down-regulation of akt/mammalian target of rapamycin signaling pathway in response to myostatin...

Down-Regulation of Akt/Mammalian Target ofRapamycin Signaling Pathway in Response toMyostatin Overexpression in Skeletal Muscle

Adel Amirouche, Anne-Cecile Durieux, Sebastien Banzet, Nathalie Koulmann,Regis Bonnefoy, Catherine Mouret, Xavier Bigard, Andre Peinnequin,and Damien Freyssenet

Pole de Recherche et d’Enseignement Supérieur Université de Lyon (A.A., R.B., D.F.), Universite Jean Monnet,Laboratoire de Physiologie de l’Exercice, Equipe d’accueil 4338, F-42023 Saint-Etienne, France; Institut d’Anatomie(A.-C.D.), Universite de Berne, 3000 Berne, Suisse; and Départment des Facteurs Humains (S.B., N.K., X.B.) etDepartement de Radiobiologie et Radiopathologie (C.M., A.P.), Centre de Recherche du Service de Sante des Armees,F-38702 La Tronche, France

Myostatin, a member of the TGF-� family, has been identified as a master regulator of embryonicmyogenesis and early postnatal skeletal muscle growth. However, cumulative evidence also sug-gests that alterations in skeletal muscle mass are associated with dysregulation in myostatin ex-pression and that myostatin may contribute to muscle mass loss in adulthood. Two major branchesof the Akt pathway are relevant for the regulation of skeletal muscle mass, the Akt/mammaliantarget of rapamycin (mTOR) pathway, which controls protein synthesis, and the Akt/forkhead boxO (FOXO) pathway, which controls protein degradation. Here, we provide further insights into themechanisms by which myostatin regulates skeletal muscle mass by showing that myostatin neg-atively regulates Akt/mTOR signaling pathway. Electrotransfer of a myostatin expression vectorinto the tibialis anterior muscle of Sprague Dawley male rats increased myostatin protein level anddecreased skeletal muscle mass 7 d after gene electrotransfer. Using RT-PCR and immunoblotanalyses, we showed that myostatin overexpression was ineffective to alter the ubiquitin-protea-some pathway. By contrast, myostatin acted as a negative regulator of Akt/mTOR pathway. Thiswas supported by data showing that the phosphorylation of Akt on Thr308, tuberous sclerosiscomplex 2 on Thr1462, ribosomal protein S6 on Ser235/236, and 4E-BP1 on Thr37/46 was attenuated7 d after myostatin gene electrotransfer. The data support the conclusion that Akt/mTOR signalingis a key target that accounts for myostatin function during muscle atrophy, uncovering a novel rolefor myostatin in protein metabolism and more specifically in the regulation of translation inskeletal muscle. (Endocrinology 150: 286–294, 2009)

Factors that decrease skeletal muscle mass can have profoundeffects on overall health and viability. Loss of muscle mass is

observed after musculoskeletal trauma, during aging, and inmany catabolic diseases such as cancer, diabetes, renal failure,respiratory insufficiency, or sepsis as well as neuromuscular dis-orders (1–4). The consequences of a reduction in muscle mass aremultiple and can include a reduction in strength and power out-

put, an increase in fatigability, and an increase in insulinresistance.

Myostatin (growth differentiation factor-8) is a TGF-� familymember that acts as a master negative regulator of skeletal mus-cle mass. Naturally occurring mutations as well as experimentalknockout of the myostatin gene lead to hypermuscular pheno-type (5–8). The biological action of myostatin is particularly well

ISSN Print 0013-7227 ISSN Online 1945-7170Printed in U.S.A.Copyright © 2009 by The Endocrine Societydoi: 10.1210/en.2008-0959 Received June 26, 2008. Accepted September 9, 2008.First Published Online September 18, 2008

Abbreviations: ARBP, acidic ribosomal phosphoprotein P0; CAPN2, calpain 2; CAPN3,calpain 3; CMV, cytomegalovirus; CycA, cyclophilin A; 4E-BP1, eukaryotic initiation fac-tor-4E binding protein1; FOXO, forkhead box protein O; MuRF-1, muscle ring finger-1;mTOR, mammalian target of rapamycin; Nedd4, neural precursor cell expressed develop-mentally down-regulated gene 4; PDK1, phosphoinositide-dependent protein kinase 1;TA, tibialis anterior; TBP, TATA box binding protein; TSC2, tuberous sclerosis complex 2;ZNF216, zinc finger protein 216.

