Redox Signalling by Nitric Oxide in Skeletal Muscle

描述（由申请人提供）：该项目的长期目标是阐明内源性一氧化氮（NO）对骨骼肌功能的控制。我们的分析表明，肌肉Ca2+通量是由肌浆网（SR）蛋白质中半胱氨酸残基的基于no的动态翻译后修饰（s -亚硝基化）调节的，这种修饰取决于肌肉氧张力（pO2），并且在基础条件下鉴定的ryanodine受体/Ca2+释放通道（RyR1）中的单个关键Cys是这种基于氧化还原的调节机制的中心位点。我们最近的研究结果表明，pO2的影响是由酶促氧传感器介导的，该传感器将不同的pO2转导为活性氧（ROS）的改变，活性氧（ROS）介导RyR1和sr其他蛋白质中调节性Cys硫醇的氧化修饰。我们的研究结果支持了兴奋-收缩耦合是由NO和ROS对肌肉pO2原位范围的共同影响自适应控制的观点。本研究的具体目的是：1)明确确定SR中O2感知的机制，特别是验证一个假设，即NADPH氧化酶的SR常驻异构体，将pO2的变化转导为ROS的改变，是传递pO2对RyR1 S-亚硝基化的影响所必需和充分的。2)鉴定RyR1内的半胱氨酸残基（也可能在肌浆网状Ca2+- atp酶内），这些残基由于pO2-感应酶的活性而被氧化修饰，以及氧化修饰的性质。3)评估pO2在体外（分离的SR和肌纤维）对RyR1功能的影响，以及对完整肌肉收缩力的影响，在小鼠制备的制剂中，鉴定的pO2感应Nox异构体已被基因删除，或从野生型小鼠中使用siRNA来敲除Nox。4)评估RyR1活性和肌肉收缩力，包括NO/pO2的影响，我们已经成功创建敲入小鼠。其中Cys3635（内源性NO的关键po2敏感靶标）被丙氨酸取代。这些目标的完成将为氧化还原调节肌肉功能的分子机制提供新的见解。此外，NO/ROS在肌肉生理学中的重要性表明，阐明它们的联合作用可能有助于我们理解一系列疾病状态的病理生理学，包括膈功能障碍、恶性低体温和中暑，其中RyR起着核心作用，以及肌肉萎缩症，其中NO的产生失调。更广泛地说，我们的实验计划应该首次确定体内氧感应的特定酶机制，该机制调节蛋白质Cys残基的po2偶联亚硝基化修饰，例如RyR1的Cys 3635在骨骼肌中。公共卫生相关性。我们的研究表明，气态分子一氧化氮在骨骼肌中产生并调节肌肉功能，而这种调节的主要位点是ryanodine受体/钙释放通道（RyR），这是驱动肌肉收缩的细胞内钙通量的主要来源。尽管分子细节尚不完全清楚，但我们已经发现肌肉氧水平控制着一氧化氮执行这种调节功能的能力。由于肌肉萎缩症通常涉及肌肉一氧化氮产生的中断，并且由于RyR功能的中断可导致许多肌肉疾病和诸如中暑等问题，因此更充分的一氧化氮和氧气水平对肌肉功能相互作用影响的模型可能会导致更好地理解一系列临床状况。

DESCRIPTION (provided by applicant): 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.