SR Calcium Release Channel Regulation in Aging Muscle

描述（由申请人提供）：骨骼肌功能下降是老年人活动性和独立性下降的主要原因。此外，肌无力也是导致福尔斯跌倒造成严重损伤的一个重要因素。因此，尽管骨骼肌无力是一个主要的公共卫生问题，但衰老中骨骼肌功能障碍的分子机制还没有得到很好的理解。 这项工作的长期目标是阐明衰老引起的骨骼肌无力的机制。肌肉收缩由Ca+经由SR Ca+释放通道/ryanodane受体（RyR 1）从肌浆网（SR）流出而引发。老年人肌肉中SR Ca+释放受到抑制。因此，不完全的收缩激活有助于肌肉无力。作为主要的SR Ca+释放途径，RyR 1功能障碍可能是衰老肌肉中Ca+释放受损的原因。 RyR 1通道的体外氧化改变了它们对生理效应物的反应。此外，老化减缓了RyR 1的周转率，可能会导致有害蛋白质修饰的积累。初步实验表明：1）年龄相关的ATP和咖啡因激活RyR 1的下降;和2）RyR 1 CaM结合结构域中的反应性半胱氨酸的修饰。因此，我推测，RyR 1从老年骨骼肌氧化修饰和重要的内源性效应器的调节受损。为了验证这一假设，目标1将确定是否老化改变调节RyR 1的腺嘌呤核苷酸和效应离子;目标2将确定老化的影响功能相互作用的RyR 1与辅助蛋白钙调蛋白;和目标3将确定年龄相关的RyR 1半胱氨酸残基的氧化程度。 这些研究将使用各种技术，包括[3 H]ryanodine和[35 S]钙调蛋白结合SR囊泡，以评估RyR 1的生理调节，单通道分析，以确定衰老改变RyR 1通道功能的分子机制，和monobromobimane测定SR囊泡和纯化RyR 1通道的游离巯基含量。这项工作将进一步加深我们对与年龄相关的肌无力的分子机制的理解，并有助于开发治疗和预防骨骼肌无力的疗法。

DESCRIPTION (provided by applicant): A decline in skeletal muscle function is a major contributor to decreased mobility and independence in the elderly. In addition, muscle weakness in a significant factor contributing to falls resulting in serious injury. Thus, although skeletal muscle weakness is a major public health concern, the molecular mechanisms underlying skeletal muscle dysfunction in aging are not well understood. The long-term objective of this work is to clarify the mechanisms underlying aging-induced skeletal muscle weakness. Muscle contraction is initiated by Ca}+ efflux from the sarcoplasmic reticulum (SR) via the SR Ca}+ release channel/ryanodane receptor (RyR1). SR Ca}+ release is depressed in muscle from aged individuals. Thus incomplete contractile activation contributes to muscle weakness. Being the major SR Ca}+ effiux pathway, RyR1 dysfunction is a likely contributor to impaired Ca}+ release in aging muscle. In vitro oxidation of RyR1 channels modifies their response to physiological effectors. In addition, aging slows the RyR1 turn-over rate potentially allowing the accumulation of detrimental protein modifications. Preliminary experiments suggest 1) an age-associated decline in ATP and caffeine activation of RyR1; and 2) modification of a reactive cysteine in the RyR1 CaM binding domain. Therefore, I hypothesize that RyR1 from aged skeletal muscle is oxidatively modified and its regulation by important endogenous effectors is impaired. To test this hypothesis, Aim 1 will determine whether aging alters the regulation of RyR1 by adenine nucleotides and effector ions; Aim 2 will determine the impact of aging on the functional interaction of RyR1 with the accessory protein calmodulin; and Aim 3 will determine the extent of age-associated oxidation of RyR1 cysteine residues. These studies will use a variety of techniques including [3H]ryanodine and [35S]calmodulin binding to SR vesicles to assess RyR1 regulation by physiological regulators, single channel analysis to determine the molecular mechanisms by which aging alters RyR1 channel function, and monobromobimane determination of the free thiol content of SR vesicles and purified RyR1 channels. This work will further our understanding of the molecular mechanisms underlying age-associated muscle weakness and aid in the development of therapies to treat and prevent skeletal muscle frailty.