G R O W T H F A C T O R S - C Y T O K I N E S

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illustrated in myostatin-null animals, which display approxi-mately a 2-fold increase in skeletal muscle mass (7). Later studieshave subsequently established that myostatin regulates the sizeand the number of muscle fibers by inhibiting myoblast prolif-eration and differentiation (9–14). The remarkable effects ofmyostatin on skeletal muscle development, together with theobservation that myostatin expression is increased in response tomuscle disuse (15–17), have suggested that myostatin may alsocontribute to the regulation of skeletal muscle mass in adult-hood. To test the biological relevance of myostatin as a regulatorof skeletal muscle mass, we previously overexpressed myostatinby gene electrotransfer into rat tibialis anterior (TA) muscle (18).We showed that in vivo myostatin overexpression induced adecrease in muscle mass, muscle fiber cross-sectional area, andmuscle protein content, thus identifying myostatin as a criticalregulator of skeletal muscle mass in adulthood.

Skeletal muscle mass is tightly regulated by the balance be-tween catabolic and anabolic processes. The ubiquitin-protea-some pathway has emerged as a critical pathway for the break-down of myofibrillar proteins in many forms of physiologicaland pathological conditions associated with severe muscle atro-phy and muscle remodeling. This is evidenced by the inductionof genes for several proteasomal subunits, forkhead box O(FOXO) transcription factors, and ubiquitin ligases such as atro-gin-1 and muscle ring finger-1 (MuRF-1) and increased proteinubiquitination and proteasome activities in response to musclemass loss (19–24). Importantly, muscle cachexia induced by iminjection of CHO cells overexpressing myostatin leads to an in-creased expression of atrogenes (atrogin-1 and MuRF-1) (25),suggesting potential functional relationships between myostatinsignaling and the regulation of the ubiquitin-proteasome pathway.

Regulation of muscle protein synthesis by the Akt/mamma-lian target of rapamycin (mTOR) signaling pathway is a criticalmediator of skeletal muscle hypertrophy and atrophy under avariety of experimental and physiological situations (for reviewsee Ref. 26). mTOR forms structurally and functionally twodistinct multiprotein complexes, mTORC1 consisting of mTOR,mLST8, and raptor, and mTORC2 consisting of mTOR,mLST8, SIN1, and rictor (recently reviewed in Ref. (27).mTORC1 is activated after a series of intracellular signaling cas-cades involving IGF-I, phosphatidylinositol 3-kinase, phosphoi-nositide-dependent protein kinase 1 (PDK1), and Akt. mTORC1phosphorylation by Akt regulates protein synthesis by phos-phorylating and inhibiting the translation inhibitor eukaryoticinitiation factor-4E binding protein 1 (4E-BP1) and by phos-phorylating and activating S6 kinase (p70S6k). Akt phosphor-ylates and inactivates tuberous sclerosis complex 2 (TSC2), re-leasing the inhibitory effect of TSC2 on mTORC1. mTORC1activation by Akt regulates protein synthesis by phosphorylatingand inhibiting the translation inhibitor, eukaryotic initiation fac-tor-4E binding protein 1 (4E-BP1), and by phosphorylating andactivatin S6 kinase (p70S6k). Finally, Akt phosphorylation bymTORC2 activates FOXO (28), thus preventing its nucleartranslocation and ultimately the expression of atrogenes such asatrogin-1 and MuRF-1 (23, 24). Regulation of FOXO transcrip-tional activity by Akt therefore establishes common reciprocalregulatory links between protein degradation and protein syn-

thesis. Importantly, myostatin has been shown to inhibit invitro the incorporation of [14C]leucine in C2C12 proliferatingmyoblasts (13), suggesting potential functional relationshipsbetween myostatin signaling and the regulation of proteintranslation.

Based on these observations, we wanted to determine whethermyostatin overexpression is able to coordinately regulate theubiquitin-proteasome pathway and mTOR pathway in skeletalmuscle. We report that myostatin overexpression was inefficientat inducing neither the expression of atrogenes (MuRF-1, atro-gin-1, Nedd4 and ZNF216) or at regulating the catalytic activ-ities of proteasome. By contrast, myostatin overexpression in-duced a marked down-regulation of Akt/mTOR signalingpathway. These findings uncover a novel role for myostatin in theregulation of translational potential in skeletal muscle.

Materials and Methods

Animal careMale Sprague Dawley rats (271.9 � 1.7 g, n � 16) were maintained

in the Animalerie Centrale de la Faculte de Medecine (Universite JeanMonnet, Saint-Etienne) under a constant 12-h light, 12-h dark cycle withfood and water ad libitum. The study was approved by the Comited’Ethique Animale de la Plate-Forme d’Experimentation Animale de laFaculte de Medecine (Universite Jean Monnet, Saint-Etienne).

Plasmid DNApcDNA-myostatin is the full-length murine myostatin cDNA into

pcDNA3.1-Zeo expression vector (11). pcDNA3.1-Zeo is the corre-sponding empty vector. pC3-Luc was generated by cloning the humanproteasome C3 subunit promoter fragment from �460 to �1 into thepGL2 basic luciferase reporter plasmid (29). pCMV-�-galactosidase wasfrom Clontech (Palo Alto, CA). Plasmids were amplified in JM109 bac-teria, purified (EndoFree Plasmid Mega Kit; QIAGEN, Valencia, CA),and dissolved in sterile endotoxin-free 0.9% saline solution.

