research article transcriptional pathways associated with...

Research ArticleTranscriptional Pathways Associated with SkeletalMuscle Changes after Spinal Cord Injury and TreadmillLocomotor Training

Celine Baligand1 Yi-Wen Chen23 Fan Ye4 Sachchida Nand Pandey2

San-Huei Lai2 Min Liu4 and Krista Vandenborne4

1Department of Physiology and Functional Genomics University of Florida Gainesville FL 32610 USA2Center for Genetic Medicine Research Childrenrsquos National Health System Northwest Washington DC 20010 USA3Department of Integrative Systems Biology George Washington University Northwest Washington DC 20010 USA4Department of Physical Therapy University of Florida Gainesville FL 32610 USA

Correspondence should be addressed to Krista Vandenborne kvandenbphhpufledu

Received 11 February 2015 Accepted 19 May 2015

Academic Editor Christian Guelly

Copyright copy 2015 Celine Baligand et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The genetic and molecular events associated with changes in muscle mass and function after SCI and after the implementation ofcandidate therapeutic approaches are still not completely known The overall objective of this study was to identify key molecularpathways activated with muscle remodeling after SCI and locomotor training We implemented treadmill training in a well-characterized rat model of moderate SCI and performed genome wide expression profiling on soleus muscles at multiple timepoints 3 8 and 14 days after SCI We found that the activity of the protein ubiquitination and mitochondrial function relatedpathways was altered with SCI and corrected with treadmill training The BMP pathway was differentially activated with earlytreadmill training as shown by Ingenuity Pathway Analysis The expression of several muscle mass regulators was modulated bytreadmill training including Fst Jun Bmpr2Actr2b and Smad3 In addition key players in fatty acids metabolism (Lpl and Fabp3)responded to both SCI induced inactivity and reloading with training The decrease in Smad3 and Fst early after the initiation oftreadmill training was confirmed by RT-PCR Our data suggest that TGF120573Smad3 signalingmay bemainly involved in the decreasein muscle mass observed with SCI while the BMP pathway was activated with treadmill training

1 Introduction

Spinal cord injury (SCI) is one of the most disabling healthproblems faced by adults today One of the physiologicalchanges secondary to SCI is progressive muscle atrophy Theloss of muscle mass after spinal cord injury has been welldocumented with patients with complete SCI having about20ndash55 muscle atrophy [1] and patients with incompleteSCI showing about 20ndash30 atrophy [2] within 6 months to1 year after SCI Muscle wasting does not only impact thecare and lifestyle of patients after SCI but also has significanthealth implications increasing the patientsrsquo risk to developsecondary complications such as bone demineralization

diabetes and cardiovascular disease [3 4] Maintenance ofmuscle mass is essential to metabolic health and is necessaryto maximize functional gain from rehabilitation strategiesafter SCI

A potential rehabilitation intervention in the treatmentof individuals with SCI is the use of repetitive locomo-tor training to promote neural plasticity This approachwas derived from animal and human studies showing thatstepping can be generated by virtue of the neuromuscularsystemrsquos responsiveness to phasic peripheral sensory infor-mation associated with locomotion in the presence of centralpattern generator (reviewed in [5]) Our group and othershave shown that locomotor training can induce substantial

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 387090 13 pageshttpdxdoiorg1011552015387090

2 BioMed Research International

recovery inmuscle size andmuscle function in transected [6]and moderate contusion injury animal models of SCI [7ndash9]Some studies suggest that this may reflect enhanced synthesisof growth factors within the central nervous system [10] butother potential activity-dependentmolecular changes remainunknown in particular the ones occurring locally in themuscle in parallel with atrophy and hypertrophy

Muscle atrophy depends on the balance between pro-tein breakdown protein synthesis rates and apoptosis Ithas been attributed to the activation of various proteindegradation pathways in several models of disuse such asdenervation unloading cachexia or aging [11 12] Pre-vious work in animal models showed that alterations inthe protein ubiquitination and energy production relatedpathways are common features of the atrophy process [13ndash16] The activation of growth factors such as insulin-likegrowth factors (IGFs) myogenic regulatory factors (MRFs)[17] transforming growth factors (TGFs) [18] and the bonemorphogenic proteins (BMPs) [19] has also been shown toplay an important role in muscle atrophy and hypertrophyHowever the activation or predominance of the differentpathways involved can be specific to the condition inducingatrophy [20] To our knowledge only one study used geneprofiling in human muscle samples to perform a generalscreening of the pathways activated in skeletal muscle afterSCI [21] However the specific molecular signaling changesthat occur after SCI and subsequent recovery andor rehabil-itation intervention remain largely unknown

Abetter understanding of themolecular events regulatingprotein synthesis and degradation after SCI and locomotortraining and their temporal relationship to changes in musclemass is of considerable clinical importance and has far-reaching implications for posttraumatic health care There-fore the overall objective of this study was to identify keymolecular pathways activated with muscle remodeling afterSCI and during locomotor training We implemented tread-mill training in a well-characterized rat model of moderateSCI [9 22ndash24] and performed genome wide expressionprofiling with microarray on soleus muscles at multiple timepoints during the course of SCI (prior and 3 8 and 14days after SCI) and during the course of a treadmill trainingintervention initiated 7 days after injury (8 and 14 days afterSCI) We used two different approaches for data analysesFirst we took an unsupervised approach and identifiedmolecular pathways affected most at each time point Wethen targeted genes that are known to be involved in muscleremodeling and may play important roles in the process

2 Material and Methods

21 Study Design In this cross-sectional study six groups ofrats were studied including a control group three SCI groupsat three time points (days 3 8 and 14) and 2 SCI groupswith treadmill training at two time points (days 8 and 14)In the latter groups the treadmill was initiated 7 days afterSCI For the analysis of changes during the course of SCIcomparisons were done with reference to control samplesfor each time point after SCI (days 3 8 and 14) For the

analysis of the effects of treadmill training on SCI samplesfrom the treadmill trained SCI rats were compared to samplescollected from SCI rats with no training Differences betweenthe trained muscles and untrained muscles at a specific timepoint (day 8 or day 14) were determined

22 Animals Thirty-six adult female Sprague Dawley rats(16 weeks of age 260ndash280 g at the beginning of the study)were obtained from Charles River Laboratories and housedin a temperature (22 plusmn 1∘C) and humidity (50 plusmn 10)controlled room with 1212 hours lightdark cycle Animalswere provided rodent chow and water ad libitum and weregiven 1 week to acclimatize to the environment Animalswere sacrificed at one of the following time points andconditions 3 days after SCI (SCI3d 119899 = 6) 8 days afterSCI (SCI8d 119899 = 6) 14 days after SCI (SCI14d 119899 = 6)8 days after SCI and after 3 treadmill training sessions(SCI8d + TM 119899 = 6) and 14 days after SCI and after 5days of repeated treadmill training sessions (SCI14d + TM)Age-matched control animals (CTR 119899 = 6) were used asthe baseline Experimental animals were given access totransgenic dough diet (Bio-Serv NJ product number S3472)placed on the bottom of the cage to ensure adequate foodintake All procedures were performed in accordance withthe US Government Principle for the Utilization and Care ofVertebrate Animals and were approved by the InstitutionalAnimal Care andUse Committee at the University of Florida

23 Spinal Cord Injury Procedure All surgical procedureswere performed under aseptic conditions Moderate con-tusion SCI was produced using a NYU-MASCIS injurydevice as previously described [25 26] Briefly the animalswere deeply anesthetized with a combination of ketamine(90mgkg body weight) and xylazine (8mgkg body weight)and a dorsal laminectomy was performed at the thoracicvertebral level T7ndashT9 to expose the spinal cord [27] Clampsattached to the spinous processes of T7 and T9 stabilized thevertebral column Contusion was produced by dropping a10 g cylinder from a height of 25mm onto the T8 segment ofthe spinal cord Analgesia was given in the form of buprinex(0025mgkg) and ketoprofen (22mgkg) once daily over thefirst 36 hrs after SCI The animals were housed individuallyand kept under vigilant postoperative care including dailyexamination for signs of distress weight loss dehydrationand bladder dysfunction Manual expression of bladders wasperformed 2-3 times daily as required and animals weremonitored for the possibility of urinary tract infection

24 Locomotor Training Quadrupedal locomotor trainingwas initiated on postoperative day 7 Training consistedof 20min stepping sessions on a treadmill Training wasperformed 3 times in the SCI8d + TM group (2 times onday 7 and one time on day 8) and was repeated twice aday for 5 days in the SCI14d minus TM group When necessarybody weight support was manually provided by the trainerThe level of body weight support was adjusted to make surethat the hindlimbs of the animals did not collapse and wasgradually removed as locomotor capability improved During

BioMed Research International 3

the first day of training assistance was provided to place therat hindpaws in plantar-stepping position during trainingTypically rats started stepping when they experienced someload on their hindlimbs

25 Tissue Collection Left soleus muscles were harvested inall groups In the SCI8d + TM and SCI14d + TM groupsmuscle samples were harvested 8 hours after the end of thelast treadmill training session Briefly rats were anesthetizedwith isoflurane (3 for induction 1-2 for maintenance)and a small dorsal midline incision was made to exposethe gastrocnemius-soleus complex The soleus was carefullyseparated from the gastrocnemius harvested and weighedThe sample was rapidly frozen in isopentane precooled inliquid nitrogen and subsequently stored at minus80∘C119905-test was used to determine the statistical significance

