Hypertrophic response of Duchenne and limb-girdle muscular dystrophies is associated with activation of Akt pathway

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

Hypertrophic response of Duchenne and limb-girdle muscular dystrophies is associated with activation of Akt pathway Angela K. Peter a , Rachelle H. Crosbie a,b,⁎ a

Department of Physiological Science, University of California Los Angeles, CA 90095, USA Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA

b

ARTICLE INFORMATION

ABS T R AC T

Article Chronology:

Dystrophic muscle undergoes repeated cycles of degeneration/regeneration, characterized

Received 10 November 2005

by the presence of hypertrophic fibers. In order to elucidate the signaling pathways that

Revised version received

govern these events, we investigated Akt activation in normal and dystrophic muscle. Akt is

23 March 2006

activated in neonatal muscle and in actively dividing myoblasts, supporting a

Accepted 27 April 2006

developmental role for Akt signaling. Akt activation was detected at very early,

Available online 22 May 2006

prenecrotic stages of disease pathogenesis, and maximal activation was observed during peak stages of muscle hypertrophy. Duchenne muscular dystrophy patients exhibit a

Keywords:

similar pattern of Akt activation. Mice with sarcoglycan-deficient muscular dystrophy

Akt

possess more severe muscle pathology and display elevated Akt signaling. However, the

DMD

highest levels of Akt activation were found in dystrophin–utrophin-deficient muscle with

mdx

very advanced dystrophy. We propose that Akt may serve as an early biomarker of disease

Muscular dystrophy

and that Akt activation mediates hypertrophy in muscular dystrophy. Current investigations

Hypertrophy

are focused on introducing constitutively active and dominant-negative Akt into prenecrotic

Regeneration

mdx mice to determine how early modification of Akt activity influences disease

IGF-1

pathogenesis. © 2006 Elsevier Inc. All rights reserved.

Abbreviations: Akt/PKB, protein kinase B DG, dystroglycan DGC, dystrophin–glycoprotein complex DMD, Duchenne muscular dystrophy GSK, glycogen synthase kinase 3 GS, glycogen synthase LGMD, limb-girdle muscular dystrophy mTOR, mammalian target of rapamycin PI(3)K, phosphoinositide 3-OH kinase SG, sarcoglycan

⁎ Corresponding author. Department of Physiological Science, University of California Los Angeles, 621 Charles E. Young Drive South, Life Sciences Building, Room 5804, Los Angeles, CA 90025, USA. Fax: +1 310 206 3987. E-mail address: [email protected] (R.H. Crosbie). 0014-4827/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2006.04.024

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Introduction The most well-characterized forms of muscular dystrophy involve mutations in the genes encoding components of the dystrophin–glycoprotein complex (DGC) (for review, see Ref. [1]). Integral and peripheral membrane components of the DGC include dystrophin, the dystroglycans (α- and β-DG), the sarcoglycans (α-, β-, γ-, and δ-SG), sarcospan, and the syntrophins [2–5]. The DGC is localized to the sarcolemma and forms a physical linkage between the extracellular matrix and the intracellular actin cytoskeleton. Dystrophin binds to β-DG and tethers the DGC to the intracellular actin cytoskeleton [2,3]. The extracellular matrix protein laminin-2 binds to the peripheral membrane protein α-DG, thereby providing structural integrity to the sarcolemma during muscle contraction [3,6]. Primary mutations in the dystrophin gene are responsible for causing Duchenne muscular dystrophy (DMD) [7–9]. Genetic mutations in either α-, β-, γ-, and δ-SG lead to autosomal-recessive limb girdle muscular dystrophy (AR-LGMD) (for review, see [10]). Absence of either dystrophin or any one of the SG proteins causes DGC instability, leading to sarcolemma damage and cycles of fiber degeneration/regeneration. It is well established that the DGC provides structural stability to the sarcolemma during contraction. However, several lines of evidence suggest that the DGC may also play a role in cell signaling. First, the DGC is associated with several signaling molecules, including Grb2 [11], nNOS [12,13], caveolin-3 [14,15], dystrobrevin [16,17] as well as regulatory kinases [18,19]. Second, gene expression studies reveal that several signaling pathways are activated in young (pre-phenotypic) dystrophin-deficient muscle [20]. Third, results from cultured muscle cells support the hypothesis that the SG subcomplex participates in bidirectional signaling with the integrins [18]. Lastly, treatment of cultured mouse myoblasts with antibodies that block dystroglycan–laminin interactions causes apoptosis accompanied by perturbations in Akt signaling [21]. The findings that Akt activation may be somehow linked to the DGC complex is intriguing given that recent investigations have demonstrated the importance of the phosphoinositide 3kinase (PI(3)K/Akt) pathway in regulation of muscle hypertropy [22]. In DMD patients, the regenerative capacity of muscle fibers eventually diminishes and myofibers undergo atrophy and necrosis [23,24]. The dystrophin-deficient mouse serves as a model of DMD. These mice are phenotypically normal at birth but undergo muscular degeneration starting at 3 weeks of age. Muscles from mdx mice undergo rounds of degeneration and regeneration accompanied by hypertrophy. Eventually, mdx mice die prematurally at ∼78 weeks of age, when regeneration can not surpass degeneration. It has been proposed that the robust regenerative capacity of mdx mice compared to DMD patients is responsible for their relatively mild phenotype [25–28]. Thus, understanding signaling mechanisms that mediate this regenerative, hypertrophic response in mdx tissue may help to identify key molecules that could serve as therapeutic targets. Akt, also known as protein kinase B (PKB), is a serine– threonine kinase that is stimulated by a number of receptor tyrosine kinases at the cell surface [29]. Activation of the PI(3)K/ Akt signaling pathway is a key modulator of skeletal muscle hypertrophy both in vitro [22,30] and in vivo [31–33]. Over-