Electrotransfer of plasmid DNAAnimals were anesthetized with an ip injection of sodium pentobar-

bital (60 mg/kg body weight). In a first experiment, the entire TA musclewas injected with 500 �g pcDNA-myostatin (0.625 �g/�l) by using a30-gauge needle. Contralateral TA muscle was similarly injected with thecorresponding empty vector (pcDNA3.1-Zeo). Thirty seconds after in-jection, eight trains of 500 electric pulses (100 �sec, 50 mA) were de-livered (30) by using GET42 electroporator (31). In a second experimentdesigned to determine whether myostatin transactivates the proteasomeC3 subunit promoter, the middle belly portion of TA muscle was injectedwith 50 �g pC3-Luc together with pcDNA-myostatin (50 �g). Con-tralateral TA muscle was similarly injected, but pcDNA3.1-Zeo was usedinstead of pcDNA-myostatin. pCMV-�-galactosidase (50 �g) was usedto correct for transfection efficiency. Thirty seconds after injection, eighttrains of 125 pulses (100 �sec, 50 mA) were applied.

Tissue collectionSeven days after gene electrotransfer, animals were anesthetized as

described above. TA muscles were removed, weighed, and stored at �80C for subsequent analyses. Animals were then killed by an overdose ofsodium pentobarbital.

Muscle fiber cross-sectional areaMuscle transverse sections (10 �m) were cut in a cryostat microtome

at �20 C and stained with hemalun-eosin-safran. For each muscle, eight

Endocrinology, January 2009, 150(1):286–294 endo.endojournals.org 287


to 11 photographs covering the entire muscle section were taken at �40magnification. Five fields consistently positioned across muscle sectionswere chosen, and the cross-sectional area of 60 fibers per fields wascounted (300 fibers per muscle). All analyses were performed with a lightmicroscope connected to a computerized image analysis system (NIHImage version 1.61).

mRNA isolation and reverse transcription reactionMuscle samples conditioned in RNALater (QIAGEN) were disrupted

in lysis buffer (QIAGEN) (5 �l/mg). mRNA was isolated from 10 mglysed tissue in 50 �l using MagNA Pure LC mRNA isolation kit II in aMagNA Pure LC instrument (Roche Applied Science, Indianapolis, IN).RT was carried out with 4 �l of mRNA solution in 10 �l using the ReverseTranscriptase Core Kit (Eurogentec, Angers, France) with 50 �M oli-go(deoxythymidine) 15 primer and ribonuclease inhibitor (2 IU).

Primer designPrimer design, optimization, and specificity checking were done as de-

scribed previously (32), with the following primer sequences: acidic ribo-somal phosphoprotein P0 (ARBP), forward 5�-CCTGCACACTCGCTTC-CTAGAG-3� and reverse 5�-CAACAGTCGGGTAGCCAATCTG-3�(GenBank NM_022402); atrogin-1, forward 5�-GCTTGTGCGATGT-TACCCAAGAA-3� and reverse 5�-GAAAGTGAGACGGAG-CAGCTCT-3� (GenBank NM_133521); calpain 2 (CAPN2), forward5�-GGTTTCAAGCTGCCCTGTCAAC-3� and reverse 5�-CCAAACACCG-CACAAAGTTGTC-3� (GenBank NM_017116); calpain 3 (CAPN3), for-ward 5�-CATCGACTTTGACAGCTTCATCTG-3� and reverse 5�-TGAT-GCCATCTCCATCCTTGTC-3� (GenBank NM_017116); cyclophilin A(CycA), forward 5�-TATCTGCACTGCCAAGACTGAGTG-3� and reverse5�-CTTCTTGCTGGTCTTGCCATTCC-3� (GenBank NM_017101);MuRF-1, forward 5�-GGGAACGACCGAGTTCAGACTATC-3� and re-

verse 5�-CCTTCACCTGGTGGCTGTTTTC-3� (GenBank NM_080903);neural precursor cell expressed developmentally down-regulated gene 4(Nedd4), forward5�-GAGGTTGTGGTCACCAACAAGAAC-3�andreverse5�-GCAGCCATTTGCTTCTGGATTC-3� (GenBank NM_012986); TATAboxbindingprotein(TBP),forward5�-GCCACGAACAACTGCGTTGAT-3�and reverse 5�-AGCCCAGCTTCTGCACAACTCTA-3� (GenBank XM_217785); zinc finger protein 216 (ZNF216), forward 5�-GCCCAAACCAAA-GAAGAACAGATG-3� and reverse 5�-TGCTTGTCAGAGTAACGGT-GAAGTC-3� (GenBank NM_001106356).

