This is an amended application to study the basic mechanisms of action of nitric oxide (NO) in mitochondrial biogenesis in the liver in sepsis. NO synthase induction is fundamental to the sepsis-induced immune response but the production reactive nitrogen and oxygen species (RNS, ROS) is a stress that drives mitochondrial dysfunction and is important in the pathogenesis of multiple organ failure (MOF). We have discovered that RNS and ROS production in experimental sepsis damage mitochondrial DNA (mtDNA), thereby impairing mitochondrial transcription, proteome fidelity, and mitochondrial function. This mitochondrial damage stimulates cellular compensation involving mitochondrial biogenesis, which requires activation of mitochondrial transcription factor A (mtTFA) and two nuclear transcription factors, nuclear respiratory factor-1 and -2 (NRF-1 and -2), and a co-activator, PGC-1. These unique cell responses, under nuclear control, signify a crucial side of mitochondrial biology modulated by RNS and ROS, and one that is pro-survival. Though we still know very little about regulation of biogenesis in inflammation or its disruption in sepsis, our data show clearly that mitochondrial pathology is not limited simply to the NO-chemistry that damages mitochondria and kills cells. Therefore, we propose to test the hypothesis that iNOS-stimulated mitochondrial biogenesis opposes cell necrosis in sepsis, and thereby, regulation of biogenesis is an important determinant of cell survival. To test this hypothesis we propose three Specific Aims:
Aim 1 : Measure the contribution of iNOS to the pathogenesis of damage to hepatic mitochondrial DNA and proteins in sepsis using wild type and iNOS knockout mice;
Aim 2 : Define the importance of iNOS in mitochondrial transcription factor A (Tfam) activation and the restoration of hepatic mtDNA copy number and transcription in wild type and iNOS knockout mice in sepsis;
Aim 3 : Determine the contribution of iNOS to nuclear transcriptional activation of biogenesis in sepsis via NRF-1 and NRF-2 expression in wild type and iNOS knockout mice. This work will provide a better mechanistic understanding of the nuclear-mitochondrial communication during the host inflammatory response, which should help foster new molecular strategies to assess the ability of mitochondrial response markers to predict MOF and to guide interventions to prevent and eventually to treat sepsis-induced MOF. ? ?
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