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Myogenin and Class II HDACs Control Neurogenic Muscle Atrophy by Inducing E3 Ubiquitin Ligases

Myogenin and Class II HDACs

Control Neurogenic Muscle Atrophy

by Inducing E3Ubiquitin Ligases

Viviana Moresi,1Andrew H.Williams,1Eric Meadows,4Jesse M.Flynn,4Matthew J.Potthoff,1John McAnally,1 John M.Shelton,2Johannes Backs,1,5William H.Klein,4James A.Richardson,1,3Rhonda Bassel-Duby,1

and Eric N.Olson1,*

1Department of Molecular Biology

2Department of Internal Medicine

3Department of Pathology

University of Texas Southwestern Medical Center,Dallas,TX75390,USA

4Department of Biochemistry and Molecular Biology,University of Texas MD Anderson Cancer Center,Houston,TX77030,USA 5Present address:Department of Cardiology,University of Heidelberg,69117Heidelberg,Germany

*Correspondence:eric.olson@https://www.wendangku.net/doc/2b1193670.html,

DOI10.1016/j.cell.2010.09.004

SUMMARY

Maintenance of skeletal muscle structure and func-tion requires innervation by motor neurons,such that denervation causes muscle atrophy.We show that myogenin,an essential regulator of muscle development,controls neurogenic atrophy.Myoge-nin is upregulated in skeletal muscle following dener-vation and regulates expression of the E3ubiquitin ligases MuRF1and atrogin-1,which promote muscle proteolysis and atrophy.Deletion of myogenin from adult mice diminishes expression of MuRF1and atrogin-1in denervated muscle and confers resis-tance to atrophy.Mice lacking histone deacetylases (HDACs)4and5in skeletal muscle fail to upregulate myogenin and also preserve muscle mass following denervation.Conversely,forced expression of myogenin in skeletal muscle of HDAC mutant mice restores muscle atrophy following denervation. Thus,myogenin plays a dual role as both a regulator of muscle development and an inducer of neurogenic atrophy.These?ndings reveal a speci?c pathway for muscle wasting and potential therapeutic targets for this disorder.

INTRODUCTION

Maintenance of muscle mass depends on a balance between protein synthesis and degradation.Innervation of skeletal muscle?bers by motor neurons is essential for maintenance of muscle size,structure,and function.Numerous disorders, including amyotrophic lateral sclerosis(ALS),Guillain-Barre′syndrome,polio,and polyneuropathy,disrupt the nerve supply to muscle,causing debilitating loss of muscle mass(referred to as neurogenic atrophy)and eventual paralysis.

Loss of the nerve supply to muscle?bers results in muscle atrophy mainly through excessive ubiquitin-mediated proteo-lysis via the proteasome pathway(Beehler et al.,2006).Other pathologic states and systemic disorders,including cancer, diabetes,fasting,sepsis,and disuse,also cause muscle atrophy through ubiquitin-dependent proteolysis(Attaix et al.,2008; Attaix et al.,2005;Medina et al.,1995;Tawa et al.,1997).The muscle-speci?c E3ubiquitin ligases MuRF1(also called Trim63)and atrogin-1(also called MAFbx or Fbxo32)are upregulated during muscle atrophy and appear to represent?nal common mediators of this process(Bodine et al.,2001;Clarke et al.,2007;Gomes et al.,2001;Kedar et al.,2004;Lecker et al.,2004;Li et al.,2004;Li et al.,2007;Willis et al.,2009). However,the precise molecular mechanisms and signaling pathways that control the expression of these key regulators of muscle protein turnover have not been fully de?ned and it remains unclear whether all types of atrophic signals control these E3ubiquitin ligase genes through the same or different mechanisms.Further understanding of the molecular pathways that regulate muscle mass is a prerequisite for the development of novel therapeutics to ameliorate muscle-wasting disorders. Myogenin is a bHLH transcription factor essential for skeletal muscle development(Hasty et al.,1993;Nabeshima et al., 1993).After birth,myogenin expression is downregulated in skeletal muscle but is reinduced in response to denervation (Merlie et al.,1994;Tang et al.,2008;Williams et al.,2009). Upregulation of myogenin in denervated skeletal muscle promotes the expression of acetylcholine receptors and other components of the neuromuscular synapse(Merlie et al.,1994; Tang and Goldman,2006;Williams et al.,2009).However,it has not been possible to address the potential involvement of myogenin in neurogenic atrophy because myogenin null mice die at birth due to failure in skeletal muscle differentiation(Hasty et al.,1993;Nabeshima et al.,1993).

