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Decreased Expression of Mitochondrial Calcium Uptake Protein 1 Leads to Skeletal Muscle Dysfunction in Septic Mice
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摘要:
目的 观察脓毒症对骨骼肌功能的影响，并探讨骨骼肌线粒体钙摄取蛋白1（mitochondrial calcium uptake protein 1, MICU1）的作用。 方法 选择SPF级健康雄性C57/BL 6J小鼠40只，随机分为4组：假手术组（Sham组，n=8）；脓毒症建模6 h组（CLP-6 h组，n=10）；脓毒症建模12 h组（CLP-12 h组，n=10）；脓毒症建模24 h组（CLP-24 h组，n=12）。采用盲肠结扎穿孔术（cecal ligation and puncture, CLP）构建脓毒症模型，Sham组小鼠仅行开腹盲肠探查。另选取SPF级小鼠20只，一侧胫前肌空转染腺相关病毒（AAV）作为对照（AAV-C），另一侧胫前肌转染AAV提高MICU1表达（AAV-M）。小鼠随机分为2组：假手术组（AAV-C-Sham，AAV-M-Sham；n=8）和脓毒症建模24 h组（AAV-C-CLP，AAV-M-CLP；n=12）。于相应时间点检测小鼠抓力以及复合肌动作电位（compound muscle action potential, CMAP）。采用ELISA测定骨骼肌炎症因子肿瘤坏死因子α（tumor necrosis factor α, TNF-α）及白细胞介素6（interleukin 6, IL-6）表达水平。通过HE染色检测骨骼肌细胞形态变化，运用Western blot检测MICU1以及肌肉萎缩相关蛋白：肌肉环状指蛋白1（muscle RING-finger containing protein 1, MuRF1）和肌肉萎缩盒F蛋白（muscle atrophy Fbox protein, MAFbx）表达水平，RT-qPCR检测骨骼肌MICU1 mRNA表达水平。 结果 与Sham组相比，CLP组小鼠体质量下降（P<0.05），且随着CLP建模时间延长，小鼠抓力下降（P<0.05）；CMAP幅值下降，持续时间和潜伏期延长（P<0.05）；骨骼肌炎症因子TNF-α、IL-6表达水平逐渐增高（P<0.05）；骨骼肌肌纤维直径及横截面积随建模时间延长逐渐下降（P<0.05）；MuRF1、MAFbx蛋白表达水平逐渐增高（P<0.05）；MICU1蛋白及mRNA表达水平逐渐下降（P<0.05）。AAV-M-Sham组与AAV-C-Sham组相比，各项指标差异均无统计学意义（ P>0.05）。AAV-M-CLP组与AAV-C-CLP组比较，小鼠抓力增高（P<0.05）；CMAP幅值增高，持续时间和潜伏期缩短（P<0.05）；肌纤维直径及横截面积增加（P<0.05）；MuRF1、MAFbx蛋白表达水平降低（P<0.05）。 结论 脓毒症导致骨骼肌功能障碍，与线粒体MICU1表达水平下降有关。 Abstract:Objective To observe the effect of sepsis on skeletal muscle function and to explore the role of skeletal muscle mitochondrial calcium uptake protein 1 (MICU1). Methods A total of 40 specific-pathogen-free (SPF) healthy male C57BL/6J mice were randomly assigned to 4 groups, a sham operation group (Sham group, n=8), a sepsis modeling 6 h group (cecal ligation and puncture [CLP]-6 h group, n=10), a sepsis modeling 12 h group (CLP-12 h group, n=10), and a sepsis modeling 24 h group (CLP-24 h, n=12). The sepsis model was established by CLP. Mice in the Sham group only underwent laparotomic exploration of the cecum. Another 20 SPF mice were selected. The tibialis anterior muscle on one side was empty-transfected with adeno-associated virus (AAV) as controls (AAV-C), and the tibialis anterior muscle on the other side was transfected with AAV to enhance MICU1 expression (AAV-M). The mice were randomly assigned to two groups, a sham operation group (AAV-C-Sham and AAV-M-Sham, n=8) and a sepsis model 24 h group (AAV-C-CLP and AAV-M-CLP, n=12). The grip strength and compound muscle action potential (CMAP) of the tibialis anterior muscle were measured in each group at the corresponding time points. The levels of inflammatory factors, including tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6), in the skeletal muscle were measured by ELISA. The morphological changes of skeletal muscle cells were observed through H&E staining. The expression levels of MICU1 and muscle atrophy-related proteins, including muscle RING-finger containing protein 1 (MuRF1) and muscle atrophy Fbox protein (MAFbx), were determined by Western blot. The expression levels of MICU1 mRNA in skeletal muscle were determined by RT-qPCR. Results Compared with mice in the Sham group, mice in the CLP group showed decreased body weight (P<0.05); their grip strength decreased with the prolongation of CLP modeling time (P<0.05); the amplitude of CMAP decreased, showing prolonged duration and latency (P<0.05); the expression levels of inflammatory factors, including TNF-α and IL-6, in skeletal muscle increased gradually (P<0.05); the fiber diameter and cross-sectional area of skeletal muscle decreased gradually with the prolongation of modeling time (P<0.05); the protein expression levels of MuRF1and MAFbx proteins increased gradually (P<0.05); the expression levels of MICU1 protein and mRNA decreased gradually (P<0.05). There was no significant difference in all indices between AAV-M-Sham and AAV-C-Sham groups (P>0.05). Compared with mice in the AAV-C-CLP group, mice in the AAV-M-CLP group showed increased grip strength (P<0.05); the amplitude of CMAP increased, showing shortened duration and latency (P <0.05); the fiber diameter and cross-sectional area of skeletal muscle increased (P<0.05); the expression levels of MuRF1and MAFbx decreased (P<0.05). Conclusion Sepsis leads to skeletal muscle dysfunction, which is related to the decrease in mitochondrial MICU1 expression. -
Key words:
- Sepsis /
- Skeletal muscles /
- Muscle weakness /
- Mitochondria /
- Mitochondrial calcium uptake protein 1
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图 1 各组小鼠抓力（A），CMAP检测结果（B），胫前肌炎症因子表达（C），HE染色（×40）、肌纤维直径及横截面积定量分析结果（D），萎缩相关蛋白（E）及MICU1蛋白表达和mRNA的表达（F）
Figure 1. Grip strength (A), CMAP (B), IL-6 and TNF-α (C), muscle fiber diameter and CSA quantification after H&E staining (×40) (D), expression of MuRF1 and MAFbx proteins (E), and MICU1 protein and mRNA (F)
CMAP: compound muscle action potential; IL-6: interleukin 6; CSA: cross-sectional area; MuRF1: muscle RING-finger containing protein 1; MAFbx: muscle atrophy Fbox protein; MICU1: mitochondrial calcium uptake protein 1. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001, vs. Sham group; # P<0.05, ## P<0.01, ### P<0.001, ####P<0.0001, vs. CLP-6 h group; ▲ P<0.05, ▲▲ P<0.01, ▲▲▲ P<0.001, vs. CLP-12 h group.
图 2 AAV干预后，各组小鼠抓力（A）、CMAP检测结果（B）、小鼠胫前肌HE染色（×40）、肌纤维直径及横截面积定量分析结果（C）、MICU1蛋白和mRNA表达（D）和胫前肌萎缩相关蛋白表达（E）
Figure 2. The grip strength (A), CMAP (B), tibialis anterior muscle fiber diameter and CSA quantification after HE staining (×40) (C), the expression of MICU1 protein (D) and mRNA MuRF1 and MAFbx proteins (E) after AAV intervention
AAV: adeno-associated virus; the other abbreviations are explained in the notes to Fig 1. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001, vs. AAV-C-Sham group; ## P<0.01, #### P<0.0001, vs. AAV-M-Sham group; ▲ P<0.05, ▲▲ P<0.01, ▲▲▲▲ P<0.0001, vs. AAV-C-CLP group.
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