Up-regulation of the peroxiredoxin-6 related metabolism of reactive oxygen species in skeletal muscle of mice lacking neuronal nitric oxide synthase

Da Silva-Azevedo, Luis; Jähne, Sebastian; Hoffmann, Christian; Stalder, Daniel; Heller, Manfred; Pries, Axel R; Zakrzewicz, Andreas; Baum, Oliver (2009). Up-regulation of the peroxiredoxin-6 related metabolism of reactive oxygen species in skeletal muscle of mice lacking neuronal nitric oxide synthase. Journal of physiology, 587(Pt 3), pp. 655-68. Oxford: Wiley-Blackwell 10.1113/jphysiol.2008.164947

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Although neuronal nitric oxide synthase (nNOS) plays a substantial role in skeletal muscle physiology, nNOS-knockout mice manifest an only mild phenotypic malfunction in this tissue. To identify proteins that might be involved in adaptive responses in skeletal muscle of knockout mice lacking nNOS, 2D-PAGE with silver-staining and subsequent tandem mass spectrometry (LC-MS/MS) was performed using extracts of extensor digitorum longus muscle (EDL) derived from nNOS-knockout mice in comparison to C57Bl/6 control mice. Six proteins were significantly (P < or = 0.05) more highly expressed in EDL of nNOS-knockout mice than in that of C57 control mice, all of which are involved in the metabolism of reactive oxygen species (ROS). These included prohibitin (2.0-fold increase), peroxiredoxin-3 (1.9-fold increase), Cu(2+)/Zn(2+)-dependent superoxide dismutase (SOD; 1.9-fold increase), heat shock protein beta-1 (HSP25; 1.7-fold increase) and nucleoside diphosphate kinase B (2.6-fold increase). A significantly higher expression (4.1-fold increase) and a pI shift from 6.5 to 5.9 of peroxiredoxin-6 in the EDL of nNOS-knockout mice were confirmed by quantitative immunoblotting. The concentrations of the mRNA encoding five of these proteins (the exception being prohibitin) were likewise significantly (P < or = 0.05) higher in the EDL of nNOS-knockout mice. A higher intrinsic hydrogen peroxidase activity (P < or = 0.05) was demonstrated in EDL of nNOS-knockout mice than C57 control mice, which was related to the presence of peroxiredoxin-6. The treatment of mice with the chemical NOS inhibitor L-NAME for 3 days induced a significant 3.4-fold up-regulation of peroxiredoxin-6 in the EDL of C57 control mice (P < or = 0.05), but did not alter its expression in EDL of nNOS-knockout mice. ESR spectrometry demonstrated the levels of superoxide to be 2.5-times higher (P < or = 0.05) in EDL of nNOS-knockout mice than in C57 control mice while an in vitro assay based on the emission of 2,7-dichlorofluorescein fluorescence disclosed the concentration of ROS to be similar in both strains of mice. We suggest that the up-regulation of proteins that are implicated in the metabolism of ROS, particularly of peroxiredoxin-6, within skeletal muscles of nNOS-knockout mice functionally compensates for the absence of nNOS in scavenging of superoxide.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > Unit Childrens Hospital > Protein- und Zellbiologie
04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Anatomy > Functional Anatomy

UniBE Contributor:

Heller, Manfred and Baum, Oliver

ISSN:

0022-3751

Publisher:

Wiley-Blackwell

Language:

English

Submitter:

Factscience Import

Date Deposited:

04 Oct 2013 15:10

Last Modified:

13 Jan 2016 16:30

Publisher DOI:

10.1113/jphysiol.2008.164947

PubMed ID:

19047200

Web of Science ID:

000262944900015

URI:

https://boris.unibe.ch/id/eprint/31010 (FactScience: 195391)

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