Histone acetylation has been implicated in denervation-dependent changes in skeletal muscle gene expression,and histone deacetylase(HDAC)inhibitors block the expression of Cell143,35–45,October1,2010a2010Elsevier Inc.35

myogenin in response to denervation(Tang and Goldman,2006). In this regard,the class IIa HDACs,HDAC4and HDAC5,which act as transcriptional repressors(Haberland et al.,2009;McKin-sey et al.,2000;Potthoff et al.,2007),are upregulated in skeletal muscle upon denervation and repress the expression of Dach2, a negative regulator of myogenin(Cohen et al.,2007;Tang et al., 2008).

To investigate the potential involvement of myogenin,HDAC4, and HDAC5in neurogenic atrophy,we performed denervation experiments in mutant mice in which these transcriptional regulators were deleted in adult skeletal muscle.We show that adult mice lacking myogenin fail to upregulate the E3ubiq-uitin ligases MuRF1and atrogin-1following denervation and are resistant to neurogenic atrophy.We demonstrate that myo-genin binds and activates the promoter regions of the MuRF1 and atrogin-1genes,in vitro and in vivo.Similar to adult mice lacking myogenin,mice lacking Hdac4and Hdac5in skeletal muscle do not upregulate myogenin following denervation and are resistant to muscle atrophy.Conversely,overexpression of myogenin in skeletal muscle is suf?cient to upregulate the expression of MuRF1and atrogin-1and promote neurogenic atrophy in mice lacking Hdac4and Hdac5.These?ndings reveal a key role of myogenin and class IIa HDACs as mediators of neurogenic atrophy and potential therapeutic targets to treat this disorder.

RESULTS

Adult Mice Lacking Myogenin Are Resistant

to Muscle Atrophy upon Denervation

To bypass the requirement of myogenin for skeletal muscle development and investigate its functions in muscle of adult mice,we used a conditional myogenin null allele(Knapp et al., 2006),which could be deleted in adult muscle with a tamox-ifen-regulated Cre recombinase transgene(Hayashi and McMa-hon,2002;Knapp et al.,2006).Tamoxifen was administered to mice at2months of age,and89%deletion of the conditional myogenin allele occurred as measured by PCR genotyping from genomic DNA1week after tamoxifen injection(see Figure S1available online).Hereafter,we refer to these mice with deletion of myogenin during adulthood as Myogà/àmice. To examine the role of myogenin in denervated skeletal muscle,the sciatic nerve was severed one month following tamoxifen administration,and muscle atrophy was assessed 14days later by weighing denervated and contralateral tibialis anterior(TA)muscles.Wild-type(WT)denervated TA showed approximately a40%decrease in weight following denervation in comparison to the contralateral TA(Figure1A).In contrast, denervated TA from Myogà/àmice showed a minimal decrease in muscle weight($20%)compared to the contralateral TA(Figure1A),suggesting that Myogà/àmice were partially resistant to muscle atrophy.Because we deleted myogenin in adult mice,muscle development and growth occurred normally prior to tamoxifen administration.As expected,the muscle weights of the nondenervated contralateral TA in Myogà/àand WT mice were similar(WT TA=37.82±0.87mg;Myogà/àTA=36.27±0.54mg;t test=0.19).Comparable resistance to atrophy was observed in the gastrocnemius and plantaris(GP) weight of Myogà/àmice(Figure1A).

Immunostaining for laminin of TA cross-sections clearly delin-eated a decrease of muscle?ber size in the WT denervated TA in comparison to the contralateral muscle,indicative of muscle atrophy(Figure1B).In contrast,the decrease in?ber size was less evident in the Myogà/àdenervated TA(Figure1B).Morpho-metric analysis of TA cross-sections highlighted a signi?cant difference in myo?ber size between WT and Myogà/àmuscles following denervation,con?rming the latter were resistant to muscle atrophy(Figure1C).

