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能力和独立性下降的主要原因。此外，肌肉无力是导致摔倒导致严重受伤的一个重要因素。因此，尽管骨骼肌无力是一个主要的公共卫生问题，但衰老过程中骨骼肌功能障碍的分子机制尚不清楚。 这项工作的长期目标是阐明衰老导致骨骼肌无力的潜在机制。肌肉收缩是由肌浆网(SR)的钙离子通过肌浆网钙离子释放通道/兰诺丹受体(RyR1)外流启动的。老年人肌肉中SR、Ca~(2+)释放受到抑制。因此，不完全的收缩激活会导致肌肉无力。作为肌浆网钙离子外流的主要途径，RyR1功能障碍可能是衰老肌肉钙释放受损的原因之一。 在体外，RyR1通道的氧化改变了它们对生理效应器的反应。此外，衰老会减缓RyR1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)RyR1的ATP和咖啡因活性随着年龄的增长而下降；以及2)RyR1CaM结合域中的活性半胱氨酸的修饰。因此，我假设来自衰老骨骼肌的RyR1被氧化修饰，其由重要内源性效应器调节的功能受损。为了验证这一假说，目标1将确定衰老是否改变腺核苷酸和效应离子对RyR1的调节；目标2将确定衰老对RyR1与辅助蛋白钙调蛋白功能相互作用的影响；目标3将确定与年龄相关的RyR1半胱氨酸残基的氧化程度。 这些研究将使用多种技术，包括[~3H]ryanodine和[35S]calmodin与SR囊泡的结合，以评估生理调节剂对RyR1的调节，单通道分析，以确定衰老改变RyR1通道功能的分子机制，以及一溴莫比胺测定SR囊泡和纯化的RyR1通道的游离硫醇含量。这项工作将进一步加深我们对年龄相关性肌肉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治疗方法。

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.