Introduction

Decorin, a small leucine-rich proteoglycan, is a component of the extracellular matrix of all collagen-containing tissues.¹ Decorin is pivotal in regulating the proper assembly of collagenous matrices and in controlling cell proliferation under various conditions.² On the basis of its ability to bind fibrillar collagen and delay in vitro fibrillogenesis, decorin is regarded as a key modulator of matrix assembly.^3,4 This proteoglycan can modulate the bioactivity of growth factors and act as a direct signaling molecule to different cells.⁵ Decorin, which is expressed at high levels in skeletal muscle during early development,⁶ also interferes with muscle cell differentiation and migration and regulates connective tissue formation in skeletal muscle.^7,8,9

Because terminal differentiation is critical for initial skeletal muscle development and regeneration after injury and disease,¹⁰ we examined decorin's role in remodeling healing skeletal muscle. We have shown that the direct injection of bovine decorin decreased muscle fibrosis and provided nearly complete functional recovery.¹¹ Decorin blocks fibrosis (mostly by inhibiting transforming growth factor (TGF)- activity), which improves muscle healing. However, the role of decorin in muscle cell differentiation and regeneration is still unknown. Although we hypothesize that decorin's effect on muscle fibrosis may indirectly impact regeneration, we were unable to exclude the possibility that decorin promotes regeneration independent of its effects on fibrosis formation.

Many studies have investigated the mechanism behind the antifibrotic effect of decorin.^3,12,13,14 Others have shown that hepatocyte growth factor increases decorin production by fibroblasts through the extracellular signal–regulated kinase 1/2, and p38 mitogen-activated protein kinase–mediated pathways.¹⁴ Decorin stimulates the growth of smooth muscle cells under specific conditions and influences the growth of epidermal cells by interacting with epidermal growth factor and its receptors.^15,16 Recent research has shown that decorin can bind both insulin-like growth factor-I and its receptor; this interaction leads to the phosphorylation of protein kinase B (Akt) and p21 expression in endothelial cells.¹⁷

Decorin also influences muscle cell behavior by interacting with p21, an important cyclin-dependent kinase inhibitor.^18,19 Follistatin and myostatin are involved in the control of muscle mass during development. These two proteins have opposite effects on muscle growth, as documented by genetic models.^21,22 Recent studies have shown that myostatin action is inhibited by decorin,²³ resulting in enhanced healing and reduced fibrosis within myostatin-null mice compared with wild-type mice.²⁴ A recent study indicates that peroxisome-proliferator-activated receptor-gamma co-activator-1 (PGC-1), is also involved in the muscle healing process and influences muscle fiber–type determination.^25,26 Decorin may also interact with PGC-1 expression in skeletal muscle after injury.

In this study, we investigated the in vitro effect of decorin on the differentiation of myoblasts (C2C12) and characterized the in vitro and in vivo behavior of myoblasts transfected with the decorin gene (CD cells). We also studied the influence that decorin over-expression had on myostatin, follistatin, PGC-1, and p21 expression. Using an adeno-associated virus (AAV) vector, we transduced the decorin gene into injured skeletal muscle to further investigate its function on muscle healing. Our overall goal in this study was to determine whether decorin could improve skeletal muscle healing by enhancing muscle regeneration independently of its antifibrotic action.

Results

Genetic engineering of myogenic cells to over-express decorin

We used lipofectin to transfect a pAAV-CMV-decorin plasmid (Figure 1a) into both 293 cells (packaging cell line) and C2C12 cells (myoblast cell line). The results of western blot analysis (Figure 1b) showed decorin in both the supernatant (culture media) and the lysate of the 293 cells 48 hours after transfection. The transfected C2C12 cells (CD clone cells) expressed decorin (Figure 1c: lane 2, 24 hours; lane 4, 48 hours; lane 5, decorin-positive control), whereas non-transfected C2C12 cells did not (Figure 1c: lane 1, 24 hours; lane 3, 48 hours). We also detected decorin in both myoblasts (C2C12) and muscle-derived stem cells after mDecorin-AAV (mDec-AAV) gene transfer in vitro (Figure 1d and e).

