The FASEB Journal • Research Communication

Nitric oxide deficiency determines global chromatinchanges in Duchenne muscular dystrophy

Claudia Colussi,* Aymone Gurtner,† Jessica Rosati,‡ Barbara Illi,* Gianluca Ragone,§

Giulia Piaggio,† Maurizio Moggio,� Costanza Lamperti,� Grazia D’Angelo,#

Emilio Clementi,#,¶ Giulia Minetti,** Chiara Mozzetta,** Annalisa Antonini,‡

Maurizio C. Capogrossi,‡ Pier Lorenzo Puri,**,†† and Carlo Gaetano‡,1

*Laboratorio di Terapia Genica e Biologia Vascolare, Istituto Cardiologico Monzino, Milan, Italy;†Laboratorio di Oncogenesi Molecolare and Rome Oncogenomic Center, Istituto Regina Elena,Rome, Italy; ‡Laboratorio di Patologia Vascolare and §Laboratorio di Oncologia Molecolare, IstitutoDermopatico dell’ Immacolata, Rome, Italy; �Fondazione Ospedale Maggiore Mangiagalli e ReginaElena Centro Dino Ferrari and ¶Dipartimento di Scienze Precliniche, Universita di Milano, Milan,Italy; #Istituto Scientifico E. Medea, Bosisio Parini, Italy; **Istituto Dulbecco Telethon at Istituto DiRicovero e Cura a Carattere Scientifico, Santa Lucia Fondazione and European Brain ResearchInstitute, Rome, Italy; and ††Burnham Institute for Medical Research, San Diego, California, USA

ABSTRACT The present study provides evidence thatabnormal patterns of global histone modification arepresent in the skeletal muscle nuclei of mdx mice andDuchenne muscular dystrophy (DMD) patients. A combi-nation of specific histone H3 modifications, includingSer-10 phosphorylation, acetylation of Lys 9 and 14, andLys 79 methylation, were found enriched in muscle biop-sies from human patients affected by DMD and in late-term fetuses, early postnatal pups, or adult mdx mice. Inthis context, chromatin immunoprecipitation experi-ments showed an enrichment of these modifications atthe loci of genes involved in proliferation or inflamma-tion, suggesting a regulatory effect on gene expression.Remarkably, the reexpression of dystrophin induced bygentamicin treatment or the administration of nitric oxide(NO) donors reversed the abnormal pattern of H3 his-tone modifications. These findings suggest an unantici-pated link between the dystrophin-activated NO signalingand the remodeling of chromatin. In this context, theregulation of class IIa histone deacetylases (HDACs) 4and 5 was found altered as a consequence of the reducedNO-dependent protein phosphatase 2A activity, indicatingthat both NO and class IIa HDACs are important forsatellite cell differentiation and gene expression in mdxmice. In conclusion, this work provides the first evidenceof a role for NO as an epigenetic regulator in DMD.—Colussi, C., Gurtner, A., Rosati, J., Illi, B., Ragone, G.,Piaggio, G., Moggio, M., Lamperti, C., D’Angelo, G.,Clementi, E., Minetti, G., Mozzetta, C., Antonini, A.,Capogrossi, M. C., Puri, P. L., Gaetano, C. Nitric oxidedeficiency determines global chromatin changes in Duch-enne muscular dystrophy. FASEB J. 23, 000–000 (2009)

Key Words: histone deacetylase � protein phosphatase � differ-entiation � myoblast � histone

Duchenne muscular dystrophy (DMD) is a progres-sive muscle-wasting disease leading to disability and

death usually in early adulthood (1). The disease resultsfrom the absence of the structural protein dystrophin,and a variety of mutations and deletions in the dystro-phin gene, located on the chromosomal band Xp21,have been identified as causing the disorder (2). DMDpathogenesis is frequently studied in the dystrophicmdx mouse model (3). Despite a complete absence ofdystrophin, mdx mice breed normally and have anormal life span. Nevertheless, the absence of dystro-phin results in muscle weakness and muscle necrosisfollowed by regeneration from satellite cells, a processthat normally starts around the fourth week of age (4).In humans and mice, the dystrophin defect has beenassociated with reduced nitric oxide (NO) production(5), chronic inflammation, and tissue degeneration,altogether leading to altered gene expression profiles(6, 7) and deficient regeneration. Although dystrophinplays an important role as a mechanical transducer (8),some evidence has recently highlighted the presence ofother signal transduction pathways, including that ofPI3K-AKT, that are altered during the mechanicalstretching or regeneration of dystrophic muscles (8–10). Notably, AKT is also implicated in the regulation ofthe intracellular NO synthesis (11), the deficiency ofwhich has a detrimental role in DMD pathogenesis.

