Experimental Physiology

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1 Exp Physiol 97.1 pp Research Paper Exercise training attenuates the hypermuscular phenotype and restores skeletal muscle function in the myostatin null mouse Antonios Matsakas 1,2, Raymond Macharia 3,AnthonyOtto 1,MohamedI.Elashry 1, Etienne Mouisel 4, Vanina Romanello 5,RobertaSartori 5,HelgeAmthor 4, Marco Sandri 5, Vihang Narkar 2 and Ketan Patel 1 1 School of Biological Sciences, University of Reading, Reading, UK 2 The University of Texas Health Science Center, Institute of Molecular Medicine, Houston, TX, USA 3 RoyalVeterinaryCollege,London,UK 4 Université Pierre et Marie Curie, Institut de Myologie, Paris, France 5 Venetian Institute of Molecular Medicine, University of Padua, Padua, Italy Experimental Physiology Myostatin regulates both muscle mass and muscle metabolism. The myostatin null (MSTN / ) mouse has a hypermuscular phenotype owing to both hypertrophy and hyperplasia of the myofibres. The enlarged muscles display a reliance on glycolysis for energy production; however, enlarged muscles that develop in the absence of myostatin have compromised force-generating capacity. Recent evidence has suggested that endurance exercise training increases the oxidative properties of muscle. Here, we aimed to identify key changes in the muscle phenotype of MSTN / mice that can be induced by training. To this end, we subjected MSTN / mice to two different forms of training, namely voluntary wheel running and swimming, and compared the response at the morphological, myocellular and molecular levels. We found that both regimes normalized changes of myostatin deficiency and restored muscle function. We showed that both exercise training regimes increased muscle capillary density and the expression of Ucp3, Cpt1α, Pdk4 and Errγ, key markers for oxidative metabolism. Cross-sectional area of hypertrophic myofibres from MSTN / mice decreased towards wild-type values in response to exercise and, in this context, Bnip3, a key autophagy-related gene, was upregulated. This reduction in myofibre size caused an increase of the nuclear-to-cytoplasmic ratio towards wild-type values. Importantly, both training regimes increased muscle force in MSTN / mice. We conclude that impaired skeletal muscle function in myostatin-deficient mice can be improved through endurance exercise-mediated remodelling of muscle fibre size and metabolic profile. (Received 31 October 2011; accepted after revision 4 November 2011; first published online 4 November 2011) Corresponding authors A. Matsakas and K. Patel: School of Biological Sciences, Hopkins Building, Whiteknights Campus, Reading, Berkshire RG6 6UB, UK. a.matsakas@gmail.com (A.M.); ketan.patel@reading.ac.uk (K.P.) A well-defined relationship exists in skeletal muscle between the size of myofibres, force generation capacity and metabolism. In a mixed muscle, fibres that rely on oxidative phosphorylation in the mitochondria to generate ATP tend to have small cross-sectional area (CSA) and high density of capillaries in order to facilitate rapid diffusion of O 2. These fibres have high fatigue threshold; however, their small CSA means that each fibre generates low levels of force. In contrast, fibres from mixed muscles that rely primarily on glycolysis show increased CSA and high force generation capacity, but have low fatigue threshold owing to the metabolic pathways that generate energy rapidly but leadtobuildupoflactate(bottinelliet al. 1991; Bruce et al. 1997; Jones et al. 2008; Krivickas et al. 2011). An experimental model that gives an insight into the molecular control of oxidative muscle phenotype is the myostatin null (MSTN / ) mouse. Genetic ablation of myostatin leads to a predominance of fasttwitch glycolytic fibres that has its origin in prenatal development (Matsakas et al. 2010b). The glycolytic DOI: /expphysiol

2 126 A. Matsakas and others Exp Physiol 97.1 pp muscle phenotype of myostatin null mice is associated with a decrease in capillary density, mitochondrial number and expression of mitochondrial enzymes, such as cytochrome c oxidase subunit 4 (McPherron etal. 1997; McPherron & Lee, 1997; Amthor et al. 2007; Lipina et al. 2010). Nevertheless, intensive interest both for potential therapeutic application and, unavoidably, for cosmetic purposes has been stimulated by the fact that muscle mass can be increased through blockade of myostatin signalling at postnatal stages (Matsakas & Diel, 2005; Bradley et al. 2008). In contrast to the canonical view of muscle structure and function, the hypertrophic muscle that develops in the absence of myostatin is not accompanied by a proportionate increase in contraction strength (i.e. tension; Mendias et al. 2006; Amthor et al. 2007; Gentry et al. 2011); however, recent evidence suggested that endurance exercise training may normalize the muscle phenotype induced by the absence of myostatin (e.g. Savage & McPherron, 2010; Matsakas et al. 2010a). In the present study, we examined the phenotypic alterations of the MSTN / mouse in response to endurance exercise training by focusing on myocellular, molecular and physiological changes in the skeletal muscle of the MSTN / mouse.akeyfactorinthe experimental design was to use two different types of endurance training, voluntary wheel running and swimming, to identify common mediators of phenotypic change. We report here that both training regimes affected key features of myostatin null muscle. We show that endurance exercise by running or swimming of myostatin null mice reduced muscle fibre size, increased muscle oxidative properties, increased capillary density and, most importantly, improved muscle force generation. By examining a panel of molecular markers, we identified changes induced by both training regimes in the expression of key molecules regulating muscle fibre size, oxidative properties of muscle and capillary density. These results demonstrate that features induced by a germ-line deletion of myostatin are not genetically locked down but can be modified by exercise. Methods Ethical approval Animals were maintained and experiments performed under license from the UK Home Office in accordance with the Animals (Scientific Procedures) Act 1986 and complied with the policy and regulations of the journal. Animal maintenance Healthy male MSTN +/+ (wild type) and MSTN / mice on a C57Bl/6 background were bred and maintained in the biological resource unit of Reading University as described previously (Matsakas et al. 2010a). Four-week-old mice were housed in standard environmental conditions (20 22 C, 12 h 12 h light dark cycle) and provided with tap water and food ad libitum. The animals were maintained according to the Guide for Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no , revised 1996) and animal wellfare regimes were approved by the University of Reading. Exercise training and specimen collection Animals were randomly assigned to a swimming group (n = 4 for each genotype), a wheel running group (n = 6 MSTN / and n = 4 MSTN +/+ ) and an untrained group housed in normal cages, which will be referred to as sedentary (n = 4 for each genotype). Both swim training and free wheel running protocol details have been reported elsewhere (Matsakas et al. 2006, 2010a). In brief, exercise training for both regimes lasted for 5 weeks. Wheel running took place in unidirectional running wheels, and running performance was comparable between genotypes, ranging 2.0 to 2.5 km day 1.Mice swam at C for 60 min day 1,fivetimesaweek. The MSTN / mice exhibited reduced tolerability for the swim training regime; however, their swimming performance significantly increased in terms of time (from 14 min during week 1 to 60 min during weeks 3 5). To minimize the acute exercise effects on gene expression, animal tissue was collected 48 h after the last swimming bout, and wheels were removed 48 h before the end of the experiment. Animals were killed by cervical dislocation according to schedule 1. Extensor digitorum longus (EDL), gastrocnemius and rectus femoris muscles were surgically removed from the hindlimbs of the mice, weighed, immersed in liquid nitrogen-chilled melting isopentane and stored at 80 C. Rodent EDL, gastrocnemius and rectus femoris muscles have previously been shown to be recruited during both swimming and running (e.g. Gruner & Altman, 1980; Laughlin et al. 1984; Roy et al. 1985, 1991; de Leon et al. 1994; Raja et al. 2003). Single myofibre isolation and immunocytochemistry Isolation of single myofibres permits the quantification of myonuclei, because all other cell types (including fibroblasts) are enzymatically removed. Extensor digitorum longus muscles were surgically removed from the tendons and digested in Dulbecco s modified Eagle s medium (DMEM) containing 0.2% (w/v) collagenase type I in fibre isolation medium (DMEM with 2% L-glutamine and 1% penicillin streptomycin), in order to obtain intact single myofibres. Single myofibres were mounted using fluorescent mounting medium

3 Exp Physiol 97.1 pp Myostatin and exercise 127 (Dako Cytomation, Ely, UK) containing 2.5 μgml 1 4,6-diamidino-2-phenylindole (DAPI) for nuclear visualization. Immunohistochemistry and histology Serialtransversemid-belly 10-μm-thick cryosections were obtained from EDL and rectus femoris muscles in intervals of 80 μm. Only the mid-belly of each muscle was used in this study, corresponding to one-third of the total muscle bulk. Frozen muscle sections were processed for fibre typing by immunohistochemical staining of myosin heavy chain (MHC) type I, IIA, IIX and IIB using the mouse monoclonal antibodies A4.840, A4.74, 6H1 and BF- F3, respectively (Developmental Studies Hybridoma Bank, Iowa City, IA, USA). Immunohistochemical staining for CD31 was performed using a rat anti-mouse monoclonal antibody (MCA2388; AbD serotec, Raleigh, NC, USA). Laminin staining was performed using a rabbit polyclonal antibody (L9393; Sigma). All primary antibodies were visualized via suitable AlexaFluor secondary antibodies from Molecular Probes (Grand Island, NY, USA). Muscle fibre oxidative capacity was determined on transverse sections by quantifying the staining intensity of succinate dehydrogenase (SDH). Briefly, samples were incubated at 37 C in darkness for 20 min in a medium containing 37 mm sodium phosphate buffer, 74 mm sodium succinate and 0.4mM tetranitroblue tetrazolium. Digital