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(The FASEB Journal. 2009;23:2131-2141.)
© 2009 FASEB

Nitric oxide deficiency determines global chromatin changes in Duchenne muscular dystrophy

Claudia Colussi*, Aymone Gurtner{dagger}, Jessica Rosati{ddagger}, Barbara Illi*, Gianluca Ragone§, Giulia Piaggio{dagger}, Maurizio Moggio||, Costanza Lamperti||, Grazia D'Angelo#, Emilio Clementi#,¶, Giulia Minetti**, Chiara Mozzetta**, Annalisa Antonini{ddagger}, Maurizio C. Capogrossi{ddagger}, Pier Lorenzo Puri**,{dagger}{dagger} and Carlo Gaetano{ddagger},1

* Laboratorio di Terapia Genica e Biologia Vascolare, Istituto Cardiologico Monzino, Milan, Italy;

{dagger} Laboratorio di Oncogenesi Molecolare and Rome Oncogenomic Center, Istituto Regina Elena, Rome, Italy;

{ddagger} Laboratorio di Patologia Vascolare and

§ Laboratorio di Oncologia Molecolare, Istituto Dermopatico dell’ Immacolata, Rome, Italy;

|| Fondazione Ospedale Maggiore Mangiagalli e Regina Elena Centro Dino Ferrari and

Dipartimento di Scienze Precliniche, Università di Milano, Milan, Italy;

# Istituto Scientifico E. Medea, Bosisio Parini, Italy;

** Istituto Dulbecco Telethon at Istituto Di Ricovero e Cura a Carattere Scientifico, Santa Lucia Fondazione and European Brain Research Institute, Rome, Italy; and

{dagger}{dagger} Burnham Institute for Medical Research, San Diego, California, USA

1 Correspondence: Laboratorio di Patologia Vascolare, Istituto Dermopatico dell’ Immacolata, Via Monti di Creta 104, 00167, Roma, Italy. E-mail: gaetano{at}idi.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study provides evidence that abnormal patterns of global histone modification are present in the skeletal muscle nuclei of mdx mice and Duchenne muscular dystrophy (DMD) patients. A combination of specific histone H3 modifications, including Ser-10 phosphorylation, acetylation of Lys 9 and 14, and Lys 79 methylation, were found enriched in muscle biopsies from human patients affected by DMD and in late-term fetuses, early postnatal pups, or adult mdx mice. In this context, chromatin immunoprecipitation experiments showed an enrichment of these modifications at the loci of genes involved in proliferation or inflammation, suggesting a regulatory effect on gene expression. Remarkably, the reexpression of dystrophin induced by gentamicin treatment or the administration of nitric oxide (NO) donors reversed the abnormal pattern of H3 histone modifications. These findings suggest an unanticipated link between the dystrophin-activated NO signaling and the remodeling of chromatin. In this context, the regulation of class IIa histone deacetylases (HDACs) 4 and 5 was found altered as a consequence of the reduced NO-dependent protein phosphatase 2A activity, indicating that both NO and class IIa HDACs are important for satellite cell differentiation and gene expression in mdx mice. In conclusion, this work provides the first evidence of a role for NO as an epigenetic regulator in DMD.


Key Words: histone deacetylase • protein phosphatase • differentiation • myoblast • histone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DUCHENNE MUSCULAR DYSTROPHY (DMD) is a progressive muscle-wasting disease leading to disability and death usually in early adulthood (1) . The disease results from the absence of the structural protein dystrophin, and a variety of mutations and deletions in the dystrophin gene, located on the chromosomal band Xp21, have been identified as causing the disorder (2) . DMD pathogenesis is frequently studied in the dystrophic mdx mouse model (3) . Despite a complete absence of dystrophin, mdx mice breed normally and have a normal life span. Nevertheless, the absence of dystrophin results in muscle weakness and muscle necrosis followed by regeneration from satellite cells, a process that normally starts around the fourth week of age (4) . In humans and mice, the dystrophin defect has been associated 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 dystrophin plays an important role as a mechanical transducer (8) , some evidence has recently highlighted the presence of other signal transduction pathways, including that of PI3K-AKT, that are altered during the mechanical stretching or regeneration of dystrophic muscles (8 9 10) . Notably, AKT is also implicated in the regulation of the intracellular NO synthesis (11) , the deficiency of which has a detrimental role in DMD pathogenesis.