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expression of insulin-like growth factor (IGF-1) induces Akt activation and rescues muscle atrophy in vivo [31]. In addition, transgenic IGF-1 overexpression in a dystrophin-deficient mouse model has been shown to increase muscle mass and force generation compared to non-transgenic dystrophindeficient controls [32]. Taken together, this evidence suggests that the PI(3)K/Akt pathway plays an important role in IGF-1 and load-induced muscle hypertrophy. Modulation of this pathway may be of therapeutic value for DMD. In the present study, we investigate effects of dystrophin- and sarcoglycan deficiency on the PI(3)K/Akt pathway.

Materials and methods Cell culture and growth curve C2C12 myoblasts, strain C3 H (American Type Cell Culture, Manassas, VA), were maintained in Dulbecco's modified essential medium (DMEM, Mediatech, Herndon, VA). Media were supplemented with 10% fetal bovine serum, 1% Lglutamine, and 1% penicillin–streptomycin (Mediatech, Herndon, VA). Cells were trypsinized after reaching 90% confluency on a 150-mm plate. Cells were then washed in phosphatebuffered saline (PBS) without CaCl2 and MgCl2 (1.4 mM NaCl, 0.27 mM KCl, 1 mM Na2HP04, and 0.18 mM dibasic phosphate, pH 7.4). 1.5 × 105 cells were then plated onto 100-mm gelatin coated plates containing DMEM media. 2 h postplating, media was changed and this time was designated as time = zero. At the zero time point and every 6 h thereafter, three 100-mm plates were trypsinized, washed, and then counted using a hemocytometer. Cells were then frozen at −80°C until analysis. At the termination of the time course experiment, frozen cells were lysed in modified RIPA lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM ethylenediaminetetraacetic acid (EDTA), 5 mM N-ethylmaleimide, 50 mM sodium fluoride, 2 mM α-glycerophosphate, 1 mM sodium orthovanadate, 100 nM okadaic acid, 5 nM microcystin LR, and 20 mM Tris–HCl, pH 7.6) supplemented with protease inhibitors (0.6 μg/ml pepstatin A, 0.5 μg/ml aprotinin, 0.5 μg/ml leupeptin, 0.75 mM benzamidine, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF)). Following incubation for 1 h at 4°C, cell lysates were clarified by centrifugation at 15,000×g for 15 min.

Animal models Wild-type (C57BL/6) and mdx breeder pairs were purchased from Jackson Laboratories (Bar Harbor, ME). Heterozygous δsarcoglycan (Sgcd) knockout males (Jackson Laboratories, Bar Harbor, ME) were bred with wild-type (C57BL/6) females until homozygous Sgcd mice [34] were obtained. All mice were housed in the UCLA Life Sciences Vivarium. Mice at various ages were euthanized by CO2 followed by cervical dislocation. To examine mdx disease progression, three mice were analyzed at 2 weeks (prenecrotic stage), 4 weeks (peak necrotic stage), and 41–59 weeks (hypertrophic stage) of age. Homozygous δ-sarcoglycan knockout mice were analyzed at 4 weeks of age. Skeletal muscles were harvested and snap frozen in liquid nitrogen. 13-week wild-type (F1B) and δ-sarcoglycan-deficient (BIO14.6) hamster skeletal muscles were obtained from Bio

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Breeders, Inc. (Watertown, MA). All tissues were stored at −80°C until analysis. All procedures were carried in accordance with guidelines set by the UCLA Institutional Animal Care and Use Committee.

