Calcium Leak-Dependent Muscle Function Loss in Aged C elegans

 描述（由申请人提供）：肌肉减少症定义为与年龄相关的肌肉质量和功能丧失，是一种新兴的健康问题，在老年人群中的患病率高达30%。目前的治疗具有低的长期疗效，并且不针对肌肉功能丧失的任何潜在原因。我们实验室最近在老年小鼠中的研究表明，Ryanodine受体（RyR 1）是一种对兴奋-收缩偶联很重要的细胞内钙释放通道，是一种潜在的治疗靶点。随着时间的推移，RyR 1经历翻译后修饰（“重塑”），导致钙稳定蛋白1（一种重要的RyR 1调节亚基）从通道中解离;这导致RyR 1开放概率增加和Ca 2+“泄漏”。这种Ca 2+“泄漏”导致附近的线粒体钙超载并产生过量的活性氧（ROS），这通过进一步重塑RyR 1加剧了泄漏。由于小鼠的寿命相对较长，我们正在测试C。线虫可以作为一个更好的模型，年龄依赖性肌肉功能丧失。C.线虫含有RyR 1同源物<$N-68和钙稳定蛋白同源物FKB-2;因此，我们假设1）FKB-2解离使<$N-68通道不稳定，导致老年线虫中致病性肌浆网（SR）钙泄漏和随后的肌无力，2）线粒体ROS氧化<$N-68通道附近并加剧体壁肌肉中的SR钙泄漏。Aim One将测试泄漏的E10 -68通道是否在C中发挥作用。肌肉无力我们首先使用免疫沉淀和免疫印迹法确定WT蠕虫中β-68重塑随年龄增加，但在FKB-2 KO蠕虫中加速。我们已经通过将WT和FKB-2 KO蠕虫与Pmyo-3：GCaMP 2蠕虫杂交确定FKB-2消耗导致降低的峰值Ca 2+瞬变和减弱的β-68咖啡因活化。在目标二中，我们将通过测量两种电子传递链（ETC）突变株，EBT-1和mev-1中的线粒体自由基来评估线粒体ROS在EBT-68功能障碍中的作用。我们已经发现，β-68重塑的时间与他们的寿命差异相关; β-1是长寿的，直到老年都没有β-68重塑，而mev-1是短命的，在生命早期就重塑了β-68通道。成功完成拟议的研究将确定病理性SR钙泄漏在年龄依赖性肌肉功能丧失中的作用。

 DESCRIPTION (provided by applicant): Sarcopenia, defined as an age-related loss of muscle mass and function, is a burgeoning health problem with a prevalence as high as 30% in the geriatric population. Current treatments have low long- term efficacy and do not target any underlying causes of muscle function loss. Recent research in aged mice in our lab has implicated the Ryanodine Receptor (RyR1), an intracellular calcium release channel important for excitation-contraction coupling, as a potential therapeutic target. Over time, RyR1 undergoes posttranslational modification ('remodeling') that causes calstabin1, an important RyR1 modulatory subunit, to dissociate from the channel; this results in increased RyR1 open probability and Ca2+ 'leak'. This Ca2+ 'leak' cause nearby mitochondria to overload with calcium and produce excess reactive oxygen species (ROS), which exacerbate the leak by further remodeling RyR1. Due to the relatively long lifespan of mice we were testing if C. elegans could serve as a better model of age- dependent muscle function loss. C. elegans contain UNC-68, the RyR1 homologue, and FKB-2, a calstabin homologue; therefore, we hypothesize that 1) FKB-2 dissociation destabilizes UNC-68 channels, resulting in pathogenic sarcoplasmic reticulum (SR) calcium leak and subsequent muscle weakness in aged nematodes and 2) mitochondrial ROS oxidizes nearby UNC-68 channels and exacerbates SR calcium leak in body wall muscle. Aim One will test whether leaky UNC-68 channels play a role in C. elegans muscle weakness. We first established that UNC-68 remodeling increases with age in WT worms, but is accelerated in FKB-2 KO worms using immunoprecipitation and immunoblot. We have determined that FKB-2 depletion results in reduced peak Ca2+ transients and blunted UNC-68 caffeine activation by crossing WT and FKB-2 KO worms with Pmyo-3: GCaMP2 worms. In Aim Two, we will assess the contribution of mitochondrial ROS in UNC-68 dysfunction by measuring mitochondrial free radicals in two electron transport chain (ETC) mutant strains, clk-1 and mev-1. We have found that the timing of UNC-68 remodeling correlates with their lifespan differences; clk-1 is long-lived and lacks UNC- 68 remodeling until old age, while mev-1 is short-lived and has remodeled UNC-68 channels earlier in life. Successful completion of the proposed studies will define the contributions of pathological SR calcium leak in age-dependent muscle function loss.