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. 2010 Aug 3;18(12):2155–2163. doi: 10.1038/mt.2010.165

Laminin-111: A Potential Therapeutic Agent for Duchenne Muscular Dystrophy

Sébastien Goudenege 1, Yann Lamarre 1, Nicolas Dumont 2, Joël Rousseau 1, Jérôme Frenette 2, Daniel Skuk 1, Jacques P Tremblay 1
PMCID: PMC2997583  PMID: 20683444

Abstract

Duchenne muscular dystrophy (DMD) still needs effective treatments, and myoblast transplantation (MT) is considered as an approach to repair damaged skeletal muscles. DMD is due to the complete loss of dystrophin from muscles. The lack of link between the contracting apparatus and the extracellular matrix leads to frequent damage to the sarcolemma triggering muscle fiber necrosis. Laminins are major proteins in the extracellular matrix. Laminin-111 is normally present in skeletal and cardiac muscles in mice and humans but only during embryonic development. In this study, we showed that intramuscular injection of laminin-111 increased muscle strength and resistance in mdx mice. We also used laminin-111 as a coadjuvant in MT, and we showed this protein decreased considerably the repetitive cycles of degeneration, inflammatory reaction, and regeneration. Moreover, MT is significantly improved. To explain the improvement, we confirmed with the same myoblast cell batch that laminin-111 improves proliferation and drastically increases migration in vitro. These results are extremely important because DMD could be treated only by the injection of a recombinant protein, a simple and safe therapy to prevent loss of muscle function. Moreover, the improvement in MT would be significant to treat the muscles of DMD patients who are already weak.

Introduction

Duchenne muscular dystrophy (DMD) is a severe muscle degenerative disease affecting ~1 out of every 3,500 male newborns, making it the most prevalent muscular dystrophy. The gene for DMD, found on the X chromosome (Xp21) encodes a 427 kDa cytoskeletal protein, called dystrophin.1 Dystrophin is required inside myofibers to link elements of the internal cytoskeleton to a complex of glycoproteins in the sarcolemma. The dystrophin-associated protein complex provides a mechanical link between the extracellular matrix and the cell cytoskeleton.2 This linkage may supply an important mechanism for anchoring myofibers to the extracellular matrix, stabilizing, and protecting the sarcolemma from the mechanical stresses that occurs during muscle contraction and relaxation. In DMD patients, this linkage is lost, rendering the sarcolemma susceptible to damage during muscle contraction/relaxation and thus producing myofiber necrosis.3,4 The subsequent inflammatory response could increase the myofiber damage.5 A mouse model for DMD exists (mdx mice) and is proving useful for furthering our understanding of this pathology.1

Various therapeutic strategies for DMD are under investigation, including gene or cell therapy, but no efficient treatment is yet available. Our laboratory is working since several years on myoblast transplantation (MT) as a potential therapy for most of the recessive dystrophies and specifically for DMD. MT is so far the only approach that has unequivocally proved in mouse experiments to be able to form new myofibers,6,7 supply new myogenic cells and form new satellite cells.8 Indeed, we have observed the probable neoformation of small myofibers in some of the patients participating in our clinical trial.9 One of these patients exhibited a cluster of ~500 potentially new dystrophin-positive myofibers. To progress toward clinical applications of MT, there are some issues of this approach that need to be improved. An interesting avenue is opened by the recent findings about the beneficial effects of some laminins. Silva-Barbosa et al. showed that local irradiation of tibialis anterior (TA) muscles from immunodeficient mice enhances the laminin content in the host muscle microenvironment and provides a better engraftment of human myoblasts.10

Many proteins are implicated in muscle stability and integrity. Among them, laminins are major proteins in the extracellular matrix. They are found predominantly in basement membranes, which are the thin sheets of extracellular matrix that underlie epithelial and endothelial cells and surround muscle cells, Schwann cells, and fat cells.3 Laminins are large heterotrimeric proteins that contain an α-chain, a β-chain, and a γ-chain. Laminin molecules are named according to their chain composition. Fourteen chain combinations have been identified in vivo. The trimeric proteins form a cross, giving a structure that can bind to other cell membrane and extracellular matrix molecules. They are secreted and incorporated into cell-associated extracellular matrices. Laminins are vital for the maintenance and survival of tissues.

Laminin-111 (α1, β1, γ1) is the most widely studied isoform. It is expressed during embryonic development but is absent in postnatal normal or dystrophic skeletal muscle. Recently, Rooney et al.11 showed that injection of laminin-111 into mdx mice increased expression of α7-integrin, stabilized the sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscle from exercised-induced damage. These results indicated that laminin-111 is a potential therapeutic agent for DMD and, together with the findings of Silva-Barbosa et al.,10 suggest that it could be an important therapeutic for DMD.

Therefore, in the present study, we wanted to further evaluate the benefits of laminin-111 to improve the muscle pathology in mdx mice, and mainly to evaluate the possibility of combining this molecule with MT.

Results

Laminin-111 increases resistance and total/specific strength in dystrophin-deficient muscle

In spite of the high molecular mass of laminin-111 (900 kDa), Rooney et al.11 showed that this was not a barrier to its systemic delivery following intraperitoneal (i.p.) administration, reaching the skeletal muscles and heart of mdx mice. We therefore tested intramuscular (i.m.) injection and i.p. injection of laminin-111 on the resistance to eccentric contractions and on the strength of the mdx extensor digitorum longus muscle (EDL). For the i.m. experiments, the right EDL muscle was injected with laminin-111 and the left EDL muscle with phosphate-buffered saline (PBS). C57BL10J mice injected i.m. or i.p. with PBS were used as normal control. For both i.m. and i.p. administration, mice were killed 24 days after the treatment to ensure complete muscle regeneration. The EDLs were prepared to evaluate the contractile properties.

