Inhibition of ATP synthase reverse activity restores energy homeostasis in mitochondrial pathologies

Abstract

The maintenance of cellular function relies on the close regulation of adenosine triphosphate (ATP) synthesis and hydrolysis. ATP hydrolysis by mitochondrial ATP Synthase (CV) is induced by loss of proton motive force and inhibited by the mitochondrial protein ATPase inhibitor (ATPIF1). The extent of CV hydrolytic activity and its impact on cellular energetics remains unknown due to the lack of selective hydrolysis inhibitors of CV. We find that CV hydrolytic activity takes place in coupled intact mitochondria and is increased by respiratory chain defects. We identified (+)-Epicatechin as a selective inhibitor of ATP hydrolysis that binds CV while preventing the binding of ATPIF1. In cells with Complex-III deficiency, we show that inhibition of CV hydrolytic activity by (+)-Epichatechin is sufficient to restore ATP content without restoring respiratory function. Inhibition of CV-ATP hydrolysis in a mouse model of Duchenne Muscular Dystrophy is sufficient to improve muscle force without any increase in mitochondrial content. We conclude that the impact of compromised mitochondrial respiration can be lessened using hydrolysis-selective inhibitors of CV.

Keywords: ATP hydrolysis; ATPase Inhibitor (ATPIF1); Complex V; epicatechin; muscular dystrophy.

Figure 1. ATP synthesis and hydrolysis coexist in the same mitochondria and can be modulated independently (see also Fig EV1 and Dataset EV1)

1. A

   Scheme representing the forward (synthesis) and reverse (hydrolysis) mode of ATP synthase and the analysis that can be performed in fresh (intact) or previously frozen samples.

2. B

   Sequential measurements of respiration and ATP hydrolysis in freshly isolated heart mitochondria. The top (OCR) and bottom (Acidification) charts were recorded simultaneously from the same sample. ATP hydrolysis is measured using the acidification channel (bottom). Oxygen consumption rate (OCR) is measured first in the presence of ADP and fueled by pyruvate (Pyr) and malate (Mal) (Blue; State 3 respiration), ADP is phosphorylated, and oxygen is consumed. A control group received pyruvate and malate alone (red; State 4), showing OCR that were lower than State 3, demonstrating that mitochondria are coupled. Following the addition of ATP, OCR remains low, but hydrolysis measured by acidification is increased. Hydrolysis is further activated by FCCP‐induced depolarization and inhibited by oligomycin. The oligomycin‐insensitive portion of acidification is attributed to carbon dioxide‐induced respiration.

3. C

   OCR of heart mitochondria respiring using Pyr + Mal in State 4 (no ADP), State 3 (presence of ADP) or State 3 plus oligomycin (n ≥ 6).

4. D

   ATP hydrolysis measured as acidification rates in heart mitochondria respiring in State 4 (no ADP), State 3 (presence of ADP) or State 3 plus oligomycin using Pyr + Mal under conditions of coupled (in the presence of ATP) or uncoupled (in the presence of FCCP) (n = 4). Note that ATP hydrolysis in coupled mitochondria is at steady state at 20% of the maximal ATP hydrolytic capacity measured in uncoupled mitochondria.

5. (E–F)

   Screening for mutations that result in increased ATP hydrolysis by CV. (E) Representative SDS‐PAGE blots of ATPIF1, SDHB, ATP5A1, and Vinculin in control and mutant patient‐derived fibroblasts. Right panel shows mean of ATPIF1 bands relative intensity normalized per ATP5A1 and represented as % of control cells (n = 2). The colored histogram illustrates the mitochondrial element impacted by each mutation. Note that ATPIF1 expression in patient cells is lower than in cells from control subjects. (F) ATP hydrolytic capacity measured in control and mutant patient derived fibroblasts (n ≥ 3). Data are normalized by the maximal CII activity (SR respiration) of the samples. Colored histogram illustrates the mitochondrial element impacted by each mutation. Note that ATP hydrolytic capacities in cells from specific patients are higher than those found in control subjects.

6. G

   Identification of binding sites in the ATPIF1 binding groove on the surface of F1‐ATP synthase (1ohh, gray) using PELE. Image shows binding sites for Quercetin and the enantiomers, (‐)‐Catechin and (+)‐Epicatechin (top); (‐)‐Epicatechin versus (+)‐Epicatechin (middle) and (+)‐Epicatechin and ATPIF1 (bottom).

