Voluntary Physical Activity Protects from Susceptibility to Skeletal Muscle Contraction–Induced Injury But Worsens Heart Function in mdx Mice
2013; Elsevier BV; Volume: 182; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2013.01.020
ISSN1525-2191
AutoresChristophe Hourdé, Pierre Joanne, Fadia Medja, Nathalie Mougenot, Adeline Jacquet, Étienne Mouisel, Alice Pannérec, Stéphane N. Hatem, Gillian Butler‐Browne, Onnik Agbulut, Arnaud Ferry,
Tópico(s)Adipose Tissue and Metabolism
ResumoIt is well known that inactivity/activity influences skeletal muscle physiological characteristics. However, the effects of inactivity/activity on muscle weakness and increased susceptibility to muscle contraction–induced injury have not been extensively studied in mdx mice, a murine model of Duchenne muscular dystrophy with dystrophin deficiency. In the present study, we demonstrate that inactivity (ie, leg immobilization) worsened the muscle weakness and the susceptibility to contraction-induced injury in mdx mice. Inactivity also mimicked these two dystrophic features in wild-type mice. In contrast, we demonstrate that these parameters can be improved by activity (ie, voluntary wheel running) in mdx mice. Biochemical analyses indicate that the changes induced by inactivity/activity were not related to fiber-type transition but were associated with altered expression of different genes involved in fiber growth (GDF8), structure (Actg1), and calcium homeostasis (Stim1 and Jph1). However, activity reduced left ventricular function (ie, ejection and shortening fractions) in mdx, but not C57, mice. Altogether, our study suggests that muscle weakness and susceptibility to contraction-induced injury in dystrophic muscle could be attributable, at least in part, to inactivity. It also suggests that activity exerts a beneficial effect on dystrophic skeletal muscle but not on the heart. It is well known that inactivity/activity influences skeletal muscle physiological characteristics. However, the effects of inactivity/activity on muscle weakness and increased susceptibility to muscle contraction–induced injury have not been extensively studied in mdx mice, a murine model of Duchenne muscular dystrophy with dystrophin deficiency. In the present study, we demonstrate that inactivity (ie, leg immobilization) worsened the muscle weakness and the susceptibility to contraction-induced injury in mdx mice. Inactivity also mimicked these two dystrophic features in wild-type mice. In contrast, we demonstrate that these parameters can be improved by activity (ie, voluntary wheel running) in mdx mice. Biochemical analyses indicate that the changes induced by inactivity/activity were not related to fiber-type transition but were associated with altered expression of different genes involved in fiber growth (GDF8), structure (Actg1), and calcium homeostasis (Stim1 and Jph1). However, activity reduced left ventricular function (ie, ejection and shortening fractions) in mdx, but not C57, mice. Altogether, our study suggests that muscle weakness and susceptibility to contraction-induced injury in dystrophic muscle could be attributable, at least in part, to inactivity. It also suggests that activity exerts a beneficial effect on dystrophic skeletal muscle but not on the heart. Duchenne muscular dystrophy is a degenerative disorder affecting both skeletal and cardiac muscles. It is caused by deficiency of dystrophin, a subsarcolemmal protein, which is thought to play a role in force transmission, sarcolemma stability, localization, and function of different proteins that trigger the damage process in its absence.1Allen D.G. Gervasio O.L. Yeung E.W. Whitehead N.P. Calcium and the damage pathways in muscular dystrophy.Can J Physiol Pharmacol. 