The long-term objective of this project is to elucidate the control of skeletal muscle function by endogenous nitric oxide (NO). Our analysis has revealed that muscle Ca2+ flux is modulated by NO-based, dynamic post- translational modification (S-nitrosylation) of cysteine residues within proteins of the sarcoplasmic reticulum (SR), that modification is conditional upon muscle oxygen tension (pO2), and that a single critical Cys within the ryanodine receptor/Ca2+-release channel (RyR1), as identified under basal conditions, is a central locus of this redox-based regulatory mechanism. Our more recent findings suggest that the influence of pO2 is mediated by an enzymatic oxygen sensor that transduces varying pO2 into altered production of reactive oxygen species (ROS), which mediate oxidative modification of regulatory Cys thiols within RyR1 and perhaps other proteins of the SR. Our results support the proposition that excitation-contraction coupling is adaptively controlled by the conjoint influence of NO and ROS over the in situ range of muscle pO2.
The specific aims of this proposal are: 1) To identify definitively the mechanism of O2 sensing within the SR, and in particular to test the hypothesis that an SR-resident isoform of NADPH oxidase, which transduces changes in pO2 into altered production of ROS, is necessary and sufficient to convey the effects of pO2 on the S- nitrosylation of RyR1. 2) To identify the cysteine residues within the RyR1 (and also, potentially, within the sarcoplasmic reticular Ca2+-ATPase) that are oxidatively modified consequent upon activity of the pO2- sensing enzyme, as well as the nature of the oxidative modification(s). 3) To assess the effects of pO2 on RyR1 function in vitro (isolated SR and myofibers) and on contractility of intact muscle, in preparations derived from mice in which the identified pO2-sensing Nox isoform has been deleted genetically, or from wild- type mice where siRNA has been employed to knock down that Nox. 4) To evaluate RyR1 activity and muscle contractility, including the effects of NO/pO2, in knock-in mice that we have successfully created, in which Cys3635 (the critical pO2-sensitive target of endogenous NO) has been replaced with alanine. Completion of these aims will provide novel insight into the molecular mechanisms of redox regulation of muscle function. Further, the emerging importance of NO/ROS in muscle physiology indicates that elucidating their conjunct actions may contribute significantly to our understanding of the pathophysiology of a range of disease states including diaphragmatic dysfunction, malignant hypothermia, and heat stroke, in which the RyR plays a central role, as well as muscular dystrophies in which NO production is dysregulated. More generally, our experimental program should for the first time identify the specific enzymatic mechanisms subserving oxygen sensing in vivo that regulates pO2-coupled nitrosylative modification of protein Cys residues, exemplified in skeletal muscle by Cys 3635 of RyR1.

Public Health Relevance

. Our research has shown that the gaseous molecule nitric oxide is produced in skeletal muscle and regulates muscle function, and that a principal locus of that regulation is the ryanodine receptor/calcium-release channel (RyR), which is the principal source of the intracellular calcium flux that drives muscle contraction. Although the molecular details are incompletely understood, we have found that muscle oxygen levels control the ability of nitric oxide to carry out this regulatory function. Because muscular dystrophies often involve disrupted production of nitric oxide by muscle, and because disrupted function of the RyR can contribute to a number of muscle diseases and to problems such as heat stroke, a more adequate model of the interacting influence of nitric oxide and oxygen levels on muscle function may lead to better understanding of a range of clinical conditions.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Skeletal Muscle and Exercise Physiology Study Section (SMEP)
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Smith, Robert A
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Case Western Reserve University
Internal Medicine/Medicine
Schools of Medicine
United States
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