Up‐regulation of enzymes involved in reactive oxygen species (ROS) metabolism in skeletal muscle of mice lacking neuronal nitric oxide synthase (nNOS)

Abstract

nNOS knockout mice do not show any phenotypic malfunctions, thus we hypothesized that cellular mechanisms exist which compensate for nNOS deficiency. In order to identify molecular compensatory mechanisms as well as to gain new insights into the function of nNOS in skeletal muscle, we searched for proteins differentially expressed in extensor digitorum longus muscle (EDL) of nNOS knockout mice compared to C57/Bl6 wild type mice, by two‐dimensional polyacrylamide electrophoresis (2D‐PAGE) and immunoblot. About 800 spots were detected on silver‐stained gels after densitometric analysis. Analysis of silver‐stained gels showed one protein with Mr of 24.5 kDa and pI of 5.9 present in EDL of nNOS−/− mice but not of C57 mice. This protein was identified as peroxiredoxin‐6 (PRX6) by LC‐MS/MS and immunoblot. Five other proteins were found to be present in higher amounts in EDL of nNOS‐knockout mice: peroxiredoxin‐3 (PRX3), prohibitin, superoxide dismutase, heat shock protein beta‐1 and nucleoside diphosphate kinase A. Up‐regulation of PRX6, PRX3 and heat shock protein beta‐1 was also observed at the mRNA level. The in vitro production rate of total ROS was significantly higher in EDL of nNOS knockout mice. Furthermore, treatment of C57 mice with the NOS inhibitor L‐NAME induced PRX6 in EDL. Induction of PRX6 in nNOS−/− mice could have a protective role in regulating levels of H202, 02‐ as well as oxidised phosholipids.