Transgenic overexpression of the α7 integrin reduces muscle pathology and improves viability in the dy^W mouse model of merosin-deficient congenital muscular dystrophy type 1A

Abstract

Merosin-deficient congenital muscular dystrophy 1A (MDC1A) is a devastating neuromuscular disease that results in children being confined to a wheelchair, requiring ventilator assistance to breathe and premature death. MDC1A is caused by mutations in the LAMA2 gene, which results in the partial or complete loss of laminin-211 and laminin-221, the major laminin isoforms found in the basal lamina of skeletal muscle. MDC1A patients exhibit reduced α7β1 integrin; however, it is unclear how the secondary loss of α7β1 integrin contributes to MDC1A disease progression. To investigate whether restoring α7 integrin expression can alleviate the myopathic phenotype observed in MDC1A, we produced transgenic mice that overexpressed the α7 integrin in the skeletal muscle of the dy^W−/− mouse model of MDC1A. Enhanced expression of the α7 integrin restored sarcolemmal localization of the α7β1 integrin to laminin-α2-deficient myofibers, changed the composition of the muscle extracellular matrix, reduced muscle pathology, maintained muscle strength and function and improved the life expectancy of dy^W−/− mice. Taken together, these results indicate that enhanced expression of α7 integrin prevents muscle disease progression through augmentation and/or stabilization of the existing extracellular matrix in laminin-α2-deficient mice, and strategies that increase α7 integrin in muscle might provide an innovative approach for the treatment of MDC1A.

Introduction

Merosin-deficient congenital muscular dystrophy 1A (MDC1A) is caused by mutations in the LAMA2 gene resulting in the production of a truncated form or complete loss of laminin-α2 protein (Helbling-Leclerc et al., 1995; Naom et al., 1997). The most severe form of MDC1A is associated with mutations that result in a complete loss of laminin-α2 protein and therefore the absence of laminin-211 and laminin-221 heterotrimers in the basal lamina. MDC1A patients exhibit severe weakness, dysmyelinating neuropathy, hypotonia, muscle atrophy and limited eye movement (Philpot et al., 1999b; Jones et al., 2001; Muntoni and Voit, 2004). Patients also exhibit feeding problems and/or respiratory difficulties and often require feeding tube placement or ventilator assistance (Philpot et al., 1999a). Most MDC1A patients are unable to walk without assistance and are confined to a wheelchair (Muntoni and Voit, 2004). A unique characteristic of MDC1A are changes in brain white-matter that are observed after 6 months of age. These changes might be associated with an increased likelihood of seizure-like activity (Philpot et al., 1999b; Jones et al., 2001; Muntoni and Voit, 2004). There is currently no cure or effective treatment for MDC1A, and patients die as early as the first decade of life (Muntoni and Voit, 2004; Mendell et al., 2006).

Laminin-211 and laminin-221 are the predominant laminin isoforms in the basal lamina of adult skeletal muscle. Laminin-211 is present at the extrajunctional sarcolemma, and laminin-221 is enriched at the myotendinous and the neuromuscular junctions (Martin et al., 1995; Burkin and Kaufman, 1999; Sasaki et al., 2002). Loss of laminin-211, in MDC1A patients and in mouse models of this disease, results in an inability of muscle fibers to adhere to the basement membrane and as a result laminin-α2-null myotubes are unstable and undergo apoptosis (Vachon et al., 1997; Kuang et al., 1999). Failed regeneration is also observed in laminin-α2-deficient myofibers, indicating the importance of the laminin-rich microenvironment for muscle repair (Kuang et al., 1999; Rooney et al., 2009b). In addition, laminin-α2 is expressed by Schwann cells in the peripheral nervous system, and lack of laminin-211 leads to reduced myelination resulting in impaired conduction velocity and peripheral neuropathy (Miyagoe-Suzuki et al., 2000; Occhi et al., 2005).

The α7β1 integrin is a primary laminin receptor expressed in skeletal, cardiac and vascular smooth muscle (Burkin et al., 1998; Burkin and Kaufman, 1999; Burkin et al., 2000). The α7 integrin transcript undergoes developmentally regulated RNA splicing to produce three alternative cytoplasmic domains, designated α7A, α7B and α7C, and two alternative extracellular domains, designated α7X1 and α7X2 (Collo et al., 1993; Song et al., 1993; Ziober et al., 1993; Hodges et al., 1997). The cytoplasmic domains differ in size, sequence and potential for signal transduction, whereas extracellular domains bind with differential affinities to laminin isoforms (Song et al., 1993; von der Mark et al., 2002). The α7B cytoplasmic domain is the largest and has a number of regions that potentially participate in signal transduction (Song et al., 1993).

The α7β1 integrin can bind to multiple laminin isoforms, including laminin-111, laminin-211, laminin-221, laminin-332, laminin-411, laminin-511, galectin-1 and fibronectin (Gu et al., 1994; Burkin and Kaufman, 1999; Patarroyo et al., 2002). Mutations in the α7-integrin-encoding gene (ITGA7) cause α7 integrin congenital myopathy, and patients exhibit delayed developmental milestones and impaired mobility (Hayashi et al., 1998). Mice that lack the α7 integrin develop muscular dystrophy that affects the myotendious junctions (Mayer et al., 1997; Hayashi et al., 1998; Nawrotzki et al., 2003; Flintoff-Dye et al., 2005). In the adult mouse, the α7β1 integrin is localized at the extrajunctional sarcolemma but is also enriched at neuromuscular and myotendinous junctions, where it mediates the adhesion of the muscle fibers to the extracellular matrix (Martin et al., 1996; Burkin et al., 1998; Burkin et al., 2000).

