Oxygen Sensitive Signaling in Primary Cardiac Fibroblast

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DESCRIPTION (provided by applicant): Oxygen concentration ranges from 12 to below 0.5% in mammalian organs under normoxic conditions with approximately 14% in arterial blood and <10% in the myocardium. During mild hypoxia, myocardial O2 drops to about 1-3%. In response to chronic moderate hypoxia, cells adjust their normoxia set-point such that reoxygenation- dependent elevation of pO2 results in "perceived hyperoxia". O2 sensing has been actively studied for hypoxia. Reoxygenation, however, has been mostly studied in the context of oxidative injury. Cardiac fibroblasts (CF) orchestrate physiological (repair) and pathological (fibrosis) tissue remodeling. This proposal rests on a striking observation that CF, isolated from adult murine ventricle, cultured in 10% or 21% O2 ("elevated O2", relative to pO2 to which cells are adjusted in vivo), compared to 3% (mildly hypoxic), exhibit reversible growth inhibition and a phenotype indicative of differentiation. The working hypothesis is that O2, even in marginal relative excess of the pO2 to which cells are adjusted, results in activation of specific signal transduction pathways that alter the phenotype and biological function of CF. To test this hypothesis and establish its significance in the reoxygenated heart, we propose the following four specific aims: AIM 1:Determine whether elevated (10-21%) ambient O2-induced growth inhibition of CF is associated with differentiation phenotype. AIM 2: Examine the significance of p21Wafl/Cip1/Sdi1-induction in elevated (10-21%) ambient O2-induced growth inhibition and differentiation of CF. AIM 3: Characterize the role of TGFbeta and p38MAPK activation in cells exposed to elevated (10-21%) ambient O2 in conferring the cellular responses outlined in Aims 1 & 2. AIM 4: Characterize perceived hyperoxia (as defined in Aims 1-3) in the ischemia-reoxygenated (I-R) heart. This proposal has three fundamentally important significances. First, it offers a fresh look at ischemia-reoxygenation biology, centrally important in numerous cardiac disorders. That marginal relative elevation in pO2, compared to pO2 to which cells are adjusted during chronic moderate hypoxia, may serve as a signal to trigger cardiac fibroblast differentiation and tissue remodeling is a novel concept relevant to fibrosis and tissue repair related to reperfusion injury. Second, this proposal postulates that CF isolated from the heart are not "blind" to a change of pO2 from <10%O2 (in vivo normoxia) to 21% O2 (widely interpreted as in vitro normoxia). This raises a significant broad-based issue of controlling ambient O2 during cell culture. Finally, an isolated cell culture model to study chronic moderate hypoxia followed by reoxygenation is offered.