Integrated analysis of the involvement of nitric oxide synthesis in mitochondrial proliferation, mitochondrial deficiency and apoptosis in skeletal muscle fibres

Abstract

Nitric oxide (NO) is an important signaling messenger involved in different mitochondrial processes but only few studies explored the participation of NO in mitochondrial abnormalities found in patients with genetic mitochondrial deficiencies. In this study we verified whether NO synthase (NOS) activity was altered in different types of mitochondrial abnormalities and whether changes in mitochondrial function and NOS activity could be associated with the induction of apoptosis. We performed a quantitative and integrated analysis of NOS activity in individual muscle fibres of patients with mitochondrial diseases, considering mitochondrial function (cytochrome-c-oxidase activity), mitochondrial content, mitochondrial DNA mutation and presence of apoptotic nuclei. Our results indicated that sarcolemmal NOS activity was increased in muscle fibres with mitochondrial proliferation, supporting the relevance of neuronal NOS in the mitochondrial biogenesis process. Sarcoplasmic NOS activity was reduced in cytochrome-c-oxidase deficient fibres, probably as a consequence of the involvement of NO in the regulation of the respiratory chain. Alterations in NOS activity or mitochondrial abnormalities were not predisposing factors to apoptotic nuclei. Taken together, our results show that NO can be considered a potential molecular target for strategies to increase mitochondrial content and indicate that this approach may not be associated with increased apoptotic events.

NO control of mitochondrial function in normal and transformed cells

“…Studies using histochemical stainings also support that nNOS is upregulated as increased NOS activity was found in several types of mtDNA mutations and the activity was higher in muscle fibers with mitochondrial proliferation (99). …”

“…Fibers with COX deficiency express eNOS but had low NOS activity in the sarcoplasm, indicating that eNOS is down-regulated probably as a regulatory mechanism to increase ATP generation (97). This down-regulation was only found in the presence of COX deficiency, as other patients with Complex II or I deficiency did not have alterations in NOS activity (99). However, these mechanisms may be more complex because a study with cultured osteosarcoma derived cybrid cells with a mitochondrial deficiency due to the m.3243A>G mutation, showed an increase of intracellular NO and nitrate/nitrite in cultured media when compared the control cells (107).…”

Over Expression of NOS2 in Ragged-red Fibers from Patients with Mitochondrial Disorders due to Mutations in mtDNA

“…NOS3 was observed on myofibrils, in the sarcoplasm and in the subsarcolemmal region of RRFs with a distribution corresponding to SDH histochemical reactivity and therefore compatible with a mitochondrial localization of the protein [25,26]. Reduced NOS activity, detected as NADPH diaphorase (NADPHd) activity, was observed in the sarcoplasm of COX deficient fibers, irrespective of their mitochondrial content [26,27]. On the other hand, NADPHd histochemical staining was increased on the sarcolemma in fibers with abnormal mitochondrial proliferation (RRFs) and in COX negative fibers, and was higher in COX deficient fibers with mitochondrial proliferation (RRFs/COX-) compared with COX deficient fibers with a normal mitochondrial content (COX-) [27].…”

“…The expression and localization of NOS1 and NOS3 have been previously investigated in skeletal muscle of patients with MDs [25][26][27]. NOS1 was detected on the sarcolemma of muscle fibers with abnormal mitochondria proliferation as well as of normal muscle fibers, contrarily to previous observations reporting a stronger staining of NOS1 in the sarcolemma region of RRFs [25,26].…”

“…Reduced NOS activity, detected as NADPH diaphorase (NADPHd) activity, was observed in the sarcoplasm of COX deficient fibers, irrespective of their mitochondrial content [26,27]. On the other hand, NADPHd histochemical staining was increased on the sarcolemma in fibers with abnormal mitochondrial proliferation (RRFs) and in COX negative fibers, and was higher in COX deficient fibers with mitochondrial proliferation (RRFs/COX-) compared with COX deficient fibers with a normal mitochondrial content (COX-) [27]. These data suggested a positive correlation between NOS and COX sarcoplasmic activity and betweenNOS1 and NOS3 expression and mitochondrial content of myofibers [25,27].…”