The Journal of Physiology · The mechanism by which increased levels of tonic ﬁring of motor...

J Physiol 590.6 (2012) pp 1427–1442 1427

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Nitric oxide synthase inhibition prevents activity-inducedcalcineurin–NFATc1 signalling and fast-to-slow skeletalmuscle fibre type conversions

Karen J. B. Martins1, Mathieu St-Louis4, Gordon K. Murdoch5, Ian M. MacLean1, Pamela McDonald1,Walter T. Dixon2, Charles T. Putman1,3 and Robin N. Michel4

1Exercise Biochemistry Laboratory, Faculty of Physical Education and Recreation, 2Department of Agricultural, Food and Nutritional Sciences and3The Centre for Neuroscience, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada T6G 2H94Cellular and Molecular Neuromuscular Physiology Laboratory, Departments of Biology, Chemistry & Biochemistry, and Exercise Science, ConcordiaUniversity, The Richard J. Renaud Science Complex, Montreal, QC, Canada H4B 1R65Animal and Veterinary Science Department, University of Idaho, Moscow, ID 83844, USA

Key points

• Exercise is known to trigger skeletal muscle structural and functional adaptations.• Control of these adaptive alterations is a complex process involving multiple signalling pathways

and levels of regulation.• The well-characterized calcineurin–nuclear factor of activated T-cells (NFATc1) signalling

pathway is involved in the regulation of activity-dependent alterations in skeletal musclemyosin heavy chain expression. Myosin heavy chain is a contractile protein that largely dictatesa muscle’s speed of contraction.

• We show that a signalling molecule called nitric oxide may be regulating alterations in myosinheavy chain expression via activity-modulated calcineurin–NFATc1 signalling.

• These findings increase our understanding of how skeletal muscle adaptive alterations areregulated.

Abstract The calcineurin–NFAT (nuclear factor of activated T-cells) signalling pathway isinvolved in the regulation of activity-dependent skeletal muscle myosin heavy chain (MHC)isoform type expression. Emerging evidence indicates that nitric oxide (NO) may play a criticalrole in this regulatory pathway. Thus, the purpose of this study was to investigate the role of NO inactivity-induced calcineurin–NFATc1 signalling leading to skeletal muscle faster-to-slower fibretype transformations in vivo. Endogenous NO production was blocked by administering L-NAME(0.75 mg ml−1) in drinking water throughout 0, 1, 2, 5 or 10 days of chronic low-frequencystimulation (CLFS; 10 Hz, 12 h day−1) of rat fast-twitch muscles (L+Stim; n = 30) and outcomeswere compared with control rats receiving only CLFS (Stim; n = 30). Western blot and immuno-fluorescence analyses revealed that CLFS induced an increase in NFATc1 dephosphorylation andnuclear localisation, sustained by glycogen synthase kinase (GSK)-3β phosphorylation in Stim,which were all abolished in L+Stim. Moreover, real-time RT-PCR revealed that CLFS induced anincreased expression of MHC-I, -IIa and -IId(x) mRNAs in Stim that was abolished in L+Stim.SDS-PAGE and immunohistochemical analyses revealed that CLFS induced faster-to-slower MHCprotein and fibre type transformations, respectively, within the fast fibre population of both Stimand L+Stim groups. The final fast type IIA to slow type I transformation, however, was preventedin L+Stim. It is concluded that NO regulates activity-induced MHC-based faster-to-slower fibretype transformations at the transcriptional level via inhibitory GSK-3β-induced facilitation of

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society DOI: 10.1113/jphysiol.2011.223370

1428 K. J. B. Martins and others J Physiol 590.6

calcineurin–NFATc1 nuclear accumulation in vivo, whereas transformations within the fast fibrepopulation may also involve translational control mechanisms independent of NO signalling.

(Received 27 October 2011; accepted after revision 3 January 2012; first published online 4 January 2012)Corresponding authors R. N. Michel: Departments of Biology, Chemistry & Biochemistry and Exercise Science, andThe Centre for Structural and Functional Genomics, Concordia University, The Richard J. Renaud Science Complex,Montreal, QC, H4B 1R6, Canada. Email: [email protected] or C. T. Putman: Email: [email protected]

Abbreviations CnA, calcineurin; CLFS, chronic low-frequency stimulation; DAPI, 4′,6-diamidino-2-phenylindole;de-PO4, dephosphorylated; eNOS, endothelial nitric oxide synthase; GSK-3β, glycogen synthase kinase-3β; MCK, musclecreatinine kinase; MHC, myosin heavy chain; NFAT, nuclear factor of activated T-cells; nNOS, neuronal nitric oxide;NO, nitric oxide; NOS, nitric oxide synthase; PGC1α, peroxisome proliferator-activated receptor gamma co-activator1-alpha; PO4, phosphorylated.

Introduction

Mammalian skeletal muscle is a heterogeneous tissuecomprised of diverse fibre populations. This fibre typediversity is, in part, attributed to the various myosin heavychain (MHC) protein isoforms that largely dictate the rateof force development, maximum sarcomeric shorteningvelocity and rate of cross-bridge cycling (Bottinelli et al.1991; Pette & Staron, 1997). Fibre types range from slow(type I) to fast (type IIA, IID(X) and IIB) in adult rodentskeletal muscle that contain the correspondingly namedMHC isoforms listed in increasing order of shorteningvelocity: MHCI, MHCIIa, MHCIId(x) and MHCIIb (Pette& Staron, 1997). In response to various contractiledemands such as exercise, skeletal muscle demonstratesremarkable adaptability or plasticity that is largely dictatedby changes in motor neuron activity. For example, chroniclow-frequency electrical stimulation (CLFS; 10 Hz) of themotor nerve mimics the tonic firing pattern typical ofslow motor neurons (Hennig & Lomo, 1985) and inducesmaximal faster-to-slower fibre type transformations in theabsence of skeletal muscle injury in the rat model (Putmanet al. 1999, 2000, 2001; Martins et al. 2006; LaFramboiseet al. 2009). This fibre type transformation generallyfollows the ‘next nearest-neighbour’ rule where fibre typesundergo a predictable pattern of transformation in thedirection of fast type IIB→IID(X)→IIA→ slow typeI (Pette & Vrbova, 1999; Pette & Staron, 2000). Thespecific signalling pathways that transduce motor neuronfiring patterns into shifts in fibre-specific gene expression,however, remain to be fully elucidated.

The mechanism by which increased levels of tonicfiring of motor neurons induce transcription ofslower, more energy-efficient, fibre-specific genes involvessustained elevations in low-amplitude intracellular [Ca2+]oscillations, which in turn stimulate a number ofkey downstream signalling pathways (for reviews seeMichel et al. 2004, 2007; Bassel-Duby & Olson, 2006).Calcineurin–NFAT (nuclear factor of activated T-cells) isone of the best characterised of these signalling pathways(Chin et al. 1998; Dunn et al. 1999, 2000, 2001; Liu

et al. 2001). Calcineurin is a Ca2+–calmodulin-dependentprotein phosphatase that dephosphorylates the fourmuscle-localised transcription factor isoforms of theNFAT family, NFATc1–c4. NFAT dephosphorylationresults in its nuclear translocation and binding to specificsequences on the promoters of target genes that induceslower, more oxidative fibre-specific phenotypes (Hoganet al. 2003; Rana et al. 2005, 2008; Meissner et al. 2007;Calabria et al. 2009), and repress expression of fast contra-ctile protein isoforms, such as TnIf, at least in slowfibres (Rana et al. 2008). Although this pathway describesthe activity-induced activation of NFAT, regulation ofthis transcription factor is complex, being subject todynamic cycles of activation (i.e. dephosphorylation andnuclear import) and deactivation (i.e. phosphorylationand nuclear export) that results in nuclear-cytoplasmicshuttling (Dunn et al. 2000, 2001; Liu et al. 2001,2005). Skeletal muscle NFAT phosphorylation can occurby several protein kinases, such as glycogen synthasekinase-3β (GSK-3β), which has been identified as animportant promoter of NFAT nuclear export (Shen et al.2007) and an inhibitor of NFAT-mediated increases in slowMHC gene expression (Jiang et al. 2006).

