ROS reduction in the dystrophic muscle cells. PGC-1α is a main regulator of mitochondrial biogenesis and function in several tissues (Handschin and Spiegelman 2006) and is regulated by PPARs and SIRT1 (Cantó and Auwerx 2009). LEDT and idebenone treatment increased SIRT1, PPARδ, and PGC-1α in the dystrophic muscle cells, in our study. In agreement with our results, previous studies have demonstrated that PBMT can induce mitochondrial biogenesis by the elevation of SIRT1 and PGC-1α in the C2C12 muscle cells (Nguyen et al. 2014) and in a transient cerebral ischemia mouse model (Salehpour et al. 2019). Recently, our research group also reported the correlation between elevated PGC-1α levels and oxidative stress reduction in the dystrophic muscle treated with antioxidants (Silva et al. 2021; Mizobuti et al. 2022). Regarding idebenone, a previous study, collaborating with our results, showed that this antioxidant can positively influence mitochondrial biogenesis by increasing PPARGC1A gene expression in human-induced pluripotent stem cells (Augustyniak et al. 2017).

However, despite the novelty of this study, one limitation must be recognized. While the beneficial effects reported after LEDT and idebenone treatment are statistically significant, the relative change when compared to the control group is low. This fact may have occurred because a single application of LEDT and only one dose of antioxidant were evaluated. In addition, the analysis period after the treatments may also have interfered with the results obtained. In view of this, we intend to evaluate new variables (e.g., several LEDT applications and different exposure periods to the antioxidant treatment) in future experiments.

Summarizing, our findings suggest that LEDT and idebenone treatment, alone or together, can modulate the calcium and mitochondrial signaling pathways, such as SLN, SERCA 1, and PGC-1α, contributing to the improvement of the dystrophic phenotype in muscle cells (Fig. 7). It is also important to highlight that data from the LEDT plus idebenone treatment showed slightly better results than those of each separate treatment in terms of SLN, OXPHOS, and SIRT-1. In addition, this work contributes to the knowledge that the combining of new approaches holds promise for better and more specific DMD treatments in the future. The combined therapies usually show a synergistic effect and act on the treatment of the secondary consequences of the muscular dystrophy (e.g., oxidative stress and mitochondrial dysfunction) and are more effective than single therapies. Therefore, the future technical advances in combined therapeutic assays will help to lead to more effective treatment for DMD.

Fig. 7.

Fig. 7

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LEDT and idebenone treatment effects on mdx muscle cells. Upregulation: blue up arrow; downregulation: red down arrow; cross-talk between sarcolipin reduction: words in green; cross-talk between PGC1-α increase: word underlined in blue

Acknowledgements

We thank Mrs. Deirdre Jane Donovan Giraldo for the English revision of the manuscript.

Funding

This research was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; #2020/09733-4), Coordenação de Pessoal de Nível Superior-Brasil (CAPES)–Finance Code 001, CNPq, and FAEPEX. G.L.R. and D.S.M. were the recipients of a CAPES fellowship. H.N.M.S., V.A.P., E.M., and M.V.C. are the recipients of a CNPq fellowship. A.G.O. received a scholarship from FAPESP (# 2018/20581-1).