Real-time quantitative PCRPCR was carried out with LC Fast Start DNA Master SYBR Green kit

(Roche Applied Science) from 0.5 �l cDNA in 20 �l [4 mM MgCl2, 0.4�M of each primer (except ARBP 0.5 �M)]. PCR was performed using aLightcycler (Roche Applied Science) for 45 cycles at 95 C for 20 sec, 53C (CAPN2 and CAPN3), 55 C (Nedd4), 57 C (ARBP and ZNF216), 58C (CycA and TBP), 59 C (MuRF-1), or 60 C (atrogin-1) for 5 sec and afinal step of 10 sec at 72 C. Crossing point values were calculated fromLightcycler Software version 3.5 (Roche Applied Science) using the sec-ond-derivative maximum method. Quantification was achieved using apool of all the cDNA samples as calibrator according to the comparativethreshold cycle method (33) with efficiency correction (34) using geo-metric average of three internal validated control genes (CycA, ARBP,and TBP) (35). The gene stability ranking was TBP and CycA/ARBP. The0.131 pairwise variations of ARBP, CycA, and TBP were below the thresh-old (0.150) that requires the inclusion of an additional normalization gene.Therefore, ARBP, CycA, and TBP were used for normalization.

Protein isolation and enzyme assaysProtein extraction was performed according to Ref. 36. Protein con-

centration was spectrophotometrically measured at 750 nm. Proteasomeenzyme activities (chymotrypsin-like, trypsin-like,and caspase-like) were fluorometrically determined(�exc � 380 nm; �exc � 460 nm) (19, 22).

Protein isolation, firefly luciferase, and�-galactosidase assays

Proteins isolation and measurement of firefly lu-ciferase activity were performed as described previ-ously (31). To correct for interindividual variations intransfection efficiency, luciferase activity was normal-ized to ß-galactosidase activity (31).

Protein isolation and immunoblotanalyses

Muscle samples were homogenized at 4 C in 20vol buffer [50 mM Tris-HCl (pH 7.4), 100 mM NaCl,2 mM EDTA, 2 mM EGTA, 50 mM �-glycerophos-phate, 50 mM sodium fluoride, 120 nM okadaic acid,3 mM benzamidine, 1 mM phenylmethylsulfonyl flu-oride, 10 �g/ml aprotinin, 10 �g/ml leupeptin, 1%Triton X-100]. Homogenates were centrifuged at12,000 � g for 20 min at 4 C. Proteins (50 �g) weresubjected to SDS-PAGE and transferred to nitrocel-lulose membranes. Blots were incubated overnightat 4 C with antibodies against Akt (1:1500; CellSignaling Technology, Beverly, MA), phospho-AktThr308 (1:1000; Cell Signaling Technology),phospho-AktSer473 (1:1500; Cell Signaling Technol-ogy), CAPN2 (1:1600; Abcam, Cambridge, MA),CAPN3 (1:100; Novocastra, Newcastle upon Tyne,UK), phospho-4E-BP-1Thr37/46 (1:1500; Cell Signal-ing Technology), FOXO1a (1:1000; Cell SignalingTechnology), phospho-FOXO1Ser256 (1:1000; CellSignaling Technology), mTOR (1:1500; Cell Signal-ing Technology), phospho-mTORSer2448 (1:1500;

FIG. 1. Myostatin overexpression induces skeletal muscle atrophy. A myostatin expression vector(pcDNA-Mstn, 500 �g) or the corresponding empty vector (pcDNA3.1-Zeo, 500 �g) waselectrotransfected into the TA muscle. Muscles were removed 7 d after gene electrotransfer. A,Myostatin is detected as a 26-kDa band corresponding to the processed dimer, and a fainter 50- to 52-kDa band that corresponds to the unprocessed full-length 375-amino-acid protein. The blot was alsoprobed with anti-�-tubulin to show gel loading. Also shown is the quantification of myostatin proteinlevel (processed dimer at 26 kDa). B, Comparison of representative samples of dissected skeletal TAmuscles. C, TA muscle weight. Values are means � SE (n � 6–8 per group). *, P � 0.05; **, P � 0.01relative to corresponding contralateral muscles.

288 Amirouche et al. Skeletal Muscle Myostatin and Akt/mTOR Signaling Endocrinology, January 2009, 150(1):286–294


Cell Signaling Technology), myostatin (1:250, sc-34781; Santa Cruz Bio-technology, Santa Cruz, CA), phospho-ribosomal protein S6Ser235/236

(1:1000; Cell Signaling Technology), TSC2 (1:250, sc-893; Santa CruzBiotechnology), phospho-TSC2Thr1462 (1:1000; Cell Signaling Technol-ogy), �-tubulin (1:3000; Abcam), and ubiquitin (1:400; BioMol Inter-national, Plymouth Meeting, PA). Incubation with corresponding horse-radish peroxidase-conjugated rabbit antimouse (1:2500; Dako,Carpinteria, CA), goat antirabbit (1:2500; Dako), rabbit antigoat (1:2000;Sigma Chemical Co., St. Louis, MO) antibodies was used for chemilumi-nescent detection of proteins (ECL; Amersham, Piscataway, NJ). The filmswere scanned and quantified using NIH Image version 1.63.

