Department of Cardiology and Cardiovascular Research Institute, General Hospital of Northern Theater Command, Shenyang, China.
Autophagy. 2021 Dec;17(12):4102-4118. doi: 10.1080/15548627.2021.1904488. Epub 2021 Apr 18.
CREG1 (cellular repressor of E1A-stimulated genes 1) is involved in tissue homeostasis and influences macroautophagy/autophagy to protect cardiovascular function. However, the physiological and pathological role of CREG1 in the skeletal muscle is not clear. Here, we established a skeletal muscle-specific knockout mouse model () by crossing the -floxed mice () with a transgenic line expressing recombinase under the muscle-specific (creatine kinase, muscle) promoter. In mice, the exercise time to exhaustion and running distance were significantly reduced compared to mice at the age of 9 months. In addition, the administration of recombinant (re)CREG1 protein improved the motor function of 9-month-old mice. Moreover, electron microscopy images of 9-month-old mice showed that the mitochondrial quality and quantity were abnormal and associated with increased levels of PINK1 (PTEN induced putative kinase 1) and PRKN/PARKIN (parkin RBR E3 ubiquitin protein ligase) but reduced levels of the mitochondrial proteins PTGS2/COX2, COX4I1/COX4, and TOMM20. These results suggested that CREG1 deficiency accelerated the induction of mitophagy in the skeletal muscle. Mechanistically, gain-and loss-of-function mutations of altered mitochondrial morphology and function, impairing mitophagy in C2C12 cells. Furthermore, HSPD1/HSP60 (heat shock protein 1) (401-573 aa) interacted with CREG1 (130-220 aa) to antagonize the degradation of CREG1 and was involved in the regulation of mitophagy. This was the first time to demonstrate that CREG1 localized to the mitochondria and played an important role in mitophagy modulation that determined skeletal muscle wasting during the growth process or disease conditions.: CCCP: carbonyl cyanide m-chlorophenylhydrazone; CKM: creatine kinase, muscle; COX4I1/COX4: cytochrome c oxidase subunit 4I1; CREG1: cellular repressor of E1A-stimulated genes 1; DMEM: dulbecco's modified eagle medium; DNM1L/DRP1: dynamin 1-like; FCCP: carbonyl cyanide p-trifluoro-methoxy phenyl-hydrazone; HSPD1/HSP60: heat shock protein 1 (chaperonin); IP: immunoprecipitation; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MFF: mitochondrial fission factor; MFN2: mitofusin 2; MYH1/MHC-I: myosin, heavy polypeptide 1, skeletal muscle, adult; OCR: oxygen consumption rate; OPA1: OPA1, mitochondrial dynamin like GTPase; PINK1: PTEN induced putative kinase 1; PPARGC1A/PGC-1α: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; PRKN/PARKIN: parkin RBR E3 ubiquitin protein ligase; PTGS2/COX2: prostaglandin-endoperoxide synthase 2; RFP: red fluorescent protein; RT-qPCR: real-time quantitative PCR; SQSTM1/p62: sequestosome 1; TFAM: transcription factor A, mitochondrial; TOMM20: translocase of outer mitochondrial membrane 20; VDAC: voltage-dependent anion channel.
CREG1（E1A 刺激基因 1 的细胞抑制剂 1）参与组织稳态，并影响巨自噬/自噬以保护心血管功能。然而，CREG1 在骨骼肌中的生理和病理作用尚不清楚。在这里，我们通过将 -floxed 小鼠（）与在肌肉特异性 （肌酸激酶，肌肉）启动子下表达重组酶的转基因系杂交，建立了骨骼肌特异性 敲除小鼠模型（）。在 9 个月大的 小鼠中，与 小鼠相比，运动至力竭的时间和跑步距离明显减少。此外，重组（re）CREG1 蛋白的给药改善了 9 个月大的 小鼠的运动功能。此外，9 个月大的 小鼠的电子显微镜图像显示线粒体质量和数量异常，并伴有 PINK1（PTEN 诱导的假定激酶 1）和 PRKN/PARKIN（parkin RBR E3 泛素蛋白连接酶）水平升高，但线粒体蛋白 PTGS2/COX2、COX4I1/COX4 和 TOMM20 水平降低。这些结果表明 CREG1 缺乏加速了骨骼肌中自噬的诱导。在机制上，和功能获得和丧失突变改变了线粒体的形态和功能，损害了 C2C12 细胞中的自噬。此外，HSPD1/HSP60（热休克蛋白 1）（401-573 aa）与 CREG1（130-220 aa）相互作用，拮抗 CREG1 的降解，并参与自噬的调节。这是首次证明 CREG1 定位于线粒体，在调节决定生长过程或疾病状态下骨骼肌消耗的自噬中发挥重要作用。：CCCP：羰基氰化物 m-氯苯腙；CKM：肌酸激酶，肌肉；COX4I1/COX4：细胞色素 c 氧化酶亚基 4I1；CREG1：E1A 刺激基因 1 的细胞抑制剂 1；DMEM：杜尔贝科改良鹰培养基；DNM1L/DRP1：dynamin 1 样；FCCP：羰基氰化物 p-三氟甲氧基苯腙；HSPD1/HSP60：热休克蛋白 1（伴侣）；IP：免疫沉淀；MAP1LC3B/LC3B：微管相关蛋白 1 轻链 3B；MFF：线粒体裂变因子；MFN2：线粒体融合蛋白 2；MYH1/MHC-I：肌球蛋白，重链 1，骨骼肌，成人；OCR：耗氧量；OPA1：OPA1，线粒体动力相关 GTP 酶；PINK1：PTEN 诱导的假定激酶 1；PPARGC1A/PGC-1α：过氧化物酶体增殖物激活受体γ，共激活因子 1α；PRKN/PARKIN：parkin RBR E3 泛素蛋白连接酶；PTGS2/COX2：前列腺素内过氧化物合酶 2；RFP：红色荧光蛋白；RT-qPCR：实时定量 PCR；SQSTM1/p62：自噬体 1；TFAM：线粒体转录因子 A；TOMM20：外线粒体膜转位酶 20；VDAC：电压依赖性阴离子通道。
