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Deficient nitric oxide signalling impairs skeletal muscle growth and performance: involvement of mitochondrial dysregulation

Deficient nitric oxide signalling impairs skeletal muscle growth and performance: involvement of... 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 mt network remodelling, and increased mitochondrial unfolded protein response (UPR ) 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 mt muscle, namely mitochondrial bioenergetics and network remodelling, UPR and autophagy. These events are likely associated with nNOSμ-dependent impairments of muscle fibre growth resulting in a deficit of muscle performance. Keywords: Nitric oxide synthase and signalling, Mitochondrial bioenergetics, Mitochondrial network, Unfolded protein response, Autophagy, Akt-mTOR pathway, Akt-FoxO3-Mul-1 axis, Fibre growth, Muscle structure, Muscle exercise * Correspondence: d.cervia@unitus.it; emilio.clementi@unimi.it 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 Full list of author information is available at the end of the article © 2014 De Palma et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. De Palma et al. Skeletal Muscle 2014, 4:22 Page 2 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 Background excitation contraction coupling and prevent atrophy Nitric oxide (NO) is a gas and a messenger with pleio- [28,29]. In addition, mitochondria are involved in regulat- tropic functions in most tissues and organs, synthesized ing autophagy [30], whose derangement plays a role in a by a family of NO synthases. NO is also generated in number of inherited muscle diseases [31-33]. Mitochon- skeletal muscle, in particular by the muscle-specific drial protein homeostasis is maintained through proper neuronal NO synthases (nNOS or NOS1) [1,2]. nNOSμ folding and assembly of polypeptides. This involves the mt is the predominant nNOS isoform in muscle and is an- mitochondrial unfolded protein response (UPR ), a stress chored to the sarcolemma as a component of the dys- response that activates transcription of nuclear-encoded trophin glycoprotein complex [3]. This enzyme produces mitochondrial chaperone genes to maintain proteins in a NO at low, physiological levels (in the pico to nanomolar folding or assembly-competent state, preventing deleteri- range) in a way controlled by second messengers [1,2]; ous protein aggregation [34-36]. its expression is increased by crush injury, muscle activ- In this study we have examined the relationship between ity and ageing [4,5]. NO has an important role in regu- the NO system, mitochondrial structure/activity and skel- lating skeletal muscle physiological activity, including etal muscle phenotype/growth/functions using a mouse excitation-contraction coupling, muscle force generation, model in which nNOSμ is absent (NOS1-/-). Also, NO- auto-regulation of blood flow, calcium homeostasis, me- induced effects and the NO pathway were dissected in tabolism and bioenergetics [2,6,7]. In addition, it is a key myogenic precursor cells. Our results indicate that the determinant in myogenesis that it regulates at several deficit in NO signalling leads in skeletal muscle to alter- key steps, especially when the process is stimulated to ations in mitochondrial morphology, bioenergetics and repair muscle damage after injury [5,8,9]. network remodelling, accompanied by defective autophagy mt The importance of NO in muscle repair also emerges and the induction of a UPR response. These events, from the observation that nNOS signalling is defective in while not severely altering the overall resting skeletal many genetically diverse skeletal muscle diseases in which muscle structure, are associated with modifications in the muscle repair is dysregulated, including Duchenne muscu- Akt-mammalian target of rapamycin (mTOR) pathway lar dystrophy, Becker muscular dystrophy, limb-girdle and Akt-FoxO3-mitochondrial E3 ubiquitin protein ligase muscular dystrophies 2C, 2D and 2E, Ullrich congenital 1 (Mul-1) axis and are sufficient to dysregulate skeletal muscular dystrophy and inflammatory myositis [3,10-13]. muscle growth and exercise performance. Based on this evidence and on the fact that the restoration of NO signalling by nNOS overexpression ameliorates muscle function [14,15], genetic and pharmacologic strat- Methods egies to boost nNOS/NO signalling in dystrophic muscle Animals are being tested with encouraging results: in particular, the NOS1-/- animals are mice homozygous for targeted combination of NO donation with non steroidal anti- disruption of the nNOS gene (strain name B6129S4- tm1Plh inflammatory activity limits muscle damage and favours NOS1 /J) that were purchased from Jackson Labora- muscle healing in vivo [16-18] such thatitis currently be- tories (Bar Harbor, Maine, USA) (stock no. 002633). In ing tested as a therapeutic for Duchenne muscular dys- this mouse line, targeted deletion of exon 2 specifically trophy in humans [19,20]. eliminates expression of nNOSμ [37]. NOS1-/- mice were The observation that nNOS is localised in close prox- crossed with the wild-type B6129 to maintain the original imity to mitochondria suggests a tight coupling between background and to obtain a colony of NOS1-/- mice and NO generation and regulation of mitochondrial respir- wild-type littermate controls, with genotyping performed ation and metabolism. The role of NO in regulating oxi- from tail clippings. Experiments were performed on male dative phosphorylation and mitochondrial biogenesis in mice at postnatal day 10 (P10) and P120. C57BL/6 wild- skeletal muscle physiology has been established [21-24]. type mice (strain name C57Bl10SnJ) were purchased from Likewise NO-dependent inhibition of mitochondrial fis- Charles River (Calco, Italy). Animals were housed in a reg- sion occurs during myogenic differentiation [25]. ulated environment (23 ± 1°C, 50 ± 5% humidity) with a How the effects of NO on mitochondria impact on 12-hour light/dark cycle (lights on at 08.00 a.m.), and pro- muscle function, however, has not been investigated yet. vided with food and water ad libitum. For specific experi- Elucidation of this aspect is relevant in view of the role ments, mice were killed by cervical dislocation. All studies that mitochondria play in muscle pathophysiology and were conducted in accordance with the Italian law on ani- may shed light on the muscular disorders in which NO mal care N° 116/1992 and the European Communities signalling is impaired [26]. In particular, increases in Council Directive EEC/609/86. The experimental pro- mitochondria number and oxidative phosphorylation ac- tocols were approved by the Ethics Committee of the tivity is relevant during differentiation [27] and the bal- University of Milano. All efforts were made to reduce both ance of fission and fusion is necessary to preserve animal suffering and the number of animals used. De Palma et al. Skeletal Muscle 2014, 4:22 Page 3 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 Mitochondrial membrane potential 225 mM sucrose, 44 mM KH PO and 6 mM EDTA. 2 4 Mitochondrial membrane potential in isolated transfected Total oxidative phosphorylation (OXPHOS)-ATP in iso- fibres from flexor digitorum brevis muscles was measured lated mitochondria was measured by the luciferin-luciferase by epifluorescence microscopy based on the accumulation method, as described, with slight modifications [25]. Briefly, of tetramethylrhodamine methyl ester (TMRM) fluores- mitochondria were plated in 96 wells and treated with cence [25,29,38]. Briefly, flexor digitorum brevis myofibres buffer-A (150 mM KCl, 25 mM Tris-HCl, 2 mM EDTA, were placed in 1 ml Tyrode’s buffer and loaded with 5 nM 0.1% bovine serum albumin, 10 mM KH PO and 0.1 mM 2 4 TMRM supplemented with 1 μM cyclosporine H for MgCl (pH 7.4) containing 0.8 M malate, 2 M glutamate, 30 minutes at 37°C. Myofibres were then observed with an 500 mM ADP, 100 mM luciferin and 1 mg/ml luciferase. Olympus IX81 inverted microscope equipped with a CellR Oligomycin (2 μg/ml) was also used to detect the presence imaging system (Olympus, Tokio, Japan). Sequential im- of glycolytic ATP. OXPHOS-ATP was measured using a ages of TMRM fluorescence were acquired every 60 - GloMax luminometer (Promega, Milan, Italy). seconds with a × 20 0.5, UPLANSL N A objective (Olympus). When indicated, oligomycin (5 μM) or the High-resolution respirometry protonophore carbonylcyanide-p-trifluoromethoxyphenyl Respiratory chain defects were assessed in tibialis anterior hydrazone (FCCP, 4 μM) was added [39]. Images were ac- and diaphragm fibre bundles using published protocols quired and stored, and analysis of TMRM fluorescence [40-42]. After transferring the tissue sample into ice-cold over mitochondrial regions of interest was performed BIOPS (10 mM CaK ethyleneglycoltetraacetic acid (EGTA) using ImageJ software (http://rsbweb.nih.gov/ij/). buffer, 7.23 mM K EGTA buffer, 0.1 μM free calcium, 20 mM imidazole, 20 mM taurine, 50 mM 2-(N-morpho- Primary myogenic cell cultures lino)ethanesulfonic acid hydrate, 0.5 mM dithiothreitol, Using published protocols [25], myogenic precursor cells 6.5 mM MgCl 6H O, 5.7 mM ATP and 15 mM phospho- 2 2 (satellite cells) were freshly isolated from the muscles of creatine (pH 7.1)), connective tissue was removed and the newborn C57BL/6 mice. When indicated, cells were ob- muscle fibres were mechanically separated. Complete per- tained from NOS1-/- mice and wild-type littermate con- meabilisation of the plasma membrane was ensured by trols. Briefly, hind limb muscles were digested with 2% gentle agitation for 30 minutes at 4°C in 2 ml of BIOPS so- collagenase-II and dispase for 10 minutes at 37°C with lution containing 50 μg/ml saponin. The fibre bundles were gentle agitation. Contamination by non-myogenic cells rinsed by agitation for 10 minutes in ice-cold mitochondrial was reduced by pre-plating the collected cells onto plas- respiration medium (MiR05; 0.5 mM EGTA, 3 mM MgCl , tic dishes where fibroblasts tend to adhere more rapidly. 60 mM K-lactobionate, 20 mM taurine, 10 mM KH PO , 2 4 Dispersed cells were then resuspended in Iscove’s modi- 20 mM Hepes, 110 mM sucrose and 1 g/l bovine serum fied Dulbecco’s medium supplemented with 20% foetal albumin (pH 7.1). The permeabilised muscle fibres were bovine serum, 3% chick embryo extract (custom made), weighedand addedtoanOxygraph-2krespiratorycham- 10 ng/ml fibroblast growth factor, 100 U/ml penicillin, ber (Oroboros Instruments, Innsbruck, Austria) containing 100 μg/ml streptomycin and 50 μg/ml gentamycin, and 2 ml of MiR06 (MiR05 supplemented with 280 U/ml cata- plated onto matrigel-coated dishes. Differentiation was lase at 37°C). Oxygen flux per muscle mass was recorded induced by changing the medium to Iscove’s modified online using DatLab software (Oroboros Instruments). Dulbecco’s medium supplemented with 2% horse serum After calibration of the oxygen sensors at air saturation, a and the antibiotics. few μlofH O were injected into the chamber to reach a 2 2 concentration of 400 μMO . In order to detect the elec- Measurement of ATP formation tron flow through CI and CII mitochondrial complexes, Tibialis anterior and diaphragm muscles were dissected, titrations of all of substrates, uncouplers and inhibitors were trimmed clean of visible fat and connective tissue, added in series as previously described [41,42]. The meas- minced with scissors and digested in ATP medium, con- urement of CIV respiration was obtained by addition of the taining 50 mM Tris-HCl (pH 7.4), 100 mM KCl, 5 mM artificial substrates N,N,N,N ’ ’-tetramethyl-p-phenylenediamine MgCl , 1.8 mM ATP, 1 mM ethylenediaminetetraacetic dihydrochloride and ascorbate [40]. Oxygen fluxes were acid (EDTA), and 0.1% collagenase type V for 10 minutes corrected by subtracting residual oxygen consumption at 37°C under strong agitation. After centrifugation, the from each measured mitochondrial steady-state. Respi- pellet was homogenised with Ultra-Turrax T10 (Ika-lab, rometry measurements were performed in duplicate on Staufen, Germany) for 10 seconds at maximum speed in each specimen. ATP medium. The mitochondrial fraction, obtained by different centrifugations (380 g and 10,000 g for five Real-time quantitative PCR minutes at 4°C), was then suspended in a mitochondria Satellite cells and muscle tissue samples were homoge- resuspension buffer containing 12.5 mM Tris acetate, nised, and RNA was extracted using the TRIzol protocol De Palma et al. Skeletal Muscle 2014, 4:22 Page 4 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 (Invitrogen-Life Technologies, Monza, Italy). Using pub- In vivo imaging using two-photon confocal microscopy lished protocols [43], after solubilisation in RNase-free Mitochondrial morphology and autophagosome forma- water, first-strand cDNA was generated from 1 μgof tion in living animals were monitored in tibialis anterior total RNA using the ImProm-II Reverse Transcription muscles transfected by electroporation with plasmids en- System (Promega). As show in Table 1, a set of primer coding pDsRed2-Mito or the LC3 protein fused to the pairs amplifying fragments ranging from 85 to 247 bp yellow fluorescent protein (YFP-LC3), as described pre- was designed to hybridise to unique regions of the ap- viously [29,38,46]. Two-photon confocal microscopy in propriate gene sequence. Real-time quantitative PCR the live, anaesthetised animals was then performed (qPCR) was performed using the SYBR Green Supermix 12 days later on in situ exposure of transfected muscles (Bio-Rad, Hercules, CA, USA) on a Roche LightCycler [29,38,46]. To allow the muscle to recover from the 480 Instrument (Roche, Basel, Switzerland). All reactions injection-induced swelling, microscopic observation was were run in triplicate. A melt-curve analysis was per- interrupted for two to five minutes. formed at the end of each experiment to verify that a single product per primer pair was amplified. As a con- Transmission electron microscopy trol experiment, gel electrophoresis was performed to Tibialis anterior muscles were dissected and fixed for verify the specificity and size of the amplified qPCR one hour in a solution containing 4% paraformaldehyde products. Samples were analysed using the Roche Light- and 0.5% glutaraldehyde in 0.1 M cacodylate buffer, Cycler 480 software and the second derivative maximum pH 7.4, immobilised on a Nunc Sylgard coated Petri dish method. The fold increase or decrease was determined (ThermoFisher Scientific, Waltham, MA, USA) to pre- relative to a calibrator after normalising to 36b4 (internal vent muscular contraction as previously described [47]. -ΔΔCT standard) through the use of the formula 2 [44]. The muscles were rinsed in the same buffer and dis- Mitochondrial DNA (mtDNA) from muscle tissue sected further into small blocks that were subsequently samples was quantified as described with slight modifi- processed for transmission electron microscopy (TEM) cations [45]. Briefly, total DNA was extracted with the as described elsewhere [48]. Briefly, the samples were QIAamp DNA mini kit (Qiagen, Milano, Italy). Twenty postfixed with osmium tetroxide (2% in cacodylate buf- ng of total DNA was assessed by qPCR. RNaseP gene fer), rinsed, en bloc stained with 1% uranyl acetate in was used as an endogenous control for nuclear DNA 20% ethanol, dehydrated and embedded in epoxy resin and the cytochrome b gene as a marker for mtDNA. (Epon 812; Electron Microscopy Science, Hatfield, PA, Primer sequences are shown in Table 1. USA) that was baked for 48 hours at 67°C. Thin sections Table 1 Primer pairs designed for qPCR analysis Name/symbol Gene accession Number Primer sequence Amplicon Atg4b NM_174874 F: 5′-ATTGCTGTGGGGTTTTTCTG-3′ 247 bp R: 5′-AACCCCAGGATTTTCAGAGG-3′ Atrogin-1 (fbxo32) NM_026346 F: 5′-GCAAACACTGCCACATTCTCTC-3′ 93 bp R: 5′-CTTGAGGGGAAAGTGAGACG-3′ Bnip3 NM_009760 F: 5′-TTCCACTAGCACCTTCTGATGA-3′ 150 bp R: 5′-GAACACGCATTTACAGAACAA-3′ Cytochrome b (mt-cytb) NC_005089 F: 5′-ACGCCATTCTACGCTCTATC-3′ 95 bp R: 5′-GCTTCGTTGCTTTGAGGTGT-3′ MuRF1 (Trim63) NM_001039048 F: 5′-ACCTGCTGGTGGAAAACATC-3′ 96 bp R: 5′-CTTCGTGTTCCTTGCACATC-3′ MUSA1 (fbxo30) NM_001168297, NM_027968 F: 5′-TCGTGGAATGGTAATCTTGC-3′ 191 bp R: 5′-CCTCCCGTTTCTCTATCACG-3′ p62 (Sqstm1) NM_011018 F: 5′-GAAGCTGCCCTATACCCACA-3′ 85 bp R: 5′-AGAAACCCATGGACAGCATC-3′ RNaseP (Rpp30) NM_019428 F: 5′-GAAGGCTCTGCGCGGACTCG-3′ 100 bp R: 5′-CGAGAGACCGGAATGGGGCCT-3′ 36b4 (Rplp0) NM_007475 F: 5′-AGGATATGGGATTCGGTCTCTTC-3′ 143 bp R: 5′-TCATCCTGCTTAAGTGAACAAACT-3′ F: forward, R: reverse. De Palma et al. Skeletal Muscle 2014, 4:22 Page 5 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 were obtained with a Leica ultramicrotome (Reichert (GeneTex, Irvine, CA, USA), goat polyclonal anti-actin Ultracut E and UC7; Leica Microsystems, Wetzlar, (I-19) or rabbit polyclonal anti-GAPDH (FL-335) primary Germany) stained with uranyl acetate and lead citrate, antibodies (Santa Cruz Biotechnology). When appropriate, and finally examined with a Philips CM10 TEM (Philips, rabbit polyclonal anti-FoxO3a (75D8), rabbit monoclonal Eindhoven, The Netherlands). Morphometric analysis of S6 ribosomal protein (54D2), rabbit polyclonal 4E-BP1 mitochondrial cristae complexity was evaluated with a (53H11), and rabbit polyclonal Akt primary antibodies stereological method. Briefly, a regular grid has been (Cell Signaling Technology) that recognise the protein in- superimposed over 10500X TEM micrographs and the dependently of its phosphorylation state were also used in number of intersections between the grid and mitochon- reprobing experiments. drial cristae was recorded. The same grid was used for all the different analysis. Confocal microscopy of myogenic precursor cells Cells were plated in eight-well Nunc LabTeck Chamber Protein isolation and western blotting slides (ThermoFisher Scientific). When indicated cells Satellite cells were harvested and homogenised for were transfected with YFP-LC3 plasmid. Transfections 10 minutes at 4°C in RIPA lysis buffer, containing 50 mM were performed with the Lipofectamine LTX with Plus Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 1% sodium reagent (Invitrogen-Life Technologies) according to the deoxycholate, 1 mM EDTA and 0.1% sodium dodecyl manufacturer’s instructions. The cells were used 24 hours sulphate (SDS). Tissue samples from muscles were homo- after transfection in the various experimental settings genised in a lysis buffer containing 20 mM Tris-HCl described. For confocal imaging, the cells were fixed in (pH 7.4), 150 mM NaCl, 1% Triton X-100, 10% glycerol, paraformaldehyde and washed in phosphate-buffered sa- 10 mM EGTA and 2% SDS. Buffers were supplemented line [50]. To prevent nonspecific background, cells were with a cocktail of protease and phosphatase inhibitors incubated in 10% goat serum/phosphate-buffered saline (cOmplete and PhosSTOP; Roche). Protein concentration followed by probing with the primary antibody mouse was determined using the bicinchoninic acid assay (Ther- monoclonal anti-cyclophillin D (Abcam). Cells were then moFisher Scientific). Using published protocols [49], SDS incubated with the secondary antibody, Alexa Fluor 546 and β-mercaptoethanol were added to samples before dye-conjugated anti-mouse IgG (Molecular Probes-Life boiling, and equal amounts of proteins (40 μg/lane) were Technologies, Monza, Italy). Slides were placed on the separated by 4% to 20% SDS-polyacrylamide gel elec- stage of a TCS SP2 Laser-Scanning Confocal microscope trophoresis (Criterion TGX Stain-free precast gels and (Leica Microsystems) equipped with an electronically Criterion Cell system; Bio-Rad). Proteins were then trans- controlled and freely definable Acousto-Optical Beam ferred onto a nitrocellulose membrane using a Bio-Rad Splitter. Images were acquired with x63 magnification Trans-Blot Turbo System. The membranes were probed oil-immersion lenses. Analyses were performed using using the following primary antibodies as indicated in the Imagetool software (Health Science Center, University of text: goat polyclonal anti-HSP60 (N-20) and rabbit poly- Texas, San Antonio, TX, USA). Images of cells express- clonal anti-MyoD (C-20) (Santa Cruz Biotechnology, ing YFP-LC3 were thresholded by using the automatic Dallas, TX, USA), mouse monoclonal anti-ClpP and rabbit threshold function. polyclonal anti-LC3B (Sigma-Aldrich, Saint Louis, MO, USA), rabbit polyclonal anti-Mul-1 (Abcam, Cambridge, Immunohistochemistry and histology UK), mouse monoclonal anti-sarcomeric myosin (MF20) Laminin and haematoxylin and eosin (H & E) staining (Developmental Studies Hybridoma Bank, Iowa City, IA, were performed as previously described [47,51]. To meas- USA), rabbit polyclonal anti-phospho-FoxO3a (Ser253), ure the cross sectional area (CSA) of myofibres, muscle rabbit polyclonal anti-phospho-S6 ribosomal protein sections were stained with an anti-laminin A antibody (Ser240/244), rabbit monoclonal anti-phospho-4E-BP1 (L1293; Sigma-Aldrich). Laminin, a cell-adhesion mol- (Thr37/46) (263B4) and rabbit polyclonal anti-phospho- ecule strongly expressed in the basement membrane of Akt (Ser473) (Cell Signaling Technology, Danvers, skeletal muscle, was detected using an appropriate sec- MA, USA). After the incubation with the appropriate ondary antibody. Morphometric analyses were performed horseradish-peroxidase-conjugated secondary antibody on sections collected from similar regions of each muscle (Cell Signaling Technology), bands were visualised using using a Leica DMI4000 B automated inverted microscope the Bio-Rad Clarity Western ECL substrate with a Bio-Rad equipped with a DCF310 digital camera. Image acquisition ChemiDoc MP imaging system. To monitor for potential was controlled by the Leica LAS AF software. The ImageJ artefacts in loading and transfer among samples in dif- software was used to determine the CSA of 1,000 to 3,000 ferent lanes, the blots were routinely treated with the individual fibres from at least two different fields for each Restore Western Blot Stripping Buffer (ThermoFisher muscle section. Four to nine sections from each muscle Scientific) and reprobed with rabbit polyclonal anti-calnexin were analysed. For histological analyses, serial muscle De Palma et al. Skeletal Muscle 2014, 4:22 Page 6 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 sections were obtained and stained in H & E following tibialis anterior muscle sections (20 to 30 from each standard procedures. The number of fibres was counted muscle) were then collected and the immunofluorescence and analysed using the ImageJ software. of EBD-positive fibres was imaged using Texas red red fil- Single myofiber isolation of hind limb muscle and nu- ter. Creatine kinase (CK) serum levels (units per litre) were clei immunofluorescence on single fibers was performed measured in blood samples obtained from the tail vein of as previously described [8]. Nuclei of 30 individual fibres mice after treadmill running. The blood was centrifuged at from each muscle were analysed. 13,000 × g at 4°C and the supernatant used to measure CK activity in an indirect colorimetric assay (Randox Labora- Whole body tension tories, Crumlin, Northern Ireland, UK) [16,18]. The whole body tension (WBT) procedure was used to determine the ability of mice to exert tension in a for- Statistics ward pulling manoeuvre that is elicited by stroking the Upon verification of normal distribution, the statistical tail of the mice [52]. The tails were connected to a Grass significance of the raw data between the groups in each FT03 transducer (Astro-Med, West Warwick, RI, USA) experiment was evaluated using the unpaired Student’s with a 4.0 silk thread (one end of the thread being tied t-test (single comparisons) or one way analysis of vari- to the tail and the other end to the transducer) [47]. ance (ANOVA) followed by the Newman-Keuls post-test Each mouse was placed into a small tube constructed of (multiple comparisons). The GraphPad Prism software a metal screen with a grid spacing of 2 mm. The mice package (GraphPad Software, La Jolla, CA, USA) was entered the apparatus and exerted a small resting ten- used. After statistics (raw data), data from different ex- sion on the transducer. Forward pulling movements periments were represented and averaged in the same were elicited by a standardised stroke of the tail with graph. The results are expressed as means ± SEM of the serrated forceps, and the corresponding forward pulling indicated n values. tensions were recorded using a Grass Polyview recording system (Astro-Med). Between 20 and 30 strokes of the Chemicals tail forward pulling tensions were generally recorded pDsRed2-Mito was a gift of Prof. Luca Scorrano (University during each session. The WBT was determined by divid- of Padova, Padova, Italy). Dispase was purchased from ing the average of the top ten or top five forward pulling Gibco-Life Technologies (Monza, Italy). TMRM and the tensions, respectively, by the body weight and represent secondary antibody for laminin experiments were obtained the maximum phasic tension that can be developed over from Molecular Probes-Life Technologies. Iscove’s modified several attempts [52]. It is important to note that treat- Dulbecco’s medium, penicillin, streptomycin, gentamycin, ments or conditions which primarily alter muscle mass horse serum, and foetal bovine serum were purchased from without changing the tension developed per unit of Euroclone (Pero, Italy). Matrigel was obtained from BD- muscle mass produce corresponding alterations in forward Bioscience (Milano, Italy). Primer pairs were obtained from pulling tension that are not associated with changes in Primmbiotech (Milano, Italy). Fibroblast growth factor was either WBT 5 or WBT 10 [52,53]. purchased from Tebu-bio (Milano, Italy). DETA-NO and KT5823 were obtained from Merck Millipore (Darmstadt, Treadmill running Germany). ODQ and cyclosporine were purchased from Animals were made to run on a standard treadmill Enzo Life Sciences (Farmingdale, NY, USA). L -arginine machine (Columbus Instruments, Columbus, OH, USA) methylester (L-NAME) and the other chemicals were pur- either on a 0% grade or tilted 10% downhill starting at a chased from Sigma-Aldrich. warm-up speed of 5 m/minute for five minutes [54]. Every subsequent five minutes, the speed was increased by 5 m/ Results minute until the mice were exhausted. Exhaustion was de- nNOSμ deficiency leads to mitochondrial dysfunction fined as the inability of the animal to return to running Mitochondrial function in skeletal muscles of adult within 10 seconds after direct contact on an electric stimu- NOS1-/- mice, that is, at P120, was dissected and com- lus grid. Running time was measured and running distance pared with that of the respective age-matched wild-type calculated. Distance is the product of time and speed of littermattes (control). Mitochondrial membrane potential the treadmill. was monitored in isolated fibres from flexor digitorum bre- As a measure of membrane permeability, the Evans blue vis muscles loaded with TMRM, a potentiometric fluores- dye (EBD) assay was used [47]. A concentration of 5 μg/μl cent dye. TMRM accumulates in the mitochondria that EBD prepared in physiological saline was injected intraven- maintain a polarised mitochondrial membrane potential. ously through the tail vein. Injections (50 μl/10 g body A latent mitochondrial dysfunction masked by the ATP weight) were performed 20 to 30 minutes after treadmill synthase operating in a reverse mode, that is, to consume running. Mice were sacrificed 24 hours after EBD injection. ATP in order to maintain the mitochondrial membrane De Palma et al. Skeletal Muscle 2014, 4:22 Page 7 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 potential, can be unveiled using the ATP synthase inhibi- the muscle bioenergetic parameters. To this end, we tor oligomycin [25]. In agreement with previous reports measured ATP generation from OXPHOS in isolated [29], addition of oligomycin to control mice fibres did not mitochondria of tibialis anterior and diaphragm muscle cause immediate changes in membrane potential even fibres. As shown in Figure 1B, total OXPHOS-generated after extensive incubation (Figure 1A). Conversely, mito- ATP was significantly lower in NOS1-/- mice when chondria in fibres of NOS1-/- mice underwent marked de- compared to control. polarisation after oligomycin. We then analysed the mitochondrial bioenergetics in We investigated whether the latent mitochondrial dys- intact fibres using an in situ approach measuring oxygen function observed in muscles of NOS1-/- mice affected consumption by high resolution respirometry. By this Figure 1 Mitochondrial metabolism is impaired in skeletal muscles of NOS1-/- mice. Fibres were isolated from different muscles of wild-type and NOS1-/- mice at P120. (A) Mitochondrial membrane potential measured in fibres isolated from flexor digitorum brevis muscles, loaded with TMRM and treated with 5 μM oligomycin (Olm) or 4 μM FCCP. TMRM staining was monitored in six to ten fibres obtained from at least three different animals per experimental group. Data are expressed by setting the initial value as 1. (B) ATP production on mitochondria isolated from tibialis anterior and diaphragm muscles, at 10 minutes after substrate addition. Data are expressed by setting the initial value as 1. (C-D) Oxygen consumption on fibres isolated from tibialis anterior and diaphragm muscles, supplied with specific CI, CII and CIV mitochondrial complex substrates, as indicated in the Methods. (E) Quantitative analysis of the mtDNA copy number. Data are expressed by normalizing mtDNA values versus nuclear DNA. Each histogram represents the data obtained from at least five different animals per experimental group. * P <0.05 and ** P <0.01 versus the respective wild-type control. De Palma et al. Skeletal Muscle 2014, 4:22 Page 8 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 approach, we found that the maximal tissue mass- showed the presence of autophagic vacuoles and multi- specific OXPHOS capacity with physiological combina- vesicular bodies, indicative of an active autophagic path- tions of CI mitochondrial complex substrates was similar way [59], in tibialis anterior and diaphragm muscles of in both tibialis anterior and diaphragm of NOS1-/- and P120 NOS1-/- mice (Figure 2B and Additional file 1: control mice (Figure 1C-D). In contrast, the CII-linked Figure S1C). The enhanced autophagy in the absence of respiratory capacity in tibialis anterior of NOS1-/- mice nNOSμ in skeletal muscle was confirmed by Western was lower than that in control muscle fibres, while no blot analysis. The appearance of a faster migrating band difference was observed in the diaphragm. In both tibi- of LC3 protein due to its lipidation and cleavage is a alis anterior and diaphragm of NOS1-/- mice the CIV- common marker of autophagy induction [58]. As shown linked respiratory capacity decreased significantly with in Figure 2G, tibialis anterior muscles of P120 NOS1-/- respect to the controls. Of interest, qPCR analysis of mice exhibited increased lipidated LC3 levels when com- mtDNA levels in tibialis anterior and diaphragm mus- pared to control mice. Similar results on LC3 conversion cles did not reveal any difference between NOS1-/- and were obtained analysing diaphragm muscle samples (see control mice (Figure 1E) suggesting that mitochondrial Additional file 1: Figure S1D). mass was not affected and defects in OXPHOS were due mt to dysfunctional mitochondria. NO signalling regulates UPR and autophagy machinery Activation of the NO-dependent enzyme guanylate nNOSμ deficiency affects mitochondrial network cyclase, with formation of cyclic guanosine monophosphate mt remodelling, UPR and autophagy (cGMP) and activation of a variety of downstream signal- Alterations in the content, shape or function of the ling cascades, including cGMP-dependent-protein kinases mitochondria have been associated with muscle homeo- (PKG), contributes significantly to mediate the physio- stasis [31,55]. To identify the changes in mitochondrial logical effects of NO in muscle [2,5,60]. To investigate the mt network morphology, tibialis anterior muscles of P120 involvement of the cGMP-dependent signalling on UPR NOS1-/- and wild-type control mice were imaged using and autophagy, myogenic precursor cells were differenti- pDsRed2-Mito, a mitochondrially targeted red fluores- ated for six hours in the absence (control) or in the pres- cent protein, by in situ two-photon confocal microscopy ence of the inhibitor of NOS L-NAME (6 mM), the [29,38,46]. NOS1-/- mice showed a disorganised mito- inhibitor of guanylate cyclase ODQ (10 μM), and the in- chondrial network (Figure 2A). Accordingly, ultrastruc- hibitor of PKG KT5823 (1 μM) [61-66]. L-NAME, ODQ tural analyses by TEM (Figure 2B and Additional file 1: and KT5823 treatment increased the expression of HSP60 Figure S1A) revealed changes in the subsarcolemmal and ClpP protein. The NO donor DETA-NO (80 μM) and mitochondria of tibialis anterior muscles of NOS1-/- mice the membrane-permeant cGMP analogue 8Br-cGMP that exhibited, in thin sections, a significant increase in (2.5 mM) [62-66] reversed the effects of L-NAME and mitochondrial surface area (Figure 2C) and a significant ODQ, respectively (Figure 3A). decrease in the density of the cristae (Figure 2D), as com- In another set of experiments, cells were transiently pared with the controls. The same evaluation was per- transfected with YFP-LC3 and then differentiated. As formed on subsarcolemmal mitochondria from diaphragm shown by confocal microscopy fluorescence analysis of muscle with similar results (data not shown). The analysis LC3 and the mitochondrial matrix-specific protein cyclo- of intermyofibrillar mitochondria (see Additional file 1: phillin D (Figure 3B), in control cells LC3 staining was dif- Figure S1B) showed a pattern of enlarged mitochondria fuse and the majority of mitochondria were in the indicating that the presence of these mitochondrial alter- elongated form, indicating myogenic differentiation [25] ations in NOS1-/- mice muscle is not restricted to the and a low rate of autophagy. L-NAME, ODQ, and KT5823 sarcolemma but is a more general phenomenon. treatment, while inducing mitochondrial fragmentation, We then analysed two downstream processes linked to resulted in LC3 localisation into dot cytoplasmic struc- mt mitochondrial stress: UPR and autophagy. In tibialis tures, as compared to the diffuse cytoplasmic distribution