Eccentric contractions occur when a muscle is contracting and lengthened simultaneously. Such forced lengthening damages the myofibers and reduces subsequent tetanic force development. Following i.p. or i.m. administration of laminin-111 in mdx mice, the percentage of force drop was always lower than for the group injected with PBS. The curves of force drop in laminin-111-treated and PBS-injected mdx mice were thus significantly different both following i.m. injections (P < 0.0018, Figure 1b) and i.p. injections (P < 0.0164, Figure 1a). However, both mdx curves (laminin-111-treated and PBS-injected mice) following i.m. injections (P < 0.0001, P < 0.0001, respectively) and i.p. injections in mice (P < 0.0001, P < 0.0001, respectively) were significantly different from normal C57BL10J mice injected with PBS. These results indicate that laminin-111 protected the muscle fibers of mdx mice from damage induced by eccentric contractions; however, it did not restore complete normality.

Figure 1.

Figure 1

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Laminin-111 increases resistance, absolute (g) and specific force (N/cm2) in dystrophin-deficient muscle. (a,b) Muscle resistance was measured by performing seven consecutive eccentric contractions. The change of tetanic force between each contraction reflects the degree of muscle damage. An eccentric contraction protocol was performed on mdx injected with laminin-111 or PBS, and on normal C57BL/10J. Systemic delivery (i.p.) of laminin-111 and intramuscular injection (i.m.) are represented, respectively, in a and b. The tetanic tension developed during the first cycle was designated as 100%. The mdx laminin-111 curves were significantly lower (percentage of force drop) than the mdx PBS control following i.m. injections (P < 0.0018, b) and i.p. injections (P < 0.0164, a). Both mdx curves (laminin-111 treated and PBS-injected mice) following i.m. injections (P < 0.0001, P < 0.0001, respectively) and i.p. injections to mice (P < 0.0001, P < 0.0001, respectively) were significantly different from normal C57BL10J. The P value refers to comparison between trend of multipoint curve laminin-111, control mdx and normal mice. (c,d) Absolute maximal muscle forces of control mdx injected with PBS, mdx injected with laminin-111, and normal C57BL/10J groups were compared. No significant difference was observed on absolute maximal force between mdx groups following i.p. injection of laminin-111 (c). A significant 41% increase of the mdx absolute maximal force (d) was observed following an i.m. injection of laminin-111 relative to the mdx muscle injected with PBS. (e,f) The specific maximal force was calculated by analyzing the absolute maximal force of a muscle in function of its mass and length. The specific maximal force of the mdx muscles injected i.p. with laminin-111 or with PBS was significantly different from the wild-type control muscles. There was, however, no significant difference of the specific maximal force between the mdx muscles injected i.p. with PBS or with laminin-111 (e). However, a significant 44% increase of the mdx specific maximal force (f) was observed following an i.m. injection of laminin-111 relative to the mdx-injected i.m. with PBS. *Statistically different results (n = 5 for i.p. injection, n = 4 for i.m. injection). i.p., intraperitoneal; PBS, phosphate-buffered saline.

The strength of C57BL10J and mdx EDL injected with laminin-111 or with PBS was also measured in vitro by stimulating the muscles at 120 Hz. Significant increases of the mdx absolute maximal force (40.6%, Figure 1d) and of the mdx specific force (43.72%, Figure 1f) were observed following i.m. injection of laminin-111. The strengths of the PBS and laminin-111-treated mdx mice were 50.2 and 72.2%, respectively, of the normal mice. However, such improvements of mdx mice were not observed following i.p. delivery of laminin-111 (Figure 1c,e), probably because the concentration of laminin-111 in the EDL was not as high as the one obtained by i.m. injection. Thus, the sole presence of laminin-111 was sufficient to protect muscles from eccentric damage, as indicated by the force drop results, and to increase the absolute or the specific force in the EDL of mdx mice.

To rule out that the strength improvement of the mdx mice was due to an injury/regeneration-based induced by the injection of laminin-111, the number of centrally nucleated fibers (CNF) was quantified (Supplementary Figure S1). After 24 days, mdx muscles injected with Hank's balanced salt solution (HBSS) showed no significant difference as compared to the control mdx without injection, respectively, 54.8 and 52.9%; thus, the process of regeneration induced by intramuscular injection seems to be over. In addition, the percentage of CNF significantly decreased after laminin-111 injection, i.e., the mdx muscles injected with HBSS had 54.8% positive CNF compared with the contralateral laminin-111-injected muscles, which has only 37.1% positive CNF (n = 5, P < 0.05). These results indicate that the intramuscular injection of laminin-111 reduced myofiber degeneration as previously shown by Rooney et al.11

Laminin-111 prevents muscle pathology in mdx mice after MT

To examine whether laminin-111 could improve MT, human myoblasts were resuspended either in 20 µl of a 500 nmol/l solution laminin-111 or in HBSS alone, and injected in the TA of Rag/mdx mice. Rag/mdx mice have an mdx dystrophic phenotype and a Rag mutation that render them immunodeficient and able to accept human grafts. The mice were killed 24 days later, and the TA muscles were prepared to analyze muscle integrity and the success of MT. Laminin-111 was detected by immunohistochemistry throughout the cryosections of muscles injected i.m. with laminin-111, but only background staining was observed in the mdx muscle injected with HBSS (Figure 2a,b). This confirmed Rooney et al.'s observation11,12 that laminin-111 injected intramuscularly was present throughout the TA muscle for at least 28 days.

Figure 2.

Figure 2

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Immunofluorescence of laminin-111. (a) The presence of laminin-111 was detected by immunofluorescence in the extracellular space of the tibialis anterior muscles of the Rag/mdx mice injected i.m. with laminin-111. (b) Only background staining was observed in Hank's balanced salt solution–injected Rag/mdx muscle. Bar = 90 µm, it applies to both a and b.

Hematoxylin and eosin staining was also used to examine the muscle structure 24 days after MT with or without laminin-111 (Figure 3). Control mdx muscles transplanted with myoblasts without laminin-111 exhibited large clusters of regenerating myofibers, identified by their small diameter, basophilic cytoplasm, mononuclear cell infiltrate, and centrally located nuclei with dispersed chromatin (Figure 3a,c,e). In contrast, 24 days after MT with laminin-111, the muscles (n = 6) did not exhibit these clusters of regenerating myofibers (Figure 3b,d,f) and thus had the appearance of wild-type muscle at low magnification. Minimal mononuclear cell infiltrate was observed in only one laminin-111-injected muscle.