Figure 1.

Decorin plasmid construction and initial transfection in vitro. (a) The decorin plasmid used for the study contained the full sequence of a mouse decorin gene inserted at the NotI site, which placed it under the control of a cytomegalovirus (CMV) promoter. (b) We transfected the plasmid into 293 cells. We observed decorin expression in both the 293 cells and their supernatant, but not in the control adeno-associated virus (AAV)–transfected (green fluorescent protein, GFP) cells. (c) Western blot analysis also revealed decorin expression in CD clone cells within different time period cultures (lane 2, 24 hours; lane 4, 48 hours), but not in C2C12 (lane 1, 24 hours; lane 3, 48 hours). We used 5 g of decorin as a positive control (lane 5). GAPDH, glyceraldehyde-3-phosphate dehydrogenase is used as a control. (d) Decorin expression in muscle-derived stem cells (MDSCs) in pellet form was low, but this was not the case in the supernatant. Both MDSCs and their cultured supernatant strongly expressed decorin after mDec-AAV gene transfer. -actin is used as a control. (e) We did not detect decorin in normal C2C12 cells, but C2C12 cells and their cultured supernatant both expressed decorin after mDec-AAV gene transfer.

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Decorin stimulates myoblast differentiation in vitro

To investigate myoblast differentiation, we compared decorin-co-cultured C2C12 cells with non-treated C2C12 cells in vitro. C2C12 cells cultured with decorin (10 g/mL) and grown in differentiation/fusion medium exhibited significantly enhanced differentiation and fusion in vitro. After 3 and 4 days of stimulating C2C12 cells with decorin, we observed a significant increase in the number of myotubes when compared with un-stimulated C2C12 (control) cells (P< 0.01 at 3 days and P< 0.05 at 4 days, respectively). However, the numbers of myotubes 5 days after treatment were not significantly different (Figure 2a). We then evaluated whether CD cells exhibited a greater propensity to undergo myogenic differentiation than did non-transfected C2C12 cells. As shown in Figure 2b (myotubes stained in mouse anti-myosin heavy chain are red) and Figure 2c, CD cells generated significantly more myotubes overall and created significantly larger myotubes than did non-transfected C2C12 cells.

Figure 2.

Decorin stimulates C2C12 differentiation in vitro. (a) Decorin treatment accelerated the differentiation and fusion of myoblasts (C2C12 cells) compared with non-treated myoblasts (C2C12 cells). (a) The cultures of decorin-treated C2C12 cells contained more myotubes at the 3- and 4-day time points than control cells. (b, c) Similarly, decorin-transfected C2C12 clone cells (CD cells) produced more myotubes than did C2C12 cells, including larger myotubes (containing more than three nuclei) in vitro. Red staining shows myosin heavy chain fluorescence after immunostaining (b).

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Decorin increases myoblast differentiation and induces myogenic gene expression in vitro

We investigated whether the CD cells expressed higher levels of myogenic genes than did non-transfected C2C12 (control) cells. Our results, shown in Figure 3a, demonstrate that decorin gene transfer led to higher expression of the myogenic genes Myf5, Myf6, MyoD, and myogenin. Desmin expression levels in CD and C2C12 cells remained similar.

Figure 3.