anterior muscles of P120 NOS1-/- mice the expression observed in control cells. The effects of L-NAME and of the nuclearly-encoded mitochondrial chaperones ODQ were prevented by DETA-NO and 8Br-cGMP, HSP60 and the protease ClpP, which correlates with the respectively. level of unfolded proteins in mitochondria [56,57], was NO control of autophagy was assessed further by analys- found to be higher than in the controls (Figure 2E). In ing the expression of relevant markers of the autophagic addition, the two-photon confocal microscopy of the signalling pathway, namely LC3, by western blotting and YFP-LC3 [29,38,46] revealed the presence of LC3- p62, Bnip3 and Atg4 by qPCR analysis [58,67]. L-NAME, positive vesicles, an established marker of autophago- ODQ and KT5823 treatments increased lipidated LC3 some formation [58], in tibialis anterior muscles of P120 conversion in differentiated satellite cells and LC3 lipida- NOS1-/- mice (Figure 2F). Furthermore, TEM analysis tion induced by L-NAME and ODQ was blocked by De Palma et al. Skeletal Muscle 2014, 4:22 Page 9 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 mt Figure 2 Mitochondrial morphology, UPR and autophagy in skeletal muscles of NOS1-/- mice. Tibialis anterior muscles were isolated from wild-type and NOS1-/- mice at P120. (A) In vivo imaging of the mitochondrial network by two-photon confocal microscopy. Muscles were transfected with the mitochondrially targeted red fluorescent protein pDsRed2-Mito. The images are representative of results obtained from at least five different animals per experimental group. Scale bar: 10 μm. (B) TEM images detecting the presence of abnormal, enlarged subsarcolemmal mitochondria (asterisks) or autophagic vacuoles (arrowheads) in NOS1-/- muscles. The inset depicts a multivesicular body in NOS1-/- fibres taken at higher magnification. The images are representative of results obtained from at least three different animals per experimental group. (C-D) Subsarcolemmal mitochondrial ultrastructure analysis by TEM. Data represent the quantification of the mitochondrial area and morphometric analysis of mitochondrial cristae complexity. Each histogram represents the data obtained from at least three different animals per experimental group. * P <0.05 and ** P <0.01 versus the respective wild-type control. (E) Western blot analysis of HSP60 and ClpP expression. Actin was used as the internal standard. The image is representative of results obtained from at least five to seven different animals per experimental group. (F) In vivo imaging of autophagosome formation by two-photon confocal microscopy. Muscles were transfected with YFP-LC3. The images are representative of results obtained from at least five different animals per experimental group. Scale bar: 10 μm. (G) Western blot analysis of LC3 lipidation. Actin was used as the internal standard. The image is representative of results obtained from at least 10 different animals per experimental group. DETA-NO or 8Br-cGMP, respectively (Figure 4A). In from the cytoplasm to the nucleus determines the direct addition, cells treated with L-NAME, ODQ and KT5823 transcriptional activation of genes essential to autopha- expressed higher levels of transcripts encoding p62, Bnip3 gosome formation, namely p62, Bnip3 and Atg4 [58,67]. and Atg4 (Figure 4B). Enhanced activity of FoxO transcription factors has also been associated with disruption of mitochondrial func- Deficient nitric oxide signalling promotes FoxO3-Mul-1 tion and organisation leading to impaired skeletal axis muscle function and development [29]. As shown in Catabolic conditions activate FoxO transcription factors, Figure 5A, C, phosphorylated FoxO3 levels in tibialis which stimulate the ubiquitin-proteasome system as a anterior and diaphragm muscles of P120 NOS1-/- mice response to skeletal muscle-wasting [31,55]. FoxO3 ac- were lower than in the controls. In addition, tibialis an- tivity is necessary and sufficient for the induction of au- terior and diaphragm muscles of NOS1-/- mice overex- tophagy in skeletal muscle [38]. FoxO3 translocation pressed the protein corresponding to mitochondrial De Palma et al. Skeletal Muscle 2014, 4:22 Page 10 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 mt Figure 3 NO signalling, UPR , and autophagy on myogenic precursor cells. Cells were differentiated for six hours in the absence (control) or in the presence of L-NAME (6 mM), ODQ (10 μM), KT5823 (1 μM), L-NAME + DETA-NO (80 μM), and ODQ +8 Br-cGMP (2.5 mM). (A) Western blot analysis of HSP60 and ClpP expression. Actin was used as the internal standard. (B) Confocal microscopy imaging of cells transfected with YFP-LC3. Mitochondrial morphology was detected by mitochondrial matrix-specific protein cyclophillin D (CypD) staining. Scale Bar: 10 μm. Images are representative of at least three to five independent experiments.. ubiquitin ligase Mul-1 (Figure 5B, D), which has been obtained with MUSA1 analysis are difficult to correlate recently reported to be upregulated in muscle through with nNOSμ deficiency. These findings indicate that the FoxO3 transcription factors and promoting mitochon- effects of nNOSμ absence on E3 ubiquitin ligases mainly drial fission, depolarization and mitophagy [68,69]. As affect expression of Mul-1 gene. shown in Figure 5E, in vitro treatment of differentiated myogenic precursor cells from wild-type control mice nNOSμ deficiency affects muscle growth with L-NAME, ODQ and KT5823 increased Mul-1 pro- We evaluated the effects of the absence of nNOSμ on tein expression. The effects induced by L-NAME and skeletal muscle phenotype. Tibialis anterior, gastrocne- ODQ were blocked by DETA-NO and 8Br-cGMP, re- mius, soleus, and extensor digitorum longus muscles were spectively. In tibialis anterior and diaphragm muscles of dissected and weighed. Since the body weight and the P120 NOS1-/- mice, qPCR analysis of other E3 ubiquitin visceral adipose tissue of NOS1-/- male mice were sig- ligases, atrogin-1 and MuRF1, involved in muscle loss nificantly lower than wild-type control (see Additional [69,70], ruled out a nNOSμ-dependent modulation of file 2: Figure S2A-B) [71] we calculated the muscle size their expression (Figure 5F, G). Also, the differences relative to body weight [72]. As shown in Figure 6A and De Palma et al. Skeletal Muscle 2014, 4:22 Page 11 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 Figure 4 NO signalling and autophagic pathway on myogenic precursor cells. (A) Western blot analysis of LC3 lipidation in cells differentiated for six hours in the absence or in the presence of L-NAME (6 mM), ODQ (10 μM), KT5823 (1 μM), L-NAME + DETA-NO (80 μM), and ODQ +8 Br-cGMP (2.5 mM). Actin was used as the internal standard. Image is representative of at least five independent experiments. (B) qPCR analysis of mRNA levels for p62, Bnip3 and Atg4 in cells differentiated for six hours in the absence (control) or in the presence of L-NAME ODQ, and KT5823. Values are expressed as the fold change over control. Each histogram represents the data obtained from at least five independent experiments. * P <0.05 versus respective control. Additional file 2: Figure S2C, the relative mass of the The examination of multiple time points was then muscles for the P120 NOS1-/- mice was significantly carried out in order to establish a possible link between lower than the relative mass of the muscles for the con- the changes in mitochondrial homeostasis and the re- trol mice. This excludes the possibility that the changes duction in muscle size. The CSA (Figure 7A-C) and the in muscle mass are simply due to an overall change in number of myonuclei (Figure 7D) of hind limb muscle size of the mice. fibres were significantly decreased in P10 NOS1-/- The overall morphology of the tibialis anterior and mice, when compared with the respective control. diaphragm muscle in P120 NOS1-/- mice was normal, Muscle growth during post-natal development (P0 to without pathological features of necrosis, macrophage P21), but not at later stages, is accompanied by a con- infiltration and centronucleated fibres (see Additional tinuous increase in the number of myonuclei resulting file 2: Figure S2D). In addition, the number of fibres in from satellite cell fusion [69,73]. As shown in Figure 7E, tibialis anterior muscles was comparable in both NOS1-/- cells exhibited lower levels of myosin and NOS1-/- and control mice (Figure 6B). By contrast, lam- MyoD, which are markers of myogenic differentiation, inin staining of tibialis anterior and diaphragm, used to as compared to control cells. Interestingly, CycloD stain- identify individual muscle fibres, revealed a significant ing of differentiating myogenic precursor cells indicated decrease in the mean CSA of tibialis anterior and dia- that the absence of nNOSμ induces diffuse mitochondrial phragm sections in P120 NOS1-/- mice when compared fragmentation (Figure 7F) [25]. Taken together, our data with control (Figure 6C-H). argue that the absence of nNOSμ induces mitochondrial De Palma et al. Skeletal Muscle 2014, 4:22 Page 12 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 Figure 5 NO signalling, FoxO3, and ubiquitin ligases. Western blot analysis of phosphorylated FoxO3 levels (pFoxO3) or mitochondrial ubiquitin ligase Mul-1 expression in tibialis anterior (A-B) and diaphragm (C-D) of wild-type and NOS1-/- mice at P120. FoxO3 or actin were used as the internal standard. The images are representative of results obtained from at least four to ten different animals per experimental group. (E) Western blot analysis of Mul-1 expression in myogenic precursor cells differentiated in the absence or in the presence of L-NAME (6 mM), ODQ (10 μM), KT5823 (1 μM), L-NAME + DETA-NO (80 μM) and ODQ +8 Br-cGMP (2.5 mM). Actin was used as the internal standard. The image is representative of at least five independent experiments. qPCR analysis of mRNA levels for atrogin-1, muRF1 and MUSA1 in tibialis anterior (F) and diaphragm (G) muscles of wild-type and NOS1-/- mice at P120. Values are expressed as the fold change over wild-type. Each histogram represents the data obtained from at least five to eight different animals per experimental group. * P <0.05 versus the respective wild-type control. fragmentation and a deficit in satellite cell fusion/differen- Using NOS1-/- mice it has been previously shown that tiation, thus impairing fibre growth. nNOS modulates the mechanism of disuse-induced atro- At P30 we found that the CSA of tibialis anterior was phy via FoxO transcription factors [75]. Our observation significantly decreased in NOS1-/- mice, when com- that at P10, P30 (see Additional file 2: Figure S2E-F) and pared with controls (Figure 8A-C). In this crucial time P120 (Figure 5E-F) NOS1-/- and control mice expressed of muscle growth we also measured the activation of the similar levels of transcripts encoding the classical atro- Akt-mTOR pathway as a positive regulator [55,69,73,74]. genes atrogin-1 and MuRF1 [69,70,75], indicates that the As shown in Figure 8D, phosphorylated levels of S6 ribo- atrophy pathways do not play a key role in the develop- somal protein, 4E-BP1 and Akt in tibialis anterior mus- ment of NOS1-/- muscles. cles of NOS1-/- mice were lower than in the controls. FoxO3 proteins are phosphorylated by Akt, which ren- nNOSμ deficiency affects muscle function ders them inactive; this may explain why phosphorylated We evaluated whether the absence of nNOSμ affected FoxO3 levels were found to be lower as well, while Mul- skeletal muscle function. The WBT measurement deter- 1 was overexpressed (Figure 8E). Of importance, both mines the total phasic forward pulling tension exerted events are correlated with muscle mitochondrial dys- by the fore and hind limb muscles and reflects the max- function and growth [29,55,68,69,73,74]. imal acute phasic force the mouse can achieve to escape De Palma et al. Skeletal Muscle 2014, 4:22 Page 13 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 Figure 6 Skeletal muscle phenotype of wild-type and NOS1-/- mice at P120. (A) Weight of tibialis anterior, gastrocnemius, soleus, and extensor digitorum longus (EDL) muscles. The muscle size is relative to body weight. Each histogram represents the data obtained from at least 10 different animals per experimental group. (B) The number of myofibres in tibialis anterior. Each histogram represents the data obtained from at least four to five different animals per experimental group. Laminin staining of tibialis anterior (C-E) and diaphragm (F-H) muscles. (C, F) Immunohistochemical images. Scale bar: 100 μm. (D, G) Representative distribution of CSA values. (E, H) Quantification of CSA. Images and quantifications represent the data obtained from at least four to seven different animals per experimental group. *P <0.05, **P <0.01, and ***P <0.001 versus the respective wild-type control. a potentially harmful event [52]. As shown in Figure 9A, significant exercise intolerance after repetitive exercise the WBT normalised for body weight in P120 NOS1-/- challenges, while control mice at day 3 showed even im- mice was significantly lower than in the wild-type con- proved exercise capacity, compared to day 1. NOS1-/- trol, consistent with an unpaired muscle specific force mice also exhibited a significantly decreased treadmill output in the absence of nNOSμ. runtime to exhaustion (Figure 9C). We also examined the muscle resistance to fatigue: we We then assessed the structure/damage of skeletal subjected NOS1-/- mice to treadmill running, that mea- muscle myofibres after exercise. TEM analysis performed sures resistance to fatigue during a forced exercise, and ex- in tibialis anterior muscles of P120 NOS1-/- mice after the amined both exercise performance and tolerance. As treadmill running showed marked ultrastructural changes, shown in Figure 9B, the total distance run by NOS1-/- as, for instance, defects in the organisation of the contract- mice during one bout of exhaustive treadmill running (day ile apparatus (sarcomere), that were observed neither in 1) was significantly lower when compared to controls. the wild-type mice nor in unchallenged NOS1-/- mice This reduction in performance of NOS1-/- mice was also (Figure 9D). The features observed in challenged NOS1-/- observed after repeated challenges: NOS1-/- mice showed mice might be a direct consequence of denervation events De Palma et al. Skeletal Muscle 2014, 4:22 Page 14 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 Figure 7 Skeletal muscle phenotype of wild-type and NOS1-/- mice at P10. (A-C) Laminin staining of hind limb muscles. (A) Immunohistochemical images. Scale bar: 100 μm. (B) Representative distribution of CSA values. (C) Quantification of CSA. Images and quantifications represent the data obtained from at least five different animals per experimental group. (D) Number of myonuclei per fibre in hind limb muscles. Each histogram represents the data obtained from at least three different animals per experimental group. (E) Western blot analysis of myosin (MF20) and MyoD expression in myogenic precursor cells isolated from wild-type and NOS1-/- mice and differentiated for increasing times. Calnexin was used as the internal standard. Images are representative of at least three independent experiments. (F) Confocal microscopy imaging of myogenic precursor cells isolated from wild-type and NOS1-/- mice and differentiated for 48 hours. Mitochondrial morphology was detected by mitochondrial matrix-specific protein cyclophillin D staining. Scale Bar: 10 μm. Images are representative of at least three independent experiments. * P <0.05 versus the respective wild-type control. as also indicated by collagen fibres deposition and motor time a link between a deficit in NO signalling, mito- end-plates lacking the presynaptic nerve ending (data not chondrial alterations and skeletal muscle impairments. shown). As shown in Figure 9E, tibialis anterior muscles The first result emerging from our analysis is that of P120 NOS1-/- mice after the treadmill running dis- nNOSμ deficiency is per se sufficient to induce profound played an increased uptake versus wild-type of EBD, which defects in mitochondria, with alterations in mitochondrial stains damaged myofibres [47]. As an in vivo indicator of distribution, shape, morphology and size accompanied by skeletal muscle damage we also analysed the serum levels a latent mitochondrial dysfunction such that energy gener- of CK, a skeletal muscle enzyme released during fibre de- ation is impaired. Nitric oxide has several key functions in generation whose activity increased in dystrophic animals mitochondria: it inhibits mitochondrial fission, induces [16,18]. As expected, in NOS1-/- mice after the treadmill mitochondrial biogenesis and controls mitochondrial re- running, the serum CK activity was found to be signifi- spiratory rate by reversible inhibition of complex IV in the cantly higher than that in the wild-type mice (Figure 9F). mitochondrial respiratory chain [25,76,77]. Furthermore, it controls the expression of several enzymes in the Krebs Discussion cycle [78]. Derangement of these mitochondrial functions This study documents that nNOSμ deficiency, while se- is most likely at the basis of the multiple mitochondrial verely altering