Declarations

Conflict of interest

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. Augustyniak J, Lenart J, Zychowicz M, Stepien PP, Buzanska L. Mitochondrial biogenesis and neural differentiation of human iPSC is modulated by idebenone in a developmental stage-dependent manner. Biogerontology. 2017;18:665–677. doi: 10.1007/s10522-017-9718-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Babu GJ, Bhupathy P, Carnes CA, Billman GE, Periasamy M. Differential expression of sarcolipin protein during muscle development and cardiac pathophysiology. J Mol Cell Cardiol. 2007;43:215–222. doi: 10.1016/j.yjmcc.2007.05.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Balakrishnan R, Mareedu S, Babu GJ. Reducing sarcolipin expression improves muscle metabolism in mdx mice. Am J Physiol Cell Physiol. 2022;322:C260–C274. doi: 10.1152/ajpcell.00125.2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bodensteiner JB, Engel AG. Intracellular calcium accumulation in Duchenne dystrophy and other myopathies: a study of 567,000 muscle fibers in 114 biopsies. Neurology. 1978;28:439–446. doi: 10.1212/wnl.28.5.439. [DOI] [PubMed] [Google Scholar]
  5. Bombardier E, Smith IC, Vigna C, Fajardo VA, Tupling AR. Ablation of sarcolipin decreases the energy requirements for Ca2 þ transport by sarco(endo)plasmic reticulum Ca2 þ -ATPases in resting skeletal muscle. FEBS Lett. 2013;587:1687–1692. doi: 10.1016/j.febslet.2013.04.019. [DOI] [PubMed] [Google Scholar]
  6. Borenfreund E, Puerner JA (1985) Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol Lett. 24(2–3):119–24. 10.1016/0378-4274(85)90046-3 [DOI] [PubMed]
  7. Briggs FN, Lee KF, Wechsler AW, Jones LR. Phospholamban expressed in slow-twitch and chronically stimulated fast-twitch muscles minimally affects calcium affinity of sarcoplasmic reticulum Ca(2þ)-ATPase. J Biol Chem. 1992;267:26056–26061. doi: 10.1016/S0021-9258(18)35716-8. [DOI] [PubMed] [Google Scholar]
  8. Buyse GM, Gueven N, McDonald CM. Idebenone as a novel therapeutic approach for Duchenne muscular dystrophy. Eur Neurol Rev. 2015;10:189–194. doi: 10.17925/ENR.2015.10.02.189. [DOI] [Google Scholar]
  9. Cantó C, Auwerx J. PGC-1alpha, SIRT1 and AMPK, an energy sensing network that controls energy expenditure. Curr Opin Lipidol. 2009;20:98–95. doi: 10.1097/MOL.0b013e328328d0a4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Emery AEH. The muscular dystrophies. Lancet. 2002;359:687–695. doi: 10.1016/S0140-6736(02)07815-7. [DOI] [PubMed] [Google Scholar]
  11. Fajardo VA, Bombardier E, Vigna C, Devji T, Bloemberg D, Gamu D, Gramolini AO, Quadrilatero J, Tupling AR. Co-expression of SERCA isoforms, phospholamban and sarcolipin in human skeletal muscle fibers. PloS One. 2013;8:e84304. doi: 10.1371/journal.pone.0084304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Freitas LF, Hamblin MR. Proposed mechanisms of photobiomodulation or low-level light therapy. IEEE J Sel Top Quantum Electron. 2016;22:7000417. doi: 10.1109/JSTQE.2016.2561201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fujii J, Lytton J, Tada M, MacLennan DH. Rabbit cardiac and slow-twitch muscle express the same phospholamban gene. FEBS Lett. 1988;227:51–55. doi: 10.1016/0014-5793(88)81412-1. [DOI] [PubMed] [Google Scholar]
  14. Gaglianone RB, Santos AT, Bloise FF, Ortiga-Carvalho TM, Costa ML, Quirico-Santos T, da Silva WS, Mermelstein C. Reduced mitochondrial respiration and increased calcium deposits in the EDL muscle, but not in soleus, from 12-week-old dystrophic mdx mice. Sci Rep. 2019;9:1986. doi: 10.1038/s41598-019-38609-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gailly P, De Backer F, Van Schoor M, Gillis JM. In situ measurements of calpain activity in isolated muscle fibres from normal and dystrophin-lacking mdx mice. J Physiol. 2007;582:1261–1275. doi: 10.1113/jphysiol.2007.132191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gamu D, Juracic ES, Hall KJ, Tupling AR. The sarcoplasmic reticulum and SERCA: a nexus for muscular adaptive thermogenesis. Appl Physiol Nutr Metab. 2020;45:1–10. doi: 10.1139/apnm-2019-0067. [DOI] [PubMed] [Google Scholar]