image analysis and morphometrics Fluorescent stained sections were examined using a Zeiss Axioimager fluorescence microscope, and images were captured using an Axiocam digital camera as described previously (Matsakas et al. 2010a). In brief, quantification of fibre number, fibre type and CSA was performed using Zeiss Axiovision software version 4.8 (Welwyn Garden City, UK) while blinded for treatment. The total EDL fibre number per animal was considered for quantifying fibre type and CSA from three sections per animal. Five hundred fibres per muscle per animal were considered for quantifying SDH-positive fibres. The SDH staining intensity of muscle transverse sections was analysed densitometrically with the public domain NIH ImageJ program. Tissue sections were digitized as grey level images, and each pixel on the computer was quantified as one of 256 grey levels upon correcting for background staining. Cell analysis for DAPI-stained myonuclei was carried out on single myofibres by live fluorescent imaging manual quantification by two independent individuals. Muscle physiology Extensor digitorum longus muscles were stimulated directly by an electric field generated between two silver electrodes placed longitudinally on either side of the muscle using an isolated constant-voltage stimulator (S48; Grass Telefactor, Slough, UK). Square-wave pulses of 2 ms duration and supramaximal stimulus intensities were used. The muscle length was adjusted to determine the optimal muscle length that produced maximal twitch tension. Tetanic contractions were elicited by stimulus trains of 500 ms duration at 10, 20, 50, 100 and 200 Hz. The EDL muscle twitch (in millinewtons), maximal tetanic force (in millinewtons) and specific force (in newtons per gram) were determined as described previously (Amthor et al. 2007; Matsakas et al. 2009). mrna transcript levels Extensor digitorum longus, gastrocnemius and rectus femoris muscles were used for studying mrna expression. Total RNA was prepared from skeletal muscle specimens using the Qiagen RNeasy Mini Kit. For EDL, 1.5 μg and, for gastrocnemius and rectus femoris, 5 μg of RNA were reverse transcribed to cdna with Invitrogen SuperScript II Reverse Transcriptase and analysed by quantitative real-time RT-PCR on an ABI7900 cycler, using the Applied Biosystems SYBRGreen PCR Master Mix. Primers were designed using the software Primer Express 3.0 (Applied Biosystems), and the primers are listed in Table S1. Quantification by means of a standard curve was performed for each primer. All data were normalized to cyclophilin (which had a coefficient of variance of 13.9% among all experimental groups). Statistical analysis Data arepresentedas means± SD. Significant differences among groups for dependent variables were detected by using two-way (genotype exercise) ANOVA. In the case of non-homogeneous variances (Lavene s test; P < 0.05) for a variable, ANOVA was performed using the square root of the observations. A χ 2 analysis was used for the detection of differences in myofibre CSA and SDH activity. Pearson s correlation and linear regression were used in order to examine whether the nuclear:cytoplasmic ratio correlated with force, as well as whether changes of the nuclear:cytoplasmic ratio might predict tetanic and specific force, respectively. Significant differences were considered for P Results Exercise training improved oxidative capacity of the myostatin null mouse, and metabolic gene-profiling Endurance training has been shown to induce an oxidative phenotype in muscle. We first tested whether the muscle from germ-line deletion of MSTN could respond in

4 128 A. Matsakas and others Exp Physiol 97.1 pp the normal manner to exercise. Succinate dehydrogenase activity is routinely used to distinguish between oxidative and non-oxidative fibres and is an indirect indicator of mitochondrial content. We found that EDL from MSTN +/+ mice did not show any significant increase in SDH activity following training by either swimming or wheel running. Examination of SDH staining of MSTN / EDL muscles prior to exercise training showed that they contained more pale fibres compared with MSTN +/+ muscles, in keeping with their shift towards a glycolytic phenotype. We found that both swimming and wheel running nearly doubled the number of intensely SDH-stained fibres in MSTN / muscles, whereas no such effect of exercise was seen on MSTN +/+ muscles (Fig. 1). We next determined whether the increase in the activity of the mitochondrion-associated enzyme SDH that was specifically induced in the muscle of MSTN / mice by exercise was accompanied by any change in expression of marker genes for the oxidative phenotype and mitochondrial biogenesis. The markers investigated were myoglobin, uncoupling protein- 3 (Ucp3), carnitine palmitoyltransferase 1α (Cpt1α) and pyruvate dehydrogenase kinase isozyme 4 (Pdk4). Myoglobin expression has previously been shown to be directly induced by nuclear factor of activated T cells (NFAT) following its activation by muscle contractionmediated release of Ca 2+ (Wittenberg, 2009). Gene expression of Ucp3 is an indicator of the content of mitochondria of a particular tissue (Jones et al. 2003). Expression