Decorin gene transfer up-regulates myogenic proteins and p21 and down-regulates myostatin during muscle cell differentiation. (a) Genetic engineering of myoblasts to express decorin influenced the expression of myogenic proteins (including Myf5/6, Myogenin, and MyoD), as shown by western blot results. However, C2C12 cells and CD clone cells expressed comparable levels of desmin. (b) We detected the presence of p21 expressed in CD clone cells. We also detected peroxisome-proliferator-activated receptor-gamma co-activator-1(PGC-1) and follistatin expressed in CD, but not C2C12, cells. In addition, CD cells exhibited lower levels of myostatin, a negative regulator of muscle mass. The induction of tumor growth factor (TGF)-1 auto-expression was also inhibited by decorin over-expression in CD cells. (c) Similar results were obtained by real-time polymerase chain reaction. Lanes 1–6 show results for C2C12 cells exposed to different concentrations of decorin (0, 0.001, 0.01, 0.1, 1.0, and 5.0 ng/ml, respectively). Lane 7 displays the test results for CD cells, which served as the positive control. We did not detect a visible change in the expression of PGC-1, follistatin, myostatin, or p21 after 12 hours of stimulation with different concentrations of decorin. PGC-1 and follistatin were up-regulated in C2C12 cells in a dose-dependent manner after 18 hours of stimulation with decorin. Myostatin was down-regulated in C2C12 cells in a dose-dependent manner after 24 hours of decorin stimulation. The concentration of p21 did not visibly change after cell stimulation with any experimental concentration of decorin over all time points. With decorin gene transfer, we found that CD cells consistently expressed follistatin, p21, and PGC-1 but were negative for myostatin. Note that -actin was selected as a positive gene control.

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Decorin up-regulates p21, follistatin, and PGC-1, but down-regulates TGF-1 and myostatin in C2C12

We also performed experiments designed to investigate the mechanism by which decorin influences the differentiation of muscle cells. We found that CD cells exhibited increased p21 expression and decreased myostatin expression (Figure 3b). The C2C12 cells can be induced to express TGF-1 in an autocrine manner, as we have previously determined;²⁷ however, the CD cells do not show any detectable expression of TGF-1 after TGF-1 stimulation. More importantly, we detected that both follistatin and PGC-1 (Figure 3b) had been up-regulated when compared with C2C12 cells. We also discovered that follistatin and PGC-1 messenger RNA were altered in C2C12 cells after decorin stimulation, as determined by real-time polymerase chain reaction. Specifically, we found that PGC-1 and follistatin increased in a dose-dependent manner after 18 hours of stimulation with decorin, and that myostatin was decreased in a dose-dependent manner after 24 hours of stimulation with decorin (Figure 3c). In CD cells, we also observed increased amounts of all three genes (p21, follistatin,and PGC-1), but a decrease in myostatin was observed (Figure 3c).

The up-regulation of follistatin, PGC-1, p21, and myogenic genes, including MyoD (Figure 3a), in CD cells could at least partially explain how decorin promotes muscle cell differentiation. Alternatively, the down-regulation of myostatin, a well-known negative regulator of muscle growth during muscle regeneration, could also benefit muscle cell differentiation.

The implantation of CD cells in skeletal muscle results in improved muscle regeneration

The implantation of CD cells within skeletal muscle resulted in significantly better muscle regeneration than that observed for control C2C12 cells, as determined 4 weeks after injection of the cells into MDX/SCID mice. Although the number of LacZ-positive muscle fibers (i.e., regenerating muscle fibers) did not differ between the groups (Figure 4a, c, and d), the diameters of the regenerating muscle fibers (e.g., dystrophin-positive myofibers) in the muscles injected with CD cells were significantly larger than those of the regenerating muscle fibers in the control muscles (Figure 4b, e, and f; dystrophin is red). The larger diameters of the dystrophin-positive muscle fibers generated by CD cells could indicate that implantation of CD cells accelerated muscle regeneration; however, we were unable to exclude the possibility that the CD cells may have a greater propensity to fuse in host myofibers than C2C12 control cells.

Figure 4.

Decorin gene transfer stimulates muscle regeneration in vivo. C2C12 cells regenerated muscle fibers after transplantation into skeletal muscle of MDX/SCID mice, as shown on both (a) LacZ- and (b) dystrophin-expressing myofibers. However, transplantation of CD clone cells, rather than C2C12 cells, resulted in larger muscle fibers in MDX/SCID mice, as shown in some of the (d) LacZ- and (e) dystrophin-positive myofibers. (c) Although there was no significant difference between the number of LacZ-labeled muscle fibers that formed in muscles transplanted with C2C12 or CD cells, (f) the transplantation of CD cells resulted in the regeneration of larger-diameter myofibers (P < 0.01).