Recently, NO has been found to be involved inepigenetic histone modifications, chromatin remod-eling, and gene expression regulation in humanendothelial cells (12). In fact, covalent modificationsof specific residues in the core histone tails areimportant in regulating chromatin configuration andtranscription factor accessibility (13). In addition tospecific histone modifications at the individual loci,

1 Correspondence: Laboratorio di Patologia Vascolare, Isti-tuto Dermopatico dell’ Immacolata, Via Monti di Creta 104,00167, Roma, Italy. E-mail: gaetano@idi.it

doi: 10.1096/fj.08-115618

10892-6638/09/0023-0001 © FASEB

The FASEB Journal article fj.08-115618. Published online April 2, 2009.

the modification of extended regions of the genomeestablishes the chromatin conformation that regu-lates the expression of cluster of genes (14, 15).While the significance of global histone modifica-tions in mammalian cells is still unclear, nevertheless,it may reflect either the activity of specific classes ofhistone modification enzymes as well as a specificresponse to pathological stimuli. In this regard, inhuman prostate cancer, the recent investigation (16)of global histone modification patterns revealed aprognostic correlation with a high incidence of tu-mor recurrence.

Although prior work (17–19) indicated the poten-tial role of epigenetic enzymes, such as histonedeacetylases (HDACs), in DMD pathogenesis, themechanism linking these enzymes to the epigeneticprofile of dystrophic muscles is still unknown. Thefunction of specific HDACs has been recently draw-ing attention as a potential therapeutic target inhuman cancer (16, 20) or in chronic inflammation(21); however, little is known about their relevance ingenetic diseases (22) and specifically during theonset and/or progression of DMD (17). In muscle,the role of HDACs and their functional counterpartthe histone acetylases (HATs) has been well charac-terized during normal growth and differentiation(23). Both enzyme families regulate the activity ofmyogenic proteins, in particular that of MEF-2 andMyoD (24). The core myogenic regulators, in fact,induce modifications of histones in the local chro-matin infrastructure through the recruitment ofHATs or HDACs on target genes during the course ofmyogenesis (25–27). Recent evidence (28 –30) as-signs to class IIa HDACs an important role in muscledifferentiation or regeneration and in the control ofcardiac hypertrophy (29, 31). We previously reportedabout the role of NO in class IIa HDAC regulation inhuman endothelial cells, providing evidence thattheir nuclear localization, complex formation, andepigenetic effects on gene expression were stronglydependent on NO synthesis and phosphatase 2A(PP2A) activation (12). Herein, we describe the identi-fication of global epigenetic alterations in mdx mice andDMD patients. These defects were functionally associatedwith the dystrophin absence and NO deficiency thatimpairs class IIa HDAC function. In this condition, thepharmacological intervention with NO donors normal-ized the histone modification profile and triggeredHDAC5 nuclear localization with a positive effect on themuscle differentiation and/or regeneration programs(12, 32, 33).

The present study unravels a functional hierarchi-cal link between the absence of dystrophin, theimpairment of NO production, the function of classIIa HDACs, and the dystrophic chromatin landscapeconformation, which provides a molecular basis forthe altered gene expression profile of dystrophicmuscles.

MATERIALS AND METHODS

Human samples and clinical assessment

Specimens from biceps brachials (biopsies 7818, 7261, 7496,6767, 7503, 6440, 8389, and 7869), quadriceps (biopsies 8006,7864, and 8040), or deltoid (biopsy 7893) muscles of patientswere frozen in isopentane, cooled in liquid nitrogen, andstored in liquid nitrogen until use. Histological and histo-chemical analyses were performed on 8-�m-thick frozen crosssections stained with the hematoxylin and eosin Gomoritrichrome, acid phosphatase, or acridine orange techniques(see Supplemental Material for clinical assessment).