Figure 3.

Figure 3

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Laminin-111 prevents muscle pathology in mdx mice after myoblast transplantation. Hematoxylin and eosin staining reveals that control mdx muscles injected with (a,c,e) myoblasts and Hank's balanced salt solution contain large clusters of regenerating myofibers (identified by their small diameter, basophilic cytoplasm, mononuclear cell infiltrate, and centrally located nuclei) compared with mdx muscles injected i.m. with (b,d,f) myoblasts and laminin-111. Pictures illustrate representative cross-sections (n = 6). a,b, Bar = 0.35 mm; c,d, bar = 160 µm; e,f, bar = 45 µm.

We also evaluated the levels of myofiber necrosis following MT. For this, we injected myoblasts with laminin-111 in the left TA muscle, whereas the right TA muscle was transplanted with myoblasts suspended only in HBSS. We also performed a control i.m. injection of laminin-111 alone, i.e., without myoblast. Evans blue dye (EBD) was injected i.p. the day before killing the treated mice. EBD uptake (Figure 4) was observed in cryosections of all mdx muscles. The mdx muscles injected with myoblasts and laminin-111 had 5.5-fold fewer EBD positive myofibers than the right muscles injected with myoblasts without laminin-111 (10 ± 3 myofibers and 56 ± 28 myofibers, respectively, n = 7, P < 0.05). Moreover, there was no significant difference between muscles injected with laminin-111 alone, and muscles injected with laminin-111 and myoblasts (10 ± 3 and 11 ± 4 myofibers, respectively). These results indicate that laminin-111 dramatically increased sarcolemmal integrity and consequently reduced myofiber necrosis in mdx mice.

Figure 4.

Figure 4

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Laminin-111 increases sarcolemmal integrity. (a) Representative cross-sections of tibialis anterior (Rag/mdx) in optic microscopy. Bar = 150 µm. (b) Evans blue dye (EBD) staining in Rag/mdx control mice, same cross-section as in a. (c) Rag/mdx muscles injected with laminin-111 alone or co-injected i.m. with laminin-111, and myoblasts had reduced EBD uptake compared with control co-injected with Hank's balanced salt solution and myoblasts. *Results are statistically different (P < 0.05; n = 7). There is no significant difference between both laminin-111 conditions with or without myoblasts.

To verify whether laminin-111 reduced the inflammatory reaction associated with myofiber necrosis, we analyzed the infiltration of inflammatory cells in mdx muscles injected i.m. either with laminin-111 or with HBSS. This was determined 24 days after the injections by immunostaining with an anti-Mac-1 antibody (Figure 5). Mac-1+ cells were observed dispersed in the interstitium but mostly inside myofiber profiles, corresponding with the process of myofiber phagocytosis that follow necrosis. Myofibers undergoing phagocytosis were observed in clusters, similar to those described above for regenerating myofibers. The intensity of the inflammatory cell infiltration and indirectly myofiber phagocytosis was thus quantified by measuring the cross-sectional area that was Mac-1+ in the muscles. This area was 90% smaller in laminin-111-treated muscles than in control HBSS-injected muscles (P < 0.01). This indicates that macrophage infiltration was drastically reduced because of the reduced muscle fiber damage.

Figure 5.

Figure 5

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Laminin-111 reduced the inflammatory reaction. Macrophage immunodetection was used to follow the inflammatory reaction in tibialis anterior muscles. (a–c) Rag/mdx control mice co-injected i.m. with HBSS and myoblasts. (b–d) Mice co-injected i.m. with laminin-111 and myoblasts. a,b, Bar = 0.4 mm; c,d, bar = 130 µm. (e) Fluorescence intensity decreased by 90% with laminin-111 compared to the control indicating that macrophage infiltration was drastically reduced. *Significant difference relative to the control (n = 6; P < 0.01). HBSS, Hank's balanced salt solution.

Thus, these experiments unambiguously proved that the presence of laminin-111 reduced considerably the repetitive cycles of necrosis, inflammatory reaction, and regeneration in mdx mice.

Laminin-111 improves the success of MT in Rag/mdx mice

We also wanted to verify whether the injection of laminin-111 concomitant with human MT in Rag/mdx host mice would increase the extent of muscle repair and thus allow the formation of more myofibers expressing human dystrophin. The presence of myofibers expressing human proteins was thus assessed 24 days after injection of myoblasts in the TA. Human dystrophin was detected with a specific monoclonal antibody (mAb) under the sarcolemma of many myofibers. Myofibers expressing human dystrophin were thus quantified to evaluate the success of MT. Figure 6a illustrates representative cross-sections of myoblast-transplanted muscles of Rag/mdx mice treated with laminin-111. The total number of human dystrophin-positive myofibers was significantly higher (33.4%) when laminin-111 was co-injected with the myoblasts than when myoblasts were injected alone (Figure 6b). Thus, injection of laminin-111 in dystrophic host mice significantly improved the success of normal MT.

Figure 6.

Figure 6

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Immunofluorescence showing dystrophin-positive myofibers. (a) Representative cross-sections of tibialis anterior muscles injected i.m. with laminin-111 and human myoblasts (Rag/mdx). Bar = 120 µm. (b) Rag/mdx muscle injected i.m. with laminin-111 contained significantly more dystrophin-positive fibers than control Rag/mdx injected i.m. with human myoblasts without laminin-111. *Statistically different results (P < 0.05; n = 8). HBSS, Hank's balanced salt solution.