  17. Gloss D, Moxley RT, 3rd, Ashwal S, Oskoui M. Practice guideline update summary: corticosteroid treatment of Duchenne muscular dystrophy: report of the Guideline Development Subcommittee of the American Academy of Neurology. Neurology. 2016;86:465–472. doi: 10.1212/WNL.0000000000002337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. González-Jamett A, Vásquez W, Cifuentes-Riveros G, Martínez-Pando R, Sáez JC, Cárdenas AM. Oxidative stress, inflammation and connexin hemichannels in muscular dystrophies. Biomedicines. 2022;10:507. doi: 10.3390/biomedicines10020507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Handschin C, Spiegelman BM. Peroxisome proliferator-activated receptor gamma coactivator 1 coactivators, energy homeostasis, and metabolism. Endocr Rev. 2006;27:728–735. doi: 10.1210/er.2002-0012. [DOI] [PubMed] [Google Scholar]
  20. Hussain T, Mangath H, Sundaram C, Anandaraj MPJS. Expression of the gene for large subunit of m-calpain is elevated in skeletal muscle from Duchenne muscular dystrophy patients. J Genet. 2000;79:77–80. doi: 10.1007/Bf02728949. [DOI] [Google Scholar]
  21. Karu TI. Mitochondrial signaling in mammalian cells activated by red and near-IR radiation. Photochem Photobiol. 2008;84:1091–1099. doi: 10.1111/j.1751-1097.2008.00394.x. [DOI] [PubMed] [Google Scholar]
  22. Kyrychenko V, Poláková E, Janícek R, Shirokova N. Mitochondrial dysfunctions during progression of dystrophic cardiomyopathy. Cell Calcium. 2015;58:186–195. doi: 10.1016/j.ceca.2015.04.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Landfeldt E, Edström J, Buccella F, Kirschner J, Lochmüller H. Duchenne muscular dystrophy and caregiver burden: a systematic review. Dev Med Child Neurol. 2018;60:987–996. doi: 10.1111/dmcn.13934. [DOI] [PubMed] [Google Scholar]
  24. Macedo AB, Mizobuti DS, Hermes TA, Mancio RD, Pertille A, Kido LA, Cagnon VHA, Minatel E. Photobiomodulation therapy for attenuating the dystrophic phenotype of Mdx mice. Photochem Photobiol. 2020;96:200–207. doi: 10.1111/php.13179. [DOI] [PubMed] [Google Scholar]
  25. Macedo AB, Moraes LHR, Mizobuti DS, Fogaca AR, Moraes FRS, Hermes TA, Pertille A, Minatel E. Low-level laser therapy (LLLT) in dystrophin-deficient muscle cells: effects on regeneration capacity, inflammation response and oxidative stress. PloS One. 2015;10:e0128567. doi: 10.1371/journal.pone.0128567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Maclennan DH, Yip CC, Iles GH, Seeman P. Isolation of sarcoplasmic reticulum proteins. Cold Spring Harb Sym. 1973;37:469–477. doi: 10.1101/SQB.1973.037.01.058. [DOI] [Google Scholar]
  27. Mareedu S, Million ED, Duan D, Babu GJ. Abnormal calcium handling in Duchenne muscular dystrophy: mechanisms and potential therapies. Front Physiol. 2021;12:647010. doi: 10.3389/fphys.2021.647010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mizobuti DS, Fogaca AR, Moraes FRS, Moraes LHR, Mancio RD, Hermes TA, Macedo AB, Valduga AH, Lourenco CC, Pereira ECL, Minatel E. Coenzyme Q10 supplementation acts as antioxidant on dystrophic muscle cells. Cell Stress Chaperones. 2019;24:1175–1185. doi: 10.1007/s12192-019-01039-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mizobuti DS, Rocha GL, Silva HNM, Covatti C, Lourenço CC, Pereira ECL, Salvador MJ, Minatel E. Antioxidant effects of bis-indole alkaloid indigo and related signaling pathways in the experimental model of Duchenne muscular dystrophy. Cell Stress Chaperones. 