of Cpt1α is a gauge of mitochondrial transport of long-chain fatty acids. Finally, Pdk4 was examined because it is a key regulator of glucose metabolism in the mitochondria. We found that either exercise regime induced a significant increase in the expression of Ucp3, Cpt1α and Pdk4 in the rectus femoris muscle of MSTN / compared with MSTN +/+ mice (Fig. 2). Expression of myoglobin was increased in the rectus femoris muscle of MSTN / mice only by swimming and not by wheel running (Fig. 2). These results show that both training regimes increase the expression of three of the four genes indicative of the oxidative profile of a tissue in MSTN / mice. We next determined the mechanisms that deliver the changes in the oxidative capacity of MSTN / mice by examining the expression of key regulators of this process. Recent work has shown that oestrogen-related receptor γ (ERRγ) is a key molecular mediator in the development of the oxidative phenotype of skeletal muscle (Rangwala et al. 2010; Narkar et al. 2011). The activity of ERRγ can be increased by the action of peroxisome proliferator-activated receptor γ-coactivator 1α (PGC-1α; Huss et al. 2002); PGC-1α is also capable of interacting with peroxisome proliferator-activated receptors (PPARs) to initiate gene expression. The following two members of the PPAR family are of particular interest: PPARα has been shown to be involved in fatty acid oxidation, whereas PPARδ is involved in energy uncoupling, as well as fatty acid uncoupling (Evans et al. 2004). We found a significant increase in the levels of Errγ and Pparα induced by swimming in the rectus femoris muscle of MSTN / mice compared with the same muscle from MSTN +/+ mice trained by swimming (Fig. 2A). Voluntary running increased the levels of Errγ, but decreased the expression of Pgc-1α and Pparα in the rectus femoris muscle of MSTN / mice compared with the same muscle from MSTN +/+ mice trained by running (Fig. 2B); therefore, ERRγ may play a central role in the development of an oxidative phenotype in response to training in MSTN / mice. We asked whether the normalization of the metabolic phenotype in MSTN / muscle was associated with a normalization of the contractile phenotype. We therefore determined the effect of exercise on MHC isoform expression in muscle fibres. The MSTN +/+ EDL muscle responded minimally to either training regime in terms of MHC expression. No significant fibre type changes were found in the EDL muscle of MSTN / mice with regard to either exercise regime (Table 1). Taken together, these findings indicate that exercise training remodels metabolic properties of the muscle independent of changes in MHC expression (i.e. fibre type). Exercise increases capillary density and transcript levels of angiogenic factors in the myostatin null mice Our profiling of muscles from MSTN / mice revealed an increase in oxidative characteristics after exercise. Previous evidence has indicated a decreased muscle capillary density in mice lacking myostatin (Rehfeldt et al. 2005). Thus, we next sought to determine whether exercise reconstitutes muscle capillarization in the MSTN / mouse. We observed an increase in the capillary number of the MSTN / EDL muscle, as evidenced by immunofluorescent staining for PECAM1 (CD31), a marker for endothelial cells. Normalization of thecapillarynumberperfibrenumberormusclegirth consistently showed an increase in response to wheel and swim training in the MSTN / mice by 85 and 40%, respectively (Fig. 3A). Capillary density (i.e. capillaries per fibre number) also increased by 10 13% in trained MSTN +/+ mice (both swimming and wheel running), but this failed to reach statistical significance (data not shown). The increase in capillary density seen in the MSTN / EDL muscle was accompanied by upregulated transcription of genes known to regulate angiogenesis, such as vascular endothelial growth factor isoform A-121 (Vegfa-121), Vegfa-165, Vegfb, neuropilin1(nrp1) and EphrinB2, by either of the two training regimes (Fig. 3B). The mrna levels for fibroblast growth factor 1 (Fgf1),

5 Exp Physiol 97.1 pp Myostatin and exercise 129 Figure 1. Exercise increases MSTN / mouse oxidative capacity in the extensor digitorum longus (EDL) muscle A, representative histological staining depicting succinate dehydrogenase (SDH) in EDL transverse sections. Scale bar represents 100 μm. B, both exercise regimes resulted in more oxidative myofibres in the MSTN / mouse hindlimb musculature compared with sedentary MSTN / mice. Five hundred fibres per EDL muscle from each animal were included in the analysis. P < versus MSTN +/+ sedentary (main effect of genotype); and #P < versus MSTN / sedentary (significant interaction).

6 130 A. Matsakas and others Exp Physiol 97.1 pp Figure 2. Effect of exercise on rectus femoris transcript levels of genes related to oxidative metabolism and mitochondrial biogenesis A, oxidative markers (upper panel) and transcriptional regulators (lower panel) in mice subjected to swimming. B, oxidative markers (upper panel) and transcriptional regulators (lower panel) in mice subjected to wheel running. #P 0.02 versus MSTN +/+ (ANOVA; significant interaction of genotype and exercise).

