Nitric oxide (NO) is a free radical and a signaling molecule in several pathways, produced by nitric oxide synthase (NOS) from the conversion of L-arginine to citrulline. Supplementation of L-arginine has been used to treat MELAS (mitochondrial encephalopathy with lactic acidosis and stroke like syndrome), a mitochondrial disease caused by the m.3243A>G mutation. Low levels of serum arginine and endothelium dysfunction have been reported in MELAS and this treatment may increase NO in endothelial cells and promote vasodilation, decreasing cerebral ischemia and strokes. Although clinical benefits have been reported, little is known about NO synthesis in MELAS. In this study we found that osteosarcoma derived cybrid cells with high levels of m.3243A>G had increased nitrite, an NO metabolite, and increased intracellular NO, demonstrated by an NO fluorescent probe (DAF-FM). Muscle vessels from patients with the same mutation had increased staining in NADPH diaphorase, suggestive of increased NOS. These results indicate increased production of NO in cells harboring the m.3243A>G, however no nitrated protein was detected by Western blotting. Further studies are necessary to clarify the exact mechanisms of L-arginine effect to determine the appropriate clinical use of this drug therapy.

Mitochondrial diseases are complex disorders caused by nuclear or mitochondrial DNA mutations, leading to oxidative phosphorylation deficiency and excessive production of reactive oxygen species (ROS). While ROS have been well established in the pathogenesis of these diseases, the role of reactive nitrogen species (RNS) remains unclear. In this study, we performed a quantitative analysis of muscle fibers to investigate the relationship between protein nitration and mitochondrial abnormalities (mitochondrial proliferation and cytochrome-c oxidase (COX) deficiency) and factors like genotype, muscle damage, and age. A total of 1961 muscle fibers (303 from 4 controls and 1658 from 29 patients with mitochondrial diseases) were analyzed by immunostaining for nitro-tyrosine. Contrary to previous findings, which identified nitro-tyrosine only in small muscle vessels, we observed a broader distribution affecting the sarcolemma and sarcoplasm. Using multivariate techniques, we identified a significant correlation between protein nitration and mitochondrial proliferation but found no associations with COX deficiency, age, muscle damage, or genotype. These findings suggest that nitrative stress may contribute to mitochondrial dysfunction or play a role in signaling processes that induce mitochondrial biogenesis. Our results provide new insights into the molecular mechanisms of mitochondrial diseases and highlight the potential relevance of protein nitration.