(119901 lt 005) of the changes in muscle mass

26 Expression Profiling GeneChip Rat Genome 230 20Array microarrays containing approximately 30000 tran-scripts were used for the expression profiling experimentStandard procedures including total RNA isolation cDNAsynthesis cRNA labeling microarray hybridization andimage acquisition were done as described in the manu-facturerrsquos protocol and our previous publications [20 28]Briefly total RNA was isolated with TRIzol reagent (Invitro-gen) and then purified with RNeasy MinElute Cleanup Kit(Qiagen) Two hundred nanograms of total RNA from eachsample was reverse-transcribed to double-stranded cDNAfollowed by in vitro cRNA synthesis using one-cycle targetlabeling and control reagents and protocol (Affymetrix)Biotin-labeled cRNAwas then purified usingGeneChip Sam-ple Cleanup Module (Affymetrix) and fragmented randomlyprior to hybridizing to the microarrays overnight Eacharray was washed and stained using the Affymetrix FluidicsStation 450 and then scanned using the GeneChip Scanner3000 The quality control criteria developed at ChildrenrsquosNational Medical Center Microarray Center for each arraywere followed [6]

Generation of hybridization signals of the microarrayswas done using Microarray Suite 50 (MAS 50) (AffymetrixCA) as previously described [21 29ndash32] After the absoluteanalysis the gene expression values were imported intoGeneSpring 110 (Silicon Genetics) for data filtering andstatistical analysis First genes were filtered with numbers ofpresent calls across the 36 arrays analyzed Genes with at least4 present calls (detected bymore than 10 of the arrays) wereselected for statistical analysis We identified 31099 probe setsthat met this filtering criterion In GeneSpring 119905-test wasperformed and probe sets showing significant (119901 lt 005)expression changes were retained for pathway analysis Noadditional fold change filters were used The comparisonswere done by comparing samples of each time point afterSCI (days 3 8 and 14) to the control time point (day 0)respectively The treadmill trained samples were comparedto the SCI samples collected at the same time point toobtain differences between the trainedmuscles and untrainedmuscles at a specific time point (day 8 and day 14)

Table 1 Primer sequences used for quantitative RT-PCR

Genes Primer Primer sequence

Gapdh Forward F-51015840-TCCGCCCCTTCCGCTGATG-31015840

Reverse R-51015840-CACGGAAGGCCATGCCAGTGA-31015840

Smad3 Forward F-51015840-AAGATACCCCCAGGCTGC-31015840

Reverse R-51015840-CTGTCTGTCTCCTGTACTC-31015840

Myf6 Forward F-51015840-CTAAGGAAGGAGGAGCAAG-31015840

Reverse R-51015840-TGTTCCAAATGCTGACTGAG-31015840

Fst Forward F-51015840-GTGTATCAAAGCAAAGTCTTG-31015840

Reverse R-51015840-GCTCATCGCAGAGAGCA-31015840

To investigate molecular networks and pathways associ-ated with gene lists in this study Ingenuity Pathway Analysis(IPA) (Ingenuity Systems) was used with default settingsto identify gene interactions and to prioritize molecularpathways differentially affected in different groups Hierar-chical clustering was performed using GeneSpring softwareto visualize transcripts showing coordinate regulation as afunction of time

The significance of the association between the genes ineach dataset and the canonical pathway was determined byFisherrsquos exact test The 119901 values were calculated to determinethe probability of the association between the genes Allprofiles have been made publicly accessible via NCBI GEO(number GSE45550) (httpwwwncbinlmnihgovgeo)

27 Reverse Transcription and Quantitative RT-PCR AnalysisReverse transcription and quantitative RT-PCR (qRT-PCR)were performed as previously described [17] Briefly totalRNA (2 120583g)was reverse-transcribed to cDNAusing oligo(dT)primer (05 120583g120583L) and reagents from Invitrogen cDNAwas amplified in triplicate in SYBR Green PCR MasterMix (Applied Biosystems) The thermal cycling conditionsincluded 95∘C for 10min followed by 40 cycles of amplifi-cation at 95∘C for 15 s and 60∘C for 1min Primer sequencesused for rat myogenic factor 6 (MYF6 6MRF4) follistatin(FST) mothers against decapentaplegic homolog 3 or SMADfamily member 3 (SMAD3) and glyceraldehyde 3-phosphatedehydrogenase (GAPDH) which served as an internal con-trol are provided in Table 1 All primers were tested fornonspecific amplicons and primer dimers by visualizing PCRproducts on 2 agarose gels before performing qRT-PCRThe ΔΔCT value method (where CT is cycle threshold) wasused to determine fold differences as described previously[17]

3 Results

31 Changes in Soleus Muscle Wet Weight after SCI andTreadmill Training Soleus muscle wet weights are presentedin Figure 1 SCI resulted in a rapid loss in muscle weight3 and 8 days after injury (minus25 119901 = 00005) By day14 muscle weight was still lower than in controls withoutreaching significance (minus16 119901 = 0056) However 5 days oftraining significantly increased muscle wet weight (SCI14d +TM 119901 = 0005)


Table 2 Five most significantly activated pathways for each comparison of the study

Groupcomparison 3 days 8 days 14 days

No treadmill trainingversus control

Protein ubiquitination pathway Mitochondrial dysfunction Mitochondrial dysfunction

Mitochondrial dysfunction Valine leucine and isoleucinedegradation

Synthesis and degradation ofketone bodies

Oxidative phosphorylation Propanoate metabolism Oxidative phosphorylationRegulation of eIF4 and p70S6Ksignaling Butanoate metabolism Citrate cycle

Ubiquinone biosynthesis Pyruvate metabolism Ephrin receptor signaling

Treadmil training versusno training

Protein ubiquitination pathway Estrogen receptor signalingAmyloid processing EIF2 signaling

BMP signaling pathway Regulation of eIF4 andp70S6K signaling

Role of BRCA1 in DNA damageresponse Glutamate metabolism

Hereditary breast cancer signaling Granzyme B signaling

14300

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8Time (days)

Sol

eus w

et w

eigh

t (m

g)

No TMTM

lowastlowast

lowast

lowast

Figure 1 Muscle wet weight was significantly decreased on days3 and 8 as compared to day 0 and significantly increased in thetreadmill training group as compared to untrained animals on day14 lowast119901 lt 005

32TheHighest Number ofDifferentially Expressed TranscriptsWas Observed 3 Days after SCI The largest number of genesdifferentially expressed compared to controls was found 3days after SCI (Figure 2) In the treadmill trained groupsthe largest changes were found on day 14 compared tountrained animals (Figure 2) In the untrained group about60 of the genes were downregulated at each time pointwhich was reversed in the treadmill training group on day8 with 63 of the genes upregulated To identify the majormolecular pathways affected in each condition IngenuityPathwayAnalysis (IPA)was performed Table 2 shows the top5 canonical pathways for each comparison

33 Protein Ubiquitination and ATP Production RelatedGenes Were Altered following SCI At the early time point

times103

1430

1

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umbe

r of p

robe

sets

in S

CI

(com

pare

d to

cont

rol)

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DownUp

Figure 2 Number of probe sets differentially expressed in controland SCI rat soleus muscles decreased 8 and 14 days after SCI

(day 3) we found that the protein ubiquitination pathwaywas largely activated Indeed 90 of the genes identifiedby IPA showed increase in gene expression In particularthe expression of many proteasome subunits (PSMs) andubiquitin specific peptidases and enzymes was increased(Suppl Table 2 in Supplementary Material available onlineat httpdxdoiorg1011552015387090) However changesin this pathway were no longer significant on days 8 and14 as compared to control levels On the other hand themitochondrial dysfunction and oxidative phosphorylationpathways were consistently ranked among the top 5 mostsignificant pathways at all time points of the study (days 38 and 14) (Table 2) More than 95 of the genes identifiedin these two pathways were downregulated 3 days after SCI(Suppl Tables 2 3 and 4) In particular the expression of theNADHdehydrogenase subunits (NDUF) genes the succinate


dehydrogenase complex subunits (SDH) genes and the ATPsynthase genes showed a decrease in gene expression

34 Treadmill Training Rapidly Reversed the Changes in Pro-tein Ubiquitination Translation Factors and MitochondrialFunction Pathway The protein ubiquitination pathway wasrapidly affected by treadmill training While the majority ofthe PSMs were upregulated on day 3 an overall significantdecrease was observed after 3 sessions of treadmill trainingIn the Eif2 and the eif4p70sk6 pathways the expression ofthe eukaryotic translation initiation factors (EIFs) subunitsand kinases underwent overall negative fold changes onday 3 (Suppl Table 2) which was corrected with training(Suppl Table 4) In addition the expression of NDUFs ATPsynthases Cox8A and Cox7A2L was upregulated in trainedanimals on day 14 also showing a corrective effect of TM onmuscle oxidative metabolism