Animals and treatments

Eight-week-old normal wild-type C57BL/10, CD1, and mdxmice were used in the experiments. Gentamicin-treated mdxmice received subcutaneous injection of the drug (34 mg/kg/d) for 21 d as described. The nonspecific NO synthaseinhibitor N-(�)-nitro-l-arginine methyl ester (L-NAME) wasgiven to CD1 mice daily in the drinking water at a dose of 12.5mg/100 ml for 28 d (34). Eighteen-day-old fetuses and 4-d-oldpups were also used. All experimental procedures wereapproved by the internal Animal Research Ethical Committee(Protocol HH39) according to the Italian Ministry of Healthand complied with the National Institutes of Health Guide forthe Care and Use of Laboratory Animals.

Cells cultures and treatments

Satellite cells from isolated muscle fibers were prepared asdescribed previously (35, 36). For further information, seeSupplemental Material.

Infections

Satellite cells were infected with either adenoviruses CMV-GFP encoding for the constitutively active isoform of endo-thelial nitric oxide synthase (eNOS S1177D) or CMV-GFP-null. In in vivo experiments, the adductor muscles wereinjected with 1 � 108 plaque-forming units/animal of virusaccording to previously published procedures (37). Experi-ments were performed at 48–72 h postinfection.

Protein analysis and immunoprecipitation

Coimmunoprecipitation experiments were performed on 1mg of tissue protein extracts after lysis in the following RIPAbuffer: 50 mM Tris-HCl (pH 8.0), 125 mM NaCl, 1 mM DTT,5 mM MgCl2, 1 mM EDTA, 10% glycerol, and 0.1% NonidetP-40 supplemented with 1 mM PMSF and protease inhibitormix using the Exactacruz immunoprecipitation system (SantaCruz Biotechnology, Santa Cruz, CA, USA). For furthertechnical details, see Supplemental Material.

Confocal analysis

Sections from adductor muscles were deparaffinized andsubjected to microwave for antigen retrieval in a solution of0.01 M sodium citrate buffer for confocal immunofluores-cence analysis. Cryosections from human quadriceps biopsiesof DMD patients, Becker muscular dystrophy (BMD) patients,and age-matched controls were also used for immunofluores-cence analysis. Sections were incubated for 1 h with 10%BSA/PBS to block nonspecific protein-binding sites andovernight at 4°C with antibodies (see Supplemental Material

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for type and dilution of antibodies). After a brief rinse,sections were incubated with FITC or tetramethylrhodamineisothiocyanate secondary antibodies (dilution 1:50; Dako,Copenhagen, Denmark). When necessary, nuclei were coun-terstained with DAPI, propidium iodide, or TOPRO3. C2C12cells or satellite cells were fixed in 4% paraformaldehyde for10 min, permeabilized with 0.1% Triton for 10 min, andsubjected to immunofluorescence protocol as indicatedabove. For further information, see Supplemental Material.

Densitometry and statistical analysis

The results of Western blotting were analyzed by ImageJ v1.28software (U.S. National Institutes of Health, Bethesda, MD,

USA). Tubulin, histone H1, or red Ponceau staining was usedas a loading control. Optical density values of specific proteinswere normalized to those of tubulin or histone H1 andcorrected for those obtained from wild-type mice, which wereconsidered equal to 1. Data represent means � se of at leastthree independent experiments. The Student’s two-tailed ttest was applied to calculate the statistic significance. Aprobability of �5% was considered significant (P�0.05).

Phosphatase assay

Phosphatase assays were performed by using the Ser/Threophosphatase assay system (Promega, Madison, WI, USA),according to the manufacturer’s instructions for the detec-

Figure 1. Confocal analysis of covalentglobal histone modifications in wild-type (WT) mouse, mdx mouse, andhuman samples. A) Top panels: confo-cal analysis of WT and mdx musclefiber nuclei from 3-mo-old adductormuscle sections (n�6) stained with antiH3K14ac antibody (green). Nuclei werecounterstained with TOPRO3. Scalebar � 25 �m. Similar results wereobtained with the anti H3K9ac anti-body (not shown). Bottom panels: 3Dmaps of the above micrographs withthe same x and y axes. Graphs showfluorescence intensity rising above theplane of the micrograph. Mean fluo-rescence intensity (MFI) was obtainedfrom analysis of 200 nuclei/sample.B) Top panel: confocal analysis showspercentages of mdx muscle fiber nu-

clei stained positive by antibodies directed toward a panel of covalent histone H3 modifications (n�8). Bottom panels:representative Western blotting analysis of the indicated histone H3 modifications in WT (left panel) and mdx (right panel)muscle extracts and relative band density evaluation (n�6). *P � 0.05. C) Percentages of muscle fiber nuclei stained positive withantibodies against a panel of histone H3 modifications in human skeletal muscle biopsies from nondystrophic (CRL) (n�6),BMD (n�5), and DMD patients (n�7). Results for each group are represented by box plot. Analysis of H3K9ac shows that BMDand DMD have higher numbers of positive nuclei than control (P�0.0007 and P�0.00008, respectively), while H3K14ac,H3K79me2, and H3K4me2 stained a higher number of nuclei only in DMD patients (P�0.0082, P�0.0053, and P�0.0023,respectively).

