Anoxia (lack of oxygen) followed by reoxygenation causes severe detrimental effects in a wide variety of medical conditions, including ischemic reperfusion injury and myocardial infarction. How animals sense anoxia- reoxygenation and prevent tissue injury are fundamental and unanswered issues. The transcription factor hypoxia inducible factor (HIF) is a key cell protector against anoxia-reoxygenation (A/R)-induced injury. The discovery of the C. elegans gene egl-9, which encodes an O2-sensing prolyl hydroxylase of HIF-1, has led to the identification of an evolutionarily conserved pathway central for maintaining O2 homeostasis in organisms from nematodes to humans. Inhibition of mammalian HIF hydroxylase homologs of EGL-9 strongly protects from myocardial ischemia and reperfusion injury. Using automated behavioral tracking under conditions of changing O2 concentrations, I discovered a locomotary behavior called the O2-ON response and have shown that the O2-ON response can model key aspects of mammalian tissue response to ischemia-reperfusion injury. EGL-9 is essential for the O2-ON response and mediates the effect of hypoxic preconditioning on the suppression of the O2-ON response. From a series of genetic screens, I discovered CYSL-1 as a new regulator of EGL-9 and a Cytochrome P450 enzyme that generates eicosanoid signaling molecules downstream of EGL-9 to control the O2-ON response. I also isolated C. elegans mutants that define additional novel regulators and targets of the EGL-9/HIF-1 pathway. The overall goal of this project is to clone the genes defined by these mutants and identify the novel conserved regulators of biological responses to A/R, which is modulated by the EGL-9 pathway, and determine the underlying molecular and cellular mechanisms. In the K99 phase of this project, I will establish and characterize C. elegans behavioral and cellular models for ischemia-reperfusion injury. In the R00 phase of this project, I will further determine the key mechanisms by which A/R causes the O2-ON response and identify novel conserved regulators and targets of the EGL-9 pathway, which mediates protection from A/R-induced cellular injury and behavioral response to A/R. Using combined molecular, cellular and behavioral analyses together with powerful genetic screens, I will systematically dissect the genetic pathways and define the fundamental mechanisms that regulate cellular and animal responses to A/R. With the support of and training opportunities provided by K99/R00, I plan to expand my current experimental and intellectual skills and develop expertise in areas of O2-related biology and diseases, which is vital to my career goal of directing an independent and successful research laboratory.
(relevance of this research to public health): Ischemic heart disease is the most common cause of adult death in the United States and in most industrialized countries around the world;ischemia-reperfusion injury remains a leading cause of organ failure associated with high morbidity and mortality. Using a novel C. elegans behavioral model of ischemia- reperfusion injury, these proposed studies should reveal fundamental conserved mechanisms of cellular responses to anoxia-reoxygenation and the molecular basis of how animal behaviors are regulated by O2 availability, as well as identify potential new therapeutic targets to help treat human disorders that involve anoxia-reoxygenation, such as ischemic reperfusion injury and myocardial infarction.