7 Exp Physiol 97.1 pp Myostatin and exercise 131 Table 1. Extensor digitorum longus (EDL) muscle fibre type percentage changes in response to a 5 week swim training or wheel running regime in MSTN +/+ and MSTN / mice Type I Type IIA Type IIX Type IIB Total EDL fibre number MSTN +/+ Sedentary 5.6 ± ± ± ± ± 112 Swimming 5.0 ± ± ± ± ± 41 Wheel 2.5 ± ± ± ± ± 91 MSTN / Sedentary 0.2 ± ± ± ± ± 97 Swimming 1.2 ± ± ± ± ± 254 Wheel 0.7 ± ± ± ± ± 162$ P 0.006, significant main effect of genotype for fibre type I, IIA, IIX, IIB and total fibre number. P = 0.04, significant main effect of exercise (swim training) on type IIX fibres and (wheel) on type I and IIA fibres. P 0.016, significantly different from MSTN +/+ sedentary (main effect of genotype). $P 0.017, significantly different from MSTN / sedentary (i.e. significant main effect of exercise). The total myofibre population of the EDL muscle from each animal was considered for the fibre type analysis. a potent mediator of angiogenesis, were significantly increased in the EDL of MSTN / mice by swimming but not by running. In line with these findings, both capillary density and transcript levels of angiogenic factors were upregulated in rectus femoris (another fast-twitch muscle) of the MSTN / mice subjected to exercise training (Fig. S1). Taken together, these data show that both training regimes restored muscle vascular supply and increased the expression of five of the six pro-angiogenic genes in the MSTN / mice. Exercise-specific effects on the hypermuscular phenotype of the myostatin null mouse We examined whether endurance training affected the myofibre size of MSTN / mice. In MSTN +/+ mice, both exercise regimes reduced CSA of EDL type IIA and IIX fibres. The type IIB fibres from MSTN +/+ mice showed a differential response to exercise; swimming decreased the size of these fibres, whereas they enlarged in response to wheel running (Fig. 4). Type IIA and IIX EDL fibres did not show CSA changes in the MSTN / mice with either exercise regime; however, both training regimes decreased the CSA of myostatin null type IIB fibres, with swimming exhibiting a more pronounced effect than wheel running (Fig. 4). As the vast majority (approximately 86%) of the total EDL fibre population are type IIB, these data indicate that exercise may attenuate the hypermuscular size of the MSTN / mouse. Changes in myofibre CSA were in line with wet muscle mass changes, leading to significantly reduced EDL, gastrocnemius and rectus femoris mass in response to swimming and reduced EDL mass in response to wheel running in the MSTN / mice (Table S2). Exercise training increased nuclear:cytoplasmic ratio in myostatin null muscle We next investigated the mechanism underpinning the myofibre size reduction detected in MSTN / mice after the training regimes. One explanation is that the fibres contain fewer myonuclei. We therefore quantified the total number of myonuclei in single muscle fibres (Fig. 5A and B).Thenumberofmyonucleipermyofibrewasunaffected in both genotypes in response to the either training regime. Estimation of the nuclear:cytoplasmic ratio showed an increase for MSTN / mice in response to both exercise regimes (Fig. 5C). Having found that the myofibres do not alter their nuclear content and yet decrease in size, we postulated that mechanisms that eliminate cellular content could explain the decrease in MSTN / muscle size brought about by the two exercise regimes. Autophagy is the major cellular route for the degradation of long-lived proteins and cytoplasmic organelles. The degree of autophagy taking place in a particular tissue can be gauged by measuring the levels of key genes regulating the process. We examined the expression of Bnip3 (inducer of autophagy; Tracy & Macleod, 2007), beclin (which controls the development of the autophagosome, the double-membraned vacuole that develops around structures targeted for breakdown), LC3 (a protein that through its ability to undergo lipidation with phosphatidylethanolamine controls the development of the membrane component of the autophagosome), ATF4 (a cysteine protease responsible for cleaving LC3 at its C-terminal arginine residue, facilitating the exposed C-terminal glycine to be conjugated to phosphatidylethanolamine; Kirisako et al. 2000) and cathepsin L (a key lyosomal cysteine endopepdidase required for the degradation of autophagosomal content; Dennemärker et al. 2010). We found a significant increase in the mrna levels of LC3 and Bnip3 in the swim-trained gastrocnemius muscle from MSTN / mice compared with swim-trained MSTN +/+ mice. A slightly different profile was revealed by the voluntary running regime, whereby a significant increase in the mrna levels of Bnip3, cathepsin L, ATF4 and beclin was found in the MSTN / mice compared with wheel-trained MSTN +/+ mice (Fig. 6). Therefore, the common denominator in terms of gene expression was the increase in Bnip3 mrna

8 132 A. Matsakas and others Exp Physiol 97.1 pp Figure 3. Effect of exercise on muscle capillary density A, representative immunofluorescent images depicting EDL muscle capillaries via CD31 staining. Scale bar represents 100 μm. Capillaries per fibre number or per unit area (20 images per group at 20 magnification) were consistently lower in the MSTN / mice in the sedentary state compared with MSTN +/+ mice and they were restored via both exercise regimes; P < compared with MSTN +/+ sedentary mice; and #P < compared with MSTN / sedentary mice. B, EDL mrna levels of known angiogenic factors Vegfa-121, Vegfa-165, Vegfb, Fgf1, Nrp1 and Ephrinb2. (ANOVA; P < 0.05 different from sedentary animals of the same genotype; and #P < 0.05 different from MSTN +/+ sedentary).