35 TGF-120573Smad and BMP Signaling Pathways Are Involvedin theMuscle Remodeling Process during the Course of SCI andEarly Treadmill Training Because the bone morphogenicproteins (BMPs) signaling pathway was significantly alteredon day 8 in the trained group (ranked number 3 119901 lt 0005)we examined the changes in gene expression of some keyplayers in this pathway and in the TGF120573 pathway To explorepossible downstream changes we also looked into the geneexpression of the different smads associated with these path-ways (Smad1 Smad3 Smad4 Smad5 Smad6 and Smad8) inparticular on day 3 and with acute training on day 8

On day 3 in the BMP pathway Smad4 (+18 119901 lt 005)Smad1 (+18 119901 lt 005) and Smad5 (+13 119901 lt 005) geneexpression was upregulated Smad3 did not show any signifi-cant changes on day 3 while the activin receptor 2B (Acvr2B)was downregulated (minus16 119901 lt 005) In parallel Smad6inhibitor of Bmpr2 activation was decreased (minus23119901 lt 005)

In SCI8d Smad1 gene expression was increased (+16fold 119901 lt 005) In addition both follistatin (Fst) and Smad3expression were increased (+25 fold 119901 lt 005 +20 fold119901 lt 005 resp) as compared to controls (Figure 3)

In SCI + TM on day 8 Fst was significantly decreased(minus15 fold 119901 lt 005) Smad3was also found to be significantlydownregulated (minus13 fold 119901 lt 005) The expression of theBMP complex 3 (Bmp3) was decreased (minus235 119901 lt 005)Importantly Bmpr2 was concomitantly overexpressed withtreadmill training as compared to SCI only (+16 fold 119901 lt005) As part of the genes reported in the BMP pathwayactivated with training on day 8 Jun followed the same largeand transient increase (+15 119901 lt 005) as seen in Bmpr2(Figure 3)

36 Genes Involved in Myogenesis and Muscle RegenerationWere Affected by Treadmill Training In addition to genesidentified by IPA we specifically studied genes involved inmyogenesis lipid metabolism and fiber type switches with aparticular focus on those that were affected by 5-day treadmilltraining

Igfbp5 modulator of IGF1 function in skeletal muscleshowed a significant decrease 3 days after SCI followed by

a large increase on day 14This upregulationwas not observedin the treadmill training group

Three days after injury dramatic changes were observedin myogenic regulatory factors (MRFs) including Myf6 andmyogenin (Myog) (Figure 4) The Myog expression patternwas not affected by treadmill training On the other handtreadmill training induced an additional increase (119901 lt 005)in Myf6 after the first 3 sessions as compared to untrainedanimals

37 Fatty Acid Metabolism and Fiber Type Switch RelatedGenes Showed High Sensitivity to SCI and Treadmill TrainingLipoprotein lipase (Lpl) and Fabp3 gene expression weredramatically decreased 3 days after SCI as compared tocontrols and maintained low expression levels on days 8 and14 after SCI While the first sessions of locomotor trainingdid not significantly affect Lpl expression it was completelyrestored on day 14 (+232 fold 119901 lt 0005) as shown inFigure 5

As early as 3 days after SCI and throughout the experi-ment several fast-twitch fiber markers genes (Mhy1 Mybphand Myh4) showed large and significant changes (Figure 6)In parallel the expression of several slow-twitch and vas-culature smooth muscles markers (Myh3 and Myl2) wasdecreased (119901 lt 004) (Figure 6) Treadmill training had asignificant reverse effect on the expression of some fiber typerelated genes such as the fast-twitchmarkersMyh1 andMyh4

38 RT-qPCRValidation To validatemicroarray findings weselected 3 genes (Fst Smad3 and Myf6) that were found tobe significantly affected by SCI and showed reversed changesafter the initial treadmill training RT-qPCR confirmed thatFst mRNA levels were significantly higher on day 8 after SCIcompared to controls (+25 fold 119901 lt 005) and decreasedin the trained group compared to untrained (Figure 7)Smad3 expression was also significantly decreased with acutetreadmill training (minus15 fold 119901 lt 005) On the other handthe increase in Myf6 in SCI on day 8 was not confirmed(Figure 7(c))

4 Discussion

The genetic and molecular events associated with changesin muscle mass and function after SCI and after the imple-mentation of candidate therapeutic approaches are still notcompletely known We used a well-characterized rat modelof moderate SCI combined with treadmill training as a reha-bilitation strategy to explore the pathways and genes involvedin these conditions The unique design of our study andthe use of genome wide analysis allowed the identificationof several major canonical pathways involved in proteinsynthesis and muscle metabolism regulation after SCI andtreadmill training In particular the activity of the proteinubiquitination and mitochondrial function related pathwayswas altered with SCI and corrected with treadmill trainingOf particular interest the BMP pathway was differentiallyactivated with early treadmill training as shown by IPA Theexpression of several muscle mass regulators was modulated


140 3 8Time after injury (days)

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essio

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(h)

Figure 3 BMPTGF120573 pathway related genes Gene expression level changes in Fst (a) Smad3 (b) Acvr2b (c) Smad1 (d) Bmpr2 (e) Smad6(f) Jun (g) and Smad4 (h) in soleus of trained and untrained SCI animals All expression levels are referenced to a control samplersquos GAPDHlevels lowastSignificantly different from controls (119901 lt 005) Significantly different from untrained animals (119901 lt 005)


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Expr

essio

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times103

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(b)

Figure 4 Myogenic regulatory factors Gene expression level changes inMyf6 (a)Myog (b) in soleus of trained and untrained SCI animalsAll expression levels are referenced to a control samplersquos GAPDH levels lowastSignificantly different from controls (119901 lt 005) Significantlydifferent from untrained animals (119901 lt 005)

1400

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3Ex

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l (au

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(b)

Figure 5 Fatty acids metabolism Gene expression level changes in Lpl (a) Fabp3 (b) in soleus of trained and untrained SCI animals Allexpression levels are referenced to a control samplersquos GAPDH levels lowastSignificantly different from controls (119901 lt 005) Significantly differentfrom untrained animals (119901 lt 005)

by treadmill training including Fst Jun Bmpr2 Actr2b andSmad3 In addition key players in fatty acids metabolism(Lpl and Fabp3) proved to be major sensors of SCI inducedinactivity and reloading with trainingThe decrease in Smad3and Fst early after the initiation of treadmill training wasconfirmed by RT-PCR Our data suggest that TGF-120573Smad3signaling may be mainly involved in the decrease in musclemass observed with SCI while the BMP pathway was acti-vated with treadmill training We also identified changes in

fiber type markers consistent with a switch towards type IIfibers with SCI and a reverse effect of treadmill training atthe gene expression level as early as 1 day after initiation

41 Acute Response from the Protein Ubiquitination Pathwayto SCI and Training The protein ubiquitination pathway isessential to the control of protein breakdown and turnoverin the cell It has been established that the activation of thispathway contributes largely to muscle wasting in multiple


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Figure 6Myosin heavy and light chain indicative of fiber type Gene expression level changes inMyh1 (a)Myhbp (b) indicative of fast fibersandMyl2 andMyh3 indicative of slow fibers in soleus of trained and untrained SCI animals All expression levels are referenced to a controlsamplersquos GAPDH levels lowastSignificantly different from controls (119901 lt 005) Significantly different from untrained animals (119901 lt 005)

conditions [33] including denervation [34 35] age andsarcopenia [36] and hindlimb suspension in rats [37] andin spinal cord injury in humans [21 38] In our modelof moderate SCI the protein ubiquitination pathway wasactivated as early as 3 days after injury with the expressionof 56 genes significantly upregulated This upregulation wasmost likely responsible for the unbalance in protein synthe-sisdegradation ratio resulting in the large muscle wastingobserved in the soleus on day 3 Conversely the expression ofgenes in the protein ubiquitination pathway was very rapidlydecreased on day 8 after only 3 sessions of treadmill trainingin SCI animals and the expression of several 20S proteasomesubunits was significantly reduced This was in accordancewith previous observations in a hindlimb suspension ratmodel [39] 10 days after reloading However in another studyin humans Reich et al [38] could not detect any reverseeffect with 24 h of reloading after lower limb suspension

Here we were able to observe a significant and early responsewithin 36 h after the first training session with a significantdecrease in the expression of the genes related to proteinubiquitination (Suppl Table 5)

42 Normalization of Mitochondrial Function Related Path-ways with Training Genes involved in the mitochondrialdysfunction and oxidative phosphorylation related pathwayswere also significantly changed Together the large numberof NUDF SDH and ATP synthases with changing geneexpression observed in SCI demonstrates a strong metabolicactivity disturbance associated with SCI that does not com-pletely recover within 14 days after surgery Mitochondrialdysfunction after SCI was observed in vivo in several studiesperformed in humans with SCI [40 41] Our own work using31Pmagnetic resonance spectroscopy to assessmitochondrialfunction in vivo in the same rat model of moderate SCI [24]


CTR SCI8d-TMSCI8d0000

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Figure 7 FST (a) SMAD3 (b) andMYF6 (c) mRNA expression in the soleus muscle of controls untrained SCI animal on day 8 after surgery(SCI8d) and trained SCI animal on day 8 after surgery (SCI8d + TM) lowast119901 lt 005

