Role of Toll-like Receptor 4 (TLR4) in Ischemic Myocardial Injury

描述（由申请人提供）：围手术期心肌缺血和梗死是大量接受心脏移植、冠状动脉旁路移植术和血运重建等各种手术的外科患者发病和死亡的主要原因。近年来的研究表明，心肌细胞凋亡或程序性细胞死亡在缺血性心肌损伤中起着重要作用。 天然免疫系统如Toll样受体4（TLR 4）代表了抵抗感染的第一道防线。除了其在宿主免疫中的关键作用之外，最近的研究已经证明TLR 4是响应于非感染性损伤的组织炎症和细胞存活的重要功能性贡献者。 我们的初步数据表明，TLR 4信号在心脏和分离的心肌细胞中对缺血性损伤的心脏保护中起着关键作用。本研究的目的是明确心肌TLR 4在缺血再灌注损伤（IRI）模型中保护心肌细胞的作用，并确定介导这些作用的下游机制。我们预计，从拟议的研究中获得的见解将作为缺血性心肌损伤管理的新的治疗方法的未来发展的基础。 该提议基于以下三个假设：1）TLR 4通过其信号蛋白IRAK-1的激活代表心脏中的重要存活机制，2）一氧化氮合酶2（NOS 2）介导TLR 4诱导的存活益处，以及3）心脏TLR 4信号传导的增强将减少心肌损伤并在IRI中产生有意义的功能性拯救。为了验证这些假设，我们将使用转基因动物以及腺病毒基因转移来操纵TLR 4和IRAK-1的心脏表达。具体目标1：我们将探讨TLR 4在体外细胞凋亡模型中保护心肌细胞的机制。在具体目标2：我们将确定如何NOS 2和NO有助于TLR 4介导的抗凋亡作用在分离的心肌细胞。在具体目标3中：我们将确定心脏（与心脏外）TLR 4在保护心脏免受IRI中的作用。在具体目标4中：我们将评估在IRI小鼠模型中操作IRAK-1的解剖和功能后果。我们将测试IRAK-1缺失或心脏IRAK-1表达（通过基因转移）对IRI的影响。 明确控制心肌细胞存活的信号通路并学会操纵这些通路可能为治疗某些心脏疾病如缺血损伤和心肌病提供新的方法。

DESCRIPTION (provided by applicant): Perioperative myocardial ischemia and infarction represent the major cause of morbidity and mortality for the large number of surgical patients who undergoes a variety of operations such as heart transplantation, coronary artery bypass grafting, and revascularization. Recent studies have demonstrated that cardiomyocyte apoptosis, or programmed cell death, plays an important role in ischemic myocardial injury. Innate immune system such as Toll-like receptor 4 (TLR4) represents the first line of defense against infection. In addition to its pivotal role in host immunity, recent studies have demonstrated that TLR4 is an important functional contributor to tissue inflammation and cell survival in response to non-infectious injury. Our preliminary data suggest that TLR4 signaling plays a critical role in cardioprotection against ischemic injury in the heart and in isolated cardiomyocytes. The goals of this proposal are to define the role of cardiac TLR4 in protecting cardiomvocvtes in models of ischemia-reperfusion injury (IRI) and to identify the downstream mechanisms that mediate these effects. We anticipate that insights gained from the proposed studies will serve as a foundation for the future development of novel therapeutic approaches for the management of ischemic myocardial injury. This proposal is based on the following three hypotheses: 1) that TLR4 activation via its signaling protein IRAK-1 represents an important survival mechanism in the heart, 2) that nitric oxide synthase 2 (NOS2) mediates the TLR4-induced survival benefits, and 3) that augmentation of cardiac TLR4 signaling will reduce myocardial damage and produce a meaningful functional rescue in IRI. To test these hypotheses, we will manipulate cardiac expression of TLR4 and IRAK-1 using genetically modified animals as well as adenoviral gene transfer. In Specific Aim 1: we will explore the mechanisms by which TLR4 protects cardiomyocytes in in vitro models of apoptosis. In Specific Aim 2: we will determine how NOS2 and NO contribute to the TLR4-mediated anti-apoptotic effects in isolated cardiomyocytes. In Specific Aim 3: we will ascertain the role of cardiac (vs. extra-cardiac) TLR4 in protecting the heart against IRI. In Specific Aim 4: we will evaluate the anatomic and functional consequences of manipulating IRAK-1 in mouse models of IRI. We will test the impact of IRAK-1 deletion or cardiac IRAK-1 expression (via gene transfer) on IRI. Defining the signaling pathways that control cardiomyocyte survival and learning to manipulate these pathways in the heart may provide novel approach for the treatment of some cardiac conditions such as ischemia injury and cardiomyopathy.