MDC1A patients, dy^−/− and dy^2J−/− mouse models have decreased levels of α7 integrin protein in their muscles, and this secondary loss of α7 integrin might contribute to disease progression (Hodges et al., 1997; Vachon et al., 1997; Peat et al., 2008). To determine whether restoring α7 integrin expression could serve as a therapeutic strategy in the treatment of MDC1A, we generated transgenic mice that overexpressed the α7 integrin in the skeletal muscle of the dy^W−/− mouse model of MDC1A. Transgenic expression of the α7 integrin increased longevity and reduced the myopathic phenotype of dy^W−/− mice. We show that these beneficial changes were achieved through augmenting and/or stabilizing the existing extracellular matrix in laminin-α2-deficient muscle. Our data demonstrate, for what we believe to be the first time, that the α7 integrin is a modifier of disease progression in laminin-α2-deficient mice and might be a novel drug target for the treatment of MDC1A.

Results

The α7B integrin is overexpressed in the muscle of dy^W−/−;itga7+ transgenic mice

To determine whether overexpression of the α7 integrin in laminin-α2-deficient muscle could alleviate disease progression, transgenic mice were generated in which rat α7BX2 integrin expression was driven by the muscle creatine kinase (Mck, also known as Ckm) promoter, which we designate as itga7+ mice. These mice were bred with dy^W+/− animals to generate dy^W−/−;itga7+ mice. Pups were genotyped at 10 days of age for the laminin-α2 mutant and wild-type alleles and the Itga7 transgene, as previously described (Kuang et al., 1998; Burkin et al., 2005; Boppart et al., 2008).

To confirm that the presence of the transgene resulted in increased α7 integrin in dy^W muscle quantitative real-time PCR (qRT-PCR) and immunoblotting were performed on the mouse tibialis anterior (TA) and gastrocnemius muscle. qRT-PCR experiments showed a 4.1-fold increase in α7 integrin transcript in the dy^W−/− muscle compared with that in wild-type muscle, whereas dy^W−/−;itga7+ mice exhibited a 17-fold increase in α7 integrin transcript in muscle compared with that in wild-type (Table 1). Immunoblotting confirmed a 2.8-fold increase in α7B integrin in the muscle of dy^W−/−;itga7+ mice (Fig. 1A,C). There was a 67.5% and 83.2% reduction in α7A integrin in the muscles of dy^W−/− and dy^W−/−;itga7+ animals respectively compared with that in wild-type (Fig. 1A,B). Analysis of other laminin-binding integrins in skeletal muscle revealed a 86% and 61.5% reduction in α3 integrin protein in dy^W−/− and dy^W−/−;itga7+ animals respectively compared with that in wild-type (Fig. 1D). Finally no significant change in β1D integrin protein was observed in any of the genotypes (Fig. 1E). Together these results indicate the MCK-α7BX2 transgene increased both the transcript and protein level expression of α7BX2 integrin in the skeletal muscle of dy^W−/− mice without altering the expression of other laminin-binding integrin isoforms and chains.

Table 1.

Changes in gene expression in dy^W−/− mice

Fig. 1.

Enhanced α7B integrin in the muscle of dy^W−/−;itga7+ mice. (A) Immunoblot showing protein levels of α7A integrin, α7B integrin, α3 integrin and β1D integrin in wild-type, dy^W−/−, and dy^W−/−;itga7+ mice. α-tubulin was used to normalize protein loading. (B) Densitometry analysis of α7A integrin indicating that both the dy^W−/− animals (*P<0.05) and the dy^W−/−;itga7+ animals have significantly reduced levels of α7A integrin (**P<0.01). (C) Densitometry showing that α7B integrin is significantly elevated in the dy^W−/−;itga7+ animals when compared with the dy^W−/− animals (*P<0.05). (D) Protein quantification for α3 integrin showing a significant reduction in protein levels in the dy^W−/− mice compared with the wild-type mice (*P<0.05) with no other significant changes. (E) No differences in β1D protein levels were observed among the three genotypes analyzed.

Transgenic expression of the α7 integrin in dy^W−/− muscle restores sarcolemmal localization of the α7β1 integrin

Previous studies have shown that loss of laminin-α2 results in reduced sarcolemmal localization of α7 integrin (Hodges et al., 1997; Vachon et al., 1997; Peat et al., 2008). Immunofluorescence was used to determine whether forced expression of α7 integrin restored sacrolemmal localization of the α7β1 integrin in skeletal muscle. Immunofluorescence analysis confirmed that, whereas wild-type mice exhibited substantial laminin-α2 in the basal lamina, dy^W−/− and dy^W−/−;itga7+ mice were negative for laminin-α2 and produced only negligible amounts of the laminin-α2 globular domain (Fig. 2A), which is consistent with previous studies (Guo et al., 2003). The localization of β1D integrin was examined in wild-type, dy^W−/− and dy^W−/−;itga7+ TA muscle (Fig. 2A). Compared with that in wild-type mice, dy^W−/− muscle exhibited a reduced sarcolemmal localization of β1D integrin. By contrast, transgenic overexpression of the α7 integrin restored sarcolemmal localization of β1D integrin in TA muscle. Spectrin levels were the same in all three genotypes and were used as a positive control for immunofluorescence.