As expected,seven days after denervation,MuRF1and atrogin-1expression was dramatically upregulated in the GP of denervated WT mice(Figure1D).Remarkably,this upregulation was signi?cantly reduced in Myogà/àdenervated GP(Figure1D), suggesting that the lack of upregulation of MuRF1and atrogin-1 in denervated Myogà/àmuscles was responsible for resistance to atrophy.Deletion of myogenin mRNA from adult Myogà/àmuscle was con?rmed by real-time PCR(Figure1D).Of note, expression of MyoD(Myod1),another bHLH myogenic regula-tory factor(Davis et al.,1987),was highly upregulated in both the contralateral and denervated GP of the Myogà/àmice,seven days after denervation(Figure1D).These data show that myoge-nin does not regulate Myod1expression following denervation. The dramatic upregulation of Myod1following denervation of Myogà/àmice,which are resistant to atrophy,also argues against a major role of Myod1in promoting neurogenic atrophy. Accordingly,Myod1null mice are not resistant to muscle atrophy following denervation(Jason O’Rourke and E.Olson,unpub-lished data).

Denervation is known to affect skeletal myo?ber composition (Herbison et al.,1979;Midrio et al.,1992;Nwoye et al.,1982; Patterson et al.,2006;Sandri et al.,2006;Sato et al.,2009). To determine whether the resistance to muscle atrophy ob-served in mice lacking myogenin was due to differences in ?ber type composition,we performed?ber type analysis of soleus muscles2weeks after denervation.Our?ndings re-vealed no difference in?ber type composition between WT and Myogà/àmice(Figure S2).These?ndings suggest that myogenin,which is upregulated following denervation,is required for maximal induction of E3ubiquitin ligase genes and neurogenic atrophy.

We next tested whether myogenin was necessary for medi-ating other forms of atrophy,such as occurs in response to fasting.As shown in Figure1E,the GP muscles of WT and Myogà/àmice displayed comparable loss in mass following a 48hr fast.We observed the upregulation of MuRF1and atrogin-1upon fasting in both WT and Myogà/àmice and vali-dated the deletion of myogenin in Myogà/àmice(Figure1F). These data clearly demonstrate that myogenin is not required for starvation atrophy,but rather is a speci?c mediator of neurogenic atrophy.

Myogenin Activates MuRF1and Atrogin-1Transcription Because upregulation of MuRF1and atrogin-1was impaired in Myogà/àmice,we analyzed the promoter regions of the MuRF1and atrogin-1genes for E boxes(CANNTG)that might confer sensitivity to myogenin.Indeed,three E boxes are located

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in the promoter of the MuRF1gene,E1(à143bp),E2(à66bp),and E3(à44bp),and one conserved E box is located 79bp upstream of the atrogin-1gene (Figure S3A).The E boxes upstream of MuRF1are contained in a genomic region near the binding site for FoxO transcription factors (Waddell et al.,2008),but several kilobases away from a region shown to be regulated by NF k B (Cai et al.,2004).The E box upstream of atro-gin-1is embedded in a region containing multiple FoxO-binding sites (Sandri et al.,2004).

To con?rm the binding of myogenin to the MuRF1and atrogin-1promoters,we performed chromatin immunoprecipitation (ChIP)assays using differentiated C2C12myotubes,as

Myogenin

Figure 1.Adult Mice Lacking Myogenin Are Resistant to Muscle Atrophy upon Denervation

(A)Percentage of TA or GP muscle weight of WT and Myog à/àmice 14days after denervation,expressed relative to contralateral muscle.*p <0.05versus WT.**p <0.005versus WT.n =4for each sample.Data are represented as mean ±standard error of the mean (SEM).

(B)Immunostaining for laminin of contralateral and denervated TA of WT and Myog à/àmice,14days after denervation.Scale bar =20microns.

(C)Morphometric analysis of contralateral and denervated TA of WT and Myog à/àmice,14days after denervation.Values indicate the mean of cross-sectional area of denervated TA ?bers as a percentage of the contralateral ?bers ±SEM.**p <0.005versus WT.n =3cross-sections.