Recently, NO has been found to be involved in epigenetic histone modifications, chromatin remodeling, and gene expression regulation in human endothelial cells (12) . In fact, covalent modifications of specific residues in the core histone tails are important in regulating chromatin configuration and transcription factor accessibility (13) . In addition to specific histone modifications at the individual loci, the modification of extended regions of the genome establishes the chromatin conformation that regulates the expression of cluster of genes (14 , 15) . While the significance of global histone modifications in mammalian cells is still unclear, nevertheless, it may reflect either the activity of specific classes of histone modification enzymes as well as a specific response to pathological stimuli. In this regard, in human prostate cancer, the recent investigation (16) of global histone modification patterns revealed a prognostic correlation with a high incidence of tumor recurrence.

Although prior work (17 18 19) indicated the potential role of epigenetic enzymes, such as histone deacetylases (HDACs), in DMD pathogenesis, the mechanism linking these enzymes to the epigenetic profile of dystrophic muscles is still unknown. The function of specific HDACs has been recently drawing attention as a potential therapeutic target in human cancer (16 , 20) or in chronic inflammation (21) ; however, little is known about their relevance in genetic diseases (22) and specifically during the onset and/or progression of DMD (17) . In muscle, the role of HDACs and their functional counterpart the histone acetylases (HATs) has been well characterized during normal growth and differentiation (23) . Both enzyme families regulate the activity of myogenic proteins, in particular that of MEF-2 and MyoD (24) . The core myogenic regulators, in fact, induce modifications of histones in the local chromatin infrastructure through the recruitment of HATs or HDACs on target genes during the course of myogenesis (25 26 27) . Recent evidence (28 29 30) assigns to class IIa HDACs an important role in muscle differentiation or regeneration and in the control of cardiac hypertrophy (29 , 31) . We previously reported about the role of NO in class IIa HDAC regulation in human endothelial cells, providing evidence that their nuclear localization, complex formation, and epigenetic effects on gene expression were strongly dependent on NO synthesis and phosphatase 2A (PP2A) activation (12) . Herein, we describe the identification of global epigenetic alterations in mdx mice and DMD patients. These defects were functionally associated with the dystrophin absence and NO deficiency that impairs class IIa HDAC function. In this condition, the pharmacological intervention with NO donors normalized the histone modification profile and triggered HDAC5 nuclear localization with a positive effect on the muscle differentiation and/or regeneration programs (12 , 32 , 33) .

The present study unravels a functional hierarchical link between the absence of dystrophin, the impairment of NO production, the function of class IIa HDACs, and the dystrophic chromatin landscape conformation, which provides a molecular basis for the altered gene expression profile of dystrophic muscles.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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 patients were frozen in isopentane, cooled in liquid nitrogen, and stored in liquid nitrogen until use. Histological and histochemical analyses were performed on 8-µm-thick frozen cross sections stained with the hematoxylin and eosin Gomori trichrome, 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 mdx mice were used in the experiments. Gentamicin-treated mdx mice received subcutaneous injection of the drug (34 mg/kg/d) for 21 d as described. The nonspecific NO synthase inhibitor N-({omega})-nitro-L-arginine methyl ester (L-NAME) was given to CD1 mice daily in the drinking water at a dose of 12.5 mg/100 ml for 28 d (34) . Eighteen-day-old fetuses and 4-d-old pups were also used. All experimental procedures were approved by the internal Animal Research Ethical Committee (Protocol HH39) according to the Italian Ministry of Health and complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Cells cultures and treatments
Satellite cells from isolated muscle fibers were prepared as described previously (35 , 36) . For further information, see Supplemental Material.

Infections
Satellite cells were infected with either adenoviruses CMV-GFP encoding for the constitutively active isoform of endothelial nitric oxide synthase (eNOS S1177D) or CMV-GFP-null. In in vivo experiments, the adductor muscles were injected with 1 x 108 plaque-forming units/animal of virus according to previously published procedures (37) . Experiments were performed at 48–72 h postinfection.

Protein analysis and immunoprecipitation
Coimmunoprecipitation experiments were performed on 1 mg of tissue protein extracts after lysis in the following RIPA buffer: 50 mM Tris-HCl (pH 8.0), 125 mM NaCl, 1 mM DTT, 5 mM MgCl2, 1 mM EDTA, 10% glycerol, and 0.1% Nonidet P-40 supplemented with 1 mM PMSF and protease inhibitor mix using the Exactacruz immunoprecipitation system (Santa Cruz Biotechnology, Santa Cruz, CA, USA). For further technical details, see Supplemental Material.

