MUSCLE PROTEIN TURNOVER AND AMINO ACID UPTAKE IN SEPSIS

以往的研究表明，脓毒症诱导的肌肉分解代谢反映了糖皮质激素调节的肌原纤维蛋白的泛素依赖的蛋白酶体降解。因为完整的肌原纤维不会被蛋白酶体降解，所以肌动蛋白和肌球蛋白有可能在被蛋白酶体泛素化和降解之前从肌原纤维中解离出来。我们将检验假设：1)脓毒症导致糖皮质激素介导的骨骼肌钙/钙蛋白酶依赖的Z带解体和释放；2)脓毒症导致N端规则通路依赖的肌肉蛋白质泛素化和分解增加，泛素结合酶E2/14k和泛素连接酶E3pha的表达和活性上调；3)在体内，蛋白酶体阻滞剂可以抑制脓毒症引起的肌肉恶病质；4)脓毒症患者的肌肉恶病质与N-末端规则通路中钙痛的表达和活性增加、肌丝释放和上调的蛋白质分解有关。大多数实验采用盲肠结扎和穿刺法建立大鼠脓毒症模型。通过分别测定酪氨酸和3-甲基组氨酸的净释放量来测量孵化肌肉中总蛋白和肌原纤维蛋白的降解率。用电子显微镜研究了肌节Z带的完整性。通过Northern和Western印迹分析，分别检测了calain和calastatin的基因和蛋白表达。通过丹曲林和地尔硫卓对脓毒症诱导的形态和代谢变化的影响来评估钙/钙蛋白酶依赖的蛋白分解的作用。糖皮质激素受体拮抗剂RU38486确定了糖皮质激素在脓毒症引起的肌肉钙水平变化和肌丝释放中的作用。为了测试N-末端规则通路的作用，测定了E2/14K和E3a的表达和活性，并在无细胞体系中使用了特异性的E3albha抑制剂。脓毒症患者的肌肉也进行了类似的测定。这一建议是新颖的，因为它表明脓毒症时肌肉恶病质是由两种不同的机制引起的，即依赖钙/钙蛋白的肌丝从肌节释放肌丝，然后在N端规则通路中肌丝泛素化，以及随后26S蛋白酶体降解泛素化的肌丝。这一假说暗示可以在两个水平上预防/治疗败血症引起的肌肉恶病质，即通过钙拮抗剂抑制肌丝释放，以及通过蛋白酶体阻滞剂抑制泛素/蛋白酶体依赖的降解释放的肌丝。

Previous studies suggest that sepsis-induced muscle catabolism reflects ubiquitin-proteasome-dependent degradation of myofibrillar proteins regulated by glucocorticoids. Because intact myofibrils are not degraded by the proteasome, it is possible that actin and myosin are dissociated from the myofibrils before they are ubiquitinated and degraded by the proteasome. We will test the hypotheses: 1) sepsis results in glucocorticoid-mediated calcium/calpain-dependent Z-band disintegration and release of myofilaments in skeletal muscle; 2) sepsis results in increased N-end rule pathway-dependent ubiquitination and breakdown of muscle proteins and upregulated expression and activity of the ubiquitin- conjugating enzyme E2/14k and ubiquitin ligase E3alpha; 3) sepsis- induced muscle cachexia can be inhibited by proteasome blocker in vivo; 4) muscle cachexia in patients with sepsis is associated with increased expression and activity of calpains, release of myofilaments and upregulated protein breakdown in the N-end rule pathway. A septic model in rats consisting of cecal ligation and puncture is used in the majority of experiments. Total and myofibrillar protein breadkdown rates are measured in incubated muscles by determining net release of tyrosine and 3-methylhistidine respectively. Integrity of sarcomeric Z-bands is studied by electron microscopy. Gene and protein expression of calpain and calpastatin are determined by Northern and Western blot analysis, respectively. The role of calcium/calpain-dependent proteolysis is assessed by the effect of dantrolene and diltiazem on sepsis-induced morphologic and metabolic changes. The role of glucocorticoids in sepsis- induced changes in muscle calcium levels and release of myofilaments is determined by the glucocorticoid receptor antagonist RU38486. To test the role of the N-end rule pathway, expression and activity of E2/14k and E3alpha are determined and specific E3albha inhibitors are used in a cell- free system. Similar determinations are performed in muscle from patients with sepsis. The proposal is novel because it suggests that muscle cachexia during sepsis is caused by two distinct mechanisms, i.e., calcium/calpain-dependent release of myofilaments from the sarcomere followed by ubiquitination of myofilaments in the N-end rule pathway and subsequent degradation of ubiquitinated filaments by the 26S proteasome. The hypothesis implies two levels at which sepsis-induced muscle cachexia may be prevented/treated, i.e., inhibition of myofilament release by treatment with a calcium antagonist and inhibition of ubiquitin/proteasome-dependent degradation of the released myofilaments by a proteasome blocker.