StatisticsData are means � SE (n � 6–8 per group). Statistical comparisons

were performed using paired t test (StatView SE�Graphics; Abacus Con-

cept, Inc., Piscataway, NJ). The 0.05 level of confidence was accepted forstatistical significance.

Results

In vivo myostatin gene electrotransfer leads tomyostatin overexpression

Whole TA muscles were electrotransfected by an im injectionof a plasmid DNA encoding the murine myostatin cDNA underthe control of a cytomegalovirus (CMV) promoter. Contralat-eral TA muscles were injected with the corresponding emptyvector. Myostatin protein expression was strongly increased in

response to myostatin gene electrotransfer (Fig. 1A).The antibody used for Western blot analysis allowsdetection of both precursor and mature forms ofmyostatin: a faint 50- to 52-kDa band correspondsto the unprocessed full-length 375-amino-acid pro-tein and the major 26-kDa band corresponds to theprocessed dimer, i.e. the active form of myostatin.This characteristic pattern was observed both inmuscles injected with control empty vector and myo-statin expression vector. However, the 75% increaseof the 26-kDa band after gene electrotransfer (P �

0.05) indicated that the construct was efficientlytranscribed leading to myostatin overexpression.

Myostatin overexpression induces atrophy ofTA muscle

TA muscles overexpressing myostatin weresmaller in size and weight (Fig. 1, B and C). Sevendays after myostatin gene electrotransfer, TA muscleweights were decreased by about 10% (P � 0.01)when compared with contralateral TA muscles. Ac-cordingly, muscle fiber cross-sectional area was alsosignificantly decreased from 2543 � 136 �m2 in con-tralateral muscles to 1805�123�m2 inmusclesover-expressing myostatin (P � 0.001). Taken together andin agreement with our previous report (18), these dataclearly demonstrate that myostatin overexpressionby gene electrotransfer induced atrophy of skeletalmuscle.

Myostatin overexpression does not alter theubiquitin-proteasome pathway butdifferentially regulates CAPN2 and CAPN3

One of the major systems involved in the regula-tion of muscle mass is the ubiquitin-proteasomepathway (37). Proteins degraded by the ubiquitin-proteasome pathway are first covalently bound toubiquitin, a process that is partly regulated by ubiq-uitin ligases (E3) (37). Several E3s have been identi-fied to be involved in skeletal muscle atrophy, in-cluding atrogin-1 (20, 38), MuRF-1 (20), and morerecently Nedd4 (39). Surprisingly, mRNA levels ofatrogin-1, MuRF-1, and Nedd4 remained un-changed in response to myostatin overexpression

FIG. 2. Ubiquitin-proteasome pathway in response to myostatin gene electrotransfer. Amyostatin expression vector (pcDNA-Mstn, 500 �g) or the corresponding empty vector(pcDNA3.1-Zeo, 500 �g) was electrotransfected into the TA muscle. A, RT-PCR analysis wasused to quantify MuRF-1, atrogin-1, Nedd4, and ZNF216 mRNA level. B, Quantification ofphosphorylated FOXO1a at Ser256 and total FOXO1a protein. Also shown are representativeimmunoblots. The same blot probed with anti-�-tubulin shows the equal loading of protein. C,A plasmid containing the human proteasome C3 subunit promoter fragment from �460 to �1into the pGL2 basic luciferase reporter plasmid (50 �g) was electrotransfected together with anexpression plasmid containing the myostatin expression vector (50 �g) or the correspondingempty vector (50 �g). pCMV-�-galactosidase (50 �g) was used to correct for transfectionefficiency. Luciferase and �-galactosidase activities were measured 7 d later. Luciferase activitywas normalized to �-galactosidase activity and expressed as percentage of contralateral musclevalues. D, Chymotrypsin-like, trypsin-like, and caspase-like enzyme activities of proteasome werefluorometrically determined. E, Immunoblot analysis of protein ubiquitination. Representativeimmunoblots are shown in the different conditions. Values are means � SE (n � 6–8 pergroup). *, P � 0.05 relative to corresponding contralateral muscles.