Confocal analysis
Sections from adductor muscles were deparaffinized and subjected to microwave for antigen retrieval in a solution of 0.01 M sodium citrate buffer for confocal immunofluorescence analysis. Cryosections from human quadriceps biopsies of DMD patients, Becker muscular dystrophy (BMD) patients, and age-matched controls were also used for immunofluorescence analysis. Sections were incubated for 1 h with 10% BSA/PBS to block nonspecific protein-binding sites and overnight at 4°C with antibodies (see Supplemental Material for type and dilution of antibodies). After a brief rinse, sections were incubated with FITC or tetramethylrhodamine isothiocyanate secondary antibodies (dilution 1:50; Dako, Copenhagen, Denmark). When necessary, nuclei were counterstained with DAPI, propidium iodide, or TOPRO3. C2C12 cells or satellite cells were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton for 10 min, and subjected to immunofluorescence protocol as indicated above. For further information, see Supplemental Material.

Densitometry and statistical analysis
The results of Western blotting were analyzed by ImageJ v1.28 software (U.S. National Institutes of Health, Bethesda, MD, USA). Tubulin, histone H1, or red Ponceau staining was used as a loading control. Optical density values of specific proteins were normalized to those of tubulin or histone H1 and corrected for those obtained from wild-type mice, which were considered equal to 1. Data represent means ± SE of at least three independent experiments. The Student’s two-tailed t test was applied to calculate the statistic significance. A probability of <5% was considered significant (P<0.05).

Phosphatase assay
Phosphatase assays were performed by using the Ser/Threo phosphatase assay system (Promega, Madison, WI, USA), according to the manufacturer’s instructions for the detection of PP2A-specific activity. Total cell and muscle extracts were performed by using a standard RIPA buffer, without phosphatase inhibitors.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
An altered global histone modification profile characterizes muscle fiber nuclei of mdx mice and DMD patients
Confocal (Fig. 1A ) and Western blotting (Fig. 1B , bottom panels) analyses revealed an increased intensity in signals associated with a global histone modification profile that distinguishes the muscles of 3-mo-old mdx mice from those of controls. These findings were substantiated by the evidence that the number of nuclei positive for global histone modifications associated with an open chromatin state, including acetylated Lys residues on H4 (panH4Kac), H3 acetylated Lys 9 (H3K9ac) and 14 (H3K14ac), dimethylated Lys 4 (H3K4me2), dimethylated Lys 79 (H3K79me2), and phosphorylated Ser-10 (H3S10p), significantly increased in mdx mice. Conversely, those histone modifications associated with compacted chromatin and/or transcription repression, including H3 dimethylation on Lys 9 (H3K9me2) and H4 trimethylation on Lys 20 (H4K20me3), were largely underrepresented, while the level of monomethylation on Lys 36 (H3K36me) was not statistically different (Fig. 1B , bottom right panel). In agreement with these observations, further immunohistological analyses showed that in ~45% of the mdx muscle fibers, nuclei heterochromatin protein 1 (HP1) did not colocalize with acetylated chromatin (Supplemental Fig. S1A), an aspect compatible with a potentially higher transcription factor accessibility to target DNA sequences (38) . Further, we investigated the presence of Ser-2-phosphorylated RNA polymerase II (Pol-IIS2p) in the nuclei of normal vs. mdx mice and found that ~70% of the mdx nuclei stained positive for this marker suggestive of active RNA synthesis (Supplemental Fig. S1B; ref. 39 ).


Figure 1
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  		Figure 1. Confocal analysis of covalent global histone modifications in wild-type (WT) mouse, mdx mouse, and human samples. A) Top panels: confocal analysis of WT and mdx muscle fiber nuclei from 3-mo-old adductor muscle sections (n=6) stained with anti H3K14ac antibody (green). Nuclei were counterstained with TOPRO3. Scale bar = 25 µm. Similar results were obtained with the anti H3K9ac antibody (not shown). Bottom panels: 3D maps of the above micrographs with the same x and y axes. Graphs show fluorescence intensity rising above the plane of the micrograph. Mean fluorescence intensity (MFI) was obtained from analysis of 200 nuclei/sample. B) Top panel: confocal analysis shows percentages of mdx muscle fiber nuclei 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 with antibodies 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 BMD and 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).