TGF120573 FST uarr BMPs

ActRIIb darr Bmpr2

Atrophy Hypertrophy

Smad2

Smad3 uarr

Smad6 darr

Smad4 uarr

Smad1 uarr

Smad5 uarrSmad8

(a)

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ActRIIb Bmpr2 uarr

Atrophy Hypertrophy

Smad2

Smad3 darr

Smad6

Smad4

Smad1

Smad5Smad8

(b)

Figure 8 Schematic of the changes in soleus muscle gene expression within the TGF120573 and BMP pathways with SCI (a) and acute responseto treadmill training (b) Panel (a) shows the combined changes observed on day 3 and 8 after SCI where several Smads (Smads 3 4 and 15)and Fst gene expression was increased compared to control in parallel with muscle atrophy Panel (b) summarizes the changes observed 8days after SCI after 3 sessions of treadmill training compared to untrained animals The changes in Smad3 and Fst were reversed and Bmpr2expression was increased suggesting a role for the BMP pathway in the initiation of muscle mass recovery process observed with treadmilltraining


showed that ATP production by mitochondria was affected 1week after SCI and recovered in 3 weeks without training Itshould be noted that in the present study no acute effect oftreadmill training on mitochondrial dysfunction and oxida-tive phosphorylation pathways related genes was detected onday 8 However our results suggest that treadmill trainingand the concomitant muscle hypertrophy are accompaniedby partial correction of the mitochondrial function pathwaygenes (NUDFs and ATP synthase) at a later time (day 14)making mitochondria a target for early therapy in moderateSCI

43 Role of TGF-120573Smad and BMP Pathways in Atrophyfollowing SCI Follistatin (FST) is well known as an inhibitorof the myostatin signaling within the TGF120573 pathway It hasbeen shown that Fst overexpression leads to a large increasein muscle mass in different animal models [42 43] Ourresults showed a large significant increase of FST mRNAlevels with SCI after 8 days It is possible that this increasereflects a protective mechanism intended to stimulate musclegrowth However our data showed that muscle wet weightdid not further decrease after the initial 28 loss on day3 One possibility is that the increase in FST mRNA levelsmay not have been sufficient to produce a recovery ofmuscle mass Interestingly we also observed a concomitantincrease in Smad3 and Smad4 gene expression downstreamthe Act2b receptor which might be responsible for atrophyIndeed phosphorylated SMAD3 can mediate the activationof ubiquitin ligases that induces proteasomal degradationof contractile proteins [44] More importantly Winbankset al [45] recently showed that SMAD3 protein expressionprevents skeletal muscle growth induced by follistatin andmay suppress AktmTORS6K signaling In addition Smad1expression downstream the BMP pathway was increasedpotentially competing with SMAD3 for the recruitment ofSMAD4 If translated at the protein level the balance betweenFST inhibition of the TGF120573 signaling and increased SMAD3and SMAD1 expression may have led to a plateau in soleusmuscle mass after the initial drop (Figure 8)

44 Role of TGF120573Smad and BMP Pathways in Hypertrophyfollowing Treadmill Training On the other hand treadmilltraining was able to restore the initial muscle mass by day 14after SCI Ourmain associated findings were a large and rapidincrease in the expression of Bmpr2 with treadmill training36 hours after initiation in parallel with the significantdecrease in FST and SMAD3 mRNA levels The increasedBmpr2 (BMPs receptor) levels could favor its activation byBMPs and the subsequent phosphorylation of Smad1 Smad5and Smad8 which in turn bind with Smad4 leading tohypertrophy [19] Whereas a decrease in FST expression hasthe ability to enable MSTN signaling and muscle growtha parallel decrease in SMAD3 protein expression couldcontribute to an increase in SMAD4rsquos availability to otherbinding proteins that can lead to hypertrophy such asSMADs 1 5 and 8 A concomitant and rapid increase inJun was observed It was shown that this transcription factoracts downstream the TGF120573 [46] and that overexpression

of JUN results in dephosphorylation of SMAD3 [47] Thisbody of observations is suggestive of a deactivation of theTGF120573Smad pathway and a larger role for the BMP axis in thehypertrophy process following SCI and treadmill training

45 Other Growth Factors (IGFs MRFs) The IGF1-Akt-mTOR pathway is known as a positive regulator of musclemass [18 48ndash50] This pathway was not highly ranked by thepresent microarray analysis However the group previouslydemonstrated the impact of moderate SCI and treadmilltraining on several IGF proteins and binding proteins butnot on MYF5 in skeletal muscle [23] Consistently with theseprevious results here we observed that Igbp5 andMyog wereoverexpressed after SCI and that Igbp5 expression levels werecorrected with treadmill training In addition we exploredthe possible impact of treadmill training on MRF4MYF6 asindicated by the microarray results and found no significantincrease in mRNA levels This discrepancy may be dueto experimental variations or slight biological differencesbetween the batches of muscle samples used for microarrayand RT-qPCR validation

46 Fatty Acid Metabolism and Fiber Type Related Genes asa Sensor of Muscle Activity in SCI and Treadmill TrainingLipoprotein lipase (LPL) is a major enzyme involved in fattyacid (FA) metabolism and transport After transport intothe cytoplasm FA binds to the fatty acid binding protein 3(FABP3) Both LPL and FABP3 have been shown to be highlysensitive to contractile activity in muscle [51 52] Here bothLpl and Fabp3 genes showed high sensitivity to SCI induceddisuse and to locomotor training The downregulation ofthese fatty acid transporters in SCI rats was consistent withthe recent observation by Long et al [53] in muscle biopsiesobtained from human subjects with SCI On the other handwe measured a large positive change in Lpl and Fabp3gene expression in response to locomotor training withexpression levels nearly corrected (92 of control levels) byday 14 Upregulation of LPL and FABP3 has been previouslydemonstrated in conjunction with increased muscle activitysuch as muscle endurance training in humans [54] reloadingafter hindlimb suspension [28] and electrostimulation indenervatedmuscles [55] in ratsWe established that treadmilltraining as performed in our study was sufficient to restorecontrol levels of gene expression within 5 days Our resultsfurther demonstrated the high sensitivity of Lpl and Fabp3gene expression to muscle activity and reloading achieved bytreadmill training leading tomuscle hypertrophy inmoderateSCI rats

Fiber type related genes also showed a great sensitivityto SCI induced disuse It has been well established in theliterature that muscle fibers distribution undergoes a shifttowards fast fibers with disuse after SCI [56] Consistent withthis we observed a fast change in the expression of myosinheavy and light chains genes with an increase in some of thefast type (Myh1Myhbp) and a decrease in the slow type (Myl2and Myh3) Interestingly the expression level of Myh1 wascorrected in response to treadmill training on day 14 showingthe effect of training on SCI muscles


In summary using genome wide analysis we were ableto identify some of the main pathways responsible for musclewasting following moderate SCI and more importantly thecorrective effect of treadmill training on these pathwaysHere we chose to focus on the BMP and TGF120573 signalingand established a key role for some of the genes within thesepathways Our observations suggest that Smad3 Bmpr2 andFst are genes of interest in the study of moderate SCI Proteinexpression and phosphorylation need to be investigated toallow further interpretation although beyond the scope ofthis study It wouldmake it possible to test the hypotheses thatFst overexpression in SCI competes with Smad3 regulationand that this effect is reversed by treadmill training leading tomusclemass recoveryMore importantly this would elucidatewhether or not treadmill training activates the BMPSmadpathway contributing to hypertrophy This would establishthe effect of BMP signaling activation and TGF120573 signaling onmuscle regeneration with treadmill training in SCI via Smad3downregulation proving early indicators of efficient reloadingin SCI rat and as a promising therapeutic approachThe bodyof data presented in this study constitutes a comprehensiveguide to future studies targeting muscle mass preservation orrecovery in moderate SCI

Disclaimer

Thecontent is solely the responsibility of the authors and doesnot necessarily represent the official views of the NationalInstitutes of Health

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was funded by the Paralyzed VeteransAdministration-2347 NIHNICHD RO1HD048051 and1R24HD050846 Yi-Wen Chen and Sachchida Nand Pandeywere partially supported by NIHNIAMS under Awardnumber 1R01AR052027 and FSHD Society under Awardnumber FSHS-82013-01

References

[1] M J Castro D F Apple Jr E A Hillegass and G A DudleyldquoInfluence of complete spinal cord injury on skeletal musclecross-sectional area within the first 6 months of injuryrdquo Euro-pean Journal of Applied Physiology and Occupational Physiologyvol 80 no 4 pp 373ndash378 1999

[2] P K Shah J E Stevens C M Gregory et al ldquoLower-extremitymuscle cross-sectional area after incomplete spinal cord injuryrdquoArchives of Physical Medicine and Rehabilitation vol 87 no 6pp 772ndash778 2006

[3] R Banerjea U Sambamoorthi F Weaver M Maney L MPogach and T Findley ldquoRisk of stroke heart attack anddiabetes complications among veterans with spinal cord injuryrdquo

Archives of Physical Medicine and Rehabilitation vol 89 no 8pp 1448ndash1453 2008

