Akt activation prevents the force drop induced by eccentric contractions in dystrophin-deficient...

Akt activation prevents the force drop inducedby eccentric contractions in dystrophin-deficientskeletal muscle

Bert Blaauw1,3, Cristina Mammucari1,3, Luana Toniolo2, Lisa Agatea1,3, Reimar Abraham3,

Marco Sandri1,3,4, Carlo Reggiani2 and Stefano Schiaffino1,3,�

1Department of Biomedical Sciences, CNR Institute of Neurosciences and 2Department of Human Anatomy

and Physiology, University of Padova, Padova, Italy, 3Venetian Institute of Molecular Medicine (VIMM), Padova,

Italy and 4Dulbecco Telethon Institute, Modena, Italy

Received July 16, 2008; Revised and Accepted August 24, 2008

Skeletal muscles of the mdx mouse, a model of Duchenne Muscular Dystrophy, show an excessive reductionin the maximal tetanic force following eccentric contractions. This specific sign of the susceptibility ofdystrophin-deficient muscles to mechanical stress can be used as a quantitative test to measure the efficacyof therapeutic interventions. Using inducible transgenesis in mice, we show that when Akt activity is increasedthe force drop induced by eccentric contractions in mdx mice becomes similar to that of wild-type mice. Thiseffect is not correlated with muscle hypertrophy and is not blocked by rapamycin treatment. The force dropinduced by eccentric contractions is similar in skinned muscle fibers from mdx and Akt-mdx mice when stretchis applied directly to skinned fibers. However, skinned fibers isolated from mdx muscles exposed to eccentriccontractions in vivo develop less isometric force than wild-type fibers and this force depression is completelyprevented by Akt activation. These experiments indicate that the myofibrillar-cytoskeletal system of dystro-phin-deficient muscle is highly susceptible to a damage caused by eccentric contraction when elongation isapplied in vivo, and this damage can be prevented by Akt activation. Microarray and PCR analyses indicatethat Akt activation induces up-regulation of genes coding for proteins associated with Z-disks and costameres,and for proteins with anti-oxidant or chaperone function. The protein levels of utrophin and dysferlin are alsoincreased by Akt activation.

INTRODUCTION

Eccentric exercise, such as downhill running, is known tocause prolonged weakness and muscle soreness resultingfrom stretch-induced muscle damage. Fast muscles aremore susceptible than slow muscles and fast muscles ofthe dystrophin-deficient mdx mouse, a model of DuchenneMuscular Dystrophy, are especially sensitive to damagecaused by eccentric contractions. Mild protocols of lengtheningcontractions, which do not affect force production in normalmouse muscles, cause a marked reduction in the maximaltetanic force produced by mdx tibialis anterior and extensordigitorum longus (EDL) but not by mdx soleus (1–3). Asimilar force drop has been observed in dystrophin-deficient

canine muscles (4). The force deficit caused by eccentriccontractions has been variably attributed to alterations atthe plasma membrane (5), membrane systems involved inexcitation–contraction coupling (6,7), myofibrils (8) and cyto-skeleton (9); however, the problem is still unresolved.

A current view is that exercise-induced injury is due toplasma membrane disruptions that produce both efflux ofcell constituents, such as creatine kinase (CK), into theblood and influx of serum components, such as albumin,into skeletal muscle fibers (10). Focal disruptions of the sarco-lemma were first detected by electron microscopy in somenon-necrotic fibers in Duchenne muscular dystrophy in 1975(11). A primary function of dystrophin would be to providemechanical reinforcement to the sarcolemma and thereby

�To whom correspondence should be addressed at: Venetian Institute of Molecular Medicine (VIMM), Via Orus 2, 35126 Padova, Italy.Tel: þ39 0497923232; Fax: þ39 0497923250; Email: [email protected]

# The Author 2008. Published by Oxford University Press. All rights reserved.For Permissions, please email: [email protected]

Human Molecular Genetics, 2008, Vol. 17, No. 23 3686–3696doi:10.1093/hmg/ddn264Advance Access published on August 27, 2008

by guest on October 7, 2013

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protect it from the membrane stresses developed duringmuscle contraction. Increased susceptibility of mdx musclesto sarcolemmal ruptures was revealed by the uptake of IgG(3) and of dyes that do not penetrate intact fibers, such asProcion Red (2), fluorescein-dextran (12) and Evans blue(13). An alternative hypothesis is that the reduction of forceassociated with the stretch contractions is dependent on theopening of stretch-activated ion channels (14). These channelsappear to be more sensitive to the effect of stretch contractionsin mdx compared to wild-type mouse, and blockers of thesechannels have been reported to improve force and reducemembrane permeability in mdx muscles (15). A commonmechanism for the force drop induced either by membranetears or by changes in surface channels could be an increasein intracellular calcium and consequent cell damage mediatedby calcium-activated proteases, such as calpains, or by othermeans. Alterations in the myofibrils, such as focal disinte-gration of sarcomere structure and Z-disk streaming, havebeen described following stretch contractions and might con-tribute to the immediate weakness induced by eccentric con-tractions. However, muscle fibers from mdx mice are notespecially susceptible to eccentric contractions when theseare applied directly in vitro to skinned fibers, suggestingthat dystrophic symptoms do not arise from factors withinmyofibrils (16).