This analysis was extended to muscle biopsies obtained from patients affected by DMD and BMD (see Supplemental Tables S1 and S2), and it was found that higher levels of H3K9ac were present in these patients as compared with the age-matched normal individuals (Fig. 1C ). Although in BMD only the H3K9ac modification was found to be higher than in controls, in contrast all the epigenetic modifications observed in mdx mice (Fig. 1C, D ) were reproducibly detected in DMD. Remarkably, none of the global histone modifications identified in mdx mice and DMD or BMD samples were detected in muscles from normal individuals (Fig. 1C ) or from fascioscapulo-humeral dystrophy patients (not shown).

Global histone modification profile of mdx mice unravels an early epigenetic alteration
We further investigated whether the altered pattern of global histone modifications detected in dystrophic adult muscles was associated with or independent from the inflammatory response that characterizes this disease. Remarkably, histone modifications similar to those found in muscles of adult mdx mice were also detected in 18-d-old fetuses as well as in 14-d-old pups well before the onset of clinically relevant signs of muscle degeneration or inflammatory infiltration (see Fig. 2A ). These modifications were also present in nonregenerating organs such as the brain and heart, suggesting that they were independent on the proliferation processes (Supplemental Fig. S2A). To further explore this aspect, experiments were performed on satellite cells isolated from mdx mice, which showed an increase in global H3K9ac, H3K14ac, H3K79me2, and H3S10p on transition to differentiation medium when compared with satellite cells obtained from wild-type mice, indicating that these global histone modifications could be caused by a mechanism inherent to mdx satellite cells and triggered by the onset of cell differentiation (Fig. 2B ).


Figure 2
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  		Figure 2. Evaluation of global histone modifications in WT and mdx fetuses, early pups, and satellite cells. A) Left panel: Western blotting analysis of histone H3K9ac in 18-d-old embryo extracts and in the adductor muscle of 14-d-old postnatal pups (n=3). Right panel: data have been normalized by densitometric analysis. Similar results were obtained with anti-acetylated H3K14ac antibody (Supplemental Fig. S4A). B) Left panel: Western blotting analysis of a number of histone H3 modifications (H3K14ac, H3K9ac, H3K79me, and H3S10p) in normal and mdx satellite cells cultured in growing (GM) or differentiation medium (DM; 3 d; n=4). Right panel: samples have been normalized by densitometric analysis. *P < 0.05.

The global histone modifications detected in mdx mice were also present at the single-gene level, as indicated by a series of quantitative chromatin immunprecipitation (ChIP-qPCR) experiments performed in vivo on the promoter region of genes known to be involved in inflammation and proliferation processes. Supplemental Fig. S2B–D shows, in fact, that the levels of H3K9ac and H3K79me2 were increased at the single-gene promoter level, whereas the inhibitory H3K9me2 was significantly decreased, suggesting that the global pattern of histone modifications is replicated at the single-gene level and may have important functional consequences for gene transcription (40).

