MG53在慢性肾脏病骨骼肌线粒体自噬及肌萎缩中的作用及机制

Skeletal muscle atrophy is an important manifestation of protein-energy wasting. The 4-6 years mortality of the chronic kidney disease (CKD) patients with skeletal muscle atrophy was 3 times higher than those who were not. But the mechanism remains unclear. It was reported that mitochondrial autophagy damage can lead to skeletal muscle atrophy, while supplement of recombinant Mitsugumin 53 (MG53) intramuscular can alleviate muscle atrophy. We found that skeletal muscle of patients with end-stage renal disease (ESRD) showed abnormal mitophagy flux and hyperactive mitophagy, and the expression of Ambra1 and MG53 in the skeletal muscle of CKD rats was significantly decreased. In vitro experiments showed that MG53 overexpression could promote the expression of Ambra1 and mitochondrial autophagy in C2C12 cells. Therefore, this research team speculate that recovery of autophagy by MG53 intervention may help remove abnormal mitochondria and relieve CKD-induced muscle atrophy..Therefore, this research intends to explore the possible mechanism of CKD environment inhibiting MG53 expression in skeletal muscle, the role of MG53 deficiency in skeletal muscle atrophy and mitochondrial autophagy in CKD mice, the effects of MG53 on skeletal muscle atrophy and mitochondrial function of CKD through intramuscular injection of recombinant MG53 into CKD mouse model; the effect of MG53 on mitochondrial autophagy through AMBRA1 by transfection plasmid into mouse satellite cells and C2C12 myoblasts; and the mechanism of MG53 maintaining mitochondrial function is systematically studied. Successful completion of this project will lay the ground work for new therapeutic target of CKD skeletal muscle atrophy.

骨骼肌萎缩是影响慢性肾脏病（CKD）患者生存质量、导致死亡率增加的重要原因之一，但机制尚未明确。文献报道线粒体自噬受损可以导致骨骼肌萎缩，而予重组MG53肌肉注射可缓解其病变。我们前期发现终末期肾病患者腹直肌线粒体自噬障碍，CKD大鼠骨骼MG53蛋白表达减少，体外实验过表达MG53可通过调节小鼠C2C12成肌细胞Ambra1表达促进线粒体自噬。据此我们提出假设：CKD环境抑制了骨骼肌MG53表达，提高MG53表达有助于改善CKD引起的骨骼肌线粒体自噬障碍和骨骼肌萎缩。藉此，本课题拟探索CKD环境抑制骨骼肌MG53表达的可能机制；通过CKD小鼠模型肌肉注射重组MG53，探讨其对CKD骨骼肌萎缩及线粒体功能的作用，在小鼠卫星细胞和C2C12成肌细胞中，调控MG53水平并检测AMBRA1的泛素化，系统研究MG53维持线粒体功能的机制，本项目的完成将为CKD骨骼肌萎缩的临床干预提供新的治疗靶点。

MG53是一种在心肌和骨骼肌特异性表达的TRIM家族成员，本课题组发现慢性肾脏疾病(CKD)小鼠骨骼肌TLR13 表达上调，与MG53蛋白表达呈负相关，我们推测TLR13是MG53下游的关键靶点，TLR13通过介导胰岛素抵抗促进CKD骨骼肌萎缩发生，在胰岛素信号受损的分解代谢条件下，靶向TLR13可提高胰岛素敏感性，防止肌肉萎缩。.我们采用5/6肾切除术构建CKD小鼠模型，发现CKD小鼠胫骨前肌TLR13表达显著上调。体外培养小鼠成肌细胞系C2C12，发现尿毒症血清可抑制C2C12细胞的活力，促进细胞TLR13表达；而抑制TLR13表达后细胞活力改善，肌管面积增加。尿毒症血清可使C2C12肌管中肌肉萎缩因子atrogin-1和MuRF-1表达水平上调，蛋白质合成减少、降解增加；而下调TLR13可抑制atrogin-1和MuRF-1表达及蛋白质降解。TLR13损害C2C12成肌细胞胰岛素信号传导；通过免疫共沉淀实验发现TLR13通过结合并激活IRF3，抑制AKT的活性，诱导胰岛素抵抗；通过双荧光素酶报告基因分析证实TLR13受转录因子ETS2调节，ETS2激活刺激TLR13表达。构建TLR13基因敲除的CKD小鼠模型，发现TLR13基因敲除改善了CKD小鼠肌肉的胰岛素抵抗,并可保护小鼠免受CKD诱导的肌肉萎缩。.我们还发现CKD大鼠骨骼肌中CAPN6 mRNA和蛋白质水平略有增加。敲除CAPN6后大鼠L6成肌细胞 LC3水平增加；而过表达CAPN6降低了LC3 水平并损害了自噬；mTOR、Raptor和α-微管蛋白表达增加。炎症细胞因子，如IL-6、TNF-α、INF-γ和脂多糖可上调 CAPN6表达，抑制 L6成肌细胞自噬并稳定mTOR 活性。表明Calpain6可能通过维持 mTOR 信号传导减少自噬，可能参与CKD导致的肌肉萎缩发生。.综上所述，我们的研究结果表明，TLR13参与了CKD介导的肌肉胰岛素抵抗和骨骼肌萎缩；在胰岛素信号受损的分解代谢条件下，靶向TLR13可能提高胰岛素敏感性，防止肌肉萎缩。另一方面，Calpain6可能通过维持 mTOR信号传导减少自噬从而促进 CKD的肌肉萎缩，是否可以作为CKD骨骼肌萎缩的干预靶点，还需要进一步的研究。项目研究成果在Cell Proliferation, BMC Nephroloy等期刊发表论著3篇。