(D)Expression of MuRF1,atrogin-1,Myogenin and Myod1in contralateral (à)and denervated (+)GP of WT and Myog à/àmice,7days after denerva-tion,detected by real-time PCR.The values are normalized to WT contralateral GP.Data are rep-resented as mean ±SEM.*p <0.05;**p <0.005versus WT.n =4for each sample.

(E)Weight of GP muscle of WT and Myog à/àmice fed (à)or fasted (+)for 48hr.Data are represented as mean ±SEM.**p <0.005versus fed GP.NS =not signi?cant.n =6for each sample.

(F)Expression of MuRF1,atrogin-1and Myogenin in fed (à)and 48hr fasted (+)GP of WT and Myog à/àmice,detected by real-time PCR.The values are normalized to WT fed GP.Data are represented as mean ±SEM.z p <0.005versus WT.**p <0.005versus fed.NS =not signi?cant.n =6for each sample.

See also Figure S4and Figure S5.

fourteen days after denervation,the TA of denervated dKO mice showed a decrease in weight of only $10%compared to the contralateral TA

(Figure 3A),revealing that the dKO mice were more resistant to muscle atrophy compared to Hdac4sk KO or Hdac5KO mice.The weight of the contralateral TA was comparable among the mice (data not shown).Similar differences were also observed among GP muscles between WT and dKO mice (Figure S5).Immunostaining for laminin 14days after denervation clearly demonstrated that the denervated TA ?bers from Hdac4sk KO and Hdac5KO mice were larger than the denervated WT ?bers and that the denervated TA from dKO mice had a minimal decrease in muscle ?ber size compared to the contralateral dKO TA (Figure 3B).Morphometric analysis on TA sections revealed that,although WT mice showed a reduction of $70%in the myo-?ber cross-sectional area between denervated and contralateral TA,Hdac4sk KO denervated TA displayed $30%reduction in myo?ber cross-sectional area.Hdac5KO denervated TA also showed a substantial reduction in myo?ber area ($50%)when compared to the contralateral TA,whereas in dKO mice this reduction was only $25%(Figure 3C).From these results,we conclude that HDAC4and HDAC5redundantly regulate skeletal muscle atrophy and mice lacking these HDACs in skeletal muscle are resistant to muscle atrophy upon denervation.

Aberrant Transcriptional Responses to Denervation in HDAC Mutant Mice

We compared the transcriptional responses to denervation in WT and dKO mice by real-time PCR analysis of denervation-responsive transcripts.As reported previously (Cohen et al.,2007;Tang et al.,2008),Dach2expression was dramatically downregulated upon denervation in WT mice.However,Dach2was only modestly downregulated in the dKO mice (Figure 4).Consistent with the repressive in?uence of Dach2on Myogenin expression,in WT mice,Myogenin and Myod1were strongly upregulated three days after denervation,as were MuRF1and atrogin-1(Figure 4).In contrast,neither Myogenin nor Myod1transcripts were upregulated following denervation of dKO

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mice (Figure 4).The upregulation of MuRF1and atrogin-1was also completely abolished in dKO denervated GP (Figure 4),sug-gesting that the lack of upregulation of MuRF1and atrogin-1in denervated dKO muscles was in part responsible for resistance to atrophy.

Myogenin Overexpression in dKO Muscle Restores Neurogenic Atrophy

To examine whether forced expression of myogenin was suf?-cient to overcome the resistance of the dKO TA muscle to dener-vation-induced atrophy,we electroporated the TA of dKO mice with either a myogenin expression plasmid or an empty expres-sion plasmid.Gene delivery ef?ciency was monitored by coelec-troporation with a GFP vector (Dona et al.,2003;Rana et al.,2004).Three days after electroporation,which is suf?cient time for the electroporated plasmids to be expressed in skeletal muscle (Dona et al.,2003),we denervated one leg of the dKO mice by cutting the sciatic nerve;the TA muscles were harvested 10days after denervation.As seen in Figure 5A,laminin immu-nostaining of dKO TA muscles clearly revealed a decrease

Figure 4.dKO Mice Show Altered Gene Expression upon Denervation

Expression of the indicated mRNAs was detected by real-time PCR in WT and dKO denervated GP and normalized to the expression in the contralateral muscle.Data are represented as mean ±SEM.**p <0.005versus dKO.n =6for each time

point.

