Physiological role for S-nitrosylation of RyR1 in skeletal muscle function and development

Excitation-contraction coupling in skeletal muscle myofibers depends upon Ca²⁺ release from the sarcoplasmic reticulum through the ryanodine receptor/Ca²⁺-release channel RyR1. The RyR1 contains ∼100 Cys thiols of which ∼30 comprise an allosteric network subject to posttranslational modification by S-nitrosylation, S-palmitoylation and S-oxidation. However, the role and function of these modifications is not understood. Although aberrant S-nitrosylation of multiple unidentified sites has been associated with dystrophic diseases, malignant hyperthermia and other myopathic syndromes, S-nitrosylation in physiological situations is reportedly specific to a single (1 of ∼100) Cys in RyR1, Cys³⁶³⁶ in a manner gated by pO₂. Using mice expressing a form of RyR1 with a Cys³⁶³⁶→Ala point mutation to prevent S-nitrosylation at this site, we showed that Cys³⁶³⁶ was the principal target of endogenous S-nitrosylation during normal muscle function. The absence of Cys³⁶³⁶ S-nitrosylation suppressed stimulus-evoked Ca²⁺ release at physiological pO₂ (at least in part by altering the regulation of RyR1 by Ca²⁺/calmodulin), eliminated pO₂ coupling, and diminished skeletal myocyte contractility in vitro and measures of muscle strength in vivo. Furthermore, we found that abrogation of Cys³⁶³⁶ S-nitrosylation resulted in a developmental defect reflected in diminished myofiber diameter, altered fiber subtypes, and altered expression of genes implicated in muscle development and atrophy. Thus, our findings establish a physiological role for pO₂-coupled S-nitrosylation of RyR1 in skeletal muscle contractility and development and provide foundation for future studies of RyR1 modifications in physiology and disease.