Dystrophin defect and NO deficiency determines global histone epigenetic modifications in skeletal muscle
The link between dystrophin deficiency and the global histone modifications detected in dystrophic muscles was investigated by restoring dystrophin expression in mdx mice. A 3-wk treatment with gentamicin restored dystrophin expression (41) , reduced the percentage of central-nucleated fibers, and improved muscle strength (Fig. 3A ; Supplemental Fig. S2E). Reexpression of dystrophin in gentamicin-treated mdx mice correlated with the restoration of the histone modification profile typical of wild-type mice (Fig. 3B ). Consistently, satellite cells isolated from mdx mice and treated in vitro with gentamicin reexpressed dystrophin (see Supplemental Fig. S3A) and showed a marked decrease in H3K9ac, as compared with untreated mdx satellite cells (Fig. 3C ). Previous studies reported on the impaired NO production in mdx muscle and isolated satellite cells (42) due to the disruption of the neuronal nitric oxide synthase (nNOS) association with the dystrophin-sarcoglycan complex (43 44 45) . Although increasing NO levels ameliorate the dystrophic phenotype in mdx mice (46) , the mechanisms linking dystrophin and NO to this morphological and functional improvement is still unclear. In this regard, experiments were performed in which the pharmacological inhibition of NO synthesis in satellite cells isolated from mdx mice reduced the effect on histone modifications in response to gentamicin-dependent dystrophin reexpression (Fig. 3D ; Supplemental Fig. S3A), while inhibition of NO synthesis by oral administration of the NO inhibitor L-NAME to normal CD1 mice caused an increase in the global histone modification pattern similar to that observed in mdx mice and DMD patients (Fig. 4A ). Furthermore, restoration of NO signaling in mdx satellite cells or in muscles of mdx mice by treatment with the NO donor diethylenetriamine/nitric oxide (DETANO) or by adenovirus-mediated expression of a constitutively active eNOS mutant (AdeNOS) (47) invariably restored a normal-like pattern of H3 acetylation (Fig. 4B ). The epigenetic role of NO was further explored at the single-gene level in a series of in vivo chromatin-immunoprecipitation from c-Fos, TNF-{alpha}, and SDF-1 promoter regions performed in the adductor muscle of mdx mice infected with AdeNOS or a control GFP adenovirus (AdGFP). Supplemental Fig. S3B, C shows that H3K9ac and H3K9me2 levels were restored to near normal in animals expressing the active eNOS.


Figure 3
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  		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 µm. Graph (bottom right) shows muscle strength recovery after dystrophin reexpression evaluated by treadmill test (n=6). B) Top panel: Western blotting analysis of H3K9ac in muscle extracts from WT, mdx, and gentamicin-treated mdx mice (n=3). Bottom panel: samples have been normalized by densitometric analysis. C) Left panel: Western blotting analysis of H3K9ac in WT and mdx isolated satellite 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. Left panel: Cells were cultured in the presence or absence of gentamicin alone or in combination with the NO inhibitors 7-nitroindazole (7N) or sodium methyl thiosourea (SMT). Right panel: normalization by densitometric analysis (n=3). *P < 0.05.


Figure 4
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  		Figure 4. Effect of NO on the global histone modifications profile. A) Image panels: immunohistological analysis of global histone modifications in normal CD1 mice after administration of the NO inhibitor L-NAME for 28 d (n=6). Scale bar = 50 µm. Graph: percentage of muscle fiber nuclei stained positive with antibodies for a panel of covalent histone H3 modifications in L-NAME-treated CD1 for 7 and 28 d compared with controls. B) Top panel: in vitro evaluation of H3K9ac in differentiated mdx satellite cells after administration of DETANO or infection with the eNOS adenovirus (immunoblots) and densitometric analysis (graph; n=4). Bottom panel: in vivo analysis of H3K9ac in adductor muscle samples from WT and mdx mice injected either 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 vitro experiments with anti-acetylated H3K14ac antibody (Supplemental Fig. S4C).

Collectively, these data place histone modifications as a key epigenetic event downstream of the NO signaling. This evidence supports the existence of a functional link between dystrophin-regulated NO production and the epigenetic events that presumably lead to the deregulated gene expression profile detected in dystrophic muscles (6) .

Defect in NO production alters class IIa HDAC and PP2A nuclear localization during muscle differentiation
Previous works (25 , 48) indicate that class IIa HDACs are important regulators of skeletal myogenesis. We (12) recently reported that NO regulates global chromatin remodeling by activating class IIa HDACs 4 and 5 in human endothelial cells. Moreover, a deficient expression of HDAC9 has been associated with an increased global histone acetylation in muscle fiber nuclei (49) . In light of this evidence, we investigated whether NO regulated class IIa HDAC 4/5 distribution during satellite cell differentiation and whether this process was altered in mdx mice. Figure 5A shows that the expression of the constitutively active eNOS mutant caused a nuclear enrichment of HDAC5 in C2C12 cells cultured in growing conditions. Similar results were obtained for HDAC4 (not shown). Therefore, we asked whether HDAC5 localization could be altered in mdx muscle fibers. Figure 5B shows that HDAC5 was underrepresented in the nucleus of mdx mice compared with normal controls. However, after eNOS expression, the number of HDAC5-positive nuclei sharply incremented, a process paralleled by the increase of HDAC5 complexed to HDAC3, suggesting a role for this molecule in the NO-dependent process of muscle fiber regeneration (Fig. 5C ). Accordingly, biochemical analysis revealed that HDAC5 was significantly hyperphosphorylated in proliferating C2C12 cells, in isolated mdx satellite cells, and in the adductor muscle fibers (Fig. 5D ). The treatment with NO donors reverted the phosphorylation status of HDAC5 in C2C12 (Fig. 5D ).