Figure 5.Ectopic Expression of Myogenin Induces Muscle Atrophy in dKO Mice Following Denervation

(A)Immunostaining for laminin (red)of cross-section of contralateral and denervated dKO TA electroporated with GFP expression plasmid and control plasmid (HDAC4/5dKO Control)or GFP plasmid and myogenin (HDAC4/5dKO +Myogenin),10days after denervation.Histology shows that the dKO denervated GFP-positive ?bers coelectroporated with myogenin are smaller than denervated GFP-positive ?bers coelectroporated with control plasmid.Scale bar =20microns.

(B)Morphometric analysis performed on GFP-positive ?bers of contralateral (à)and denervated (+)dKO TA muscles electroporated with GFP expression plasmid and control plasmid (Control)or GFP plasmid and myogenin (Myogenin),10days after denervation.Values indicate the mean of cross-sectional area of GFP-positive muscle ?bers as a percentage of the contralateral control ?bers ±SEM.*p <0.05versus control.n =7for each condition.

(C)Expression of Myogenin ,MuRF1,and atrogin-1in contralateral (à)and denervated (+)dKO TA muscles electroporated with GFP plasmid and a control plasmid (Control)or GFP plasmid and myogenin (Myogenin),10days after denervation.Values are normalized to the expression in the contralateral control muscles.Data are represented as mean ±SEM.*p <0.05versus control.n =3for each sample.See also Figure S6.

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myo?ber size in the denervated TA of dKO mice overexpressing myogenin compared to the denervated dKO TA electroporated with the control vector.Morphometric analysis performed on GFP-positive myo?bers showed a signi?cant decrease in the size of myo?bers of the denervated dKO TA electroporated with myogenin versus control vector (Figure 5B).Real-time PCR analysis validated the overexpression of Myogenin in elec-troporated TA muscle of dKO mice and showed an upregulation of the expression of MuRF1and atrogin-1(Figure 5C),con?rming the myogenin-dependent regulation of the E3ubiquitin ligases.The potential role of myogenin in driving muscle atrophy was further investigated by overexpressing myogenin in the TA muscle of WT mice.Morphometric analysis performed on GFP-positive myo?bers showed no signi?cant size difference between myo?bers electroporated with control or myogenin expression plasmid (Figures S6A and S6B).Real-time PCR anal-ysis validated the overexpression of myogenin in electroporated TA muscle of WT mice and showed an upregulation of the expression of MuRF1and atrogin-1(Figure S6C).Taken together,these ?ndings demonstrate that overexpression of myo-genin is necessary but not suf?cient to induce muscle atrophy.DISCUSSION

The results of this study demonstrate a key role of myogenin,well known for its function as an essential regulator of myogenesis,in controlling neurogenic atrophy.Myogenin promotes muscle atrophy upon denervation by directly activating the expression of MuRF1and atrogin-1,which encode E3ubiquitin ligases responsible for muscle proteolysis.Upregulation of Myogenin in response to denervation is controlled by a transcriptional pathway in which HDAC4and 5are initially induced and,in turn,repress the expression of Dach2(Tang and Goldman,2006),a negative regulator of Myogenin (Figure 6).

It is generally accepted that muscle atrophy occurs when proteolysis exceeds protein synthesis (Eley and Tisdale,2007;Glass,2003;Mammucari et al.,2008;Sandri et al.,2004).Up-regulation of myogenin in response to denervation has been proposed as an adaptive mechanism to prevent muscle

atrophy

Figure 6.Model for Neurogenic Atrophy

Denervation of skeletal muscle results in the upregulation of HDAC4and HDAC5,which represses Dach2,a negative regulator of myogenin,resulting in Myogenin expression.Myogenin activates the expression of MuRF1and atro-gin-1,two E3ubiquitin ligases that participate in the proteolytic pathway resulting in muscle atrophy.Myoge-nin also regulates miR-206,which establishes a negative feedback loop to repress HDAC4expression and promote reinnervation.