7. H

   Representative Seahorse profiles measuring ATP hydrolytic activity (millipH units, mpH) in isolated heart mitochondria in the presence of the indicated concentrations of EPI, using oligomycin (OLIGO) as an inhibitory control.

8. I

   EPI reduces maximal hydrolytic capacity of CV. Maximal ATP hydrolysis capacity measured in frozen heart mitochondria in the presence of EPI (100 nM) and using oligomycin (1 μM) as control (n ≥ 7).

9. J

   State 3 and maximal respiration driven by pyruvate + malate in isolated mitochondria from mouse heart. EPI was added to the respiration media at the indicated concentrations (n ≥ 3). Note that EPI does not affect maximal respiration but has a small effect on ATP synthesis‐dependent OCR.

Data information: For each biological replicate, technical replicates were averaged. Data represent average ± SEM. Unpaired t‐test versus Ctrl or untreated conditions shows statistical differences depicted by P‐value. Source data are available online for this figure.

Figure EV1. ATP hydrolysis can be measured in fresh and frozen mitochondria (linked to main Fig 1). Also, see Appendix Supplementary Methods

1. Representative acidification rate traces of heart mitochondria respiring with the indicated substrates either in State 4 (no ADP) or State 3 (plus ADP) fueled by Pyruvate + Malate.

2. State 3 and ATP hydrolytic activity in fresh heart mitochondria untreated or treated with proteinase K (PK) (n = 4).

3. Overview of the ATP hydrolysis assay (HyFS). Representative trace of OCR (top) of a sample measured with the substrates of interest. Representative acidification rate (ECAR channel) trace from the same assay (bottom).

4. Effects of increasing concentrations of EPI in maximal CI, CII and ATP hydrolytic activity measured in frozen mouse heart mitochondria (n = 4).

Data information: Each point represents a biological sample replicate. For each biological replicate, technical replicates were averaged. Data represent average ± SEM. Two‐way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by P‐value.

Figure 2. EPI inhibits ATP hydrolysis by direct binding to CV

1. CV in gel ATP hydrolytic activity in intact mitochondria incubated in the presence of the indicated concentrations of EPI. EPI was added to samples of isolated mouse heart mitochondria that were then lysed by freeze/thaw and digitonin. In gel ATP hydrolysis was allowed to proceed for 3 h (top). Coomassie staining is shown as loading control (bottom). Oligomycin (oligo) was used as a control for ATP synthesis and ATP hydrolysis.

2. Quantification of in gel ATP hydrolytic activity of panel A under the indicated EPI concentrations (n ≥ 5).

3. Representative blot (left) and quantification (right) showing CV assembly by BNGE in mouse heart mitochondria treated with EPI as in (A) (n ≥ 3).

4. CV in gel ATP hydrolytic activity in permeabilized mouse heart mitochondria treated with EPI. EPI and oligo were added after mitochondria were permeabilized. In gel activity is shown after 3 h incubation (top). Coomassie staining was used as loading control (bottom).

5. Quantification of CV in gel hydrolytic activity of panel (D) (n = 6).

6. CV in gel ATP hydrolytic activity in mouse heart mitochondria where EPI or oligo were added in the assay buffer after mitochondrial complexes and supercomplexes were separated by BNGE. CV in gel ATP hydrolytic activity is shown after 3 h incubation (top). Coomassie staining was used as loading control (bottom).

7. CV in gel ATP hydrolytic activity quantification of panel (F) (n = 5).

Data information: For each biological replicate, technical replicates were averaged. Data represent average ± SEM. (B) Two‐way ANOVA followed by Dunnett's multiple comparisons test and (E, G) unpaired t‐test show statistical differences depicted by P‐value. Source data are available online for this figure.