Other activity-dependent signalling pathways canalso co-regulate the transition of fast-twitch fibrestoward slower more energy-efficient phenotypes, asdemonstrated by the expression dependence of slow(TnIs) and fast (TnIf) isoforms of Troponin-I topatterned electrical activity (Nakayama et al. 1996;Rana et al. 2005). Another signalling intermediateinvolved in fibre remodelling is the transcriptionalco-activator peroxisome proliferator-activated receptorgamma co-activator 1-alpha (PGC1α), which is highlyexpressed in slow type I fibres (Wu et al. 1999; Lin et al.2002) and displays considerable plasticity by increasing itsexpression levels in response to endurance exercise (Baaret al. 2002; Terada et al. 2002; Russell et al. 2003). PGC1α isinduced by various upstream signals, such as p38-MAPK(Akimoto et al. 2005; Wright et al. 2007), CaMK andcalcineurin (Handschin et al. 2003), and possibly MEF2(Czubryt et al. 2003; Vissing et al. 2008). PGC1α has also

C© 2012 The Authors. The Journal of Physiology C© 2012 The Physiological Society

J Physiol 590.6 NO involvement in skeletal muscle fibre type transformations 1429

been shown to be induced by AMPK, but signals trans-mitted through this mechanism are restricted to metabolicgenes (Terada et al. 2002; Zong et al. 2002; Putman et al.2003; Suwa et al. 2006), and do not display regulatorycontrol over expression of contractile proteins such asmyosin heavy chains (MHC) (Putman et al. 2003).

Nitric oxide (NO) is a ubiquitous signalling moleculethat is controlled at the synthesis level by NO synthase(NOS), which is, in turn, regulated by Ca2+–calmodulinbinding (Stamler & Meissner, 2001). Increased NOSactivity and resultant NO production occur in responseto muscle contraction, as well as CLFS, and are involvedin a number of important regulatory processes withinthis tissue (Reiser et al. 1997; Stamler & Meissner,2001; McConell & Wadley, 2008). It has, for example,been demonstrated that NO production is reduced inDuchenne muscular dystrophy patients (Grozdanovic &Baumgarten, 1999) but exposure to NO donors improvesmuscle myoblast differentiation and regeneration withindystrophic muscle fibres (Pisconti et al. 2006; Brunelli et al.2007; Colussi et al. 2008, 2009). The AKT pathway has alsobeen shown to be important for NO synthesis (Dimmeler& Zeiher, 1999), and NO seems to be a requirement forincreased activity of histone deacetylases, which in turnregulate activation of the myogenic transcription factorsMEF2 and MyoD (Sartorelli et al. 1999; Lu et al. 2000;Naya et al. 2000).

NO has also been directly linked to mitochondrialbiogenesis, and the increased potential for terminal sub-strate oxidation. Exercise, for example, is known toincrease eNOS, which in turn induces the expressionof PGC1α, an important intermediary signal leadingto mitochondrial biogenesis (see review by Nisoli& Carruba, 2006). Likewise, nNOS activity and NOproduction are known to increase in response to electricalstimulation (Reiser et al. 1997; McConell & Wadley, 2008).Recently, Drenning et al. (2008, 2009) showed that NOis also associated with inhibitory phosphorylation ofGSK-3β, facilitation of NFATc1 nuclear accumulation,and increased slow MHCI mRNA expression in responseto Ca2+-ionophore treatment in vitro. Collectively, theseobservations suggest that CLFS-induced faster-to-slowerfibre type transformations may be regulated by bothcalcineurin and NO working synergistically to promoteNFATc1 nuclear accumulation in vivo. Therefore, thepurpose of the present study was to test the hypothesisthat pharmacological inhibition of NOS activity wouldprevent CLFS-induced skeletal muscle NFATc1 nuclearaccumulation and subsequent faster-to-slower fibre typetransformations in vivo. Our findings indicate thatNO regulates MHC-related faster-to-slower fibre typeconversions at the transcriptional level via neuralactivity-modulated nuclear accumulation of NFATc1in vivo involving both calcineurin and GSK-3β whereastransformations within the fast fibre population may also

involve translational control mechanisms independent ofNO signalling.

Methods

Ethical approval

All animal procedures were carried out in accordancewith the guidelines of the Canadian and UK Councilsfor Animal Care and received ethical approval from theUniversity of Alberta and Concordia University.

Experimental design and use of animals

Sixty adult male Wistar rats (Charles River Laboratories,Montreal, PQ, Canada) were individually housed undercontrolled environmental conditions (22◦C with 12:12 hlight–dark cycle) and consumed standard rat chow andwater or an aqueous solution of L-NAME ad libitum,which was measured and replaced daily. Animals in theexperimental groups received L-NAME (0.75 mg ml−1)in drinking water throughout 0, 1, 2, 5 or 10 days ofCLFS. CLFS (10 Hz, 12 h day−1) was applied across theperoneal nerve thereby stimulating the fast-twitch tibialisanterior and extensor digitorum longus (L+Stim; n = 6animals per group). Outcome measures were comparedwith control rats receiving only CLFS of matched timepoints (Stim; n = 6 animals per group). The applicationof CLFS has been shown to elicit a compensatory effect inthe contralateral control muscles due to increased weightbearing (Putman et al. 2000); therefore comparisons weremade to animals receiving only a sham operation of theleft leg (Control). As a post hoc consideration, mixedfast-twitch plantaris muscles of wild type (WT-C57/BL6)and transgenic mice that over-express constitutively activecalcineurin (MCK-CnA∗-Tg) were collected and servedas positive controls for NFATc1 nuclear localizationexperiments (Dunn et al. 2000; Chakkalakal et al. 2004).

Systemic inhibition of nitric oxide synthase activity

The pharmacological inhibition of NOS was achievedby administering the competitive non-isoform-specificNOS inhibitor L-NAME (Sigma-Aldrich, Oakville, ON,Canada) daily in the drinking water of animals starting2 days prior to the onset of stimulation and continuingfor the duration of the study. An L-NAME concentrationof 0.75 mg ml−1 was used that resulted in a daily dose of∼100 mg (kg body mass)−1. Body mass was recorded dailythroughout the experimental period. This dose has beenshown to effectively inhibit NOS activity in the rat (Smithet al. 2002; Sellman et al. 2006).



Chronic low-frequency stimulation

CLFS (10 Hz, impulse width 380 μs, 12 h day−1) wasapplied across the left common peroneal nerve as pre-viously described (Simoneau & Pette, 1988). Briefly,while animals were under general anaesthesia (75 mg (kgbody wt)−1 ketamine, 10 mg (kg body wt)−1 xylazine and0.5 mg (kg body wt)−1 acepromazine maleate via intra-peritoneal injection), bipolar electrodes were implantedlateral to the common peroneal nerve of the left hindlimb, externalised at the dorsal intrascapular region,and connected to a small, portable stimulator. Animalswere allowed to recover for 7 days before the onset ofstimulation.

Muscle sampling

Upon completion of the stimulation period, rats wereanaesthetised as before and the tibialis anterior andextensor digitorum longus were excised from bothhind limbs and frozen in melting isopentane cooled inliquid N2 (–159◦C). Muscles were subsequently storedin liquid N2 (–196◦C). The anaesthetised animals werethen killed after all muscles were collected with anoverdose of Euthanyl (100 mg (kg body wt)−1 via intra-peritoneal injection) (Bimedia-MTC Animal Health Inc.,Cambridge, ON, Canada), followed by exsanguination.For mice, all surgical procedures were performed understerile conditions on animals anaesthetized (1.2 μl (g)−1

I.M.) with 100 mg (ml)−1 ketamine hydrochloride and20 mg ml−1 xylaxine in a volume ratio of 1.6:1. Mice werekilled by cervical dislocation.

Western blot analyses

Western blot analyses were performed as previouslydescribed (Dunn et al. 2000, 2001). Briefly, for extractionof whole cell protein, rat extensor digitorum longussamples were homogenized in 1 ml of RIPA buffer (1%(v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate,0.1% (w/v) SDS, 10 μg (ml)−1 aprotinin, 10 μg (ml)−1

leupeptin, 1 mM phenylmethylsulfonyl fluoride, 10 mM

sodium fluoride, 1 mM sodium orthovanadate in PBS,pH 7.4). Homogenates were centrifuged at 20,000 g for30 min at 4◦C. Protein concentrations were determinedin order to ensure equal loading of samples betweenlanes (Bradford, 1976). Samples were subsequentlydiluted in modified Laemmli lysis buffer and boiled for5 min (Laemmli, 1970). Each sample (150 μg of totalprotein) was subjected to 6% SDS–PAGE electrophoresis(Mini-PROTEAN 3 system; Bio-Rad Laboratories,Mississauga, ON, Canada) and then transferred to a poly-vinylidene fluoride membrane (Millipore, Bedford, MA,USA). Transfer efficiency and a second evaluation of equalsample loading were confirmed by Ponceau-S staining of

membrane-bound proteins. Membranes were incubatedin blocking solution (5% powdered milk, 0.1% Tween-20in TBS, pH 8.0) for 1 h and then incubated overnightat 4◦C with rabbit monoclonal anti-phospho-GSK-3β(Ser9) (clone 5B3; Cell Signalling Technologies, Beverly,MA, USA) that was diluted in blocking solution (1:2000),or rabbit monoclonal anti-GSK-3β (clone 27C10; CellSignalling Technologies) diluted in blocking dilution(1:2000), or with mouse monoclonal anti-NFATc1(clone 7A6; Santa Cruz Biotechnology, Santa Cruz,CA, USA) also diluted in blocking solution (1:200).Membranes were washed with 0.1% Tween-20 in TBSand then incubated for 1 h with anti-mouse monoclonal(1:1000; Sigma-Aldrich) or anti-rabbit polyclonal (1:2000;Cell Signalling Technologies) horseradish peroxidasesecondary antibody conjugates diluted in blockingsolution and washed as before. The protein–antibodycomplex was revealed by chemiluminescence usingthe Immobilon Western Chemiluminescence Substratekit (Millipore). Membranes were reprobed with poly-clonal rabbit anti-α-tubulin (1:2000; Cell SignallingTechnologies), which served as the internal control andfurther confirmed equal loading. Immunoreactivity oftubulin was visualized as described above after incubationwith anti-rabbit horseradish peroxidase secondary anti-body conjugates in blocking solution (1:2000; CellSignalling Technologies).