the structure and bioenergetics potential deficits we observed in NOS1-/- mice. of skeletal muscle mitochondria does not impact signifi- Of importance, we found that this overall mitochon- cantly on the overall resting muscle structure, apart from drial dysfunction was accompanied both in intact myofi- reducing muscle mass and the CSA of the myofibres of bres in vivo and in isolated satellite cells in vitro by an mt specific muscles. When the muscle is exposed to work- enhanced UPR response. It has been hypothesised that mt loads, however, the consequences of nNOSμ deficiency the UPR is activated prior to the induction of autoph- become apparent, with a significantly reduced resistance agy [79]; in particular, that the autophagy pathway is ac- of the muscles accompanied by increased sensitivity to tivated when mitochondria cannot maintain a polarised mt exercise-induced damage. This establishes for the first membrane potential despite UPR activation. We found De Palma et al. Skeletal Muscle 2014, 4:22 Page 15 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 Figure 8 Skeletal muscle phenotype of wild-type and NOS1-/- mice at P30. (A-C) Laminin staining of tibialis anterior muscles. (A) Immunohistochemical images. Scale bar: 100 μm. (B) Representative distribution of CSA values. (C) Quantification of CSA. Images and quantifications represent the data obtained from at least five different animals per experimental group. Western blot analysis in tibialis anterior: (D) phosphorylated S6, 4E-BP1 and Akt levels, (E) phosphorylated FoxO3 levels or mitochondrial ubiquitin ligase Mul-1 expression. S6, 4E-BP1, Akt, FoxO3 or actin were used as the internal standard. The images are representative of results obtained from at least four different animals per experimental group. *P <0.05 versus the respective wild-type control. mt that the increase in UPR was accompanied by autoph- controls, although they did not show any pathological agy and increased expression of molecules relevant to features reminiscent of muscle damage, such as inflam- autophagic signalling, namely p62, Bnip3 and Atg4. This mation, necrosis or fibrosis. Similar morphological data suggests that nNOSμ deficiency leads to a sufficiently se- were obtained in male NOS1-/- mice backcrossed onto vere mitochondrial deficit that cannot be restored by the B6129 background (our experimental model) [71] or mt mt UPR . The enhanced autophagic and UPR response backcrossed onto the C57BL/6 background [80], al- were normalised when the cGMP-dependent signalling though in the latter model no difference in tibialis anterior was activated, indicating that these events are controlled muscle mass relative to body mass was reported. That by NO via its physiological second messenger cGMP. thedecreaseinmusclemassis due to mechanisms other The second relevant information is that an altered NO than the decrease in body mass was recently suggested system leads to impairment of muscle function that is using NOS1-/- mdx mice [72]. The deficiency of nNOSμ selective to specific parameters and unmasked during is also accompanied by muscle ageing [81] and fibre exercise. In particular we found that skeletal muscles in growth was prevented in the NOS1-/- mice model of the absence of nNOSμ are smaller relative to the rest of skeletal muscle hypertrophy [82] and NOS1-/- mdx the body, thus indicating that muscle mass decrease was mice [72]. In a recent study, no difference in the weight not simply attributable to a generalised decreased body and CSA of tibialis anterior muscles from NOS1-/- and mass tissues (including adipose tissue) and likely due to control was also reported but the animal background a specific reduction in the size of the muscle fibres was not indicated [83]. Discrepancies in these studies themselves. In agreement with this, NOS1-/- mice mus- may be explained, at least in part, by strain-specific cles (that is, tibialis anterior and diaphragm) displayed modulation of the nNOSμ-regulated phenotype, a hy- smaller myofibre CSA when compared to littermate pothesis substantiated by the observation, by the same De Palma et al. Skeletal Muscle 2014, 4:22 Page 16 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 Figure 9 Skeletal muscle function in wild-type and NOS1-/- mice. (A) WBT measurements determined by dividing the average of the top ten or top five forward pulling tensions, respectively, by the body weight. (B) Running distance calculated during one bout of exhaustive treadmill running (day 1) and after repeated challenges (days 2 and 3). (C) Treadmill runtime to exhaustion calculated as the averages obtained at day 1 to 3. Each histogram represents the data obtained from at least four to five different animals per experimental group. (D) TEM analysis performed in tibialis anterior muscles of both unchallenged (no run) and challenged (exhaustive running) mice. The images are representative of results obtained from at least three different animals per experimental group. (E) EBD uptake in tibialis anterior muscles after the treadmill running. Scale Bar: 100 μm. The images are representative of results obtained from at least four different animals per experimental group. (F) CK serum levels (units per litre) of mice after treadmill running. Each histogram represents the data obtained from at least four different animals per experimental group. *P <0.05, **P <0.01 and ***P <0.001 versus the respective wild-type control. WBT and treadmill running were performed on animals at P120. group, that morphological data differed between NOS1-/- The functional studies revealed two important aspects mice backcrossed onto the C57BL/6 and the B6129 back- of the role of NO in skeletal muscle. Firstly, the fact that ground [37,71,80]. NOS1-/- mice in our in vivo experiments exhibited a De Palma et al. Skeletal Muscle 2014, 4:22 Page 17 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 deficit in forward pulling tension and resistance to performance and provide an indication of the mechanism fatigue during a forced exercise indicates that nNOSμ is responsible for the impaired fibre growth resulting in a def- important to maintain skeletal muscle strength and the icit of muscle performance. In particular, nNOSμ absence animal’s ability to perform in repetitive exercise training. altered mitochondrial homeostasis in myogenic precursor Our results in vivo are in line with a previous study with cells with a decrease in the number of myonuclei per fibres an in situ approach reporting that nNOSμ-deficient tibi- and impaired muscle development at early stages of growth. alis anterior muscles exhibit a reduced force production This also suggests that fusion of myogenic precursor cells and a specific deficit in adapting to exercise and develop during perinatal myogenesis is impaired. Accordingly, NO profound fatigue upon repeated contraction [71]. An ex- has been shown to stimulate the ability of myogenic precur- cessive fatigue has been also observed in NOS1-/- mice sor cells to become activated and fuse to each other and wild-type mice treated with a nNOS inhibitor [12]. [5,8,85]. There is a general agreement that mitochondria A specific and intrinsic deficit in muscle force produc- change when the myoblasts differentiate into myotubes tion has been recently reported in NOS1-/- mdx mice, [27]. Also, NO maintains functional mitochondria and this although muscle fatigue was unaffected by nNOS deple- permits differentiation of myogenic precursor cells in vitro tion [72]. Secondly, our data on muscle phenotype and [25]. At the signalling level, the Akt-mTOR pathway and CK measurements after treadmill running indicate that Akt-FoxO3-Mul-1 axis are involved in skeletal muscle nNOSμ deficiency induces muscle degeneration/damage growth/wasting, autophagy and mitochondrial dysfunction post-exercise. This raises the possibility that nNOSμ- [29,31,38,46,55,58,67-69,73,74]. Of interest, Mul-1 has been deprived muscles cannot activate protective responses. recently reported to be upregulated during muscle wasting, Accordingly, NOS1-/- mdx mice displayed increased possibly via an autophagic mechanism involving FoxO3 susceptibility to eccentric contraction-induced muscle transcription factors [68]. Our data indicate the relevance damage [72]. In addition, expression of a muscle-specific of the above signalling pathways and that they are con- nNOS transgene prevents muscle membrane injury dur- trolled by NO. We observed an inhibition of the Akt- ing modified muscle use [84]. In this respect, there is a mTOR pathway in the absence of nNOSμ.Concomitantly, general agreement that NO produced by nNOS plays an the Akt-FoxO3-Mul-1 axis was also dysregulated. In important role in muscle repair in chronic conditions addition, the inhibition of the nNOS/NO/cGMP/PKG [5,8,9] although the use of NOS1-/- mice suggested that system induced the transcriptional activity of FoxO3 nNOS is not essential to functional recovery after acute and increased Mul-1 expression. These events are likely injury [80]. associated with nNOSμ-dependent impairments of The third important observation is the correlation be- muscle fibre growth. tween mitochondrial defects and muscle impairment. We cannot exclude that failure of other NO-dependent Alterations in the content, shape or function of the mito- action involving, for instance, the vascular system, may chondria appear to occur in damaged muscle and inhib- have contributed to the functional and structural defects ition of mitochondrial fission protects from muscle loss we observed in skeletal muscle. Extensor digitorum longus during fasting [29]. Recent findings have also underlined of NOS1-/- mice revealed an altered capillary-to-fibre ra- the crucial role of autophagy in the control of muscle mass tio but not changes in the capillary ultrastructure or the and functions [29,31,55,69]. Autophagy derangement is in- hemodynamics at basal conditions [86]. Noteworthy, NO volved in a number of inherited muscle diseases [31-33]. generated by sarcolemmal nNOSμ normally acts as a para- Of interest, mitochondria are involved in regulating au- crine signal that optimises blood flow in the working tophagy [30]. In addition, skeletal muscle was shown to be muscle [12,87,88] and the protective vasodilating action is sensitive to the physiological stressors that trigger the impaired in the contracting muscles of NOS1-/- mice mt mt UPR [35,36] and UPR is activated in skeletal muscle [12,89]. In this respect, the lack of this vasodilating action during exercise as part of an adaptive response to exercise in NOS1-/- mice has been suggested to affect muscle per- training [54]. Here, we raise the possibility that mitochon- formance [71]. Results obtained in NOS1-/- mice with dif- mt drial dysfunction, UPR and autophagy are functionally ferent cardiac injuries indicated a protective role of nNOS, related to each other and promoted by a single event, that although an opposite effect cannot be excluded [90,91]. is, the deficit in NO signalling, thus suggesting that the The deficit in exercise performance of NOS1-/- muscles association of altered mitochondrial homeostasis and may be the consequence, at least in part, of a decreased muscle phenotype/performance in NOS1-/- mice is not oxygen delivery following blood flow impairment. coincidental. The experiments we carried-out in myogenic precursor Conclusions cells and NOS1-/- mice during critical stages of muscle Muscle exercise performance is a complex physiological development are consistent with an association of al- process that can occur by many different mechanisms tered mitochondrial homeostasis and muscle phenotype/ and NO has long been described to be relevant among De Palma et al. Skeletal Muscle 2014, 4:22 Page 18 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 them [2]. Our study now suggests that the relevance of EA, TT, SR, VR, SC, VC and PP acquired and analysed the data. CM, MTB and MS analysed the data and revised the manuscript. CP participated in the NO also resides in the fact that it regulates key homeo- design of the study, analysed and interpreted the data, and revised the static mechanisms in skeletal muscle, namely mitochon- manuscript. DC and EC participated in the design and the coordination of mt drial bioenergetics and network remodelling, UPR and the study, analysed and interpreted the data, drafted and revised the manuscript, and wrote the final version of the manuscript. All authors read autophagy. Although NOS1-/- mice do not display the and approved the final manuscript. overt features of myopathies, such as muscle degener- ation, reactive regeneration and replacement of muscle Authors’ information with fibroadipous tissue [92,93], we clearly show that al- CDP is a post-doctoral research associate. FM and SP are PhD students. SR is a terations of the NO system significantly impair muscle research fellow. EA, TT, VR, SC and VC are post-doctoral research fellows. PP is a graduate medical student. MTB is a Senior Researcher. CM is a Professor of fibre growth, thus resulting in a deficit of muscle force Human Anatomy. MS is a Professor of Pathology. CP is a Professor of and the ability to sustain prolonged exercise. This aspect Pharmacology. DC is a Professor of Physiology. EC is a Professor of may explain why NO deficiency contributes to muscle Pharmacology and the Head of the Pharmacology group. impairment in degenerative disease of the muscle, such as muscular dystrophies. Acknowledgements We thank Laura Pozzi (Scientific Institute IRCCS Eugenio Medea, Bosisio Parini, Lecco, Italy) for technical help. We are grateful to Prof. Luca Scorrano (University Additional files of Padova, Padova, Italy) for providing us with pDsRed2-Mito. This work was supported by: “Ministero della Salute”“Giovani Ricercatori 2011-2012” grant to C. Additional file 1: Figure S1. Mitochondrial ultrastructure and LC3 D.P and “Ricerca corrente 2014” grant to E.C.; “Ministero dell’Istruzione, Università lipidation in skeletal muscles of wild-type and NOS1-/- mice. (A) TEM i eRicerca”, PRIN2010-2011 grants to E.C. and D.C.; European Community’s mages of subsarcolemmal mitochondria of tibialis anterior muscles. Scale framework programme FP7/2007-2013 under the agreement n°223098 bar: 0.1 μm. (B) TEM images of intermyofibrillar mitochondria of tibialis (OPTISTEM) and n°241440 (ENDOSTEM) to E.C. The funders had no role in study anterior muscles. Scale bar: 1 μm. (C) TEM images of diaphragm muscles design, data collection and analysis, decision to publish, or preparation of the detecting the presence of autophagic vacuoles (arrowheads) in NOS1-/- manuscript. fibres. TEM images are representative of results obtained from at least three different animals per experimental group. (D) Western blot analysis Author details of LC3 lipidation in diaphragm muscles of wild-type and NOS1-/- mice. Unit of Clinical Pharmacology, National Research Council-Institute of GAPDH was used as internal standard. The image is representative of Neuroscience, Department of Biomedical and Clinical Sciences “Luigi results obtained from at least 10 different animals per experimental Sacco”, University Hospital “Luigi Sacco”, Università di Milano, Milano, Italy. 2 3 group. Analyses were performed on animals at P120. Scientific Institute IRCCS Eugenio Medea, Bosisio Parini, Italy. Dulbecco Additional file 2: Figure S2. Weight, muscle structure and muscle Telethon Institute at Venetian Institute of Molecular Medicine, Padova, Italy. expression of ubiquitin ligases in wild-type and NOS1-/- mice. Body National Research Council-Institute of Neuroscience, Department of Medical (A) and visceral adipose tissue (VAT) (B) weight. Each histogram Biotechnology and Translational Medicine, Università di Milano, Milano, Italy. 5 6 represents the data obtained from at least three to eight different animals CNI@NEST, Italian Institute of Technology, Pisa, Italy. Unit of Morphology, per experimental group. *P <0.05, and **P <0.01 versus the respective Department of Biomedical and Clinical Sciences “Luigi Sacco”, Università di wild-type control. (C) Pictures of tibialis anterior, soleus, and gastrocnemius Milano, Milano, Italy. Department of Biomedical Science, Università di Padova, muscles. The image is representative of at least 10 different animals per Padova, Italy. Department for Innovation in Biological, Agro-food and Forest experimental group. (D) Histological sections of tibialis anterior and Systems, Università della Tuscia, Viterbo, Italy. diaphragm muscles stained with H & E. The images are representative of results obtained from at least five different animals per experimental group. Received: 26 June 2014 Accepted: 18 November 2014 Scale bar: 100 μm. Analyses were performed on animals at P120. qPCR analysis of mRNA levels for atrogin-1 and muRF1 in hind limb muscles at P10 (E) and tibialis anterior muscles at P30 (F). 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Hum Mol Genet 1998, 7:823–829. doi:10.1186/s13395-014-0022-6 Cite this article as: De Palma et al.: Deficient nitric oxide signalling impairs skeletal muscle growth and performance: involvement of mitochondrial dysregulation. Skeletal Muscle 2014 4:22. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