Stimulation of muscle growth has been explored as a stra-tegy to prevent muscle wasting and increase muscle strengthin dystrophin-deficient skeletal muscle. Loss of myostatin, anegative regulator of muscle growth that causes muscle hyper-trophy, attenuates the severity of muscular dystrophy in mdxmice (17). Increased muscle strength was induced in mdxmuscles by treatment with anti-myostatin antibodies (18) orby pharmacological blockade with a myostatin propeptide(19). However, reduction in muscle force induced by lengthen-ing contractions was not improved by myostatin blockade(18). Indeed, following two lengthening contractions, fastmuscles from myostatin null mice had a greater force deficitcompared to control mice (20). Muscle force was likewiseincreased in mdx muscles by IGF-1 treatment (21) or by cross-ing mdx mice with transgenic mice expressing a local form ofIGF-1 (mIGF-1) in skeletal muscle (22). The decrement inmuscle force caused by eccentric contractions was similar inmdx and mdx-IGF-1 transgenic mice when compared at thesame stimulation frequency, whereas some protection againstdamage was afforded when comparisons were made at equi-valent forces (different stimulation frequency) (22).

IGF-1 activates Akt/PKB kinase and the increased phos-phorylation of Akt has been demonstrated in mdx-IGF-1 trans-genic mice (22), suggesting that the Akt pathway may beinvolved in mediating the beneficial effects of IGF-1. ActivatedAkt induces muscle hypertrophy in vivo by stimulating proteinsynthesis via the kinase mTOR (23–25) and blocks proteindegradation by inhibiting the transcription factor FoxO, whichcontrols both the ubiquitin-proteasome pathway (26,27) andthe autophagy-lysosome pathway (28,29). However, the effectof Akt activation in dystrophin-deficient muscles has not beenexplored. Here, we have used an inducible muscle-specificAkt transgenic model to determine whether Akt activation isable to reduce the decrease in force induced by lengtheningcontractions in mdx skeletal muscle. We find that activated

Akt prevents both the force drop of whole muscles in vivoand also the force drop of skinned fibers isolated from mdxmuscles exposed to eccentric contractions in vivo. The effectof Akt is not correlated with muscle hypertrophy and is notdependent on mTOR. Gene expression analyses show that acti-vated Akt induces increased protein level of utrophin and dys-ferlin and up-regulation of genes coding for proteinsassociated with Z-disks and costameres, as well as proteinswith anti-oxidant or chaperone function.

RESULTS

Generation of mdx mice bearing an inducible activatedAkt in skeletal muscle

Mice expressing an Akt inducible transgene in skeletal musclewere obtained by crossing a transgenic line bearing a silentAkt-ER, containing Akt1 fused to a src myristoylation signaland a mutated estrogen receptor hormone binding domain,with a transgenic line expressing Cre recombinase under thecontrol of a myosin light chain 1 fast promoter (28). Thesemice were crossed with mdx mice resulting in an inducibleAkt transgenic line in a mdx background, hereafter referred toas Akt-mdx (Fig. 1A). Adult (about 3-month-old) Akt-mdxmice were treated for 3 weeks every other day either withtamoxifen, to induce Akt activation, or with oil vehicle ascontrol. Akt activation following tamoxifen treatment wasdemonstrated using antibodies specific for phospho-Akt S473(Fig. 1B). This activation occurs in both fast and slow skeletalmuscles of Akt-mdx mice, since the myosin light chain 1 fastpromoter driving the Cre recombinase is active in all murineskeletal muscles during early development (30).

Akt activation leads to muscle hypertrophy but does notaffect the pathological phenotype of mdx muscles

As shown in Figure 1C, Akt activation for 3 weeks led to avariable increase in muscle weight in the gastrocnemius(þ22+ 9%), EDL (þ84+ 17%) and soleus muscles(þ54+ 17%), as well as other skeletal muscles examined,including the diaphragm (not shown). During this period,body weight increased by 20+ 3% in the tamoxifen-treatedgroup when compared with 7+ 2% in control animals.Muscle fiber size was correspondingly increased in tamoxifen-compared to oil-treated mice; however, fiber size was evenmore variable compared to control due to the large hyper-trophy of some fibers (Fig. 1D). The proportion of fiberswith central nuclei was similar in tamoxifen- and oil-treatedanimals (Fig. 1E). After Evans blue injection, a wide variabi-lity in the distribution of positive fibers among differentmuscles and among different areas of the same muscle wasobserved in both experimental groups, similar to thatdescribed in mdx mice (31). However, we noted a consistenttendency for positive fibers to be grouped in clusters in oil-treated mice, as in mdx mice, whereas positive fibers wereusually diffusely distributed in tamoxifen-treated mice (Sup-plementary Material, Fig. 1A). A similar staining patternwas observed using antibodies against IgG (SupplementaryMaterial, Fig. 1A). The proportion of Evans blue positivefibers was similar in tamoxifen- and oil-treated mice.

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No significant change was likewise seen in the proportion ofregenerating fibers or type I and IIA fibers, as determinedby immunocytochemistry with myosin heavy chain-specificantibodies (not shown). Serum creatine kinase levels werenot significantly different between oil- and tamoxifen-treatedmice (Supplementary Material, Fig. 1B).

Akt activation does not increase muscle force but preventsthe force deficit induced in mdx muscles by eccentriccontractions

Gastrocnemius muscles from tamoxifen- and oil-treated micewere stimulated through the nerve at different frequencies,ranging from a single twitch to a completely fused tetanus, toassess force production. As shown in Figure 2A, no significantdifference in force was observed when force was normalizedfor muscle mass. Twitch properties (time to peak and half-relaxation) were also identical in the two groups (not shown).