2010; 88: 83-91Crossref PubMed Scopus (135) Google Scholar, 2Chan S. Head S.I. The role of branched fibres in the pathogenesis of Duchenne muscular dystrophy.Exp Physiol. 2011; 96: 564-571Crossref PubMed Scopus (52) Google Scholar, 3Gumerson J.D. Michele D.E. The dystrophin-glycoprotein complex in the prevention of muscle damage.J Biomed Biotechnol. 2011; 2011: 210797Crossref PubMed Scopus (82) Google Scholar, 4Lynch G.S. Role of contraction-induced injury in the mechanisms of muscle damage in muscular dystrophy.Clin Exp Pharmacol Physiol. 2004; 31: 557-561Crossref PubMed Scopus (39) Google Scholar The skeletal muscle from mdx mice, a widely used mouse model for Duchenne muscular dystrophy, exhibits a muscle weakness (ie, reduced specific maximal force: absolute maximal force generated relative to muscle cross-sectional area or weight).5Dellorusso C. Crawford R.W. Chamberlain J.S. Brooks S.V. Tibialis anterior muscles in mdx mice are highly susceptible to contraction-induced injury.J Muscle Res Cell Motil. 2001; 22: 467-475Crossref PubMed Scopus (174) Google Scholar, 6Lynch G.S. Hinkle R.T. Chamberlain J.S. Brooks S.V. Faulkner J.A. Force and power output of fast and slow skeletal muscles from mdx mice 6-28 months old.J Physiol. 2001; 535: 591-600Crossref PubMed Scopus (254) Google Scholar, 7Hayes A. Williams D.A. Contractile function and low-intensity exercise effects of old dystrophic (mdx) mice.Am J Physiol. 1998; 274: C1138-C1144PubMed Google Scholar, 8Pastoret C. Sebille A. Time course study of the isometric contractile properties of mdx mouse striated muscles.J Muscle Res Cell Motil. 1993; 14: 423-431Crossref PubMed Scopus (35) Google Scholar Mdx muscle is also more fragile [ie, susceptible to damage caused by lengthening (eccentric) contraction (contraction with passive stretch)]. Lengthening contractions cause a marked reduction in force generation in fast-type muscles, but not slow-type muscles, in mdx mice.9Head S.I. Williams D.A. Stephenson D.G. Abnormalities in structure and function of limb skeletal muscle fibres of dystrophic mdx mice.Proc Biol Sci. 1992; 248: 163-169Crossref PubMed Scopus (116) Google Scholar, 10Moens P. Baatsen P.H. Marechal G. Increased susceptibility of EDL muscles from mdx mice to damage induced by contractions with stretch.J Muscle Res Cell Motil. 1993; 14: 446-451Crossref PubMed Scopus (272) Google Scholar Not surprisingly, dystrophin deficiency deteriorates voluntary physical activity in mdx mice. Several studies have reported that voluntary wheel running is reduced in mdx mice compared with wild-type mice.11Dupont-Versteegden E.E. McCarter R.J. Katz M.S. Voluntary exercise decreases progression of muscular dystrophy in diaphragm of mdx mice.J Appl Physiol. 1994; 77: 1736-1741PubMed Google Scholar, 12Carter G.T. Wineinger M.A. Walsh S.A. Horasek S.J. Abresch R.T. Fowler Jr., W.M. Effect of voluntary wheel-running exercise on muscles of the mdx mouse.Neuromuscul Disord. 1995; 5: 323-332Abstract Full Text PDF PubMed Scopus (77) Google Scholar Because it is well established that activity plays an important role in skeletal muscle physiological characteristics,13Pette D. Vrbova G. What does chronic electrical stimulation teach us about muscle plasticity?.Muscle Nerve. 1999; 22: 666-677Crossref PubMed Scopus (208) Google Scholar, 14Harridge S.D. Plasticity of human skeletal muscle: gene expression to in vivo function.Exp Physiol. 2007; 92: 783-797Crossref PubMed Scopus (129) Google Scholar, 15Fitts R.H. Riley D.R. Widrick J.J. Physiology of a microgravity environment invited review: microgravity and skeletal muscle.J Appl Physiol. 