Quantification of NFATc1

Consistent with previous findings in rodent skeletalmuscle (Dunn et al. 2000, 2001), multiple NFATc1bands ranging from 80–100 kDa in size were detected onWestern blots of rat extensor digitorum longus whole cellextracts (Fig. 1A). Pretreatment of samples with alkalinephosphatase (AP) increased the prevalence of only onelower molecular weight species of NFATc1 (∼85 kDa;Fig. 1A, see +AP lane), corresponding to the mostdephosphorylated form of this protein. The density ofthe most-dephosphorylated (de-PO4; ∼85 kDa) and mostphosphorylated (PO4; ∼95 kDa) bands of NFATc1 weredetermined using Fluorchem software (Cell Biosciences,Santa Clara, CA, USA) and expressed as a ratio for eachsample.

Immunofluorescent detection of NFATc1

Rat tibialis anterior muscles were mounted in embeddingmedium (Tissue-Tek O.C.T. Compound, Sakura Finetek,Torrance, CA, USA) and 10-μm-thick transverse frozensections were collected from the mid-belly of eachmuscle and transferred onto Superfrost Plus microscopeslides (Fisher Scientific, Ottawa, ON, Canada) at –20◦C.The immunostaining protocol was modified from Xiao



Table 1. Rat specific real-time reverse-transcriptase polymerase chain reaction primers and probes

Target Forward primer Reverse primer Probe

MHCI 5′-GCAGTTGGATGAGCGACTCA-3′ 5′-TCCTCAATCCTGGCGTTGA-3′ 5′-AGAAGGACTTTGAGTTAAAT-3′

MHCIIa 5′-GGCGGCAAGAAGCAGATC-3′ 5′-TTCCGCTTCTGCTCACTCTCT-3′ 5′-AGGCCAGAGTGCGTG-3′

MHCIId(x) 5′-GGCGGCAAGAAGCAGATC-3′ 5′-TTCGTTTTCAACTTCTCCTTCAAGT-3′ 5′-AGGCCAGGGTCCG-3′

MHCIIb 5′-GGCGGCAAGAAGCAGATC-3′ 5′-TTTTCCACCTCGTTTTCAAGCT-3′ 5′-TGGAGGCCAGAGTGA-3′

et al. (2008). Specifically, sections were fixed with 2%(v/v) paraformaldehyde for 20 min (Sigma-Aldrich)and washed three times in PBS. Sections were thenblocked and permeablized, with 2% (v/v) normal goatserum (Sigma-Aldrich) and 0.2% (v/v) Triton X-100(Sigma-Aldrich), respectively, both in PBS for 1 h each.Sections were incubated overnight at 4◦C with mono-clonal mouse anti-NFATc1 primary antibody (Santa CruzBiotechnology) at a 1:200 dilution in PBS containing 1%(v/v) normal goat serum and 0.05% (v/v) Triton X-100.Slides were then washed as before and goat anti-mouseAlexa Fluor 488 secondary antibody (Invitrogen, LifeSciences, Burlington, ON, Canada) was applied for 1 h atroom temperature in PBS containing 1% (v/v) normalgoat serum and 0.05% (v/v) Triton X-100. Slides werewashed as before, air-dried and mounted in Vectashieldcontaining 1.43 nM 4′,6-diamidino-2-phenylindole(DAPI; Vector Laboratories, Burlington, ON, Canada).Control experiments omitting primary antibodiesrevealed absent or very low-level background staining.Frozen sections of plantaris obtained from WT andMCK-CnA∗-Tg were stained in parallel, and served aspositive controls (Dunn et al. 2000). Images were acquiredon an Olympus BX60 fluorescent microscope (Olympus,Centre Valley, PA, USA) using Image-Pro Plus software(Media Cybernetics, Bethesda, MD, USA). A total area of0.08 mm2 was analysed in each rat.

Myosin heavy chain mRNA analyses by real-timereverse transcriptase-polymerase chain reaction

Patterns of MHC isoform expression in tibialis anteriormuscles were analysed at the mRNA level using real-timeRT-PCR (Martins et al. 2009). TRIzol RNA extractionwas performed according to manufacturer’s instructions(Invitrogen). The concentrations and purity of RNAextracts were evaluated by measuring the absorbanceat 260 and 260/280 nm, respectively, using a NanoDropND 1000 system (Rose Scientific Ltd, Edmonton, AB,Canada). Synthesis of cDNA was performed according toan established procedure (Martins et al. 2009). Sampleswere diluted to 1 μg (μl)−1 and reverse transcriptionwas performed for 1 h at 37◦C with oligo (dT12−18)primers (Invitrogen) and moloney murine leukemiavirus DNA polymerase (Invitrogen). Primers (Invitrogen)

and Taqman-MGB probes (Applied Biosystems, FosterCity, CA, USA) were designed with the EuropeanMolecular Biology Laboratory–European BioinformaticsInstitute and aligned using Clustal W for rat MHCI(X15939), MHCIIa (L13606), MHCIId(x) (XM 213345)and MHCIIb (L24897) (Table 1). Real-time PCR wasperformed on 1 μl cDNA samples, in duplicate, usingan ABI 7900HT thermocycler (Applied Biosystems). 18SrRNA (Applied Biosytems) was used as the endogenouscontrol. Relative changes in MHC isoform gene expressionwere determined using the 2−��Ct method of analysis(Livak & Schmittgen, 2001). Inter-assay variation wasevaluated by repeated analysis of a known sample oneach 96-well plate and was confirmed to be negligible.Additionally, the amplification efficiencies of the MHCisoforms and 18S were similar.

Electrophoretic analyses of myosin heavy chainprotein isoforms

Quantitative MHC protein isoform analyses werecompleted as previously described (Hamalainen & Pette,1996; Putman et al. 2004). Briefly, frozen powdered tibialisanterior muscles were homogenised in an ice-cold buffercontaining 100 mM NaP2O7 (pH 8.5), 5 mM EGTA, 5 mM

MgCl2, 0.3 mM KCl, 10 mM DTT (Sigma-Aldrich) and5 mg ml−1 of a protease inhibitor cocktail (Complete,Roche Diagnostics, Indianapolis, IN, USA). Samples werethen centrifuged at 12,000 g for 5 min at 4◦C; super-natants were diluted 1:1 with glycerol and stored at –20◦Cuntil analysed. Prior to gel loading, muscle extracts werediluted in modified Laemmli lysis buffer to a concentrationof 0.2 μg (μl)−1 and boiled for 6 min (Laemmli, 1970).Samples (1 μg total protein per lane) were electro-phoresed (275 V for 24 h at 8◦C) in duplicate on 7% (w/v)SDS-PAGE gels containing glycerol, under denaturingconditions. Gels were then fixed and MHC isoforms weredetected by silver staining and evaluated by integrateddensitometry (ChemiGenius, GeneSnap and GeneTools,Syngene, UK).