Repeated lengthening (eccentric) contractions are known tolead to a decrease in isometric force in fast muscles, which ishigher in dystrophic than in wild-type muscle. We used a pro-tocol of 20 eccentric contractions applied in vivo, as describedin the materials and Methods section, and assessed the forcedeficit, namely the decrease in maximum force expressed asa percentage of initial force. As shown in Figure 2B, this pro-tocol led to a force drop of 38+ 3% in the gastrocnemiusmuscles of mdx mice but only 14+ 1% in control muscles(Fig. 2B). Representative records of the responses of controland mdx muscles are illustrated in Supplementary Material,Fig. 2. To ascertain that this force drop was not due to meta-bolic fatigue, we performed the same protocol withoutlengthening the muscle and found no significant decrease inisometric force (not more than 5%) either in control or dys-trophic muscle (Fig. 2E).

Next, we repeated the eccentric contraction protocol in theAkt-mdx mice. Mice treated with oil vehicle showed a 34+4%decrease in isometric force, a reduction comparable to thatmeasured in mdx mice. In contrast, tamoxifen treatmentcompletely prevented the additional force drop induced by

Figure 2. Effect of Akt activation on muscle force production and response toeccentric contractions in mdx mice. (A) Force measurements performed in vivoshow that Akt activation leads to no significant change in normalized force ofthe gastrocnemius muscle in tamoxifen-treated mice (n ¼ 20). (B) In vivorecordings during eccentric contractions reveal a much greater force drop(measured as percent of initial force after 20 eccentric contractions in vivo)in mdx compared to wild-type C57 muscles (n ¼ 18). (C) Eccentric contrac-tions in oil- and tamoxifen-treated Akt-mdx muscles show that the forcedrop is reduced to levels of wild-type mice by Akt activation (right)(n ¼ 20). (D) Summary of the percentage remaining force after 20 eccentriccontractions in the four experimental groups. (E) No significant force dropis seen with isometric contractions not accompanied by stretch (n ¼ 12 oil;n ¼ 7 tam). (F) The reduction in force induced in mdx muscles by eccentriccontractions is not modified by tamoxifen treatment for 3 weeks (n ¼ 10).

Figure 1. Generation and characterization of muscle-specific inducible Akt-mdxmice. (A) Schematic representation of the generation of a muscle-specific indu-cible Akt transgenic line and its crossing with mdx mice, giving rise to Akt-mdxmice. (B) Immunoblot analysis of protein extracts from gastrocnemius muscle ofAkt-mdx mice treated with tamoxifen for 3 weeks. Tamoxifen treatment inducesphosphorylation and protein stabilization of Akt-ER (Akt fused to mutated estro-gen receptor) while the amount of endogenous Akt protein is unaffected, whereasphosphorylated Akt-ER is undetectable in oil-treated controls. (C) Akt activationfor 3 weeks leads to muscle hypertrophy both in fast Gastrocnemius and EDLmuscles, as well as in slow soleus muscle. Each bar represents the percentageincrease in weight of muscles from tamoxifen treated mice versus controls(n ¼ 12 for each muscle, � P , 0.05) (D) Hematoxilin and Eosin staining ofoil-treated and tamoxifen treated Akt-mdx gastrocnemius muscles. Note thepresence of central nuclei and variable fiber diameter after 3 weeks of Aktactivation (n ¼ 6). (E) The proportion of fibers with central nuclei is similar inmuscles from oil- and tamoxifen-treated mice.

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lengthening contractions in dystrophic muscles (Fig. 2C). Infact after eccentric contractions, the force fell by 16+ 2% intamoxifen-treated Akt-mdx mice, a value very close to thatmeasured in wild-type C57 mice. This effect is not due totamoxifen per se, because tamoxifen treatment of mdx micedid not affect their response to eccentric contractions(Fig. 2F). The effect of Akt activation was also apparentlynot dependent on muscle hypertrophy, because no significantcorrelation was seen between the muscle weight/body weightratio of singles muscles and their residual isometric forceafter eccentric contractions (Supplementary Material, Fig. 3).

Effect of lengthening contractions on skinned fibers

The striking effect of Akt activation in preventing the forcedrop induced by lengthening contractions in mdx musclesraises the question of the mechanism of this protectiveeffect. Sarcolemmal ruptures are usually considered as themain culprit; however, we did not find any significant differ-ence in the percentage of Evans blue positive fibers aftereccentric contractions between oil- and tamoxifen-treatedAkt-mdx mice (Supplementary Material, Fig. 4). To determinewhether the myofibrillar-cytoskeletal system is involved in thereduced force drop induced by Akt activation in mdx muscles,we examined single permeabilized muscles fibers (skinnedfibers). It was previously reported that mdx muscle fibers arenot especially susceptible to eccentric contractions whenthese are applied directly in vitro to skinned fibers (16).Indeed, we confirmed that after two stretches applied on maxi-mally activated skinned fibers, force deficits do not differamong fibers from gastrocnemius muscles of C57, mdx, oil-and tamoxifen-treated Akt-mdx mice (Fig. 3A).

Next, we compared skinned fibers dissected from musclespreviously subjected to eccentric contractions in vivo. As acontrol, we used fibers taken from the gastrocnemius muscleof the controlateral leg not exposed to eccentric contractions.Whereas muscle fibers from wild-type C57 mice did notshow any significant decrease in force after eccentric contrac-tions in vivo, mdx muscle fibers showed a 28+ 13% lowerforce than controlateral fibers (Fig. 3B). Interestingly, wedid not observe any bimodal distribution of force drop inmdx muscle fibers. These findings support the notion that themyofibrillar-cytoskeletal system of mdx muscles is alteredwhen eccentric contractions are performed in intact musclefibers in vivo. The same experiment was then performed intamoxifen- and oil-treated Akt-mdx mice. As shown inFig. 3C, fibers from oil-treated mice showed a significantforce decrease of 35+ 11%, similar to that of mdx fibers,which was completely prevented by Akt activation.

