MOLECULAR PATHOGENESIS OF CARDIAC DYSFUNCTION

这项建议的目的是确定感染性休克和热创伤后发生的心功能障碍的分子机制。以往的工作表明，心功能障碍是由细胞因子肿瘤坏死因子-α(TNF)介导的，肿瘤坏死因子-α是由心肌细胞在心肌局部产生的。这项建议利用新的分子和遗传策略来研究肿瘤坏死因子的有害作用的机制，并开发与肿瘤坏死因子相关的心脏贡献的治疗方法。首先，我们将研究转基因小鼠，在转基因小鼠中，肿瘤坏死因子仅由心肌细胞结构性表达。这些小鼠出现严重的心脏功能障碍、心肌病、心肌炎和心力衰竭，这与人类的心脏收缩功能障碍类似。通过将这些转基因动物培育到靶向干扰iNOS(诱导型一氧化氮合酶)、IRAK(IL-1受体相关激酶)和ICAM-1/P-选择素的小鼠，以及通过药物抑制特定的途径，我们将定量地确定iNOS、IL-1和迁移的白细胞在心力衰竭发病机制中的作用。心脏表型的主要特征将是在体外对小鼠心脏进行朗宁多夫灌流；功能的验证性纵向分析将通过心电门控磁共振成像在体内完成。生理学发现将与存活、死后组织学和心脏基因表达模式相关。接下来，我们将通过开发心脏特异的、可受饮食四环素调节的二元转基因系统来优化转基因动物模型。通过这个系统，我们将确定肿瘤坏死因子的作用是否与表达剂量和持续时间有关。我们将描述由肿瘤坏死因子诱导的次级细胞因子的级联反应。我们还将确定低水平的瞬时表达的肿瘤坏死因子是否可能是进化适应性的，并对随后的心脏损伤起到保护作用。通过了解肿瘤坏死因子阻碍心肌功能的分子机制，将有可能开发出特定的、有针对性的治疗策略，用于治疗脓毒症、烧伤创伤和其他与肿瘤坏死因子相关的心脏疾病，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.