Immunohistochemistry for myosin heavy chainprotein isoforms

Tibialis anterior muscles were mounted as before and10-μm-thick transverse frozen sections were collected



from the mid-belly of each muscle at –20◦C. Immuno-staining was completed according to an establishedprotocol (Putman et al. 2001, 2003). Briefly, sections werewashed once in PBS with 0.1% (v/v) Tween-20 (PBS-T),twice in PBS and then incubated for 15 min in 3% (v/v)H2O2 in methanol. Serial sections stained for BA-D5,SC-71 or BF-35 were incubated for 1 h in a blockingsolution (BS-1: 1% (w/v) bovine serum albumin and10% (v/v) horse serum in PBS-T, pH 7.4) containingavidin-D blocking reagent (Vector Laboratories). Serialsections stained for BF-F3 were incubated in a similarblocking solution, with the exception that goat serumwas replaced with horse serum (BS-2). Sections wereprobed with monoclonal antibodies directed againstadult MHC isoforms (Schiaffino et al. 1988, 1989)harvested from the supernatants of hybridoma cell linesobtained from the American Type Culture Collection(Manassas, VA, USA): BA-D5 (IgG, anti-MHCI), SC-71(IgG, anti-MHCIIa) and BF-F3 (IgM, anti-MHCIIb).Clone BF-35 (purified IgG, staining all MHCs exceptMHCIId(x)) was a generous gift from Dr S. Schiaffino(Padova, Italy). Sections were incubated overnight at4◦C with a primary antibody that was diluted in itscorresponding blocking solution containing a biotinblocking reagent (Vector Laboratories). Biotinylated horseanti-mouse-IgG (BA-D5; SC-71; BF-35) or biotinylatedgoat anti-mouse-IgM (BF-F3) was applied for 1 h.Sections were again washed and incubated withVectastain ABC reagent according to the manufacturer’sinstructions (Vector Laboratories) and reacted with0.07% (w/v) diaminobenzidine, 0.05% (v/v) H2O2 and0.03% (w/v) NiCl2 in 50 mM Tris-HCl (pH 7.5). Allsections were subsequently dehydrated, cleared andmounted in Entellan (Merck, Darmstadt, Germany).Analysis was completed with a Leitz Diaplan micro-scope (Ernst Leitz Wetzlar GmbH, Germany) fitted witha Pro-Series high performance charge-coupled digitalcamera (Cohu, San Diego, CA, USA), Image-Pro Plussoftware (Media Cybernetics) and a custom-designedanalytical program (Putman et al. 2000). A similarnumber of fibres were examined from three representativeareas (i.e. deep, middle and superficial regions) ineach rat (i.e. 1345 fibres per rat). Type I, IIA andIIB fibres were identified by positive staining, andtype IID(X) fibres were identified by the absence ofstaining.

Statistical analyses

Data are summarised as means ± SEM. Differencesbetween group means were assessed using a two-wayANOVA (i.e. treatment (Stim or L+Stim) × days ofstimulation (0, 1, 2, 5 or 10 days)). When a significantF ratio was found, mean values were compared using the

Newman–Keuls post hoc analysis. Data in which a priorihypotheses were established and the direction of changespredicted in advance were analysed by the Student’sone-tailed t test. Differences were considered significantat P < 0.05. There were no differences between the leftand right legs of Control, as determined by the t test fordependent samples, therefore left and right leg data werepooled.

Results

Animal weights

Animals initially weighed 318 ± 3 g and gained 29 ± 6 gduring 10 days of stimulation. Additionally, animalweights did not differ between Stim and L+Stim groupsat matched time points (i.e. 0, 1, 2, 5 and 10 days).

NFATc1 phosphorylation status and localisation

To test the hypothesis that NOS inhibition pre-vents activity-induced skeletal muscle NFATc1nuclear accumulation in vivo, we measured boththe phosphorylation status and nuclear localisation ofNFATc1 in rat fast-twitch muscles exposed to CLFS.Western blots of rat extensor digitorum longus extractsshowed a multiple banding pattern for NFATc1 rangingfrom 80 to 100 kDa, reflecting the phosphorylation statusand post-translational modifications of this transcriptionfactor (Fig. 1A). CLFS induced a marked increase in totalNFATc1 protein after 2 days of stimulation comparedwith Control which was not observed in L+Stim animals.We then identified the most dephosphorylated (de-PO4)and phosphorylated (PO4) forms of this protein andcompared the ratio of their expression across experimentalconditions (Fig. 1B). CLFS induced a rapid yet transientdephosphorylation of NFATc1 as shown by a 40–50%increase in the ratio of NFATc1 de-PO4-to-PO4 in 2-and 5-day-stimulated animals, respectively, returning toControl levels by day 10. This effect was abrogated inanimals that received L-NAME treatment throughout the10 day time course of stimulation. Likewise, as detectedby immunohistochemistry in rat tibialis anterior tissuesections (Fig. 2A and B), CLFS induced a 2.6-fold increasein the proportion of NFATc1-positive nuclei by 5 daysof stimulation, returning to baseline Control values by10 days, while L-NAME treatment blocked this effect(Fig. 2C). Taken together, these results suggest thatNOS activity is required for CLFS-induced increasesin NFATc1 nuclear accumulation. Analysis of frozentissue cross-sections of the fast plantaris obtained fromWT and transgenic mice overexpressing constitutivelyactive CnA∗ (Dunn et al. 2000; Chakkalakal et al. 2004)served as a comparative positive control for NFATc1



nuclear localization experiments (Fig. 2D). Note the peakproportion of NFATc1-positive nuclei after 10 days ofStim was comparable to levels observed in CnA∗ mice, aswere baseline levels in their respective controls.

GSK-3β phosphorylation status

To better understand the involvement of NOS activityin the regulation of activity-induced NFATc1 nuclearaccumulation and MHC mRNA expression in vivo, wemeasured phosphorylated and total GSK-3β protein byWestern blot analysis in rat fast-twitch muscles exposed toCLFS (Fig. 3A). Quantification of Western blots revealedthat CLFS induced a rapid increase in both phosphorylatedGSK-3β (Fig. 3B) and the ratio of phosphorylated to totalGSK-3β (Fig. 3C) from day 1 of stimulation that remainedsustained to beyond 5 days, compared with Control levels.L-NAME treatment abrogated this effect throughout thestimulation period (Fig. 3B). These results suggest thatNOS activity may be involved in promoting CLFS-inducedincreases in NFATc1 nuclear accumulation via inhibitoryphosphorylation of GSK-3β, thus suppressing NFATc1phosphorylation and subsequent nuclear export.

Myosin heavy chain mRNA isoform expression

To test the hypothesis that NOS inhibition pre-vents activity-induced faster-to-slower fibre type trans-formations in vivo, we first measured MHC isoforms atthe mRNA level in rat fast-twitch muscles exposed toCLFS. As detected by real-time PCR in rat tibialis anteriorextracts, CLFS induced an increase in the expressionof most MHC mRNAs after 5 days of stimulation, withfurther increases attaining values that were 16.0-fold(MHCI) (Fig. 4A), 25.1-fold (MHCIIa) (Fig. 4B) and8.6-fold (MHCIId(x)) (Fig. 4C) by 10 days of stimulationcompared with Control. However, MHCIIb mRNA levelswere not changed by the 10 day stimulation protocol(Fig. 4D). L-NAME treatment blocked the CLFS-inducedincreases in MHC mRNA isoform expressions throughoutthe 10 days of stimulation (Fig. 4A and C). Collectively,these data indicate that NOS activity is essential foractivity-induced increases in relatively slower MHCmRNA isoforms that are up-regulated in response to CLFSin rat fast-twitch muscles.

Myosin heavy chain protein isoform and fibre typeexpression

To further investigate the effects of NOS inhibition onactivity-induced changes in MHC expression in vivo,we measured MHC isoforms at the protein level aswell as fibre type proportions in rat fast-twitch musclesexposed to CLFS. Quantitative MHC protein content

of whole muscle extracts was measured by SDS-PAGE(Fig. 5A) and detailed fibre type analysis was assessed byimmunohistochemistry specific for the various MHC iso-forms (Figs 6 and 7). Regardless of treatment condition,the first significant changes in the whole muscle MHCprotein isoform pattern were detected by 5 days ofstimulation where the relative contents of MHCIIa andMHCIIb increased and decreased, respectively (Fig. 5B).These reciprocal changes progressed with continuedstimulation such that by 10 days of stimulation the

Figure 1. NFATc1 phosphorylation status in rat fast-twitchmuscleA, Western blots of NFATc1 in whole cell extracts prepared from ratextensor digitorum longus muscles. α-Tubulin served as a loadingcontrol for total protein. B, ratio of the most de-PO4-to-most-PO4

NFATc1 protein bands. Values are means ± SEM; n = 6 animals pergroup. Bracket indicates groups within each treatment conditionthat were pooled; n = 12 animals per group. Statistical symbolsindicate: Cdifferent from Control (i.e. 0 day (d) Stim); ∗different fromL+Stim of the matched time point of stimulation(P < 0.05).