Deficient nitric oxide signalling impairs skeletal muscle growth and performance: involvement of mitochondrial dysregulation

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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 mt network remodelling, and increased mitochondrial unfolded protein response (UPR ) 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 mt muscle, namely mitochondrial bioenergetics and network remodelling, UPR and autophagy. These events are likely associated with nNOSμ-dependent impairments of muscle fibre growth resulting in a deficit of muscle performance. Keywords: Nitric oxide synthase and signalling, Mitochondrial bioenergetics, Mitochondrial network, Unfolded protein response, Autophagy, Akt-mTOR pathway, Akt-FoxO3-Mul-1 axis, Fibre growth, Muscle structure, Muscle exercise * Correspondence: d.cervia@unitus.it; emilio.clementi@unimi.it 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 Full list of author information is available at the end of the article © 2014 De Palma et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. De Palma et al. Skeletal Muscle 2014, 4:22 Page 2 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 Background excitation contraction coupling and prevent atrophy Nitric oxide (NO) is a gas and a messenger with pleio- [28,29]. In addition, mitochondria are involved in regulat- tropic functions in most tissues and organs, synthesized ing autophagy [30], whose derangement plays a role in a by a family of NO synthases. NO is also generated in number of inherited muscle diseases [31-33]. Mitochon- skeletal muscle, in particular by the muscle-specific drial protein homeostasis is maintained through proper neuronal NO synthases (nNOS or NOS1) [1,2]. nNOSμ folding and assembly of polypeptides. This involves the mt is the predominant nNOS isoform in muscle and is an- mitochondrial unfolded protein response (UPR ), a stress chored to the sarcolemma as a component of the dys- response that activates transcription of nuclear-encoded trophin glycoprotein complex [3]. This enzyme produces mitochondrial chaperone genes to maintain proteins in a NO at low, physiological levels (in the pico to nanomolar folding or assembly-competent state, preventing deleteri- range) in a way controlled by second messengers [1,2]; ous protein aggregation [34-36]. its expression is increased by crush injury, muscle activ- In this study we have examined the relationship between ity and ageing [4,5]. NO has an important role in regu- the NO system, mitochondrial structure/activity and skel- lating skeletal muscle physiological activity, including etal muscle phenotype/growth/functions using a mouse excitation-contraction coupling, muscle force generation, model in which nNOSμ is absent (NOS1-/-). Also, NO- auto-regulation of blood flow, calcium homeostasis, me- induced effects and the NO pathway were dissected in tabolism and bioenergetics [2,6,7]. In addition, it is a key myogenic precursor cells. Our results indicate that the determinant in myogenesis that it regulates at several deficit in NO signalling leads in skeletal muscle to alter- key steps, especially when the process is stimulated to ations in mitochondrial morphology, bioenergetics and repair muscle damage after injury [5,8,9]. network remodelling, accompanied by defective autophagy mt The importance of NO in muscle repair also emerges and the induction of a UPR response. These events, from the observation that nNOS signalling is defective in while not severely altering the overall resting skeletal many genetically diverse skeletal muscle diseases in which muscle structure, are associated with modifications in the muscle repair is dysregulated, including Duchenne muscu- Akt-mammalian target of rapamycin (mTOR) pathway lar dystrophy, Becker muscular dystrophy, limb-girdle and Akt-FoxO3-mitochondrial E3 ubiquitin protein ligase muscular dystrophies 2C, 2D and 2E, Ullrich congenital 1 (Mul-1) axis and are sufficient to dysregulate skeletal muscular dystrophy and inflammatory myositis [3,10-13]. muscle growth and exercise performance. Based on this evidence and on the fact that the restoration of NO signalling by nNOS overexpression ameliorates muscle function [14,15], genetic and pharmacologic strat- Methods egies to boost nNOS/NO signalling in dystrophic muscle Animals are being tested with encouraging results: in particular, the NOS1-/- animals are mice homozygous for targeted combination of NO donation with non steroidal anti- disruption of the nNOS gene (strain name B6129S4- tm1Plh inflammatory activity limits muscle damage and favours NOS1 /J) that were purchased from Jackson Labora- muscle healing in vivo [16-18] such thatitis currently be- tories (Bar Harbor, Maine, USA) (stock no. 002633). In ing tested as a therapeutic for Duchenne muscular dys- this mouse line, targeted deletion of exon 2 specifically trophy in humans [19,20]. eliminates expression of nNOSμ [37]. NOS1-/- mice were The observation that nNOS is localised in close prox- crossed with the wild-type B6129 to maintain the original imity to mitochondria suggests a tight coupling between background and to obtain a colony of NOS1-/- mice and NO generation and regulation of mitochondrial respir- wild-type littermate controls, with genotyping performed ation and metabolism. The role of NO in regulating oxi- from tail clippings. Experiments were performed on male dative phosphorylation and mitochondrial biogenesis in mice at postnatal day 10 (P10) and P120. C57BL/6 wild- skeletal muscle physiology has been established [21-24]. type mice (strain name C57Bl10SnJ) were purchased from Likewise NO-dependent inhibition of mitochondrial fis- Charles River (Calco, Italy). Animals were housed in a reg- sion occurs during myogenic differentiation [25]. ulated environment (23 ± 1°C, 50 ± 5% humidity) with a How the effects of NO on mitochondria impact on 12-hour light/dark cycle (lights on at 08.00 a.m.), and pro- muscle function, however, has not been investigated yet. vided with food and water ad libitum. For specific experi- Elucidation of this aspect is relevant in view of the role ments, mice were killed by cervical dislocation. All studies that mitochondria play in muscle pathophysiology and were conducted in accordance with the Italian law on ani- may shed light on the muscular disorders in which NO mal care N° 116/1992 and the European Communities signalling is impaired [26]. In particular, increases in Council Directive EEC/609/86. The experimental pro- mitochondria number and oxidative phosphorylation ac- tocols were approved by the Ethics Committee of the tivity is relevant during differentiation [27] and the bal- University of Milano. All efforts were made to reduce both ance of fission and fusion is necessary to preserve animal suffering and the number of animals used. De Palma et al. Skeletal Muscle 2014, 4:22 Page 3 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 Mitochondrial membrane potential 225 mM sucrose, 44 mM KH PO and 6 mM EDTA. 2 4 Mitochondrial membrane potential in isolated transfected Total oxidative phosphorylation (OXPHOS)-ATP in iso- fibres from flexor digitorum brevis muscles was measured lated mitochondria was measured by the luciferin-luciferase by epifluorescence microscopy based on the accumulation method, as described, with slight modifications [25]. Briefly, of tetramethylrhodamine methyl ester (TMRM) fluores- mitochondria were plated in 96 wells and treated with cence [25,29,38]. Briefly, flexor digitorum brevis myofibres buffer-A (150 mM KCl, 25 mM Tris-HCl, 2 mM EDTA, were placed in 1 ml Tyrode’s buffer and loaded with 5 nM 0.1% bovine serum albumin, 10 mM KH PO and 0.1 mM 2 4 TMRM supplemented with 1 μM cyclosporine H for MgCl (pH 7.4) containing 0.8 M malate, 2 M glutamate, 30 minutes at 37°C. Myofibres were then observed with an 500 mM ADP, 100 mM luciferin and 1 mg/ml luciferase. Olympus IX81 inverted microscope equipped with a CellR Oligomycin (2 μg/ml) was also used to detect the presence imaging system (Olympus, Tokio, Japan). Sequential im- of glycolytic ATP. OXPHOS-ATP was measured using a ages of TMRM fluorescence were acquired every 60 - GloMax luminometer (Promega, Milan, Italy). seconds with a × 20 0.5, UPLANSL N A objective (Olympus). When indicated, oligomycin (5 μM) or the High-resolution respirometry protonophore carbonylcyanide-p-trifluoromethoxyphenyl Respiratory chain defects were assessed in tibialis anterior hydrazone (FCCP, 4 μM) was added [39]. Images were ac- and diaphragm fibre bundles using published protocols quired and stored, and analysis of TMRM fluorescence [40-42]. After transferring the tissue sample into ice-cold over mitochondrial regions of interest was performed BIOPS (10 mM CaK ethyleneglycoltetraacetic acid (EGTA) using ImageJ software (http://rsbweb.nih.gov/ij/). buffer, 7.23 mM K EGTA buffer, 0.1 μM free calcium, 20 mM imidazole, 20 mM taurine, 50 mM 2-(N-morpho- Primary myogenic cell cultures lino)ethanesulfonic acid hydrate, 0.5 mM dithiothreitol, Using published protocols [25], myogenic precursor cells 6.5 mM MgCl 6H O, 5.7 mM ATP and 15 mM phospho- 2 2 (satellite cells) were freshly isolated from the muscles of creatine (pH 7.1)), connective tissue was removed and the newborn C57BL/6 mice. When indicated, cells were ob- muscle fibres were mechanically separated. Complete per- tained from NOS1-/- mice and wild-type littermate con- meabilisation of the plasma membrane was ensured by trols. Briefly, hind limb muscles were digested with 2% gentle agitation for 30 minutes at 4°C in 2 ml of BIOPS so- collagenase-II and dispase for 10 minutes at 37°C with lution containing 50 μg/ml saponin. The fibre bundles were gentle agitation. Contamination by non-myogenic cells rinsed by agitation for 10 minutes in ice-cold mitochondrial was reduced by pre-plating the collected cells onto plas- respiration medium (MiR05; 0.5 mM EGTA, 3 mM MgCl , tic dishes where fibroblasts tend to adhere more rapidly. 60 mM K-lactobionate, 20 mM taurine, 10 mM KH PO , 2 4 Dispersed cells were then resuspended in Iscove’s modi- 20 mM Hepes, 110 mM sucrose and 1 g/l bovine serum fied Dulbecco’s medium supplemented with 20% foetal albumin (pH 7.1). The permeabilised muscle fibres were bovine serum, 3% chick embryo extract (custom made), weighedand addedtoanOxygraph-2krespiratorycham- 10 ng/ml fibroblast growth factor, 100 U/ml penicillin, ber (Oroboros Instruments, Innsbruck, Austria) containing 100 μg/ml streptomycin and 50 μg/ml gentamycin, and 2 ml of MiR06 (MiR05 supplemented with 280 U/ml cata- plated onto matrigel-coated dishes. Differentiation was lase at 37°C). Oxygen flux per muscle mass was recorded induced by changing the medium to Iscove’s modified online using DatLab software (Oroboros Instruments). Dulbecco’s medium supplemented with 2% horse serum After calibration of the oxygen sensors at air saturation, a and the antibiotics. few μlofH O were injected into the chamber to reach a 2 2 concentration of 400 μMO . In order to detect the elec- Measurement of ATP formation tron flow through CI and CII mitochondrial complexes, Tibialis anterior and diaphragm muscles were dissected, titrations of all of substrates, uncouplers and inhibitors were trimmed clean of visible fat and connective tissue, added in series as previously described [41,42]. The meas- minced with scissors and digested in ATP medium, con- urement of CIV respiration was obtained by addition of the taining 50 mM Tris-HCl (pH 7.4), 100 mM KCl, 5 mM artificial substrates N,N,N,N ’ ’-tetramethyl-p-phenylenediamine MgCl , 1.8 mM ATP, 1 mM ethylenediaminetetraacetic dihydrochloride and ascorbate [40]. Oxygen fluxes were acid (EDTA), and 0.1% collagenase type V for 10 minutes corrected by subtracting residual oxygen consumption at 37°C under strong agitation. After centrifugation, the from each measured mitochondrial steady-state. Respi- pellet was homogenised with Ultra-Turrax T10 (Ika-lab, rometry measurements were performed in duplicate on Staufen, Germany) for 10 seconds at maximum speed in each specimen. ATP medium. The mitochondrial fraction, obtained by different centrifugations (380 g and 10,000 g for five Real-time quantitative PCR minutes at 4°C), was then suspended in a mitochondria Satellite cells and muscle tissue samples were homoge- resuspension buffer containing 12.5 mM Tris acetate, nised, and RNA was extracted using the TRIzol protocol De Palma et al. Skeletal Muscle 2014, 4:22 Page 4 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 (Invitrogen-Life Technologies, Monza, Italy). Using pub- In vivo imaging using two-photon confocal microscopy lished protocols [43], after solubilisation in RNase-free Mitochondrial morphology and autophagosome forma- water, first-strand cDNA was generated from 1 μgof tion in living animals were monitored in tibialis anterior total RNA using the ImProm-II Reverse Transcription muscles transfected by electroporation with plasmids en- System (Promega). As show in Table 1, a set of primer coding pDsRed2-Mito or the LC3 protein fused to the pairs amplifying fragments ranging from 85 to 247 bp yellow fluorescent protein (YFP-LC3), as described pre- was designed to hybridise to unique regions of the ap- viously [29,38,46]. Two-photon confocal microscopy in propriate gene sequence. Real-time quantitative PCR the live, anaesthetised animals was then performed (qPCR) was performed using the SYBR Green Supermix 12 days later on in situ exposure of transfected muscles (Bio-Rad, Hercules, CA, USA) on a Roche LightCycler [29,38,46]. To allow the muscle to recover from the 480 Instrument (Roche, Basel, Switzerland). All reactions injection-induced swelling, microscopic observation was were run in triplicate. A melt-curve analysis was per- interrupted for two to five minutes. formed at the end of each experiment to verify that a single product per primer pair was amplified. As a con- Transmission electron microscopy trol experiment, gel electrophoresis was performed to Tibialis anterior muscles were dissected and fixed for verify the specificity and size of the amplified qPCR one hour in a solution containing 4% paraformaldehyde products. Samples were analysed using the Roche Light- and 0.5% glutaraldehyde in 0.1 M cacodylate buffer, Cycler 480 software and the second derivative maximum pH 7.4, immobilised on a Nunc Sylgard coated Petri dish method. The fold increase or decrease was determined (ThermoFisher Scientific, Waltham, MA, USA) to pre- relative to a calibrator after normalising to 36b4 (internal vent muscular contraction as previously described [47]. -ΔΔCT standard) through the use of the formula 2 [44]. The muscles were rinsed in the same buffer and dis- Mitochondrial DNA (mtDNA) from muscle tissue sected further into small blocks that were subsequently samples was quantified as described with slight modifi- processed for transmission electron microscopy (TEM) cations [45]. Briefly, total DNA was extracted with the as described elsewhere [48]. Briefly, the samples were QIAamp DNA mini kit (Qiagen, Milano, Italy). Twenty postfixed with osmium tetroxide (2% in cacodylate buf- ng of total DNA was assessed by qPCR. RNaseP gene fer), rinsed, en bloc stained with 1% uranyl acetate in was used as an endogenous control for nuclear DNA 20% ethanol, dehydrated and embedded in epoxy resin and the cytochrome b gene as a marker for mtDNA. (Epon 812; Electron Microscopy Science, Hatfield, PA, Primer sequences are shown in Table 1. USA) that was baked for 48 hours at 67°C. Thin sections Table 1 Primer pairs designed for qPCR analysis Name/symbol Gene accession Number Primer sequence Amplicon Atg4b NM_174874 F: 5′-ATTGCTGTGGGGTTTTTCTG-3′ 247 bp R: 5′-AACCCCAGGATTTTCAGAGG-3′ Atrogin-1 (fbxo32) NM_026346 F: 5′-GCAAACACTGCCACATTCTCTC-3′ 93 bp R: 5′-CTTGAGGGGAAAGTGAGACG-3′ Bnip3 NM_009760 F: 5′-TTCCACTAGCACCTTCTGATGA-3′ 150 bp R: 5′-GAACACGCATTTACAGAACAA-3′ Cytochrome b (mt-cytb) NC_005089 F: 5′-ACGCCATTCTACGCTCTATC-3′ 95 bp R: 5′-GCTTCGTTGCTTTGAGGTGT-3′ MuRF1 (Trim63) NM_001039048 F: 5′-ACCTGCTGGTGGAAAACATC-3′ 96 bp R: 5′-CTTCGTGTTCCTTGCACATC-3′ MUSA1 (fbxo30) NM_001168297, NM_027968 F: 5′-TCGTGGAATGGTAATCTTGC-3′ 191 bp R: 5′-CCTCCCGTTTCTCTATCACG-3′ p62 (Sqstm1) NM_011018 F: 5′-GAAGCTGCCCTATACCCACA-3′ 85 bp R: 5′-AGAAACCCATGGACAGCATC-3′ RNaseP (Rpp30) NM_019428 F: 5′-GAAGGCTCTGCGCGGACTCG-3′ 100 bp R: 5′-CGAGAGACCGGAATGGGGCCT-3′ 36b4 (Rplp0) NM_007475 F: 5′-AGGATATGGGATTCGGTCTCTTC-3′ 143 bp R: 5′-TCATCCTGCTTAAGTGAACAAACT-3′ F: forward, R: reverse. De Palma et al. Skeletal Muscle 2014, 4:22 Page 5 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 were obtained with a Leica ultramicrotome (Reichert (GeneTex, Irvine, CA, USA), goat polyclonal anti-actin Ultracut E and UC7; Leica Microsystems, Wetzlar, (I-19) or rabbit polyclonal anti-GAPDH (FL-335) primary Germany) stained with uranyl acetate and lead citrate, antibodies (Santa Cruz Biotechnology). When appropriate, and finally examined with a Philips CM10 TEM (Philips, rabbit polyclonal anti-FoxO3a (75D8), rabbit monoclonal Eindhoven, The Netherlands). Morphometric analysis of S6 ribosomal protein (54D2), rabbit polyclonal 4E-BP1 mitochondrial cristae complexity was evaluated with a (53H11), and rabbit polyclonal Akt primary antibodies stereological method. Briefly, a regular grid has been (Cell Signaling Technology) that