CYTOPROTECTIVE EFFECTS OF INFLAMMATION MEDIATED MEMBRANE REPAIR

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。我们先前已经表明，肿瘤坏死因子（TNF）的细胞保护作用是由1型（TNFR 1）和2型（TNFR 2）TNF受体传递的。注意到TRAF 2（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传导蛋白，我们已经在分离的心肌细胞以及具有心脏限制性TRAF 2过表达的转基因小鼠（MHC-TRAF 2小鼠）和显性阴性TRAF 2的转基因小鼠（MHC-TRAF 2-DN小鼠）中进行了实验，这些实验已经鉴定了TRAF 2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TRAF 2的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。因此，我们建议本申请集中于通过确定dysferlin是否是介导这些作用所必需的和/或足够的来描绘TRAF 2介导的膜修复作为I/R损伤中的新型细胞保护机制的作用。具体目标1将检验以下假设：TRAF 2在离体I/R损伤中的细胞保护作用至少部分是通过dysferlin介导的心肌细胞质膜完整性维持介导的。具体目标2将测试（1）TRAF 2在体内I/R损伤中的细胞保护作用是否至少部分通过dysferlin介导的心肌细胞质膜完整性的维持来介导（2），以及TRAF 2和dysferlin的有益作用是否在体内界定组织损伤时伴随着更快的心肌炎症消退时间。具体目标3将测试（1）TRAF 2的细胞保护作用是否至少部分地通过在分离的新生大鼠心肌细胞中增强的胞吐膜修复和增强的膜完整性介导;和（2）诱导性多能干细胞（iPSC）从来自dysferlin病患者的真皮成纤维细胞产生的衍生人心肌细胞在缺氧/复氧后的胞吐作用受损和细胞死亡增加体外损伤。我们期望特异性目标1 - 3的结果将提供关于TRAF 2介导的心脏细胞保护机制的明确信息，并使我们能够确定dysferlin（一种新型Ca++依赖性应急反应基因）是否是介导TRAF 2在I/R损伤后的细胞保护作用所必需和/或足够的。 公共卫生相关性：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检查炎症如何允许心肌细胞在心脏病发作后修复其细胞膜来理解心脏中炎症的有益作用的机制，并因此可能导致开发帮助心脏更好地承受心脏损伤的新疗法。

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. 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.