Figure 2. Immunofluorescent analysis of NFATc1 localisation in rat fast-twitch muscleA, representative high magnification Z-stacked photomicrographs of NFATc1-positive (arrows in ‘+’ panel rows)or NFATc1-negative (arrowheads in ‘–’ panel row) nuclei. Sections of rat tibialis anterior muscles were stained forNFATc1 (green) and nuclei visualized with DAPI (red). Note the perinuclear staining of NFATc1 in all panels. Barrepresents 6.25 μm. B, representative photomicrographs of Stim- and L+Stim-treated rat tibialis anterior musclesat 0, 1, 2, 5 and 10 days of stimulation. Sections were stained as in A. Arrows point to nuclei identified asNFATc1-positive from high magnification images. Bar represents 50 μm. C, proportion of NFATc1-positive nuclei inStim- and L+Stim-treated rat tibialis anterior muscles. D, proportion of NFATc1-positive nuclei in plantaris musclesof wild type (WT) and transgenic mice overexpressing constitutively active calcineurin (CnA∗) as a comparativecontrol (see Methods). Values are means ± SEM; n = 6 animals per group for Stim and L+Stim and n = 3 animalsper group for WT and CnA∗. Statistical symbols indicate: Cdifferent from Control (i.e. 0 days Stim); ∗different fromL+Stim of the matched time point of stimulation; ‡CnA∗ different from wild type (P < 0.05).



Figure 3. GSK-3β phosphorylation status in rat fast-twitch muscleA, representative Western blot of phosphorylated GSK-3β and GSK3b in whole cell extracts prepared from ratextensor digitorum longus muscles; α-tubulin served as a loading control for total protein. B, phosphorylatedGSK-3β expressed relative to control. C, ratio of phosphorylated GSK-3β/GSK3b expressed relative to control.Values are means ± SEM; n = 6 animals per group. Statistical symbols indicate: Cdifferent from Control (i.e. 0 daysStim; P < 0.05); ∗different from L+Stim of the matched time point of stimulation (P < 0.05).

Figure 4. Patterns of MHC mRNA isoform expression in rat fast-twitch muscleFold changes in MHCI (A), MHCIIa (B), MHCIId(x) (C) and MHCIIb (D) mRNA expression levels in rat tibialis anteriormuscles as determined by the 2−��Ct method of analysis. Values are means ± SEM; n = 6 animals per group.Statistical symbols indicate: Cdifferent from Control (i.e. 0 days Stim); ∗different from L+Stim of the matched timepoint of stimulation (P < 0.05).



relative content of MHCIIa had increased 1.9-fold, witha concomitant 1.3-fold decrease in MHCIIb, comparedto Control (Fig. 5B; main effects P = 0.0002). Similarly,by 10 days of stimulation, the proportion of fibresexpressing MHCIIa was 1.2-fold greater compared withControl (Fig. 7B; main effect P = 0.03). More importantly,10 days of stimulation induced 2.0-fold increases in therelative content of MHCI and in the proportion of fibresexpressing MHCI compared with Control, a response thatwas blocked by L-NAME treatment (Figs 5B and 7A).Taken together, these results suggest that NOS activityappears necessary for activity-induced increases in MHCIprotein expression and type I fibre transformation. On theother hand, NOS activity does not appear to be requiredfor CLFS-induced MHC protein isoform or fibre typeconversions within the fast fibre population over the 10 dayduration of the present study.

Discussion

NO has been established as an important signallingmolecule in skeletal muscle (Stamler & Meissner, 2001). In

response to increased nerve-mediated muscle contraction,such as is induced by CLFS, Ca2+–calmodulin-dependentNOS activity and resultant NO production increases(Stamler & Meissner, 2001). It has been reportedrecently that NOS activity is also required for Ca2+

ionophore-induced NFATc1 nuclear accumulation andMHCI mRNA expression in vitro (Drenning et al. 2008,2009). Therefore, the purpose of the present studywas to investigate the involvement of NOS activityin CLFS-induced NFATc1 nuclear accumulation andsubsequent faster-to-slower skeletal muscle fibre typetransformations in vivo. Multiple early time pointsof stimulation were chosen because CLFS-inducedfaster-to-slower MHC-based isoform transformations areshown to begin at the mRNA level after 3 days and atthe protein level after 5 days, responses that continueto change through 10 days of stimulation (Jaschinskiet al. 1998). Our study reports the novel finding thatnerve activity-induced faster-to-slower MHC isoformtransformations are transcriptionally regulated by NOSactivity. Specifically, we show that the NOS inhibitorL-NAME prevented CLFS-induced increases in slowMHCI and fast MHCIIa and IId(x) mRNAs that are

Figure 5. Myosin heavy chain proteinisoform distribution in rat fast-twitchmuscleA, example of the electrophoretic methodused to quantify MHC isoform compositionof rat tibialis anterior muscles. B,percentage of MHCI, MHCIIa, MHCIId(x)and MHCIIb protein isoform content in rattibialis anterior muscles, as determined bydensitometric evaluation of duplicate gels.Values are means ± SEM; n = 6 animals pergroup. Statistical symbols indicate:Cdifferent from Control (i.e. 0 days Stim);∗different from L+Stim of the matchedtime point of stimulation; §main effect ofdays of stimulation (P < 0.05).



typically observed during fibre conversions (Fig. 4). Onthe other hand, we found that L-NAME only inhibitedactivity-induced accumulation of slow MHCI protein iso-forms and type I fibre conversions (Figs 5–7, respectively),suggesting that MHC transformations within the fast fibrepopulation also involve translational control mechanismsindependent of NO signalling. In association with thesechanges, we found that NOS activity is necessary foractivity-induced inhibitory phosphorylation of GSK-3β(Fig. 3), and increases in calcineurin-dependent NFATc1dephosphorylation (Fig. 1), both contributing to thenuclear localisation of this transcription factor (Fig. 2).Collectively, the current data extend previous in vitrofindings (Drenning et al. 2008, 2009) and denotethe importance of NO as a regulator of MHC-basedfaster-to-slower fibre type conversions at the trans-criptional level via nerve activity-modulated NFATc1nuclear accumulation in vivo.

Chronic low-frequency stimulation andcalcineurin–NFATc1 signalling

In order to elicit a pronounced stimulus forfaster-to-slower fibre type transformations in the absenceof muscle fibre injury and regeneration, CLFS wasemployed using the rat model (Putman et al. 1999). CLFSis a model of muscle training that mimics the electricaldischarge pattern of slow motor neurons innervatingslow-twitch muscles (Hennig & Lomo, 1985), causingCa2+–calcineurin-dependent NFAT dephosphorylationand nuclear translocation (Dunn et al. 2000, 2001;Liu et al. 2001; Tothova et al. 2006; Shen et al.2007; Drenning et al. 2008; Calabria et al. 2009), andfaster-to-slower fibre type transformations, as exemplifiedin the current study. Unlike other rodent exercise models,however, CLFS synchronously recruits all targeted motorunits, including those not normally activated duringsub-maximal exercise training (Pette & Staron, 2000).

Figure 6. Immunohistochemical detection of myosin heavy chain isoforms in serial cross-sections of ratfast-twitch musclePhotomicrographs of representative MHC isoform immunohistochemistry of rat tibialis anterior muscle. Immuno-stains for MHCI (clone BA-D5), MHCIIa (clone SC-71), MHCIId(x) (clone BF-35; staining all MHCs except MHCIId(x))and MHCIIb (clone BF-F3). The bar located lower right represents 100 μm.



In doing so, the adaptive potential of CLFS-targetedmuscles is maximally challenged. Also, the standardisedand highly reproducible conditions of CLFS allowsfor activity-induced faster-to-slower phenotypic changesto occur in a well-defined time-dependent manner(Pette & Staron, 1997; Jaschinski et al. 1998). None-theless, despite these unique properties of CLFS, theextent and time course of neural activation-dependentNFATc1 dephosphorylation and nuclear localisation thatwe report here were almost identical to those observedafter compensatory overload of mouse plantaris muscle,another model effecting major fibre conversions towardsslower, more highly oxidative, phenotypes (Dunn et al.1999, 2000, 2001). Indeed, under physiological overloadconditions, NFATc1 dephosphorylation was marked asearly as 1 day and peaked between 5–7 days post-overload,with nuclear NFATc1 peaking at 5 days (Dunn et al. 2001).This neural activation-dependent dephosphorylation andnuclear localisation of NFATc1 was found to be calcineurindependent since it was abolished in mice administrated thecalcineurin blockers cyclosporine A or FK506 (Dunn et al.2001).

Nitric oxide involvement in faster-to-slower fibretype transformations

Several lines of evidence show that the calcineurin–NFATsignalling pathway is involved in the promotion

and maintenance of a slower, more energy-efficient,fibre-specific phenotype (as reviewed by Michel et al.2004, 2007; Liu et al. 2005). Emerging evidenceindicates that NO may also be involved in thisregulatory pathway. To our knowledge, only the Criswelllaboratory has investigated the involvement of NOin faster-to-slower fibre type transformations to date(Smith et al. 2002; Sellman et al. 2006; Drenning et al.2008, 2009). They initially reported that L-NAMEprevented the up-regulation of MHCI mRNA andprotein in rat plantaris muscles following 5 and 14 daysof functional overload, respectively (Smith et al. 2002;Sellman et al. 2006). More recently, their laboratoryhas demonstrated that NOS activity is necessary forCa2+-induced NFATc1 nuclear accumulation andincreased MHCI mRNA expression in vitro (Drenninget al. 2008, 2009). Our present results further theseobservations in vivo by showing that NOS activity is notonly required for CLFS-induced increases in NFATc1dephosphorylation (Fig. 1) and nuclear localisation(Fig. 2), but also plays a critical role in the expressionof both slow and fast MHC transcript levels duringactivity-induced faster-to-slower fibre transformations(i.e. MHCIIb→MHCIId(x)→MHCIIa→MHCI)(Fig. 4).