Fig. 2.

Transgenic expression of α7 integrin restores sarcolemmal location in dy^W−/− skeletal muscle. (A) Immunofluorescence using TA muscle from 4-week-old male mice was used to confirm the absence of laminin-α2 protein in dy^W−/− muscle. Wild-type TA muscle shows sarcolemmal staining for laminin-α2, which is not present in dy^W−/− or dy^W−/−;itga7+ animals. Sections were also incubated with anti-β1D antibody to visualize β1D integrin. (B) High-resolution confocal microscopy of TA muscle using rhodamine-labeled WGA, to highlight the sarcolemma, and CDB347 antibody, to visualize α 7B integrin, was performed. The increased yellow colocalization signal in the dy^W−/−;itga7+ animals indicates restoration of sarcolemmal localization of the α7 integrin. Scale bars: 20 μm (A); 30 μm (B).

To examine whether transgenic overexpression restored the sarcolemmal localization of the α7 integrin in muscle, TA cryosections were stained for α7B integrin (green) and with rhodamine-labeled wheat germ agglutinin (WGA) (red), which binds N-acetylglucosamine-labeled sugar residues in the basal lamina, and high-resolution confocal microscopy was used to assess sarcolemmal localization of α7 integrin (Fig. 2B). Wild-type and dy^W−/−;itga7+ muscle exhibited colocalization of the α7 integrin and WGA (indicated by the yellow staining), as well as sharp sarcolemmal staining for α7 integrin (Fig. 2B). By contrast, the dy^W−/− animals showed little colocalization between α7 integrin and WGA, and did not show sharp α7 integrin sarcolemmal localization (Fig. 2B). Together these data indicate that transgenic expression of the α7 integrin in dy^W−/− muscle restored sarcolemmal localization of the α7β1 integrin in laminin-α2 deficient muscle.

Transgenic overexpression of α7 integrin increases the longevity of dy^W−/− mice

MDC1A patients and the dy^W−/− mouse model exhibit severe muscle wasting that results in reduced life expectancy (Muntoni and Voit, 2004; Mendell et al., 2006). We next investigated whether transgenic overexpression of α7 integrin improved the longevity of dy^W−/− animals. Kaplan–Meier survival analysis revealed that 50% of dy^W−/− mice died by 70 days of age (Fig. 3A). By contrast, 50% of dy^W−/−;itga7+ mice survived beyond 140 days of age (Fig. 3A). The oldest dy^W−/− mouse lived to 132 days, whereas the oldest dy^W−/−;itga7+ animal lived to 318 days, which represents a 2.4-fold increase in life expectancy (Fig. 3A). These results indicate that increasing α7 integrin expression in skeletal muscle substantially improves the life expectancy of laminin-α2-deficient mice.

Fig. 3.

Transgenic expression of α7 integrin in the skeletal muscle of dy^W−/− mice increases longevity but not weight gain. (A) Kaplan–Meier survival curve shows dy^W−/−;itga7+ (n=9) animals have a median life expectancy that is 2.0-fold longer than their dy^W−/− (n=15) littermates (P<0.05). (B) Analysis of body weights shows that wild-type mice are heavier than both dy^W−/−;itga7+ and dy^W−/− animals at all ages analyzed (***P<0.001).

MDC1A patients exhibit reduced body weight and muscle strength (Muntoni and Voit, 2004; Mendell et al., 2006). We next examined whether transgenic expression of the α7 integrin improved the body weight and muscle strength and function of dy^W−/− mice. Compared with that of wild-type mice, both dy^W−/− and dy^W−/−;itga7+ mice had reduced body weight at 3, 8 and 12 weeks of age with no difference between these laminin-α2-deficient mice (Fig. 3B). This indicates that the transgene is not effective at restoring body weight to that of wild-type animals.

We next examined muscle strength. Forelimb grip strength was measured to analyze overall forelimb muscle strength. Compared with 4-week-old wild-type mice, both dy^W−/− and dy^W−/−;itga7+ mice exhibited a 36.9% and 30.8% reduction respectively in forelimb grip strength compared with that of wild-type mice, with no difference between these laminin-α2-null animals (Fig. 4A). However, when the animals were re-evaluated at 8 weeks of age, dy^W−/− animals showed a 55% reduction in grip strength compared with that of wild-type mice, whereas dy^W−/−;itga7+ mice showed only a 24.8% reduction in grip strength compared with that in wild-type (Fig. 4A). This indicates that while the dy^W−/− animals progressively got weaker, enhanced levels of α7 integrin in the dy^W−/−;itga7+ animals preserved muscle strength over time.

Fig. 4.