(Hyatt et al.,2003;Ishido et al.,2004).On the contrary,we demonstrate here that myogenin directly regulates MuRF1and atrogin-1,which promote the loss of muscle mass in response to denervation,revealing a mechanistic basis for neurogenic muscle atrophy and a previously unrecognized function for myogenin in this pathological process.

Recently,we showed that microRNA (miR)206is also upregu-lated in denervated skeletal muscle via a series of conserved E boxes that bind myogenin (Williams et al.,2009).miR-206,in turn,represses expression of HDAC4and controls a retrograde signaling pathway that promotes reinnervation of denervated myo?bers (Figure 6).Thus,skeletal muscle responds to denerva-tion by activating an elaborate network of transcriptional and epigenetic pathways,involving positive and negative feedback loops,which modulate nerve-muscle interactions and muscle growth and function (Figure 6).

Dual Roles of Myogenin in Muscle Development and Atrophy

Our ?ndings reveal the gene regulatory circuitry for muscle development is redeployed in adulthood to control aspects of muscle disease and stress responsiveness.Thus,myogenin can exert opposing effects on skeletal muscle—either promoting differentiation or degradation—depending on the developmental or pathological setting.These contrasting activities of myogenin likely re?ect differential modulation by signaling pathways and cofactors that enable myogenin to regulate distinct sets of target genes.

Similar to myogenin,Dach2is a transcription factor involved in both muscle development and muscle atrophy.Dach2is expressed in the developing somites prior to the onset of myogenesis and has been shown to regulate myogenic speci?-cation by interacting with the Eya2and Six1transcription factors (Heanue et al.,1999;Kardon et al.,2002).Indeed,Dach proteins are required for activation of Six1targets (Li et al.,2003),sug-gesting a possible role of Dach proteins in the Six1-mediated regulation of muscle development (Laclef et al.,2003)or ?ber type speci?cation (Grifone et al.,2004).Following denervation,Dach2plays a role in connecting neuronal activity with myogenin expression (Cohen et al.,2007;Tang and Goldman,2006;Tang et al.,2008).

The ?nding that forced expression of myogenin in HDAC4/5mutant mice is suf?cient to restore muscle atrophy following denervation indicates that myogenin is a key downstream mediator of the proatrophic functions of these HDACs.It is

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noteworthy,however,that the blockade to muscle atrophy and E3ligase expression imposed by the combined deletion of HDACs4and5is more pronounced than in Myogà/àmice. This suggests the existence of additional downstream targets of these HDACs that promote neurogenic atrophy.We also note that forced overexpression of myogenin in innervated skel-etal muscle was not suf?cient to induce muscle atrophy (Figure S6)(Hughes et al.,1999).These?ndings indicate that myogenin is necessary,but not suf?cient,to regulate the genetic program for muscle atrophy and imply the existence of additional denervation-dependent signals that potentiate the ability of myogenin to promote atrophy.

MyoD,like myogenin,is upregulated in response to denerva-tion(Figure4and(Charge et al.,2008;Hyatt et al.,2003;Ishido et al.,2004).In Myogà/àmice,Myod1expression is dramatically elevated compared to WT muscles and is super-induced in response to denervation(Figure1D).The observation that Myod1null mice are not resistant to muscle atrophy following denervation(Jason O’Rourke and E.Olson,unpublished data) demonstrates a negligible role for Myod1in neurogenic atrophy and points to myogenin as the major myogenic bHLH factor involved in this process.This is consistent with the?nding that, although MyoD and myogenin bind the same DNA consensus sequences,they regulate distinct sets of target genes(Blais et al.,2005;Cao et al.,2006).

A Myogenin-Dependent Transcriptional Pathway

for Muscle Atrophy

We show,both in vivo using denervated muscles and in vitro using differentiated C2C12cells,that myogenin binds the endogenous MuRF1and atrogin-1promoters.We observed a decrease in myogenin expression and binding to these E3 ubiquitin ligase promoters between days3and day7after dener-vation(Figure2B and Figure4),suggesting an especially impor-tant role of myogenin in triggering the transcriptional cascade leading to atrophy.Consistent with our?nding that myogenin regulates MuRF1and atrogin-1expression,these E3ubiquitin ligases are upregulated upon C2C12differentiation(Figure S3B) (Spencer et al.,2000),a process known to be regulated by myogenin.Although it is well established that MuRF1and atro-gin-1function in driving skeletal muscle atrophy(Bodine et al., 2001;Clarke et al.,2007;Gomes et al.,2001;Kedar et al., 2004;Lecker et al.,2004;Li et al.,2004;Li et al.,2007;Willis et al.,2009),their potential roles in myogenesis have not been explored.Considering the important role of ubiquitination in regulating proteolysis,endocytosis,signal transduction(Hicke, 2001),and transcription(Salghetti et al.,2001),it will be inter-esting to investigate the potential involvement of MuRF1and atrogin-1in muscle development and regeneration. Therapeutic Implications

