Peroxynitrite (ONOO-) formed by the diffusion-limited reaction of superoxide with nitric oxide is a major mechanism of oxidative damage in the heart. Isolated heart muscle exposed to micromolar concentrations of peroxynitrite for only a few minutes exhibit many of the pathological changes characteristic of clinical heart disease, including injury to mitochondria, loss of contractile function and irreversible increases in diastolic stiffness. One of the major consequences of peroxynitrite attack is the selective nitration of tyrosine residues in specific proteins. Myocytes in rat heart become nitrated quickly during ischemia/reperfusion and inhibition of nitric oxide production both preserves myocardial function and reduces tyrosine nitration. In the human heart, similar patterns of myocyte nitration occur after ischemia, sepsis and myocarditis. Quantitative HPLC analysis revealed 1-7% of protein-bound tyrosines are nitrated in septic patients, showing nitration is a significant pathological process in humans. Western blots demonstrate that a surprisingly limited number of proteins are vulnerable to tyrosine nitration by peroxynitrite. The principal investigator has shown that manganese superoxide dismutase, actin and intermediate filaments are nitrated in human autopsy material and that the function of these proteins is markedly diminished by peroxynitrite in vitro. Electrospray mass spectrometry has revealed that peroxynitrite preferentially nitrates certain tyrosine motifs in hydrophilic regions common to many structural proteins. The principal investigator hypothesizes that tyrosine nitration of structural proteins by peroxynitrite contributes to myocardial injury.
His first aim i s to quantitatively assess the magnitude of tyrosine nitrosation in ischemic rat heart and to directly measure peroxynitrite using a novel probe they have developed.
The second aim i s to identify which specific proteins are nitrated in rat and human heart and determine by tandem mass spectrometry which tyrosines in these proteins are nitrated.
The third aim i s to characterize how nitration affects the function of actin and then other structural proteins to assess how nitration can affect myocontractile proteins. These studies are a new and novel approach to understanding the specific mechanisms by which biologically generated oxidants injure the heart and allow us to identify major targets for peroxynitrite attack in vivo.