The dy^W−/−;itga7+ transgenic mice maintain muscle strength and exhibit increased motor activity. (A) Box and whiskers plot of forelimb grip strength at four weeks of age showing that dy^W−/− and dy^W−/−;itga7+ mice are weaker than the wild-type animals (***P<0.001). At 8 weeks of age wild-type and dy^W−/−;itga7+ animals (*P<0.05) are significantly stronger than dy^W−/− mice. (B) Box and whiskers plot of activity, as measured by the amount of active time over a 5-minute period. At 4 weeks of age there is no significant difference between any of the genotypes analyzed (P>0.05). At 8 weeks of age wild-type and dy^W−/−;itga7+ mice were significantly more active than dy^W−/− animals. There was no significant difference in the activity levels of 8 week old wild-type and dy^W;itga7+ mice. (C) Box and whiskers plot of stand-up activity over a 5-minute period. At 4 weeks of age there were no significant differences between the genotypes. At 8 weeks of age, wild-type and dy^W;itga7+ animals exhibited significantly more stand ups compared to dy^W−/− animals. There was no significant difference in stand up activity between the dy^W;itga7+ and wild-type animals. *P<0.05; **P<0.01; ****P<0.0001. For each plot the box represents 1st quartile, mean and 3rd quartile and the whiskers represent minimum and maximum values.

We next evaluated two different measures of activity in 4- and 8-week-old animals. Measurement of overall activity over a 5-minute period when a mouse is introduced into a new cage revealed that at 4 weeks of age there was no significant difference between wild-type, dy^W−/− and dy^W−/−;itga7+ mice (Fig. 4B). By contrast, at 8 weeks of age, wild-type and dy^W−/−;itga7+ mice animals exhibited a 1.8-fold and 1.5-fold increase in active time respectively compared with that of dy^W−/− animals. There was no significant difference between the wild-type and dy^W−/−;itga7+ animals (Fig. 4B). Finally, stand-up activity of animals introduced to a new cage over a 5-minute period was assessed on mice capable of standing up. At four weeks of age there was no significant difference between the genotypes (Fig. 4C). At eight weeks of age wild-type and dy^W−/−;itga7+ animals exhibited a 5.2-fold and 4.2-fold increase in stand-up activity compared with that of dy^W−/− animals (Fig. 4C). Together these results indicate transgenic expression of the α7 integrin maintained muscle strength and activity in the dy^W−/− mouse model of MDC1A.

Transgenic overexpression of α7 integrin reduces skeletal muscle pathology in dy^W−/− mice

To determine whether enhanced α7 integrin expression prevented skeletal muscle pathology in laminin-α2-deficient mice, TA muscle cryosections from 4-week-old wild-type, dy^W−/− and dy^W−/−;itga7+ mice were stained with hematoxylin and eosin. Compared with that in wild-type mice, dy^W−/− TA muscle showed greater variation in myofiber size, presence of centrally located nuclei (CLN), fibrosis and inflammatory cell infiltrate (Fig. 5A). The dy^W−/−;itga7+ muscle exhibited less muscle pathology, including fewer myofibers with CLN, less inflammatory infiltrate and less variation in myofiber size (Fig. 5A).

Fig. 5.

Reduced muscle disease in laminin-α2-deficient mice that transgenically express α7 integrin. (A) Hematoxylin and eosin staining of sections from TA muscles from 4-week-old mice shows more muscle pathology in dy^W−/− mice compared with that in wild-type. The TA muscle of dy^W−/−;itga7+ mice exhibited reduced muscle pathology. Scale bar: 20 μm. (B) The percentage of myofibers containing CLN were used to evaluate muscle repair in TA muscles from 4-week-old mice. Compared with that in wild-type, dy^W−/− showed a 19-fold increase in the percentage of myofibers containing CLN. By contrast, dy^W−/−;itga7+ TA muscle showed a 66.5% reduction in the percentage of myofibers with CLN (***P<0.001). (C) Myofiber cross-sectional area revealed dy^W−/−;itga7+ animals have a fiber size distribution closer to that of wild-type animals than to dy^W−/− mice (supplementary material Fig. S1).

The requirement for muscle repair was determined by evaluating the percentage of myofibers containing CLN. CLN are an indicator of muscle degeneration and regeneration, which requires satellite cell activation to repair damaged myofibers (Xu et al., 2007). Compared with 4-week-old wild-type mice, which showed few myofibers with CLN, 4-week-old dy^W−/− TA muscle exhibited a 4.8-fold increase in myofibers with CLN (Fig. 5B). There was a 66.5% reduction in the percentage of CLN in the dy^W−/−;itga7+ compared with that in the dy^W−/− animals, although the level of CLN in these mice was still elevated compared with that in wild-type mice (Fig. 5B). These results indicate transgenic expression of the α7 integrin in the skeletal muscle results in reduced muscle pathology and fewer myofibers with CLN than in dy^W−/− mice.

MDC1A patients and dy^W−/− mice exhibit substantial myofiber size variation and the presence of a large number of hypotrophic muscle fibers. Measurements of myofiber cross-sectional area were used to determine whether transgenic expression of the α7 integrin improved this aspect of muscle pathology. Compared with that of wild-type, with a mean myofiber cross-sectional area of 16.1 μm², dy^W−/− TA muscle had a mean myofiber cross-sectional area of 9.5 μm², with a curve shifted to the left indicating increased numbers of hypotrophic muscle fibers (Fig. 5C). By contrast, TA myofibers in dy^W−/−;itga7+ mice had a mean cross-sectional area of 11.5 μm² (Fig. 5C). These results indicate that transgenic expression of the α7 integrin in skeletal muscle of laminin-α2-null mice reduced the variation in myofiber size and improved the myofiber cross-sectional area. At the maximum frequency of occurrence (i.e. that most commonly observed), the dy^W−/− mice had significantly smaller fibers than the wild-type mice. There was no significant difference between the dy^W−/− and dy^W−/−;itga7+ or between the dy^W−/−;itga7+ and wild-type mice (supplementary material Fig. S1). This indicates transgenic mice that overexpress the α7 integrin in muscle have fewer hypotrophic fibers compared with dy^W−/− animals and are more similar to wild-type.

