The Role of Tfam in Mitochondrial Biogenesis following sepsis

描述（由申请人提供）：目的：该NRSA奖学金的目的是研究线粒体转录因子A （Tfam）在介导线粒体生物发生和预防败血症器官功能障碍中的作用，并促进Keenan博士作为一名科学家的发展。背景：脓毒症是美国第十大死亡原因，也是多器官功能障碍综合征的最常见原因。近年来，线粒体损伤在败血症的病理生理学中起着重要作用。线粒体生物发生，或恢复或增加细胞中功能性线粒体质量的过程，对败血症后的细胞恢复至关重要，并与危重患者的生存有关。它受多种刺激启动的双基因组转录程序调控，包括炎症和败血症，以响应增加的细胞能量需求。Tfam是一种核编码蛋白，促进线粒体DNA的转录和复制。它是线粒体生物发生程序的一个组成部分，但其在介导线粒体生物发生和预防败血症后器官功能障碍中的作用尚未明确。假设：假设在脓毒性攻击时肝脏Tfam的选择性丧失会抑制线粒体生物发生，导致肝细胞死亡增加，肝功能衰竭恶化和免疫抑制。方法：采用tfam敲除小鼠模型对上述假设进行验证。具体来说，Tfam在肝细胞中会有条件地沉默。然后，在实验小鼠和野生型对照小鼠腹膜植入金黄色葡萄球菌浸透的纤维蛋白凝块，诱导脓毒症。在脓毒症诱导后48小时内的不同时间点处死小鼠，取肝脏。通过实时PCR、Western blot和组织学分析，线粒体生物发生的激活、相关的抗炎途径以及肝细胞损伤和功能障碍的程度将被确定。预期结果：预计Tfam的缺失将导致线粒体生物发生减少，器官损伤和功能障碍增加，以及相关抗炎途径的过度激活。这将证明Tfam对脓毒症中线粒体生物发生和器官保护的激活至关重要。这一假设的证实将为未来的实验以及靶向治疗的发展奠定基础，以改善败血症的预后。

DESCRIPTION (provided by applicant): Objective: The goal of this NRSA fellowship is to investigate the role of mitochondrial transcription factor A (Tfam) in mediating mitochondrial biogenesis and preventing organ dysfunction in sepsis and to promote the development of Dr. Keenan as a scientist. Background: Sepsis is the 10th leading cause of death in the United States and the most common cause of multiple organ dysfunction syndromes. In recent years, it has become clear that mitochondrial damage is central to the pathophysiology of sepsis. Mitochondrial biogenesis, or the process of restoring or increasing functional mitochondrial mass in the cell, is critical for cellular recovery following sepsis and has been linked to surviva in critically ill patients. It is regulated by a bi-genomic transcriptional program initiated by a diverse array of stimuli, including inflammation and sepsis, in response to increased cellular energy requirements. Tfam is a nuclear encoded protein that facilitates transcription and replication of mitochondrial DNA. It is a component of the mitochondrial biogenesis program, but its role in mediating mitochondrial biogenesis and preventing organ dysfunction following sepsis has not been characterized. Hypothesis: It is hypothesized that the selective loss of hepatic Tfam upon septic challenge will inhibit mitochondrial biogenesis resulting in increased hepatocyte cell death, worsening hepatic failure, and immune suppression. Methods: The hypothesis will be tested by using a Tfam-knockout mouse model. Specifically, Tfam will be conditionally silenced in the hepatocytes. Sepsis will then be induced by the peritoneal implantation of a S. aureus impregnated fibrin-clot in experimental and wild-type control mice. The mice will be sacrificed at different time points over 48 hours from the time of induction of sepsis and the livers will be harvested. Using real-time PCR, Western blot, and histologic analysis, the activation of mitochondrial biogenesis, related anti- inflammatory pathways, and the extent of hepatocyte damage and dysfunction will be determined. Expected Results: It is expected that the loss of Tfam will result in reduced mitochondrial biogenesis increased organ damage and dysfunction as well as hyper-activation of linked anti-inflammatory pathways. This will demonstrate Tfam essential for the activation of mitochondrial biogenesis and organ protection in sepsis. Confirmation of this hypothesis will form the basis for future experimentation as well as the development of targeted therapies to improve outcomes in sepsis.