The effect of Akt activation in preventing the force deficitinduced in mdx muscles by eccentric contractions is notdependent on mTOR

Activation of Akt induces activity of mTOR which mediatesthe hypertrophic effect of activated Akt in skeletal muscle.In fact rapamycin, a selective inhibitor of mTOR, blocksmuscle hypertrophy induced by functional overload (23) andmuscle growth of regenerating muscle (25). Therefore, weused rapamycin to determine whether the response of

tamoxifen-treated Akt-mdx mice to eccentric contractions isdependent on mTOR. We observe that the ‘protective’ effectof Akt activation is readily reversible, so that if Akt-mdxmice treated with tamoxifen for 2 weeks are shifted to oilvehicle during the third week, their response to eccentric con-tractions is comparable to that of mdx mice. Therefore, weexamined the response of mice treated for 2 weeks withtamoxifen and the third week with tamoxifen plus rapamycin(Fig. 4A). The efficacy of rapamycin in blocking the mTORpathway was demonstrated by the complete inhibition of thephosphorylation of the mTOR effector S6 in tamoxifen-treatedAkt-mdx muscles (Fig. 4B). As shown in Figure 4C and D, theresponse of muscles from rapamycin-treated mice to eccentriccontractions was essentially similar to that of tamoxifen-treated mice, namely the force drop was completely prevented.

Gene expression changes induced by Akt activation

How does Akt activation affect the response to eccentric con-tractions? The finding that short-term tamoxifen treatment,even 1 week treatment, has no significant effect (our unpub-lished observations) suggests that reprogramming of geneexpression is involved in this process. As an initial steptowards identifying the mechanism responsible for the protective

Figure 3. Effect of lengthening contractions on skinned fibers. (A) Forcereduction recorded after two eccentric contractions applied in vitro toskinned fibers from gastrocnemius muscle of C57 (wild-type), mdx and oil-or tamoxifen-treated Akt-mdx mice. No significant difference is seenbetween the various groups (n ¼ 30 fibers per group). (B) Recordings oftension (i.e. force normalized for cross-sectional area) produced by skinnedfibers from C57 (n ¼ 21 fibers) or mdx (n ¼ 31 fibers) muscles either subjectedto eccentric stimulation in vivo (ecc) or not stimulated previously (ctrl). Noteforce drop in skinned fibers from mdx but not wild-type animals. (C) Same asB using fibers from oil-treated (n ¼ 38 fibers) or tamoxifen-treated (n ¼ 33fibers) Akt-mdx muscles. Note that the force drop induced by eccentric con-tractions in vivo in oil-treated mice is completely prevented by Akt activationin tamoxifen-treated mice.


effect of Akt activation, we performed whole genome micro-array analysis, followed by quantitative RT–PCR of selectedgenes and western blotting of a few gene products. Weespecially focused on changes in components associated tothe sarcolemmal and/or myofibrillar cytoskeleton. Geneexpression profiling showed that Akt activation causesup-regulation of genes coding for proteins associated to theZ-disks, such as desmin and muscle LIM protein, or costa-meres, such as several integrins (Table 1). Also dysferlin, aprotein involved in plasma membrane repair, showed a1.9-fold increase (not shown). The up-regulation of many ofthese genes was confirmed by quantitative RT–PCR analysis(Fig. 5A) and an increased protein content of desmin and dys-ferlin was demonstrated by western blotting (Fig. 6). Next weexamined the expression of utrophin, the autosomal homo-logue of dystrophin, since it is known that an utrophin trans-gene can reduce the dystrophic pathology in mdx mice (32).While mRNA levels of utrophin were not increased intamoxifen-treated Akt-mdx muscles (Fig. 5A), western blot-ting analysis showed a 2-fold increase in utrophin proteincontent in tamoxifen-treated compared to oil-treated Akt-mdx mice (Fig. 6). We also checked whether dystrophinexpression is changed after Akt activation. It has been reportedthat proteasome inhibitors can rescue the expression of dystro-phin in mdx mice (33) and we have previously shown that acti-vated Akt blocks the ubiquitin-proteasome pathway in skeletalmuscle by inhibiting the transcription factor FoxO3 (26).However, using an antibody specific for the N-terminaldomain of dystrophin, we found no significant difference indystrophin expression between oil- and tamoxifen-treatedmice, only occasional revertant muscle fibers being detectedin the two groups (Supplementary Material, Fig. 5).

Microarray results showed that a number of genes coding forproteins with anti-oxidant or chaperone function were alsoup-regulated by Akt activation (Table 1). RT–PCR analysisconfirmed the up-regulation of different metallothioneins,

glutaredoxin, sulfiredoxin 1, thioredoxin reductase 3 andaB-crystallin (Fig. 5B).

DISCUSSION

Dystrophin-deficient muscles are unable to sustain the mech-anical stress induced by eccentric contractions and show an

Figure 4. The protective effect of Akt activation against eccentric contraction-induced force drop is not blocked by rapamycin. (A) Scheme of the experimentalplan involving tamoxifen treatment for 2 weeks and either tamoxifen plusrapamycin (rapa) or oil during the third week. (B) Immunoblotting analysisshows that rapamycin treatment completely blocks the phosphorylation of theAkt target S6. (C) In vivo recordings during eccentric contractions reveal amuch greater force drop in muscles from control (tamoxifen followed by oil)compared to rapamycin treated mice (n¼ 8). (D) Percentage remaining forceafter 20 eccentric contractions in the two experimental groups.