3EPIGENETIC ANALYSIS OF DYSTROPHIC MUSCLE

tion of PP2A-specific activity. Total cell and muscle extractswere performed by using a standard RIPA buffer, withoutphosphatase inhibitors.

RESULTS

An altered global histone modification profilecharacterizes muscle fiber nuclei of mdx mice andDMD patients

Confocal (Fig. 1A) and Western blotting (Fig. 1B,bottom panels) analyses revealed an increased intensity

in signals associated with a global histone modificationprofile that distinguishes the muscles of 3-mo-old mdxmice from those of controls. These findings weresubstantiated by the evidence that the number of nucleipositive for global histone modifications associated withan open chromatin state, including acetylated Lys resi-dues on H4 (panH4Kac), H3 acetylated Lys 9 (H3K9ac) and14 (H3K14ac), dimethylated Lys 4 (H3K4me2), dimethy-lated Lys 79 (H3K79me2), and phosphorylated Ser-10(H3S10p), significantly increased in mdx mice. Con-versely, those histone modifications associated withcompacted chromatin and/or transcription repression,

Figure 2. Evaluation of global histone modifica-tions in WT and mdx fetuses, early pups, andsatellite cells. A) Left panel: Western blottinganalysis of histone H3K9ac in 18-d-old embryoextracts and in the adductor muscle of 14-d-oldpostnatal pups (n�3). Right panel: data havebeen normalized by densitometric analysis. Sim-ilar results were obtained with anti-acetylatedH3K14ac antibody (Supplemental Fig. S4A).B) Left panel: Western blotting analysis of anumber of histone H3 modifications (H3K14ac,H3K9ac, H3K79me, and H3S10p) in normal andmdx satellite cells cultured in growing (GM) ordifferentiation medium (DM; 3 d; n�4). Rightpanel: samples have been normalized by densi-tometric analysis. *P � 0.05.

Figure 3. Effect of dystrophin reexpression on global histone H3 modifications. A) Dystrophin immunostaining (top left)performed on muscle sections from WT, mdx, and gentamicin-treated mdx mice for 21 d. Scale bar � 25 �m. Graph (bottomright) shows muscle strength recovery after dystrophin reexpression evaluated by treadmill test (n�6). B) Top panel: Westernblotting analysis of H3K9ac in muscle extracts from WT, mdx, and gentamicin-treated mdx mice (n�3). Bottom panel: sampleshave been normalized by densitometric analysis. C) Left panel: Western blotting analysis of H3K9ac in WT and mdx isolatedsatellite cells cultured in the presence or absence of gentamicin for 10 d and induced to differentiate. Right panel:normalization by densitometric analysis. Similar results were obtained with anti-acetylated H3K14ac antibody (Supplemental Fig.S4B). D) Western blotting analysis of H3K9ac in satellite cells isolated from WT-, mdx-, and gentamicin-treated mdx mice. Leftpanel: Cells were cultured in the presence or absence of gentamicin alone or in combination with the NO inhibitors7-nitroindazole (7N) or sodium methyl thiosourea (SMT). Right panel: normalization by densitometric analysis (n�3). *P �0.05.

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including H3 dimethylation on Lys 9 (H3K9me2) andH4 trimethylation on Lys 20 (H4K20me3), were largelyunderrepresented, while the level of monomethylationon Lys 36 (H3K36me) was not statistically different(Fig. 1B, bottom right panel). In agreement with theseobservations, further immunohistological analysesshowed that in �45% of the mdx muscle fibers, nucleiheterochromatin protein 1 (HP1) did not colocalizewith acetylated chromatin (Supplemental Fig. S1A), anaspect compatible with a potentially higher transcrip-tion factor accessibility to target DNA sequences (38).Further, we investigated the presence of Ser-2-phosphorylated RNA polymerase II (Pol-IIS2p) in thenuclei of normal vs. mdx mice and found that �70% ofthe mdx nuclei stained positive for this marker sugges-tive of active RNA synthesis (Supplemental Fig. S1B;ref. 39).