recognise the protein in- superimposed over 10500X TEM micrographs and the dependently of its phosphorylation state were also used in number of intersections between the grid and mitochon- reprobing experiments. drial cristae was recorded. The same grid was used for all the different analysis. Confocal microscopy of myogenic precursor cells Cells were plated in eight-well Nunc LabTeck Chamber Protein isolation and western blotting slides (ThermoFisher Scientific). When indicated cells Satellite cells were harvested and homogenised for were transfected with YFP-LC3 plasmid. Transfections 10 minutes at 4°C in RIPA lysis buffer, containing 50 mM were performed with the Lipofectamine LTX with Plus Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 1% sodium reagent (Invitrogen-Life Technologies) according to the deoxycholate, 1 mM EDTA and 0.1% sodium dodecyl manufacturer’s instructions. The cells were used 24 hours sulphate (SDS). Tissue samples from muscles were homo- after transfection in the various experimental settings genised in a lysis buffer containing 20 mM Tris-HCl described. For confocal imaging, the cells were fixed in (pH 7.4), 150 mM NaCl, 1% Triton X-100, 10% glycerol, paraformaldehyde and washed in phosphate-buffered sa- 10 mM EGTA and 2% SDS. Buffers were supplemented line [50]. To prevent nonspecific background, cells were with a cocktail of protease and phosphatase inhibitors incubated in 10% goat serum/phosphate-buffered saline (cOmplete and PhosSTOP; Roche). Protein concentration followed by probing with the primary antibody mouse was determined using the bicinchoninic acid assay (Ther- monoclonal anti-cyclophillin D (Abcam). Cells were then moFisher Scientific). Using published protocols [49], SDS incubated with the secondary antibody, Alexa Fluor 546 and β-mercaptoethanol were added to samples before dye-conjugated anti-mouse IgG (Molecular Probes-Life boiling, and equal amounts of proteins (40 μg/lane) were Technologies, Monza, Italy). Slides were placed on the separated by 4% to 20% SDS-polyacrylamide gel elec- stage of a TCS SP2 Laser-Scanning Confocal microscope trophoresis (Criterion TGX Stain-free precast gels and (Leica Microsystems) equipped with an electronically Criterion Cell system; Bio-Rad). Proteins were then trans- controlled and freely definable Acousto-Optical Beam ferred onto a nitrocellulose membrane using a Bio-Rad Splitter. Images were acquired with x63 magnification Trans-Blot Turbo System. The membranes were probed oil-immersion lenses. Analyses were performed using using the following primary antibodies as indicated in the Imagetool software (Health Science Center, University of text: goat polyclonal anti-HSP60 (N-20) and rabbit poly- Texas, San Antonio, TX, USA). Images of cells express- clonal anti-MyoD (C-20) (Santa Cruz Biotechnology, ing YFP-LC3 were thresholded by using the automatic Dallas, TX, USA), mouse monoclonal anti-ClpP and rabbit threshold function. polyclonal anti-LC3B (Sigma-Aldrich, Saint Louis, MO, USA), rabbit polyclonal anti-Mul-1 (Abcam, Cambridge, Immunohistochemistry and histology UK), mouse monoclonal anti-sarcomeric myosin (MF20) Laminin and haematoxylin and eosin (H & E) staining (Developmental Studies Hybridoma Bank, Iowa City, IA, were performed as previously described [47,51]. To meas- USA), rabbit polyclonal anti-phospho-FoxO3a (Ser253), ure the cross sectional area (CSA) of myofibres, muscle rabbit polyclonal anti-phospho-S6 ribosomal protein sections were stained with an anti-laminin A antibody (Ser240/244), rabbit monoclonal anti-phospho-4E-BP1 (L1293; Sigma-Aldrich). Laminin, a cell-adhesion mol- (Thr37/46) (263B4) and rabbit polyclonal anti-phospho- ecule strongly expressed in the basement membrane of Akt (Ser473) (Cell Signaling Technology, Danvers, skeletal muscle, was detected using an appropriate sec- MA, USA). After the incubation with the appropriate ondary antibody. Morphometric analyses were performed horseradish-peroxidase-conjugated secondary antibody on sections collected from similar regions of each muscle (Cell Signaling Technology), bands were visualised using using a Leica DMI4000 B automated inverted microscope the Bio-Rad Clarity Western ECL substrate with a Bio-Rad equipped with a DCF310 digital camera. Image acquisition ChemiDoc MP imaging system. To monitor for potential was controlled by the Leica LAS AF software. The ImageJ artefacts in loading and transfer among samples in dif- software was used to determine the CSA of 1,000 to 3,000 ferent lanes, the blots were routinely treated with the individual fibres from at least two different fields for each Restore Western Blot Stripping Buffer (ThermoFisher muscle section. Four to nine sections from each muscle Scientific) and reprobed with rabbit polyclonal anti-calnexin were analysed. For histological analyses, serial muscle De Palma et al. Skeletal Muscle 2014, 4:22 Page 6 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 sections were obtained and stained in H & E following tibialis anterior muscle sections (20 to 30 from each standard procedures. The number of fibres was counted muscle) were then collected and the immunofluorescence and analysed using the ImageJ software. of EBD-positive fibres was imaged using Texas red red fil- Single myofiber isolation of hind limb muscle and nu- ter. Creatine kinase (CK) serum levels (units per litre) were clei immunofluorescence on single fibers was performed measured in blood samples obtained from the tail vein of as previously described [8]. Nuclei of 30 individual fibres mice after treadmill running. The blood was centrifuged at from each muscle were analysed. 13,000 × g at 4°C and the supernatant used to measure CK activity in an indirect colorimetric assay (Randox Labora- Whole body tension tories, Crumlin, Northern Ireland, UK) [16,18]. The whole body tension (WBT) procedure was used to determine the ability of mice to exert tension in a for- Statistics ward pulling manoeuvre that is elicited by stroking the Upon verification of normal distribution, the statistical tail of the mice [52]. The tails were connected to a Grass significance of the raw data between the groups in each FT03 transducer (Astro-Med, West Warwick, RI, USA) experiment was evaluated using the unpaired Student’s with a 4.0 silk thread (one end of the thread being tied t-test (single comparisons) or one way analysis of vari- to the tail and the other end to the transducer) [47]. ance (ANOVA) followed by the Newman-Keuls post-test Each mouse was placed into a small tube constructed of (multiple comparisons). The GraphPad Prism software a metal screen with a grid spacing of 2 mm. The mice package (GraphPad Software, La Jolla, CA, USA) was entered the apparatus and exerted a small resting ten- used. After statistics (raw data), data from different ex- sion on the transducer. Forward pulling movements periments were represented and averaged in the same were elicited by a standardised stroke of the tail with graph. The results are expressed as means ± SEM of the serrated forceps, and the corresponding forward pulling indicated n values. tensions were recorded using a Grass Polyview recording system (Astro-Med). Between 20 and 30 strokes of the Chemicals tail forward pulling tensions were generally recorded pDsRed2-Mito was a gift of Prof. Luca Scorrano (University during each session. The WBT was determined by divid- of Padova, Padova, Italy). Dispase was purchased from ing the average of the top ten or top five forward pulling Gibco-Life Technologies (Monza, Italy). TMRM and the tensions, respectively, by the body weight and represent secondary antibody for laminin experiments were obtained the maximum phasic tension that can be developed over from Molecular Probes-Life Technologies. Iscove’s modified several attempts [52]. It is important to note that treat- Dulbecco’s medium, penicillin, streptomycin, gentamycin, ments or conditions which primarily alter muscle mass horse serum, and foetal bovine serum were purchased from without changing the tension developed per unit of Euroclone (Pero, Italy). Matrigel was obtained from BD- muscle mass produce corresponding alterations in forward Bioscience (Milano, Italy). Primer pairs were obtained from pulling tension that are not associated with changes in Primmbiotech (Milano, Italy). Fibroblast growth factor was either WBT 5 or WBT 10 [52,53]. purchased from Tebu-bio (Milano, Italy). DETA-NO and KT5823 were obtained from Merck Millipore (Darmstadt, Treadmill running Germany). ODQ and cyclosporine were purchased from Animals were made to run on a standard treadmill Enzo Life Sciences (Farmingdale, NY, USA). L -arginine machine (Columbus Instruments, Columbus, OH, USA) methylester (L-NAME) and the other chemicals were pur- either on a 0% grade or tilted 10% downhill starting at a chased from Sigma-Aldrich. warm-up speed of 5 m/minute for five minutes [54]. Every subsequent five minutes, the speed was increased by 5 m/ Results minute until the mice were exhausted. Exhaustion was de- nNOSμ deficiency leads to mitochondrial dysfunction fined as the inability of the animal to return to running Mitochondrial function in skeletal muscles of adult within 10 seconds after direct contact on an electric stimu- NOS1-/- mice, that is, at P120, was dissected and com- lus grid. Running time was measured and running distance pared with that of the respective age-matched wild-type calculated. Distance is the product of time and speed of littermattes (control). Mitochondrial membrane potential the treadmill. was monitored in isolated fibres from flexor digitorum bre- As a measure of membrane permeability, the Evans blue vis muscles loaded with TMRM, a potentiometric fluores- dye (EBD) assay was used [47]. A concentration of 5 μg/μl cent dye. TMRM accumulates in the mitochondria that EBD prepared in physiological saline was injected intraven- maintain a polarised mitochondrial membrane potential. ously through the tail vein. Injections (50 μl/10 g body A latent mitochondrial dysfunction masked by the ATP weight) were performed 20 to 30 minutes after treadmill synthase operating in a reverse mode, that is, to consume running. Mice were sacrificed 24 hours after EBD injection. ATP in order to maintain the mitochondrial membrane De Palma et al. Skeletal Muscle 2014, 4:22 Page 7 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 potential, can be unveiled using the ATP synthase inhibi- the muscle bioenergetic parameters. To this end, we tor oligomycin [25]. In agreement with previous reports measured ATP generation from OXPHOS in isolated [29], addition of oligomycin to control mice fibres did not mitochondria of tibialis anterior and diaphragm muscle cause immediate changes in membrane potential even fibres. As shown in Figure 1B, total OXPHOS-generated after extensive incubation (Figure 1A). Conversely, mito- ATP was significantly lower in NOS1-/- mice when chondria in fibres of NOS1-/- mice underwent marked de- compared to control. polarisation after oligomycin. We then analysed the mitochondrial bioenergetics in We investigated whether the latent mitochondrial dys- intact fibres using an in situ approach measuring oxygen function observed in muscles of NOS1-/- mice affected consumption by high resolution respirometry. By this Figure 1 Mitochondrial metabolism is impaired in skeletal muscles of NOS1-/- mice. Fibres were isolated from different muscles of wild-type and NOS1-/- mice at P120. (A) Mitochondrial membrane potential measured in fibres isolated from flexor digitorum brevis muscles, loaded with TMRM and treated with 5 μM oligomycin (Olm) or 4 μM FCCP. TMRM staining was monitored in six to ten fibres obtained from at least three different animals per experimental group. Data are expressed by setting the initial value as 1. (B) ATP production on mitochondria isolated from tibialis anterior and diaphragm muscles, at 10 minutes after substrate addition. Data are expressed by setting the initial value as 1. (C-D) Oxygen consumption on fibres isolated from tibialis anterior and diaphragm muscles, supplied with specific CI, CII and CIV mitochondrial complex substrates, as indicated in the Methods. (E) Quantitative analysis of the mtDNA copy number. Data are expressed by normalizing mtDNA values versus nuclear DNA. Each histogram represents the data obtained from at least five different animals per experimental group. * P <0.05 and ** P <0.01 versus the respective wild-type control. De Palma et al. Skeletal Muscle 2014, 4:22 Page 8 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 approach, we found that the maximal tissue mass- showed the presence of autophagic vacuoles and multi- specific OXPHOS capacity with physiological combina- vesicular bodies, indicative of an active autophagic path- tions of CI mitochondrial complex substrates was similar way [59], in tibialis anterior and diaphragm muscles of in both tibialis anterior and diaphragm of NOS1-/- and P120 NOS1-/- mice (Figure 2B and Additional file 1: control mice (Figure 1C-D). In contrast, the CII-linked Figure S1C). The enhanced autophagy in the absence of respiratory capacity in tibialis anterior of NOS1-/- mice nNOSμ in skeletal muscle was confirmed by Western was lower than that in control muscle fibres, while no blot analysis. The appearance of a faster migrating band difference was observed in the diaphragm. In both tibi- of LC3 protein due to its lipidation and cleavage is a alis anterior and diaphragm of NOS1-/- mice the CIV- common marker of autophagy induction [58]. As shown linked respiratory capacity decreased significantly with in Figure 2G, tibialis anterior muscles of P120 NOS1-/- respect to the controls. Of interest, qPCR analysis of mice exhibited increased lipidated LC3 levels when com- mtDNA levels in tibialis anterior and diaphragm mus- pared to control mice. Similar results on LC3 conversion cles did not reveal any difference between NOS1-/- and were obtained analysing diaphragm muscle samples (see control mice (Figure 1E) suggesting that mitochondrial Additional file 1: Figure S1D). mass was not affected and defects in OXPHOS were due mt to dysfunctional mitochondria. NO signalling regulates UPR and autophagy machinery Activation of the NO-dependent enzyme guanylate nNOSμ deficiency affects mitochondrial network cyclase, with formation of cyclic guanosine monophosphate mt remodelling, UPR and autophagy (cGMP) and activation of a variety of downstream signal- Alterations in the content, shape or function of the ling cascades, including cGMP-dependent-protein kinases mitochondria have been associated with muscle homeo- (PKG), contributes significantly to mediate the physio- stasis [31,55]. To identify the changes in mitochondrial logical effects of NO in muscle [2,5,60]. To investigate the mt network morphology, tibialis anterior muscles of P120 involvement of the cGMP-dependent signalling on UPR NOS1-/- and wild-type control mice were imaged using and autophagy, myogenic precursor cells were differenti- pDsRed2-Mito, a mitochondrially targeted red fluores- ated for six hours in the absence (control) or in the pres- cent protein, by in situ two-photon confocal microscopy ence of the inhibitor of NOS L-NAME (6 mM), the [29,38,46]. NOS1-/- mice showed a disorganised mito- inhibitor of guanylate cyclase ODQ (10 μM), and the in- chondrial network (Figure 2A). Accordingly, ultrastruc- hibitor of PKG KT5823 (1 μM) [61-66]. L-NAME, ODQ tural analyses by TEM (Figure 2B and Additional file 1: and KT5823 treatment increased the expression of HSP60 Figure S1A) revealed changes in the subsarcolemmal and ClpP protein. The NO donor DETA-NO (80 μM) and mitochondria of tibialis anterior muscles of NOS1-/- mice the membrane-permeant cGMP analogue 8Br-cGMP that exhibited, in thin sections, a significant increase in (2.5 mM) [62-66] reversed the effects of L-NAME and mitochondrial surface area (Figure 2C) and a significant ODQ, respectively (Figure 3A). decrease in the density of the cristae (Figure 2D), as com- In another set of experiments, cells were transiently pared with the controls. The same evaluation was per- transfected with YFP-LC3 and then differentiated. As formed on subsarcolemmal mitochondria from diaphragm shown by confocal microscopy fluorescence analysis of muscle with similar results (data not shown). The analysis LC3 and the mitochondrial matrix-specific protein cyclo- of intermyofibrillar mitochondria (see Additional file 1: phillin D (Figure 3B), in control cells LC3 staining was dif- Figure S1B) showed a pattern of enlarged mitochondria fuse and the majority of mitochondria were in the indicating that the presence of these mitochondrial alter- elongated form, indicating myogenic differentiation [25] ations in NOS1-/- mice muscle is not restricted to the and a low rate of autophagy. L-NAME, ODQ, and KT5823 sarcolemma but is a more general phenomenon. treatment, while inducing mitochondrial fragmentation, We then analysed two downstream processes linked to resulted in LC3 localisation into dot cytoplasmic struc- mt mitochondrial stress: UPR and autophagy. In tibialis tures, as compared to the diffuse cytoplasmic distribution anterior muscles of P120 NOS1-/- mice the expression observed in control cells. The effects of L-NAME and of the nuclearly-encoded