2000; 89: 823-839PubMed Google Scholar it is possible that inactivity could contribute to muscle weakness and the higher susceptibility to contraction-induced injury observed in mdx mice. Moreover, it could be expected that activity would mitigate these two dystrophic features. These assumptions are based on the facts that inactivity and activity can reduce and increase maximal specific force of both dystrophic and healthy muscles,16Hayes A. Williams D.A. Beneficial effects of voluntary wheel running on the properties of dystrophic mouse muscle.J Appl Physiol. 1996; 80: 670-679Crossref PubMed Scopus (102) Google Scholar, 17Salazar J.J. Michele D.E. Brooks S.V. Inhibition of calpain prevents muscle weakness and disruption of sarcomere structure during hindlimb suspension.J Appl Physiol. 2010; 108: 120-127Crossref PubMed Scopus (46) Google Scholar, 18Call J.A. Voelker K.A. Wolff A.V. McMillan R.P. Evans N.P. Hulver M.W. Talmadge R.J. Grange R.W. Endurance capacity in maturing mdx mice is markedly enhanced by combined voluntary wheel running and green tea extract.J Appl Physiol. 2008; 105: 923-932Crossref PubMed Scopus (80) Google Scholar, 19Litvinova K.S. Shenkman B.S. Influence of hindlimb suspension on calcium-induced contraction characteristics in dystrophin-deficient animals.J Gravit Physiol. 2007; 14: P91-P92PubMed Google Scholar respectively, whereas inactivity and activity can result in higher and lower susceptibility to contraction-induced injury, respectively, at least in healthy muscles.17Salazar J.J. Michele D.E. Brooks S.V. Inhibition of calpain prevents muscle weakness and disruption of sarcomere structure during hindlimb suspension.J Appl Physiol. 2010; 108: 120-127Crossref PubMed Scopus (46) Google Scholar, 20Gosselin L.E. Attenuation of force deficit after lengthening contractions in soleus muscle from trained rats.J Appl Physiol. 2000; 88: 1254-1258PubMed Google Scholar, 21Warren G.L. Hayes D.A. Lowe D.A. Williams J.H. Armstrong R.B. Eccentric contraction-induced injury in normal and hindlimb-suspended mouse soleus and EDL muscles.J Appl Physiol. 1994; 77: 1421-1430PubMed Google Scholar The purpose of this study is to better understand the contribution of inactivity to muscle weakness and the higher susceptibility to contraction-induced injury observed in dystrophic mdx skeletal muscle. To our knowledge, the role of inactivity in the context of muscular dystrophy has not yet been studied. Our general hypothesis is that muscle weakness and susceptibility to contraction-induced injury could be attributable, at least in part, to inactivity in mdx mice. The first corollary to our primary hypothesis is that muscle weakness and susceptibility to contraction-induced injury would be worsened by inactivity in mdx mice. A second corollary is that inactivity, at least in part, would mimic these two dystrophic features in wild-type mice. A third corollary is that muscle weakness and susceptibility to contraction-induced injury would be improved by activity (voluntary wheel running) in mdx mice. Our results reveal that inactivity and activity have detrimental and beneficial effects, respectively, on muscle weakness and susceptibility to contraction-induced injury in dystrophic muscle. All procedures were performed in accordance with national and European legislations. Female mdx mice (mdx, C57BL/10ScSc-DMDmdx/J) and age-matched wild-type female control mice (C57) were used. Mice were randomly divided into different control and experimental groups. Two groups of inactive mdx (mdx + staple) and C57 (C57 + staple) mice at 5 months of age were formed, in which one hind limb was immobilized for 2 weeks by stapling the foot.22Caron A.Z. Drouin G. Desrosiers J. Trensz F. Grenier G. A novel hindlimb immobilization procedure for studying skeletal muscle atrophy and recovery in mouse.J Appl Physiol. 