Laminin-111 improves myoblast proliferation

In order to explain the improvement of MT observed in vivo, we studied the effects of laminin-111 on myoblast proliferation and migration in vitro. We first studied proliferation. After plating, the total number of myoblasts doubled in 24 hours and doubled again in the next 16 hours in normal proliferation medium (20% serum). The proliferation was very fast, and thus, it was difficult to observe an effect. However, an improved proliferation induced by laminin-111 was observed but not statistically significant at all time points (data not shown). For this reason, the proliferation test was repeated in a condition in which the myoblasts did not proliferate as rapidly, i.e., in a low serum medium (2% serum). The total number of myoblasts was measured at different times (30, 72, and 96 hours) after the beginning of treatment. As shown in Figure 7, an increased proliferation was observed with laminin-111 at all time points (18 and 20%, respectively, after 30 and 72 hours, P < 0.05), and this enhancement was maximum at 96 hours (28%, P < 0.01). To rule out the possibility that laminin-111 is not increasing proliferation per se, but is instead preventing withdrawal from the cell cycle, i.e., preventing premature differentiation, a differentiation test has been realized (Supplementary Figure S2). After 2 days in differentiation medium, no significant difference was observed on the fusion index. After 3 and 4 days, fusion percentages were significantly higher for myoblasts treated by laminin-111 than control myoblasts; thus, the observed increased proliferation was not due to a reduced fusion. From these observations, it was concluded that the presence in the culture medium of laminin-111 had no toxic effects on myoblasts in vitro and increased myoblast proliferation.

Figure 7.

Figure 7

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Laminin-111 stimulated cell proliferation measured by the CyQUANT assay. The fluorescence intensity of the CyQUANT GR dye was measured at different times (0, 30, 72, and 96 hours) to evaluate the myoblast proliferation in a low serum medium (2% serum) (n = 3). An increased proliferation was observed at all time intervals (18 and 20%, respectively, after 30 and 72 hours, P < 0.05), and this enhancement was maximum at 96 hours (28%, P < 0.01). *Statistically different results.

Laminin-111 improves drastically myoblast migration in a serum-free medium

We also studied the effect of laminin-111 on myoblast migration. Myoblasts were loaded into the upper compartment of a Transwell migration chamber. As revealed by colorimetric analysis (Figure 8), laminin-111 enhanced considerably cell migration compared with the control condition without laminin-111, i.e., when laminin-111 was added to the serum-free medium, the quantity of myoblasts that migrated through the 8 µm pore-size polycarbonate membrane to reach the lower chamber of the Transwell system increased by 751% in reference to the migration in the absence of laminin-111 (n = 5, P < 0.01). These results show the strong promigratory effects of laminin-111 in vitro and suggest that laminin-111 co-injected with myoblasts might promote their migration in vivo and potentially their fusion outside the injection site following MT in skeletal muscle.

Figure 8.

Figure 8

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Laminin-111 improved the overall migration rate of human myoblasts in serum-free medium. 3 × 104 cells/100 µl were loaded into the upper chamber of a Transwell migration assay. The medium in the upper chamber was complemented or not with laminin-111 at 100 nmol/l, whereas the lower chamber contained only serum-free medium. The migrating cells were stained after 14 hours of incubation. Representative pictures of the colored cells are shown in (a, serum-free medium) and (b, laminin-111). (c) Histogram shows that laminin-111 enhanced considerably cell migration compared with the control without laminin-111. *Statistically different results (P < 0.01, n = 5).

Discussion

No effective treatment for muscular dystrophy (DMD in particular) currently exists, but two main therapeutic avenues are being pursued by investigators: gene therapy (such as exon skipping or genetic correction by viral vectors or plasmids) and cell therapy. Normal MT is one potential cell therapy, which leads to the formation of dystrophin-positive myofibers in dystrophic muscles, resulting from the fusion of transplanted myoblasts with each other or with the host-damaged myofibers. Delivery of normal dystrophin genes by the transplantation of normal myoblasts results in the long-term restoration of this protein. Indeed, the transplanted myoblasts fuse with the host myofibers and introduce in them nuclei containing the normal dystrophin gene.7 The success of MT, however, is restricted by the fact that the implanted myoblasts fuse essentially with the myofibers around the injection trajectories, and this could be due to the limited amount of regenerating myofibers present in a given period in mdx mice and in DMD patients.13 Despite its genetic and biochemical homology to DMD,1,14 the mdx mouse has limitations as a model of this disease. As DMD patients, the mdx mice suffer extensive degeneration of their skeletal muscle fibers.15 This is most remarkable between 20 and 100 days but persists, less evidently, throughout their life.16,17,18 For these reasons, our experiments to observe morphological state of muscles were done between 1 month and 2 months when the repetitive cycles of necrosis, inflammatory reaction, and regeneration in mdx mice are most important. The dystrophin-associated protein complex provides a mechanical link between the extracellular matrix and the cell cytoskeleton. Previously, it has been established that extracellular components have a clear effect on skeletal muscle development.19,20 Laminins are major proteins in the extracellular matrix and are essential for normal myogenesis.21,22 Among laminins, laminin-111 is present only during embryonic development in skeletal and cardiac muscles in mice and humans. As tissues mature, it disappears and is replaced by other isoforms of laminin.

Two publications have recently reported beneficial effects of laminins in mice. First, Silva-Barbosa et al. showed that local irradiation of TA muscles from immunodeficient mice enhances the laminin content in the host muscle microenvironment and provides a better engraftment of human myoblasts.10 Second, Rooney et al.11 showed that injection of laminin-111 into mdx mice increased expression of α7-integrin, stabilized the sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscles from exercised-induced damage. Together, these results indicated that laminin-111 could be an important therapeutic agent for DMD, whereas the first one suggested that laminin could improve the success of MT.