Human muscle samples Normal and DMD muscle samples were obtained in accordance with approval from the Institutional Review Board. Dystrophin staining was normal in controls, but was absent in DMD biopsies. DMD 1 patient was age 8 months at biopsy, DMD 2 age 3 years at time of biopsy.

Subcellular Proteome Extraction Kit (EMD Biosciences, San Diego CA). Protease inhibitor cocktail and Benzonase®, an unspecified nuclease, were included. A 20X phosphatase inhibitor cocktail (2 μM okadaic acid, 100 nM microcystin LR, 20 mM sodium orthovanadate, 1 M sodium fluoride, 40 mM βglycerophosphate, and 20 mM EDTA) was added to each extraction buffer. The subcellular fractions were quantified for protein concentration and analyzed by immunoblot analysis. All subcellular fractions were stored at −80°C.

Immunofluorescence

Frozen skeletal muscle samples were crushed in liquid nitrogen with a mortar and pestle and immediately homogenized on ice in modified RIPA lysis buffer with phosphatase inhibitors (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 5 mM N-ethylmaleimide, 50 mM sodium fluoride, 2 mM β-glycerophosphate, 1 mM sodium orthovanadate, 100 nM okadaic acid, 5 nM microcystin LR, and 20 mM Tris–HCl, pH 7.6). Prior to homogenization, protease inhibitors (0.6 μg/ml pepstatin A, 0.5 μg/ml aprotinin, 0.5 μg/ml leupeptin, 0.75 mM benzamidine, and 0.1 mM PMSF) were added to the lysis buffer. Homogenates were rocked at 4°C for 1 h and clarified by centrifugation at 15,000 ×g for 15 min. Clarified tissue lysates were stored at −80°C.

Transverse muscle cryosections (7 μm) from control and DMD patients were prepared with a Leica CM 3050S cryostat (Leica Microsystems, Bannockburn, IL). Sections were blocked with 3% BSA in PBS for 1 h at RT and then incubated with immunohistochemistry specific primary antibody against PAkt (Ser 473) (Cell Signaling Technologies) at a 1:10 dilution at 4°C for 12 h. After washing the sections with PBS, sections were incubated with FITC anti-rabbit conjugated secondary antibody (Vector Laboratories Inc, Burlingame, CA) diluted 1:500 for 1 h at RT. Sections were washed and mounted using VectaShield (Vector Laboratories Inc). Secondary antibody alone was used as a negative control. All sections were visualized using the Axioplan 2 fluorescent microscope (Carl Zeiss Inc, Thornwood, NY), and digitized images were captured under identical conditions using Axiovision 3.0 software (Carl Zeiss Inc).

Immunoblot analysis

Histology

Samples were quantified for protein concentration using the DC Protein Assay® (Bio-Rad, Hercules, CA). Equal concentrations of protein samples were resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes (Millipore Corp., Billerica, MA) for subsequent immunoblotting. Primary antibodies against Akt, P-Akt (Ser 473), and P-GSK3β were purchased from Cell Signaling Technologies (Beverly, MA) and used at a dilution of 1:750. P-p70S6K (Cell Signaling Technologies) was used at a 1:250 dilution. The monoclonal c-JUN (Cell Signaling Technologies) antibody was diluted 1:500. Horseradish peroxidase-conjugated anti-rabbit IgG and antimouse IgG (Amersham Pharmacia Biotech, Piscataway, NJ) secondary antibodies were used at a 1:3000 dilution. Enhanced chemiluminescence with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) was used to develop immunoblots. AlphaImager® (Alpha Innotech, San Leandro, CA) was employed to quantitate the density of protein bands on immunoblots developed within the linear range of the autoradiography film. Data are represented percentages relative to wild-type control levels (100%). Averages are plotted with standard error. Statistical analysis was performed using the t test function in SigmaPlot® (Systat Software, Inc., Point Richmond, CA).

For hematoxylin and eosin (H&E) staining, muscle sections were incubated with hematoxylin for 3 minutes, washed with water for 1 min, incubated with eosin for 3 min, dehydrated in solutions of 70%, 80%, 90% and 100% ethanol and then incubated in xylene for a total of 6 min. Stained sections were mounted with permount. All supplies for the H&E staining were purchased from Fisher Scientific (Fairlawn, NJ).