2009; 106: 2049-2059Crossref PubMed Scopus (80) Google Scholar A group of active mdx (mdx + wheel) mice at 4 to 5 weeks of age were placed in separate cages containing a wheel and were allowed to run 4 to 4.5 months ad libitum. At the age of 5.5 months, these mdx and C57 mice were studied. In a second series of experiments, we analyzed the effect of activity in female C57 mice. For this, active mice, aged 4 weeks, were placed in cages with a wheel and studied 3 months later, at the age of 4 months. Muscle weakness and susceptibility to contraction-induced injury were evaluated by measuring the in situ tibialis anterior (TA) muscle contraction in response to nerve stimulation, as previously described.23Koo T. Malerba A. Athanasopoulos T. Trollet C. Boldrin L. Ferry A. Popplewell L. Foster H. Foster K. Dickson G. Delivery of AAV2/9-microdystrophin genes incorporating helix 1 of the coiled-coil motif in the C-terminal domain of dystrophin improves muscle pathology and restores the level of alpha1-syntrophin and alpha-dystrobrevin in skeletal muscles of mdx mice.Hum Gene Ther. 2011; 22: 1379-1388Crossref PubMed Scopus (47) Google Scholar, 24Hoogaars W.M. Mouisel E. Pasternack A. Hulmi J.J. Relizani K. Schuelke M. Schirwis E. Garcia L. Ritvos O. Ferry A. 't Hoen P.A. Amthor H. Combined effect of AAV-U7-induced dystrophin exon skipping and soluble activin type IIB receptor in mdx mice.Hum Gene Ther. 2012; 23: 1269-1279Crossref PubMed Scopus (31) Google Scholar, 25Mouisel E. Vignaud A. Hourde C. Butler-Browne G. Ferry A. Muscle weakness and atrophy are associated with decreased regenerative capacity and changes in mTOR signaling in skeletal muscles of venerable (18-24-month-old) dystrophic mdx mice.Muscle Nerve. 2010; 41: 809-818Crossref PubMed Scopus (41) Google Scholar Mice were anesthetized using pentobarbital (60 mg/kg i.p.). Body temperature was maintained at 37°C using radiant heat. The knee and foot were fixed with pins and clamps, and the distal tendon of the muscle was attached to a lever arm of a servomotor system (model 305B, Dual-Mode Lever; Aurora Scientific, Aurora, ON, Canada) using a silk ligature. The sciatic nerve was proximally crushed and distally stimulated by a bipolar silver electrode using supramaximal square wave pulses of 0.1-millisecond duration. We measured the absolute maximal force that was generated during isometric contractions in response to electrical stimulation (frequency, 75 to 150 Hz; train of stimulation, 500 milliseconds). Absolute maximal force was determined at the length at which maximal tension was obtained during the tetanus. Absolute maximal force was normalized to the muscle mass as an estimate of specific maximal force. Susceptibility to contraction-induced injury was estimated from the force decrease resulting from lengthening contraction-induced injury. The sciatic nerve was stimulated for 700 milliseconds (frequency, 150 Hz). A maximal isometric contraction of the TA muscle was initiated during the first 500 milliseconds. Then, muscle lengthening (10% initial length) at a velocity of 5.5 mm/second (0.85 fiber lengths per second) was imposed during the last 200 milliseconds. All isometric contractions were made at an initial length. Nine lengthening contractions of the TA muscles were performed, each separated by a 60-second rest period. Maximal isometric force was measured 1 minute after each lengthening contraction and expressed as a percentage of the initial maximal force. After contractile measurements, the animals were euthanized with an overdose of pentobarbital, and muscles and hearts were weighed. Echocardiography was performed on lightly anesthetized mice under isoflurane (induction with 2% isoflurane and 100% O2, and maintained with 0.5% to 100% O2). Noninvasive measurements of left ventricular dimensions were evaluated using echocardiography-Doppler (Vivid 7 Dimension/Vivid7 PRO; GE Medical System Co, Vélizy, France) with a probe emitting ultrasounds