In the present study, we evaluated the benefits of laminin-111 to improve the muscle pathology in mdx mice, and specifically resistance and strength. We also combined MT and intramuscular injection of laminin-111 into Rag/mdx mice, and our observations demonstrated that this molecule can indeed be used as a coadjuvant for improving MT. In order to have a significant effect on overall muscle physiology, the next step will be to optimize the procedure of MT combined with the laminin-111 treatment. Many improvements may be considered. Notably, a pretreatment with laminin-111 or different concentrations of laminin-111 during MT could be used. MT could also be done after an i.p. treatment with laminin-111 at an optimal concentration. All these experiments are in progress in our laboratory. Considering the function of laminin during myogenesis, it is not surprising that this molecule also enhances the engraftment of myoblasts within regenerating skeletal muscle. In order to explain the improvement of MT observed in vivo, we studied the effects of laminin-111 on myoblast proliferation and migration in vitro (using the same myoblast cell batch used for the in vivo experiments). Our results indicated that the presence in the culture medium of laminin-111 had no toxic effects in vitro and increased myoblast proliferation. These results confirm previous reports showing that laminins are implicated in both expansion and differentiation of myogenic cells.23,24,25,26 As previously mentioned, we also studied the effect of laminin-111 on myoblast migration in vitro. The first clinical trials of MT were based on the hope that some myoblasts injected at isolated points in a skeletal muscle of the patient would proliferate and migrate throughout the muscle, fusing with most of the myofibers. One of the problems limiting the success of MT is that the implanted myoblasts fuse generally only with the myofibers damaged by the injections. This observation was attributed to the absence of migration of the myoblasts toward the myofibers between the injection trajectories. This problem was clearly observed for the first time in monkeys, when myoblasts labeled with a β-galactosidase transgene were injected in muscles using injection trajectories ≥2 mm apart.27,28 After 1 month, rows of β-galactosidase+ myofibers were observed in the muscle biopsies, correlating with the previous injection trajectories. However, no β-galactosidase+ myofibers were observed between the injection trajectories. This observation was attributed to the absence of migration of the myoblasts toward the myofibers between the injection trajectories. Therefore, an increased migration could help to reduce the number of injection sites during the MT for the treatment of DMD patients. Here, we showed that laminin-111 increased drastically myoblast migration in vitro; thus, its co-injection with myoblasts might promote their migration in vivo and potentially their fusion outside the injection site following MT in skeletal muscle. Observations of Silva-Barbosa et al.10 supported this hypothesis and indicated that human myoblasts are much more dispersed with a local enhancement of laminin by irradiation. Laminins and laminin-111 peptides are known to play a clear role in the migration process of different cell types.29,30,31,32 Future experiments will be done in dystrophic dogs to verify whether myoblasts co-injected with laminin-111 migrate more in these dystrophic muscles.

Understanding the mechanisms by which laminin-111 increases migration could be very helpful to further improve the process. A possible mechanism could be through the interaction between laminin and matrix metalloproteinases (MMPs). Indeed, an increased expression of MMP-3 has been observed in fibroblasts upon binding to laminin-111 (refs. 33,34). MMPs are a family of serine proteases involved in the remodeling and maintenance of the extracellular matrix.35 MMP-3 is a typical MMP and plays an important role in the activation of other MMPs, such as proMMP-1, 7, 8, and 9 (refs. 36,37). It also plays an important role in tissue remodeling. Werle38 demonstrated that MMP-3 is implicated in the remodeling of the extracellular matrix at the neuromuscular junction. Moreover, Nishimura et al.39 used a MMP inhibition model to demonstrate that MMP-3 is associated with an increased migration in muscle cells.

Two potential mechanisms of cell death exist (apoptosis and necrosis). Anoikis is a special form of apoptosis observed after the disruption of interactions between cells or between cells and the extracellular matrix. In an intact organism, anoikis prevents the survival of the cells located in inadequate places. Transplantation of myoblasts with extracellular matrix components such as laminin-111 could reduce anoikis and consequently improve MT. Indeed, Bouchentouf et al. showed that the co-injection of myoblasts with fibronectin in mouse muscle increased their survival by about 28%, whereas their co-injection with vitronectin increased their proliferation by 40%.40 Necrosis occurs when a cell suffers irreversible damage that produces metabolic impairments leading to death. Early membrane damage, leading to the influx of extracellular molecules and ions, is characteristic of necrosis. EBD uptake indicated that intramuscular injection of the laminin-111 protein with MT strongly increased sarcolemmal integrity and reduced myofiber degeneration in mdx mice. As a consequence, the inflammatory reaction characterized by macrophage infiltration was drastically reduced and the mdx muscle exhibited almost no cluster of regenerating myofibers. The presence of laminin-111 in the extracellular matrix permits to link it to the predominant laminin-binding integrin in skeletal muscle, integrin α7β1 (ref. 22), which is present in the sarcolemma, thus replacing the mdx mice that links between the matrix and the membrane normally ensured by the dystrophin complex. These new links protect the muscle fibers from damage during contraction. Our data confirmed that the presence of laminin-111 reduced considerably the repetitive cycles of degeneration, inflammatory reaction, and regeneration in mdx mice.

The ultimate goal of therapy for DMD is to replace the missing subsarcolemmal protein, dystrophin, which, it is suggested, will enhance the functional capacity of the muscle. In a study of Mueller et al.,41 the mass and functional capacity of the EDL and TA muscles of adult mdx mice that received intramuscular injections of either normal primary myoblasts or muscle-derived stem cells were evaluated 9 weeks after cell transfer. No significant alterations in mass or in absolute or specific force generation have been found in mdx EDL or TA muscles after cellular therapy with either protocol. More recently, Rousseau et al.42 showed that the dystrophin restoration by MT is not sufficient by itself to increase the strength of the muscle. The main problem of various potential therapies to treat DMD is that they will not allow the restoration of strength in weak muscles that have already been too much destroyed by the degenerative progression of the disease. Our study is one of the first to clearly indicate that laminin-111 protected the muscle fibers from damage induced by eccentric contractions (as indicated by the force drop results) and also increased the absolute or the specific force in the EDL of mdx muscles. There are so far very few potential treatments, which have improved the strength of mdx muscles. To confirm our short-time results (i.e., 3–4 weeks after treatment) and to rule out a transient effect, the strength and resistance will be analyzed in our next experiments after a 1-year treatment. This will permit to determine how much the laminin-111-treated mdx mice have improved relative to untreated mdx mice and how close they are to normal mice. We are currently treating mice (starting with mice that were 1 month old), every month, i.e., i.m. or i.p.; analyses will be done after 1 year.