This analysis was extended to muscle biopsies ob-tained from patients affected by DMD and BMD (seeSupplemental Tables S1 and S2), and it was found thathigher levels of H3K9ac were present in these patients

as compared with the age-matched normal individuals(Fig. 1C). Although in BMD only the H3K9ac modifi-cation was found to be higher than in controls, incontrast all the epigenetic modifications observed inmdx mice (Fig. 1C, D) were reproducibly detectedin DMD. Remarkably, none of the global histone mod-ifications identified in mdx mice and DMD or BMDsamples were detected in muscles from normal individ-uals (Fig. 1C) or from fascioscapulo-humeral dystrophypatients (not shown).

Global histone modification profile of mdx miceunravels an early epigenetic alteration

We further investigated whether the altered pattern ofglobal histone modifications detected in dystrophicadult muscles was associated with or independent fromthe inflammatory response that characterizes this dis-ease. Remarkably, histone modifications similar tothose found in muscles of adult mdx mice were also

Figure 4. Effect of NO on the global histone modifications profile. A) Image panels: immunohistological analysis of globalhistone modifications in normal CD1 mice after administration of the NO inhibitor L-NAME for 28 d (n�6). Scale bar � 50�m. Graph: percentage of muscle fiber nuclei stained positive with antibodies for a panel of covalent histone H3 modificationsin L-NAME-treated CD1 for 7 and 28 d compared with controls. B) Top panel: in vitro evaluation of H3K9ac in differentiatedmdx satellite cells after administration of DETANO or infection with the eNOS adenovirus (immunoblots) and densitometricanalysis (graph; n�4). Bottom panel: in vivo analysis of H3K9ac in adductor muscle samples from WT and mdx mice injectedeither with the GPF or eNOS adenovirus (immunoblots) and densitometric analysis of the normalized samples (graph; WT:n�4; mdx/GFP: n�4; mdx/eNOS: n�4). *P � 0.05; #P � 0.01. Similar results were obtained for both in vivo and in vitroexperiments with anti-acetylated H3K14ac antibody (Supplemental Fig. S4C).

5EPIGENETIC ANALYSIS OF DYSTROPHIC MUSCLE

detected in 18-d-old fetuses as well as in 14-d-old pupswell before the onset of clinically relevant signs ofmuscle degeneration or inflammatory infiltration (seeFig. 2A). These modifications were also present innonregenerating organs such as the brain and heart,suggesting that they were independent on the prolifer-ation processes (Supplemental Fig. S2A). To furtherexplore this aspect, experiments were performed onsatellite cells isolated from mdx mice, which showed anincrease in global H3K9ac, H3K14ac, H3K79me2, andH3S10p on transition to differentiation medium whencompared with satellite cells obtained from wild-typemice, indicating that these global histone modificationscould be caused by a mechanism inherent to mdxsatellite cells and triggered by the onset of cell differ-entiation (Fig. 2B).

The global histone modifications detected in mdxmice were also present at the single-gene level, asindicated by a series of quantitative chromatin immun-precipitation (ChIP-qPCR) experiments performed invivo on the promoter region of genes known to beinvolved in inflammation and proliferation processes.Supplemental Fig. S2B–D shows, in fact, that the levelsof H3K9ac and H3K79me2 were increased at the single-gene promoter level, whereas the inhibitory H3K9me2was significantly decreased, suggesting that the globalpattern of histone modifications is replicated at thesingle-gene level and may have important functionalconsequences for gene transcription (40).

Dystrophin defect and NO deficiency determinesglobal histone epigenetic modifications in skeletalmuscle

The link between dystrophin deficiency and the globalhistone modifications detected in dystrophic muscleswas investigated by restoring dystrophin expression inmdx mice. A 3-wk treatment with gentamicin restoreddystrophin expression (41), reduced the percentage ofcentral-nucleated fibers, and improved muscle strength(Fig. 3A; Supplemental Fig. S2E). Reexpression ofdystrophin in gentamicin-treated mdx mice correlatedwith the restoration of the histone modification profiletypical of wild-type mice (Fig. 3B). Consistently, satellitecells isolated from mdx mice and treated in vitro withgentamicin reexpressed dystrophin (see SupplementalFig. S3A) and showed a marked decrease in H3K9ac, ascompared with untreated mdx satellite cells (Fig. 3C).Previous studies reported on the impaired NO produc-tion in mdx muscle and isolated satellite cells (42) dueto the disruption of the neuronal nitric oxide synthase(nNOS) association with the dystrophin-sarcoglycancomplex (43–45). Although increasing NO levels ame-liorate the dystrophic phenotype in mdx mice (46), themechanisms linking dystrophin and NO to this mor-phological and functional improvement is still unclear.In this regard, experiments were performed in whichthe pharmacological inhibition of NO synthesis insatellite cells isolated from mdx mice reduced the effect

