CYTOPROTECTIVE EFFECTS OF INFLAMMATION MEDIATED MEMBRANE REPAIR

这项研究的长期目标是描绘促炎细胞因子的细胞保护作用。 在心肌缺血再灌注(I/R)损伤中。我们之前已经证明，细胞保护作用 肿瘤坏死因子(TNF)由1型(TNFR1)和2型(TNFR2)肿瘤坏死因子受体传递。 注意到TRAF2(肿瘤坏死因子受体相关因子2)是唯一一种 对于这两种受体的共同之处，我们已经在分离的心肌细胞中进行了实验，以及转基因 心脏限制性过表达TRAF2和显性阴性TRAF2的小鼠(MHC-TRAF2小鼠) (MHC-TRAF2-dN小鼠)已确定TRAF2在肿瘤坏死因子介导的过程中起着不可或缺的作用 细胞保护性信号。为了进一步确定TRAF2细胞保护作用的机制 对MHC-TRAF2和MHC-TRAF2-DN小鼠的心脏进行了比较基因表达谱分析 发现了一种独特的“紧急反应基因”，称为异铁蛋白，它负责维持钙离子。 依赖心肌细胞质膜完整性。因此，我们建议将目前的重点放在 TRAF2介导的膜修复作为一种新的细胞保护机制的应用 在I/R损伤中，通过确定是否有必要和/或足够的干扰素来调节这些影响。 特异性目标1将检验TRAF2在体外I/R损伤中的细胞保护作用的假设 至少部分地通过去铁蛋白介导的心肌细胞质膜的维持来介导 正直。特异性靶点2将测试(1)TRAF2在体内I/R损伤中的细胞保护作用 至少部分地通过去铁蛋白介导的心肌细胞质膜的维持来介导 完整性(2)以及TRAF2和deferlin在界定组织损伤方面是否有好处 活体心肌炎症消退时间较快。《特定目标3》将测试 (1)TRAF2的细胞保护作用至少部分是通过增强胞吐膜介导的 新生大鼠心肌细胞的修复和增强膜完整性；和(2)可诱导 多能干细胞(IPSCs)来源于真皮成纤维细胞的人心肌细胞 铁代谢障碍的患者在缺氧后胞吐功能受损，细胞死亡增加 体外复氧损伤。我们期望具体目标1-3的结果将提供明确的 关于TRAF2介导的心脏细胞保护机制的信息，以及允许 US确定一种新的钙离子依赖的紧急反应基因deferlin是否必要和/或 足以介导TRAF2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.