Figure 3. EPI competes with ATPIF1 on binding to CV (see also Fig EV2)

1. A–G

   Binding of purified recombinant ATPIF1‐GFP to CV. Mitochondrial membranes were disrupted by freeze–thaw and exposed to ATPIF1‐GFP at different ratios of mitochondrial (mt) protein/ATPIF1 purified protein. Binding was assessed in BNGE by quantifying ATPIF1‐GFP fluorescence. (A) BNGE assessed for ATPIF1‐GFP binding (top); CV hydrolytic activity (middle) and protein loading (bottom). (B) Quantification of in gel CV–ATP hydrolytic activity shown in panel (A). On the left‐hand side are the levels of CV–bound ATPIF1 and on the right‐hand side is CV–ATP hydrolytic activity normalized to the levels of fully assembled CV (n = 3). (C, D) Representative acidification rate profile showing the effects on CV–ATP hydrolytic activity (C) and its quantification (D) (n = 4). (E–G) Binding competition assay between ATPIF1‐GFP and EPI to CV. (E) BNGE assessed for ATPIF1‐GFP binding (top); CV hydrolytic activity (middle) and protein loading (bottom). (F, G) Quantification of gels shown in panel (E). (F) Quantification of ATPIF1 bound to monomeric CV under the indicated treatments (n = 3). (G) Quantification of in gel ATP hydrolytic activity under the indicated treatments (n = 3). Note that EPI reduces ATPIF1 binding while maintaining the same inhibitory effect on ATP hydrolysis.

2. H

   Plot illustrating the lack of correlation of ATPIF1 expression levels with EPI effect in inhibiting maximal CV–ATP hydrolytic activity. Each point represents a biological sample replica.

Data information: For each biological replicate, technical replicates were averaged. Data represent average ± SEM. (B, D, G) Two‐way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by P‐value. (F) Unpaired t‐test show statistical differences depicted by P‐value. (H) Simple linear regression including R ² and P‐value. Source data are available online for this figure.

Figure EV2. EPI binds to ATPIF1 pocket in CV (linked to main Fig 3)

1. A

   CV assembly in heart mitochondria incubated in the presence of different concentrations of ATPIF1‐GFP. CII (SDHA) was used as loading control.

2. B–D

   Representative Seahorse traces showing the effects of increasing concentrations of ATPIF1‐GFP added to mitochondria on maximal CI, CII and CIV (B, C) activity measured in frozen mouse heart mitochondria and its quantification (D) (n = 4).

3. E

   CV assembly in heart mitochondria incubated with ATPIF1, EPI or both. CII (SDHA) was used as loading control.

4. F

   Representative blots showing the competition assay between ATPIF1‐GFP and EPI for binding to CV tetramer (CVt) (left) and their quantification (right).

5. G

   Expression levels of ATP5A1 and ATPIF1 in the indicated bovine CV preparations.

6. H–J

   CV in gel ATP hydrolytic activity from purified bovine CV preparations under the indicated EPI and oligo concentrations; CV monomer (H) CV tetramer (I), and oligomer mix 2 (J). CVm: CV monomer; CVt: CV tetramer. CV in gel activity is measured after O/N incubation or 3h (J) (left blot) and after stopping the activity with 50% methanol (middle blot). Western blot for ATP5A1 was used as loading control (right blot).

7. K

   Expression levels of ATP5A1 and ATPIF1 in mouse tissue lysates. Vinculin is used as loading control.

Data information: For each biological replicate, technical replicates were averaged. Data represent average ± SEM.

Figure 4. In mitochondrial disease models, EPI inhibits ATP hydrolysis, increasing cellular ATP content without affecting respiration (see also Fig EV3)

1. A, B

   PLA of CV and ATPIF1 produces a red fluorescence signal where anti‐ATPIF1 and anti‐ATP5A1 antibodies are localized at a distance < 10 nm of each other. Anti‐TOMM20 (green) was used to reveal mitochondrial architecture. (A) Representative high‐resolution Airyscan confocal images showing control fibroblasts treated with EPI for 24 h. Maximum intensity projection is shown. Scale bars: 20 and 5 μm in zoomed image. Note that PLA reveals the spatial distribution of ATPIF1 binding along the mitochondrial length. (B) Analysis of ATPIF1‐CV complexes occurrence quantified as PLA dots per μm³of mitochondria. Effect of EPI is shown relative to untreated (n = 2 from > 50 replicates per n).

2. C

   Effect of EPI on CV ATP hydrolytic capacity in cells overexpressing either ATPIF1^WT or its continuously active form, ATPIF1^H49K. Hydrolytic capacity is measured by the acidification rates and is normalized to maximal oxygen consumption on succinate + rotenone (Max OCR on SR). Effect of EPI is shown as % of control cells, untreated with EPI. Cells were treated for 24 h with 50 nM EPI (n = 4). Note that in cells overexpressing ATPIF1, EPI treatment does not result in further inhibition of ATP hydrolysis.

