CYTOPROTECTIVE EFFECTS OF INFLAMMATION MEDIATED MEMBRANE REPAIR

描述(由申请人提供)：这项研究的长期目标是描述促炎细胞因子在心肌缺血再灌注损伤中的细胞保护作用。我们先前已经证明，肿瘤坏死因子(TNF)的细胞保护作用是通过1型(TNFR1)和2型(TNFR2)肿瘤坏死因子受体来传递的。注意到TRAF2(肿瘤坏死因子受体相关因子2)是两种受体共有的唯一信号蛋白，我们在分离的心肌细胞以及心脏限制性过表达TRAF2的转基因小鼠(MHC-TRAF2小鼠)和显性阴性TRAF2的转基因小鼠(MHC-TRAF2-DN小鼠)中进行了实验，证实了TRAF2在肿瘤坏死因子介导的细胞保护信号中起着不可或缺的作用。为了进一步确定TRAF2的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，并鉴定了一个独特的被称为disferlin的“紧急反应基因”，它负责维持钙依赖的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，通过确定是否有必要和/或充分地介导这些作用。具体目的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干扰蛋白介导的维持心肌细胞质膜完整性的假说。特异性目标2将测试(1)TRAF2在活体I/R损伤中的细胞保护作用是否至少部分是通过差异蛋白介导的维持心肌细胞质膜完整性来介导的(2)以及在体内TRAF2和差异蛋白在界定组织损伤方面的有利作用是否伴随着更快的心肌炎症消退时间。特殊目的3将测试(1)TRAF2的细胞保护作用是否至少部分地通过增强新生大鼠心肌细胞的胞吐修复和增强膜完整性来介导；以及(2)可诱导多能干细胞(IPSCs)来源的人心肌细胞来自于来自铁代谢障碍患者的真皮成纤维细胞，在体外低氧复氧损伤后，TRAF2的胞吐功能受损并增加了细胞死亡。我们期望特定的AIMS 1-3的结果将为TRAF2介导的心脏细胞保护机制提供明确的信息，并使我们能够确定一个新的钙依赖的紧急反应基因dyferlin是否必要和/或足以介导TRAF2在I/R损伤后的细胞保护作用。 与公共健康相关：研究表明，心脏病发作后恢复心脏血流后发生的炎症既有有益的影响，也有有害的影响。本申请中提出的研究旨在通过研究炎症如何使心肌细胞在心脏病发作后修复其细胞膜来了解炎症对心脏有益影响的机制，从而可能导致开发新的治疗方法，帮助心脏更好地抵御心脏损伤。

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.