Figure 5. Genes up-regulated by Akt activation. Quantitative RT–PCR analy-sis reveals significant up-regulation of genes coding for proteins associatedwith Z-disks and costameres (A) and for proteins with anti-oxidant and chaper-one function (B) induced by Akt activation (P , 0.05, n ¼ 3).

Table 1. Genes up-regulated by Akt activationa

Gene Symbol Fold D

Genes coding for proteins associated withZ-disks and costameres

Keratin 8 Krt8 3.1Integrin a9 Itga9 2.8Muscle LIM protein (MLP)/cysteine and

glycine-rich protein 3Csrp3 2.4

Integrin a5 Itga5 2.2PDZ and LIM domain 5 Pdlim5 2.1Ankyrin repeat domain 2 Ankrd2 2.1Integrin aV Itgav 2.0Integrin b1 Itgb1 1.9PDZ and LIM domain 3 Pdlim3 1.9Calsarcin 1 Myoz2 1.6Desmin Des 1.6

Genes coding for proteins with anti-oxidantand chaperone-like function

Metallothionein 3 Mt3 11.0Metallothionein 2 Mt2 4.1Metallothionein 1 Mt1 4.0Heat shock protein 1 (Hsp25) Hspb1 2.8Glutathione S-transferase, a1/a2 Gsta1/2 2.8Glutaredoxin Glrx 2.4Sulfiredoxin 1 Srxn1 2.2Thioredoxin reductase 3 Txnrd3 2.2Crystallin aB (Hsp5) Cryab 2.0

a Gastrocnemius muscles from Akt-mdx mice treated with tamoxifen for3 weeks were compared with muscles from the silent Akt line not crossedwith the mlc1f-Cre line but bred onto mdx background and treated withtamoxifen for 3 weeks.


excessive force drop which is probably responsible for theactivity-dependent damage seen in dystrophic muscles (5).Thus, the reduction of the force drop caused by eccentric con-tractions has been used as a quantitative test to measure theefficacy of therapeutic interventions, such as utrophin overe-xpression (34). The results reported here show that Akt acti-vation prevents completely the exacerbated deleterious effectof eccentric contractions on mdx muscle function. Severalaspects of these findings deserve discussion: (i) the protectiveeffect of Akt is not correlated with muscle hypertrophy and isnot mediated by the mTOR pathway; (ii) the protective effectof Akt is accompanied by changes in distribution but not inproportion of Evans blue positive fibers; (iii) Akt activationprevents the force drop in skinned fibers from musclesexposed to eccentric contractions in vivo; (iv) Akt activationcauses an increase of utrophin, dysferlin, proteins associatedwith Z-disks and costameres, and proteins with anti-oxidantand chaperone function.

Akt activation, muscle hypertrophy and prevention ofcontraction-induced force deficit

The notion that muscular dystrophy can be alleviated by pro-moting muscle growth was initially proposed based on theeffect of muscle-specific IGF-1 expression (22) and myostatininactivation or blockade (17,18). However, no protection(myostatin blockade) or limited protection (IGF-1 overexpres-sion) against contraction-induced injury was observed underthese conditions. Other studies have shown that virus-mediated IGF-1 overexpression in mdx muscles led toincreased muscle mass but no protection against mechanicalinjury (35). On the other hand, systemic administrationof low dose IGF-1 for 8 week, that causes a shift towards

a more oxidative fiber-type profile but not hypertrophy, wasfound to partially reduce contraction-induced force drop inmdx muscles (36).

We report here that muscle-specific Akt activation in aninducible transgenic expression model is able to provide acomplete protection against eccentric contraction-dependentinjury, reducing the force drop to levels comparable to thoseseen in normal muscles. This effect is independent of hyper-trophy, because we found no significant correlation betweenincrease in muscle weight and degree of protection, and isnot mediated via mTOR, a major Akt effector and determinantof Akt-dependent muscle hypertrophy (23), because it was notprevented by rapamycin treatment. Furthermore, we didnot observe any change in the fiber-type profile during the3 week period of Akt activation. Clearly, other Akt targetsmust be involved in this effect.

The protective effect of Akt is not accompanied by changesin the proportion of Evans blue positive fibers

Previous studies on isolated mdx and normal muscles chal-lenged by forced lengthening during tetanic contractions invitro in the presence of procion orange showed that penetrationof dye occurs in a greater number of fibers in mdx muscles(13,37). Infection of mdx muscles with an adenovirus contain-ing a minidystrophin gene showed an intermediate number oflabeled fibers and a correspondingly intermediate force dropafter eccentric contraction, so that a linear correlation wasobserved between the force drop and the proportion ofprocion orange positive fibers (37). These findings supportedthe notion that the contraction-induced force deficit resultsfrom sarcolemmal ruptures. However, an alternative inter-pretation is that both the force deficit and the fraction ofprocion orange positive fibers are simultaneously but indepen-dently reduced by the successful minidystrophin transfection.However, different results were obtained when eccentriccontractions were induced in vivo. The reduction in susce-ptibility to contraction-induced damage in vivo after long-termlow dose IGF-1 administration was not linked to changes inEvans blue uptake (36). Accordingly, the greater force dropobserved in muscles from dystrophin-deficient canine musclescompared to control did not correlate with the proportion offibers showing Evan blue penetration (4). We also find no sig-nificant difference in the proportion of Evans blue positivefibers between tamoxifen-treated Akt-mdx muscles, whichshow complete protection against eccentric contraction injury,and oil-treated controls, which show a much greater forcedrop, comparable to that of mdx muscles. It remains to be seenwhether the difference between results in isolated muscles andmuscles in situ are due to the different tracer used. Procionorange is a low molecular weight molecule and might penetrateeven slightly damaged muscle fibers, whereas Evans blue isknown to bind albumin and thus act as macromolecular tracer,which may need larger sarcolemmal ruptures to penetratemuscle fibers. Anyway, our findings with skinned fibers indicatethat the force drop induced by eccentric contractions is notexplicable in terms of ‘injured’ versus ‘non-injured’ fibers,since we did not observe any bimodal distribution of forcedrop in mdx muscle fibers. Thus ‘sarcolemmal ruptures’ do notseem to be required to produce the eccentric functional damage.