3. D

   Effect of EPI on the mitochondrial matrix ATP pool (mtATP) under conditions where mitochondrial respiration is inhibited by Antimycin A (AA). mtATP content measured as fluorescence intensity per mitochondria area and shown as % of control untreated (n = 3). Control fibroblasts were incubated for 30 min with DMSO or EPI in different concentrations of AA. Mitochondrial area was quantified using MTG staining.

4. E

   mtATP content measured in human fibroblasts from control and mitochondrial disease patients, after 30 min treatments with increasing concentrations of EPI as indicated. Analysis shows the mean intensity of mtATP probe in MTG area normalized per % of correspondent untreated cells (n = 3).

5. F–H

   Effects of EPI treatment on fibroblasts with a CIII assembly defect (P1:BCS1L) and fibroblast with a CV ATP synthesis defect (ATP6). (F) Total cellular ATP content shown as % of control untreated cells. Cells were treated for 24 h (n = 3). (G) Cell proliferation rates shown as % change from 0 h (untreated) to 72 h after treatment (n = 3). (H) Basal OCR vs ECAR in Control, CIII‐ and CV‐deficient patient fibroblasts treated with and without 50 nM EPI (n = 3).

Data information: (A–H) In all cases, for each biological replicate, technical replicates were averaged. Data represent average ± SEM. Two‐way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by P‐value. (G) Unpaired t‐test versus untreated conditions shows statistical differences depicted by P‐value. Source data are available online for this figure.

Figure EV3. Screening of EPI effect and inhibition of ATP hydrolysis in mitochondrial disease models (linked to main Fig 4)

1. A

   Representative SDS‐PAGE blots for ATPIF1, HA, SDHB, ATP5A, and Vinculin showing the overexpression of ATPIF1‐WT and H49K in Control (Ctrl) and CIII‐deficient cells (P1:BCS1L).

2. B

   Representative confocal images showing mitochondrial localization of overexpressed ATPIF1‐WT and H49K in Ctrl and CIII‐deficient fibroblasts, labeled with anti‐HA (green) antibody and DAPI (nuclei – blue). Maximum intensity projection is shown. Scale bars: 20 μm.

3. C

   Basal respiration (left) and acidification rates (right) in control fibroblasts before and after the direct injection of the indicated concentrations of Antimycin A (AA) (n = 4).

4. D

   Basal respiration in control fibroblasts before and after the direct injection of the indicated concentrations of oligomycin.

5. E, F

   Membrane potential measured using TMRE (average intensity) in Mitotracker Green (mitochondria area) normalized per % of untreated control: (E) control fibroblasts incubated for 30 min with AA plus DMSO, 100 nM EPI or 1 μM Oligo (n = 3); and (F) control fibroblasts incubated for 30 min with AA plus increased concentrations of Oligomycin (Oligo) (n = 3).

6. G

   Basal respiration (OCR) in control fibroblasts before and after the direct injection of the indicated concentrations of EPI (n = 4).

7. H

   Total ATP content measured in fibroblasts from patients with different mutations in mitochondrial proteins by luciferase assay and normalized by % of luminescence from control cells (n = 3).

8. I

   Extracellular lactate levels measured as % of the untreated control from Control (Ctrl), CIII-(P1:BCS1L) and CV‐deficient (ATP6) fibroblasts treated with and without 50 nM EPI (n = 3). Control cells grown in galactose media (GAL) were included as a positive control for low levels of lactate.

Data information: In all cases, data represent average ± SEM. Two‐way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by P‐value.

Figure EV4. EPI replaces ATPIF1 to block ATP hydrolysis in mitochondrial disease models (linked to main Fig 5)

1. A

   Representative BNGE blots (top) and quantification (bottom) of CIII₂, CIII₂ + CIV showing the distribution of CIII complexes and supercomplexes in control and CIII‐deficient cells treated with and without 50 nM EPI for 24 h. Samples were immunoblotted with UQCRC2 antibody. SDHB was used as a loading control.

2. B

   Representative BNGE blots (top) and quantification (bottom) of CV monomer (CVm) supercomplex in control and CV‐deficient cells treated with and without 50 nM EPI for 24 h. Samples were immunoblotted with ATP5A antibody. SDHB was used as a loading control.

3. C, D

   (C) Representative BNGE blots and quantification (D) of ATPIF1 relative intensity normalized per amount of CV and represented as % of control untreated cells in fibroblasts (n = 3). SDHB was used as a loading control.