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mDec-AAV gene transfer promotes muscle regeneration and reduces fibrosis

Better muscle regeneration was observed within mDec-AAV-treated muscle (Figure 5a and b) than within non-mDec-AAV-treated muscle at 2 weeks after injury (Figure 5c and d). Histological analysis of total collagen deposition 4 weeks after injury revealed that mDec-AAV-injected muscles contained less fibrous scar tissue in the injured area than did non-treated control muscles (Figure 5f, h, and j; collagen deposition areas are blue). We also observed that decorin stimulated skeletal muscle regeneration 4 weeks after laceration injury. We found that mDec-AAV-injected muscles contained more centronucleated (regenerating) myofibers and less scar tissue 4 weeks after injury than did control muscles (Figure 5e, g, and i).

Figure 5.

mDec-AAV vector gene therapy in injured muscle prevents fibrosis and promotes muscle regeneration. Decorin-treated muscle exhibits a greater number of regenerating myofibers than the control muscle at all time points (a–d 2 weeks; e–h 4 weeks). (i) The mDec-AAV-injected muscle contained significantly higher numbers of centronucleated (regenerated) myofibers than did control (sham-injected) muscle at 4 weeks after therapy. We also found that decorin gene therapy minimized fibrosis in injured skeletal muscle. We used Masson's trichrome staining to reveal collagen in injured skeletal muscle, the results of which show that (f) mDec-AAV-injected muscle contained significantly less fibrosis in the injured area than did the (h) control muscle at (j) 4 weeks after injury.

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Discussion

Results from these experiments show that decorin is able to activate the differentiation of skeletal muscle cells (C2C12) in vitro and enhances muscle regeneration in two mouse models in vivo. The mechanism behind decorin's accelerated muscle healing is not yet known; however, our results demonstrate that decorin up-regulates the expression of PGC-1, follistatin, p21, and a variety of myogenic proteins (including MyoD) but down-regulates myostatin expression. These results, in addition to decorin's ability to neutralize the effects of TGF-1, likely explain the beneficial action that decorin has on muscle cell differentiation and muscle regeneration.

Our previous studies have demonstrated that myogenic cells (including muscle-derived stem cells) in injured muscle can differentiate into fibrotic cells and that TGF-1 is a major stimulator of this differentiation.^27,28 Using different animal models of muscle injury, we have investigated biological approaches to prevent fibrosis and thereby improve muscle healing.^{11,27,29,30,31} We have used various molecules, such as decorin, that impede fibrosis by blocking TGF-1 to facilitate the near-complete recovery of injured skeletal muscle.¹¹ The ability of decorin to inhibit TGF-1 activity is the likely mechanism by which this molecule blocks fibrosis formation. However, our results indicate that the improved muscle healing observed after decorin treatment is due to both its inhibiting effect on fibrosis and its stimulating effect on muscle regeneration. Figure 6 summarizes the potential effect of decorin on muscle healing.

Figure 6.

Schematic of the potential effect of decorin on muscle healing. Decorin may improve muscle healing through various pathways: inhibition of tumor growth factor (TGF)-1, up-regulation of follistatin, peroxisome-proliferator-activated receptor-gamma co-activator-1(PGC-1), p21, and myogenic genes (such as MyoD), and down-regulation of myostatin expression. MDSCs, muscle-derived stem cells.

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The repair of injured skeletal muscle occurs through the activation of muscle precursor cells located between the basal lamina and the sarcolemma, including satellite cells and stem cells.³² The activation and growth of these cells are regulated by various growth factors released by infiltrating lymphocytes, injured myofibers, and the extracellular matrix.^10,32 Some growth factors, such as insulin-like growth factor-1 and hepatocyte growth factor, can stimulate precursor cell proliferation and differentiation by increasing the transcriptional activity of the muscle basic helix–loop–helix.^33,34,35,36 Healing and organizational processes are dependent upon the extra- and intracellular signaling that induces the expression of myogenic genes, including MyoD, Myf5, and myosin.^37,38 When properly stimulated, precursor cells fuse with one another or with local myofibers to repair the damaged muscle.³⁹

