Proteomics study of oxidative stress and Src kinase inhibition in H9C2 cardiomyocytes: a cell model of heart ischemia-reperfusion injury and treatment
Proteomics study of oxidative stress and Src kinase inhibition in H9C2 cardiomyocytes: a cell model of heart ischemia-reperfusion injury and treatment
复制标题
DOI：
10.1016/j.freeradbiomed.2010.04.001
复制
发表时间：
2010-07-01
影响因子：
7.4
通讯作者：
Chan, Hong-Lin
中科院分区：
文献类型：
--
作者：
Chou, Hsiu-Chuan;Chen, Yi-Wen;Chan, Hong-Lin
蛋白质磷酸化在信号转导途径中发挥着至关重要的作用，这些信号转导途径调节基因表达、代谢、细胞粘附和响应氧化应激的细胞存活。在本研究中，我们使用过氧化氢处理 H9C2 大鼠心肌细胞作为心脏缺血再灌注损伤中氧化应激的模型。我们发现氧化应激会诱导这种细胞类型中多种蛋白质的强烈酪氨酸磷酸化。然后使用抗磷酸酪氨酸亲和纯化和 LC-MS/MS 的磷酸蛋白质组学方法来鉴定这种应激诱导的磷酸化的蛋白质靶标。鉴定出二十三种酪氨酸磷酸化蛋白，其中大多数已知与细胞-细胞连接、肌动蛋白细胞骨架和细胞粘附相关。这表明氧化应激可能对细胞间连接和细胞骨架产生深远影响，从而影响细胞粘附、形态和存活。重要的是，Src 激酶被证明是这些事件的主要上游调节因子。免疫荧光研究、荧光激活细胞分选和基于细胞的测定被用来证明氧化应激诱导的细胞粘附结构和细胞骨架的修饰、诱导的脱粘附和细胞凋亡的增加，这些变化可以通过 Src 激酶抑制剂 PP1 的治疗来逆转。这些数据证明了 Src 激酶在氧化应激诱导的心肌细胞磷酸化和细胞损伤中的关键作用，并表明靶向该激酶可能是预防心脏缺血再灌注损伤的有效策略。 (C) 2010 Elsevier Inc. 保留所有权利。
Protein phosphorylation plays a crucial role in the signal transduction pathways that regulate gene expression, metabolism, cell adhesion, and cell survival in response to oxidative stress. In this study, we have used hydrogen peroxide treatment of H9C2 rat cardiomyocytes as a model of oxidative stress in heart ischemia-reperfusion injury. We show that oxidative stress induces a robust tyrosine phosphorylation of multiple proteins in this cell type. A phosphoproteomics approach using anti-phosphotyrosine affinity purification and LC-MS/MS was then used to identify the protein targets of this stress-induced phosphorylation. Twenty-three tyrosine-phosphorylated proteins were identified, with the majority known to be associated with cell-cell junctions, the actin cytoskeleton, and cell adhesion. This suggested that oxidative stress may have a profound effect on intercellular connections and the cytoskeleton to affect cell adhesion, morphology, and survival. Importantly, Src kinase was shown to be a major upstream regulator of these events. Immunofluorescence studies, fluorescence-activated cell sorting, and cell-based assays were used to demonstrate oxidative stress-induced modification of cell adhesion structures and the cytoskeleton, induced de-adhesion, and increased apoptosis, which were reversed by treatment with the Src kinase inhibitor PP1. These data demonstrate the critical role of Src kinase in oxidative stress-induced phosphorylation and cell damage in cardiomyocytes and suggest that targeting this kinase may be an effective strategy for preventing ischemia-reperfusion injury in the heart. (C) 2010 Elsevier Inc. All rights reserved.