MUSCLE PROTEIN TURNOVER AND AMINO ACID UPTAKE IN SEPSIS

以往的研究表明，脓毒症诱导的肌肉catalysis反映了糖皮质激素调节的泛素-蛋白酶体依赖性的肌原纤维蛋白降解。 因为完整的肌原纤维不被蛋白酶体降解，所以肌动蛋白和肌球蛋白可能在被蛋白酶体泛素化和降解之前从肌原纤维解离。 我们将测试假设：1）脓毒症导致糖皮质激素介导的钙/钙蛋白酶依赖性Z带分解和骨骼肌中肌丝的释放; 2）脓毒症导致增加的N-末端规则途径依赖性泛素化和肌肉蛋白的分解以及泛素缀合酶E2/14 k和泛素连接酶E3 α的上调表达和活性; 3）脓毒症诱导的肌肉恶病质可被体内蛋白酶体阻断剂抑制; 4）脓毒症患者的肌肉恶病质与钙蛋白酶的表达和活性增加、肌丝的释放和N-末端规则途径中的蛋白质分解上调相关。 在大多数实验中使用由盲肠结扎和穿孔组成的大鼠脓毒症模型。 通过分别测定酪氨酸和3-甲基组氨酸的净释放，在孵育的肌肉中测量总蛋白和肌原纤维蛋白的面包化速率。 用电子显微镜研究肌节Z带的完整性。 分别通过北方和Western印迹分析测定钙蛋白酶和钙蛋白酶抑制素的基因和蛋白表达。 通过丹曲林和地尔硫卓对脓毒症诱导的形态学和代谢变化的影响来评估钙/钙蛋白酶依赖性蛋白水解的作用。 糖皮质激素在脓毒症诱导的肌肉钙水平变化和肌丝释放中的作用由糖皮质激素受体拮抗剂RU 38486决定。 为了测试N-末端规则途径的作用，测定E2/14 k和E3 α的表达和活性，并在无细胞系统中使用特异性E3 α抑制剂。 在脓毒症患者的肌肉中进行类似的测定。 该提议是新颖的，因为它表明败血症期间的肌肉恶病质是由两种不同的机制引起的，即，肌丝从肌节的钙/钙蛋白酶依赖性释放，随后是肌丝在N-末端规则途径中的泛素化和随后的26 S蛋白酶体对泛素化的肌丝的降解。该假设暗示了可以预防/治疗脓毒症诱导的肌肉恶病质的两个水平，即，通过用钙拮抗剂处理抑制肌丝释放和通过蛋白酶体阻断剂抑制释放的肌丝的泛素/蛋白酶体依赖性降解。

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.