Myofilament Function in Human Heart Failure

描述（由申请人提供）：本项目的核心假设是，非缺血性心力衰竭患者心功能的进行性下降反映了心脏收缩蛋白的翻译后修饰，特别是细丝蛋白、肌钙蛋白I、T和调节肌球蛋白轻链（MLC2）。为了验证这一假设，我们将从两组不同的患者中收集心肌样本：i)接受冠状动脉搭桥手术的糖尿病患者和非糖尿病患者，ii)接受心脏手术（心室改造或移植）的特发性扩张型心肌病患者，以及作为对照，接受瓣膜置换术或搭桥手术的正常心室功能患者。我们将测试皮肤细胞中肌丝功能障碍的程度，并确定PP1A去磷酸化是否会消除样品组之间的差异（目的1）。接下来，我们将把肌丝功能和临床状态与收缩蛋白异构体分布和磷酸化状态联系起来，特别关注肌钙蛋白复合物的组成部分和调节肌球蛋白轻链（目的2）。最后，将在病理细胞的背景下提取肉瘤蛋白，并用经过修饰的重组蛋白代替，以概括非病理情况（目标3）。该项目的总体目标是确定特定的和可修改的生化界面，可能允许合理治疗非缺血性心肌功能障碍。

DESCRIPTION (provided by applicant): The central hypothesis that underlies this project is that the progressive decline in cardiac function seen in patients with non-ischemic heart failure is a reflection of the post-translational modification of cardiac contractile proteins, specifically the thin filaments proteins, troponin I, T and the regulatory myosin light chain (MLC2). To test this hypothesis, we will collect myocardial samples from two distinct groups: i) patients with and without diabetes undergoing coronary bypass surgery, ii) patients with idopathic dilated cardiomyopathy undergoing cardiac surgery (either ventricular modification or tranplantation) and, as controls patients with normal ventricular function undergoing valve replacement or bypass surgery. We will test the extent of myofilament dysfunction in skinned cells and determine whether dephosphorylation by PP1A will eliminate differences between sample groups (aim 1). Next, we will correlate myofilament function and clinical status with contractile protein isoform distribution and phosphorylation status, paying particular attention to the components of the troponin complex and the regulatory myosin light chain (aim 2). Finally, sarcomeric proteins will be extracted in the context of the pathologic cell and replaced with recombinant proteins that have been modified so as to recapitulate the non-pathologic situation (aim 3). The overall goal of the project is to identify specific and modifiable biochemical interfaces that might allow rational treatment of nonischemic cardiac muscle dysfunction.