Protein preparation

Subcellular Localization Subcellular protein extraction of skeletal muscles from wildtype (C57BL/6) and mdx mice was performed as described in the manufacturer's protocol included with the ProteoExtract™

DGC purification The DGC was extracted using 1% digitonin (Biosynth, Naperville, IL) from wild-type (C57BL/6) skeletal muscle and applied to succinylated wheat germ agglutinin (sWGA) column (Vector Laboratories Inc, Burlingame, CA) affinity chromatography, as described previously [35]. The sWGA sepharose was washed with 4 column volumes of 0.1% digitonin and the DGC was subsequently eluted from the column with N-acetyl glucosamine. The eluate was concentrated to 500 μl and 200 μl was applied to 5–30% sucrose gradients and subjected to centrifugation at 4°C for 2 h at 150,000 ×g in a Beckman Optima L-90K ultracentrifuge using an SW41 rotor (Beckman, Palo Alto, California). Following centrifugation, the sucrose gradient was fractionated into 14 fractions (Biocomp Gradient Station ip, Fredericton, NB, Canada). 70 μl of each fraction was electrophoresed by 10% SDS-PAGE, transferred to nitrocellulose, and probed with antibodies to the DGC and Akt. The α-DG antibody clone IIH6C4 (Upstate Cell Signaling Solutions, Lake Placid, NY) was used at a 1:1000 dilution. α-, γ-, and δ-SG antibodies (Novocastra Laboratories Ltd./Vision BioSystems Inc., Norwell,

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MA) were used at a 1:10 dilution. Akt primary antibody (Cell Signaling Technologies) was diluted 1:750. Horseradish peroxidase-conjugated anti-mouse IgG, anti-rabbit IgG (Amersham Pharmacia Biotech), and anti-mouse IgM (Upstate Cell Signaling Solutions) secondary antibodies were used at a 1:3000 dilution. Detection was completed as described above.

Results and discussion

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points represent the lag phase (Fig. 1B). Upon exponential growth, Akt and P-Akt levels increased rapidly and were maintained during active cell division (Figs. 1A and B). In order to determine if Akt activation was also evident during in vivo muscle development, skeletal muscles from wild-type mice at various ages were analyzed for Akt activation. Akt and P-Akt were dramatically elevated in 2- and 4-week-old muscle compared to muscle isolated from adult mice (Fig. 1C). These data support a role for Akt signaling during cell growth for both cultured myoblasts and wild-type skeletal muscle.

Akt expression and activation in normal muscle In order to investigate how loss of the DGC impacts Akt signaling during each stage of mdx dystrophy, Akt activation in normal muscle was characterized. We first examined total Akt levels and Akt phosphorylation in actively dividing, cultured C2C12 myoblasts. C2C12 cells are a well-established murine skeletal muscle cell line that can be maintained as undifferentiated, mono-nuclear cells [36]. Myoblasts were examined for Akt expression at various time points after plating. During the first 30 h, Akt and P-Akt were detected at very low levels (Fig. 1A). Analysis of growth rates revealed that these time

Fig. 1 – Activation of Akt expression and phosphorylation during muscle cell growth. (A) C2C12 myoblast cells were harvested every 6 h after plating. Clarified cell lysates (54 μg) were separated on 10% SDS-PAGE and transferred to nitrocellulose membranes. Identical nitrocellulose membranes were probed with antibodies against Akt and P-Akt as indicated. (B) C2C12 growth curve indicating cell number (×105) per plate. Cells were counted at 6-h increments. (C) Muscle lysates (60 μg) from 2-week-old (2 weeks), 4-week-old (4 weeks), and adult (adult) wild-type mice were analyzed by immunoblotting. Identical nitrocellulose membranes were probed for Akt and P-Akt, as indicated.

Analysis of Akt signaling components in dystrophin-deficient mdx skeletal muscle The mdx mouse is the best-characterized model of dystrophindeficient muscular dystrophy. The mdx mouse lacks the dystrophin protein due to a point mutation in exon 23 that introduces a premature stop codon in the dystrophin gene [37]. These mice display progressive muscular dystrophy that can be categorized into three main stages of disease: prenecrotic (<3 weeks of age), peak necrotic (3–5 weeks of age), and hypertrophic (>6 weeks of age) [38]. In order to investigate Akt activation during progressive muscular dystrophy, we examined skeletal muscle from control and mdx mice at these stages of disease. Immunoblots of total skeletal muscle lysates from mdx mice at prenecrotic stages were stained with antibodies to Akt and P-Akt. Total Akt protein levels are identical between mdx and control mice at this age and only modest elevations in PAkt are detectable in young mdx mice (Fig. 2). In order to evaluate downstream effectors of Akt, we analyzed P-GSK3β and P-p70S6K, which represent distinct forks in the Akt signaling axis (for schematic of Akt pathway, see Fig. 9). Phosphorylation of GSK3β by P-Akt inhibits GSK3β activity, leading to an overall decrease in glycogen synthesis. P-Akt activates the mammalian target of rapamaycin (mTOR), which in turn phosphorylates P-p70S6K and results in increased protein synthesis [39]. Inhibition of GSK3β and activation of mTOR contribute to myofiber hypertrophy [22]. Upregulation of P-Akt and P-p70S6K was observed in prenecrotic mdx mice, but GSK3β was not significantly altered in young mdx mice compared to age-matched controls (Fig. 2). Results for prenecrotic tissue show no significant difference in the activation of the GSK3β pathway but do show dramatic activation of the P-p70S6K pathway (Fig. 2). During prenecrotic stages, muscle morphology is normal despite the loss of dystrophin. Interestingly, Akt signaling is altered during the prenecrotic stage of the disease, suggesting that the hypertrophic response of mdx muscle is initiated early in the pathogenic process. We purpose that analysis of P-Akt could serve as an early biomarker for muscular dystrophy. Mice at ages representing peak necrotic (4 weeks) and hypertrophic (adult) stages were examined for perturbations in Akt signaling. We find that total Akt protein levels are dramatically increased in these later stages of disease (Figs. 3 and 4). Levels of Akt are 2.3-fold (4 weeks) and 1.9-fold (adult) higher in mdx mice relative to controls (Fig. 4B). Mdx mice at both of these stages exhibit a significant increase of levels of P-Akt in comparison to age-matched controls. Akt protein levels are significantly upregulated in peak necrotic