Figure 5. NO treatment regulates class IIa HDAClocalization and complex formation in vitro andin vivo. A) Confocal immunofluorescence per-formed on C2C12 myoblasts showing HDAC5(red) nuclear localization following adenovirus-dependent transduction of GFP (AdGFP; top pan-els) or active eNOS (AdeNOS; bottom panels).Nuclei are counterstained with TOPRO3 (blue).B) Confocal analysis of HDAC5 (green) per-formed on adductor muscle sections obtainedfrom WT and mdx mice infected with eNOS orGFP adenoviruses. Nuclei were counterstainedwith TOPRO3 (blue). Scale bars � 25 �m (A); 50�m (B). C) Coimmunoprecipitation and Westernblotting analysis of complex formation among

HDAC4 and 5 and HDAC3 in WT, mdx, and mdx overexpressing active eNOS. D) Top panels: Western blotting analysis ofHDAC5 phosphorylation in C2C12 treated with DETANO (left panel), performed in total protein extracts obtained fromdifferentiated WT and mdx satellite cells (middle panel) and in WT or mdx adductor muscle extracts (right panel). Bottompanels: relative densitometric values. *P � 0.05.

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on histone modifications in response to gentamicin-dependent dystrophin reexpression (Fig. 3D; Supple-mental Fig. S3A), while inhibition of NO synthesis byoral administration of the NO inhibitor L-NAME tonormal CD1 mice caused an increase in the globalhistone modification pattern similar to that observed inmdx mice and DMD patients (Fig. 4A). Furthermore,restoration of NO signaling in mdx satellite cells or inmuscles of mdx mice by treatment with the NO donordiethylenetriamine/nitric oxide (DETANO) or by ade-novirus-mediated expression of a constitutively activeeNOS mutant (AdeNOS) (47) invariably restored anormal-like pattern of H3 acetylation (Fig. 4B). Theepigenetic role of NO was further explored at thesingle-gene level in a series of in vivo chromatin-immunoprecipitation from c-Fos, TNF-�, and SDF-1promoter regions performed in the adductor muscle ofmdx mice infected with AdeNOS or a control GFPadenovirus (AdGFP). Supplemental Fig. S3B, C showsthat H3K9ac and H3K9me2 levels were restored to nearnormal in animals expressing the active eNOS.

Collectively, these data place histone modifications asa key epigenetic event downstream of the NO signaling.This evidence supports the existence of a functionallink between dystrophin-regulated NO production andthe epigenetic events that presumably lead to the deregu-lated gene expression profile detected in dystrophicmuscles (6).

Defect in NO production alters class IIa HDAC andPP2A nuclear localization during muscledifferentiation

Previous works (25, 48) indicate that class IIa HDACsare important regulators of skeletal myogenesis. We

(12) recently reported that NO regulates global chro-matin remodeling by activating class IIa HDACs 4 and 5in human endothelial cells. Moreover, a deficient ex-pression of HDAC9 has been associated with an in-creased global histone acetylation in muscle fiber nu-clei (49). In light of this evidence, we investigatedwhether NO regulated class IIa HDAC 4/5 distributionduring satellite cell differentiation and whether thisprocess was altered in mdx mice. Figure 5A shows thatthe expression of the constitutively active eNOS mutantcaused a nuclear enrichment of HDAC5 in C2C12 cellscultured in growing conditions. Similar results wereobtained for HDAC4 (not shown). Therefore, we askedwhether HDAC5 localization could be altered in mdxmuscle fibers. Figure 5B shows that HDAC5 was under-represented in the nucleus of mdx mice compared withnormal controls. However, after eNOS expression, thenumber of HDAC5-positive nuclei sharply incremented, aprocess paralleled by the increase of HDAC5 complexedto HDAC3, suggesting a role for this molecule in theNO-dependent process of muscle fiber regeneration(Fig. 5C). Accordingly, biochemical analysis revealedthat HDAC5 was significantly hyperphosphorylated inproliferating C2C12 cells, in isolated mdx satellite cells,and in the adductor muscle fibers (Fig. 5D). Thetreatment with NO donors reverted the phosphoryla-tion status of HDAC5 in C2C12 (Fig. 5D).