Muscle regeneration, the key event in muscle healing, is often incomplete, particularly in severely injured muscle.^{10,11,27,28,32,40} The overgrowth of the extracellular matrix leads to significant local fibrosis (i.e., fibrous scar formation) in the injured area, which can impede the formation of normal muscle fibers. The presence of fibrous scar tissue in injured muscle results in incomplete functional recovery and a propensity for re-injury.⁴¹ Muscle regeneration and fibrosis in injured muscle often occur simultaneously and thus compete with one another during the muscle healing process.^32,36,40 A persistent imbalance between collagen biosynthesis and degradation contributes to hypertrophic scar formation and fibrosis in many tissues.^42,43 Several studies have revealed high levels of collagen in injured regions of skeletal muscle, and shown that inhibition of collagen deposition reduced the formation of scar tissue in injured skeletal muscle.^27,28,30,31

Interactions between decorin and TGF-1 have been observed in many tissues, and researchers have used various animal models to study the antifibrotic effect of decorin.^3,11,13,44 Researchers have also shown that hepatocyte growth factor can increase the level of decorin expression in fibroblasts, perhaps by activating the extracellular signal–regulated kinase 1/2 and p38 mitogen-activated protein kinase–mediated pathways.¹⁴ Such findings could explain the antifibrotic effect of hepatocyte growth factor in a variety of tissues.^14,45,46,47 In endothelial cells, decorin binds with both insulin-like growth factor-I and its receptor to influence cell behavior.¹⁷ Decorin can also control and suppress cancer growth and invasion, presumably by influencing the biological activity of growth factors such as TGF-1, platelet-derived growth factor, vascular endothelial growth factor, and epidermal growth factor, all of which are released by cancer cells.^5,15 These decorin-induced effects appear to be mediated, at least in part, by a specific interaction between the decorin protein core and the epidermal growth factor receptor.^15,16 This interaction triggers a signal cascade that results in activation of mitogen-activated protein kinase, mobilization of intracellular calcium, up-regulation of p21, and, ultimately, the suppression of tumor growth.^18,19

Cell cycle exit and the differentiation of muscle cells are coordinated by p21, which is essential for normal myogenic progenitor cell differentiation and skeletal muscle regeneration. Studies have indicated that p21 is necessary for MyoD-induced activity in cells, allowing them to enter into and be stabilized in a post-mitotic state. Since MyoD plays a central role in the differentiation of muscle cells, TGF-1 controls myostatin-related regulation of myogenesis in muscle cells by down-regulating both p21 and MyoD. In this study, we determined that the treatment of myoblasts with decorin down-regulated the expression of myostatin, which might influence p21 and myogenic protein expression. In addition, myostatin and follistatin interact directly in the skeletal muscle system. Follistatin can inhibit myostatin, leading to muscle differentiation in a concentration-dependent manner.²¹ PGC-1, which is expressed in several tissues, including brown fat and the skeletal muscle of mammals, activates mitochondrial biogenesis and oxidative metabolism.²⁶ PGC-1 is a principal factor involved in determining muscle fiber type in injured skeletal muscle and is involved in exercise-induced mitochondrial biogenesis.⁴⁸ In this experiment, we observed that decorin treatment increased PGC-1 expression in skeletal muscle cells. Combined with our previous research results, our current findings suggest that decorin not only acts as an antifibrotic agent but also enhances muscle regeneration in skeletal muscle.

Successful muscle differentiation during limb development requires decorin expression.⁶ Previous findings have shown that decorin can improve muscle healing by inhibiting fibrosis and that myoblasts and muscle satellite cells expressing decorin in an injured site regenerated damaged myofibers faster than the controls.^11,27 The results of this study demonstrate that decorin is also a potent stimulator of skeletal muscle regeneration. Myoblasts expressing decorin differentiated and fused to form myotubes and myofibers at a significantly higher rate than did normal myoblasts in vitro and in vivo. We attribute this enhanced differentiation to the up-regulation of p21, follistatin, PGC-1, and myogenic gene expression and the down-regulation of TGF-1 and myostatin. These results provide at least a partial explanation of the way in which decorin promotes muscle regeneration and may explain why there is such a high level of decorin expression in developing skeletal muscle. It is possible that decorin increases muscle fiber growth and limits the overgrowth of connective tissues. These findings indicate that decorin could be very useful in promoting the healing of muscles damaged by injury or disease.

