Korean J Physiol Pharmacol.  2016 Mar;20(2):201-211. 10.4196/kjpp.2016.20.2.201.

NecroX-5 protects mitochondrial oxidative phosphorylation capacity and preserves PGC1alpha expression levels during hypoxia/reoxygenation injury

• Thu VT
• Kim HK
• Long LT
• Nyamaa B
• Song IS
• Thuy TT
• Huy NQ
• Marquez J
• Kim SH
• Kim N
• Ko KS
• Rhee BD
• Han J

• KMID: 2388677
• DOI: http://doi.org/10.4196/kjpp.2016.20.2.201

Abstract

Although the antioxidant and cardioprotective effects of NecroX-5 on various in vitro and in vivo models have been demonstrated, the action of this compound on the mitochondrial oxidative phosphorylation system remains unclear. Here we verify the role of NecroX-5 in protecting mitochondrial oxidative phosphorylation capacity during hypoxia-reoxygenation (HR). Necrox-5 treatment (10 microM) and non-treatment were employed on isolated rat hearts during hypoxia/reoxygenation treatment using an ex vivo Langendorff system. Proteomic analysis was performed using liquid chromatography-mass spectrometry (LC-MS) and non-labeling peptide count protein quantification. Real-time PCR, western blot, citrate synthases and mitochondrial complex activity assays were then performed to assess heart function. Treatment with NecroX-5 during hypoxia significantly preserved electron transport chain proteins involved in oxidative phosphorylation and metabolic functions. NecroX-5 also improved mitochondrial complex I, II, and V function. Additionally, markedly higher peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC1alpha) expression levels were observed in NecroX-5-treated rat hearts. These novel results provide convincing evidence for the role of NecroX-5 in protecting mitochondrial oxidative phosphorylation capacity and in preserving PGC1alpha during cardiac HR injuries.

Keyword

Hypoxia; Mitochondria; NecroX; Oxidative phosphorylation; PCG1alpha

MeSH Terms

Animals
Anoxia
Blotting, Western
Citric Acid
Electron Transport
Heart
Mitochondria
Oxidative Phosphorylation*
Peroxisomes
Rats
Real-Time Polymerase Chain Reaction
Spectrum Analysis
Citric Acid

Figure

• 

  Fig. 1 Functional groups and protein-protein interactions of proteins identified in control, HR, and NecroX-5 treated rat hearts.(A) A Venn diagram illustrates all possible logical relationships between proteins identified in control, HR, and NecroX-5 rat hearts. (B) A protein-protein interaction network built on identified proteins illustrates their proposed interactions in various cellular processes. (C) Functional protein groups were determined by interactive merging of initially defined groups based on the number or percentage of genes provided per term. (D) Identified proteins are presented based on their molecular functions in up- and down-regulated biological processes (Bonferonni corrected p-values).

• 

  Fig. 2 Quantitative real-time-PCR validation of LC-MS-mediated protein identification.A histogram shows the ratio of summarized representative protein and mRNA expression levels between HR and NecroX-5 treated hearts. n=3~5 for each group, †p<0.05. Ndufv2: NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial; Idh3a: Isocitrate dehydrogenase [NAD] subunit alpha, mitochondrial; Ndufa1: NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 1; Ndufb8: NADH dehydrogenase (Ubiquinone) 1 beta subcomplex 8; Idh1: Isocitrate dehydrogenase [NADP] cytoplasmic; Hyou1: hypoxia upregulated 1; Ak2: Isoform 1 of Adenylate kinase 2, mitochondrial; Lum: Lumican; Cabc1: Chaperone activity of bc1 complex-like, mitochondrial; Nexn: Nexilin.

• 

  Fig. 3 PGC1α mRNA and protein expression levels in control, HR, and NecroX-5 treated hearts.Real-time PCR and Western blot were performed to assess mRNA expression (A) and protein expression (B) of PGC1α normalized to α-tubulin in rat hearts. Cardiac tissue was selected from untreated HR hearts and HR hearts treated with 10 µM NecroX-5. n=3~6 for each group; *p<0.05 vs. control, †p<0.05 vs. HR.

• 

  Fig. 4 Mitochondrial complex and citrate synthase activities in HR and NecroX-5 treated hearts.Graphs illustrated the activity of mitochondrial complexes I~V (mOD/min) with the relative Vmax curves of complex I~V activity (OD) (A~E, upper panels) and mitochondrial citrate synthase activity (F) in untreated HR hearts and NecroX-5 treated HR hearts. n=4~5 for each groups; *p<0.05.

• 

  Fig. 5 Proposed function of NecroX-5 during HR injury.ETC, electron transport chain; HR, hypoxia/reoxygenation.

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