9 Exp Physiol 97.1 pp Myostatin and exercise 133 levels in the gastrocnemius of MSTN / mice following training. Exercise training improved force-generating capacity in the myostatin null animals We next aimed to examine the function of the trained MSTN / mice by measuring the contraction profile of EDL muscle. Twitch, tetanic and specific force remained unaffected in the MSTN +/+ mice in response to both exercise regimes. On the contrary, there was a significant increase in the twitch, tetanic and specific force of the MSTN / EDL muscle in response to swimming, as well as a significant increase in tetanic and specific force in response to wheel running (Fig. 7A; significant main effect Figure 4. Effect of exercise on myofibre cross-sectional area (CSA) of the EDL muscle Both exercise regimes elicited a shift towards smaller fibres (P 0.01) in the MSTN +/+ mice except for type IIB, which showed a slight hypertrophy with regard to the swim training regime (χ 2 = 140, d.f. = 13, P < 0.001). The MSTN / type IIB fibres were significantly smaller with either exercise regime (χ 2 = 813, d.f. = 15, P < and χ 2 = 212, d.f. = 14, P < for swim and wheel, respectively), with swim training resulting in greater CSA reductions. The CSA of type IIA and IIX remained unaffected. In addition, baseline comparisons between sedentary MSTN +/+ and MSTN / mice confirmed the hypermuscular phenotype of the latter (χ 2 = 576, d.f. = 15, P < 0.001). The total myofibre population of the EDL from each animal was considered for the CSA analysis.

10 134 A. Matsakas and others Exp Physiol 97.1 pp of exercise and significant interaction). These findings indicate that exercise training has beneficial effects on the function of the MSTN / skeletal muscle, restoring forcegenerating capacity to the levels of the MSTN +/+ mice. Moreover, we detected a significant correlation between the nuclear:cytoplasmic ratio and both tetanic and specific force for the MSTN / mice (Fig. 7B). Linear regression analysis indicated that the nuclear:cytoplasmic ratio may predict tetanic and specific force for the MSTN / mice (P < 0.001, respectively). Discussion In this study, we demonstrate that key phenotypic alterations of skeletal muscle that develop in constitutive myostatin knockout mice normalize through endurance exercise. The use of wheel running or swim training increased the oxidative metabolic phenotype, increased capillary density, decreased muscle fibre size and, most importantly, improved the force generation. Skeletal muscle function is related to its structure and the metabolic processes that maintain the tissue (Jones et al. 2008; Krivickas et al. 2011). Mixed muscles that are required to generate high levels of tension for short periods tend to have large muscle fibres that rely on glycolytic pathways for energy. In contrast, low-tension but sustained contracting muscles are composed of muscle fibres that rely on oxidative phosphorylation for generating ATP (Bottinelli et al. 1991; Bruce et al. 1997). Myostatin signalling acts to co-ordinate structure and metabolism by simultaneously suppressing muscle fibre enlargement Figure 5. Effect of exercise on nuclear:cytoplasmic ratio and correlation between the myonuclear domain and the tetanic and specific force in the MSTN / mice A, representative immunofluorescent image depicting an isolated single fibre from the EDL muscle stained with DAPI to visualize the myonuclei. Scale bar represents 50 μm. B, the total number of myonuclei per myofibre did not change in either genotype in response to swim training, but there was a significant interaction in response to wheel running, resulting in an increase for the MSTN / and decrease for the MSTN +/+ mice (P = 0.002). C, estimation of the nuclear:cytoplasmic ratio (DAPI/μm 2 ) revealed a significant upregulation for the MSTN / mice in response to both regimes, originating mainly in the reduction of myofibre CSA.