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progression. Mdx/utrn−/− mice exhibit severe kyphosis and muscle wasting and die between 4 and 14 weeks of age [41,42]. Mdx:utrn−/− mice were analyzed at 4 weeks. Mdx:utrn−/− muscle exhibits levels of activation that exceed that of agematched mdx mice (Fig. 8C). This further substantiates the hypothesis that Akt is perturbed in response to dystrophic disease pathology.

Analysis of Akt signaling in DMD

Fig. 2 – Mild activation of Akt in prenecrotic mdx mice. Skeletal muscle lysates (50 μg) from 2-week-old wild-type (WT) and mdx (mdx) mice were separated on 10% SDS-PAGE and transferred to nitrocellulose membranes. Nitrocellulose membranes were separately stained with antibodies against Akt, P-Akt, P-70S6K, and P-GSK3, as indicated. The P-70S6K polyclonal antibody also cross-reacted with P-85S6K (P-70S6K is indicated by an asterisk). Coomassie blue staining of total protein is shown on the right panel (CB Stain). Molecular size standards are indicated on the left (×103 Da).

and hypertrophic mdx muscle. However, phosphorylation of Akt is not coordinately regulated with total Akt protein expression, as illustrated by the mild elevation of P-Akt in mdx muscle (Fig. 4B). This has led us to hypothesize that activation of Akt in mdx, while significantly increased from wild-type age-matched controls, maybe somewhat defective in mdx mice. Immunoblots of total skeletal muscle from mdx and control mice were probed with antibodies to P-GSK3β and P-p70S6K. These downstream effectors of the Akt pathway were affected in response to amplified Akt activation (Figs. 3 and 4). P-p70S6K was highly elevated in 4 weeks mdx mice (2.4-fold) and moderately elevated in adult mdx mice (1.6-fold) (Fig. 4B). Likewise, decreased inhibition of the GSK3β pathway was observed in mdx mice during these later dystrophic stages (Figs. 3 and 4). Taken together, these data suggest that activation of Akt in dystrophin-deficient mice appears to stimulate the mTOR-dependent pathway in concert with disease progression while the mTOR-independent/GSK3β axis is not significantly altered. Although mdx mice represent an accurate genetic model for DMD, they lack the disease severity that is characteristic of DMD. Mdx mice exhibit modest muscle weakness and have near normal life spans. Furthermore, mdx muscle does not develop severe myofibrosis and cardiomyopathy, which is evident at end-stage DMD [25–28]. One possible explanation for the mild phenotype of mdx mice is that a homologous protein, utrophin, compensates for the loss of dystrophin (for review, see [40]). Dystrophin- and utrophin-null mice (mdx: utrn−/−) more closely mimic DMD pathology and disease

In order to determine whether our findings extend to human disease, we analyzed muscle from several DMD patients. Western blot analysis revealed a similar pattern of Akt perturbation compared to that observed in mdx tissues. Total Akt protein and P-Akt levels were upregulated in DMD muscle compared to normal tissue (Fig. 5A). Indirect immunofluorescence with P-Akt antibodies was performed on transverse cryosections of DMD and normal individuals to more closely evaluate P-Akt expression at the myofiber level. Serial H&E sections are shown for both control and DMD samples. P-Akt was barely detected in control muscle samples (Fig. 5B). However, DMD muscle exhibited very bright cytosolic staining for P-Akt in nearly all fibers examined (Fig. 5B).

