Anti-inflammatory Signaling of RNA-binding Protein, Tristetraprolin, During Myocardial Infarction

项目摘要 心肌梗死（MI）在再灌注治疗后几乎立即启动动态细胞反应。 缺血再灌注损伤（IRI）早期炎症反应启动免疫细胞级联反应， 信号传导网络，其有助于免疫细胞募集和细胞外基质（ECM）降解。的 IRI的反常现象导致大量细胞死亡，并且是不利的心室收缩的主要决定因素。 重塑使用一种新的时空基因表达的全转录组分析，我们确定， RNA结合蛋白Tristetraprolin（TTP）对几种关键蛋白质的表达具有调节作用， 炎性细胞因子和与活性氧（ROS）相关的基因。然而，TTP在早期- 尽管其抗炎特性已得到充分证实，但MI的发病尚不清楚。本文中描述的研究 该提案旨在阐明TTP在早发性MI中的心脏保护机制。S100 A8和S100 A9法案 作为损伤相关分子模式（DAMP），其在IRI 3天后高度表达。要求1 研究TTP对S100 A8/S100 A9表达和功能的调节作用。TTP敲除（TTP- KO）和TTP过表达（TTP-OE）的骨髓细胞系将用于确定TTP对细胞凋亡的影响。 S100 A8/S100 A9表达。6个特异性AA残基的突变已显示消除了 mRNA分子。将产生TTP突变细胞系（TTP-Δ1-6），以确定TTP是否介导 S100 A8/S100 A9通过mRNA不稳定或上游调控信号传导效应。TTP的功能 S100 A8/S100 A9与Ca ~（2+）有效性密切相关。因此，Ca 2+浓度将作为 环境变量TTP还显示减少SPP 1和Cybb（NOX 2）表达，抑制 IRI后ROS产生、氧化应激和线粒体功能障碍。因此，目标2将探索 体外模拟（sIRI）对TTP-KO和TTP-OE骨髓细胞中ROS产生的影响。线粒体 还将在心肌细胞中测量暴露于条件培养基后的功能和ATP产生。 sIR 1刺激的TTP-KO和TTP-OE骨髓细胞。最后，目的3将研究骨髓特异性TTP的作用。 在减轻体内不利的心室重构中的表达。为了验证这一点，TTP用一种 LysMCre切除后的136 bp floxed位点分别产生骨髓特异性TTP-KO和TTP-OE小鼠。 实验小鼠将通过临时LAD结扎暴露于IRI治疗。超声心动图将定期 用于监测心脏功能和心室重塑。最后，我们将研究治疗 通过引入表达TTP的腺相关病毒载体9的时间依赖性TTP递送的潜力 在IRI 3天后，使用AAV 9或用TTP-OE巨噬细胞过继转移。同样的测量和 将进行评估以确定每种治疗的功效。这项研究背后的基本原理是 充分理解TTP的抗炎特性和治疗早发性MI的潜力。 具体而言，TTP表达对炎症、线粒体功能障碍和心肌病的影响。

PROJECT SUMMARY Myocardial infarction (MI) initiates a dynamic cellular response almost immediately after reperfusion therapy. Inflammation during the early stages of ischemia reperfusion injury (IRI) initiates a cascade of immune cell signaling networks which contribute to immune cell recruitment and extracellular matrix (ECM) degradation. The paradoxical phenomenon of IRI results in massive cell death and is a primary determinant of adverse ventricular remodeling. Using a novel transcriptome-wide analysis of spatiotemporal gene expression, we determined that the RNA-binding protein, Tristetraprolin (TTP) has regulator effects on the expression of several key pro- inflammatory cytokines and genes related to reactive oxygen species (ROS). However, the role of TTP in early- onset MI is unknown despite its well-established anti-inflammatory properties. The research described in this proposal seeks to elucidate the cardioprotective mechanisms of TTP in early-onset MI. S100A8 and S100A9 act as damage associated molecular patterns (DAMPs) that are highly expressed after 3 days of IRI. Aim 1 investigates the regulatory role of TTP on S100A8/S100A9 expression and functionality. TTP knockout (TTP- KO) and TTP overexpressing (TTP-OE) in myeloid cell lines will be used to determine the effects of TTP on S100A8/S100A9 expression. Mutations to 6 specific AA residues has been shown to abrogate TTP binding of mRNA molecules. TTP mutant cell lines (TTP-Δ1-6) will be generated to determine whether TTP mediates S100A8/S100A9 signaling effects through mRNA destabilization or upstream regulation. The function of TTP and S100A8/S100A9 are closely tied to Ca2+ availability. Therefore, Ca2+ concentration will tested as an environmental variable. TTP has also been shown to diminish SPP1 and Cybb (NOX2) expression, inhibiting ROS production, oxidative stress, and mitochondrial dysfunction following IRI. Therefore, Aim 2 will explore the effects of simulated (sIRI) on ROS production in TTP-KO and TTP-OE myeloid cells in vitro. Mitochondrial function and ATP production will also be measured in cardiomyocytes upon exposure to conditioned media of sIRI-stimulated, TTP-KO and TTP-OE myeloid cells. Lastly Aim 3 will investigate the role of myeloid-specific TTP expression in mitigating adverse ventricular remodeling in vivo. To test this, TTP floxed mice and mice with a 136 bp floxed site upon LysMCre excision generate myeloid-specific TTP-KO and TTP-OE mice respectively. Experimental mice will be exposed to IRI treatment by temporary LAD ligation. Echocardiography will be regularly performed to monitor heart function and ventricular remodeling. Lastly, we will investigate the therapeutic potential of time-dependent TTP delivery by introduction of TTP-expressing adeno-associated viral vector-9 (AAV9) or adoptive transfer with TTP-OE macrophages after 3 days of IRI. The same measurements and assessments will be made to determine the efficacy of each treatment. The rationale behind this research is a sound comprehension of the anti-inflammatory properties and therapeutic potential of TTP in early-onset MI. Specifically, the effects of TTP expression on inflammation, mitochondrial dysfunction, and cardiomyopathy.