4. E

   Chart shows PLA dots/μm³ of mitochondria in fibroblasts normalized as % of Ctrl values (n = 2).

5. F

   Representative confocal images showing fibroblasts treated with EPI for 24 h, labeled with anti‐ATPIF1 and anti‐ATP5A1 (PLA in red) and anti‐TOMM20 (green) antibodies. Maximum intensity projection is shown. Scale bars: 20 and 5 μm.

Data information: In all cases, data represent average ± SEM. Two‐way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by P‐value.

Figure 5. EPI replaces and mimics ATPIF1 inhibition of ATP hydrolysis, and is most effective where ATPIF1 levels are low (see also Fig EV4)

1. A–D

   PLA of CV and ATPIF1 (red). Anti‐TOMM20 (green) was used to reveal mitochondrial architecture. (A) Representative confocal micrographs showing fibroblasts treated with EPI for 24 h. Maximum intensity projection is shown. Scale bars: 20 and 5 μm zoomed images. (B) Analysis of ATPIF1‐CV complexes occurrence quantified as PLA dots per μm³of mitochondria in patient fibroblasts normalized in % of Ctrl untreated (n = 2 from > 50 replicates per n). (C) Effect of EPI on ATPIF1‐CV complexes occurrence quantified as PLA dots per μm³ under the indicated conditions. Values are shown as relative to control untreated. (D) Correlation between maximal ATP hydrolytic capacity and CV‐bound ATPIF1. Correlation is shown for control and mutant fibroblasts treated with DMSO or EPI. Note that ATP hydrolysis is increased where the levels of CV‐bound ATPIF1 is decreased (BCS1L), and that in presence of EPI the biggest decrease in hydrolysis is observed in low ATPIF1 binding condition.

2. E–G

   Effect of EPI on CIII‐deficient (BCS1L) cells stable‐expressing ATPIF1^WT or ATPIF1^H49K. (E) CV ATP hydrolytic capacity measured by the acidification rate and normalized maximal respiration. Maximal respiration was determined using the OCR channel (Max OCR on SR). Effect of EPI is shown as % of control cells, untreated with EPI. Cells were treated for 24 h with 50 nM EPI (n ≥ 4). (F) mtATP content measured as fluorescence intensity per mitochondria area and shown as % of control untreated (n = 5). Mitochondrial area was quantified using MTG staining. Cells were treated for 30 min. (G) Total cellular ATP content shown as % of untreated cells. Cells were treated for 24 h (n ≥ 4).

Data information: In all cases, data represent average ± SEM. For each biological replicate, technical replicates were averaged. Two‐way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by P‐value. Source data are available online for this figure.

Figure 6. CV–ATP hydrolysis is increased in mdx mice, a model of Duchenne Muscular Dystrophy (see also Fig EV5)

1. A–D

   D2.mdx mice, harboring the mdx mutation in a DBA/2 J genetic background (D2.mdx) were subjected to exercise‐induced gastrocnemius injury by stimulating muscle contraction (eccentric injury). Levels of CV, ATPIF1 and maximal CV ATP hydrolytic capacity were measured. (A) Representative western blot image (left) and quantification (right) showing CV (ATP5A1) and CII (SDHA) levels in gastrocnemius homogenate in WT and mdx mice after 24 h of eccentric injury (inj). Vinculin was used as loading control. Note that only in mdx mice eccentric injury results in reduced CV content. (B) Maximal CV ATP hydrolytic capacity per CV content measured in gastrocnemius homogenates from WT and mdx mice 24 h after eccentric injury. Note that hydrolytic capacity per CV was increased in mdx mice subjected to injury. (C) Analysis of levels of the cleaved OPA1 in gastrocnemius homogenate in WT and mdx mice 24 h after eccentric injury. (D) Cytochrome c release in gastrocnemius supernatants of WT and mdx at 24 h post‐injury measured by ELISA. Note that induction of OPA1 cleavage and Cyt C release in response to injury occur only in the mdx mice. From (A–D), n = 4. For each biological replicate, technical replicates were averaged. Data represent average ± SEM. Two‐way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by P‐value.

Source data are available online for this figure.

