Muscle Protein Turnover and Amino Acid Uptake in Sepsis

描述（由申请人提供）：先前的研究表明，脓毒症诱导的肌肉萎缩与肌肉钙水平升高相关，糖皮质激素是脓毒症诱导的肌肉蛋白水解的主要介质。当前项目的总体假设是糖皮质激素增加肌肉钙水平，脓毒症和糖皮质激素通过钙依赖机制增加肌肉蛋白水解。这在三个具体目标中进行了测试。特定目的1检验脓毒症和糖皮质激素增加肌肉钙水平和钙-钙调蛋白激酶II（CaMK II）和钙蛋白酶活性的假设。特异性目的2检验了以下假设：脓毒症和糖皮质激素诱导的肌肉萎缩相关转录因子C/EBPp和8以及NF-κ B的激活受钙水平升高以及CaMK II和钙蛋白酶激活的调节。具体目标3测试的假设，脓毒症和糖皮质激素诱导的肌肉蛋白水解和泛素连接酶atrogin-1和MuRF 1的上调调节钙水平的增加和激活的CaMK II和钙蛋白酶。在两种肌肉萎缩模型中进行研究，即，在败血症大鼠和地塞米松处理的培养肌管中。在先前的几项研究中，我们发现盲肠结扎穿孔（CLP）诱导的大鼠脓毒症和地塞米松处理培养的肌管导致蛋白质降解增加和泛素-蛋白酶体-蛋白水解途径的基因表达上调。通过使用钙螯合剂BAPTA或钙“拮抗剂”维拉帕米和丹曲林检查钙的作用。通过使用特异性抑制剂或通过用表达酶的质粒或天然钙蛋白酶抑制剂钙蛋白酶抑制剂转染肌细胞来测试CaMK II和钙蛋白酶的作用。该项目很重要，因为它将提供有关脓毒症和糖皮质激素诱导的肌肉萎缩的早期和起始机制的信息，如果我们的假设是正确的，钙调节机制可能成为预防和治疗脓毒症和其他分解代谢条件下肌肉萎缩的重要治疗靶点。相关性：脓毒症、癌症或损伤患者肌肉质量损失。在这些和其他分解代谢条件下的肌肉消耗显著导致发病率和死亡率。拟议的实验将确定钙和钙调节机制在脓毒症肌肉萎缩发展中的作用。增加对肌肉萎缩机制的了解将有助于改善这种衰弱状况的预防和治疗。

DESCRIPTION (provided by applicant): Previous studies suggest that sepsis-induced muscle wasting is associated with increased muscle calcium levels and that glucocorticoids are the predominant mediator of sepsis-induced muscle proteolysis. The overall hypothesis of the current project is that glucocorticoids increase muscle calcium levels and that sepsis and glucocorticoids increase muscle proteolysis through calcium-dependent mechanisms. This is tested in 3 specific aims. Specific Aim 1 tests the hypothesis that sepsis and glucocorticoids increase muscle calcium levels and calcium-calmodulin protein kinase II (CaMK II) and calpain activities. Specific Aim 2 tests the hypothesis that sepsis- and glucocorticoid-induced activation of the muscle wasting-associated transcription factors C/EBPp and 8 and NF-kB is regulated by increased calcium levels and activation of CaMK II and calpains. Specific Aim 3 tests the hypothesis that sepsis- and glucocorticoid-induced muscle proteolysis and upregulation of the ubiquitin ligases atrogin-1 arid MuRF1 are regulated by increased calcium levels and activation of CaMK II and calpains. Studies are performed in two models of muscle wasting, i.e., in septic rats and in dexamethasone-treated cultured myotubes. In several previous studies, we have found that sepsis in rats induced by cecal ligation and puncture (CLP) and treatment of cultured myotubes with dexamethasone result in increased protein degradation and upregulated gene expression of the ubiquitin-proteasome-proteolytic pathway. The role of calcium is examined by using the calcium chelator BAPTA or the calcium "antagonists" verapamil and dantrolene. The roles of CaMK II and calpains are tested by using specific inhibitors or by transfecting myocytes with plasmids expressing the enzymes or the natural calpain inhibitor calpastatin. The project is important because it will provide information about early and initiating mechanisms of sepsis- and glucocorticoid-induced muscle wasting, if our hypothesis is correct, calcium-regulated mechanisms may become important therapeutic targets in the prevention and treatment of muscle wasting in sepsis and other catabolic conditions. Relevance: Patients with sepsis, cancer, or injury suffer loss of muscle mass. Muscle wasting in these and other catabolic conditions contributes significantly to morbidity and mortality. The proposed experiments will define the role of calcium and calcium-regulated mechanisms in the development of muscle-wasting in sepsis. Increased understanding of the mechanisms driving muscle wasting will help improve prevention and treatment of this debilitating condition.