2022;27:417–429. doi: 10.1007/s12192-022-01282-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Moore TM, Lin AJ, Strumwasser AR, Cory K, Whitney K, Ho T, Ho T, Lee JL, Rucker DH, Nguyen CQ, Yackly A, Mahata SK, Wanagat J, Stiles L, Turcotte LP, Crosbie RH, Zhou Z. Mitochondrial dysfunction is an early consequence of partial or complete dystrophin loss in mdx mice. Front Physiol. 2020;11:690. doi: 10.3389/fphys.2020.00690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Moxley RT, 3rd, Pandya S, Ciafaloni E, Fox DJ, Campbell K. Change in natural history of Duchenne muscular dystrophy with long-term corticosteroid treatment: Implications for Management. J Child Neurol. 2010;25(9):1116–1129. doi: 10.1177/0883073810371004. [DOI] [PubMed] [Google Scholar]
  32. Muscoli C, Fresta M, Cardile V, Palumbo M, Renis M, Puglisi G, Paolino D, Nisticò S, Rotiroti D, Mollace V. Ethanol-induced injury in rat primary cortical astrocytes involves oxidative stress: effect of idebenone. Neurosci Lett. 2002;329:21–24. doi: 10.1016/s0304-3940(02)00567-0. [DOI] [PubMed] [Google Scholar]
  33. Nguyen LM, Malamo AG, Larkin-Kaiser KA, Borsa PA, Adhihetty PJ. Effect of near-infrared light exposure on mitochondrial signaling in C 2 C 12 muscle cells. Mitochondrion. 2014;14:42–48. doi: 10.1016/j.mito.2013.11.001. [DOI] [PubMed] [Google Scholar]
  34. Periasamy M, Kalyanasundaram A. SERCA pump isoforms: their role in calcium transport and disease. Muscle Nerve. 2007;35:430–442. doi: 10.1002/mus.20745. [DOI] [PubMed] [Google Scholar]
  35. Pertille A, Carvalho CLT, Matsumura CY, Neto HS, Marques MJ. Calcium-binding proteins in skeletal muscles of the mdx mice: potential role in the pathogenesis of Duchenne muscular dystrophy. Int J Exp Pathol. 2010;91:63–71. doi: 10.1111/j.1365-2613.2009.00688.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rando TA, Blau HM. Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. J Cell Biol. 1994;125:1275–1287. doi: 10.1083/jcb.125.6.1275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Robert V, Massimino ML, Tosello V, Marsault R, Cantini M, Sorrentino V, Pozzan T. Alteration in calcium handling at the subcellular level in mdx myotubes. J Biol Chem. 2001;276:4647–4651. doi: 10.1074/jbc.M006337200. [DOI] [PubMed] [Google Scholar]
  38. Rocha GL, Mizobuti DS, Silva HNM, Covatti C, Lourenço CC, Salvador MJ, Pereira EL, Minatel E. Multiple LEDT wavelengths modulate the Akt signaling pathways and attenuate pathological events in mdx dystrophic muscle cells. Photochem Photobiol Sci. 2022;21:1257–1272. doi: 10.1007/s43630-022-00216-0. [DOI] [PubMed] [Google Scholar]
  39. Salehpour F, Ahmadian N, Rasta SH, Farhoudi M, Karimi P, Sadigh-Eteghad S. Transcranial low-level laser therapy improves brain mitochondrial function and cognitive impairment in D-galactose–induced aging mice. Neurobiol Aging. 2017;58:140–150. doi: 10.1016/j.neurobiolaging.2017.06.025. [DOI] [PubMed] [Google Scholar]
  40. Salehpour F, Farajdokht F, Erfani M, Sadigh-Eteghad S, Shotorbani SS, Hamblin MR, Karimi P, Rasta SH, Mahmoudi J. Transcranial near-infrared photobiomodulation attenuates memory impairment and hippocampal oxidative stress in sleepdeprived mice. Brain Res. 2018;1682:36–43. doi: 10.1016/j.brainres.2017.12.040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Salehpour F, Farajdokht F, Mahmoudi J, Erfani M, Farhoudi M, Karimi P, Rasta SH, Sadigh-Eteghad S, Hamblin MR, Gjedde A. Photobiomodulation and coenzyme Q10 treatments attenuate cognitive impairment associated with model of transient global brain ischemia in artificially aged mice. Front Cell Neurosci. 2019;13:74. doi: 10.3389/fncel.2019.00074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Salehpour F, Mahmoudi J, Kamari F, Sadigh-Eteghad S, Rasta SH, Hamblin MR. Brain photobiomodulation therapy: a narrative review. Mol Neurobiol. 