Akt is not a core component of the DGC Taken together, our data show that Akt is increased in both mdx mice and DMD patients. If Akt interacts with the DGC, then loss of dystrophin may directly affect Akt expression and activation. Alternatively, changes in Akt may be a secondary effect of disease pathology. In order to distinguish between these two possibilities, we sought to determine whether Akt is

Fig. 3 – Mdx muscle at peak necrotic stage display dramatic upregulation of Akt signaling. Muscle lysates (60 μg) from wild-type (WT) and mdx (mdx) mice at 4 weeks of age were analyzed by immunoblotting. Immunoblots were probed with antibodies to Akt, P-Akt, P-70S6K, and P-GSK3. Coomassie blue staining of total protein is shown on the right panel (CB Stain). Molecular size standards are indicated on the left (×103 Da).

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digitonin-solubilized skeletal muscle membranes (S1 and S2), which is the starting material for DGC purification (Fig. 6B). However, Akt was not retained on the sWGA column, as evident by its location in the sWGA void (Fig. 6B). These data demonstrate that Akt is separated from the DGC during the first purification step and that Akt is not a core member of the

Fig. 4 – Adult mdx mice display hypertrophy and maintain dramatic elevations in Akt signaling. (A) Immunoblot analysis was performed on skeletal muscle extracts (60 μg) from adult (>41 weeks of age) wild-type and mdx mice. Blots containing wild-type (WT) and mdx (mdx) lysates were probed with antibodies to Akt, P-Akt, P-70S6K, and P-GSK3. Coomassie blue staining of total protein is shown on the right panel (CB Stain). Molecular size standards are indicated on the left (×103 Da). (B) Quantification of immunoblot data. Differences in Akt signaling between WT and mdx muscle was analyzed by densitometry. Values are represented as percentages relative to WT levels (100%). Averaged values represent three sets of tissue analyzed at each age. Error bars represent standard error of the mean. At 2 weeks of age, P-70S6K was not detected in WT tissue. *P < 0.05, **P < 0.01.

an integral component of the DGC. The DGC complex was purified from mouse skeletal muscle, as previously described [35]. Sucrose gradient fractions from the final step of the DGC purification were analyzed by Western blotting with antibodies to specific components of the DGC and Akt. The DGC components were found in fractions 1 through 4 (Fig. 6A). Even after overexposure of the immunoblots, we were unable to detect Akt in any of the sucrose gradient fractions (Fig. 6A). To determine the fate of Akt during DGC isolation, samples were analyzed at each step of the purification. Immunoblots of digitonin-solubilized membranes (S1 and S2), sWGA void (sWGA void), sWGA washes (Wash 1–4) as well as the sWGA eluate were analyzed for Akt protein. Akt was present in

Fig. 5 – DMD patients exhibit elevated P-Akt levels. (A) Muscle lysates (60 μg) from an unaffected control (control) and two DMD patients (DMD 1 and DMD 2) were separated on 10% SDS-PAGE and transferred to nitrocellulose membranes. Nitrocellulose membranes were probed with Akt and P-Akt antibodies. Equal protein loading was confirmed by total protein staining (data not shown). (B) Localization of P-Akt in control and DMD muscle. Transverse muscle cryosections from an unaffected control (control) and two DMD patients (DMD 1 and DMD 2) were stained with antibodies to P-Akt. Serial sections were stained with hematoxylin and eosin (H&E) to visualize muscle histology. Asterisks indicate identical fibers within serial sections. Scale bar, 100 μm.

