Evaluation of protein nitration in mitochondrial deficiency

Nitric oxide (NO) performs an important role in the regulation of several mitochondrial pathways, however it can also lead to nitrosative stress. The aim of this study was to evaluate the presence of protein nitration in muscle and cells with mitochondrial deficiency. We studied muscle biopsy specimens of patients with mitochondrial DNA (mtDNA) mutations (m.3243A>G, N=4; large scale deletions, N=4), muscle samples with no mitochondrial abnormalities (controls) and cybrid cells (with normal mtDNA and with the m.3243A>G mutation). Protein nitration was detected by immunofluorescence with anti-3-nitrotyrosine antibody. We also evaluated NO synthesis (NADPH diaphorase), Complex II and IV activities by histochemistry in muscle sections. NO production in cultured cells was assessed by espectrophotometry (Griess assay) and with a fluorimetric NO intracellular marker (DAF-FM). Only two muscle samples with m.3243A>G had positive immunoreactivity to the anti-3-nitrotyrosine antibody, located in muscle fibers with mitochondrial proliferaton and increased NOS. Cybrid cells with the same mutation also had nitrated protein in cytoplasm and higher concentrations of NO, demonstrated by increased production of nitrate/nitrite and detection of the intracelular NO marker. Our findings are suggestive that protein nitration can be associated with the m.3243A>G mutation, probably due to a higher NO production.