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Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle

Abstract

Duchenne muscular dystrophy is characterized by progressive muscle weakness and early death resulting from dystrophin deficiency. Loss of dystrophin results in disruption of a large dystrophin glycoprotein complex, leading to pathological calcium (Ca2+)-dependent signals that damage muscle cells1,2,3,4,5. We have identified a structural and functional defect in the ryanodine receptor (RyR1), a sarcoplasmic reticulum Ca2+ release channel, in the mdx mouse model of muscular dystrophy that contributes to altered Ca2+ homeostasis in dystrophic muscles. RyR1 isolated from mdx skeletal muscle showed an age-dependent increase in S-nitrosylation coincident with dystrophic changes in the muscle. RyR1 S-nitrosylation depleted the channel complex of FKBP12 (also known as calstabin-1, for calcium channel stabilizing binding protein), resulting in 'leaky' channels. Preventing calstabin-1 depletion from RyR1 with S107, a compound that binds the RyR1 channel and enhances the binding affinity of calstabin-1 to the nitrosylated channel, inhibited sarcoplasmic reticulum Ca2+ leak, reduced biochemical and histological evidence of muscle damage, improved muscle function and increased exercise performance in mdx mice. On the basis of these findings, we propose that sarcoplasmic reticulum Ca2+ leak via RyR1 due to S-nitrosylation of the channel and calstabin-1 depletion contributes to muscle weakness in muscular dystrophy, and that preventing the RyR1-mediated sarcoplasmic reticulum Ca2+ leak may provide a new therapeutic approach.

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Figure 1: RyR1 is S-nitrosylated and depleted of calstabin-1 in mdx mice.
Figure 2: iNOS immunoprecipitates and co-localizes with RyR1, and S-nitrosylation of RyR1 depletes the channel of calstabin-1.
Figure 3: S107 treatment prevents calstabin-1 depletion from the RyR1 complex, improves grip strength and reduces muscle damage.
Figure 4: S107 treatment decreases Ca2+ leak and increases muscle force and voluntary exercise in mdx mice.

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Acknowledgements

This work was supported in part by a grant from the Leducq Foundation. We thank J. Shan for assistance with analyses of histologic sections and J. Fauconnier for help with voluntary exercise measurements in mice.

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Contributions

A.M.B. conducted experiments and wrote the manuscript, S.R. performed biochemistry experiments, C.C. assisted with mouse experiments, M.M. performed immunohistochemistry, X.L. and L.R. assisted with histology, S.M. and A.L. performed muscle and calcium experiments, and A.R.M. conceived, designed and directed the project, analyzed data and wrote the final version of the manuscript.

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Correspondence to Andrew R Marks.

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Bellinger, A., Reiken, S., Carlson, C. et al. Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle. Nat Med 15, 325–330 (2009). https://doi.org/10.1038/nm.1916

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