What could be the reason why strength and resistance were increased by laminin-111? The muscles of mdx mice and DMD patients are highly susceptible to contraction-induced injury, and normal use of muscle induces significant sarcolemmal damage in mdx mice. Our results confirmed and expanded results of Rooney et al. that intramuscular injection of laminin-111 protein dramatically increased sarcolemmal integrity and reduced myofiber degeneration. Laminin-111, in fact, ensures the presence of new mechanical links between the extracellular matrix and the sarcolemma. This protects the sarcolemma from stress-induced damages during muscle contraction. Thus, at any given time in the presence of laminin-111, there are fewer muscle fibers that are undergoing degeneration/regeneration. Consequently, there are more muscle fibers capable of contracting normally and this probably explains the increase of strength that we have observed with laminin-111. Another justification suggested by Rooney et al. is that the mechanism underlying the protection by laminin-111 in mdx muscle involved elevated levels of compensatory proteins. They demonstrated a fourfold increase in α7-integrin, which has been shown to be therapeutic in dystrophic mice and 30% of increased utrophin protein compared to mdx control.

In conclusion, our results support previous observations that laminin-111 can by itself stop the physiopathology of a dystrophinopathy in mice. These results are extremely important, if confirmed clinically, because in this case, DMD and maybe other related muscular dystrophies could be treated to stop or at least slow down the progression of the disease, only by the injection of a recombinant protein, a simple pharmacological approach that could be rapidly applied to many patients. Our results also indicate that laminin-111 can improve MT. This would be important to treat the muscles of DMD patients who are already weak. In fact, laminin-111 treatment and restoration of dystrophin by the transplantation of normal myoblasts may be two complementary treatments, which both permit to make new links between the extracellular matrix and the sarcolemma, thus making the muscle fibers less vulnerable during contraction/relaxation.

Materials and Methods

The reagents were purchased from the following companies: fetal bovine serum from Biomedia (Drummondville, Québec, Canada); penicillin/streptomycin, trypsin from Gibco (Burlington, Ontario, Canada); HBSS, Dulbecco's modified Eagle's medium, gelatine, EBD from Sigma-Aldrich (St Louis, MO); hematoxylin from Laboratoire Mat (Beauport, Québec, Canada); eosin from Fisher Scientific (Fair Lawn, NJ); rat mAb anti-laminin-α1 from Chemicon International (Temecula, CA) (MAB 1903), the rat anti-mouse Mac-1 mAb was produced as supernatant in our laboratory, goat anti-rat IgG conjugated with Alexa 546, goat anti-rabbit IgG conjugated with Alexa 488 from Molecular Probes (Eugene, OR), mouse mAb for human and dog dystrophin [MANDYS104 (CIND, Oswestry, UK)], streptavidin-Cy3 from Sigma-Aldrich, goat anti-mouse biotinylated antibody and goat anti-rat biotinylated antibody from Dako Diagnostics (Mississauga, Ontario, Canada). CyQUANT cell proliferation assay kits were bought from Molecular Probes. 24-well Transwell assay dishes were purchased from Corning Life Sciences (Acton, MA). Natural mouse laminin-111 (α1, β1, γ1) purified from Engelbreth–Holm–Swarm mouse sarcoma cells was purchased from Invitrogen (Burlington, Ontario, Canada). All the experiments were approved by the animal care committee of the CHUL (Centre Hospitalier de l'Université Laval). Mdx mice (dystrophic mouse model with dystrophinopathy on a C57BL10J genetic background) and normal C57BL10J mice were purchased from Jackson Laboratory (Bar Harbor, ME) and reproduced in our animal facility. The Rag/mdx mice were produced in our laboratory by crossing mdx mice with Rag‐/‐ mice. Two- and eight-month-old mice were used in our experiments.

Laminin-111 protein treatment. Natural mouse laminin-111 (α1, β1, γ1) protein was injected i.m. into the left EDL muscles of 8-month-old mdx mice (8 µl, 1 mg/ml). The contralateral right EDL muscles were injected with PBS and served as a control (n = 5). For systemic delivery, 1 mg/kg laminin-111 protein in PBS was injected i.p. and tissues were harvested for analysis. Control mdx mice were injected with the same volume of PBS. After 24 days, mice were used for study muscle contractile properties (n = 4). Eight-month-old C57BL10J mice were used as normal control.

Muscle preparation for physiological measurements. Mice were injected i.p. with buprenorphine (0.1 mg/kg) as analgesic and anesthetized with pentobarbital (30 mg/kg). The EDL was prepared as described previously.43,44 Briefly, the EDL was carefully dissected and placed in an organ bath maintained at 30 °C. The muscle was incubated with oxygenated Krebs–Ringer's solution (137 mmol/l NaCl, 5 mmol/l KCl, 2 mmol/l CaCl2, 24.7 mmol/l NaHCO3, 2 mmol/l MgSO4, 1.75 mmol/l NaH2PO4, and 2 g/l dextrose, pH 7.4) supplemented with carbogen (95% O2 and 5% CO2). The proximal tendon was fixed in a stationary clamp with a 3-O suture (Harvard Apparatus, St Laurent, Quebec, Canada). The distal tendon was connected to a dual-mode level arm system 305B-LR (Aurora Scientific, Aurora, Ontario, Canada) that provided control of force and positioning of the motor arm so that both dynamic and isometric muscle contractions could be elicited. The stimulations were delivered by a pair of electrodes at both sides of the muscle. The force generated by the muscle was measured by the LabView-based DMC program (Dynamic Muscle Control and Data Acquisition; Aurora Scientific). Data were analyzed by the LabView-based DMA program (Dynamic Muscle Data Analysis).