(Fig. 2A). The mRNA level of ZNF216, a ubiquitin-binding pro-tein (40), also remained unchanged. Furthermore, protein levelof FOXO1a, a transcriptional activator of atrogin-1 expression(23), as well the phosphorylated inactive form of FOXO1a re-mained unchanged (Fig. 2B). Promoter transactivation of theproteasome C3 subunit, an �-subunit of the core proteolyticcomplex, was even reduced in response to myostatin overexpres-sion (Fig. 2C). Finally, proteasome enzyme activities (Fig. 2D) aswell as protein ubiquitination profile (Fig. 2E) all remained un-changed in response to myostatin overexpression. Therefore,from the analysis of upstream regulators of proteasome(FOXO1a, atrogin-1, MuRF-1, Nedd4, and ZNF216) and pro-teasome components (C3 subunit, trypsin-like, caspase-like, andchymotrypsin-like activities) as well as ubiquitination profile,the picture emerging is that muscle mass loss induced by myo-statin overexpression does not involve up-regulation of protea-somal degradation.

To further determine the potential role of other proteolyticpathways in response to myostatin overexpression, we nextdetermined the mRNA level of the Ca2�-activated proteases,CAPN2 and CAPN3 (Fig. 3A). CAPN2 mRNA level was in-creased in response to myostatin overexpression (P � 0.05).By contrast, CAPN3 mRNA level was significantly decreased.However when measured at the protein level, only CAPN3remained significantly affected by myostatin overexpression(Fig. 3B). The CAPN3 antibody recognizes a 90- to 95-kDaprotein corresponding to the expected molecular weight of thefull-length enzyme. Bands at 55– 60 and 35– 40 kDa were alsodetected, which presumably correspond to autolysis products(41). Only the full-length enzyme was significantly decreasedby myostatin overexpression.

Myostatin overexpression down-regulates Akt/mTORsignaling

The Akt/mTOR signaling pathway plays a major role in theregulation of protein synthesis by controlling the phosphoryla-tion level of several translation factors in multiple situations as-sociated with gain or loss of muscle mass (reviewed in Refs. 26and 42). A decrease in Akt/mTOR signaling pathway may betherefore a mechanism contributing to muscle mass loss in re-sponse to myostatin overexpression. Total protein content ofAkt/mTOR signaling intermediates such as Akt, TSC2, and mTORremained unchanged (Fig. 4A). Akt phosphorylation at Thr308,which is a direct target of PDK1, was significantly decreased byabout 25% (P � 0.05) 7 d after myostatin gene electrotransfer (Fig.4B). Importantly, Akt phosphorylation at Ser473, which is not di-rectly phosphorylated by PDK1, but is rather the target ofmTORC2 (28), remained unchanged. Although no significantchange was noted for mTOR phosphorylation on Ser2448, phos-phorylation of TSC2Thr1462, ribosomal protein S6Ser235/236, and4E-BP1Thr37/46 were all significantly decreased in response to myo-statin gene electrotransfer. Particularly, S6 phosphorylation atSer235/236 was decreased by about 40%. Finally, the protein levelof regulated in development and DNA damage response 1, a neg-ative regulator of mTOR (43), was not modified in response tomyostatin overexpression (data not shown). Taken together, thesedata suggest that myostatin overexpression down-regulates theAkt/mTOR signaling pathway in skeletal muscle.

Discussion

Cumulative evidence strongly suggests that alterations in skeletalmuscle mass are associated with dysregulation in myostatin ex-

pression (15–17) and that myostatin may contributeto muscle mass loss (18). However, whether thesechanges involve alterations in protein degradationand synthesis is currently unknown. Here, we inves-tigated the molecular response of the ubiquitin-pro-teasome and Akt/mTOR pathways in response tomyostatin overexpression in skeletal muscle. Ourstudy shows that myostatin overexpression does notalter the ubiquitin-proteasome pathway but identi-fies myostatin as a negative regulator of the Akt/mTOR pathway. Altogether these data uncover anovel role for myostatin in protein metabolism and,more specifically, in the regulation of proteinsynthesis.

A previous study in male Fischer 344 rats haddescribed that electrotransfer of a myostatin shorthairpin RNA plasmid, which reduced myostatinmRNA and protein expression, significantly de-creased TA muscle weight and fiber size (44). Bycontrast, the present study was designed to force theexpression of myostatin in rat TA skeletal muscle bygene electrotransfer of a myostatin expression plas-mid. Seven days after gene electrotransfer, myostatinexpression was increased by 1.8-fold and musclemass was decreased by 10%, illustrating that the

FIG. 3. Expression of CAPN2 and CAPN3 in response to myostatin gene electrotransfer. TAmuscles electrotransfected with a myostatin expression vector (pcDNA-Mstn, 500 �g) or thecorresponding empty vector (pcDNA3.1-Zeo, 500 �g) were removed 7 d after geneelectrotransfer. A, CAPN2 and CAPN3 mRNA levels were determined by RT-PCR analysis. B,Quantification of CAPN2 and CAPN3 protein level. Also shown are representative immunoblots.The same blot probed with anti-�-tubulin indicates gel loading. CAPN3 was detected as a full-size CAPN3 at 90–95 kDa and two degradation products at 55–60 and 30–35 kDa (41). Valuesare means � SE (n � 6–8 per group). *, P � 0.05; **, P � 0.01 relative to correspondingcontralateral muscles.



present protocol could be used as a powerful in vivo model tostudy the mechanisms involved in myostatin-induced skeletalmuscle atrophy. Our data are also in agreement with a previousreport showing that male transgenic mice that selectively over-express myostatin protein in skeletal muscle had 18–24% lowerhind- and forelimb muscle weight and 18% reduction in quad-riceps and gastrocnemius fiber cross-sectional area when com-pared with wild-type male mice (45).

