RUOLO DELL'OSSIDO NITRICO NELLA REGOLAZIONE DEL DESTINO DELLE CELLULE STAMINALI MUSCOLARI

Nitric oxide (NO) is a free radical synthesized in various cells from L-arginine by a family of enzymes called the nitric oxide syntheses (NOS). Two NOS are constitutively expressed, the type I NOS, also classified as neuronal NOS and the type III or endothelial NOS. The type II, the inducible NOS, is expressed in inflammatory cells and is up-regulated in many pathological conditions. Their activity can be pharmacologically inhibited in vitro and in vivo by L-NAME while the NO effect can be mimicked by synthetic NO donor compounds. In the skeletal muscle NO is produced by a muscle specific isoform of neuronal NOS, the NOSmu, that is localized to the sarcolemma by association with alfa-synuclein into the dystrophin glycoprotein complex. Loss of dystrophin leads to NOS mislocalization and activity down-regulation; the absence of NO thus resulting is considered one of the causes of muscle damage in Duchenne Muscular Dystrophy. NO exerts different functions in muscle: it regulates muscle contraction, couples energy supply with demand, and controls force generation and resistance to fatigue; in addition, it regulates satellite cells activation and fusion. Satellite cells are a small quiescent population of myoblast precursors responsible for growth, maintenance and repair of postnatal skeletal muscle. They become activated in response to damage, start to proliferate and differentiate to form new fibres; some of this cells exit from the cell cycle and revert to quiescence by a self renewal mechanism which is essential for muscle homeostasis. Our laboratory has recently demonstrated that NO in association with non steroidal anti-inflammatory drugs has therapeutic effects in skeletal muscle dystrophy, controlling damage and regeneration. In this work we have evaluated the effect of NO in the regulation of satellite cells self-renewal, and we have studied the importance of this effect in maintenance of skeletal muscle regeneration capacity after different types of injuries. We have carried out in vitro experiments using single fibre cultures treated with the NO donor drug SIN-1 or the NOS inhibitor, L-NAME, and single myofibres isolated from age matched nNOS null mice; and in vivo analyses of two different model of muscle damage. For the chronic waste model we have used alfa-sarcoglycan null mice that shows many characters of Duchenne muscular dystrophy including the absents of a functional NOS, To investigate acute damage we have induced cardiotoxin (CTX) damage. In both models NO or the NOS inhibitor L-NAME were supplied the diet and we have also analysed nNOS null mice as genetic-related control. The results of our experiments demonstrate that NO ameliorates the muscle function and morphology enhancing regeneration and that this effect can be maintained long term. NO appears to increase the number of quiescent satellite cells involved in self-renewal mechanism, thus avoiding satellite cells pool exhaustion. NO exerts this effect at least in part by modulation of WNT pathway. Results obtained encourage the development of NO-based therapeutic strategies in skeletal muscle diseases treatment.