Transgenic α7 integrin expression alters the composition of the extracellular matrix in laminin-α2-deficient muscle

The loss of laminin-211 and laminin-221 in the muscle extracellular matrix is the underlying cause of muscle disease in MDC1A. Given that the α7 integrin is the primary laminin receptor in muscle we next determined the mechanism by which increased α7β1 integrin rescued dy^W−/− mice in the absence of its laminin-211–laminin-221 ligand. qRT-PCR was used to examine the expression profile of genes encoding an array of extracellular matrix proteins in the gastrocnemius muscle of 4-week-old wild-type, dy^W−/− and dy^W−/−;itga7+ mice. qRT-PCR revealed that dy^W−/− mice exhibited increased levels of several transcripts: disintegrin and metalloproteinase with thrombospondin motifs 5 (Adamts5), agrin (Agrn), collagen 6A1 (Col6a1), galectin-1 (Lgals1), galectin-3 (Lgals3), matrix metalloproteinase 2 (Mmp2), integrin α3 (Itga3), integrin α6 (Itga6), integrin α7 (Itga7), laminin-α4 (Lama4), laminin-α5 (Lama5), nidogen 1 (Nid1), tenascin C (Tnc), tissue inhibitor of metalloproteinase 1 (Timp1) and tissue inhibitor of metalloproteinase 2 (Timp2), compared with that in wild-type (Table 1). Transgenic expression of the α7 integrin in dy^W−/−;itga7+ mice resulted in reduced levels of agrin and Mmp2 transcripts compared with dy^W−/− mice (Table 1). Transgenic expression of the α7 integrin in dy^W−/−;itga7+ mice resulted in increased levels of transcripts for Col6A1, Lgals1, Lgals3, Itga3, Itga6, Itga7, Tnc and Timp1 compared with that in dy^W−/− mice (Table 1).

The α7β1 integrin has been shown to interact with galectin-1 in cultured muscle cells (Gu et al., 1994), and we next determined whether transgenic expression of the α7 integrin altered expression of galectin-1 and -3 in the muscle of laminin-α2-null mice. Compared with that in wild-type mice, the level of galectin-1 transcript was increased 9.2-fold in dy^W−/− muscle and 12.1-fold in dy^W−/−;itga7+ animals (Table 1). This increase in galectin-1 transcript correlated with a 1.8-fold increase in galectin-1 protein in dy^W−/−;itga7+ animals compared with that in wild-type (Fig. 6A). These results indicate an increase in galectin-1 protein in the gastrocnemius muscle of dy^W−/−;itga7+ animals.

Fig. 6.

Transgenic expression of α7 integrin in the absence of laminin-α2 increased expression of galectin-1 and -3 in the muscle of dy^W−/− mice. (A) Immunoblot of galectin-1 protein (50 μg protein lysate was used) showing that 4-week-old dy^W−/−;itga7+ animals have a 1.4-fold increase in galectin-1 protein compared with wild-type and dy^W−/− mice (*P<0.05). (B) An immunoblot of galectin-3 protein required differential protein loading across the genotypes as follows: wild-type, 100 μg; dy^W−/−, 50 μg; and dy^W−/−;itga7+, 25 μg. Quantitative analysis revealed that 4-week-old dy^W−/− mice have 2-fold more galectin-3 compared with that in wild-type animals (**P<0.01). The dy^W−/−;itga7+ animals have 7-fold and 2.9-fold more galectin-3 protein compared with that in both wild-type and dy^W−/− animals, respectively (***P<0.001).

Galectin-3 transcript was increased 70-fold and 80-fold in 4-week-old dy^W−/− and dy^W−/−;itga7+ muscle, respectively, compared with that in wild-type mice (Table 1). This increase in galectin-3 transcript resulted in a 2-fold increase in galectin-3 protein in dy^W−/− mice and a 7-fold increase in galectin-3 protein in dy^W−/−;itga7+ animals compared with that in wild-type (Fig. 6B). These results indicate that loss of laminin-α2 results in increased galectin-3 in the muscle extracellular matrix of dy^W−/− mice and that transgenic expression of α7 integrin further enhanced the levels of galectin-3 in laminin-α2-deficient muscle.

In laminin-α2-deficient muscle, tenascin C is normally localized at the myotendinous junctions and has been shown to be enriched at extrajunctional sites, which correlate with regions of muscle regeneration (Ringelmann et al., 1999). qRT-PCR was used to examine whether transgenic overexpression of the α7 integrin altered the expression of tenascin C in the muscle of dy^W−/− mice. qRT-PCR confirmed a 28-fold increase in Tnc transcript in the gastrocnemius muscle of dy^W−/− mice and a 49-fold increase in Tnc transcript in dy^W−/−;itga7+ gastrocnemius muscle compared with that in wild-type (Table 1). These results indicate transgenic expression of the α7 integrin augmented Tnc transcription in laminin-α2-null muscle.