[4] C P Elder D F Apple C S Bickel R A Meyer and G ADudley ldquoIntramuscular fat and glucose tolerance after spinalcord injurymdasha cross-sectional studyrdquo Spinal Cord vol 42 no12 pp 711ndash716 2004

[5] S Rossignol andA Frigon ldquoRecovery of locomotion after spinalcord injury some facts and mechanismsrdquo Annual Review ofNeuroscience vol 34 pp 413ndash440 2011

[6] R D de Leon J A Hodgson R R Roy and V R EdgertonldquoLocomotor capacity attributable to step training versus spon-taneous recovery after spinalization in adult catsrdquo Journal ofNeurophysiology vol 79 no 3 pp 1329ndash1340 1998

[7] K L Caudle EH BrownA Shum-Siu et al ldquoHindlimb immo-bilization in a wheelchair alters functional recovery followingcontusive spinal cord injury in the adult ratrdquoNeurorehabilitationand Neural Repair vol 25 no 8 pp 729ndash739 2011

[8] K Fouad G A S Metz DMerkler V Dietz andM E SchwabldquoTreadmill training in incomplete spinal cord injured ratsrdquoBehavioural Brain Research vol 115 no 1 pp 107ndash113 2000

[9] M Liu P Bose G A Walter F J Thompson and K Vanden-borne ldquoA longitudinal study of skeletal muscle following spinalcord injury and locomotor trainingrdquo Spinal Cord vol 46 no 7pp 488ndash493 2008

[10] J Frystyk ldquoExercise and the growth hormone-insulin-likegrowth factor axisrdquoMedicine and Science in Sports and Exercisevol 42 no 1 pp 58ndash66 2010

[11] S H Lecker V Solomon W E Mitch and A L Gold-berg ldquoMuscle protein breakdown and the critical role of theubiquitin-proteasome pathway in normal and disease statesrdquoThe Journal of Nutrition vol 129 no 1 pp 227Sndash237S 1999

[12] W E Mitch and A L Goldberg ldquoMechanisms of diseasemechanisms of muscle wasting the role of the ubiquitin-proteasome pathwayrdquoTheNewEngland Journal ofMedicine vol335 no 25 pp 1897ndash1905 1996

[13] J Batt J Bain J Goncalves et al ldquoDifferential gene expressionprofiling of short and long term denervatedmusclerdquoTheFASEBJournal vol 20 no 1 pp 115ndash117 2006

[14] S H Lecker R T Jagoe A Gilbert et al ldquoMultiple types ofskeletal muscle atrophy involve a common program of changesin gene expressionrdquoThe FASEB Journal vol 18 no 1 pp 39ndash512004

[15] A Raffaello P Laveder C Romualdi et al ldquoDenervation inmurine fast-twitch muscle short-term physiological changesand temporal expression profilingrdquo Physiological Genomics vol25 no 1 pp 60ndash74 2006

[16] H Tang W M W Cheung F C F Ip and N Y Ip ldquoIdentifi-cation and characterization of differentially expressed genes indenervated musclerdquo Molecular and Cellular Neurosciences vol16 no 2 pp 127ndash140 2000

[17] Y-W Chen M J Hubal E P Hoffman P D Thompson andP M Clarkson ldquoMolecular responses of human muscle toeccentric exerciserdquo Journal of Applied Physiology vol 95 no 6pp 2485ndash2494 2003

[18] S Schiaffino K A Dyar S Ciciliot B Blaauw and M SandrildquoMechanisms regulating skeletal muscle growth and atrophyrdquoThe FEBS Journal vol 280 no 17 pp 4294ndash4314 2013

[19] R Sartori E Schirwis B Blaauw et al ldquoBMP signaling controlsmuscle massrdquo Nature Genetics vol 45 no 11 pp 1309ndash13212013


[20] P Bialek C Morris J Parkington et al ldquoDistinct proteindegradation profiles are induced by different disuse models ofskeletal muscle atrophyrdquo Physiological Genomics vol 43 no 19pp 1075ndash1085 2011

[21] M L Urso Y-W Chen A G Scrimgeour P C Lee K FLee and P M Clarkson ldquoAlterations in mRNA expression andprotein products following spinal cord injury in humansrdquo TheJournal of Physiology vol 579 no 3 pp 877ndash892 2007

[22] D M Basso M S Beattie and J C Bresnahan ldquoGradedhistological and locomotor outcomes after spinal cord contu-sion using the NYU weight-drop device versus transectionrdquoExperimental Neurology vol 139 no 2 pp 244ndash256 1996

[23] M Liu J E Stevens-Lapsley A Jayaraman et al ldquoImpact oftreadmill locomotor training on skeletal muscle IGF1 and myo-genic regulatory factors in spinal cord injured ratsrdquo EuropeanJournal of Applied Physiology vol 109 no 4 pp 709ndash720 2010

[24] P K Shah F Ye M Liu et al ldquoIn vivo 31P NMR spectroscopyassessment of skeletal muscle bioenergetics after spinal cordcontusion in ratsrdquo European Journal of Applied Physiology vol114 no 4 pp 847ndash858 2014

[25] D M Basso M S Beattie J C Bresnahan et al ldquoMASCISevaluation of open field locomotor scores effects of experienceand teamwork on reliability Multicenter Animal Spinal CordInjury Studyrdquo Journal of Neurotrauma vol 13 no 7 pp 343ndash359 1996

[26] M Liu P Bose G A Walter D K Anderson F J Thomp-son and K Vandenborne ldquoChanges in muscle T2 relaxationproperties following spinal cord injury and locomotor trainingrdquoEuropean Journal of Applied Physiology vol 97 no 3 pp 355ndash361 2006

[27] F J Thompson P J Reier C C Lucas and R Parmer ldquoAlteredpatterns of reflex excitability subsequent to contusion injury ofthe rat spinal cordrdquo Journal of Neurophysiology vol 68 no 5pp 1473ndash1486 1992

[28] L Bey and M T Hamilton ldquoSuppression of skeletal musclelipoprotein lipase activity during physical inactivity a molecu-lar reason to maintain daily low-intensity activityrdquo The Journalof Physiology vol 551 no 2 pp 673ndash682 2003

[29] J G Burniston THMeek S N Pandey et al ldquoGene expressionprofiling of gastrocnemius of lsquominimusclersquo micerdquo PhysiologicalGenomics vol 45 no 6 pp 228ndash236 2013

[30] Y-W Chen K Nagaraju M Bakay et al ldquoEarly onset ofinflammation and later involvement of TGF120573 in Duchennemuscular dystrophyrdquo Neurology vol 65 no 6 pp 826ndash8342005

[31] Y-W Chen P Zhao R Borup and E P Hoffman ldquoExpressionprofiling in the muscular dystrophies identification of novelaspects of molecular pathophysiologyrdquo Journal of Cell Biologyvol 151 no 6 pp 1321ndash1336 2000

[32] M Dixit E Ansseau A Tassin et al ldquoDUX4 a candidategene of facioscapulohumeral muscular dystrophy encodes atranscriptional activator of PITX1rdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 104 no46 pp 18157ndash18162 2007

[33] R T Jagoe and A L Goldberg ldquoWhat do we really know aboutthe ubiquitin-proteasome pathway inmuscle atrophyrdquoCurrentOpinion in Clinical Nutrition and Metabolic Care vol 4 no 3pp 183ndash190 2001

[34] H M Argadine N J Hellyer C B Mantilla W-Z Zhan andG C Sieck ldquoThe effect of denervation on protein synthesis anddegradation in adult rat diaphragm musclerdquo Journal of AppliedPhysiology vol 107 no 2 pp 438ndash444 2009

[35] R Medina S S Wing A Haas and A L Goldberg ldquoActivationof the ubiquitin-ATP-dependent proteolytic system in skeletalmuscle during fasting and denervation atrophyrdquo BiomedicaBiochimica Acta vol 50 no 4ndash6 pp 347ndash356 1991

[36] M Altun H C Besche H S Overkleeft et al ldquoMuscle wastingin aged sarcopenic rats is associated with enhanced activityof the ubiquitin proteasome pathwayrdquoThe Journal of BiologicalChemistry vol 285 no 51 pp 39597ndash39608 2010

[37] D Taillandier E Aurousseau D Meynial-Denis et al ldquoCoor-dinate activation of lysosomal Ca2+-activated and ATP-ubiquitin-dependent proteinases in the unweighted rat soleusmusclerdquo Biochemical Journal vol 316 no 1 pp 65ndash72 1996

[38] K A Reich Y-W Chen P D Thompson E P Hoffman andP M Clarkson ldquoForty-eight hours of unloading and 24 h ofreloading lead to changes in global gene expression patternsrelated to ubiquitination and oxidative stress in humansrdquoJournal of Applied Physiology vol 109 no 5 pp 1404ndash1415 2010

[39] E Vazeille A Codran A Claustre et al ldquoThe ubiquitin-proteasome and the mitochondria-associated apoptotic path-ways are sequentially downregulated during recovery afterimmobilization-inducedmuscle atrophyrdquoTheAmerican Journalof Physiology Endocrinology andMetabolism vol 295 no 5 ppE1181ndashE1190 2008

