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.

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

A
CV assembly in heart mitochondria incubated in the presence of different concentrations of ATPIF1-GFP. CII (SDHA) was used as loading control. 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). E CV assembly in heart mitochondria incubated with ATPIF1, EPI or both. CII (SDHA) was used as loading control. F Representative blots showing the competition assay between ATPIF1-GFP and EPI for binding to CV tetramer (CVt) (left) and their quantification (right). G Expression levels of ATP5A1 and ATPIF1 in the indicated bovine CV preparations. 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). 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 AE SEM. Figure EV3. Screening of EPI effect and inhibition of ATP hydrolysis in mitochondrial disease models (linked to main Fig 4). Data information: In all cases, data represent average AE SEM. Two-way ANOVA followed by S ıd ak'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).
A Representative BNGE blots (top) and quantification (bottom) of CIII 2 , CIII 2 + 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. 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. 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. E Chart shows PLA dots/lm 3 of mitochondria in fibroblasts normalized as % of Ctrl values (n = 2). 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 lm.
Data information: In all cases, data represent average AE SEM. Two-way ANOVA followed by S ıd ak's multiple comparisons test shows statistical differences depicted by P-value. Figure EV6. In vivo and in vitro ATP hydrolysis inhibition by EPI in mdx mice and DMD cell lines (linked to main Fig 7).
A 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). B 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). C 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). D Maximal ATP hydrolytic capacity in myotubes normalized by CV levels after 24 h of treatment with vehicle or 50 nM EPI (n ≥ 3). E 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 AE SEM. Two-way ANOVA followed by S ıd ak's multiple comparisons test shows statistical differences depicted by P-value.