Redox Signalling by Nitric Oxide in Skeletal Muscle

描述（由申请人提供）：本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+流量是由NO为基础的，动态的翻译后修饰调制肌浆网（SR）蛋白质内半胱氨酸残基的（S-亚硝基化），该修饰取决于肌肉氧张力（pO2），以及在基础条件下鉴定的兰尼碱受体/Ca2+释放通道（RyR1）内的单个关键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的影响是由酶氧传感器介导的，该传感器将不同的pO2转换为活性氧（ROS）的产生改变，其介导RyR1和可能SR的其他蛋白质内的调节性Cys硫醇的氧化修饰。我们的结果支持以下命题：收缩偶联通过NO和ROS在肌肉pO2的原位范围内的联合影响而自适应地控制。该提议的具体目的是：1）明确鉴定SR内的O2传感机制，特别是检验以下假设：将pO2的变化转换为ROS产生改变的NADPH氧化酶的SR驻留同种型对于传达pO2对RyR1的S-亚硝基化的影响是必要的和足够的。2)鉴定RyR1内（以及肌浆网Ca2 +-ATP酶内）的半胱氨酸残基，这些半胱氨酸残基在pO2敏感酶活性后被氧化修饰，以及氧化修饰的性质。3)评估pO2对体外RyR1功能（分离的SR和肌纤维）以及对完整肌肉收缩性的影响，这些影响来自已鉴定的pO2敏感型Nox同种型已被基因删除的小鼠或已使用siRNA敲低该Nox的野生型小鼠。4)为了评估RyR1活性和肌肉收缩性，包括NO/pO2的影响，在我们成功创建的基因敲入小鼠中，Cys3635（内源性NO的关键pO2敏感靶点）已被丙氨酸取代。这些目标的完成将提供新的见解氧化还原调节肌肉功能的分子机制。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对一系列疾病状态的病理生理学的理解，这些疾病状态包括脑功能障碍、恶性体温过低和中暑，其中RyR起着核心作用，以及NO产生失调的肌营养不良症。更一般地说，我们的实验计划应首次确定具体的酶促机制subserving在体内的氧传感，调节pO2-偶联亚硝基修饰的蛋白质Cys残基，例如在骨骼肌的RyR1的Cys 3635。 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，并且该调节的主要位点是兰尼碱受体/钙释放通道（RyR），其是驱动肌肉收缩的细胞内钙通量的主要来源。虽然分子细节还不完全清楚，但我们已经发现肌肉氧水平控制一氧化氮执行这种调节功能的能力。由于肌营养不良症往往涉及破坏生产的一氧化氮的肌肉，因为破坏功能的RyR可以有助于一些肌肉疾病和问题，如中暑，一个更充分的模型的相互作用的影响，一氧化氮和氧气水平对肌肉功能可能会导致更好地了解一系列的临床条件。

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.