Cytoprotective Cytokine Signaling and Reperfusion Injury

描述（由申请人提供）：本研究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。该实验室的研究表明，肿瘤坏死因子（TNF）的细胞保护作用由1型（TNFR 1）和2型（TNFR 2）TNF受体传递。值得注意的是，两种受体共有的唯一信号蛋白是一种称为TRAF2的信号蛋白，（肿瘤坏死因子受体相关因子2），我们已经在分离的心肌细胞中进行了实验，以及心脏限制性过表达TRAF2的转基因小鼠（MHC-TRAF2小鼠）和显性负性TRAF2蛋白（MHC-TRAF2小鼠），其已经鉴定了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，并确定了一个独特的"应急反应基因"，称为dysferlin，其负责维持Ca ++依赖性心肌细胞质膜的完整性。考虑到在缺血再灌注（I/R）损伤的情况下增强的膜通透性和Ca ++过载的中心作用，我们提出将本申请集中在描绘TRAF2信号传导的细胞保护作用上，并将寻求解决dysferlin是否是介导这些细胞保护作用所必需的和/或足够的。因此，我们建议提出并回答一系列三个逻辑问题。首先，TRAF2的细胞保护作用是通过激活1型NF-κ B介导的吗？B途径，如果是，p65和p50同源或异源二聚体参与（具体目标1）？第二，dysferlin是否是TRAF2在I/R损伤后的细胞保护作用所必需的（具体目标2）？第三，TRAF2和/或dysferlin在缺氧-复氧损伤后的离体心肌细胞中的细胞保护作用的细胞机制是什么（具体目标3）？我们期望特异性目的1 - 3的结果将提供关于心脏中TRAF2介导的细胞保护信号通路下游的明确信息，并使我们能够确定dysferlin（一种新型Ca ++依赖性应急反应基因）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：心肌缺血一段时间后的心肌再灌注可被视为"喜忧参半"。“也就是说，一方面，由于参与再灌注的心肌恢复而产生的明显益处;然而，另一方面，也存在有害的再灌注依赖性效应，限制了所有再灌注策略的有效性，这可能导致心力衰竭。本申请中提出的研究的意义在于将（1）集中于鉴定在自然界中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 [TRAF 2]的信号传导），和（2）研究通过缺血再灌注损伤后增强的膜修复赋予心肌细胞细胞保护的新方法。

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.