[40] K K McCully T K Mulcahy T E Ryan and Q Zhao ldquoSkeletalmuscle metabolism in individuals with spinal cord injuryrdquoJournal of Applied Physiology vol 111 no 1 pp 143ndash148 2011

[41] J L Olive J M Slade G A Dudley and K K McCully ldquoBloodflow and muscle fatigue in SCI individuals during electricalstimulationrdquo Journal of Applied Physiology vol 94 no 2 pp701ndash708 2003

[42] AMHaidet L Rizo CHandy et al ldquoLong-term enhancementof skeletal muscle mass and strength by single gene admin-istration of myostatin inhibitorsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 105 no11 pp 4318ndash4322 2008

[43] S-J Lee andA CMcPherron ldquoRegulation ofmyostatin activityand muscle growthrdquo Proceedings of the National Academy ofSciences of the United States of America vol 98 no 16 pp 9306ndash9311 2001

[44] S Lokireddy C McFarlane X Ge et al ldquoMyostatin inducesdegradation of sarcomeric proteins through a Smad3 signal-ing mechanism during skeletal muscle wastingrdquo MolecularEndocrinology vol 25 no 11 pp 1936ndash1949 2011

[45] C E Winbanks J L Chen H Qian et al ldquoThe bone morpho-genetic protein axis is a positive regulator of skeletal musclemassrdquo The Journal of Cell Biology vol 203 no 2 pp 345ndash3572013

[46] L Li J-S Hu and E N Olson ldquoDifferent members of the junproto-oncogene family exhibit distinct patterns of expression inresponse to type beta transforming growth factorrdquo The Journalof Biological Chemistry vol 265 no 3 pp 1556ndash1562 1990

[47] A Raffaello GMilan EMasiero et al ldquoJunB transcription fac-tor maintains skeletal muscle mass and promotes hypertrophyrdquoThe Journal of Cell Biology vol 191 no 1 pp 101ndash113 2010

[48] D J Glass ldquoPI3 kinase regulation of skeletal muscle hypertro-phy and atrophyrdquo Current Topics in Microbiology and Immunol-ogy vol 346 no 1 pp 267ndash278 2010

[49] E Latres A R Amini A A Amini et al ldquoInsulin-likegrowth factor-1 (IGF-1) inversely regulates atrophy-inducedgenes via the phosphatidylinositol 3-kinaseAktmammaliantarget of rapamycin (PI3KAktmTOR) pathwayrdquo The Journalof Biological Chemistry vol 280 no 4 pp 2737ndash2744 2005


[50] J M Sacheck A Ohtsuka S C McLary and A L Gold-berg ldquoIGF-I stimulates muscle growth by suppressing proteinbreakdown and expression of atrophy-related ubiquitin lig-ases atrogin-1 and MuRF1rdquo American Journal of PhysiologyEndocrinology and Metabolism vol 287 no 4 pp E591ndashE6012004

[51] R B Simsolo J M Ong and P A Kern ldquoThe regulationof adipose tissue and muscle lipoprotein lipase in runners bydetrainingrdquo The Journal of Clinical Investigation vol 92 no 5pp 2124ndash2130 1993

[52] E Ernicka E Smol J Langfort and M Gorecka ldquoTimecourse of changes in lipoprotein lipase activity in rat skeletalmuscles during denervation-reinnervationrdquo Journal of AppliedPhysiology vol 92 no 2 pp 535ndash540 2002

[53] Y C Long E Kostovski H Boon N Hjeltnes A Krook andUWidegren ldquoDifferential expression of metabolic genes essentialfor glucose and lipid metabolism in skeletal muscle from spinalcord injured subjectsrdquo Journal of Applied Physiology vol 110 no5 pp 1204ndash1210 1985

[54] B Schmitt M Fluck J Decombaz et al ldquoTranscriptionaladaptations of lipid metabolism in tibialis anterior muscle ofendurance-trained athletesrdquo Physiological Genomics vol 15 pp148ndash157 2004

[55] E Smol E Zernicka D Czarnowski and J Langfort ldquoLipopro-tein lipase activity in skeletal muscles of the rat effects ofdenervation and tenotomyrdquo Journal of Applied Physiology vol90 no 3 pp 954ndash960 2001

[56] S Ciciliot A C Rossi K A Dyar B Blaauw and S SchiaffinoldquoMuscle type and fiber type specificity in muscle wastingrdquoInternational Journal of Biochemistry and Cell Biology vol 45no 10 pp 2191ndash2199 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


recovery inmuscle size andmuscle function in transected [6]and moderate contusion injury animal models of SCI [7ndash9]Some studies suggest that this may reflect enhanced synthesisof growth factors within the central nervous system [10] butother potential activity-dependentmolecular changes remainunknown in particular the ones occurring locally in themuscle in parallel with atrophy and hypertrophy

Muscle atrophy depends on the balance between pro-tein breakdown protein synthesis rates and apoptosis Ithas been attributed to the activation of various proteindegradation pathways in several models of disuse such asdenervation unloading cachexia or aging [11 12] Pre-vious work in animal models showed that alterations inthe protein ubiquitination and energy production relatedpathways are common features of the atrophy process [13ndash16] The activation of growth factors such as insulin-likegrowth factors (IGFs) myogenic regulatory factors (MRFs)[17] transforming growth factors (TGFs) [18] and the bonemorphogenic proteins (BMPs) [19] has also been shown toplay an important role in muscle atrophy and hypertrophyHowever the activation or predominance of the differentpathways involved can be specific to the condition inducingatrophy [20] To our knowledge only one study used geneprofiling in human muscle samples to perform a generalscreening of the pathways activated in skeletal muscle afterSCI [21] However the specific molecular signaling changesthat occur after SCI and subsequent recovery andor rehabil-itation intervention remain largely unknown

Abetter understanding of themolecular events regulatingprotein synthesis and degradation after SCI and locomotortraining and their temporal relationship to changes in musclemass is of considerable clinical importance and has far-reaching implications for posttraumatic health care There-fore the overall objective of this study was to identify keymolecular pathways activated with muscle remodeling afterSCI and during locomotor training We implemented tread-mill training in a well-characterized rat model of moderateSCI [9 22ndash24] and performed genome wide expressionprofiling with microarray on soleus muscles at multiple timepoints during the course of SCI (prior and 3 8 and 14days after SCI) and during the course of a treadmill trainingintervention initiated 7 days after injury (8 and 14 days afterSCI) We used two different approaches for data analysesFirst we took an unsupervised approach and identifiedmolecular pathways affected most at each time point Wethen targeted genes that are known to be involved in muscleremodeling and may play important roles in the process

2 Material and Methods

21 Study Design In this cross-sectional study six groups ofrats were studied including a control group three SCI groupsat three time points (days 3 8 and 14) and 2 SCI groupswith treadmill training at two time points (days 8 and 14)In the latter groups the treadmill was initiated 7 days afterSCI For the analysis of changes during the course of SCIcomparisons were done with reference to control samplesfor each time point after SCI (days 3 8 and 14) For the

analysis of the effects of treadmill training on SCI samplesfrom the treadmill trained SCI rats were compared to samplescollected from SCI rats with no training Differences betweenthe trained muscles and untrained muscles at a specific timepoint (day 8 or day 14) were determined

22 Animals Thirty-six adult female Sprague Dawley rats(16 weeks of age 260ndash280 g at the beginning of the study)were obtained from Charles River Laboratories and housedin a temperature (22 plusmn 1∘C) and humidity (50 plusmn 10)controlled room with 1212 hours lightdark cycle Animalswere provided rodent chow and water ad libitum and weregiven 1 week to acclimatize to the environment Animalswere sacrificed at one of the following time points andconditions 3 days after SCI (SCI3d 119899 = 6) 8 days afterSCI (SCI8d 119899 = 6) 14 days after SCI (SCI14d 119899 = 6)8 days after SCI and after 3 treadmill training sessions(SCI8d + TM 119899 = 6) and 14 days after SCI and after 5days of repeated treadmill training sessions (SCI14d + TM)Age-matched control animals (CTR 119899 = 6) were used asthe baseline Experimental animals were given access totransgenic dough diet (Bio-Serv NJ product number S3472)placed on the bottom of the cage to ensure adequate foodintake All procedures were performed in accordance withthe US Government Principle for the Utilization and Care ofVertebrate Animals and were approved by the InstitutionalAnimal Care andUse Committee at the University of Florida

23 Spinal Cord Injury Procedure All surgical procedureswere performed under aseptic conditions Moderate con-tusion SCI was produced using a NYU-MASCIS injurydevice as previously described [25 26] Briefly the animalswere deeply anesthetized with a combination of ketamine(90mgkg body weight) and xylazine (8mgkg body weight)and a dorsal laminectomy was performed at the thoracicvertebral level T7ndashT9 to expose the spinal cord [27] Clampsattached to the spinous processes of T7 and T9 stabilized thevertebral column Contusion was produced by dropping a10 g cylinder from a height of 25mm onto the T8 segment ofthe spinal cord Analgesia was given in the form of buprinex(0025mgkg) and ketoprofen (22mgkg) once daily over thefirst 36 hrs after SCI The animals were housed individuallyand kept under vigilant postoperative care including dailyexamination for signs of distress weight loss dehydrationand bladder dysfunction Manual expression of bladders wasperformed 2-3 times daily as required and animals weremonitored for the possibility of urinary tract infection

