title: Deficient nitric oxide signalling impairs skeletal muscle growth and performance: involvement of mitochondrial dysregulation.
abstract: BACKGROUND
  Nitric oxide (NO), generated in skeletal muscle mostly by the neuronal NO synthases (nNOSμ), has profound effects on both mitochondrial bioenergetics and muscle development and function. The importance of NO for muscle repair emerges from the observation that nNOS signalling is defective in many genetically diverse skeletal muscle diseases in which muscle repair is dysregulated. How the effects of NO/nNOSμ on mitochondria impact on muscle function, however, has not been investigated yet.
  
  
  METHODS
  In this study we have examined the relationship between the NO system, mitochondrial structure/activity and skeletal muscle phenotype/growth/functions using a mouse model in which nNOSμ is absent. Also, NO-induced effects and the NO pathway were dissected in myogenic precursor cells.
  
  
  RESULTS
  We show that nNOSμ deficiency in mouse skeletal muscle leads to altered mitochondrial bioenergetics and network remodelling, and increased mitochondrial unfolded protein response (UPR(mt)) and autophagy. The absence of nNOSμ is also accompanied by an altered mitochondrial homeostasis in myogenic precursor cells with a decrease in the number of myonuclei per fibre and impaired muscle development at early stages of perinatal growth. No alterations were observed, however, in the overall resting muscle structure, apart from a reduced specific muscle mass and cross sectional areas of the myofibres. Investigating the molecular mechanisms we found that nNOSμ deficiency was associated with an inhibition of the Akt-mammalian target of rapamycin pathway. Concomitantly, the Akt-FoxO3-mitochondrial E3 ubiquitin protein ligase 1 (Mul-1) axis was also dysregulated. In particular, inhibition of nNOS/NO/cyclic guanosine monophosphate (cGMP)/cGMP-dependent-protein kinases induced the transcriptional activity of FoxO3 and increased Mul-1 expression. nNOSμ deficiency was also accompanied by functional changes in muscle with reduced muscle force, decreased resistance to fatigue and increased degeneration/damage post-exercise.
  
  
  CONCLUSIONS
  Our results indicate that nNOSμ/NO is required to regulate key homeostatic mechanisms in skeletal muscle, namely mitochondrial bioenergetics and network remodelling, UPR(mt) and autophagy. These events are likely associated with nNOSμ-dependent impairments of muscle fibre growth resulting in a deficit of muscle performance.
authors.affiliation: Unit of Clinical Pharmacology, National Research Council-Institute of Neuroscience, Department of Biomedical and Clinical Sciences "Luigi Sacco", University Hospital "Luigi Sacco", Università di Milano, Milano, Italy.
authors.forename: Clara
authors.initials: C
authors.lastname: De Palma
authors.affiliation: Unit of Clinical Pharmacology, National Research Council-Institute of Neuroscience, Department of Biomedical and Clinical Sciences "Luigi Sacco", University Hospital "Luigi Sacco", Università di Milano, Milano, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Morisi
authors.affiliation: Unit of Clinical Pharmacology, National Research Council-Institute of Neuroscience, Department of Biomedical and Clinical Sciences "Luigi Sacco", University Hospital "Luigi Sacco", Università di Milano, Milano, Italy.
authors.forename: Sarah
authors.initials: S
authors.lastname: Pambianco
authors.affiliation: Scientific Institute IRCCS Eugenio Medea, Bosisio Parini, Italy.
authors.forename: Emma
authors.initials: E
authors.lastname: Assi
authors.affiliation: Unit of Clinical Pharmacology, National Research Council-Institute of Neuroscience, Department of Biomedical and Clinical Sciences "Luigi Sacco", University Hospital "Luigi Sacco", Università di Milano, Milano, Italy.
authors.forename: Thierry
authors.initials: T
authors.lastname: Touvier
authors.affiliation: Scientific Institute IRCCS Eugenio Medea, Bosisio Parini, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Russo
authors.affiliation: Unit of Clinical Pharmacology, National Research Council-Institute of Neuroscience, Department of Biomedical and Clinical Sciences "Luigi Sacco", University Hospital "Luigi Sacco", Università di Milano, Milano, Italy.
authors.forename: Cristiana
authors.initials: C
authors.lastname: Perrotta
authors.affiliation: Dulbecco Telethon Institute at Venetian Institute of Molecular Medicine, Padova, Italy.
authors.forename: Vanina
authors.initials: V
authors.lastname: Romanello
authors.affiliation: Dulbecco Telethon Institute at Venetian Institute of Molecular Medicine, Padova, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Carnio
authors.affiliation: National Research Council-Institute of Neuroscience, Department of Medical Biotechnology and Translational Medicine, Università di Milano, Milano, Italy ; CNI@NEST, Italian Institute of Technology, Pisa, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Cappello
authors.affiliation: Unit of Clinical Pharmacology, National Research Council-Institute of Neuroscience, Department of Biomedical and Clinical Sciences "Luigi Sacco", University Hospital "Luigi Sacco", Università di Milano, Milano, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Pellegrino
authors.affiliation: Unit of Morphology, Department of Biomedical and Clinical Sciences "Luigi Sacco", Università di Milano, Milano, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Moscheni
authors.affiliation: Scientific Institute IRCCS Eugenio Medea, Bosisio Parini, Italy.
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Bassi
authors.affiliation: Dulbecco Telethon Institute at Venetian Institute of Molecular Medicine, Padova, Italy ; Department of Biomedical Science, Università di Padova, Padova, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Sandri
authors.affiliation: Unit of Clinical Pharmacology, National Research Council-Institute of Neuroscience, Department of Biomedical and Clinical Sciences "Luigi Sacco", University Hospital "Luigi Sacco", Università di Milano, Milano, Italy ; Department for Innovation in Biological, Agro-food and Forest Systems, Università della Tuscia, Viterbo, Italy.
authors.forename: Davide
authors.initials: D
authors.lastname: Cervia
authors.affiliation: Unit of Clinical Pharmacology, National Research Council-Institute of Neuroscience, Department of Biomedical and Clinical Sciences "Luigi Sacco", University Hospital "Luigi Sacco", Università di Milano, Milano, Italy ; Scientific Institute IRCCS Eugenio Medea, Bosisio Parini, Italy.
authors.forename: Emilio
authors.initials: E
authors.lastname: Clementi
keywords: Akt-FoxO3-Mul-1 axis; Akt-mTOR pathway; Autophagy; Fibre growth; Mitochondrial bioenergetics; Mitochondrial network; Muscle exercise; Muscle structure; Nitric oxide synthase and signalling; Unfolded protein response