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 thought for decades 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 very recently proven to be a signaling molecule in its own right, affecting soluble guanylyl cyclase and cytochrome P450 activities, heat shock protein 70 and heme oxygenase-1 expression, and protecting against cardiac ischemia-reperfusion injury. Based on this recent evidence, we are seeking to characterize 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, then 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 24 hours later, 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, followed by Coomassie or silver staining. Protein spots were imaged and quantitated using PDQuest" software, excised and subjected to in-gel trypsin digestion. 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 peptide mass fingerprinting analysis was conducted by submission of peak lists to MASCOT. Using 2D gel electrophoresis, we have created preliminary 2D reference maps of cardiac proteomes of control Wistar rats and those systemically administered physiologic and therapeutic levels of nitrite. We have confirmed the identity of over a hundred isolated protein spots through in-gel digestion followed by MALDI-TOF MS and peptide mass fingerprinting or by LC-MS/MS analyses. Quantitative comparative analyses have revealed significant changes to cardiac protein expression upon treatment with nitrite. These changes consisted of both up- and down-regulation of steady-state protein levels, as well as apparent alterations in post-translational protein modification and have included proteins involved in cell structure, energy metabolism, redox balance, and chaperone activity, among others. Additionally, we have measured metabonomic changes and changes to the cellular redox status that we have correlated to these proteomics data. Specifically, we have detected changes in the ratio of reduced to oxidized ascorbate and glutathione, as well as changes in the levels of S-nitroso, N-nitroso, and heme-nitroso species in the tissues. The results obtained using this combined proteomics/metabonomics approach 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, due to its stability in vivo, may be able to act in a long-range endocrine fashion to establish this protective tone in the heart. Our integrated proteomic and metabonomic approach is a step toward elucidating the scope and mechanism of cardioprotection and the potential activity of this biological compound 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 manuscript describing our results is being prepared.