PROTEOMICS, METABONOMICS OF EFFECTS OF NITRITE TREATMENT ON THE HEART

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Nitrite, stable and abundant in vivo, was long thought to be biologically inert at physiological concentrations. Formed endogenously through oxidation of nitric oxide (an ubiquitous signaling molecule known to elicit broad biological effects) and derived extensively from dietary sources, it has itself recently proven to be a signaling molecule, affecting soluble guanylyl cyclase and cytochrome P450 activities, heat shock protein 70 and heme oxygenase-1 expression, and protecting against cardiac ischemia-reperfusion injury. We are therefore engaged in characterizing the impact of changes in systemic nitrite availability on the cardiac proteome, using 2D gel-based differential display followed by LC-MS/MS or MALDI-TOF MS and peptide mass fingerprinting analyses, and correlate these data to metabonomic studies. Male Wistar rats administered a single intraperitoneal injection of sodium nitrite (0.1, 1 and 10 mg/kg) were anesthetized after 24 h, perfused free of blood and their cardiac tissue harvested. Immediately after homogenization the concentrations of NO-related metabolites were determined by gas phase chemiluminescence and HPLC. Tissue aliquots were denatured in IEF buffer and subjected to 2D-PAGE analysis, with Coomassie or silver staining. Protein spots were imaged and quantitated using PDQuest" software, excised and subjected to in-gel trypsin digestion. The resulting peptides were eluted, de-salted and analyzed by MALDI-TOF MS or by LC-MS and MS/MS. Spectra were analyzed with MoverZ" or MassLynx" and ProteinLynx" software, and PMF analysis was conducted. We have created preliminary 2D-PAGE reference maps of cardiac proteomes of control and nitrite-administered Wistar rats. We have identified more than 100 isolated protein spots by PMF and/or LC-MS/MS analyses. Quantitative comparative analyses have revealed significant changes to cardiac protein expression upon treatment with nitrite, including both up- and down-regulation of steady-state protein levels, and apparent alterations in post-translational protein modification. The proteins are involved in cell structure, energy metabolism, redox balance, and chaperone activity, etc. We also measured metabonomic changes and changes to the cellular redox status and correlated these to the proteomics data. We detected changes in the ratio of reduced/oxidized ascorbate and glutathione, and in the levels of S- and N-nitroso, and heme-nitroso species in the tissues. The results indicate specific changes in expression of cellular stress response proteins triggered by nitrite that may confer resistance to further oxidative insults. Our data suggest that nitrite may be able to act in a long-range endocrine fashion to establish this protective tone in the heart. Our approach is a step toward elucidating the scope and mechanism of cardioprotection and the potential activity of NO with newly discovered importance in human health and medicine. Our data may have direct and immediate implications for current experimental therapeutic uses of nitrite and may provoke a reassessment of the impact of daily dietary intake of nitrite. A poster describing the results was presented at the 2007 ASMS meeting and the work was included in lectures at the HUPO meeting in Seoul in October, 2007 and the Biochemical Society symposium on "Bioanalysis related to oxidative stress" at the Univ of Exeter, UK, April 2008. A manuscript describing these results has recently been published in Circulation Research.