24 Locomotor Training Quadrupedal locomotor trainingwas initiated on postoperative day 7 Training consistedof 20min stepping sessions on a treadmill Training wasperformed 3 times in the SCI8d + TM group (2 times onday 7 and one time on day 8) and was repeated twice aday for 5 days in the SCI14d minus TM group When necessarybody weight support was manually provided by the trainerThe level of body weight support was adjusted to make surethat the hindlimbs of the animals did not collapse and wasgradually removed as locomotor capability improved During




















3 Results
















14300

50

100

150

200

250

8Time (days)

Sol

eus w

et w

eigh

t (m

g)

No TMTM

lowastlowast

lowast

lowast




times103

1430

1

2

3

4

5N

umbe

r of p

robe

sets

in S

CI

(com

pare

d to

cont

rol)

8Time (days)

DownUp

















4 Discussion




0

02

04

06

08

1

Folli

stat

in ex

pres

sion

leve

l (au

)

times103

(a)


0

1

2

3

4

5

Smad3

Expr

essio

n le

vel (

au)

times103

(b)

0

02

04

06

08

Acvr2b

Expr

essio

n le

vel (

au)


lowast

lowastlowast

times103

(c)

0

05

1

15

Smad1

Expr

essio

n le

vel (

au)


lowast

times103

(d)

0

05

1

15

Bmpr2

Expr

essio

n le

vel (

au)


times103

(e)


0

005

01

015

02

025

Smad6

Expr

essio

n le

vel (

au)

lowast

times103

(f)


0

5

10

15

Jun

Expr

essio

n le

vel (

au)

times103

No TMTM

(g)


2

25

3

35

4

45

Smad4

Expr

essio

n le

vel (

au)

lowast

times103

No TMTM

(h)



1400

5

10

15

20

25


Myf6

Expr

essio

n le

vel (

au)

lowastlowast

times103

No TMTM

(a)

1400

2

4

6

8

10


Myog

Expr

essio

n le

vel (

au)

times103

No TMTM

(b)


1400

10

20

30

40


Lpl

Expr

essio

n le

vel (

au)

lowastlowast

lowast

times103

No TMTM

(a)

1400

20

40

60


FABP

3Ex

pres

sion

leve

l (au

)

lowastlowast

lowast

times103

No TMTM

(b)






1400

10

20

30

40

50


Myh1

Expr

essio

n le

vel (

au)

lowast

lowast

lowast

times103

(a)

1400

10

20

30

40

50


Myhbp

Expr

essio

n le

vel (

au)

lowast lowast

times103

(b)

1400

05

1

15

2


Myl2

Expr

essio

n le

vel (

au)

No TMTM

times103

(c)

No TMTM

1400

10

20

30

40


Myh3

Expr

essio

n le

vel (

au)

times103

(d)







0001

0002

0003

FST

expr

essio

n le

vel

lowast

lowast

(a)


001

002

003

SMA

D3

expr

essio

n le

vel

lowast

lowast

(b)


002

004

006

008

010

MYF

6 ep

ress

ion

leve

ls

lowast lowast

(c)



ActRIIb darr Bmpr2

Atrophy Hypertrophy

Smad2

Smad3 uarr

Smad6 darr

Smad4 uarr

Smad1 uarr

Smad5 uarrSmad8

(a)


ActRIIb Bmpr2 uarr

Atrophy Hypertrophy

Smad2

Smad3 darr

Smad6

Smad4

Smad1

Smad5Smad8

(b)












Disclaimer




Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















3 Results
















14300

50

100

150

200

250

8Time (days)

Sol

eus w

et w

eigh

t (m

g)

No TMTM

lowastlowast

lowast

lowast




times103

1430

1

2

3

4

5N

umbe

r of p

robe

sets

in S

CI

(com

pare

d to

cont

rol)

8Time (days)

DownUp

















4 Discussion




0

02

04

06

08

1

Folli

stat

in ex

pres

sion

leve

l (au

)

times103

(a)


0

1

2

3

4

5

Smad3

Expr

essio

n le

vel (

au)

times103

(b)

0

02

04

06

08

Acvr2b

Expr

essio

n le

vel (

au)


lowast

lowastlowast

times103

(c)

0

05

1

15

Smad1

Expr

essio

n le

vel (

au)


lowast

times103

(d)

0

05

1

15

Bmpr2

Expr

essio

n le

vel (

au)


times103

(e)


0

005

01

015

02

025

Smad6

Expr

essio

n le

vel (

au)

lowast

times103

(f)


0

5

10

15

Jun

Expr

essio

n le

vel (

au)

times103

No TMTM

(g)


2

25

3

35

4

45

Smad4

Expr

essio

n le

vel (

au)

lowast

times103

No TMTM

(h)



1400

5

10

15

20

25


Myf6

Expr

essio

n le

vel (

au)

lowastlowast

times103

No TMTM

(a)

1400

2

4

6

8

10


Myog

Expr

essio

n le

vel (

au)

times103

No TMTM

(b)


1400

10

20

30

40


Lpl

Expr

essio

n le

vel (

au)

lowastlowast

lowast

times103

No TMTM

(a)

1400

20

40

60


FABP

3Ex

pres

sion

leve

l (au

)

lowastlowast

lowast

times103

No TMTM

(b)






1400

10

20

30

40

50


Myh1

Expr

essio

n le

vel (

au)

lowast

lowast

lowast

times103

(a)

1400

10

20

30

40

50


Myhbp

Expr

essio

n le

vel (

au)

lowast lowast

times103

(b)

1400

05

1

15

2


Myl2

Expr

essio

n le

vel (

au)

No TMTM

times103

(c)

No TMTM

1400

10

20

30

40


Myh3

Expr

essio

n le

vel (

au)

times103

(d)







0001

0002

0003

FST

expr

essio

n le

vel

lowast

lowast

(a)


001

002

003

SMA

D3

expr

essio

n le

vel

lowast

lowast

(b)


002

004

006

008

010

MYF

6 ep

ress

ion

leve

ls

lowast lowast

(c)



ActRIIb darr Bmpr2

Atrophy Hypertrophy

Smad2

Smad3 uarr

Smad6 darr

Smad4 uarr

Smad1 uarr

Smad5 uarrSmad8

(a)


ActRIIb Bmpr2 uarr

Atrophy Hypertrophy

Smad2

Smad3 darr

Smad6

Smad4

Smad1

Smad5Smad8

(b)












Disclaimer




Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















14300

50

100

150

200

250

8Time (days)

Sol

eus w

et w

eigh

t (m

g)

No TMTM

lowastlowast

lowast

lowast




times103

1430

1

2

3

4

5N

umbe

r of p

robe

sets

in S

CI

(com

pare

d to

cont

rol)

8Time (days)

DownUp

















4 Discussion




0

02

04

06

08

1

Folli

stat

in ex

pres

sion

leve

l (au

)

times103

(a)


0

1

2

3

4

5

Smad3

Expr

essio

n le

vel (

au)

times103

(b)

0

02

04

06

08

Acvr2b

Expr

essio

n le

vel (

au)


lowast

lowastlowast

times103

(c)

0

05

1

15

Smad1

Expr

essio

n le

vel (

au)


lowast

times103

(d)

0

05

1

15

Bmpr2

Expr

essio

n le

vel (

au)


times103

(e)


0

005

01

015

02

025

Smad6

Expr

essio

n le

vel (

au)

lowast

times103

(f)


0

5

10

15

Jun

Expr

essio

n le

vel (

au)

times103

No TMTM

(g)


2

25

3

35

4

45

Smad4

Expr

essio

n le

vel (

au)

lowast

times103

No TMTM

(h)



1400

5

10

15

20

25


Myf6

Expr

essio

n le

vel (

au)

lowastlowast

times103

No TMTM

(a)

1400

2

4

6

8

10


Myog

Expr

essio

n le

vel (

au)

times103

No TMTM

(b)


1400

10

20

30

40


Lpl

Expr

essio

n le

vel (

au)

lowastlowast

lowast

times103

No TMTM

(a)

1400

20

40

60


FABP

3Ex

pres

sion

leve

l (au

)

lowastlowast

lowast

times103

No TMTM

(b)






1400

10

20

30

40

50


Myh1

Expr

essio

n le

vel (

au)

lowast

lowast

lowast

times103

(a)

1400

10

20

30

40

50


Myhbp

Expr

essio

n le

vel (

au)

lowast lowast

times103

(b)

1400

05

1

15

2


Myl2

Expr

essio

n le

vel (

au)

No TMTM

times103

(c)

No TMTM

1400

10

20

30

40


Myh3

Expr

essio

n le

vel (

au)

times103

(d)







0001

0002

0003

FST

expr

essio

n le

vel

lowast

lowast

(a)


001

002

003

SMA

D3

expr

essio

n le

vel

lowast

lowast

(b)


002

004

006

008

010

MYF

6 ep

ress

ion

leve

ls

lowast lowast

(c)