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tide-dependent protein kinase 1 or 2 (PDK1 or PDK2) at Thr-308 and Ser-473. P-Akt can phosphorylate downstream cytosolic substrates (i.e., GSK3β/p70S6K) or translocate to the nucleus (for review, see [43] and [44]). In the nucleus, P-Akt is thought to regulate the activity of FKHR, FKHRL1, and AFX, which are members of the forkhead transcription factor family [45–53]. These transcription factors promote transcription of antiapoptotic genes such as Fas-L, IGRBP1, and Bim. Akt-mediated phosphorylation of these transcription factors is thought to either facilitate their exportation from the nucleus or to prevent their re-entry into the nucleus (for review, see [43] and [44]). In cardiomyocytes, Akt activation has been linked to enhanced cell survival through inhibition of apoptosis [54–56]. Unfortunately, prolonged constitutive activation of Akt in cardiomyocytes causes unwanted cardiomyocyte hypertrophy or increased myofibril density [57, 58]. Interestingly, nuclear targeting of P-Akt in cardiomyocytes has recently been shown to inhibit apoptosis in cardiomyocytes without causing unwanted cardiomyocyte hypertrophy or myofibril density [59]. In order to determine whether P-Akt translocates to the nucleus during dystrophin-deficiency, we performed subcellular fractionation on adult wild-type and mdx skeletal muscle. Skeletal muscle lysates were separated into four fractions including cytosol, membrane, nuclear and cytoskeleton. To verify the purity of nuclear preparations, samples from each fraction were analyzed by immunoblotting with antibodies to c-JUN, a nuclear marker (Fig. 7). Akt and P-Akt localized to the cytosolic fraction in wild-type and mdx skeletal muscle, even in overexposed blots (Fig. 7). We did not detect Akt in nuclear fractions of either wild-type or mdx muscle suggesting that Fig. 6 – Akt is not a core component of the DGC. (A) The DGC was purified from adult mouse skeletal muscle and centrifuged through a 5–30% linear sucrose gradient. Fractions from the sucrose gradient were separated by SDS-PAGE, transferred to nitrocellulose, and stained with antibodies to dystroglycan (α-DG), the sarcoglycans (α- and δ-SG) and Akt. The DGC components are present in fractions 1–4. Akt was not detected in any fraction of the sucrose gradient, even on overexposed blots. (B) Akt separates from the DGC complex during the first step of DGC purification. Protein samples from each step of the DGC purification were analyzed by immunoblotting with Akt antibodies. Samples for the DGC preparation included solubilized membranes (S1 and S2), sWGA void, sWGA washes (Wash 1–4), and the sWGA elute. Akt is present in S1, S2 and the sWGA void. As previously established, the DGC components are retained on the sWGA column (data not shown). Molecular size standards are indicated on the left (×103 Da).

DGC complex. These data do not exclude the possibility of transient or indirect interactions between Akt and the DGC.

Akt is retained in the cytosol in mdx muscle Upon growth factor stimulation at the cell surface, the lipid product of PI(3)K recruits Akt to the cell surface through a pleckstrin-like homology domain. Following translocation to the cell membrane, Akt is phosphorylated by 3-phosphoinosi-

Fig. 7 – Akt is not translocated to the nucleus in dystrophin-deficient muscle. Muscle from adult wild-type and mdx mice was biochemically separated into the following subcellular fractions: cytosol (lane 1), membrane and organelle (lane 2), nuclear (lane 3) and cytoskeleton (lane 4). Each protein fraction (30 μg) was separated on 10% SDS-polyacrylamide gels, transferred to nitrocellulose, and probed with antibodies to Akt, P-Akt, and c-JUN. Akt and P-Akt were located predominantly in the cytosolic fractions of wild-type muscle. The localization of P-Akt was not perturbed in mdx mice. The P-Akt blots were overexposed to determine if there was any detectable protein in the nuclear fraction. The c-JUN antibody served as a control for nuclear enrichment.

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activated Akt remains in the cytosol after activation, regardless of disease pathology. These data are consistent with the cytosolic localization of P-Akt in DMD patient samples (Fig. 5B).

Analysis of Akt signaling components in SG-deficient skeletal muscle Autosomal-recessive limb-girdle muscular dystrophies (ARLGMD) resulting from SG-deficiency have a wide range of onset and disease progression. AR-LGMD type 2C-2F represent a family of diseases caused by primary mutations in the SG genes (reviewed by [60–62]). The SG subcomplex is composed of four distinct single-pass transmembrane glycoproteins, referred to as α-, β-, γ-, and δ-SG. In general, loss of one SG results in loss of the entire, tetrameric SG subcomplex [63–66], although there are a few reported cases of partial SG subcomplex loss [65,67,68]. Mice with targeted deletions in δSG also exhibit severe cardiomyopathy associated with exercise-induced microconstrictions of the vasculature [34,65,67–71]. Likewise, the BIO14.6 hamster exhibits severe muscular dystrophy and cardiomyopathy as a result of a large deletion in the δ-SG gene [72,73]. The entire SG subcomplex is absent from the sarcolemma in the BIO14.6 hamster although dystroglycan and dystrophin are expressed at normal levels at the sarcolemma [68,74]. Characteristics of hypertrophic muscular dystrophy can be detected in BIO14.6 hamsters beginning at postnatal day 10 [75]. In order to determine whether SG-deficient muscular dystrophies exhibit the similar profiles of Akt perturbation, skeletal and cardiac muscle homogenates