Figure 6. Increased dysferlin, desmin and utrophin induced by Akt activation.Immunoblotting analyses show increased expression of dysferlin (A), desmin(B) and utrophin (C) in muscles from tamoxifen-treated compared to oil-treated mice. The corresponding quantitative analyses are shown in the rightpanels (n ¼ 10 for utrophin; n ¼ 8 for dysferlin; n ¼ 8 for desmin).


Akt activation prevents the force drop in skinned fibersfrom muscles exposed to eccentric contractions in vivo

A major original finding of this study is that skinned fibersfrom mdx muscles exposed in vivo to eccentric contractionsdisplay a marked reduction in force. Interestingly, this effectis not seen when mdx skinned fibers are exposed to eccentriccontractions directly in vitro, in agreement with the previousstudy (16). The decreased force production of skinned fiberslikely reflects an alteration of myofibrillar or cytoskeletal pro-teins causing either dysfunction of the myofibrillar contractilemachinery or uncoordinated contraction of the sarcomeres dueto cytoskeletal disruption. In mdx fibers, prepared frommuscles exposed in vivo to eccentric contractions, moresevere and extensive contractile impairment are observed.This could be due to disruption of myofibrillar or cytoskeletalproteins by mechanical stress or degradation of structural pro-teins caused by some process that takes place when mdxmuscle fibers are subjected to eccentric contractions in situ.Calpain activation or changes produced by reactive oxygenspecies have been considered in recent studies (38,39). What-ever the cause, the fact that all mdx skinned fibers showed amarked force drop is not compatible with the view that thereduction in force induced in mdx muscles by eccentric con-tractions is due to the presence of a proportion of ‘injured’fibers with sarcolemmal disruption leading to dye uptake.The contractile impairment is apparently distributed amongstmost mdx fibers dissected from muscles exposed in vivo toeccentric contractions. It was previously noted that the percen-tage of mdx muscle fibers penetrated by procion orange fol-lowing eccentric contraction in vitro is lower than thepercentage of the force drop, for example �10% procionorange positive fibers were found in muscles showing aforce drop of over 40% (5). It was suggested that this mis-match may be due to the restricted longitudinal diffusion ofthe dye and an underestimation of the number of sarcolemmaldisruptions when only few cross sections of the muscles areexamined (5). However, analyses of serial sections indicatethat dye uptake is present ‘over long segments (up to a fewmm) of the same fiber’ (40). The results of the present studysuggest that most if not all mdx muscle fibers are damagedby eccentric contractions, including fibers not penetrated bythe dye. We can speculate that wide sarcolemmal disruptions,responsible of dye penetration, are not the cause of forcedeficit but possibly a more severe consequence of the sameprocess that leads to reduction of force after eccentric stress.

Mechanism responsible for the protectiveeffect of Akt activation

Transgenic experiments showed that the dystrophin-relatedprotein, utrophin, is able to functionally compensate for dystro-phin and to prevent muscular dystrophy in mdx mice (32). Inter-estingly, high levels of utrophin expression are not required forrecovery, a 2-fold or 3-fold increase being sufficient to amelio-rate the dystrophic phenotype. We observed a 2-fold increase inutrophin protein levels after Akt activation, whereas the utro-phin mRNA levels were unchanged, suggesting the existenceof post-transcriptional control of utrophin gene expression(41). However, it is not clear whether the resistance to stretch

induced by Akt activation can be accounted for by utrophinup-regulation, because studies using an inducible transgenicsystem showed that the force drop after eccentric contractionwas significantly reduced when utrophin was induced at birth,while no recovery was observed when utrophin was inducedat 30 days after birth (34). The increased dysferlin proteinlevels may be involved in the effect of Akt, given the role ofdysferlin in plasma membrane repair (42), although it has notbeen established whether dysferlin up-regulation is able to com-pensate for dystrophin deficiency.

The up-regulation of genes coding for different componentsassociated with the Z-disk may also be relevant to the effect ofAkt. Z-disks are thicker in slow compared to fast muscles, andslow muscles are known to be less susceptible to the effect ofeccentric contractions. Interestingly, some of the up-regulatedgenes are selectively expressed in slow-twitch-oxidativemuscle: this is true for muscle LIM protein (43), aB-crystallin(44) and calsarcin 1 (45). Muscle LIM protein is up-regulatedby eccentric contractions both in mouse (46) and humanmuscles (47), and the same is true for ankirin repeat domain2 (46,48). The small heat shock proteins Hspb1/Hsp25 (homo-logous to Hsp27 in human) and aB-crystallin respond imme-diately to eccentric exercise by binding to cytoskeletal/myofibrillar proteins (49,50). In the heart, muscle LIMprotein is essential in the adaptation to biomechanical stress(51). Up-regulation of integrins may also be relevant, as trans-genic overexpression of integrin a7b1 was found to amelioratethe dystrophic phenotype in mdx mice (52).

