The Role of Tfam in Mitochondrial Biogenesis following sepsis

描述（由申请人提供）：目的：该NRSA奖学金的目标是研究线粒体转录因子A（Tfam）在介导线粒体生物合成和预防脓毒症器官功能障碍中的作用，并促进Keenan博士作为科学家的发展。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，线粒体损伤在脓毒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，对于脓毒症后的细胞恢复至关重要，并且与危重患者的存活有关。它是由一个双基因组转录程序启动的一系列不同的刺激，包括炎症和脓毒症，以响应增加的细胞能量需求。Tfam是一种促进线粒体DNA转录和复制的核编码蛋白。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脓毒症后器官功能障碍中的作用尚未得到表征。假设：假设脓毒症激发后肝Tfam的选择性损失将抑制线粒体生物合成，导致肝细胞死亡增加、肝衰竭恶化和免疫抑制。方法：将通过使用Tfam敲除小鼠模型来检验该假设。具体而言，Tfam将在肝细胞中条件性沉默。然后通过腹膜植入S.金黄色葡萄球菌浸渍的纤维蛋白凝块在实验和野生型对照小鼠中。在诱导脓毒症后48小时内的不同时间点处死小鼠，并收获肝脏。使用实时PCR、蛋白质印迹和组织学分析，将确定线粒体生物合成的激活、相关抗炎途径以及肝细胞损伤和功能障碍的程度。预期结果：预计Tfam的缺失将导致线粒体生物合成减少、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对于脓毒症中线粒体生物合成和器官保护的激活至关重要。这一假设的证实将成为未来实验的基础，以及发展靶向治疗以改善脓毒症的结局。

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.