mitochondrial chaperones ODQ were prevented by DETA-NO and 8Br-cGMP, HSP60 and the protease ClpP, which correlates with the respectively. level of unfolded proteins in mitochondria [56,57], was NO control of autophagy was assessed further by analys- found to be higher than in the controls (Figure 2E). In ing the expression of relevant markers of the autophagic addition, the two-photon confocal microscopy of the signalling pathway, namely LC3, by western blotting and YFP-LC3 [29,38,46] revealed the presence of LC3- p62, Bnip3 and Atg4 by qPCR analysis [58,67]. L-NAME, positive vesicles, an established marker of autophago- ODQ and KT5823 treatments increased lipidated LC3 some formation [58], in tibialis anterior muscles of P120 conversion in differentiated satellite cells and LC3 lipida- NOS1-/- mice (Figure 2F). Furthermore, TEM analysis tion induced by L-NAME and ODQ was blocked by De Palma et al. Skeletal Muscle 2014, 4:22 Page 9 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 mt Figure 2 Mitochondrial morphology, UPR and autophagy in skeletal muscles of NOS1-/- mice. Tibialis anterior muscles were isolated from wild-type and NOS1-/- mice at P120. (A) In vivo imaging of the mitochondrial network by two-photon confocal microscopy. Muscles were transfected with the mitochondrially targeted red fluorescent protein pDsRed2-Mito. The images are representative of results obtained from at least five different animals per experimental group. Scale bar: 10 μm. (B) TEM images detecting the presence of abnormal, enlarged subsarcolemmal mitochondria (asterisks) or autophagic vacuoles (arrowheads) in NOS1-/- muscles. The inset depicts a multivesicular body in NOS1-/- fibres taken at higher magnification. The images are representative of results obtained from at least three different animals per experimental group. (C-D) Subsarcolemmal mitochondrial ultrastructure analysis by TEM. Data represent the quantification of the mitochondrial area and morphometric analysis of mitochondrial cristae complexity. Each histogram represents the data obtained from at least three different animals per experimental group. * P <0.05 and ** P <0.01 versus the respective wild-type control. (E) Western blot analysis of HSP60 and ClpP expression. Actin was used as the internal standard. The image is representative of results obtained from at least five to seven different animals per experimental group. (F) In vivo imaging of autophagosome formation by two-photon confocal microscopy. Muscles were transfected with YFP-LC3. The images are representative of results obtained from at least five different animals per experimental group. Scale bar: 10 μm. (G) Western blot analysis of LC3 lipidation. Actin was used as the internal standard. The image is representative of results obtained from at least 10 different animals per experimental group. DETA-NO or 8Br-cGMP, respectively (Figure 4A). In from the cytoplasm to the nucleus determines the direct addition, cells treated with L-NAME, ODQ and KT5823 transcriptional activation of genes essential to autopha- expressed higher levels of transcripts encoding p62, Bnip3 gosome formation, namely p62, Bnip3 and Atg4 [58,67]. and Atg4 (Figure 4B). Enhanced activity of FoxO transcription factors has also been associated with disruption of mitochondrial func- Deficient nitric oxide signalling promotes FoxO3-Mul-1 tion and organisation leading to impaired skeletal axis muscle function and development [29]. As shown in Catabolic conditions activate FoxO transcription factors, Figure 5A, C, phosphorylated FoxO3 levels in tibialis which stimulate the ubiquitin-proteasome system as a anterior and diaphragm muscles of P120 NOS1-/- mice response to skeletal muscle-wasting [31,55]. FoxO3 ac- were lower than in the controls. In addition, tibialis an- tivity is necessary and sufficient for the induction of au- terior and diaphragm muscles of NOS1-/- mice overex- tophagy in skeletal muscle [38]. FoxO3 translocation pressed the protein corresponding to mitochondrial De Palma et al. Skeletal Muscle 2014, 4:22 Page 10 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 mt Figure 3 NO signalling, UPR , and autophagy on myogenic precursor cells. Cells were differentiated for six hours in the absence (control) or in the presence of L-NAME (6 mM), ODQ (10 μM), KT5823 (1 μM), L-NAME + DETA-NO (80 μM), and ODQ +8 Br-cGMP (2.5 mM). (A) Western blot analysis of HSP60 and ClpP expression. Actin was used as the internal standard. (B) Confocal microscopy imaging of cells transfected with YFP-LC3. Mitochondrial morphology was detected by mitochondrial matrix-specific protein cyclophillin D (CypD) staining. Scale Bar: 10 μm. Images are representative of at least three to five independent experiments.. ubiquitin ligase Mul-1 (Figure 5B, D), which has been obtained with MUSA1 analysis are difficult to correlate recently reported to be upregulated in muscle through with nNOSμ deficiency. These findings indicate that the FoxO3 transcription factors and promoting mitochon- effects of nNOSμ absence on E3 ubiquitin ligases mainly drial fission, depolarization and mitophagy [68,69]. As affect expression of Mul-1 gene. shown in Figure 5E, in vitro treatment of differentiated myogenic precursor cells from wild-type control mice nNOSμ deficiency affects muscle growth with L-NAME, ODQ and KT5823 increased Mul-1 pro- We evaluated the effects of the absence of nNOSμ on tein expression. The effects induced by L-NAME and skeletal muscle phenotype. Tibialis anterior, gastrocne- ODQ were blocked by DETA-NO and 8Br-cGMP, re- mius, soleus, and extensor digitorum longus muscles were spectively. In tibialis anterior and diaphragm muscles of dissected and weighed. Since the body weight and the P120 NOS1-/- mice, qPCR analysis of other E3 ubiquitin visceral adipose tissue of NOS1-/- male mice were sig- ligases, atrogin-1 and MuRF1, involved in muscle loss nificantly lower than wild-type control (see Additional [69,70], ruled out a nNOSμ-dependent modulation of file 2: Figure S2A-B) [71] we calculated the muscle size their expression (Figure 5F, G). Also, the differences relative to body weight [72]. As shown in Figure 6A and De Palma et al. Skeletal Muscle 2014, 4:22 Page 11 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 Figure 4 NO signalling and autophagic pathway on myogenic precursor cells. (A) Western blot analysis of LC3 lipidation in cells differentiated for six hours in the absence or in the presence of L-NAME (6 mM), ODQ (10 μM), KT5823 (1 μM), L-NAME + DETA-NO (80 μM), and ODQ +8 Br-cGMP (2.5 mM). Actin was used as the internal standard. Image is representative of at least five independent experiments. (B) qPCR analysis of mRNA levels for p62, Bnip3 and Atg4 in cells differentiated for six hours in the absence (control) or in the presence of L-NAME ODQ, and KT5823. Values are expressed as the fold change over control. Each histogram represents the data obtained from at least five independent experiments. * P <0.05 versus respective control. Additional file 2: Figure S2C, the relative mass of the The examination of multiple time points was then muscles for the P120 NOS1-/- mice was significantly carried out in order to establish a possible link between lower than the relative mass of the muscles for the con- the changes in mitochondrial homeostasis and the re- trol mice. This excludes the possibility that the changes duction in muscle size. The CSA (Figure 7A-C) and the in muscle mass are simply due to an overall change in number of myonuclei (Figure 7D) of hind limb muscle size of the mice. fibres were significantly decreased in P10 NOS1-/- The overall morphology of the tibialis anterior and mice, when compared with the respective control. diaphragm muscle in P120 NOS1-/- mice was normal, Muscle growth during post-natal development (P0 to without pathological features of necrosis, macrophage P21), but not at later stages, is accompanied by a con- infiltration and centronucleated fibres (see Additional tinuous increase in the number of myonuclei resulting file 2: Figure S2D). In addition, the number of fibres in from satellite cell fusion [69,73]. As shown in Figure 7E, tibialis anterior muscles was comparable in both NOS1-/- cells exhibited lower levels of myosin and NOS1-/- and control mice (Figure 6B). By contrast, lam- MyoD, which are markers of myogenic differentiation, inin staining of tibialis anterior and diaphragm, used to as compared to control cells. Interestingly, CycloD stain- identify individual muscle fibres, revealed a significant ing of differentiating myogenic precursor cells indicated decrease in the mean CSA of tibialis anterior and dia- that the absence of nNOSμ induces diffuse mitochondrial phragm sections in P120 NOS1-/- mice when compared fragmentation (Figure 7F) [25]. Taken together, our data with control (Figure 6C-H). argue that the absence of nNOSμ induces mitochondrial De Palma et al. Skeletal Muscle 2014, 4:22 Page 12 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 Figure 5 NO signalling, FoxO3, and ubiquitin ligases. Western blot analysis of phosphorylated FoxO3 levels (pFoxO3) or mitochondrial ubiquitin ligase Mul-1 expression in tibialis anterior (A-B) and diaphragm (C-D) of wild-type and NOS1-/- mice at P120. FoxO3 or actin were used as the internal standard. The images are representative of results obtained from at least four to ten different animals per experimental group. (E) Western blot analysis of Mul-1 expression in myogenic precursor cells differentiated in the absence or in the presence of L-NAME (6 mM), ODQ (10 μM), KT5823 (1 μM), L-NAME + DETA-NO (80 μM) and ODQ +8 Br-cGMP (2.5 mM). Actin was used as the internal standard. The image is representative of at least five independent experiments. qPCR analysis of mRNA levels for atrogin-1, muRF1 and MUSA1 in tibialis anterior (F) and diaphragm (G) muscles of wild-type and NOS1-/- mice at P120. Values are expressed as the fold change over wild-type. Each histogram represents the data obtained from at least five to eight different animals per experimental group. * P <0.05 versus the respective wild-type control. fragmentation and a deficit in satellite cell fusion/differen- Using NOS1-/- mice it has been previously shown that tiation, thus impairing fibre growth. nNOS modulates the mechanism of disuse-induced atro- At P30 we found that the CSA of tibialis anterior was phy via FoxO transcription factors [75]. Our observation significantly decreased in NOS1-/- mice, when com- that at P10, P30 (see Additional file 2: Figure S2E-F) and pared with controls (Figure 8A-C). In this crucial time P120 (Figure 5E-F) NOS1-/- and control mice expressed of muscle growth we also measured the activation of the similar levels of transcripts encoding the classical atro- Akt-mTOR pathway as a positive regulator [55,69,73,74]. genes atrogin-1 and MuRF1 [69,70,75], indicates that the As shown in Figure 8D, phosphorylated levels of S6 ribo- atrophy pathways do not play a key role in the develop- somal protein, 4E-BP1 and Akt in tibialis anterior mus- ment of NOS1-/- muscles. cles of NOS1-/- mice were lower than in the controls. FoxO3 proteins are phosphorylated by Akt, which ren- nNOSμ deficiency affects muscle function ders them inactive; this may explain why phosphorylated We evaluated whether the absence of nNOSμ affected FoxO3 levels were found to be lower as well, while Mul- skeletal muscle function. The WBT measurement deter- 1 was overexpressed (Figure 8E). Of importance, both mines the total phasic forward pulling tension exerted events are correlated with muscle mitochondrial dys- by the fore and hind limb muscles and reflects the max- function and growth [29,55,68,69,73,74]. imal acute phasic force the mouse can achieve to escape De Palma et al. Skeletal Muscle 2014, 4:22 Page 13 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 Figure 6 Skeletal muscle phenotype of wild-type and NOS1-/- mice at P120. (A) Weight of tibialis anterior, gastrocnemius, soleus, and extensor digitorum longus (EDL) muscles. The muscle size is relative to body weight. Each histogram represents the data obtained from at least 10 different animals per experimental group. (B) The number of myofibres in tibialis anterior. Each histogram represents the data obtained from at least four to five different animals per experimental group. Laminin staining of tibialis anterior (C-E) and diaphragm (F-H) muscles. (C, F) Immunohistochemical images. Scale bar: 100 μm. (D, G) Representative distribution of CSA values. (E, H) Quantification of CSA. Images and quantifications represent the data obtained from at least four to seven different animals per experimental group. *P <0.05, **P <0.01, and ***P <0.001 versus the respective wild-type control. a potentially harmful event [52]. As shown in Figure 9A, significant exercise intolerance after repetitive exercise the WBT normalised for body weight in P120 NOS1-/- challenges, while control mice at day 3 showed even im- mice was significantly lower than in the wild-type con- proved exercise capacity, compared to day 1. NOS1-/- trol, consistent with an unpaired muscle specific force mice also exhibited a significantly decreased treadmill output in the absence of nNOSμ. runtime to exhaustion (Figure 9C). We also examined the muscle resistance to fatigue: we We then assessed the structure/damage of skeletal subjected NOS1-/- mice to treadmill running, that mea- muscle myofibres after exercise. TEM analysis performed sures resistance to fatigue during a forced exercise, and ex- in tibialis anterior muscles of P120 NOS1-/- mice after the amined both exercise performance and tolerance. As treadmill running showed marked ultrastructural changes, shown in Figure 9B, the total distance run by NOS1-/- as, for instance, defects in the organisation of the contract- mice during one bout of exhaustive treadmill running (day ile apparatus (sarcomere), that were observed neither in 1) was significantly lower when compared to controls. the wild-type mice nor in unchallenged NOS1-/- mice This reduction in performance of NOS1-/- mice was also (Figure 9D). The features observed in challenged NOS1-/- observed after repeated challenges: NOS1-/- mice showed mice might be a direct consequence of denervation events De Palma et al. Skeletal Muscle 2014, 4:22 Page 14 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 Figure 7 Skeletal muscle phenotype of wild-type and NOS1-/- mice at P10. (A-C) Laminin staining of hind limb muscles. (A) Immunohistochemical images. Scale bar: 100 μm. (B) Representative distribution of CSA values. (C) Quantification of CSA. Images and quantifications represent the data obtained from at least five different animals per experimental group. (D) Number of myonuclei per fibre in hind limb muscles. Each histogram represents the data obtained from at least three different animals per experimental group. (E) Western blot analysis of myosin (MF20) and MyoD expression in myogenic precursor cells isolated from wild-type and NOS1-/- mice and differentiated for increasing times. Calnexin was used as the internal standard. Images are representative of at least three independent experiments. (F) Confocal microscopy imaging of myogenic precursor cells isolated from wild-type and NOS1-/- mice and differentiated for 48 hours. Mitochondrial morphology was detected by mitochondrial matrix-specific protein cyclophillin D staining. Scale Bar: 10 μm. Images are representative of at least three independent experiments. * P <0.05 versus the respective wild-type control. as also indicated by collagen fibres deposition and motor time a link between a deficit in NO signalling, mito- end-plates lacking the presynaptic nerve ending (data not chondrial alterations and skeletal muscle impairments. shown). As shown in Figure 9E, tibialis anterior muscles The first result emerging from our analysis is that of P120 NOS1-/- mice after the treadmill running dis- nNOSμ deficiency is per se sufficient to induce profound played an increased uptake versus wild-type of EBD, which defects in mitochondria, with alterations in mitochondrial stains damaged myofibres [47]. As an in vivo indicator of distribution, shape, morphology and size accompanied by skeletal muscle damage we also analysed the serum levels a latent mitochondrial dysfunction such that energy gener- of CK, a skeletal muscle enzyme released during fibre de- ation is impaired. Nitric oxide has several key functions in generation whose activity increased in dystrophic animals mitochondria: it inhibits mitochondrial fission, induces [16,18]. As expected, in NOS1-/- mice after the treadmill mitochondrial biogenesis and controls mitochondrial re- running, the serum CK activity was found to be signifi- spiratory rate by reversible inhibition of complex IV in the cantly higher than that in the wild-type mice (Figure 9F). mitochondrial respiratory chain [25,76,77]. Furthermore, it controls the expression of several enzymes in the Krebs Discussion cycle [78]. Derangement of these mitochondrial functions This study documents that nNOSμ deficiency, while se- is most likely at the basis of the multiple mitochondrial verely altering the structure and bioenergetics potential deficits we observed in NOS1-/- mice. of skeletal muscle mitochondria does not impact