PP2A is a key phosphatase involved in the regulationof HDAC nuclear import/export and many other cel-lular functions, including satellite cell differentiation(12, 47, 50). We found that in mdx mice, PP2A activitywas significantly reduced, as compared with normalcontrols, either in isolated satellite cells or in wholeadductor muscle lysates (Fig. 6A, top panels). Remark-ably, in satellite cells, the addition of the NO donor

Figure 6. Evaluation of PP2A activity in WT and mdxskeletal muscle and satellite cells. A) Top panels: phospha-tase activity assay was performed in satellite cells (leftpanel) and in adductor muscle lysates (right panel) ob-tained from WT and mdx mice (n�3). Bottom panels: PP2A activity induced by the NO donor DETANO in normal satellitecells before and after SV40 small-T antigen expression (left panel) and effect of NO on PP2A activity in C2C12 myoblastscultured in the presence or absence of the phosphatase inhibitor okadaic acid (right panel; n�4). *P � 0.05. B) Confocalanalysis showing HDAC5 (green) and PP2A (red) distribution in WT and mdx satellite cells before and after 4 or 16 h ofdifferentiation (4 and 16 h time points). a, e, i, o) HDAC5. b, f, l, p) PP2A. c, g, m, q) Merge. d, h, n, r) Higher magnification.Nuclei were counterstained with TOPRO3 (blue). Scale bar � 50 �m.

7EPIGENETIC ANALYSIS OF DYSTROPHIC MUSCLE

DETANO increased PP2A-dependent activity, an effectprevented by small-T expression (Fig. 6A, bottom left).This effect was also present in C2C12 myoblasts andabolished by the phosphatase inhibitor okadaic acid(Fig. 6A, bottom right). Figure 6B shows that in normalsatellite cells, PP2A and HDAC5 both translocate intothe nucleus within a few hours on induction of differ-entiation, a process impaired in mdx-derived cells.Similar results were obtained with HDAC4 (not shown).

Impairment of satellite cell differentiation onHDACIIa inhibition

Production of NO in satellite cells occurs rapidly oninduction of differentiation (51), and it is important formyoblast fusion (33). Figure 7A (top) shows, as ex-

pected, that wild-type satellite cells are impaired intheir differentiation capacity when exposed to differen-tiation medium in the presence of the NO inhibitorL-NAME. Notably, similar results have been obtained byusing the class IIa HDAC inhibitor MC1568 (52, 53), asalso indicated by the significant reduction in myosinheavy chain expression (Fig. 7A, bottom). During mus-cle differentiation, the expression of the proto-onco-gene c-Fos, which is known to inhibit differentiation insatellite cells (54), is rapidly repressed as a directconsequence of MyoD and myogenin activation (54–56). Figure 7B shows that in the presence of MC1568,the c-Fos protein level remained elevated within thefirst 24 h on induction of differentiation in wild-typesatellite cells compared with their untreated controls.This effect was paralleled by the increase in H3K9ac

Figure 7. Effect of class IIa HDAC inhibition on satellite cell differentiation. A) Image panels: four representativemicrophotographs depicting WT satellite cells differentiated in the absence (top left) or presence (bottom left) of the NOdonor DETANO, the NOS inhibitor L-NAME (top right) or the class IIa HDAC inhibitor MC1568 (bottom right). Bottompanels: Western blotting analysis of MHC expression in satellite cells induced to differentiate in the presence of DETANO,L-NAME or MC1568 (immunoblots; n�3) and densitometric analysis (graph). B) Top panel: Western blotting analysis of c-Fosand H3K9ac expression in control and MC1568-treated WT satellite cells during a time course after induction of differentiation.Middle and bottom panels: densitometric analyses (n�3). *P � 0.05.

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(Fig. 7B). Altogether, these results support the hypoth-esis that PP2A activity and HDAC 4/5 function, bothregulated by NO, are important for the epigeneticcontrol of chromatin landscape as well as the process ofdifferentiation.