11 Exp Physiol 97.1 pp Myostatin and exercise 135 and the reliance on glycolytic metabolism. The outcome of the present study, however, shows that exercise can modulate the phenotype of muscle from animals that lack myostatin; therefore, the oxidative phenotype is not only promoted by myostatin signalling but also by exercise. Here, we show that exercise alters the metabolic profile of MSTN / muscle by increasing, hence normalizing, the oxidative phenotype. We have demonstrated that both swimming and running increased the number of myofibres with high SDH activity in MSTN / mice but not in MSTN +/+ animals. One possible explanation for this finding is that a certain population of fibres in both muscles have a low threshold to upregulate SDH activity.duringnormaldevelopment,activin-receptoriib signalling, initiated by myostatin, is required to induce SDH activity, and myostatin deletion results in a smaller number of SDH-positive fibres. We suggest that exercise activates alternative pathways to initiate SDH expression in the low-threshold fibres in MSTN / mice. The dramatic increase in SDH profile of the MSTN / muscle following exercise was not accompanied by substantial changes in MHC isoform expression. Changes in SDH activity reflect an increase in expression of the gene. For MHC profiling, however, we need to take into account that the pre-existing MHC prior to training must be replaced by any newly formed isomer. In fact, it has been shown that SDH expression can be upregulated very rapidly, within a few days following exercise, with a short enzyme half-life (McPhail & Cunningham, 1975; Suzuki et al. 2001); however, turnover rates for MHC are in the order of many weeks (Papageorgopoulos et al. 2002). The gene-profiling analysis gives an insight into the mechanism that regulates the oxidative phenotype induced by exercise in the MSTN / mice. We show Figure 6. Effect of exercise on muscle gene expression A, swim training-related mrna changes. P 0.01 versus MSTN +/+ (ANOVA; significant interaction of genotype and exercise). B, wheel running increased the mrna levels of the vast majority of autophagy-related genes in the gastrocnemius of MSTN / mice. P < versus MSTN +/+ (ANOVA; significant interaction of genotype and exercise).

12 136 A. Matsakas and others Exp Physiol 97.1 pp that of the panel of markers investigated, only one gene, Errγ, was significantly induced by both training regimes in MSTN / mice. Errγ is a member of the oestrogen-related receptor family and has been shown to be upregulated by exercise. Overexpression of this gene promotes SDH activity, mitochondrial enlargement, angiogenesis and slow MHC expression (Rangwala et al. 2010). Important from this study is the finding that the receptor is constitutively active in the absence of a ligand (Greschik et al. 2002), and the oxidative phenotype can be stimulated without massive increase in ligand (including PGC-1α) concentration (Willy et al. 2004). The robust increase in SDH-positive fibres in response to both regimes is not in line with the magnitude of the oxidative gene expression response, which was lower in MSTN / mice subjected to wheel exercise. One possibility is that it might reflect the use of two different muscle beds (EDL and rectus femoris) with similar fibre type composition, but differing recruitment patterns during swimming or running as based on tissue-specific blood flow patterns Figure 7. Effect of exercise on tetanic and specific force of the EDL muscle A, twitch, tetanic and specific force was unaffected in the MSTN +/+ mice in response to either the swim training or the wheel running regime. On the contrary, there was a significant main effect of exercise for tetanic and specific force in the MSTN / mice, followed by a significant interaction in response to exercise (both swimming and wheel running). B, a positive correlation was apparent between the nuclear:cytoplasmic ratio and both tetanic and specific force for the MSTN / mice (P < 0.01). Linear regression analysis revealed that nuclear:cytoplasmic ratios may predict (P < 0.001) both tetanic and specific force in the MSTN / mice.