One frequent feature of muscle atrophy is an increaseddegradation of muscle proteins through the activation of theubiquitin-proteasome pathway (reviewed in Refs. 26, 42, and46). In the present study, we failed to detect any increase in theexpression (mRNA or protein level) of upstream regulators ofproteasome including FOXO1a, atrogin-1, MuRF-1, Nedd4,and ZNF216 as well as proteasome components (transacti-vation of C3 subunit and trypsin-, caspase-, and chymotryp-sin-like enzyme activities) or ubiquitination profile, clearlyindicating that muscle mass loss induced by myostatin over-expression does not involve up-regulation of proteasomalfunction. This may apparently contrast with previous reportsshowing that an increase in the expression of atrophy-relatedgenes (atrogin-1 and MuRF-1) occurs in mice injected im withCHO cells expressing myostatin (25) and that knockout of

myostatin gene prevents the expression of atro-genes in response to dexamethasone treatment(47). The differences between these experimentaldesigns (muscle cachexia induced by im injectionof cells expressing myostatin in mice and dexa-methasone treatment in myostatin knockout mice)and the one used in the present study (gene elec-trotransfer of a myostatin expression vector in rat)may explain the apparent discrepancies betweenthese studies and our results. First, the possibilitythat activation of ubiquitin-proteasome pathwaycould be detected only after a long-term myostatintreatment (25) could be considered. Second, wecannot also totally exclude the possibility thatmyostatin could differentially influence proteaso-mal function in mice and rats. However, the mainconcern is that muscle cachexia as well as dexa-methasone treatment are both well known to trig-ger ubiquitin-proteasome-dependent degradation(20, 21, 48). Particularly, overexpression of myo-statin by CHO cells (25) substantially contributeto muscle atrophy by secreting some cachectic fac-tors that may increase proteasomal degradation,suggesting that the myostatin pathway is synergis-tic with other pathways in this study (49).

As previously reported by others in skeletalmuscle under different situations, including exper-imentally induced muscle cachexia (50) and pro-gressive dystrophy and amyotrophic lateral scle-rosis (51) as well as eccentric exercise-inducedmuscle damage (52), we found an increase inCAPN2 mRNA level and a decrease in CAPN3mRNA level. These observations have led to theconcept that CAPN3 could negatively regulate

CAPN2 and alternatively that CAPN2 could negatively reg-ulate CAPN3. However, such opposite regulation was notreproduced at the protein level because CAPN3 protein levelwas still slightly but significantly decreased, whereas CAPN2protein level remained unchanged, suggesting the existence ofa posttranscriptional regulation of CAPN2 expression. A reductionin CAPN3 mRNA or protein level has been previously reported inconditionassociatedwithmusclemass loss suchasdenervation (53,54) and cachexia (50, 55). Importantly, the recovery of skeletalmuscle mass after unloading partly depends on CAPN3 expression(56). In CAPN3-deficient mice, a boost in muscle mass and an in-crease in absolute force were obtained in response to the inhibitionof myostatin by adeno-associated virus-mediated expression of amutated propeptide (57). The decrease in CAPN3 expression bymyostatin overexpression may be therefore a possible mech-anism that contributes to the effect of myostatin on skeletalmuscle loss. More studies are clearly needed to obtain a pic-ture of the role of CAPN3 in response to myostatin overex-pression. Finally, the possibility that other proteolytic sys-tems, such as CAPN1 and the autophagy-lysosome system,can be involved in response to myostatin overexpression alsoneeds to be investigated.

FIG. 4. Down-regulation of Akt/mTOR signaling pathway in response to myostatinoverexpression. TA muscles were electrotransfected with either a myostatin expression vector(pcDNA-Mstn, 500 �g) or the corresponding empty vector (pcDNA3.1-Zeo, 500 �g). Muscleswere removed 7 d after gene electrotransfer and analyzed for protein level by immunoblotting.A, Immunoblot quantification using antibodies recognizing Akt, TSC2, mTOR, and �-tubulin.Representative immunoblots are also shown. B, Immunoblot quantification using phospho-AktSer473, phospho-AktThr308, phospho-TSC2Thr1462, phospho-mTORSer2448, phospho-ribosomalprotein S6Ser235/236, phospho-4E-BP-1Thr37/46, and �-tubulin antibodies. Representativeimmunoblots are also shown. Values are means � SE (n � 6–8 per group). *, P � 0.05; **, P �0.01 relative to corresponding contralateral muscles.



