Published in:
01-12-2020 | Muscular Dystrophy | Research article
iNOS is not responsible for RyR1 S-nitrosylation in mdx mice with truncated dystrophin
Published in: BMC Musculoskeletal Disorders | Issue 1/2020
Login to get accessAbstract
Background
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. 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.
Methods
Three- 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.
Results
mdx 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.
Conclusion
Similar 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.