13 Exp Physiol 97.1 pp Myostatin and exercise 137 (e.g. Laughlin et al. 1984). Alternatively, gene expression profiles may be sensitive to the specific exercise protocol used (e.g. LeMoine et al. 2010). Myostatin deficiency is known to reduce muscle capillary density (Rehfeldt et al. 2005), whereas exercise increases capillary density (i.e. capillaries per myofibre number; e.g. Waters et al. 2004). Here, we confirm that the MSTN / mouse has decreased capillary density in the sedentary state, which can, however, be increased following either exercise regime. The increases in muscle capillary density (i.e. capillaries per myofibre number) in the trained MSTN / animals cannot be attributed solely to the observed CSA loss, because the number of fibres counted for this purpose was not significantly different among groups, although trained MSTN / mice had consistently slightly lower values (data in Fig. S1 for rectus femoris). In line with these morphological observations, Vegf and Errγ genes have been recognized as potent angiogenic factors (e.g. Olfert et al. 2010; Narkar et al. 2011). In particular, Errγ is a recently recognized transcriptional regulator that determines intrinsic vascular features and oxidative metabolism in the skeletal muscle, in part via the induction of Vegfa (Arany et al. 2008; Narkaret al. 2011). Elevated transcript levels of these factors suggest a beneficial effect on the angiogenic programme of the MSTN / mice after exercise, which was further justified by the upregulation of Nrp1 (a cell surface coreceptor for VEGF; Neufeld et al. 1999) and EphrinB2 (a selective marker for arterial vessels and neovascularization during remodelling of adult microvessels; Galeetal. 2001). Intriguingly, Fgf1 (a growth factor known to promote endothelial cell proliferation; Friesel & Maciaq, 1995) was only significantly changed through swim training and not wheel running. The observed muscle remodelling at the vascular level may have a beneficial effect in nutrient and oxygen delivery affecting muscle metabolism. These results also highlight the rapid nature of the muscle angiogenic programme, as the capillary density was improved by exercise in 5 weeks, in contrast to the slower rate of MHC isoform remodelling. Both exercise regimes revealed significant increases in tetanic and specific force in the EDL muscle of the MSTN / mice. In particular, tetanic force was found to be 150% higher in the MSTN / mice with regard to both regimes, reaching the levels of sedentary MSTN +/+ mice. Accordingly, specific force showed increases of approximately 150% in the MSTN / mice in response to either regime. Several studies have associated the forcegenerating capacity of a muscle with the maintenance of the nuclear:cytoplasmic ratio (Roy et al. 1999; Amthor et al. 2009). Previous evidence has indicated that despite increased muscle size in the MSTN / mouse, the number of myonuclei per myofibre is not proportionally enhanced, leading to a reduced nuclear:cytoplasmic ratio (or enhanced myonuclear domain, i.e. fibre area per myonucleus; Amthor et al. 2009). Here, we report a significant increase of the nuclear:cytoplasmic ratio (i.e. reduction of the myonuclear domain) in the MSTN / mice in response to exercise, which correlates with improvements of tetanic and specific force. Indirect evidence on the relationship between muscle force and nuclear:cytoplasmic ratio has also been previously demonstrated using the hypoplastic muscle model of the Meox2 / mouse, where a reduction in myonuclear domain size (i.e. enhanced nuclear:cytoplasmic ratio) was followed by an increase in specific force (Otto et al. 2010). It seems, thus, that the exercise training-induced myofibre CSA loss was able to normalize the myonuclear domain of the MSTN / mouse and restore muscle function. In addition, from a biomechanical point of view, a pennate muscle generates less force than a parallel-fibred muscle of comparable size (Degens et al. 2009). It can be speculated thattheobservedlossinmyofibrecsainthemstn / mice might have modulated the pennation angle of the fibres and thus affected the muscle force, although this has to be established experimentally. The observed myofibre CSA loss could be delivered through autophagy. It seems that autophagy, a conserved homeostatic process that carries out degradation of damaged organelles or other cytoplasmic components and allows for cell survival (Mizushima et al. 2008; Sandri, 2010), is downregulated in the myostatin null mouse in the sedentary state. Thus, autophagy is reduced in sedentary MSTN / mice, which could explain unfavourable accumulation of material and organelles, such as the previously observed tubular aggregates, and thereby profoundly hamper muscle contractility (Amthor et al. 2007). Interestingly, although both training regimes caused a decrease in muscle mass, we found that Bnip3 alone from the panel of autophagic markers was induced by both forms of exercise in the MSTN / mice. Collectively, we found that both forms of exercise induced many similar changes in the muscle of MSTN / mice when compared with tissue from sedentary animals of the same genotype. (e.g. increase in number of SDHpositive fibres, increase in capillary density and the normalization of specific force); however, the degree of change in muscle phenotype induced by the two training regimes was not always the same. For example, of the two training regimes, swimming induced the greatest decrease in type IIB fibre CSA. In contrast, wheel running caused a greater increase in capillary density than swim training. These results imply that the differing forms of exercise may have both a qualitative and a quantitative effect on muscle phenotype. Furthermore, our results intimate that there is a large degree of phenotypic plasticity between different parameters, in that a large change in one feature is not necessarily accompanied by equal changes in other traits. At present, we do not fully understand the molecular mechanisms that regulate each parameter,

14 138 A. Matsakas and others Exp Physiol 97.1 pp but the present study indicates that they are extremely sensitive and are able to distinguish between two forms of training. One possibility is that gene expression profiles may vary according to the exercise protocol used, resulting in sensitive temporal and spatial gene expression patterns, as reported for other experimental models (e.g. LeMoine et al. 2010). However, we note that despite these variations in cellular phenotype, the two forms of training delivered the same improvement in muscle function (e.g. force). Importantly, these results highlight the prerequisite to investigate a host of features in order to establish muscle phenotype. In conclusion, endurance exercise training appears to significantly modify the skeletal muscle phenotype of the myostatin-deficient mouse, improving its forcegenerating capacity. 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