Materials and Methods

Gene transfection and transfer. An AAV-mDecorin plasmid, which encodes for a mouse decorin sequence under the control of the cytomegalovirus promoter (Figure 1), was used for gene transfection. This plasmid also contains a neomycin resistance gene to enable G418 selection. The AAV-mDecorin plasmid was transfected into 293 packaging cells and C2C12 cells with lipofectin; clone cells were selected for treatment of the cells with G418 (500 g/mL) (Gibco BRL, Grand Island, NY) for 2 weeks. The selected decorin-transfected C2C12 clone cells (CD cells) were cultured in Dulbecco's modified Eagle's medium (Gibco BRL, Grand Island, NY) containing the same concentration of G418 for the remainder of the project.

The mDec-AAV vector was produced by co-transfection methods described previously by Dr. Xiao.⁴⁹ Muscle-derived stem cells and myoblasts (C2C12 cells) were each grown to 50–60% confluency. Fresh Dulbecco's modified Eagle's medium (without fetal bovine serum or penicillin/streptomycin) containing the mDec-AAV vector (5 10⁴ particles/cell) was then added directly to the cells. The cultures were incubated at 37 °C in a 5% CO₂ incubator for 1 hour. Normal culture medium (Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% Abs) was then added for another 24 hours, at which point the cells were collected for analysis of decorin expression by western blotting.

Differentiation of myoblasts and immunocytochemistry. Three different groups of cells (C2C12 cells, C2C12 cells cultured with decorin, and CD cells) were seeded into 12-well plates containing proliferation medium.²⁸ All cells were transferred into serum-free medium 12 hours later to induce differentiation. The myotubes that formed in the cultures were counted daily for 5 days, and the numbers were compared among the groups. We considered myotubes containing three or more nuclei to be large myotubes in vitro. At different time points, the cells were fixed with cold acetone (3 minutes) for immunostaining. Mouse anti-myosin heavy chain antibody (Novocastra Lab) at a 1:200 dilution was applied for 1 hour at room temperature (RT). The primary antibody was detected using anti-mouse-Cy3, 1:250 for 45 minutes at RT. Results were analyzed by fluorescent microscopy (Nikon microscope, Nikon, Melville, New York).

Real-time polymerase chain reaction. Total RNA was extracted from the treated and non-treated C2C12 cells using a Nucleospin column (Clontech, Mountan View, CA), and the complementary DNA was synthesized with SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA), both according to manufacturer's instructions. Primers specific for myostatin, follistatin, p21, and PGC-1were designed using Oligo software (OligoPerfect Designer; Invitrogen, Carlsbad, CA). The protocol for amplification was as follows: 94 °C for 30 seconds, 58 °C for 30 seconds, and 72 °C for 30 seconds for 30 cycles. Polymerase chain reaction products were separated by size in a 1.5% agarose gel.

Western blot analysis. C2C12 and CD cells were lysed when cell density reached 70% confluency. The samples were separated on a 12% sodium dodecyl sulfate–polyacrylamide electrophoresis gel and transferred to nitrocellulose membranes used to perform immunostaining. The primary antibodies were anti-decorin (a gift from Dr. Fisher of the National Institutes of Health), anti-TGF-1 (4 g/mL; BD Pharmingen, San Diego, CA), anti-p21 (BD Pharmingen, San Diego, CA), anti-myf5, anti-myf6, anti-MyoD, and anti-myogenin (Santa Cruz Bio, Santa Cruz, CA), all at concentrations of 1:1,000, and anti-myostatin, anti-follistatin (Chemicon, Temecula, CA), and anti-desmin (Sigma, St. Louis, MO), all at concentrations of 1:2,000 for 1 hour at RT. Mouse anti--actin and anti-glyceraldehyde-3-phosphate dehydrogenase (Sigma, St. Louis, MO) were used for protein quantification and were diluted to 1:8,000. The secondary anti-rat horseradish peroxidase or anti-rabbit horseradish peroxidase (Pierce, Rockford, IL) was used at a concentration of 1:5,000 for 1 hour. Peroxidase activity was determined by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ), and the positive bands were detected on X-ray film. Northern Eclipse software v.6.0 (Empix Imaging, Mississauga, Canada) was used to evaluate all results.