Immunofluorescence was used to confirm the qRT-PCR and immunoblotting results for several proteins. Immunofluorescence also demonstrated increased extracellular galectin-1, galectin-3 and tenascin C in the extracellular matrix, with galectin-3 and tenascin C being more prevalent in the dy^W−/−;itga7+ mice (Fig. 7). Immunostaining demonstrated reduced MMP2 and increased TIMP1 in the extracellular matrix of the dy^W−/−;itga7+ mice compared with that in the dy^W−/− mice (Fig. 7). These results support the idea that overexpression of the α7 integrin results in both augmentation and stabilization of the existing extracellular matrix in dy^W−/−;itga7+ animals.

Fig. 7.

Enhanced expression of α7 integrin augments the extracellular matrix and slows matrix turnover in the dy^W−/− mouse. Immunfluorescence was used to evaluate extracellular matrix proteins and associated proteins. Galectin-1 was found to be elevated in both the dy^W−/− and dy^W−/−;itga7+ animals when compared with that in the wild-type. The dy^W−/−;itga7+ animals had the greatest amount of staining for galectin-3 with some staining present in the dy^W−/− animals and none in wild-type controls. Tenascin C was found in areas of inflammation in the dy^W−/− animals, whereas it was located more dispersedly in the dy^W−/−;itga7+ animals (arrows). Matrix metalloproteinase 2 (MMP2) was highest in the dy^W−/− animals. Overexpression of α7 integrin led to reduced staining for MMP2 in the dy^W−/−;itga7+ animals. Conversely, Tissue inhibitor of metalloproteinase 1 (TIMP1) was found to be elevated by enhanced α7 integrin expression in the dy^W−/−; itga7+ mice compared with both dy^W−/− and wild-type animals. Scale bar: 20 μm.

Inflammatory infiltrate is reduced in dy^W muscle with enhanced α7 integrin

Inflammatory cell infiltration (particularly by the monocyte or macrophage line) is a hallmark of MDC1A (Pegoraro et al., 1996). To determine whether overexpression of the α7 integrin reduced inflammation in dy^W−/−;itga7+ muscle, CD11b was used as a marker for the presence of macrophages in skeletal muscle (Fig. 8A). Compared with that in the 4-week-old wild-type TA muscle, dy^W−/− TA muscle exhibited an 11.1-fold increase in CD11b-positive cells (Fig. 8B). By contrast, 4-week-old dy^W−/−;itga7+ showed a 55.3% decrease in the number of CD11b-positive cells compared with that in dy^W−/− TA muscle (Fig. 8B). These results indicate transgenic overexpression of the α7 integrin reduced the level of macrophage-mediated inflammation in dy^W−/− muscle.

Fig. 8.

Enhanced expression of α7 integrin reduces inflammation in dy^W−/− muscle. (A) Immunofluorescence of TA muscles from 4-week-old mice with anti-CD11b antibody, a marker for macrophages. Compared with wild-type mice, dy^W−/− mice have an increased number of CD11b-positive macrophages. The dy^W−/−;itga7+ animals have less inflammatory cell infiltration compared with dy^W−/− animals. Scale bar: 20 μm. (B) Quantification revealed that dy^W−/− mice have a 2.4-fold increase in CD11b-positive macrophages in TA muscle compared with the number in wild-type mice. By contrast, dy^W−/−;itga7+ animals exhibited a 55.3% decrease in CD11b positive macrophages in TA muscle compared with the number in dy^W−/− mice. ***P<0.001 for all groups.

Transgenic expression of α7 integrin prevents muscle disease progression in the diaphragm of dy^W mice

MDC1A patients exhibit severe restrictive respiratory syndrome and require ventilator assistance to breathe as a result of severe diaphragm muscle pathology (Muntoni and Voit, 2004). Histological analysis and measurements of myofiber area were used to examine whether transgenic expression of the α7 integrin prevented the onset of severe diaphragm muscle pathology. Hematoxylin and eosin studies revealed that transgenic expression of the α7 integrin in 4-week-old dy^W−/− diaphragm muscle resulted in reduced mononuclear cell infiltrate, hypotrophic muscle fibers, CLN and fibrosis (Fig. 9A).

Fig. 9.

Enhanced α7 integrin expression improves diaphragm pathology in 4-week-old dy^W−/− mice. (A) Hematoxylin and eosin staining of diaphragm muscle from 4-week-old dy^W−/− mice exhibit variation in myofiber size, presence of CLN and fibrosis. By contrast, the dy^W−/−;itga7+ diaphragm shows reduced muscle pathology. Scale bar: 20 μm. (B) Cross-sectional myofiber area of the diaphragm muscle of 4-week-old animals shows that the dy^W−/− ;itga7+ mice have a myofiber cross-sectional area more similar to wild-type animals than that observed in dy^W−/− mice (see also supplementary material Fig. S1).

Analysis of myofiber cross-sectional areas confirmed the improvement in the muscle pathology observed in the histological studies. Compared with wild-type mice, which have peak myofiber cross-sectional area of between 3.5–4.5 μm², dy^W−/− muscle exhibited a large number of hypotrophic muscle fibers, with a peak myofiber area of only 2 μm² (Fig. 9B). By contrast, dy^W−/−;itga7+ diaphragm myofibers exhibited a peak myofiber area of between 3.5 and 5 μm² and a curve more similar to that of wild-type (Fig. 9B). At the maximum frequency myofiber area, all three groups were significantly different from one another (supplementary material Fig. S1). These results indicate that transgenic expression of the α7 integrin prevents muscle disease progression in the diaphragm of laminin-α2-null mice.