A striking feature of the present study was the markedattenuation of Akt/mTOR pathway in response to myostatinoverexpression. Whereas protein level of Akt, TSC2, andmTOR remained unchanged, the phosphorylation status ofAktThr308, TSC2Thr1462, 4E-BP1Thr37/46, and ribosomal pro-tein S6Ser235/236 were decreased. One should note that Aktphosphorylation at Ser473 remained unchanged. However,recent evidence suggests that although phosphorylation at thissite is a good estimate of Akt activity, Ser473 of Akt is notphosphorylated by PDK1 but is rather the target of mTORC2(28), the multiprotein complex consisting of mTOR, mLST8,SIN1, and rictor. Therefore, regulation of Akt activity in re-sponse to myostatin overexpression would be more dependenton PDK1 activity rather than mTORC2. Surprisingly, mTORphosphorylation at Ser2448 remained statistically unchanged.Therefore, our results do not support a response of this site-specific phosphorylation of Ser2448 in response to myostatinoverexpression. However, this finding does not preclude theinvolvement of mTOR-mediated signaling. Previous researchhas demonstrated that mutation of this phosphorylation siteto a nonphosphorylated alanine residue does not prevent theinsulin or Akt stimulation of S6K and 4E-BP1 phosphoryla-tion in HEK293 cells (58). This may therefore explain whyboth S6 and 4E-BP1 are less phosphorylated together with theobservation that mTOR phosphorylation at Ser2448 is un-changed. Furthermore, the occurrence of other mTOR phos-phorylation sites, such as Thr2446, which integrate negativeregulatory influence from AMP-activated protein kinase (59),or Ser2481, which is an autophosphorylation whose functionis unknown (60), can participate to regulate mTOR activity.Taken together, these data indicate a reduced signaling flux(reduction in phosphorylation level) of the pathway in re-sponse to 7 d myostatin overexpression. Our data are in agree-ment with the observation that myostatin inhibited in vitro theincorporation of [14C]leucine in C2C12 myoblasts (13) andare consistent with a down-regulation of muscle protein syn-thesis in response to myostatin overexpression. Dissecting theroles of myostatin Smad signaling and identifying the cofac-tors that mediate the Smad-dependent regulation of Akt/mTOR signaling are intriguing questions that require atten-tion in the coming years.

Stimulation of Akt is also known to coordinately regulatemuscle protein synthesis and degradation (through FOXO inhi-bition) (23, 61, 62). However, we did not observe such a coor-dinated regulation. This differential regulation between proteinsynthesis and degradation may indicate the existence of distinctregulatory influence on the pathway depending on the biologicaland/or experimental context. Indeed, Akt phosphorylation atSer473 measured in the present study is rather the target ofmTORC2 and mTORC2-dependent phosphorylation of Akt atSer473 is required for the regulation of FOXO (28). In thepresent study, the unchanged phosphorylation status of Akt atSer473 could thus explain the unchanged phosphorylation ofFOXO1a and the unchanged expression of atrogens. BecauseAkt phosphorylation at Ser473 is the target of mTORC2 (28),our data also suggest that mTORC2 would be insensitive tomyostatin overexpression.

In conclusion, our findings in this report establish a novellink between myostatin and Akt/mTOR signaling pathwaythat potentially contributes to the regulation of translation inskeletal muscle. These results combined with the observationthat myostatin expression is altered in many physiological andpathological situations suggest that protein translation maybe also down-regulated in these situations.

Acknowledgments

Henri Benoit, Aurelia Defour, Francois Favier, Vanessa Jahnke, andAndrea Perez are gratefully acknowledged for their comments. FabriceCognasse (Groupe Immunité des Muqueuses et Agents PathogènesEquipe d’Accueil 3064), Bodvael Fraysse (Institut National de la Sante dela Recherche Medicale Unite 582, Institut de Myologie), and LeonardFeasson (Laboratoire de Physiologie de l’Exercice Equipe d’Accueil4338) are acknowledged for the kind provision of �-tubulin, CAPN2,and CAPN3 antibodies, respectively. Romain Saulnier is acknowledgedfor his help in animal care.

Address all correspondence and requests for reprints to: DamienFreyssenet, Laboratoire de Physiologie de l’Exercice, Faculte de Me-decine, 15 rue Ambroise Pare, 42023 Saint Etienne Cedex 2, France.E-mail: [email protected].

This work was supported by the Association Francaise contre lesMyopathies Grant 2005.0214 (to D.F.). A.A. is supported by an Allo-cation Doctorale de Recherche de la Region Rhone-Alpes (Cluster 11,Handicap Vieillissement Neurosciences).

Disclosure Statement: The authors have nothing to disclose.

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