Oxidative stress is increased in muscles from Duchenne Mus-cular Dystrophy patients and mdx mice and various moleculesassociated with anti-oxidant function are increased in dys-trophic muscle (53). Using cultured myotubes from mdx andnormal mice, Rando et al. (54) found that dystrophin-deficientcells were more susceptible to oxidative stress. Evidence foroxidative stress in mdx muscles even prior to the onset of thedegenerative process was supported by the finding that theexpression of genes coding for antioxidant enzymes washigher in the pre-necrotic mdx muscles and the level of lipid per-oxidation were greater (55). A recent study shows that the anti-oxidant N-acetylcysteine reduced the force deficit associatedwith stretch-induced muscle damage (38). Therefore, theup-regulation of several genes associated with anti-oxidantfunction induced by Akt activation could be involved in the pro-tection against the effect of eccentric contractions.

CONCLUSION

In summary, we report here that Akt activation prevents com-pletely the excessive force drop induced by eccentric contrac-tions in dystrophin-deficient mdx muscles. Such completeprotection was not observed in previous studies, except aftergene replacement strategy with dystrophin or after utrophinoverexpression since early developmental stages (32,56). Onthe other hand, other aspects of the dystrophic phenotype,such as high levels of serum creatine kinase or the appearanceof Evans blue labeled fibers, are not prevented by Akt activationat least within the time frame considered in the present study. Itis possible that some aspects of the dystrophin-deficient pheno-type, such as susceptibility to mechanical stress, are directly


dependent on dystrophin deficiency, whereas other aspects ofmuscle pathology are dependent on sarcoglycan deficiency.This interpretation is consistent with the finding thatg-sarcoglycan deficiency causes muscle degeneration, but nodecrease in muscle force after eccentric contractions (57). Arecent study reported that five genes have greater than 2-foldhigher expression in mouse models of sarcoglycanopathy com-pared with dystrophinopathy: these include calsarcin1/myoze-nin 2, ankirin repeat domain 2, heat shock protein 1-like,muscle LIM protein and connective tissue growth factor (58).Interestingly, all these genes are up-regulated by Akt activation:connective tissue growth factor shows a 2.9-fold increase andheat shock protein 1-like a 1.6-fold increase (for the otherthree genes, see Table 1). It was recently pointed out that ‘. . .different therapeutic strategies may be necessary to combatthe mechanical, signaling, and immune related mechanismsthat lead to dystrophy’ (59). Our study opens the way to identifypotential targets to combat the mechanical disfunction inducedby dystrophin deficiency.

MATERIALS AND METHODS

Transgenic model

The muscle-specific inducible Akt transgenic mouse line wasdescribed elsewhere (28). In brief, this line was obtained bycrossing a transgenic line expressing a silent Akt-ER, contain-ing the Akt1 coding sequence containing a src myristoylationsignal fused to a modified estrogen receptor hormone bindingdomain (silent Akt line), with a line expressing Cre recombi-nase under the control of a myosin light chain 1 fast promoter(mlc1f-Cre line). This muscle-specific inducible Akt trans-genic line was bred onto the dystrophin deficient mdxbackground to produce the Akt-mdx line (Fig. 1A). Akt acti-vation was induced by injecting 1.5 mg of tamoxifen i.p.every other day for 3 weeks. Tamoxifen, which binds themutated estrogen receptor, induces Akt phosphorylation andactivation. Control animals received the vehicle (sunfloweroil) for the same period of time. Mdx and wild-type C57bl6mice were also used in some experiments.

Muscle histology and Evans blue uptake

Muscle histology was examined on cryosections stained withHematoxylin and Eosin. As a measure for membrane per-meability, the vital dye Evans blue was used. Oil- andtamoxifen-treated mice (n ¼ 6 per group) were injected i.p.with a solution containing 2% Evans blue in phosphate-buffered saline (pH 7.5), the injection volume being 0.5% rela-tive to body mass. The solution was sterilized by passagethrough membrane filters with a 0.2 mm pore size andmuscles were removed 18 h after dye injection. SimilarEvans Blue injections were given to mice subjected toeccentric contractions; in this case, muscles were examined1 h after exercise.

Creatine kinase assay

To evaluate the amount of creatine kinase present in the blood,samples of blood were obtained by peri-orbital bleeding.

Creatine kinase content was measured in the serum by anindirect colorimetric assay (Sentinel Diagnostics kit).

Western blotting and immunohistochemistry

We used antibodies specific for phospho-Akt1 (phosphory-lated Ser47, 1:50; Cell Signaling), Akt, phospho-S6, pan-actin(all 1:1000; Cell Signaling), utrophin (1:50 Iowa Hybridomabank), desmin (1:1000 Sigma), dysferlin (1:1000 Novocastra)and dystrophin N-terminus (gift from J. Chamberlain). HRP-,FITC-, Cy3- and Cy2-conjugated secondary antibodies wereobtained from Bio-Rad.

