MEF2C参与失重诱导肌肉萎缩发生的机理研究

MEF2C (Myocyte enhancer factor 2C) is a very important amyotrophy-related protein. It was found that the MEF2C knockout mice suffered muscular dystrophy severely, and the previous experiments revealed that MEF2C was the most significantly down-regulated protein in the muscular dystrophy tissue under weightlessness. The applicant verified in recent experiment that MEF2C was also down-regulated under simulated weightlessness condition. These results showed that MEF2C is closely related with muscular dystrophy under weightlessness. However, the detailed mechanism is still unclear. Since investigation on the interactions between MEF2C and its correlated proteins can provide us fundamental knowledge about muscular dystrophy under weightlessness, the applicant hereby proposes to conduct the following researches: ① screen out the proteins that interact directly with MEF2C, select 2-3 of those that are most significantly regulated by the gravity, and investigate the effects of gravity on their expression and consequently on the related cell biological indicators; ② determine the three-dimensional structure of MEF2C, and locate its binding sites through which the above selected proteins are connected. This reseach will be helpful to elucidate the molecular mechanism of muscular dystrophy under weightlessness, and provide us with information of target proteins for drug design and theoretical basis aiming at curing the muscular dystrophy suffered not only by the taikonauts but also by the common people.

MEF2C（Myocyte enhancer factor 2C）是一种十分重要的肌肉萎缩相关蛋白，MEF2C基因敲除小鼠会出现明显的肌肉萎缩症状。前人研究表明，该蛋白是失重引起的肌肉萎缩组织中下调最显著的蛋白。申请者实验也证实，在模拟失重条件下，骨骼肌细胞中MEF2C表达量显著减少。这些结果充分证明它与失重条件下的肌肉萎缩发生密切相关，但具体机制并不清楚。研究MEF2C及其相关蛋白之间的相互作用，将为解释失重条件下的肌肉萎缩现象提供理论依据。基于此，本项目拟开展如下研究：①筛选与MEF2C直接作用的蛋白，确定其中受重力影响最显著的2-3种关键作用蛋白，研究其在重力环境下的表达规律及对相关细胞生物学指标的影响；②解析MEF2C的空间结构，确定其与关键直接蛋白的结合位点。本研究将有助于阐明失重诱导肌肉萎缩发生的分子机制，并有望为解决航天员和地面人群的肌肉萎缩问题提供重要的药物靶点和理论基础。

肌肉萎缩是一种受力学环境影响的疾病，特别突出的表现是航天员在失重环境下出现明显的肌肉萎缩症状。解决肌肉萎缩问题，无论对保障航天员身体健康和载人航天事业的顺利发展，还是对地面人群肌肉萎缩疾病的治疗都是十分必要的。MEF2C（Myocyte enhancer factor 2C）是一种十分重要的肌肉萎缩相关蛋白，MEF2C基因敲除小鼠会出现明显的肌肉萎缩症状。前人研究表明，该蛋白是失重引起的肌肉萎缩组织中下调最显著的蛋白。基于此，我们开展了如下研究：①研究MEF2C蛋白的表达对成肌细胞相关指标的影响，我们通过转染MEF2C真核重组载体和特异性的siRNA到成肌细胞C2C12中，发现MEF2C可以显著增加C2C12细胞的铺展面积，促进细胞的增殖和增加细胞周期S期所占的比例等，进一步通过qPCR技术，筛选了受MEF2C表达影响最为显著的直接作用蛋白，说明MEF2C与肌肉萎缩发生确实直接相关；②筛选模拟失重环境下受重力影响最显著的关键作用蛋白，在细胞水平和动物水平构建模拟失重环境的模型，我们发现三维随机回转组中，MyoD，HDAC4，Notch2，Myogenin，SP1，Calpain1，CyclinD1，PPAR，ERK5和Myoglubin等都发生了显著变化。在尾悬吊组大鼠的比目鱼中，MyoD，HDAC4，Desmin，MAML1，CyclinD1和Sox18都发生了显著变化。其中，MyoD为失重环境下MEF2C诱发肌肉萎缩发生的关键直接作用蛋白；③确定MEF2C与其关键直接作用蛋白的结合位点，通过筛选MEF2C蛋白的表达和纯化条件，得到了纯度较高的蛋白，初步筛选到了MEF2C的蛋白质晶体。经过分子模拟预测到MEF2C与MyoD蛋白复合物的结构，并得到了两种蛋白的关键作用位点。其中MEF2C 的37位缬氨酸，38位亮氨酸，42位谷氨酸到44位甘氨酸，以及48位苯丙氨酸到66位亮氨酸。MyoD的45位赖氨酸到61位甘氨酸，39位脯氨酸，40位天冬酰胺，62位谷丙酰胺和63位甘氨酸等，这些都是MEF2C和MyoD的直接结合位点。本研究将有助于阐述失重诱导肌肉萎缩发生的分子机制，并有望为解决航天员和地面人群的肌肉萎缩问题提供重要的药物靶点和理论基础。