CYTOPROTECTIVE EFFECTS OF INFLAMMATION MEDIATED MEMBRANE REPAIR

这项研究的长期目标是阐明促炎细胞因子的细胞保护作用 在心肌缺血再灌注损伤中的作用。我们先前已经证明， 肿瘤坏死因子（TNF）由1型（TNFR 1）和2型（TNFR 2）TNF受体两者传递。 注意到TRAF 2（肿瘤坏死因子受体相关因子2）是唯一的信号蛋白， 我们在分离的心肌细胞中进行了实验，以及转基因的心肌细胞。 心脏限制性过表达TRAF 2的小鼠（MHC-TRAF 2小鼠）和显性阴性TRAF 2 （MHC-TRAF 2-DN小鼠），其已经确定TRAF 2在TNF介导的免疫应答中的不可或缺的作用。 细胞保护信号为了进一步确定TRAF 2细胞保护作用的机制，我们 在MHC-TRAF 2和MHC-TRAF 2-DN小鼠的心脏中进行比较基因表达谱分析， 发现了一种独特的“紧急反应基因”，称为dysferlin，负责维持Ca++ 依赖于心肌细胞质膜完整性。因此，我们建议将目前的重点放在 TRAF 2介导的膜修复作为一种新的细胞保护机制的作用 在I/R损伤中，通过确定dysferlin是否是介导这些作用所必需的和/或足够的。 具体目标1将检验TRAF 2在离体I/R损伤中的细胞保护作用是 至少部分通过dysferlin介导的心肌细胞质膜的维持介导 完整特异性目的2将测试（1）TRAF 2在体内I/R损伤中的细胞保护作用是否是 至少部分通过dysferlin介导的心肌细胞质膜的维持介导 完整性（2）以及TRAF 2和dysferlin在界定组织损伤方面的有益作用是否与TRAF 2和dysferlin的作用有关。 体内伴随着更快的时间解决心肌炎症。第3章测试是否 (1)TRAF 2的细胞保护作用至少部分是通过增强胞吐膜介导的， 在分离的新生大鼠心肌细胞中的修复和增强的膜完整性;和（2）诱导的 多能干细胞（iPSC）衍生的人心肌细胞，其由来自以下的皮肤成纤维细胞产生： 患有dysferlin病的患者在缺氧后具有受损的胞吐和增加的细胞死亡 体外复氧损伤。我们预计，具体目标1 - 3的结果将提供明确的 关于心脏中TRAF 2介导的细胞保护机制的信息，以及允许 我们确定dysferlin，一种新的Ca++依赖性应急反应基因，是否是必要的和/或 足以介导TRAF 2在I/R损伤后的细胞保护作用。

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.