The temporal relationship between CLFS and activationof target genes in our study followed a pattern typicallyobserved in excitable cells, whereby waves of increasedexpression are decoded by downstream elements and are

Figure 7. The proportion of fibres expressing the various adult MHC isoforms in rat fast-twitch muscleThe proportion of fibres expressing MHCI (A), MHCIIa (B), MHCIId(x) (C) and MHCIIb (D). Values are means ± SEM;n = 6 animals per group. Statistical symbols indicate: Cdifferent from Control (i.e. 0 days Stim); ∗different fromL+Stim of the matched time point of stimulation; §main effect of days of stimulation (P < 0.05).



often subjected to inhibitory feedback loops, in order toprevent sustained pathway activation (Michel et al. 2004).The time course of signalling changes by CLFS that wereport is gradual, with averages reaching significance in theorder of NFATc1 dephosphorylation (at day 2), NFATc1nuclear import and localisation (at day 5) and activationof NFACTc1 MHC gene targets (at day 10). The apparentlag between the events of NFATc1 dephosphorylationand NFATc1 nuclear import is consistent with theformer being a prerequisite for the latter, but is alsoinfluenced by dynamic nuclear–cytoplasmic NFATc1shuttling, albeit at a much slower rate due to inhibitionof nuclear GSK-3β. The timely progressive activation ofMHC gene targets by NFATc1, from day 5 to day 10,thus represents a key event towards fast-to-slow fibretype transformations. Further evidence indicates suchsignalling is associated with enhanced mRNA stability ofthe MHC target genes (Chakkalakal et al. 2008). Thus,it is probable that the faster-to-slower fibre adaptiveresponse continues for a period of time well after theinitial signalling events are no longer detectable. Themechanisms regulating adaptive changes in MHC mRNAbeyond 10 days (Martins et al. 2006) most certainlycontinue to involve the low-amplitude intracellular [Ca2+]oscillations responsible for sustained calcineurin–NFATsignalling (Chin et al. 1998; Dunn et al. 1999, 2000,2001; Liu et al. 2001; Michel et al. 2004, 2007). Wecannot, however, preclude the possibility that NO mayalso continue to influence those same gene targets duringlonger periods of CLFS, and in other models of increasedmuscle activity.

Calcineurin–NFAT signalling has also been implicatedin the shift from the fastest MHCIIb and IId(x) mRNA iso-forms to the slower fast MHCIIa mRNA isoform (Dunnet al. 1999, 2001; Allen et al. 2001; Allen & Leinwand,2002; Calabria et al. 2009). For example, administrationof the calcineurin inhibitors cyclosporine A and FK506 isshown to prevent overload-induced IIb→IId(x)→IIa→Ifibre conversions at the mRNA and protein levels, aswell as to prevent dephosphorylation of NFATc1, inthe fast plantaris muscle of mice (Dunn et al. 1999,2001). Furthermore, activated calcineurin or NFAT over-expression preferentially activated the MHCIIa promoterto a greater extent than the other two fast MHC isoforms(i.e. MHCIIb and MHCIId(x)) (Allen et al. 2001; Allen &Leinwand, 2002). This is consistent with our finding thatan effect of Stim was not observed on the fast MHCIIbmRNA, whilst the MHCIIa isoform displayed the greatestincrease in mRNA expression. Collectively, our findingssuggest that NO may be regulating stimulation-inducedfibre type conversions of at least three MHC isoformsat the transcriptional level via neural activity-modulatedcalcineurin–NFATc1 nuclear accumulation in vivo.

The mechanisms by which NO may be promotingCLFS-induced NFAT nuclear accumulation and increases

in slower MHC mRNA isoforms remain to beelucidated. Given we found L-NAME treatment preventedactivity-induced increases in the phosphorylation ofGSK-3β and NFATc1 nuclear accumulation, it is possiblethat NO may be regulating the transcriptional activity ofMHC isoforms by GSK-3β inhibition, thus suppressingNFATc1 phosphorylation and nuclear export, in responseto in vivo stimulation. Our results are supported bythe recent in vitro findings that showed NO-donortreatment induced increased phosphorylation of GSK-3βand augmented NFAT-dependent transcriptional activity,while L-NAME prevented this effect in C2C12 myotubesthat were exposed to a Ca2+ ionophore (Drenning et al.2008). Conversely, we cannot rule out the possibilitythat NO could also be involved in calcineurin-dependentNFAT dephosphorylation and nuclear import duringin vivo stimulation. While GSK-3β inhibition does notcause NFATc1 nuclear accumulation in resting fibres(Shen et al. 2006), NO donor treatment has been shownto increase NFATc1 nuclear accumulation and MHCImRNA expression in control fibres to the same extent asCa2+ ionophore treatment in primary myotube cultures(Drenning et al. 2009). Thus, although NO is associatedwith inhibitory phosphorylation of GSK-3β, it appearsthat NO may also promote NFATc1 nuclear import. Itshould be noted that NFATc1 and GSK-3β exhibited trans-ient increased activity in the early phase of CLFS, returningto control levels by 10 days of stimulation, during whichtime the up-regulation of slower MHC isoform mRNAswas still occurring. Previous studies have also reportedsimilar transient increases in calcineurin downstreamsignalling factors and gene targets during the early phaseof functional overload-induced faster-to-slower fibre typetransitions (Dunn et al. 1999, 2001; Miyazaki et al.2004). Therefore, since L-NAME treatment preventedCLFS-induced up-regulation of MHC mRNA isoformsthroughout the 10 day stimulation period, NO may beregulating additional downstream targets and signallingpathways during the later phase of faster-to-slowerfibre type transitions. Further studies are required tofully delineate the pathway by which NO facilitatesactivity-induced NFATc1 nuclear accumulation and MHCisoform expression.

Our data also suggest that nerve activity-dependentMHC-based transformations may not be limited tothe transcriptional regulation of these contractileproteins. Even though inhibition of NOS activity pre-vented the activity-induced up-regulation of slowerMHC mRNA isoforms, conversions within the fastfibre population still occurred at the protein levelin stimulated L-NAME-treated muscles (Figs 5–7).Therefore, activity-induced transformations within thefast fibre population may be further subjected to trans-lational control mechanisms independent of NO signallingvia the NFATc1 pathway.



Conclusions

Control of adult skeletal muscle phenotype andadaptive plasticity is a complex process involvingmultiple signalling pathways and levels of regulation.Our novel findings provide further insight into themechanisms underlying MHC-based faster-to-slowerphenotypic transformations in vivo. Results of the currentstudy show that NFATc1 dephosphorylation, nuclearaccumulation and subsequent up-regulation of relativelyslower, more energy-efficient, MHC mRNA isoformsinvolved in CLFS-induced fibre transformations aredependent on NOS activity in rat fast-twitch skeletalmuscles. These results support the hypothesis that NOcontributes to regulation of activity-induced MHC-basedfaster-to-slower fibre type transformations at the trans-criptional level by acting synergistically with calcineurinand GSK-3β to promote NFATc1 nuclear accumulationin vivo. Additionally, activity-induced transformationswithin the fast fibre population may be further subjectedto translational control mechanisms independent of NOsignalling upon NFATc1.

References

Akimoto T, Pohnert SC, Li P, Zhang M, Gumbs C, RosenbergPB et al. (2005). Exercise stimulates PGC-1a transcription inskeletal muscle through activation of the p38 MAPKpathway. J Biol Chem 280, 19587–19593.

Allen DL & Leinwand LA (2002). Intracellular calcium andmyosin isoform transitions. Calcineurin andcalcium-calmodulin kinase pathways regulated preferentialactivation of the IIa myosin heavy chain promoter. J BiolChem 277, 45323–45330.

Allen DL, Sartorius CA, Sycuro LK & Leinwand LA (2001).Different pathways regulate expression of the skeletal myosinheavy chain genes. J Biol Chem 276, 43524–43533.

Baar K, Wende AR, Jones TE, Marison M, Nolte LA, Chen Met al. (2002). Adaptations of skeletal muscle to exercise: rapidincrease in the transcriptional coactivator PGC-1. FASEB J16, 1879–1886.

Bassel-Duby R & Olson EN (2006). Signaling pathways inskeletal muscle remodeling. Annu Rev Biochem 75,19–37.