Figure 5
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  		Figure 5. NO treatment regulates class IIa HDAC localization and complex formation in vitro and in vivo. A) Confocal immunofluorescence performed on C2C12 myoblasts showing HDAC5 (red) nuclear localization following adenovirus-dependent transduction of GFP (AdGFP; top panels) or active eNOS (AdeNOS; bottom panels). Nuclei are counterstained with TOPRO3 (blue). B) Confocal analysis of HDAC5 (green) performed on adductor muscle sections obtained from WT and mdx mice infected with eNOS or GFP adenoviruses. Nuclei were counterstained with TOPRO3 (blue). Scale bars = 25 µm (A); 50 µm (B). C) Coimmunoprecipitation and Western blotting analysis of complex formation among HDAC4 and 5 and HDAC3 in WT, mdx, and mdx overexpressing active eNOS. D) Top panels: Western blotting analysis of HDAC5 phosphorylation in C2C12 treated with DETANO (left panel), performed in total protein extracts obtained from differentiated WT and mdx satellite cells (middle panel) and in WT or mdx adductor muscle extracts (right panel). Bottom panels: relative densitometric values. *P < 0.05.

PP2A is a key phosphatase involved in the regulation of HDAC nuclear import/export and many other cellular functions, including satellite cell differentiation (12 , 47 , 50) . We found that in mdx mice, PP2A activity was significantly reduced, as compared with normal controls, either in isolated satellite cells or in whole adductor muscle lysates (Fig. 6A , top panels). Remarkably, in satellite cells, the addition of the NO donor DETANO increased PP2A-dependent activity, an effect prevented by small-T expression (Fig. 6A , bottom left). This effect was also present in C2C12 myoblasts and abolished by the phosphatase inhibitor okadaic acid (Fig. 6A , bottom right). Figure 6B shows that in normal satellite cells, PP2A and HDAC5 both translocate into the nucleus within a few hours on induction of differentiation, a process impaired in mdx-derived cells. Similar results were obtained with HDAC4 (not shown).


Figure 6
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  		Figure 6. Evaluation of PP2A activity in WT and mdx skeletal muscle and satellite cells. A) Top panels: phosphatase activity assay was performed in satellite cells (left panel) and in adductor muscle lysates (right panel) obtained from WT and mdx mice (n=3). Bottom panels: PP2A activity induced by the NO donor DETANO in normal satellite cells before and after SV40 small-T antigen expression (left panel) and effect of NO on PP2A activity in C2C12 myoblasts cultured in the presence or absence of the phosphatase inhibitor okadaic acid (right panel; n=4). *P < 0.05. B) Confocal analysis showing HDAC5 (green) and PP2A (red) distribution in WT and mdx satellite cells before and after 4 or 16 h of differentiation (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 µm.

Impairment of satellite cell differentiation on HDACIIa inhibition
Production of NO in satellite cells occurs rapidly on induction of differentiation (51) , and it is important for myoblast fusion (33) . Figure 7A (top) shows, as expected, that wild-type satellite cells are impaired in their differentiation capacity when exposed to differentiation medium in the presence of the NO inhibitor L-NAME. Notably, similar results have been obtained by using the class IIa HDAC inhibitor MC1568 (52 , 53) , as also indicated by the significant reduction in myosin heavy chain expression (Fig. 7A , bottom). During muscle differentiation, the expression of the proto-oncogene c-Fos, which is known to inhibit differentiation in satellite cells (54) , is rapidly repressed as a direct consequence of MyoD and myogenin activation (54 55 56) . Figure 7B shows that in the presence of MC1568, the c-Fos protein level remained elevated within the first 24 h on induction of differentiation in wild-type satellite cells compared with their untreated controls. This effect was paralleled by the increase in H3K9ac (Fig. 7B ). Altogether, these results support the hypothesis that PP2A activity and HDAC 4/5 function, both regulated by NO, are important for the epigenetic control of chromatin landscape as well as the process of differentiation.