Discussion

Merosin-deficient congenital muscular dystrophy (MDC1A) is considered one of the most common congenital muscular dystrophies. Classic MDC1A is associated with a complete loss of laminin-α2 and has the most severe clinical signs with severe muscle weakness, hypotonia and muscle atrophy. Feeding and respiratory difficulties are common, and patients often require either feeding tube placement or positive pressure ventilation. Maximal motor activity is often unsupported sitting with few patients achieving independent ambulation (Muntoni and Voit, 2004). There is currently no cure or effective treatment for MDC1A and patients often die at a young age from respiratory insufficiency (Mendell et al., 2006).

The observation of reduced α7 integrin in MDC1A muscle has led to the proposal that the secondary loss of α7 integrin contributes to the myopathic phenotype observed in MDC1A patients and laminin-α2-null mice (Hodges et al., 1997; Vachon et al., 1997; Peat et al., 2008). Because α7 integrin congenital myopathy does not phenocopy MDC1A, disease progression must involve more than loss of the α7 integrin receptor; however, given that the α7β1 integrin binds to multiple ligands, restoring α7β1 integrin receptor in skeletal muscle might improve muscle integrity and reduce the myopathy associated with disease progression.

Previous studies have shown a loss of α7 integrin from the skeletal muscle of dy^−/− and dy^2J−/− mouse models of MDC1A (Hodges et al., 1997; Vachon et al., 1997; Gawlik et al., 2006). Our results show the dy^W−/− muscle exhibits reduced α7 integrin expression and loss of sarcolemmal localization of the α7β1 integrin. Studies have suggested transcriptional regulation between laminin and α7 integrin in muscle (Hodges et al., 1997) and the small amount of truncated laminin-α2 globular domain produced in dy^W−/− muscle (Guo et al., 2003), which might be sufficient to promote some α7 integrin expression but insufficient for the correct sarcolemmal localization in muscle. In this study, we show that transgenic overexpression of the α7 integrin in dy^W−/− mice resulted in sarcolemmal localization of the α7β1 integrin. Restoring sarcolemmal localization of the α7β1 integrin reduced muscle pathology, maintained muscle strength and increased survival of dy^W−/− mice. Taken together, our data support the hypothesis that secondary loss of α7 integrin contributes to muscle disease progression and that restoring sarcolemmal localization might reduce the myopathy in MDC1A.

Studies have indicated that the α7β1 integrin might act to modulate the organization and deposition of the muscle extracellular matrix (Colognato et al., 1999; Rooney et al., 2009a). To investigate the mechanism by which enhanced α7 integrin expression improved the myopathic phenotype in the absence of the laminin-211–laminin-221 ligand, we examined changes in levels of other extracellular matrix proteins and enzymes that regulate the extracellular matrix. MDC1A patients and laminin-α2-deficient mice have increased laminin-α4 and laminin-α5 chains; however, previous studies have indicated that the increase in these laminin isoforms is unlikely to rescue the myopathic phenotype (Patton et al., 1997; Cohn and Campbell, 2000; Gawlik et al., 2004). Transgenic expression of α7 integrin did not further increase α7A integrin isoform or α3 integrin in skeletal muscle, indicating that these laminin and integrin isoforms do not play a role in the rescue of laminin-α2-deficient mice that overexpress α7 integrin in skeletal muscle.

Our analysis of extracellular matrix proteins in the dy^W−/− muscle basal lamina revealed that transgenic expression of the α7 integrin resulted in reduced expression of Mmp2 and increased levels of Timp1. Mmp2 is associated with the breakdown of the basal lamina, whereas Timp1 acts to inhibit the activity of matrix metalloproteinases, such as Mmp2 (Chen and Li, 2009). Reduced Mmp2 and increased Timp1 in the muscle of α7 integrin transgenic dy^W−/− mice indicate that increased α7β1 integrin acts to stabilize the basal lamina by affecting the expression of enzymes that degrade and remodel the muscle extracellular matrix.

The α7β1 integrin has been shown to directly bind galectin-1 (Gu et al., 1994). Galectin-1 (L-14) is a lactoside-binding protein that has been shown to inhibit myoblast adhesion by blocking the interaction of laminin to integrins. Galectin-3 has also been shown to mediate cell adhesion and regulate the cell signaling pathways involved in apoptosis, cell proliferation, inflammation and gene transcription (Dumic et al., 2006). Galectin-3 binds extracellular matrix proteins, including laminin and tenascin C, and has been shown to bind directly to integrins. In breast carcinoma cells, galectin-3 has been shown to mediate β1 integrin endocytosis (Furtak et al., 2001). In this study, we show that both galectin-1 and -3 are increased in dy^W−/− muscle and further increased in dy^W−/−;itga7+ skeletal muscle that transgenically overexpress the α7 integrin. These results suggest that, in the absence of laminin-211, expression of galectin-1 and -3 are increased and might serve as biomarkers for disease progression. Because the α7β1 integrin has been previously shown to bind galectin-1, our results suggest that rescue of laminin-α2-deficient muscle by overexpression of the α7 integrin is mediated through interactions of the α7β1 integrin with galectin-1 and/or galectin-3 to stabilize myofibers.