Bottinelli R, Schiaffino S & Reggiani C (1991). Force–velocityrelations and myosin heavy chain isoform compositions ofskinned fibres from rat skeletal muscle. J Physiol 437,655–672.

Bradford MM (1976). A rapid and sensitive method for thequatitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal Biochem 72,248–254.

Brunelli S, Sciorati C, D’Antona G, Innocenzi A, Covarello D,Galvez BG et al. (2007). Nitric oxide release combined withnonsteroidal antiinflammatory activity prevents musculardystrophy pathology and enhances stem cell therapy. ProcNatl Acad Sci U S A 104, 264–269.

Calabria E, Ciciliot S, Moretti I, Garcia M, Picard A, Dyar KAet al. (2009). NFAT isoforms control activity-dependentmuscle fiber type specification. Proc Natl Acad Sci U S A 106,13335–13340.

Chakkalakal JV, Harrison MA, Carbonetto S, Chin E, MichelRN, Jasmin BJ (2004). Stimulation of calcineurin signalingattenuates the dystrophic pathology in mdx mice. Hum MolGenet 13, 379–388.

Chakkalakal JV, Miura P, Belanger G, Michel RN &Jasmin BJ (2008). Modulation of utrophin A mRNA stabilityin fast versus slow muscles via an AU-rich element andcalcineurin signaling. Nucleic Acids Res 36,826–838.

Chin ER, Olson EN, Richardson JA, Yang Q, Humphries C,Shelton JM et al. (1998). A calcineurin-dependenttranscriptional pathway controls skeletal muscle fiber type.Genes Dev 12, 2499–2509.

Colussi C, Gurtner A, Rosati J, Illi B, Ragone G, Piaggio G et al.(2009). Nitric oxide deficiency determines global chromatinchanges in Duchenne muscular dystrophy. FASEB J 23,2131–2141.

Colussi C, Mozzetta C, Gurtner A, Illi B, Rosati J, Straino Set al. (2008). HDAC2 blockade by nitric oxide and histonedeacetylase inhibitors reveals a common target in Duchennemuscular dystrophy treatment. Proc Natl Acad Sci U S A 105,19183–19187.

Czubryt MP, McAnally J, Fishman GI & Olson EN (2003).Regulation of peroxisome proliferator-activated receptorgamma coactivator 1 alpha (PGC-1α) and mitochondrialfunction by MEF2 and HDAC5. Proc Natl Acad Sci U S A100, 1711–1716.

Dimmeler S & Zeiher AM (1999). Nitric oxide – an endothelialcell survival factor. Cell Death Differ 6, 964–968.

Drenning JA, Lira VA, Simmons CG, Soltow QA, Sellman JE &Criswell DS (2008). Nitric oxide facilitates NFAT-dependenttranscription in mouse myotubes. Am J Physiol Cell Physiol294, C1088–C1095.

Drenning JA, Lira VA, Soltow QA, Canon CN, Valera LM,Brown DL & Criswell DS (2009). Endothelial nitric oxidesynthase is involved in calcium-induced Akt signaling inmouse skeletal muscle. Nitric Oxide 21, 192–200.

Dunn SE, Burns JL & Michel RN (1999). Calcineurin isrequired for skeletal muscle hypertrophy. J Biol Chem 274,21908–21912.

Dunn SE, Chin ER & Michel RN (2000). Matching ofcalcineurin activity to upstream effectors is critical forskeletal muscle fiber growth. J Cell Biol 151, 663–672.

Dunn SE, Simard AR, Bassel-Duby R, Williams RS & MichelRN (2001). Nerve activity-dependent modulation ofcalcineurin signaling in adult fast and slow skeletal musclefibers. J Biol Chem 276, 45243–45254.

Grozdanovic Z & Baumgarten HG (1999). Nitric oxidesynthase in skeletal muscle fibers: a signaling component ofthe dystrophin-glycoprotein complex. Histol Histopathol 14,243–256.

Hamalainen N & Pette D (1996). Slow-to-fast transitions inmyosin expression of rat soleus muscle by phasichigh-frequency stimulation. FEBS Lett 399,220–222.



Handschin C, Rhee J, Lin J, Tarr PT & Spiegelman BM (2003).An autoregulatory loop controls peroxisomeproliferator-activated receptor gamma coactivator 1αexpression in muscle. Proc Natl Acad Sci U S A 100,7111–7116.

Hennig R & Lomo T (1985). Firing patterns of motor units innormal rats. Nature 314, 164–166.

Hogan PG, Chen L, Nardone J & Rao A (2003). Transcriptionalregulation by calcium, calcineurin, and NFAT. Genes Dev 17,2205–2232.

Jaschinski F, Schuler MJ, Peuker H & Pette D (1998). Changesin myosin heavy chain mRNA and protein isoforms of ratmuscle during forced contractile activity. Am J Physiol CellPhysiol 274, C365–C370.

Jiang H, Li H & DiMario JX (2006). Control of slow myosinheavy chain 2 gene expression by glycogen synthase kinaseactivity in skeletal muscle fibers. Cell Tissue Res 323,489–494.

Laemmli UK (1970). Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature 227,680–685.

LaFramboise WA, Jayaraman RC, Bombach KL, Ankrapp DP,Krill-Burger JM, Sciulli CM et al. (2009). Acute molecularresponse of mouse hindlimb muscles to chronic stimulation.Am J Physiol Cell Physiol 297, C556–C570.

Lin J, Wu H, Tarr PT, Zhang CY, Wu Z, Boss O et al. (2002).Transcriptional co-activator PGC-1α drives the formation ofslow-twitch muscle fibres. Nature 418, 797–801.

Liu Y, Cseresnyes Z, Randall WR & Schneider MF (2001).Activity-dependent nuclear translocation and intranucleardistribution of NFATc in adult skeletal muscle fibers. J CellBiol 155, 27–39.

Liu Y, Shen T, Randall WR & Schneider MF (2005). Signalingpathways in activity-dependent fiber type plasticity in adultskeletal muscle. J Muscle Res Cell Motil 26, 13–21.

Livak KJ & Schmittgen TD (2001). Analysis of relative geneexpression data using real-time quantitative PCR and the2−��CT method. Methods 25, 402–408.

Lu J, McKinsey TA, Zhang CL & Olson EN (2000). Regulationof skeletal myogenesis by association of the MEF2transcription factor with class II histone deacetylases. MolCell 6, 233–244.

McConell GK & Wadley GD (2008). Potential role of nitricoxide in contraction-stimulated glucose uptake andmitochondrial biogenesis in skeletal muscle. Clin ExpPharmacol Physiol 35, 1488–1492.

Martins KJ, Gordon T, Pette D, Dixon WT, Foxcroft GR,MacLean IM & Putman CT (2006). Effect of satellite cellablation on low-frequency-stimulated fast-to-slowfibre-type transitions in rat skeletal muscle. J Physiol 572,281–294.

Martins KJ, Murdoch GK, Shu Y, Harris RL, Gallo M, DixonWT et al. (2009). Satellite cell ablation attenuates short-termfast-to-slow fibre type transformations in rat fast-twitchskeletal muscle. Pflugers Arch 458, 325–335.

Meissner JD, Umeda PK, Chang KC, Gros G & Scheibe RJ(2007). Activation of the beta myosin heavy chain promoterby MEF-2D, MyoD, p300, and the calcineurin/NFATc1pathway. J Cell Physiol 211, 138–148.

Michel RN, Chin ER, Chakkalakal JV, Eibl JK & Jasmin BJ(2007). Ca2+/calmodulin-based signalling in the regulationof the muscle fibre phenotype and its therapeutic potentialvia modulation of utrophin A and myostatin expression.Appl Physiol Nutr Metab 32, 921–929.

Michel RN, Dunn SE & Chin ER (2004). Calcineurin andskeletal muscle growth. Proc Nutr Soc 63, 341–349.

Miyazaki M, Hitomi Y, Kizaki T, Ohno H, Haga S & TakemasaT (2004). Contribution of the calcineurin signaling pathwayto overload-induced skeletal muscle fiber-type transition. JPhysiol Pharmacol 55, 751–764.

Nakayama M, Stauffer J, Cheng J, Banerjee-Basu S, WawrousekE & Buonanno A (1996). Common core sequences are foundin skeletal muscle slow- and fast-fiber-type-specificregulatory elements. Mol Cell Biol 16, 2408–2417.

Naya FJ, Mercer B, Shelton J, Richardson JA, Williams RS &Olson EN (2000). Stimulation of slow skeletal muscle fibergene expression by calcineurin in vivo. J Biol Chem 275,4545–4548.

Nisoli E & Carruba MO (2006). Nitric oxide and mitochondrialbiogenesis. J Cell Sci 119, 2855–2862.