Figure 7
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  		Figure 7. Effect of class IIa HDAC inhibition on satellite cell differentiation. A) Image panels: four representative microphotographs depicting WT satellite cells differentiated in the absence (top left) or presence (bottom left) of the NO donor DETANO, the NOS inhibitor L-NAME (top right) or the class IIa HDAC inhibitor MC1568 (bottom right). Bottom panels: 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-Fos and 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.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A large body of evidence documents that NO production is deficient in DMD patients and mdx mice (42 , 43 , 57 , 58) . In this context, the reconstitution of NO synthesis by transgene expression or in vitro and in vivo treatment with NO donors has been found to be beneficial, since in mdx mice it ameliorates histology and muscle strength (32 , 33 , 46 , 59) . In models of skeletal muscle degeneration/regeneration based on denervation, a significant down-regulation of nNOS has also been described (60) paralleled by histone hyperacetylation (48 , 49) , suggesting a potential functional link between NO production and the epigenetic regulation of chromatin changes. The evidence that NO is involved in class I and IIa HDAC regulation in different systems (12 , 17 , 61) prompted us to speculate that NO deficiency in DMD could alter the epigenetic environment of muscle fibers with an impact on their gene expression profile. Dystrophin, which mediates interactions among several intracellular molecules and the extracellular matrix and regulates the production of NO in skeletal muscle, may play an important role in this context (62) .

In the present study, we report that the NO deficiency of mdx mice generates a global reduction in PP2A-dependent activity associated with an increased phosphorylation of HDAC5. In this condition, the presence of HDAC5 in complexes with HDAC3 was significantly reduced. This deficit was restored to normal on NO treatment. The role of class II HDACs in histone acetylation is currently unclear. Members of class IIa, however, play an important role in regulation of several pathophysiological processes in the skeletal muscle and heart (23) . Remarkably, in the cardiac muscle, class IIa HDACs are able to counteract the prohypertrophic effect of class I HDACs, which are involved in the pathogenesis of cardiac hypertrophy and failure (31) . Hence, corepressors may provide a novel molecular mechanism secondary to the primary absence of dystrophin that causes DMD pathogenesis.

The evidence that class IIa HDAC activity is required for appropriate satellite cell differentiation is in line with previous reports (48) enlightening a positive role of these enzymes during muscle regeneration. Indeed, the HDAC activity in the early phases of satellite cell conversion to myoblasts seems to be involved in the regulation of genes, including c-Fos down-regulation, which is an important prerequisite for the muscle differentiation program to take over from the proliferative phase during regeneration. Notably, c-Fos, the down-regulation of which is prevented in the presence of the class IIa specific inhibitor MC1568, is significantly overexpressed in mdx muscle and satellite cells, possibly as a consequence of NO deficiency and/or alterations in PP2A-HDAC function.

In conclusion, this study provides evidence that in mdx mice and DMD patients, global histone modifications, putatively associated with an open chromatin configuration, are present in large quantity and are paralleled by an alteration in class IIa HDAC function. Administration of NO donors to dystrophic mice ameliorated the epigenetic profile and induced the formation of HDAC complexes similar to those found in normal controls. Overall, the evidence described in our study revealed a connection between NO signaling and the altered epigenetic profile present in dystrophin-deficient muscles, indicating an epigenetic contribution to the pathogenesis and progression of DMD.


   ACKNOWLEDGMENTS
 
This work has been partially supported by Fondo per gli Investimenti della Ricerca di Base grant RBLA035A4X-1-FIRB to M.C.C.; UE FP6 grant UE-LHSB-CT-04-502988 to M.C.C.; Association Francçaise contre les Myopathies (AFM) grants MNM2-06 to C.G and DdT2-06 to M.C.C.; MDA grant 88202 to C.G.; Telethon grant GGP07006 and an AFM grant to E.C; and Telethon project GTB07001E and Eurobiobank project QLTR-2001-02769 to M.M. G.P. was supported by Associazione Italiana per la Ricerca sul Cancro, ISS-ACC, and Ministero della Sanita grant ICS-120.4/RA00-90-R.F.02/184. P.L.P was supported by a Telethon special grant, the Muscular Dystrophy Association, Parent Project Onlus, and Compagnia San 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 Dulbecco Telethon Institute. C.C. is a Ph.D. student at the "Scienze Endocrino-Metaboliche e Endocrino-Chirurgiche" School of the Chair of Endocrinology, Catholic University, Rome 00165, Italy.

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


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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