Numerous disorders,including motor neuron disease,fasting, cancer cachexia,and sarcopenia,cause muscle atrophy and the E3ubiquitin ligase genes are thought to function as?nal common mediators of different atrophic stimuli.Myogenin is upregulated upon denervation and spinal cord isolation(Hyatt et al.,2003),but is not induced in response to other forms of atrophy,such as fasting,cancer cachexia,or diabetes(Lecker et al.,2004;Sacheck et al.,2007).In this regard,we have found that Myogà/àmice display a normal loss of skeletal muscle mass in response to fasting,further demonstrating that myogenin is dedicated to neurogenic atrophy and sensing the state of motor innervation.The fact that MuRF1and atrogin-1are upregulated in other atrophy conditions in the absence of myogenin upregu-lation(Lecker et al.,2004;Sacheck et al.,2007)strongly suggests that other transcription factors known to regulate the expression of these ubiquitin ligases,such as the FoxO family or NF k B(Bodine et al.,2001;Sandri et al.,2004;Waddell et al.,2008),play a role in driving muscle atrophy in a myoge-nin-independent manner.

Our?nding that myogenin,in addition to HDAC4and HDAC5, acts as a regulator of neurogenic muscle atrophy through the activation of E3ubiquitin ligases provides a new perspective on potential therapies for muscle wasting disorders.Class II HDACs are regulated by a variety of calcium-dependent signaling pathways that control their nuclear export through signal-dependent phosphorylation(Backs et al.,2008;McKinsey et al.,2000).In a pathological condition such as muscle denerva-tion,HDAC4and HDAC5are upregulated,shuttle into the myonuclei adjacent to neuromuscular junctions(Cohen et al., 2007),and are critical regulators of muscle atrophy.Modulation of the activity of class II HDACs,through pharmacologic inhibi-tion compatible with the maintenance of steady-state transcrip-tion of genes regulated by class II HDACs,may represent a new strategy for ameliorating muscle atrophy following denervation. EXPERIMENTAL PROCEDURES

Mouse Lines

Mice used in this study are described in the Extended Experimental Proce-dures.

Denervation

In anaesthetized adult mice,the sciatic nerve of the left leg was cut and a3mm piece was excised.The right leg remained innervated and was used as control. Mice were sacri?ced after3,7,10,or14days.

DNA Delivery by Electroporation

For gene delivery by electroporation,adult dKO mice were anesthetized;TA muscles exposed,injected with30m g of DNA in a solution of5%mannitol, and immediately subjected to electroporation.Electroporation was performed by delivering10electric pulses of20V each(?ve with one polarity followed by ?ve with inverted polarity).A pair of335mm Genepaddle electrodes(BTX, San Diego,CA)placed on opposite sides of the muscle was used to deliver the electric pulses.pCMV-Snap25-GFP(provided by Tullio Pozzan,University of Padua,Padua,Italy)was used in a1:1ratio with pcDNA3.1(Invitrogen)or EMSV-myogenin plasmid(Rana et al.,2004).

Immunohistochemistry

Cryosections of TA or soleus were?xed in4%paraformaldehyde in PBS for 10min at4 C and washed in PBS.After incubating30min with0.1% Triton X-100in PBS,the samples were?xed for1hr in15%goat serum in PBS supplemented with M.O.M.Mouse IgG blocking reagent(Vector Labora-tories)(BB)at room temperature.Primary antibodies were incubated overnight at4 C(1:100dilution of rabbit polyclonal anti-laminin antibody;1:16000 anti-type I myosin heavy chain(MHC)(Sigma).Primary antibodies were detected by Alexa Fluor-488or-555goat anti-rabbit antibody(Invitrogen) diluted1:800in BB.DAB staining(Vector Laboratories)was used on soleus muscle for detecting type I MHC.Soleus muscles were used for metachro-matic ATPase staining as described elsewhere(Ogilvie and Feeback,1990).