Pette D & Staron RS (1997). Mammalian skeletal muscle fibertype transitions. Int Rev Cytol 170, 143–223.

Pette D & Staron RS (2000). Myosin isoforms, muscle fibertypes, and transitions. Microsc Res Tech 50,500–509.

Pette D & Vrbova G (1999). What does chronic electricalstimulation teach us about muscle plasticity? Muscle Nerve22, 666–677.

Pisconti A, Brunelli S, Di PM, De PC, Deponti D, Baesso S et al.(2006). Follistatin induction by nitric oxide through cyclicGMP: a tightly regulated signaling pathway that controlsmyoblast fusion. J Cell Biol 172, 233–244.

Putman CT, Dixon WT, Pearcey J, MacLean IM, Jendral MJ,Kiricsi M et al. (2004). Chronic low-frequency stimulationup-regulates uncoupling protein-3 in transforming ratfast-twitch skeletal muscle. Am J Physiol Regul Integr CompPhysiol 287, R1419–R1426.

Putman CT, Dusterhoft S & Pette D (1999). Changes in satellitecell content and myosin isoforms in low-frequency-stimulated fast muscle of hypothyroid rat. J Appl Physiol 86,40–51.

Putman CT, Dusterhoft S & Pette D (2000). Satellite cellproliferation in low-frequency stimulated fast muscle ofhypothyroid rat. Am J Physiol Cell Physiol 279,C682–C690.

Putman CT, Kiricsi M, Pearcey J, MacLean IM, Bamford JA,Murdoch GK et al. (2003). AMPK activation increasesuncoupling protein-3 expression and mitochondrial enzymeactivities in rat muscle without fibre type transitions. JPhysiol 551, 169–178.

Putman CT, Sultan KR, Wassmer T, Bamford JA, Skorjanc D &Pette D (2001). Fiber-type transitions and satellite cellactivation in low-frequency-stimulated muscles of youngand aging rats. J Gerontol A Biol Sci Med Sci 56,B510–B519.

Rana ZA, Gundersen K & Buonanno A (2008).Activity-dependent repression of muscle genes by NFAT.Proc Natl Acad Sci U S A 105, 5921–5926.



Rana ZA, Gundersen K, Buonanno A & Vullhorst D (2005).Imaging transcription in vivo: distinct regulatory effects offast and slow activity patterns on promoter elements fromvertebrate troponin I isoform genes. J Physiol 562, 815–828.

Reiser PJ, Kline WO & Vaghy PL (1997). Induction of neuronaltype nitric oxide synthase in skeletal muscle by chronicelectrical stimulation in vivo. J Appl Physiol 82, 1250–1255.

Russell AP, Feilchenfeldt J, Schreiber S, Praz M, Crettenand A,Gobelet C et al. (2003). Endurance training in humans leadsto fiber type-specific increases in levels of peroxisomeproliferator-activated receptor-gamma coactivator-1 andperoxisome proliferator-activated receptor-alpha in skeletalmuscle. Diabetes 52, 2874–2881.

Sartorelli V, Puri PL, Hamamori Y, Ogryzko V, Chung G,Nakatani Y et al. (1999). Acetylation of MyoD directed byPCAF is necessary for the execution of the muscle program.Mol Cell 4, 725–734.

Schiaffino S, Gorza L, Pitton G, Saggin L, Ausoni S, Sartore S &Lomo T (1988). Embryonic and neonatal myosin heavychain in denervated and paralyzed rat skeletal muscle. DevBiol 127, 1–11.

Schiaffino S, Gorza L, Sartore S, Saggin L, Ausoni S, Vianello Met al. (1989). Three myosin heavy chain isoforms in type 2skeletal muscle fibres. J Muscle Res Cell Motil 10, 197–205.

Sellman JE, Deruisseau KC, Betters JL, Lira VA, Soltow QA,Selsby JT & Criswell DS (2006). In vivo inhibition of nitricoxide synthase impairs upregulation of contractile proteinmRNA in overloaded plantaris muscle. J Appl Physiol 100,258–265.

Shen T, Cseresnyes Z, Liu Y, Randall WR & Schneider MF(2007). Regulation of the nuclear export of the transcriptionfactor NFATc1 by protein kinases after slow fibre typeelectrical stimulation of adult mouse skeletal muscle fibres. JPhysiol 579, 535–551.

Shen T, Liu Y, Cseresnyes Z, Hawkins A, Randall WR &Schneider MF (2006). Activity- and calcineurin-independentnuclear shuttling of NFATc1, but not NFATc3, in adultskeletal muscle fibers. Mol Biol Cell 17, 1570–1582.

Simoneau J-A & Pette D (1988). Species-specific effects ofchronic nerve stimulation upon tibialis anterior muscle inmouse, rat, guinea pig and rabbit. Pflugers Arch 412, 86–92.

Smith LW, Smith JD & Criswell DS (2002). Involvement ofnitric oxide synthase in skeletal muscle adaptation to chronicoverload. J Appl Physiol 92, 2005–2011.

Stamler JS & Meissner G (2001). Physiology of nitric oxide inskeletal muscle. Physiol Rev 81, 209–237.

Suwa M, Egashira T, Nakano H, Sasaki H & Kumagai S (2006).Metformin increases the PGC-1α protein and oxidativeenzyme activities possibly via AMPK phosphorylation inskeletal muscle in vivo. J Appl Physiol 101, 1685–1692.

Terada S, Goto M, Kato M, Kawanaka K, Shimokawa T &Tabata I (2002). Effects of low-intensity prolonged exerciseon PGC-1 mRNA expression in rat epitrochlearis muscle.Biochem Biophys Res Commun 296, 350–354.

Tothova J, Blaauw B, Pallafacchina G, Rudolf R, Argentini C,Reggiani C & Schiaffino S (2006). NFATc1nucleocytoplasmic shuttling is controlled by nerveactivity in skeletal muscle. J Cell Sci 119, 1604–1611.

Vissing K, McGee SL, Roepstorff C, Schjerling P, Hargreaves M& Kiens B (2008). Effect of sex differences on human MEF2regulation during endurance exercise. Am J PhysiolEndocrinol Metab 294, E408–E415.

Wright DC, Geiger PC, Han DH, Jones TE & Holloszy JO(2007). Calcium induces increases in peroxisomeproliferator-activated receptor gamma coactivator-1α andmitochondrial biogenesis by a pathway leading to p38mitogen-activated protein kinase activation. J Biol Chem282, 18793–18799.

Wu Z, Puigserver P, Andersson U, Zhang C, Adelmant G,Mootha V et al. (1999). Mechanisms controllingmitochondrial biogenesis and respiration through thethermogenic coactivator PGC-1. Cell 98, 115–124.

Xiao L, Coutu P, Villeneuve LR, Tadevosyan A, Maguy A, Le BSet al. (2008). Mechanisms underlying rate-dependentremodeling of transient outward potassium current incanine ventricular myocytes. Circ Res 103, 733–742.

Zong H, Ren JM, Young LH, Pypaert M, Mu J, Birnbaum MJ &Shulman GI (2002). AMP kinase is required formitochondrial biogenesis in skeletal muscle in response tochronic energy deprivation. Proc Natl Acad Sci U S A 99,15983–15987.

Author contributions

K.J.B.M. and R.N.M. conceived the conceptual framework ofthe study and designed the experiments. Experiments wereperformed by K.J.B.M. and M.St-L. Animal surgeries and tissueextractions were performed by K.J.B.M., M.St-L. and R.N.M.Biochemical and immunocytochemical analyses were performedby K.J.B.M., M.St-L., G.K.M., I.M.M. and P.M. K.J.B.M., M.St-L.,W.T.D, C.T.P. and R.N.M. contributed to the interpretationof data. K.J.B.M. wrote the manuscript with M.St-L., C.T.P.and R.N.M. All authors approved the final version of themanuscript.

Acknowledgements

This study was funded by research grants from the NaturalSciences and Engineering Council of Canada (NSERC; C.T.P.and R.N.M.), the Alberta Heritage Foundation for MedicalResearch (AHFMR; C.T.P.), the Alberta Agricultural ResearchInstitute (W.T.D.), the Canadian Institutes of Health Research(CIHR; R.N.M.), the Canada Research Chairs Program (CRC;R.N.M.) and the Canadian Foundation for Innovation (CFI;R.N.M.). K.J.B.M. was supported by NSERC and AHFMRgraduate scholarships. R.N.M. is a Canada Research Chair Tier1 in Cellular and Molecular Neuromuscular Physiology. C.T.P.is a Senior Scholar of AHFMR. The authors thank Dr TessaGordon from the University of Alberta for use of laboratoryspace. We thank Drs Richard Schultz (Univ. of Alberta), Eva R.Chin (Univ. of Maryland), and Alisa Piekny (Concordia Univ.)for their expert advice.


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