2018;55:6601–6636. doi: 10.1007/s12035-017-0852-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Shirokova N, Niggli E. Cardiac phenotype of Duchenne muscular dystrophy: Insights from cellular studies. J Mol Cell Cardiol. 2013;58:217–224. doi: 10.1016/j.yjmcc.2012.12.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Silva AAO, Leal-Junior EC, D’Avila KAL, Serra AJ, Albertini R, Franca CM, Nishida JA, Carvalho PTC. Pre-exercise low-level laser therapy improves performance and levels of oxidative stress markers in mdx mice subjected to muscle fatigue by high-intensity exercise. Lasers Med Sci. 2015;30:1719–1727. doi: 10.1007/s10103-015-1777-7. [DOI] [PubMed] [Google Scholar]
  45. Silva HNM, Covatti C, Rocha GL, Mizobuti DS, Mâncio RD, Hermes TA, Kido LA, Cagnon VHA, Pereira ECL, Minatel E. Oxidative stress, inflammation, and activators of mitochondrial biogenesis: tempol targets in the diaphragm muscle of exercise trained-mdx mice. Front Physiol. 2021;12:649793. doi: 10.3389/fphys.2021.649793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Spencer MJ, Croall DE, Tidball JG. Calpains are activated in necrotic fibers from mdx dystrophic mice. J Biol Chem. 1995;270:10909–10914. doi: 10.1074/jbc.270.18.10909. [DOI] [PubMed] [Google Scholar]
  47. Sundaram JS, Rao VM, Meena AK, Anandaraj MP. Altered expression, intracellular distribution and activity of lymphocyte calpain II in Duchenne muscular dystrophy. Clin Chim Acta. 2006;373:82–87. doi: 10.1016/j.cca.2006.05.004. [DOI] [PubMed] [Google Scholar]
  48. Tanihata J, Nagata T, Ito N, Saito T, Nakamura A, Minamisawa S, Aoki Y, Ruegg UT, Takeda S. Truncated dystrophin ameliorates the dystrophic phenotype of mdx mice by reducing sarcolipin-mediated SERCA inhibition. Biochem Biophys Res Commun. 2018;505:51–59. doi: 10.1016/j.bbrc.2018.09.039. [DOI] [PubMed] [Google Scholar]
  49. Tupling AR. The decay phase of Ca2+ transients in skeletal muscle: regulation and physiology. Appl Physiol Nutr Metab. 2009;34:373–376. doi: 10.1139/H09-033. [DOI] [PubMed] [Google Scholar]
  50. Tupling AR, Bombardier E, Gupta SC, Hussain D, Vigna C, Bloemberg D, Quadrilatero J, Trivieri MG, Babu GJ, Backx PH, Periasamy M, MacLennan DH, Gramolini AO. Enhanced Ca2 þ transport and muscle relaxation in skeletal muscle from sarcolipinnull mice. Am J Physiol Cell Physiol. 2011;301:C841–C849. doi: 10.1152/ajpcell.00409.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Valduga AH, Mizobuti DS, Moraes FSR, Mâncio RD, Moraes LHR, Hermes TA, Macedo AB, Minatel E. Protection of dystrophic muscle cells using idebenone correlates with the interplay between calcium, oxidative stress and inflammation. Int J Exp Pathol. 2023;104:4–12. doi: 10.1111/iep.12463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Vangheluwe P, Schuermans M, Zádor E, Waelkens E, Raeymaekers L, Wuytack F. Sarcolipin and phospholamban mRNA and protein expression in cardiac and skeletal muscle of different species. Biochem J. 2005;389:151–159. doi: 10.1042/BJ20050068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Voit A, Patel V, Pachon R, Shah V, Bakhutma M, Kohlbrenner E, McArdle JJ, Dell’Italia LJ, Mendekk JR, Xie L, Hajjar RJ, Duan D, Fraidenraich D, Babu GJ. Reducing sarcolipin expression mitigates Duchenne muscular dystrophy and associated cardiomyopathy in mice. Nat Commun. 2017;8:1068. doi: 10.1038/s41467-017-01146-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wu KD, Lytton J. Molecular cloning and quantification of sarcoplasmic reticulum Ca(2+)-ATPase isoforms in rat muscles. Am J Physiol Cell Physiol. 1993;264:C333–C341. doi: 10.1152/ajpcell.1993.264.2.C333. [DOI] [PubMed] [Google Scholar]

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