from 9- to 14-MHz frequency. The two-dimensionally guided Time Motion mode recording (parasternal long-axis view) of the left ventricle (LV) provided the following measurements: diastolic and systolic septal and posterior wall thicknesses, internal end-diastolic diameter (LVEDD) and end-systolic diameter (LVESD), and heart rate. Each set of measurements was obtained from the same cardiac cycle. At least three sets of measurements were obtained from three different cardiac cycles. Fractional shortening was calculated by the following formula: [(LVEDD − LVESD)/LVEDD] × 100. LV myocardial volume, LV end-diastolic volume (EDV), and end-systolic volume (ESV) were calculated using a half-ellipsoid model of the LV. From these volumes, LV ejection fraction was calculated by the following formula: [(EDV − ESV)/EDV] × 100. Heart rates during measurements were 537.3 ± 12.8 (C57), 559.1 ± 14.9 (mdx), and 519.3 ± 18.1 (mdx + wheel) beats per minute (P > 0.05). Transverse serial sections (8 μm thick) of the TA muscles were obtained using a cryostat, in the midbelly region. Some of the sections were processed for histological analysis according to standard protocols (stained for H&E, Sirius red, and DAPI). Others were used for immunohistochemistry, as previously described.26Trollet C. Anvar S.Y. Venema A. Hargreaves I.P. Foster K. Vignaud A. Ferry A. Negroni E. Hourde C. Baraibar M.A. 't Hoen P.A. Davies J.E. Rubinsztein D.C. Heales S.J. Mouly V. van der Maarel S.M. Butler-Browne G. Raz V. Dickson G. Molecular and phenotypic characterization of a mouse model of oculopharyngeal muscular dystrophy reveals severe muscular atrophy restricted to fast glycolytic fibres.Hum Mol Genet. 2010; 19: 2191-2207Crossref PubMed Scopus (65) Google Scholar, 27Agbulut O. Vignaud A. Hourde C. Mouisel E. Fougerousse F. Butler-Browne G.S. Ferry A. Slow myosin heavy chain expression in the absence of muscle activity.Am J Physiol Cell Physiol. 2009; 296: C205-C214Crossref PubMed Scopus (31) Google Scholar, 28Joanne P. Hourde C. Ochala J. Cauderan Y. Medja F. Vignaud A. Mouisel E. Hadj-Said W. Arandel L. Garcia L. Goyenvalle A. Mounier R. Zibroba D. Sakamato K. Butler-Browne G. Agbulut O. Ferry A. Impaired adaptive response to mechanical overloading in dystrophic skeletal muscle.PLoS One. 2012; 7: e35346Crossref PubMed Scopus (22) Google Scholar In brief, frozen unfixed sections were blocked for 1 hour in phosphate buffered saline (PBS) plus 2% bovine serum albumin and 2% sheep serum, and incubated for 30 minutes with mouse Fab 1:100 in PBS. Sections were then incubated overnight with primary antibodies against neonatal myosin heavy chain (MHC-nn). After washes in PBS, sections were incubated for 1 hour with secondary antibodies. After washes in PBS, slides were finally mounted in Fluoromont (Southern Biotech, Nanterre, France). To evaluate the amount of fibrosis, hearts were fixed in 4% buffered formalin, dehydrated in ethanol and acetone, impregnated with methyl salicylate, and then embedded in paraffin. Transverse sections (6 μm thick) were cut, mounted on a slide, and stained with Sirius red, which identifies collagen. We measured the cross-sectional area occupied by Sirius red–stained interstitial tissue. Images were captured using a digital camera. The muscles were extracted on ice for 60 minutes in four volumes of extracting buffer (pH 6.5), as previously described.29Butler-Browne G.S. Whalen R.G. Myosin isozyme transitions occurring during the postnatal development of the rat soleus muscle.Dev Biol. 1984; 102: 324-334Crossref PubMed Scopus (264) Google Scholar After centrifugation, the supernatants were diluted 1:1 (v/v) with glycerol and stored at −20°C. MHC isoforms were separated on 8% polyacrylamide gels, which were made in the Bio-Rad (Marnes-la-Coquette, France) mini-Protean