MOLECULAR PATHOGENESIS OF CARDIAC DYSFUNCTION

这项建议的目的是确定在感染性休克和热创伤后发生的心功能障碍的分子机制。先前的研究表明，心功能障碍是由细胞因子肿瘤坏死因子- α (TNF)介导的，该因子由心肌细胞在心肌中局部产生。本建议利用新的分子和遗传策略来研究TNF有害作用的机制，并开发与TNF相关的心脏贡献的治疗方法。首先，我们将研究TNF仅由心肌细胞组成表达的转基因小鼠。这些小鼠发展为严重的心功能障碍、心肌病、心肌炎和心力衰竭，模仿人类的心脏收缩功能障碍。通过将这些转基因动物培育到靶向破坏iNOS（诱导性一氧化氮合酶）、IRAK （IL-1受体相关激酶）和ICAM-1 / p -选择素的小鼠身上，以及通过药物抑制特定途径，我们将定量确定iNOS、IL-1和转运白细胞在心肌衰竭发病机制中的作用。心脏表型将主要通过离体小鼠心脏的体外Langendorff灌注来表征；通过心电图门控核磁共振成像在体内完成功能的纵向验证分析。生理结果将与生存、死后组织学和心脏基因表达模式相关。下一步，我们将优化转基因动物模型，建立一个心脏特异性的、可由膳食四环素调节的二元转基因系统。通过这个系统，我们将确定TNF的作用是否与剂量和表达时间有关。我们将描述TNF诱导的次级细胞因子级联反应。我们还将确定低水平的、短暂的TNF表达是否具有进化适应性，并对随后的心脏损伤起到保护作用。通过了解TNF阻碍心肌表现的分子机制，将有可能开发出特异性的、有针对性的治疗策略，用于治疗败血症、烧伤创伤和其他与TNF相关的心脏病，如心肌病、心肌炎和缺血性心脏病。

The goal of this proposal is to determine the molecular mechanisms of cardiac dysfunction that occurs during septic shock and following thermal trauma. Previous work has demonstrated that cardiac dysfunction is mediated by the cytokine tumor necrosis factor-alpha (TNF), which is produced locally in the myocardium by cardiac myocytes. This proposal utilizes novel molecular and genetic strategies to investigate the mechanisms of TNF's detrimental effects and to develop therapeutic approaches for TNF-related cardiac contributions. First, we will study transgenic mice in which TNF is constitutively expressed only by cardiac myocytes. These mice develop profound cardiac dysfunction, cardiomyopathy, myocarditis, and cardiac failure which mimics cardiac contractile dysfunction in humans. By breeding these transgenic animals to mice which have undergone targeted disruption of iNOS (inducible nitric oxide synthase), IRAK (IL-1 receptor associated kinase), and ICAM-1 / P-selectin, as well as by pharmacological inhibition of specific pathways, we will quantitatively determine the involvement of iNOS, IL-1, and transmigrated leukocytes in the pathogenesis of myocardial failure. Cardiac phenotype will be characterized primarily by in vitro Langendorff perfusion of isolated mouse hearts; confirmatory longitudinal analysis of function will be accomplished in vivo by ECG-gated MRI imaging. Physiologic findings will be correlated with survival, post-mortem histology, and the pattern of cardiac gene expression. Next, we will optimize the transgenic animal model by developing a binary transgene system which is cardiac specific, and regulatable by dietary tetracycline. Through this system, we will determine if the effects of TNF are related to dose and duration of expression. We will describe the cascade of secondary cytokines induced by TNF. We will also determine whether low-level, transient expression of TNF may be evolutionary adaptive, and serve a protective role against subsequent cardiac insults. By understanding the molecular mechanisms by which TNF impedes myocardial performance, it will be possible to develop specific, targeted therapeutic strategies for the treatment of sepsis, burn trauma, and other TNF-related cardiac conditions such as cardiomyopathy, myocarditis, and ischemic heart disease.