Isometric contraction protocol. Once dissected and placed in the bath, the muscle was initially set at a resting tension of 1 g for 10 minutes. Lo was determined as the muscle length at which the maximal twitch force was elicited. Absolute muscle force was determined by stimulating the muscle for 500 ms at frequencies of 80, 120, and 150 Hz. Absolute muscle force represents the total force generated by the muscle, whereas specific muscle force also accounts for muscle mass and fiber length. Therefore, after force measurements, muscle length was measured, tendons were removed, and the muscles were weighed. The optimal fiber length (Lf) was determined by multiplying Lo by the Lf/Lo ratio of 0.44. Specific muscle force (N/cm2) is calculated with the formula: [Absolute muscle force (in g) × optimal fiber length (in mm) × muscle density (in mg/mm3)]/muscle mass (in mg).

Eccentric contraction protocol. Before beginning the eccentric contraction protocol, the muscle was rested 10 minutes. Briefly, the muscle was stimulated at 150 Hz for 700 ms. After 500 ms stimulation, the muscle was lengthened to 110% Lo at 0.5 Lo/s for 200 ms. When stimulation ended, the muscle length was reset to Lo at the same rate. This stimulation–stretch cycle was repeated every 2 minutes for a total of 7 cycles. To determine the percentage of the force drop after each contraction, the maximal isometric tetanic force developed during the first 500 ms of the first stimulation was considered as 100%. The formula (F1‐Fn)/F1 was used to calculate the percentage of tetanic force loss at each cycle; F1 is the tetanic force developed during the first cycle and Fn represents the tetanic force obtained during the nth cycle.

MT. Primary normal human myoblasts were obtained and proliferated as described previously.45,46 The day of transplantation, cells were trypsinized and washed first in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and secondly in HBSS, before being resuspended in 20 µl of laminin-111 (500 nmol/l) or HBSS. The left and right TAs were surgically exposed and 0.7 million cells were injected in several sites (10–15) throughout each muscle. Rag/mdx mice (two months old) received two types of transplantation: (i) left TA: transplanted with myoblasts in the laminin-111 solution, (ii) right TA: transplanted with myoblasts in HBSS. Another group of mice was injected in one TA only with HBSS (without myoblast or laminin-111) as a negative control. The fusion of human myoblasts with the mouse muscle fibers was assessed 24 days after injection by detecting the presence of human dystrophin. Necrosis, inflammatory reaction, and regeneration were also analyzed.

Evaluation of damaged myofibers: EBD labeling. EBD, a vital stain, was administered i.p. (0.1 mg/10 g of total body weight) to each mouse 20 hours before the killing. EBD dye has the capacity to infiltrate only muscle fibers with a damaged membrane. All mice were killed by CO2 inhalation. The TAs were removed, snap-frozen in liquid nitrogen and stored at ‐80 °C until sectioning. Eight transverse serial cryostat sections (12 µm) were obtained throughout each muscle and observed under fluorescence microscope. The whole cross-sectional area and the total area labeled by EBD were photographed, and the number of EBD-stained fibers corresponding to damaged fibers was counted.

Hematoxylin and eosin staining. Serial 10 µm sections were cut with a cryostat and stained with hematoxylin and eosin. Tissue sections were fixed in ice-cold 95% ethanol for 2 minutes followed by 70% ethanol for 2 minutes and then rehydrated in running water for 5 minutes. The tissue sections were then stained with hematoxylin 2 minutes and rinsed in water for 5 minutes. Tissue sections were stained in eosin solution for 1 minute. They were then dehydrated in ice-cold 70% and 100% ethanol for 30 seconds each. Tissue sections were then cleared in toluene prior to mounting with Permount (Fisher, Ottawa, Ontario, Canada). The percentage of CNF was determined by counting ~1,500 fibers for each condition, n = 5.

Immunohistochemistry. TAs of Rag/mdx mice were removed 24 days after MT. Frozen muscle cross-sections were blocked in PBS with 10% fetal bovine serum for 30 minutes and then incubated 1 hour with the mouse mAb for human dystrophin (MANDYS104), diluted 1:10). Finally, muscle sections were incubated 1 hour with a goat anti-mouse IgG biotinylated antibody (diluted 1:150) and labeled with streptavidin-Cy3 (diluted 1:500). Cross-sections were washed with PBS before and after incubation with both antibodies, and all incubations were performed at room temperature. A rat mAb was used to detect laminin-111 diluted 1:250. Muscle cross-sections were blocked in PBS with 10% fetal bovine serum, 10% goat serum, and 2% bovine serum albumin overnight at 4 °C and then incubated 1 hour with the primary antibody. Muscle sections were incubated 1 hour with a goat anti-rat IgG biotinylated antibody (diluted 1:150) and labeled with streptavidin-Cy3. Inflammatory cells were detected by a 1-hour incubation with a rat anti-mouse Mac-1 mAb, followed by a 30-minute incubation with an anti-rat IgG antibody coupled to Alexa 546.

Cell proliferation assay. The cell proliferation assay was performed using a CyQUANT kit, which measures the nucleic acid content in the test samples. The cells were harvested after various treatment times and stored at ‐80 °C until the analysis. The frozen microplates were then thawed at room temperature and the CyQUANT GR dye/cell lysis buffer was added. After incubating for 5 minutes, the fluorescence was measured (excitation/emission: 495/520 nm) using a microplate reader.

Transwell migration assay. 24-well Transwell assay dishes were used to examine myogenic cell migration. Briefly, this modified Boyden chamber consists in a well (lower compartment) containing an insert (upper compartment). The upper and lower compartments are separated by an 8 µm pore-size polycarbonate membrane. Prior to cell seeding, the Transwell permeable supports were preincubated for 2 hours in the appropriate medium to improve cell attachment and spreading. Myoblasts were harvested and suspended in serum-free medium at a concentration of 3 × 105 cells/ml. The cell suspension (100 µl) were then loaded into the upper chamber of a Transwell containing 0.6 ml of serum-free medium in the lower chamber. To assess chemokinesis, the cells were suspended in the upper chamber in a medium containing 100 nmol/l laminin-111. Following a 14-hour incubation period at 37 °C, the inserts were removed and washed three times in PBS. Cells remaining on the upper surface of the insert (nonmigrated cells) were removed gently using a cotton swab, and cells on the lower surface were stained for 20 minutes in a solution containing 0.4% crystal violet, 2% ethanol, and 0.1% ammonium oxalate. After staining, inserts were rinsed three times by dipping into PBS and left to air-dry. Colored cells that had migrated through the pores of the polycarbonate membrane were visualized using a stereomicroscope. Digital pictures were taken using a Nikon Coolpix 4500 camera (Nikon Canada, Mississauga, Ontario, Canada), and the amount of colored cells was quantified using the NIH Image software.