II Dual slab cell system, as previously described.30Agbulut O. Noirez P. Beaumont F. Butler-Browne G. Myosin heavy chain isoforms in postnatal muscle development of mice.Biol Cell. 2003; 95: 399-406Crossref PubMed Scopus (199) Google Scholar The gels were migrated for 31 hours at 72 V (constant voltage) at 4°C. After migration, the gels were silver stained. The positions of the different MHC bands were confirmed by using Western blot analysis using antibodies directed against different MHC isoforms. The gels were scanned using a video acquisition system, and bands were quantified by densitometric software (Multi Gauge; Fujifilm, Clichy, France). Total RNA was extracted from TA muscle using TRIzol (Life Technologies, Saint-Aubin, France) and Lysing Matrix D tubes (MP Biomedicals, Illkirch, France) following the manufacturer's instructions. From 750 ng of extracted RNA, the first-strand cDNA was then synthesized using the SuperScript III First-Strand kit (Invitrogen–Life Technologies) with random hexamer primer and according to the manufacturer's instructions. Relative quantification PCR analysis was then performed with green intercalating dye PCR technology using the LightCycler 1536 Real-Time PCR System (Roche Diagnostics, Meylan, France) on the platform Génotypage et Séquençage (Centre de Recherche de l'Institut du Cerveau et de la Moelle épinière–Université Pierre et Marie Curie/INSERM UMRS975/Centre National de la Recherche Scientifique UMR7225, Paris, France). The reaction was performed in duplicate for each sample in a 2-μL reaction volume made up of 1 μL of LightCycler 1536 DNA Green Master (Roche Applied Science) containing 500 nmol/L each of the forward and reverse primer and 1 μL of diluted (1:25) cDNA. The 1536-well plates were handled using Bravo Automated Liquid Handling Platform (Agilent Technologies, Massy, France) and sealed using a PlateLoc Thermal Microplate Sealer (Agilent Technologies). The thermal profile for BrightGreen Dye real-time quantitative PCR (qPCR) was 95°C for 1 minute, followed by 50 cycles at 95°C for 2 seconds and 60°C for 30 seconds. Primer sequences used in this study are available on request. The gene expressions of HPRT and P0 were used as the reference transcripts. Groups were statistically compared using variance analysis. If necessary, a subsequent Bonferroni post hoc test was also performed. For groups that did not pass tests of normality and equal variance, nonparametric tests were used (Kruskal-Wallis and Wilcoxon). Values are given as means ± SEM. By analyzing in situ TA muscle force production in response to nerve stimulation, we confirmed the important impact of the absence of dystrophin in mdx mice. The specific maximal force was decreased by 18% in mdx mice compared with C57 mice (P < 0.05) (Figure 1A). Inactivity that was induced by 2 weeks' stapling worsened specific maximal force (−24%) in mdx + staple compared with mdx (P < 0.05) (Figure 1A). Similarly, inactivity decreased specific maximal force in C57 + staple (−21%) mice compared with C57 mice (P < 0.05) (Figure 1A). To determine whether this result might be attributed to a reduction in the removal of damaged organelles and misfolded proteins (ie, impaired autophagy31Grumati P. Coletto L. Sabatelli P. Cescon M. Angelin A. Bertaggia E. Blaauw B. Urciuolo A. Tiepolo T. Merlini L. Maraldi N.M. Bernardi P. Sandri M. Bonaldo P. Autophagy is defective in collagen VI muscular dystrophies, and its reactivation rescues myofiber degeneration.Nat Med. 2011; 16: 1313-1320Crossref Scopus (423) Google Scholar and protein proteolysis via the ubiquitin proteasome system),32Mendias C.L. Kayupov E. Bradley J.R. Brooks S.V. Claflin D.R. Decreased specific force and power production of muscle fibers from myostatin-deficient mice are associated with a suppression of protein degradation.J Appl Physiol. 