In vivo gastrocnemius mechanics

Gastrocnemius muscle contractile performance was measuredin vivo using a 305B muscle lever system (Aurora ScientificInc.) in mice anaesthetized with a mixture of Xilazine andZoletil. Mice were placed on a thermostatically controlledtable, the knee was kept stationary and the foot was firmlyfixed to a footplate, which was connected to the shaft of themotor. Contraction was elicited by electrical stimulation ofthe sciatic nerve. Teflon-coated seven multi-stranded steelwires (AS 632, Cooner Sales, Chatsworth, CA, USA) wereimplanted with sutures on either side of the sciatic nerveproximally to the knee before its branching. At the distalends of the two wires, the insulation was removed, while theproximal ends were connected to a stimulator (Grass S88).In order to avoid recruitment of the dorsal flexor muscles,the common peroneal nerve was cut. The torque developedduring isometric contractions was measured at stepwiseincreasing stimulation frequency, with pauses of at least 30 sbetween stimuli to avoid effects due to fatigue. Duration ofthe trains never exceeded 600 ms. Force developed byplantar flexor muscles was calculated by dividing torque bythe lever arm length (taken as 2.1 mm). Eccentric contractionswere analysed to study muscle damage. Muscle lengtheningwas achieved by moving the foot backward at a velocity of40 mm/s while the gastrocnemius was stimulated with a fre-quency sufficient to induce full tetanic fusion (100 Hz). Thefootplate was moved 200 ms after initiation of stimulationtrain, thus eccentric pull occurs during the isometric plateauof the tetanus. The range of movement during the pull was cal-culated to be 308, clearly inside physiological limits of move-ment for the foot. Total duration of tetanic stimulation waslimited to 600 ms, assuring no sag of force. This protocolwas repeated 20 times taking the decrease in the isometricforce plateau (before beginning of the stretch) as an indicationfor muscle damage, with pauses of 30 s.

In vitro mechanics on gastrocnemius skinned fibers

Samples dissected from the superficial layers of the gastrocne-mius exposed to eccentric contractions or from the contralat-eral non-stimulated muscle were immersed in ice coldskinning solution. Single fibers were manually dissectedfrom fiber bundles under a stereomicroscope (10–20� magni-fication). At the end of the dissection, fibers were bathed for30 min in skinning solution containing 1% Triton X-100to ensure complete membrane solubilization. Segments of


1–2 mm length were then cut from the fibers and light alumi-num clips were applied at both ends. In each fiber segment, iso-metric tension (Po) was measured during maximal activationsat 208C, pCa ¼ 4.8, sarcomere length¼ 2.75 mm. Digitizedimages of each fiber were taken with a camera connectedwith the microscope at 360� magnification. Cross-sectionalarea was calculated from the average of three diameters,spaced at equal intervals along the length of the fibersegment, assuming a circular shape. The compositions of thesolutions used (skinning, relaxing, pre-activating and activa-ting) were as described (60).

To study the effect of eccentric contraction applied directlyto skinned fibers in vitro, the following protocol was adopted.In each fiber segment, isometric tension (Po) was measuredduring two maximal activations at 208C, pCa ¼ 4.8. Twoeccentric contractions were induced by stretching the fibersegment at the speed of 0.15 l/s for 0.7 s during maximal acti-vation. After this, two maximal activations were induced inisometric conditions. To determine the damage to the fiber,isometric tension generated in two activations after the twostretches was expressed in relation to isometric tensionbefore the two stretches.

Microarray and PCR analyses

Gastrocnemius muscles from three Akt-mdx mice treated withtamoxifen for 3 weeks were used for these experiments. Aktactivation in these samples was verified by immunoblotting.RNA was isolated from the three samples using Trizol fol-lowed by Rneasy (Qiagen) clean up and pooled at equimolarratios. In preliminary experiments, we found that tamoxifenper se causes significant changes in gene expression in skeletalmuscle. Therefore, we generated mice from the silent Akt linenot crossed with the mlc1f-Cre line but bred onto mdx back-ground and treated these mice with tamoxifen for 3 weeks.RNA was isolated from gastrocnemius muscles of thesemice and a pool of three samples was used as control. Comp-lementary RNAs, prepared and labeled according to standardAffymetrix protocols, were hybridized to Affymetrix MouseGenome 430 2.0 Arrays. Expression values were summarizedusing MAS 5.0 and differentially expressed genes determinedusing Excel. To identify enriched gene groups, we utilizedErmineJ (61) and searched for enrichment in gene ontologyterms of molecular component or biological function amongthe up- or downregulated genes.

Quantitative RT–PCR

For utrophin mRNA expression, total RNA was prepared fromskeletal muscles using Promega SV Total RNA Isolation kit.Complementary DNA generated with Invitrogen SuperScriptIII Reverse Transcriptase was analysed by quantitative real-time RT–PCR using Qiagen QuantiTect SYBR Green PCRKit on an ABI Prism 7000. Utrophin expression was detectedwith the following primers: Fw TTCAGTGACATCAT-TAAGTCCAGATCT, Rv TCTTGGAAAATCGAGCGTT-TATC. Data were normalized to GAPDH expression. Forgenes selected from the microarray analysis, we used thesame RNA that was isolated for the DNA microarrays andprepared cDNA using Invitrogen SuperScript III Reverse

Transcriptase. Real-time PCR was performed using the PCRMaster Mix (ABI) and ready-made TaqMan expressionassays. Expression values were normalized to b-actinexpression and showed as fold increase in Akt-mdx samplescompared to mdx samples.

Statistics

All data are expressed as means+SEMs. Differences betweengroups were assessed using student’s t-test. Significance wasdefined as a P-value of less than 0.05 (95% confidence).

FUNDING

This work was supported by grants from Telethon, Italy(GGP04227 to S.S. and TCP04009 to M.S.), the EuropeanUnion (MYORES LSHG-CT-2004-511978 to S.S., EXGEN-ESIS LSHM-CT-2004-005272 to S.S.), the Italian Ministryof Education, University and Research (PRIN 2004 to C.R.and S.S.) and from Italian Space Agency (ASI, OSMAproject to M.S., C.R. and S.S.).

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG Online.

ACKNOWLEDGEMENTS

We thank Tom Sato for the transgenic line expressing silentAkt and Steven Burden for the transgenic line expressingCre recombinase. Antibody to dystrophin was a generousgift of Dr J. Chamberlain. The technical assistance ofA. Picard and C. Argentini is gratefully acknowledged.

Conflict of Interest statement. None declared.

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