signifi- Of importance, we found that this overall mitochon- cantly on the overall resting muscle structure, apart from drial dysfunction was accompanied both in intact myofi- reducing muscle mass and the CSA of the myofibres of bres in vivo and in isolated satellite cells in vitro by an mt specific muscles. When the muscle is exposed to work- enhanced UPR response. It has been hypothesised that mt loads, however, the consequences of nNOSμ deficiency the UPR is activated prior to the induction of autoph- become apparent, with a significantly reduced resistance agy [79]; in particular, that the autophagy pathway is ac- of the muscles accompanied by increased sensitivity to tivated when mitochondria cannot maintain a polarised mt exercise-induced damage. This establishes for the first membrane potential despite UPR activation. We found De Palma et al. Skeletal Muscle 2014, 4:22 Page 15 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 Figure 8 Skeletal muscle phenotype of wild-type and NOS1-/- mice at P30. (A-C) Laminin staining of tibialis anterior muscles. (A) Immunohistochemical images. Scale bar: 100 μm. (B) Representative distribution of CSA values. (C) Quantification of CSA. Images and quantifications represent the data obtained from at least five different animals per experimental group. Western blot analysis in tibialis anterior: (D) phosphorylated S6, 4E-BP1 and Akt levels, (E) phosphorylated FoxO3 levels or mitochondrial ubiquitin ligase Mul-1 expression. S6, 4E-BP1, Akt, FoxO3 or actin were used as the internal standard. The images are representative of results obtained from at least four different animals per experimental group. *P <0.05 versus the respective wild-type control. mt that the increase in UPR was accompanied by autoph- controls, although they did not show any pathological agy and increased expression of molecules relevant to features reminiscent of muscle damage, such as inflam- autophagic signalling, namely p62, Bnip3 and Atg4. This mation, necrosis or fibrosis. Similar morphological data suggests that nNOSμ deficiency leads to a sufficiently se- were obtained in male NOS1-/- mice backcrossed onto vere mitochondrial deficit that cannot be restored by the B6129 background (our experimental model) [71] or mt mt UPR . The enhanced autophagic and UPR response backcrossed onto the C57BL/6 background [80], al- were normalised when the cGMP-dependent signalling though in the latter model no difference in tibialis anterior was activated, indicating that these events are controlled muscle mass relative to body mass was reported. That by NO via its physiological second messenger cGMP. thedecreaseinmusclemassis due to mechanisms other The second relevant information is that an altered NO than the decrease in body mass was recently suggested system leads to impairment of muscle function that is using NOS1-/- mdx mice [72]. The deficiency of nNOSμ selective to specific parameters and unmasked during is also accompanied by muscle ageing [81] and fibre exercise. In particular we found that skeletal muscles in growth was prevented in the NOS1-/- mice model of the absence of nNOSμ are smaller relative to the rest of skeletal muscle hypertrophy [82] and NOS1-/- mdx the body, thus indicating that muscle mass decrease was mice [72]. In a recent study, no difference in the weight not simply attributable to a generalised decreased body and CSA of tibialis anterior muscles from NOS1-/- and mass tissues (including adipose tissue) and likely due to control was also reported but the animal background a specific reduction in the size of the muscle fibres was not indicated [83]. Discrepancies in these studies themselves. In agreement with this, NOS1-/- mice mus- may be explained, at least in part, by strain-specific cles (that is, tibialis anterior and diaphragm) displayed modulation of the nNOSμ-regulated phenotype, a hy- smaller myofibre CSA when compared to littermate pothesis substantiated by the observation, by the same De Palma et al. Skeletal Muscle 2014, 4:22 Page 16 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 Figure 9 Skeletal muscle function in wild-type and NOS1-/- mice. (A) WBT measurements determined by dividing the average of the top ten or top five forward pulling tensions, respectively, by the body weight. (B) Running distance calculated during one bout of exhaustive treadmill running (day 1) and after repeated challenges (days 2 and 3). (C) Treadmill runtime to exhaustion calculated as the averages obtained at day 1 to 3. Each histogram represents the data obtained from at least four to five different animals per experimental group. (D) TEM analysis performed in tibialis anterior muscles of both unchallenged (no run) and challenged (exhaustive running) mice. The images are representative of results obtained from at least three different animals per experimental group. (E) EBD uptake in tibialis anterior muscles after the treadmill running. Scale Bar: 100 μm. The images are representative of results obtained from at least four different animals per experimental group. (F) CK serum levels (units per litre) of mice after treadmill running. Each histogram represents the data obtained from at least four different animals per experimental group. *P <0.05, **P <0.01 and ***P <0.001 versus the respective wild-type control. WBT and treadmill running were performed on animals at P120. group, that morphological data differed between NOS1-/- The functional studies revealed two important aspects mice backcrossed onto the C57BL/6 and the B6129 back- of the role of NO in skeletal muscle. Firstly, the fact that ground [37,71,80]. NOS1-/- mice in our in vivo experiments exhibited a De Palma et al. Skeletal Muscle 2014, 4:22 Page 17 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 deficit in forward pulling tension and resistance to performance and provide an indication of the mechanism fatigue during a forced exercise indicates that nNOSμ is responsible for the impaired fibre growth resulting in a def- important to maintain skeletal muscle strength and the icit of muscle performance. In particular, nNOSμ absence animal’s ability to perform in repetitive exercise training. altered mitochondrial homeostasis in myogenic precursor Our results in vivo are in line with a previous study with cells with a decrease in the number of myonuclei per fibres an in situ approach reporting that nNOSμ-deficient tibi- and impaired muscle development at early stages of growth. alis anterior muscles exhibit a reduced force production This also suggests that fusion of myogenic precursor cells and a specific deficit in adapting to exercise and develop during perinatal myogenesis is impaired. Accordingly, NO profound fatigue upon repeated contraction [71]. An ex- has been shown to stimulate the ability of myogenic precur- cessive fatigue has been also observed in NOS1-/- mice sor cells to become activated and fuse to each other and wild-type mice treated with a nNOS inhibitor [12]. [5,8,85]. There is a general agreement that mitochondria A specific and intrinsic deficit in muscle force produc- change when the myoblasts differentiate into myotubes tion has been recently reported in NOS1-/- mdx mice, [27]. Also, NO maintains functional mitochondria and this although muscle fatigue was unaffected by nNOS deple- permits differentiation of myogenic precursor cells in vitro tion [72]. Secondly, our data on muscle phenotype and [25]. At the signalling level, the Akt-mTOR pathway and CK measurements after treadmill running indicate that Akt-FoxO3-Mul-1 axis are involved in skeletal muscle nNOSμ deficiency induces muscle degeneration/damage growth/wasting, autophagy and mitochondrial dysfunction post-exercise. This raises the possibility that nNOSμ- [29,31,38,46,55,58,67-69,73,74]. Of interest, Mul-1 has been deprived muscles cannot activate protective responses. recently reported to be upregulated during muscle wasting, Accordingly, NOS1-/- mdx mice displayed increased possibly via an autophagic mechanism involving FoxO3 susceptibility to eccentric contraction-induced muscle transcription factors [68]. Our data indicate the relevance damage [72]. In addition, expression of a muscle-specific of the above signalling pathways and that they are con- nNOS transgene prevents muscle membrane injury dur- trolled by NO. We observed an inhibition of the Akt- ing modified muscle use [84]. In this respect, there is a mTOR pathway in the absence of nNOSμ.Concomitantly, general agreement that NO produced by nNOS plays an the Akt-FoxO3-Mul-1 axis was also dysregulated. In important role in muscle repair in chronic conditions addition, the inhibition of the nNOS/NO/cGMP/PKG [5,8,9] although the use of NOS1-/- mice suggested that system induced the transcriptional activity of FoxO3 nNOS is not essential to functional recovery after acute and increased Mul-1 expression. These events are likely injury [80]. associated with nNOSμ-dependent impairments of The third important observation is the correlation be- muscle fibre growth. tween mitochondrial defects and muscle impairment. We cannot exclude that failure of other NO-dependent Alterations in the content, shape or function of the mito- action involving, for instance, the vascular system, may chondria appear to occur in damaged muscle and inhib- have contributed to the functional and structural defects ition of mitochondrial fission protects from muscle loss we observed in skeletal muscle. Extensor digitorum longus during fasting [29]. Recent findings have also underlined of NOS1-/- mice revealed an altered capillary-to-fibre ra- the crucial role of autophagy in the control of muscle mass tio but not changes in the capillary ultrastructure or the and functions [29,31,55,69]. Autophagy derangement is in- hemodynamics at basal conditions [86]. Noteworthy, NO volved in a number of inherited muscle diseases [31-33]. generated by sarcolemmal nNOSμ normally acts as a para- Of interest, mitochondria are involved in regulating au- crine signal that optimises blood flow in the working tophagy [30]. In addition, skeletal muscle was shown to be muscle [12,87,88] and the protective vasodilating action is sensitive to the physiological stressors that trigger the impaired in the contracting muscles of NOS1-/- mice mt mt UPR [35,36] and UPR is activated in skeletal muscle [12,89]. In this respect, the lack of this vasodilating action during exercise as part of an adaptive response to exercise in NOS1-/- mice has been suggested to affect muscle per- training [54]. Here, we raise the possibility that mitochon- formance [71]. Results obtained in NOS1-/- mice with dif- mt drial dysfunction, UPR and autophagy are functionally ferent cardiac injuries indicated a protective role of nNOS, related to each other and promoted by a single event, that although an opposite effect cannot be excluded [90,91]. is, the deficit in NO signalling, thus suggesting that the The deficit in exercise performance of NOS1-/- muscles association of altered mitochondrial homeostasis and may be the consequence, at least in part, of a decreased muscle phenotype/performance in NOS1-/- mice is not oxygen delivery following blood flow impairment. coincidental. The experiments we carried-out in myogenic precursor Conclusions cells and NOS1-/- mice during critical stages of muscle Muscle exercise performance is a complex physiological development are consistent with an association of al- process that can occur by many different mechanisms tered mitochondrial homeostasis and muscle phenotype/ and NO has long been described to be relevant among De Palma et al. Skeletal Muscle 2014, 4:22 Page 18 of 21 http://www.skeletalmusclejournal.com/content/4/1/22 them [2]. Our study now suggests that the relevance of EA, TT, SR, VR, SC, VC and PP acquired and analysed the data. CM, MTB and MS analysed the data and revised the manuscript. CP participated in the NO also resides in the fact that it regulates key homeo- design of the study, analysed and interpreted the data, and revised the static mechanisms in skeletal muscle, namely mitochon- manuscript. DC and EC participated in the design and the coordination of mt drial bioenergetics and network remodelling, UPR and the study, analysed and interpreted the data, drafted and revised the manuscript, and wrote the final version of the manuscript. All authors read autophagy. Although NOS1-/- mice do not display the and approved the final manuscript. overt features of myopathies, such as muscle degener- ation, reactive regeneration and replacement of muscle Authors’ information with fibroadipous tissue [92,93], we clearly show that al- CDP is a post-doctoral research associate. FM and SP are PhD students. SR is a terations of the NO system significantly impair muscle research fellow. EA, TT, VR, SC and VC are post-doctoral research fellows. PP is a graduate medical student. MTB is a Senior Researcher. CM is a Professor of fibre growth, thus resulting in a deficit of muscle force Human Anatomy. MS is a Professor of Pathology. CP is a Professor of and the ability to sustain prolonged exercise. This aspect Pharmacology. DC is a Professor of Physiology. EC is a Professor of may explain why NO deficiency contributes to muscle Pharmacology and the Head of the Pharmacology group. impairment in degenerative disease of the muscle, such as muscular dystrophies. Acknowledgements We thank Laura Pozzi (Scientific Institute IRCCS Eugenio Medea, Bosisio Parini, Lecco, Italy) for technical help. We are grateful to Prof. Luca Scorrano (University Additional files of Padova, Padova, Italy) for providing us with pDsRed2-Mito. This work was supported by: “Ministero della Salute”“Giovani Ricercatori 2011-2012” grant to C. Additional file 1: Figure S1. Mitochondrial ultrastructure and LC3 D.P and “Ricerca corrente 2014” grant to E.C.; “Ministero dell’Istruzione, Università lipidation in skeletal muscles of wild-type and NOS1-/- mice. (A) TEM i eRicerca”, PRIN2010-2011 grants to E.C. and D.C.; European Community’s mages of subsarcolemmal mitochondria of tibialis anterior muscles. Scale framework programme FP7/2007-2013 under the agreement n°223098 bar: 0.1 μm. (B) TEM images of intermyofibrillar mitochondria of tibialis (OPTISTEM) and n°241440 (ENDOSTEM) to E.C. The funders had no role in study anterior muscles. Scale bar: 1 μm. (C) TEM images of diaphragm muscles design, data collection and analysis, decision to publish, or preparation of the detecting the presence of autophagic vacuoles (arrowheads) in NOS1-/- manuscript. fibres. TEM images are representative of results obtained from at least three different animals per experimental group. (D) Western blot analysis Author details of LC3 lipidation in diaphragm muscles of wild-type and NOS1-/- mice. Unit of Clinical Pharmacology, National Research Council-Institute of GAPDH was used as internal standard. The image is representative of Neuroscience, Department of Biomedical and Clinical Sciences “Luigi results obtained from at least 10 different animals per experimental Sacco”, University Hospital “Luigi Sacco”, Università di Milano, Milano, Italy. 2 3 group. Analyses were performed on animals at P120. Scientific Institute IRCCS Eugenio Medea, Bosisio Parini, Italy. Dulbecco Additional file 2: Figure S2. Weight, muscle structure and muscle Telethon Institute at Venetian Institute of Molecular Medicine, Padova, Italy. expression of ubiquitin ligases in wild-type and NOS1-/- mice. Body National Research Council-Institute of Neuroscience, Department of Medical (A) and visceral adipose tissue (VAT) (B) weight. Each histogram Biotechnology and Translational Medicine, Università di Milano, Milano, Italy. 5 6 represents the data obtained from at least three to eight different animals CNI@NEST, Italian Institute of Technology, Pisa, Italy. Unit of Morphology, per experimental group. *P <0.05, and **P <0.01 versus the respective Department of Biomedical and Clinical Sciences “Luigi Sacco”, Università di wild-type control. (C) Pictures of tibialis anterior, soleus, and gastrocnemius Milano, Milano, Italy. Department of Biomedical Science, Università di Padova, muscles. The image is representative of at least 10 different animals per Padova, Italy. Department for Innovation in Biological, Agro-food and Forest experimental group. (D) Histological sections of tibialis anterior and Systems, Università della Tuscia, Viterbo, Italy. diaphragm muscles stained with H & E. The images are representative of results obtained from at least five different animals per experimental group. Received: 26 June 2014 Accepted: 18 November 2014 Scale bar: 100 μm. Analyses were performed on animals at P120. qPCR analysis of mRNA levels for atrogin-1 and muRF1 in hind limb muscles at P10 (E) and tibialis anterior muscles at P30 (F). 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Hum Mol Genet 1998, 7:823–829. doi:10.1186/s13395-014-0022-6 Cite this article as: De Palma et al.: Deficient nitric oxide signalling impairs skeletal muscle growth and performance: involvement of mitochondrial dysregulation. Skeletal Muscle 2014 4:22. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

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Skeletal MuscleSpringer Journals

Published: Dec 12, 2014

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