2011; 111: 185-191Crossref PubMed Scopus (57) Google Scholar Bnip3, LC3, and atrogin-1 gene expressions were analyzed by qPCR. We found that inactivity did not repress these genes in mdx + staple mice and C57 + staple mice (P > 0.05) (Figure 1B). Absolute maximal force did not differ between mdx and C57 mice (P > 0.05) (Figure 2A). Inactivity reduced absolute maximal force in both strains (P < 0.05), but the reduction was lower for mdx + staple mice (−24%) than C57 + staple mice (−45%) (P < 0.05). This can be explained by the fact that, in contrast to the C57 + staple group, muscle weight was not reduced by inactivity in mdx + staple mice (P > 0.05) (Figure 2, B and C). The absence of atrophy in the mdx + staple mice was not caused by a strain difference in the effect of inactivity on the expression of genes controlling either autophagy and proteolysis (Figure 1B) or muscle growth (GDF8, Fst, REDD1, and REDD233McPherron A.C. Lawler A.M. Lee S.J. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member.Nature. 1997; 387: 83-90Crossref PubMed Scopus (3303) Google Scholar, 34Lee S.J. McPherron A.C. Regulation of myostatin activity and muscle growth.Proc Natl Acad Sci U S A. 2001; 98: 9306-9311Crossref PubMed Scopus (1312) Google Scholar, 35Miyazaki M. Esser K.A. Cellular mechanisms regulating protein synthesis and skeletal muscle hypertrophy in animals.J Appl Physiol. 2009; 106: 1367-1373Crossref PubMed Scopus (146) Google Scholar) (Figure 2D). GDF8 gene expression increased in both mdx + staple (97%) and C57 + staple (59%) mice. The immediate maximal force decrease after lengthening contractions is a widely used measure of the magnitude of muscle damage caused by contraction. There was a force decrease after the third (36%), sixth (60%), and ninth (72%) lengthening contraction in mdx mice, but not in C57 mice (P < 0.05) (Figure 3A), indicating a susceptibility to contraction-induced injury in mdx mice. Inactivity increased the force decrease after the third (46%) and sixth (75%) lengthening contraction in mdx + staple compared with mdx mice (P < 0.05) but had no effect in C57 + staple mice (P > 0.05) (Figure 3A). The susceptibility to contraction-induced injury in mdx + staple mice was not related to an increase in strain (force enhancement during stretch) induced by inactivity (Figure 3B). By using qPCR, we first analyzed the expression of some gene markers of fiber type (encoding the following proteins: MHC, nuclear factor for activated T cell, peroxisome proliferator-activated receptor-γ coactivator, Sirt1, and type 2 sarco/endoplasmic reticulum Ca2+-ATPase) because fast/glycolytic fiber is more fragile than slow/oxidative muscle fiber.9Head S.I. Williams D.A. Stephenson D.G. Abnormalities in structure and function of limb skeletal muscle fibres of dystrophic mdx mice.Proc Biol Sci. 1992; 248: 163-169Crossref PubMed Scopus (116) Google Scholar, 10Moens P. Baatsen P.H. Marechal G. Increased susceptibility of EDL muscles from mdx mice to damage induced by contractions with stretch.J Muscle Res Cell Motil. 1993; 14: 446-451Crossref PubMed Scopus (272) Google Scholar We found that inactivity increased the expression of the gene encoding MHC-2b protein in both mdx + staple mice (71%) and C57 + staple mice (99%) (P < 0.05) (Figure 4A). By using gel electrophoretic separation of MHC protein isoforms, we accordingly found that MHC-2a protein expression was reduced (from 10.5% to 6.5%) in mdx + staple mice compared with mdx mice (P < 0.05) (Figure 4, B and C). Next, we examined the gene expression of utrophin,36Squire S. Raymackers J.M. Vandebrouck C. Potter A. Tinsley J. Fisher R. Gillis J.M. Davies K.E. Prevention of pathology in mdx mice by expression of utrophin: analysis using an inducible transgenic expression system.Hum Mol Genet. 