Animal experiments. All animal experiments were approved by the Children's Hospital of Pittsburgh. The Animal Research Committee at the authors' institution approved all experimental protocols (No. 15/03).

Group 1: C2C12 and CD cell transplantation. Twenty-four female MDX/SCID mice (C57BL/10ScSn-Dmd^mdx crossed with C57BL/6J-Prkdc^scid/SzJ, 6–8 weeks of age) were used for the C2C12 and CD cell transplantation. C2C12 and CD clone cells were transduced with a retrovirus vector encoding for LacZ.²⁷ LacZ-positive CD cells (1 10⁶) were injected into the left gastrocnemius muscles (GMs); the same quantity of LacZ-positive C2C12 cells were injected into the right GMs as a control. At various times after injection, mice were killed, and the GMs were collected for histological analysis by LacZ staining and immunohistochemistry to stain for dystrophin-positive myofibers.

Group 2: mDec-AAV gene therapy administered to injured skeletal muscle. Twenty mice (C57BL6J^+/+, 6 weeks old; Jackson Laboratory, Bar Harbor, ME) were used for these experiments. The mDec-AAV vector (2 10¹¹ particles in 20 L of Dulbecco's modified Eagle's medium) was injected directly into the left GM of each mouse; the contralateral leg was injected with the same volume of phosphate-buffered saline (20 L) as a control. One week after injection, both GMs were lacerated in accordance with our previously described muscle injury model.^11,27,28,40 Mice were killed at different time points (5 days and 1, 2, 3, and 4 weeks after injury), and the GMs were collected for histological analysis by either hematoxylin and eosin or Masson's trichrome staining. The regeneration and fibrous scar tissue formation in the two groups were compared.

Immunohistochemical analysis. Serial 10-m cryostat sections were prepared using standard techniques.^27,28 For immunohistochemistry, the slides were fixed with formalin (4%) for 5 minutes after LacZ staining, and then blocked with donkey serum (10%) for 1 hour. Rabbit anti-dystrophin antibody (Abcam, Cambridge, MA) was applied to the slides at a 1:300 dilution for 60 minutes at RT. The second antibody, goat anti-rabbit IgG (Alexa Fluor^® 488; Molecular Probes, Eugene, OR), was used at a concentration of 1:200 for 45 minutes at RT. Negative controls were performed concurrently with all immunohistochemical staining. The nuclei of the sections were revealed using 4',6'-diamidino-2-phenylindole hydrochloride staining (Sigma, St. Louis, MO), and fluorescent microscopy was used to visualize the results as described above.

Statistical analysis. LacZ-positive myofibers were counted in 10 representative sections. Both the diameter and number of LacZ- and dystrophin-positive myofibers were assessed at different time points in each group. The statistical significance of differences between the various groups was determined using a t-test or one-way or two-way analysis of variance.

References

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Acknowledgments

The authors wish to thank James Cummins, Marcelle Pellerin, and Jing Zhou (Stem Cell Research Center, Children's Hospital of Pittsburgh, Pittsburgh, PA) for their technical assistance, Paul Robbins (Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA) for his contribution of the LacZ retrovirus vector, and Ryan Sauder, Shannon Bushyeager, and David Humiston (Stem Cell Research Center) for their excellent editorial assistance with this manuscript. The authors also gratefully acknowledge the financial support of the Department of Defense in the form of grant W81XWH-06-01-0406 and the National Institutes of Health in the form of NIH grant R01 AR47973.