Figure EV5. ATP hydrolysis is increased in mdx eccentric injury model 1 h postinjury (linked to main Fig 6)

1. Representative western blot (right) and quantification (left) showing CV (ATP5A1) and CII (SDHA) levels in gastrocnemius homogenate in WT and mdx mice 1 h after eccentric injury. Vinculin was used as loading control (n = 4).

2. Maximal ATP hydrolysis capacity per total CV measured in frozen gastrocnemius homogenate in WT and mdx mice after 1 h of eccentric injury.

3. Analysis of OPA1 levels and isoforms (right) and quantification (left) in gastrocnemius homogenate in WT and mdx mice after 1 h of eccentric injury.

4. Cytochrome c release in gastrocnemius supernatants of WT and mdx 1 h postinjury measured by western blot. From (A–D), n = 4.

Data information: For each biological replicate, technical replicates were averaged. Data represent average ± SEM. Two‐way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by P‐value.

Figure 7. Inhibition of ATP hydrolysis by EPI treatment partially restores muscle function in a mouse model of Duchenne's muscular dystrophy (DMD) (see also Fig EV6)

1. A–H

   D2.mdx mice were treated with EPI orally twice daily, with doses ranging from 0.5 to 15 mg/kg/day. After 13 days of treatment, mice were subjected to exercise‐induced gastrocnemius injury by stimulating muscle contraction and the effect of EPI on muscle force, CV ATP hydrolytic capacity, OPA1 cleavage and Cyt C release were measured. (A) Plantarflexor force quantification after eccentric injury. (B) Percentage of injury inflicted loss of force. (C) Maximal CV ATP hydrolytic capacity per CV content in gastrocnemius homogenates from mdx mice treated with vehicle or EPI measured 24 h postinjury. (D) Correlation between muscle force and maximal CV ATP hydrolytic capacity per CV content. Each experimental group is represented with different color. Note that increased CV ATP hydrolytic capacity correlates with reduced muscle force. (E) Levels of cleaved OPA1 in gastrocnemius homogenate from mdx mice subjected to the indicated combinations of injury and EPI treatment. (F) Cytochrome c release measured in supernatants from gastrocnemius muscle homogenates of mdx mice treated with vehicle or EPI 24 h post eccentric injury. (G) Detection and quantification of myofiber membrane integrity or damage following injury using Evans Blue dye (EBD) in gastrocnemius muscle from vehicle and EPI treated mdx mice. Damaged fiber membranes allow dye entry that appears as red color. Yellow asterisks indicate damage fibers (n ≥ 4). (H) Principal Component Analysis (PCA) of the effect of EPI treatment on the recovery from injury in gastrocnemius muscle of mdx mice. Note that EPI‐injured treated group cluster closer to the EPI or vehicle control group than to the vehicle‐injured group. From (A–F), n = 8.

Data information: For each biological replicate, technical replicates were averaged. Data represent average ± SEM. Two‐way ANOVA followed by Šídák's multiple comparisons test (C, E, F) and unpaired t‐tests (A, B, G) shows statistical differences depicted by P‐value. (D) Simple linear regression including P‐value. Source data are available online for this figure.

Figure EV6. In vivo and in vitro ATP hydrolysis inhibition by EPI in mdx mice and DMD cell lines (linked to main Fig 7)

1. Representative western blot showing CV (ATP5A1) and CII (SDHA) levels in gastrocnemius homogenate in mdx vehicle or EPI treated 24 h after eccentric injury (left). Quantification of the protein levels (right). Vinculin was used as loading control (n = 8).

2. Cytochrome c release in gastrocnemius supernatants of mdx vehicle or EPI treated 24 h after eccentric injury measured by western blot (n = 8). Quantification of the protein levels (right).Vinculin was used as loading control (n = 8).

3. Representative western blot showing CV (ATP5A1) and CII (SDHA) levels in DMD cell lines after 24 h treatment with 50 nM EPI (left). Quantification of the protein levels versus vinculin versus control untreated (right). Vinculin was used as loading control (n ≥ 3).

4. Maximal ATP hydrolytic capacity in myotubes normalized by CV levels after 24 h of treatment with vehicle or 50 nM EPI (n ≥ 3).

5. Cell membrane stability as measured by creatine kinase (CK) release in myotubes treated 24 h with vehicle or 50 nM EPI (n ≥ 3).

Data information: Each point represents a biological replicate. For each biological replicate, technical replicates were averaged. Data represent average ± SEM. Two‐way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by P‐value.