Fig. 8 – Akt activation is increased in a sarcoglycan-deficient model for limb-girdle muscular dystrophy. (A) Muscle from adult control (F1B) and SG-deficient (BIO14.6) hamster was analyzed for total Akt and P-Akt levels. The BIO14.6 hamsters exhibit severe, hypertrophic muscular dystrophy and cardiomyopathy due to a large deletion in the δ-SG gene [76,77]. Skeletal muscle lysates (60 μg) from wild-type (F1B) and BIO14.6 (BIO14.6) were separated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Nitrocellulose transfers were stained with antibodies against Akt and P-Akt. (B) Cardiac muscle lysates (60 μg) from control (F1B) and BIO14.6 (BIO14.6) hamsters were analyzed by immunoblotting for Akt and P-Akt. (C) Quantification of immunoblot data. Levels of total Akt and P-Akt in WT and BIO14.6 skeletal and cardiac muscles were analyzed by densitometry. Averaged values represent three sets of tissue analyzed for skeletal and cardiac muscles. Error bars represent standard error of the mean. Values are represented as percentages relative to WT levels (100%). *P < 0.05 vs. WT normalized value. **P < 0.01 vs. WT normalized value. (D) 60 μg of skeletal muscle lysates from control (WT), mdx (mdx), δ-SG-deficient (Sgcd), and mdx: utrophin double null (mdx:utrn−/−) 4-week-old mice. Identical nitrocellulose membranes were probed with antibodies to Akt and P-Akt as indicated. Coomassie blue staining of total protein is shown on the right panel (CB Stain). Molecular size standards are indicated on the left (×103 Da).

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from control (F1B) and SG-deficient (BIO 14.6) hamsters were analyzed by immunoblotting. We found that both Akt and PAkt levels were significantly elevated in BIO14.6 skeletal muscle relative to control, F1B muscle (Figs. 8A and C). Similarly, BIO14.6 cardiac tissues exhibited a 1.8-fold increase in Akt and a 2.2-fold increase in P-Akt compared to nondisease F1B control hearts (Figs. 8B and C). These results are similar to mdx mice at the onset of disease pathogenesis. We were unable to evaluate GSK3β or p70S6K in the BIO14.6 muscle

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due to manufacturer's recommendation that the currently available commercial antibodies do not cross-react with hamster tissue. δ-SG knockout (Sgcd) mice display the characteristic cardiomyopathy and muscle weakness originally characterized in the BIO14.6 hamster [34,65,67–71]. In agreement with the BIO14.6 data, Akt activation was elevated in the Sgcd mouse model when compared to control and mdx age-matched tissue (Fig. 8D). Furthermore, Sgcd mice displayed Akt activation levels higher than that observed for agematched mdx mice. In the current study, we report the activation of Akt signaling molecules in both dystrophin- and SG-deficient muscle (Fig. 9). It is likely that destabilization of the DGC indirectly influences the Akt pathway, since Akt is not a core component of the DGC complex. These findings strongly suggest that amplification of Akt phosphorylation occurs as part of the regenerative response and likely mediates the hypertrophic response in both SG- and dystrophin-deficient muscular dystrophy. This response is

similar to that reported for IGF-1 mediated muscle hypertrophy, which rescues mdx pathology [32]. Robust muscle regeneration in mdx mice compared to DMD patients has been proposed as one explanation for their relatively mild phenotype [25–28]. These results lead to the proposal that vigorous activation of hypertrophic pathways during prenecrotic stages of disease might reduce the severity of pathology or extend the prenecrotic stage of disease. Future studies will be focused on introducing constitutively active Akt and dominant-negative Akt into young mdx mice to determine how early modification of Akt activity influences disease pathogenesis.

Acknowledgments We thank the members of the Duchenne Muscular Dystrophy Research Center (UCLA) for their comments and support. A.K. Peter was supported by the Molecular, Cellular and Integrative

Fig. 9 – Schematic diagram of Akt/PKB activation in hypertrophic dystrophin- and sarcoglycan-deficient muscular dystrophies. Schematic illustration of Akt signaling in wild-type, mdx, and Sgcd muscle. The entire DGC complex is lost from the sarcolemma in mdx mice, as depicted by lighter shading of the DGC components. Sgcd muscle maintains dystrophin and the DG expression, but the SG-SSPN subcomplex is absent from the membrane. Results presented in the current report demonstrate that the Akt pathway is upregulated in dystrophin- and SG-deficient muscle. Phosphorylation of Akt/PKB leads to activation of mTOR-dependent (p70S6K) and mTOR-independent (GSK) pathways. Activation of the Akt signaling axis causes amplification of protein synthesis and myofiber hypertrophy. Biochemical fractionation shows that Akt is not translocated to the nucleus in hypertrophic, mdx muscle. Current studies are underway to determine whether activation of hypertrophic pathways during prenecrotic stages of disease would reduce the severity of pathology or extend the prenecrotic stage of disease.

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Physiology pre-doctoral training fellowship (NIH: T32 GM65823). This research was also supported in part by a grant from the NIH (AR48179-01 to R.H.C.) and the Muscular Dystrophy Association (MDA3704 to R.H.C.).

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