2002; 11: 3333-3344Crossref PubMed Scopus (131) Google Scholar α dystroglycan (Dag137Fougerousse F. Bartoli M. Poupiot J. Arandel L. Durand M. Guerchet N. Gicquel E. Danos O. Richard I. Phenotypic correction of alpha-sarcoglycan deficiency by intra-arterial injection of a muscle-specific serotype 1 rAAV vector.Mol Ther. 2007; 15: 53-61Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), syntrophin (Snta1), dystrobrevin (Dtna23Koo T. Malerba A. Athanasopoulos T. Trollet C. Boldrin L. Ferry A. Popplewell L. Foster H. Foster K. Dickson G. Delivery of AAV2/9-microdystrophin genes incorporating helix 1 of the coiled-coil motif in the C-terminal domain of dystrophin improves muscle pathology and restores the level of alpha1-syntrophin and alpha-dystrobrevin in skeletal muscles of mdx mice.Hum Gene Ther. 2011; 22: 1379-1388Crossref PubMed Scopus (47) Google Scholar), α 7 β 1 integrin (Itga738Han R. Kanagawa M. Yoshida-Moriguchi T. Rader E.P. Ng R.A. Michele D.E. Muirhead D.E. Kunz S. Moore S.A. Iannaccone S.T. Miyake K. McNeil P.L. Mayer U. Oldstone M.B. Faulkner J.A. Campbell K.P. Basal lamina strengthens cell membrane integrity via the laminin G domain-binding motif of alpha-dystroglycan.Proc Natl Acad Sci U S A. 2009; 106: 12573-12579Crossref PubMed Scopus (121) Google Scholar), cytoplasmic g-actin (Actg139Baltgalvis K.A. Jaeger M.A. Fitzsimons D.P. Thayer S.A. Lowe D.A. Ervasti J.M. Transgenic overexpression of gamma-cytoplasmic actin protects against eccentric contraction-induced force loss in mdx mice.Skelet Muscle. 2011; 1: 32Crossref Scopus (23) Google Scholar), γ 2 actin (Actg240Seto J.T. Lek M. Quinlan K.G. Houweling P.J. Zheng X.F. Garton F. MacArthur D.G. Raftery J.M. Garvey S.M. Hauser M.A. Yang N. Head S.I. North K.N. Deficiency of alpha-actinin-3 is associated with increased susceptibility to contraction-induced damage and skeletal muscle remodeling.Hum Mol Genet. 2011; 20: 2914-2927Crossref PubMed Scopus (81) Google Scholar), and desmin,41Sam M. Shah S. Friden J. Milner D.J. Capetanaki Y. Lieber R.L. Desmin knockout muscles generate lower stress and are less vulnerable to injury compared with wild-type muscles.Am J Physiol Cell Physiol. 2000; 279: C1116-C1122PubMed Google Scholar which have been shown to be related to susceptibility to contraction-induced injury. However, we found no major change in these genes, with inactivity in both mdx + staple mice and C57 + staple mice (P < 0.05) (Supplemental Figure S1A). We also checked whether changes in gene expression of NADPH oxidase, subunit gp91phox (Cybb), NADPH oxidase, subunit gp67phox (Ncf242Whitehead N.P. Yeung E.W. Froehner S.C. Allen D.G. Skeletal muscle NADPH oxidase is increased and triggers stretch-induced damage in the mdx mouse.PLoS One. 2011; 5: e15354Crossref Scopus (139) Google Scholar), transient receptor potential canonical 1 gene (TRCP143Zhang B.T. Whitehead N.P. Gervasio O.L. Reardon T.F. Vale M. Fatkin D. Dietrich A. Yeung E.W. Allen D.G. Pathways of Ca2+ entry and cytoskeletal damage following eccentric contractions in mouse skeletal muscle.J Appl Physiol. 2012; 112: 2077-2086Crossref PubMed Scopus (52) Google Scholar), stromal interacting molecule 1 (Stim144Reutenauer-Patte J. Boittin F.X. Patthey-Vuadens O. Ruegg U.T. Dorchies O.M. Urocortins improve dystrophic skeletal muscle structure and function through both PKA- and Epac-dependent pathways.Am J Pathol. 2012; 180: 749-762Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), junctophilin 1 (Jph1), and Jph245Corona B.T. Balog E.M. Doyle J.A. Rupp J.C. Luke R.C. Ingalls C.P. Junctophilin damage contributes to early strength deficits and EC coupling failure after eccentric contractions.Am J Physiol Cell
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