CYTOPROTECTIVE EFFECTS OF INFLAMMATION MEDIATED MEMBRANE REPAIR

描述（由申请人提供）：本研究计划的长期目标是描述促炎细胞因子在心肌缺血再灌注（I/R）损伤中的细胞保护作用。我们之前已经证明肿瘤坏死因子（TNF）的细胞保护作用是由1型（TNFR1）和2型（TNFR2） TNF受体传递的。注意到TRAF2（肿瘤坏死因子受体相关因子2）是两种受体共同的唯一信号蛋白，我们在分离的心肌细胞以及TRAF2心脏受限过表达的转基因小鼠（MHC-TRAF2小鼠）和TRAF2显性阴性（MHC-TRAF2- dn小鼠）中进行了实验，这些实验已经确定了TRAF2在TNF介导的细胞保护信号传导中不可或缺的作用。为了进一步确定TRAF2细胞保护作用的机制，我们在MHC-TRAF2和MHC-TRAF2- dn小鼠的心脏中进行了比较基因表达谱分析，并确定了一种独特的“紧急反应基因”，称为异常铁素，负责维持钙离子依赖性心肌细胞质膜的完整性。因此，我们建议将目前的应用重点放在描绘TRAF2介导的膜修复作为一种新的细胞保护机制在I/R损伤中的作用上，通过确定异草素在介导这些作用方面是否必要和/或充分。特异性目的1将验证TRAF2在体外I/R损伤中的细胞保护作用，至少部分是通过异干扰素介导的心肌细胞质膜完整性的维持来介导的。特异性目的2将测试(1)TRAF2在体内I/R损伤中的细胞保护作用是否至少部分是通过异铁素介导的心肌细胞质膜完整性的维持来介导的(2)以及TRAF2和异铁素在体内划定组织损伤方面的有益作用是否伴随着更快的心肌炎症消退时间。特异性Aim 3将测试(1)TRAF2的细胞保护作用是否至少部分是通过增强离体大鼠新生心肌细胞的胞外膜修复和增强膜完整性介导的；(2)体外缺氧再氧损伤后，诱导多能干细胞（iPSCs）衍生的人类心肌细胞在体外缺氧再氧损伤后会导致胞外功能受损和细胞死亡增加。我们期望特异性Aims 1 - 3的结果将提供关于TRAF2介导的心脏细胞保护机制的明确信息，并允许我们确定异常素（一种新的依赖于Ca++的紧急反应基因）是否必要和/或充分地介导TRAF2在I/R损伤后的细胞保护作用。

DESCRIPTION (provided by applicant): The long-term goal of this research initiative is to delineate the cytoprotective role of proinflammatory cytokines in myocardial ischemia reperfusion (I/R) injury. We have previously shown that the cytoprotective effects of tumor necrosis factor (TNF) are conveyed by both the type 1 (TNFR1) and type 2 (TNFR2) TNF receptors. Noting that TRAF2 (tumor necrosis factor receptor associated factor 2) was the only signaling protein that was common to both receptors, we have performed experiments in isolated cardiac myocytes, as well as transgenic mice with cardiac restricted overexpression of TRAF2 (MHC-TRAF2 mice) and a dominant negative TRAF2 (MHC-TRAF2-DN mice) that have identified an indispensable role for TRAF2 in TNF mediated cytoprotective signaling. To further identify the mechanisms for the cytoprotective effects of TRAF2 we performed comparative gene expression profiling in the hearts of MHC-TRAF2 and MHC-TRAF2-DN mice and identified a unique "emergency response gene" termed dysferlin, that is responsible for maintaining Ca++ dependent cardiac myocyte plasma membrane integrity. Accordingly, we propose to focus the present application on delineating the role of TRAF2 mediated membrane repair as a novel cytoprotective mechanism in I/R injury, by determining whether dysferlin is necessary and/or sufficient for mediating these effects. Specific Aim 1 will test the hypothesis that the cytoprotective effects of TRAF2 in I/R injury ex vivo are mediated, at least in part, through dysferlin-mediated maintenance of cardiac myocyte plasma membrane integrity. Specific Aim 2 will test whether (1) the cytoprotective effects of TRAF2 in I/R injury in vivo are mediated, at least in part, through dysferlin-mediated maintenance of cardiac myocyte plasma membrane integrity (2) and whether the beneficial effects of TRAF2 and dysferlin with respect to delimiting tissue injury in vivo are accompanied by faster time of resolution of myocardial inflammation. Specific Aim 3 will test whether (1) the cytoprotective effects of TRAF2 are mediated, at least in part, through enhanced exocytotic membrane repair and enhanced membrane integrity in isolated rat neonatal cardiac myocytes; and (2) inducible pluripotent stem cells (iPSCs) derived human cardiac myocytes generated from dermal fibroblasts from patients with dysferlinopathy have impaired exocytosis and increased cell death following hypoxia reoxygenation injury in vitro. We expect that the results of Specific Aims 1 - 3 will provide definitive information with respect to the mechanisms for TRAF2-mediated cytoprotection in the heart, as well as allow us to determine whether dysferlin, a novel Ca++ dependent emergency response gene, is necessary and/or sufficient to mediate the cytoprotective effects of TRAF2 following I/R injury. PUBLIC HEALTH RELEVANCE: Studies have shown that the inflammation that occurs after a restoration of blood flow to the heart after a heart attack has both beneficial and harmful effects. The studies proposed in the present application are designed to understand the mechanisms for the beneficial effects of inflammation in the heart by examining how inflammation allows heart muscle cells to repair their cell membranes after a heart attack, and may thus lead to the development of new therapies that help the heart to better withstand heart injury.