Abstract

BackgroundPrevious research indicated that nitric oxide synthase (NOS) is the key molecule for S-nitrosylation of ryanodine receptor 1 (RyR1) in DMD model mice (mdx mice) and that both neuronal NOS (nNOS) and inducible NOS (iNOS) might contribute to the reaction because nNOS is mislocalized in the cytoplasm and iNOS expression is higher in mdx mice. We investigated the effect of iNOS on RyR1 S-nitrosylation in mdx mice and whether transgenic expression of truncated dystrophin reduced iNOS expression in mdx mice or not.MethodsThree- to 4-month-old C57BL/6 J, mdx, and transgenic mdx mice expressing exon 45–55-deleted human dystrophin (Tg/mdx mice) were used. We also generated two double mutant mice, mdx iNOS KO and Tg/mdx iNOS KO to reveal the iNOS contribution to RyR1 S-nitrosylation. nNOS and iNOS expression levels in skeletal muscle of these mice were assessed by immunohistochemistry (IHC), qRT-PCR, and Western blotting. Total NOS activity was measured by a citrulline assay. A biotin-switch method was used for detection of RyR1 S-nitrosylation. Statistical differences were assessed by one-way ANOVA with Tukey-Kramer post-hoc analysis.Resultsmdx and mdx iNOS KO mice showed the same level of RyR1 S-nitrosylation. Total NOS activity was not changed in mdx iNOS KO mice compared with mdx mice. iNOS expression was undetectable in Tg/mdx mice expressing exon 45–55-deleted human dystrophin, but the level of RyR1 S-nitrosylation was the same in mdx and Tg/mdx mice.ConclusionSimilar levels of RyR1 S-nitrosylation and total NOS activity in mdx and mdx iNOS KO demonstrated that the proportion of iNOS in total NOS activity was low, even in mdx mice. Exon 45–55-deleted dystrophin reduced the expression level of iNOS, but it did not correct the RyR1 S-nitrosylation. These results indicate that iNOS was not involved in RyR1 S-nitrosylation in mdx and Tg/mdx mice muscles.

Highlights

  • Previous research indicated that nitric oxide synthase (NOS) is the key molecule for S-nitrosylation of ryanodine receptor 1 (RyR1) in Duchenne muscular dystrophy (DMD) model mice and that both neuronal NOS and inducible NOS might contribute to the reaction because nNOS is mislocalized in the cytoplasm and iNOS expression is higher in mdx mice

  • We revealed that mdx and mdx iNOS KO mice showed the same level of RyR1 S-nitrosylation

  • Tg/mdx and Tg/mdx iNOS KO mice showed nNOS mislocalization because mRNA and protein expression of iNOS was suppressed in Tg/mdx mice and weak even in mdx mice we assessed the mRNA expression of iNOS by quantitative RT-PCR (qRT-PCR)

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Summary

Introduction

Previous research indicated that nitric oxide synthase (NOS) is the key molecule for S-nitrosylation of ryanodine receptor 1 (RyR1) in DMD model mice (mdx mice) and that both neuronal NOS (nNOS) and inducible NOS (iNOS) might contribute to the reaction because nNOS is mislocalized in the cytoplasm and iNOS expression is higher in mdx mice. Becker muscular dystrophy (BMD), in which the reading frame in the DMD gene is not altered, is similar to DMD, but the progression of symptoms is slower and less severe than DMD because BMD patients have truncated but partially functional dystrophin [2]. Intracellular Ca2+ overload leads to muscle contracture, mitochondrial dysfunction, and activation of proteases. These are the key factors of muscle degeneration and necrosis in DMD [3]. Bellinger et al showed that RyR1 is S-nitrosylated in mdx muscle and that inducible NOS (iNOS) plays an important role in this reaction [4]

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