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. 2014 Jun 3;19(6):1034-41.
doi: 10.1016/j.cmet.2014.04.002. Epub 2014 May 8.

Pharmacological Inhibition of poly(ADP-ribose) Polymerases Improves Fitness and Mitochondrial Function in Skeletal Muscle

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Free PMC article

Pharmacological Inhibition of poly(ADP-ribose) Polymerases Improves Fitness and Mitochondrial Function in Skeletal Muscle

Eija Pirinen et al. Cell Metab. .
Free PMC article

Abstract

We previously demonstrated that the deletion of the poly(ADP-ribose)polymerase (Parp)-1 gene in mice enhances oxidative metabolism, thereby protecting against diet-induced obesity. However, the therapeutic use of PARP inhibitors to enhance mitochondrial function remains to be explored. Here, we show tight negative correlation between Parp-1 expression and energy expenditure in heterogeneous mouse populations, indicating that variations in PARP-1 activity have an impact on metabolic homeostasis. Notably, these genetic correlations can be translated into pharmacological applications. Long-term treatment with PARP inhibitors enhances fitness in mice by increasing the abundance of mitochondrial respiratory complexes and boosting mitochondrial respiratory capacity. Furthermore, PARP inhibitors reverse mitochondrial defects in primary myotubes of obese humans and attenuate genetic defects of mitochondrial metabolism in human fibroblasts and C. elegans. Overall, our work validates in worm, mouse, and human models that PARP inhibition may be used to treat both genetic and acquired muscle dysfunction linked to defective mitochondrial function.

Figures

Figure 1
Figure 1. Paribs protect from HFD-induced metabolic complications
Ten-wk-old male C57Bl/6J mice were challenged with HFD supplemented with either vehicle (DMSO; Veh) or MRL-45696 (50 mg/kg/day) (n=10/group). (A) Body weight gain during 18wks of HFD. (B) Fat mass was measured using Echo-MRI. (C–D) A comprehensive laboratory animal monitoring system was used to evaluate VO2 (C) and activity (D) after 7wks of HFD. (E) Food intake measured by averaging weekly food consumption during HFD. (F) Liver 8-oxo-dG content, as indicator of DNA damage, (G) muscle lipid peroxidation-derived aldehyde, 4-hydroxy-2-nonenal, and (H) muscle total poly(ADP-ribose) (PAR) contents were measured in vehicle and MRL-45696-treated mice (n=7/group) (I–J) Total intracellular (I) and mitochondrial (J) NAD+ levels in gastrocnemius of refed vehicle and MRL-45696-treated mice (n=5–10/group). (K) SIRT1, acetylated FOXO1 and total FOXO1 protein levels were assessed in total homogenates from quadriceps of chow-diet fed mice. (L) The acetylation status of Ndufa9 immunoprecipates was tested as a marker of SIRT3 activity. Values are shown as mean+/−SEM. * indicates statistical significant difference vs. respective Veh group. *, p<0.05; **, p<0.01; ***, p<0.001. This figure is complemented by Figures S1 and S2, and Table S1.
Figure 2
Figure 2. Paribs enhance exercise capacity and muscle mitochondrial function
Chow fed male C57BL/6J and congenic SIRT1skm−/− mice (n=5–10/group) treated with either vehicle (DMSO; Veh) and/or MRL-45696 (50 mg/kg/day) were subjected to (A) endurance treadmill test after 13wks treatments, (B) respirometry analysis of permeabilized EDL muscle fibers (CI, complex I; CII, complex II; ETS, electron transport system) and (C) CS activity measurement. (D) Oleic acid oxidation rate in the muscles of Veh and MRL-45696-treated mice after 18wks treatment (n=5–7/group). (E) Mitochondria DNA abundance in quadriceps of Veh and MRL-45696-treated mice (n=8/group). Results are expressed as mitochondrial DNA amount (16S) relative to genomic DNA (UCP2). (F) Cytochrome c oxidase (COX), succinate dehydrogenase (SDH) staining in gastrocnemius of Veh and MRL-45696-treated mice. Soleus is indicated by an arrow. (G) Protein levels of MHCI and IIb were evaluated, using heat shock protein 90 (Hsp90) as loading control (H) Myosin heavy chain I staining (MHCI) of gastrocnemius of Veh and MRL-45696-treated mice. Values are shown as mean+/−SEM. * indicates statistical significant difference vs. respective Veh group. *, p<0.05; **, p<0.01; ***, p<0.001. This figure is complemented by Figure S3.
Figure 3
Figure 3. Paribs induce UPRmt in muscle by increasing mitochondrial protein translation
(A) Blue-Native Page using isolated mitochondria from quadriceps of vehicle (DMSO; Veh) and MRL-45696 treated mice. A non-specific band was used as loading control. (B) Mitochondrial and (C) cytosolic protein translation measured in MEFs after 100nM MRL-45696 treatment for 40hr. (D) Quantification of protein translation rates. (E) MTCOI, SDHA, ClpP and Hsp60 protein levels were evaluated in quadriceps from Veh and MRL-45696 mice, using tubulin as a loading control. (F) Quantification of mitonuclear and UPRmt markers. *, p<0.05 and ***<0.001. This figure is complemented by Figure S4.
Figure 4
Figure 4. Parp activity negatively correlates with energy expenditure in mouse populations and its inhibition improves mitochondrial function in worm and human models of mitochondria dysfunction
(A) Expression of Parp-1 in the quadriceps of 37 strains of BXD mice. Each bar represents mRNA from a pool of ~5 animals per strain. Extreme strains and the parental strains are labelled. (B) Top: Correlation between quadriceps muscle Parp-1 expression and phenotypes collected from BXD mice. Night VO2 was measured though indirect calorimetry. VO2 improvement represents the increase in VO2max after 10 days of voluntary exercise. Bottom: Correlation between quadriceps muscle Parp-1 and muscle fiber type genes in the same dataset. (C) Total PAR content at day 2 of adulthood (n=~500 worms/sample) in mev-1(kn1) complex II-deficient C. elegans after water and 100nM MRL-45696 treatment. (D) Respiration at day 3 (n=10 worms/well, 19 wells/group) and (E) movement at day 1, 3 and 5 of adulthood (n=37–90 worms/group). (F) H2O2 (500µM)-induced PARylation after 48hr pretreatment with either vehicle (Veh) or 100nM MRL-45696 in NDUFS1 mutant human fibroblasts. (G–J) NAD+ levels (G), O2 consumption rates (H), oleic acid oxidation (I) and CS activity (J) in human NDUFS1 mutant fibroblasts after Veh and 100nM MRL-45696 for 48hr (n=3–12 samples/group). (K) H2O2 (500µM)-induced PAR content after 24hr pretreatment with either Veh or 10nM MRL-45696 in human primary myotubes. (L–O) NAD+ levels (L), O2 consumption rates (M), oleic acid oxidation (N) and CS activity (O) in human primary myotubes after Veh or 10nM MRL-45696 treatment for 72hr (n=6–12 samples/group). * indicates statistical significant difference vs. respective Veh group. *, p<0.05; **, p<0.01; ***, p<0.001.

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