TROPONIN MODULATION IN HEART FAILURE

描述(改编自申请人的描述)：建议的实验 这里测试了一种假设，即结构、功能和 心肌肌钙蛋白I(CTnI)和cTnT的调节参与了进化 和心力衰竭的终末期病理学。长期目标是知道如何使用 肌丝重构和共价调制在心肌梗死的发生发展中的作用 人类心脏肥大/衰竭。目标#1、#2和#3解决以下问题： 终末期人类心脏肌丝的张力和经济性能否改变 用重组蛋白取代TN复合体的组分可挽救故障 蛋白质？人类心脏细丝改变的性质是什么？ 失败？CTnI上特定位点的蛋白磷酸化有变化吗？ 还是cTnT的同工异构体？这些变化是如何改变力量和ATPase速率的 在重组制剂中？目标4解决了这样一个问题：什么是 CTnI上蛋白激酶C(PKC)位点在骨肉瘤发生发展中的特殊作用 对压力超负荷的血流动力学应激反应的肥大和衰竭？ 实现这些目标的方法包括使用交换程序 在完整的力产生晶格中的细丝蛋白质，并为 从心脏组织小样本中分离TN复合体。重组蛋白是 在未磷酸化状态下制备，并在PKC处特异性磷酸化 和PKA站点。凝胶电泳、免疫印迹和抗体 识别磷酸化形式的cTn1被用来检测Tn的变化 很复杂。目的：检测cTnI上特定的PKC位点在肿瘤细胞进化中的作用。 肥大和衰竭，携带转基因表达突变形式的 CTn1(在Ser 43和Ser 45缺乏PKC位点)和慢骨骼TnI(缺乏 抑制肽中Thr 144处的PKC位点)在压力下被强调 超载。这些实验提供了关于 心力衰竭及其药理开发的潜在价值 抑制PKC途径的途径。

DESCRIPTION (adapted from the applicant's description): Experiments proposed here test hypothesis that alterations in the structure, function, and regulation of cardiac troponin I (cTnI) and cTnT contribute to the evolution and end-stage pathology of heart failure. The long term objective is know how myofilament remodeling and covalent modulation play a role in the evolution of hypertrophy/failure in human hearts. Aims #1, #2, and #3 address the questions: Can altered tension and economy of myofilaments from human hearts in end-stage failure be rescued by replacing components of the Tn complex with recombinant proteins? What is the nature of the thin filament alterations in human heart failure? Are there changes in protein phosphorylation of specific sites on cTnI or isoform population of cTnT? How do these changes alter force and ATPase rate in reconstituted preparations? Aim #4 addresses the question: What is the specific role of protein kinase C (PKC) sites on cTnI in the development of hypertrophy and failure in response to hemodynamic stress of pressure overload? The approach to these objectives involves the use of procedures for exchanging thin filament proteins in the intact force generating lattice, and for isolating Tn complex from small heart tissue samples. Recombinant proteins are prepared in an unphosphorylated state and specifically phosphorylated at PKC and PKA sites. Gel electrophoresis, immunoblotting and antibodies that recognize phosphorylated forms of cTnl are used to detect changes in the Tn complex. To test the role of specific PKC sites on cTnI in the evolution of hypertrophy and failure, mice harboring transgenes expressing a mutant form of cTnl (lacking PKC sites at Ser 43 and Ser 45) and slow skeletal TnI (lacking a PKC site at Thr 144 in the inhibitory peptide) are stressed by pressure overload. These experiments provide crucial information on the mechanisms of heart failure and on the potential value of developing pharmacological approaches to the inhibition of the PKC pathway.