Statistical analysis. All statistical analyses were performed using repeated measures analysis of variance. Following a significant effect of any source of variation, multiple comparisons were done in order to evaluate which factor differed from the others. The false discovery rate technique was used to maintain the global type I error rate at the desired level. The normality assumption was verified using the Shapiro–Wilk's statistic, and the homogeneity of variances was verified graphically with the residual plots. The interpretation of the results was done at the 0.05 or 0.01 levels of significance, and all analysis was done using SAS software (SAS Institute, Cary, NC). Specifically, for Figure 1a, statistical analyses were performed by a statistical expert at Laval University, the P value refers to comparison between trend of multipoint curve laminin-111, control mdx, and normal mice. All data are expressed as means ± SEM and are representative of at least three separate experiments.

SUPPLEMENTARY MATERIAL Figure S1. Quantification of centrally nucleated fibers (CNF). Quantification of CNF 24 days after an i.m. injection with HBSS revealed no significant difference with muscles not injected with HBSS. However, at the same time period, there were fewer CNF after an i.m. injection of laminin-111 compared with the mdx muscles not injected or injected with HBSS (n = 5, P<0.05). Figure S2. Fusion index in vitro. (a) Immunofluorescence showing myosin heavy chain positive myotubes with laminin-111 treatment. Scale bar = 150 μm. For myogenic differentiation, the medium was replaced by 1.5 g/l glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 2% horse serum when the cells reached 90% confluence. (b) The figure illustrates the progression of the average fusion index for 3 independent experiments with or without laminin-111 (100 nM) present in the culture medium. The fusion index was calculated as the ratio of the number of nuclei inside myotubes to the total number of nuclei × 100 at days 1, 2, 3 and 4 of myogenic differentiation. The number of nuclei is the average number of nuclei counted in 20 independent and randomly chosen microscope fields. A myotube was defined by the presence of at least three nuclei within a continuous cell membrane. Results are statistically different at day 3 (n=3, P < 0.05) and day 4 (n = 3, P < 0.01). The * indicate statistically different results.

Acknowledgments

We thank Julie-Rose St-Pierre, Jean-Philippe Mathieu, and Rosalie Giguere for efficient technical assistance. We thank Marlyne Goulet for providing the rat anti-mouse Mac-1. We thank Glenn E Morris and Le Thanh Lam (MRIC Biochemistry Group, Wrexham, UK) for providing the MANDYS104 antibody. This work was supported by grants from the Association Française contre les Myopathies, the Jesse's Journey Foundation for Gene and Cell Therapy of Canada, Muscular Dystrophy Canada and the Canadian Institute of Health Research.

Supplementary Material

Figure S1.

Quantification of centrally nucleated fibers (CNF). Quantification of CNF 24 days after an i.m. injection with HBSS revealed no significant difference with muscles not injected with HBSS. However, at the same time period, there were fewer CNF after an i.m. injection of laminin-111 compared with the mdx muscles not injected or injected with HBSS (n = 5, P < 0.05).

Click here for additional data file. (138.9KB, tiff)
Figure S2.

Fusion index in vitro. (a) Immunofluorescence showing myosin heavy chain positive myotubes with laminin-111 treatment. Scale bar = 150 μm. For myogenic differentiation, the medium was replaced by 1.5 g/l glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 2% horse serum when the cells reached 90% confluence. (b) The figure illustrates the progression of the average fusion index for 3 independent experiments with or without laminin-111 (100 nM) present in the culture medium. The fusion index was calculated as the ratio of the number of nuclei inside myotubes to the total number of nuclei × 100 at days 1, 2, 3 and 4 of myogenic differentiation. The number of nuclei is the average number of nuclei counted in 20 independent and randomly chosen microscope fields. A myotube was defined by the presence of at least three nuclei within a continuous cell membrane. Results are statistically different at day 3 (n = 3, P < 0.05) and day 4 (n = 3, P < 0.01). The * indicate statistically different results.

Click here for additional data file. (11.1MB, tiff)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Quantification of centrally nucleated fibers (CNF). Quantification of CNF 24 days after an i.m. injection with HBSS revealed no significant difference with muscles not injected with HBSS. However, at the same time period, there were fewer CNF after an i.m. injection of laminin-111 compared with the mdx muscles not injected or injected with HBSS (n = 5, P < 0.05).

Click here for additional data file. (138.9KB, tiff)
Figure S2.

Fusion index in vitro. (a) Immunofluorescence showing myosin heavy chain positive myotubes with laminin-111 treatment. Scale bar = 150 μm. For myogenic differentiation, the medium was replaced by 1.5 g/l glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 2% horse serum when the cells reached 90% confluence. (b) The figure illustrates the progression of the average fusion index for 3 independent experiments with or without laminin-111 (100 nM) present in the culture medium. The fusion index was calculated as the ratio of the number of nuclei inside myotubes to the total number of nuclei × 100 at days 1, 2, 3 and 4 of myogenic differentiation. The number of nuclei is the average number of nuclei counted in 20 independent and randomly chosen microscope fields. A myotube was defined by the presence of at least three nuclei within a continuous cell membrane. Results are statistically different at day 3 (n = 3, P < 0.05) and day 4 (n = 3, P < 0.01). The * indicate statistically different results.

Click here for additional data file. (11.1MB, tiff)

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