骨骼肌纤维的钙稳态调控在冬眠达乌尔黄鼠抗废用性肌萎缩中的作用及其机制研究

Hibernators are used as a natural model of anti-disuse muscle atrophy and can provide a valuable perspective on the prevention of muscle disuse-associated diseases, the underlying mechanism of which is important to clarify not only in the field of physiological ecology, but also in the fields of aerospace, clinical, sports and rehabilitation medicine. Recent studies have shown that the calcium homeostasis disruption characterized by the cytosolic calcium overload plays an important role in disuse muscle atrophy. Our previous studies on hibernating Daurian ground squirrels (Spermophilus dauricus) suggest that the regulation of cytosolic calcium homeostasis could be pivotal to the mechanism of muscle atrophy resistance. In present study, the soleus in slow muscle, the extensor digitorum longus, semitendinosus and plantaris muscles in fast muscle and gastrocnemius in mixed muscle are selected for representative of different kinds of skeletal muscles. With comparative analysis of skeletal muscles from hindlimb unloading ground squirrels and rats, the changes in calcium concentration and the relevant signal transduction mechanism in the cytoplasm, sarcoplasmic reticulum, mitochondria and nuclei in different skeletal muscles of different hibernation states are detected and systematically analyzed in order to explore the regulation mechanism of cytosolic calcium homeostasis in skeletal muscles of ground squirrels. Moreover, the combination of transcriptomic approach allows for a thorough investigation of differentially expressed calcium-regulated genes during hibernation. This is the first attempt to explore the potential mechanisms counteracting disuse muscle atrophy in hibernators from the calcium homeostatic perspective, which would provide insights into possible approaches to prevent and treat disuse muscle atrophy in human.

冬眠动物是一种天然的抗废用性肌萎缩动物模型。阐明其发生机制是生理生态学领域亟待研究的重要课题，对航天、临床、运动及康复医学等领域骨骼肌废用相关疾病的防治亦有重要的借鉴意义。研究表明，钙稳态失衡是废用性肌萎缩的重要发生机制之一。我们前期关于冬眠达乌尔黄鼠的研究提示，对骨骼肌纤维胞浆钙稳态的调控很可能是其抗废用性肌萎缩的重要机制之一。本项目拟以达乌尔黄鼠为研究对象，通过对冬眠不同时期不同类型骨骼肌纤维的细胞质、肌质网、线粒体及细胞核中钙离子浓度的变化与信号转导机制的系统研究，辅以转录组学分析冬眠期差异表达的钙离子调控基因，并与后肢去负荷黄鼠和后肢去负荷大鼠骨骼肌相关指标进行比较，多层面多角度探讨冬眠黄鼠骨骼肌纤维胞浆钙稳态的调控机制。首次尝试从钙稳态的角度系统研究冬眠动物抗废用性肌萎缩的可能机制，并有望为人类废用性肌萎缩防治措施的研发提供新思路。

本研究对我们前期提出的冬眠动物冬眠期抗废用性肌萎缩机制的钙稳态学说进行了实验验证。并对冬眠不同时期不同类型骨骼肌细胞的氧供、氧化应激水平及抗氧化应激机制进行了研究。主要结果如下：.对达乌尔黄鼠(Spermophilus dauricus)骨骼肌细胞膜上钙转运蛋白的研究发现，阵间觉醒时肌膜上介导胞外Ca2+进入胞内的通道SOCE与其调节因子ORAI1及STIM1的共定位系数显著升高，觉醒后再入眠时又出现下降，肌膜钙泵PMCA3蛋白表达水平无显著变化，提示，胞外钙离子通过SOCE的内流水平随冬眠-阵间觉醒循环出现周期性升降变化，觉醒再入眠时下调胞外的钙内流水平可能是该时期骨骼肌细胞胞浆钙水平下降的机制之一。.对肌浆网钙和胞浆钙的相关研究发现，肌浆网参与了冬眠期胞浆钙水平的波动性变化；冬眠期肌浆网钙通道（RYR1）及钙泵（SERCA1）蛋白表达与活性或开放几率均显著升高。其中RYR1的高表达与升高的开放几率可能是冬眠期骨骼肌细胞胞浆钙水平升高的机制之一，SERCA1的高表达与升高的活性可能是胞浆钙水平周期性恢复的重要机制之一。.对线粒体钙的研究发现，冬眠期骨骼肌线粒体钙内向转运体（MCU）和钙通道（Letm1）的蛋白表达均上调，线粒体钙浓度出现轻度升高，但无细胞凋亡现象，说明未达到钙超载水平。冬眠动物良好的线粒体钙稳态维持及调控能力，使其避免了线粒体钙超载引起的细胞凋亡。.此外，对氧化应激的相关研究发现，冬眠期其骨骼肌可通过激活Nrf2/Keap1通路，上调抗氧化酶的表达及活性，防止活性氧积累，进而避免骨骼肌氧化损伤。同时发现，比目鱼肌可通过增强抗氧化应激能力防止氧化应激的产生；而趾长伸肌则维持无氧酵解为主的呼吸方式来避免氧化应激的产生，提示黄鼠不同收缩类型的骨骼肌有着不同的抗氧化应激策略。.本研究首次从钙稳态的角度系统研究了冬眠动物抗废用性肌萎缩的机制，结果发现，冬眠达乌尔黄鼠骨骼肌具有良好的胞浆钙稳态以及修复钙稳态失衡的能力。为我们提出的冬眠动物冬眠期抗废用性肌萎缩机制的钙稳态学说提供了多方面的实验证据。