Cytoprotective Cytokine Signaling and Reperfusion Injury

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。本实验室的研究表明，肿瘤坏死因子(TNF)的细胞保护作用是通过1型(TNFR1)和2型(TNFR2)肿瘤坏死因子受体传递的。注意到这两种受体共有的唯一信号蛋白是一种称为TRAF2(肿瘤坏死因子受体相关因子2)的信号蛋白，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)和显性负TRAF2蛋白(MHC-TRAF2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名为disferlin的“紧急反应基因”，它负责维持钙离子依赖的心肌细胞质膜的完整性。鉴于膜通透性增加和钙超载在缺血再灌注(I/R)损伤中的核心作用，我们建议将目前的应用重点放在描述TRAF2信号的细胞保护作用上，并将寻求解决是否有必要和/或充分地介导这些细胞保护作用的问题。因此，我们建议问并回答一系列三个合乎逻辑的问题。首先，TRAF2的细胞保护作用是否通过激活1型NF-B途径实现，如果是的话，是否涉及p65和p50同源或异源二聚体(特异性目标1)？第二，TRAF2对I/R损伤后的细胞保护作用(特异性目标2)是否需要干扰素？第三，在缺氧-复氧损伤后，TRAF2和/或去铁蛋白对心肌细胞的细胞保护作用的细胞机制是什么(特定目标3)？我们期望特定的AIMS 1-3的结果将提供关于TRAF2下游介导的心脏细胞保护信号通路的明确信息，并使我们能够确定一个新的钙依赖的紧急反应基因deferlin是否必要和/或足以介导TRAF2的细胞保护作用。公共卫生相关性：缺血一段时间后的心肌再灌注可能被认为是“喜忧参半”。也就是说，一方面有明显的好处，因为恢复的心肌参与再灌注；但另一方面，也有有害的再灌注依赖效应，限制了所有再灌注策略的有效性，这可能导致心力衰竭。本研究的意义在于：(1)研究本质上高度保守的细胞保护机制(通过肿瘤坏死因子受体相关因子2[TRAF2]传递信号)；(2)研究一种新的方法，通过加强缺血再灌注损伤后的膜修复来增强心肌细胞的细胞保护作用。

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. Studies from this laboratory have 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 the only signaling protein that was common to both receptors was a signaling protein termed TRAF2 (tumor necrosis factor receptor associated factor 2), 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 protein (MHC-TRAF2 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. Given the central role of enhanced membrane permeability and Ca++ overload in the setting of ischemia reperfusion (I/R) injury, we propose to focus the present application on delineating the cytoprotective effects of TRAF2 signaling and will seek to address whether dysferlin is necessary and/or sufficient for mediating these cytoprotective effects. Accordingly, we propose to ask and answer a series of three logical questions. First, are the cytoprotective effects of TRAF2 mediated by activation of the type 1 NF-?B pathway, and if so are p65 and p50 homo or heterodimers involved (Specific Aim 1)? Second, is dysferlin necessary for the cytoprotective effects of TRAF2 following I/R injury (Specific Aim 2)? Third, what are the cellullar mechanisms responsible for cytoprotective effects of TRAF2 and/or dysferlin in isolated cardiac myocytes following hypoxia- reoxygenation injury (Specific Aim 3)? We expect that the results of Specific Aims 1 - 3 will provide definitive information with respect to the cytoprotective signaling pathways that are downstream from TRAF2 mediated 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. PUBLIC HEALTH RELEVANCE: Myocardial reperfusion following a period of ischemia may be regarded as a "mixed blessing." That is, on the one hand there is the clear cut benefit that occurs as the result of recovery of heart muscle that attends reperfusion; however, on the other hand there are also deleterious reperfusion dependent effects that limit the effectiveness of all reperfusion strategies, which can lead to heart failure. The significance of the studies proposed in the present application is that will (1) focus on identifying cytoprotective mechanisms that have been highly conserved in nature (signaling through tumor necrosis factor receptor associated factor 2 [TRAF2]), and (2) study a novel method for conferring cytoprotection in cardiac myocytes through enhanced membrane repair following ischemia reperfusion injury.