Task Progress:
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Histological analysis. We have begun to analyze cardiac morphology and cardiac oxidative stress of mice exposed to space radiation. Heart samples obtained from Dr. Azzam’s lab have been prepared for histochemical analysis, which have been obtained from mice exposed to 40Ca20+ or 16O ions, at 20 or 40 cGy, either in a single bolus or in fractionated irradiation. Currently, immunohistochemistry is being optimized for using antibodies that recognize protein carbonylation, which is a marker of oxidatively damaged proteins, and 4-hydroxynonenal (4HNE), which is produced by lipid peroxidation that can form adducts with proteins. As mitochondria are a primary source for producing reactive oxygen species (ROS), we are particularly interested in examining mitochondrial stress response proteins, which we predict will be upregulated during space radiation-induced oxidative stress. We will also look for evidence of major markers of radiation-induced cardiovascular disease such as fibrosis, inflammation, atherosclerosis, and other vascular changes.
Analysis of proteins mediating the mitochondrial stress response. We have examined mediators of the mitochondrial stress response. Our preliminary results show that 2 keys mediators are upregulated -- the mitochondrial ATP-dependent Lon protease, which is a mitochondrial protein quality control protease and mitochondrial transcription factor A (TFAM), which is the master regulator of mitochondrial DNA (mtDNA) maintenance, expression, and transmission. Using immunoblot analysis, upregulation was observed in cardiac tissue from mice that have been exposed to 40Ca20+ at 20 cGy of 1.35 GeV/n ions (LET ~85 keV/µm), delivered either acutely as a single bolus, or, in 3 fractions (1 fraction/day over three consecutive days to simulate low dose-rate IR. Under these conditions, we have also shown by Oxyblot analysis that there is an increase in oxidatively damaged protein in response to 40Ca20+ at 20 cGy fractionated, and H+ 20 cGy administered acutely. In addition to mediating the mitochondrial stress response, the upregulation of Lon and TFAM may also reveal the possibility that space radiation induced stress stimulates a Lon- and TFAM- dependent reprogramming of the bioenergetic and metabolic flux within cardiac myocytes.
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