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Staining of transgenic lines positive for b-galactosidase was performed on GP muscles,as previously described(Williams et al.,2009).

Morphometric Analysis

Myo?ber area was assessed on TA cryosections using ImageJ software (https://www.wendangku.net/doc/2b1193670.html,/ij/)(NIH).Three H&E-stained cross-sections from three different mice for each genotype were analyzed.Between100and350GFP-positive?bers were analyzed for each electroporated TA muscle.The values are calculated as the percentage of the average of the cross-sectional area of each TA over the average cross-sectional area of the contralateral TA?bers.

RNA Isolation and RT-PCR

Total RNA was isolated from GP muscles using Trizol reagent(Invitrogen) following the manufacturer’s instructions.Three micrograms of RNA was con-verted to cDNA using random primers and Superscript III reverse transcriptase (Invitrogen).Gene expression was assessed using real-time PCR with the ABI PRISM7000sequence detection system and TaqMan or with SYBR green Master Mix reagents(Applied Biosystems).Real-time PCR values were normalized with glyceraldehyde-3-phosphate dehydrogenase(GAPDH).

A list of Taqman probes and Sybr Green primers are available in the Extended Experimental Procedures.

Plasmid Constructs

A list of the plasmids used in this study is available in the Extended Experi-mental Procedures.

Cell Culture

COS cells were grown in DMEM supplemented with10%fetal bovine serum (FBS)and antibiotics(100U/ml penicillin and100m g/ml streptomycin). C2C12myoblasts were grown in DMEM supplemented with20%FBS and antibiotics and differentiated in DMEM supplemented with2%horse serum and antibiotics.

Chromatin Immunoprecipitation Assay

ChIP assays were performed using C2C12myotubes at day six of differentia-tion or using TA muscles three and seven days after denervation with the ChIP assay kit(Upstate)following the manufacturer’s instructions.Chromatin was immunoprecipitated with antibodies against immunogloblulin G(Sigma)or my-ogenin(M-225;Santa Cruz).The sequences of the ChIP primers are available in the Extended Experimental Procedures.

Luciferase assay

C2C12transfections were performed using Lipofectamine2000(Invitrogen)as previously described(Mercer et al.,2005).COS cells were plated and trans-fected12hr later using FuGENE(Roche Applied Science)following the manu-facturer’s instructions.The MuRF1and atrogin-1reporter plasmid cloning strategy is described in the Extended Experimental Procedures.Luciferase assays were performed with the Luciferase Assay kit(Promega)according to the manufacturer’s instructions.

Site-Directed Mutagenesis

Mutations were introduced into E boxes E2and E3of the MuRF1promoter region and in the E box of the atrogin-1promoter by using the QuikChange II Site-Directed Mutagenesis Kit(Stratagene).The same E box mutations as those used in electrophoretic mobility shift assays were introduced within each E box site in the promoters.

Statistical Analysis

Data are presented as mean±standard error of the mean(SEM).Statistical signi?cance was determined using two-tailed t test with a signi?cance level minor of0.05.SUPPLEMENTAL INFORMATION

Supplemental Information includes Extended Experimental Procedures and six?gures and can be found with this article online at doi:10.1016/j.cell. 2010.09.004.

ACKNOWLEDGMENTS

We thank Marco Sandri for scienti?c input,Cheryl Nolen and Svetlana Bezprozvannaya for technical assistance,Jose Cabrera for graphics,and Jennifer Brown for editorial assistance.Work in the laboratory of E.N.O.was supported by grants from the National Institutes of Health and the Robert A.Welch Foundation(grant number I-0025).W.H.K.was supported by a grant from the Muscular Dystrophy Association and the Robert A.Welch Foundation.J.B.was supported by the Deutsche Forschungsgemeinschaft (BA2258/1-1).

Received:April20,2010

Revised:June1,2010

Accepted:August20,2010

Published:September30,2010

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