DISCUSSION

A large body of evidence documents that NO produc-tion is deficient in DMD patients and mdx mice (42, 43,57, 58). In this context, the reconstitution of NOsynthesis by transgene expression or in vitro and in vivotreatment with NO donors has been found to bebeneficial, since in mdx mice it ameliorates histologyand muscle strength (32, 33, 46, 59). In models ofskeletal muscle degeneration/regeneration based ondenervation, a significant down-regulation of nNOS hasalso been described (60) paralleled by histone hyper-acetylation (48, 49), suggesting a potential functionallink between NO production and the epigenetic regu-lation of chromatin changes. The evidence that NO isinvolved in class I and IIa HDAC regulation in differentsystems (12, 17, 61) prompted us to speculate that NOdeficiency in DMD could alter the epigenetic environ-ment of muscle fibers with an impact on their geneexpression profile. Dystrophin, which mediates interac-tions among several intracellular molecules and theextracellular matrix and regulates the production ofNO in skeletal muscle, may play an important role inthis context (62).

In the present study, we report that the NO defi-ciency of mdx mice generates a global reduction inPP2A-dependent activity associated with an increasedphosphorylation of HDAC5. In this condition, thepresence of HDAC5 in complexes with HDAC3 wassignificantly reduced. This deficit was restored to nor-mal on NO treatment. The role of class II HDACs inhistone acetylation is currently unclear. Members ofclass IIa, however, play an important role in regulationof several pathophysiological processes in the skeletalmuscle and heart (23). Remarkably, in the cardiacmuscle, class IIa HDACs are able to counteract theprohypertrophic effect of class I HDACs, which areinvolved in the pathogenesis of cardiac hypertrophyand failure (31). Hence, corepressors may provide anovel molecular mechanism secondary to the primaryabsence of dystrophin that causes DMD pathogenesis.

The evidence that class IIa HDAC activity is requiredfor appropriate satellite cell differentiation is in linewith previous reports (48) enlightening a positive roleof these enzymes during muscle regeneration. Indeed,the HDAC activity in the early phases of satellite cellconversion to myoblasts seems to be involved in theregulation of genes, including c-Fos down-regulation,which is an important prerequisite for the muscledifferentiation program to take over from the prolifer-ative phase during regeneration. Notably, c-Fos, thedown-regulation of which is prevented in the presenceof the class IIa specific inhibitor MC1568, is signifi-

cantly overexpressed in mdx muscle and satellite cells,possibly as a consequence of NO deficiency and/oralterations in PP2A-HDAC function.

In conclusion, this study provides evidence that inmdx mice and DMD patients, global histone modifica-tions, putatively associated with an open chromatinconfiguration, are present in large quantity and areparalleled by an alteration in class IIa HDAC function.Administration of NO donors to dystrophic mice ame-liorated the epigenetic profile and induced the forma-tion of HDAC complexes similar to those found innormal controls. Overall, the evidence described in ourstudy revealed a connection between NO signaling andthe altered epigenetic profile present in dystrophin-deficient muscles, indicating an epigenetic contribu-tion to the pathogenesis and progression of DMD.

This work has been partially supported by Fondo per gliInvestimenti della Ricerca di Base grant RBLA035A4X-1-FIRBto M.C.C.; UE FP6 grant UE-LHSB-CT-04-502988 to M.C.C.;Association Franc�aise contre les Myopathies (AFM) grantsMNM2-06 to C.G and DdT2-06 to M.C.C.; MDA grant 88202to C.G.; Telethon grant GGP07006 and an AFM grant to E.C;and Telethon project GTB07001E and Eurobiobank projectQLTR-2001-02769 to M.M. G.P. was supported by Associazi-one Italiana per la Ricerca sul Cancro, ISS-ACC, and Minis-tero della Sanita grant ICS-120.4/RA00-90-R.F.02/184. P.L.Pwas supported by a Telethon special grant, the MuscularDystrophy Association, Parent Project Onlus, and CompagniaSan Paolo di Torino. C.M. is a recipient of an AFM fellowship,and G.M. is a recipient of a Parent Project Onlus fellowship.P.L.P. is an Associate Telethon scientist at the DulbeccoTelethon Institute. C.C. is a Ph.D. student at the “ScienzeEndocrino-Metaboliche e Endocrino-Chirurgiche” Schoolof the Chair of Endocrinology, Catholic University, Rome00165, Italy.

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Received for publication September 17, 2008.Accepted for publication February 12, 2009.

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