ActRIIb darr Bmpr2

Atrophy Hypertrophy

Smad2

Smad3 uarr

Smad6 darr

Smad4 uarr

Smad1 uarr

Smad5 uarrSmad8

(a)


ActRIIb Bmpr2 uarr

Atrophy Hypertrophy

Smad2

Smad3 darr

Smad6

Smad4

Smad1

Smad5Smad8

(b)












Disclaimer




Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















4 Discussion




0

02

04

06

08

1

Folli

stat

in ex

pres

sion

leve

l (au

)

times103

(a)


0

1

2

3

4

5

Smad3

Expr

essio

n le

vel (

au)

times103

(b)

0

02

04

06

08

Acvr2b

Expr

essio

n le

vel (

au)


lowast

lowastlowast

times103

(c)

0

05

1

15

Smad1

Expr

essio

n le

vel (

au)


lowast

times103

(d)

0

05

1

15

Bmpr2

Expr

essio

n le

vel (

au)


times103

(e)


0

005

01

015

02

025

Smad6

Expr

essio

n le

vel (

au)

lowast

times103

(f)


0

5

10

15

Jun

Expr

essio

n le

vel (

au)

times103

No TMTM

(g)


2

25

3

35

4

45

Smad4

Expr

essio

n le

vel (

au)

lowast

times103

No TMTM

(h)



1400

5

10

15

20

25


Myf6

Expr

essio

n le

vel (

au)

lowastlowast

times103

No TMTM

(a)

1400

2

4

6

8

10


Myog

Expr

essio

n le

vel (

au)

times103

No TMTM

(b)


1400

10

20

30

40


Lpl

Expr

essio

n le

vel (

au)

lowastlowast

lowast

times103

No TMTM

(a)

1400

20

40

60


FABP

3Ex

pres

sion

leve

l (au

)

lowastlowast

lowast

times103

No TMTM

(b)






1400

10

20

30

40

50


Myh1

Expr

essio

n le

vel (

au)

lowast

lowast

lowast

times103

(a)

1400

10

20

30

40

50


Myhbp

Expr

essio

n le

vel (

au)

lowast lowast

times103

(b)

1400

05

1

15

2


Myl2

Expr

essio

n le

vel (

au)

No TMTM

times103

(c)

No TMTM

1400

10

20

30

40


Myh3

Expr

essio

n le

vel (

au)

times103

(d)







0001

0002

0003

FST

expr

essio

n le

vel

lowast

lowast

(a)


001

002

003

SMA

D3

expr

essio

n le

vel

lowast

lowast

(b)


002

004

006

008

010

MYF

6 ep

ress

ion

leve

ls

lowast lowast

(c)



ActRIIb darr Bmpr2

Atrophy Hypertrophy

Smad2

Smad3 uarr

Smad6 darr

Smad4 uarr

Smad1 uarr

Smad5 uarrSmad8

(a)


ActRIIb Bmpr2 uarr

Atrophy Hypertrophy

Smad2

Smad3 darr

Smad6

Smad4

Smad1

Smad5Smad8

(b)












Disclaimer




Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



0

02

04

06

08

1

Folli

stat

in ex

pres

sion

leve

l (au

)

times103

(a)


0

1

2

3

4

5

Smad3

Expr

essio

n le

vel (

au)

times103

(b)

0

02

04

06

08

Acvr2b

Expr

essio

n le

vel (

au)


lowast

lowastlowast

times103

(c)

0

05

1

15

Smad1

Expr

essio

n le

vel (

au)


lowast

times103

(d)

0

05

1

15

Bmpr2

Expr

essio

n le

vel (

au)


times103

(e)


0

005

01

015

02

025

Smad6

Expr

essio

n le

vel (

au)

lowast

times103

(f)


0

5

10

15

Jun

Expr

essio

n le

vel (

au)

times103

No TMTM

(g)


2

25

3

35

4

45

Smad4

Expr

essio

n le

vel (

au)

lowast

times103

No TMTM

(h)



1400

5

10

15

20

25


Myf6

Expr

essio

n le

vel (

au)

lowastlowast

times103

No TMTM

(a)

1400

2

4

6

8

10


Myog

Expr

essio

n le

vel (

au)

times103

No TMTM

(b)


1400

10

20

30

40


Lpl

Expr

essio

n le

vel (

au)

lowastlowast

lowast

times103

No TMTM

(a)

1400

20

40

60


FABP

3Ex

pres

sion

leve

l (au

)

lowastlowast

lowast

times103

No TMTM

(b)






1400

10

20

30

40

50


Myh1

Expr

essio

n le

vel (

au)

lowast

lowast

lowast

times103

(a)

1400

10

20

30

40

50


Myhbp

Expr

essio

n le

vel (

au)

lowast lowast

times103

(b)

1400

05

1

15

2


Myl2

Expr

essio

n le

vel (

au)

No TMTM

times103

(c)

No TMTM

1400

10

20

30

40


Myh3

Expr

essio

n le

vel (

au)

times103

(d)







0001

0002

0003

FST

expr

essio

n le

vel

lowast

lowast

(a)


001

002

003

SMA

D3

expr

essio

n le

vel

lowast

lowast

(b)


002

004

006

008

010

MYF

6 ep

ress

ion

leve

ls

lowast lowast

(c)



ActRIIb darr Bmpr2

Atrophy Hypertrophy

Smad2

Smad3 uarr

Smad6 darr

Smad4 uarr

Smad1 uarr

Smad5 uarrSmad8

(a)


ActRIIb Bmpr2 uarr

Atrophy Hypertrophy

Smad2

Smad3 darr

Smad6

Smad4

Smad1

Smad5Smad8

(b)












Disclaimer




Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1400

5

10

15

20

25


Myf6

Expr

essio

n le

vel (

au)

lowastlowast

times103

No TMTM

(a)

1400

2

4

6

8

10


Myog

Expr

essio

n le

vel (

au)

times103

No TMTM

(b)


1400

10

20

30

40


Lpl

Expr

essio

n le

vel (

au)

lowastlowast

lowast

times103

No TMTM

(a)

1400

20

40

60


FABP

3Ex

pres

sion

leve

l (au

)

lowastlowast

lowast

times103

No TMTM

(b)






1400

10

20

30

40

50


Myh1

Expr

essio

n le

vel (

au)

lowast

lowast

lowast

times103

(a)

1400

10

20

30

40

50


Myhbp

Expr

essio

n le

vel (

au)

lowast lowast

times103

(b)

1400

05

1

15

2


Myl2

Expr

essio

n le

vel (

au)

No TMTM

times103

(c)

No TMTM

1400

10

20

30

40


Myh3

Expr

essio

n le

vel (

au)

times103

(d)







0001

0002

0003

FST

expr

essio

n le

vel

lowast

lowast

(a)


001

002

003

SMA

D3

expr

essio

n le

vel

lowast

lowast

(b)


002

004

006

008

010

MYF

6 ep

ress

ion

leve

ls

lowast lowast

(c)



ActRIIb darr Bmpr2

Atrophy Hypertrophy

Smad2

Smad3 uarr

Smad6 darr

Smad4 uarr

Smad1 uarr

Smad5 uarrSmad8

(a)


ActRIIb Bmpr2 uarr

Atrophy Hypertrophy

Smad2

Smad3 darr

Smad6

Smad4

Smad1

Smad5Smad8

(b)












Disclaimer




Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1400

10

20

30

40

50


Myh1

Expr

essio

n le

vel (

au)

lowast

lowast

lowast

times103

(a)

1400

10

20

30

40

50


Myhbp

Expr

essio

n le

vel (

au)

lowast lowast

times103

(b)

1400

05

1

15

2


Myl2

Expr

essio

n le

vel (

au)

No TMTM

times103

(c)

No TMTM

1400

10

20

30

40


Myh3

Expr

essio

n le

vel (

au)

times103

(d)







0001

0002

0003

FST

expr

essio

n le

vel

lowast

lowast

(a)


001

002

003

SMA

D3

expr

essio

n le

vel

lowast

lowast

(b)


002

004

006

008

010

MYF

6 ep

ress

ion

leve

ls

lowast lowast

(c)



ActRIIb darr Bmpr2

Atrophy Hypertrophy

Smad2

Smad3 uarr

Smad6 darr

Smad4 uarr

Smad1 uarr

Smad5 uarrSmad8

(a)


ActRIIb Bmpr2 uarr

Atrophy Hypertrophy

Smad2

Smad3 darr

Smad6

Smad4

Smad1

Smad5Smad8

(b)












Disclaimer




Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



0001

0002

0003

FST

expr

essio

n le

vel

lowast

lowast

(a)


001

002

003

SMA

D3

expr

essio

n le

vel

lowast

lowast

(b)


002

004

006

008

010

MYF

6 ep

ress

ion

leve

ls

lowast lowast

(c)



ActRIIb darr Bmpr2

Atrophy Hypertrophy

Smad2

Smad3 uarr

Smad6 darr

Smad4 uarr

Smad1 uarr

Smad5 uarrSmad8

(a)


ActRIIb Bmpr2 uarr

Atrophy Hypertrophy

Smad2

Smad3 darr

Smad6

Smad4

Smad1

Smad5Smad8

(b)












Disclaimer




Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











Disclaimer




Acknowledgments


References



































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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