Although tenascin C is normally enriched at myotendinous junctions in wild-type muscle, in dy^−/− laminin-α2-deficient mice, tenascin C is found around regenerating myofibers, sites of inflammation and extrajunctional sites (Ringelmann et al., 1999). Our study shows that transgenic expression the α7 integrin resulted in increased tenascin C transcript in dy^W−/− muscle. Because transgenic expression of the α7 integrin results in a reduced percentage of myofibers with CLN (and therefore reduced muscle regeneration) and decreased inflammation, these results indicate that increased α7β1 integrin stabilizes the existing extracellular matrix, including tenascin C, in dy^W−/− muscle.

Approaches towards the treatment of MDC1A have targeted apoptosis in mouse models, through the inhibition of Bax, transgenic expression of the anti-apoptotic protein Bcl-2 and treatment with the anti-apoptotic drugs doxycycline (Girgenrath et al., 2004; Girgenrath et al., 2009) or omigapil (Erb et al., 2009). Another approach has focused on restoring or improving the muscle extracellular matrix through transgenic expression, in mouse models, of laminin-α1 (Gawlik et al., 2004; Gawlik et al., 2006), laminin-α2 (Kuang et al., 1998), mini-agrin (Bentzinger et al., 2005) and N-acetylgalactosamine transferase (Xu et al., 2007). Our results indicate that increased expression of the α7β1 integrin receptor in muscle improves adhesion of myofibers to the endogenous basal lamina to reduce muscle disease progression and maintain muscle strength in the dy^W−/− mouse model of MDC1A. Results from this study indicate that compounds that increase α7 integrin expression in laminin-α2-deficient muscle might provide a novel therapeutic approach for the treatment of MDC1A.

Materials and Methods

Generation of laminin-α2-deficient mice that overexpress α7 integrin in skeletal muscle

All experiments involving mice were approved by the University of Nevada, Reno Institutional Animal Care and Use Committee. Transgenic α7 integrin dy^W−/− mice were generated by breeding mice that overexpressed the α7BX2 integrin in skeletal muscle (Burkin et al., 2001) with dy^W+/− animals (Kuang et al., 1998). Resultant pups, which were heterozygous for the laminin-α2 mutant allele and positive for the α7BX2 transgene were bred to dy^W+/− mice. The male pups from these matings included dy^W+/+;itga7–(wild-type), dy^W−/−;itga7–(dy^W−/−) (laminin-α2-deficient) and dy^W−/−;itga7+ (laminin-α2-deficient and overexpressing the α7BX2 integrin) mice. Male littermates were used as controls for all experiments. Genomic DNA was isolated from tail biopsies taken at 10 days of age using the Wizard SV genomic dna purification system (Promega). PCR was used as previously described to detect the laminin-α2 allele and the α7BX2 transgene (Kuang et al., 1998; Burkin et al., 2005; Boppart et al., 2008). The dy^W+/− mice were a gift from Eva Engvall via Paul Martin (The Ohio State University, Columbus, OH).

Isolation of skeletal muscle

Four-week-old wild-type, dy^W−/− and dy^W−/−;itga7+ male mice were killed by CO₂ asphyxiation followed by cervical dislocation in accordance with a protocol approved by the University of Nevada Reno Animal Care and Use Committee. Skeletal muscles were dissected and flash frozen in liquid-nitrogen-cooled isopentane. Tissues were stored at −80°C.

Western blot analysis

Gastrocnemius muscles from 4-week-old male mice were pulverized with a mortar and pestle cooled in liquid nitrogen. Protein was extracted in RIPA buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM Na₃VO₄, 10 mM NaF, 0.5% Triton X-100, 0.5% NP50, 10% glycerol, 2 mM PMSF and a 1:200 dilution of Protease Inhibitor Cocktail Set III) and quantified using a Bradford assay (Bio-Rad). Proteins were separated by SDS-PAGE. The α7 integrin was detected with a 1:1000 dilution of anti-α7B antibody overnight. Integrin α7A was detected using a 1:1000 dilution of CDB 345 antibody overnight (a gift from Stephen Kauffman, University of Illinois, Urbana-Champaign, IL). Integrin α3 was quantified using the AB1920 antibody (Chemicon). The β1D integrin was visualized using an anti-β1D-antibody (a gift from Woo Keun Song, Gwanju Institute for Science and Technology, South Korea) overnight. All primary antibodies were followed by a goat anti-rabbit-IgG secondary antibody (1:5000, Li-Cor Biosciences) for 1 hour. Galectin-1 was detected with a 1:1000 dilution of H00003956-D01P (Abnova, Walnut, CA). Galectin-3 was detected with a 1:1000 dilution of ab53082 (Abcam, Cambridge, MA). Immunoblots were normalized by using a 1:5000 dilution of an anti-α-tubulin antibody (AbCam) followed by a 1:5000 dilution of goat anti-mouse-IgG secondary antibody. Band intensities were determined with an Odyssey Imaging System.

Immunofluorescence

Cryosections (8 μm) of 4-week-old male tibialis anterior (TA) muscle were cut using a LeicaCM 1850 cryostat and mounted onto pre-cleaned Surgipath slides. Sections were fixed using 4% paraformaldehyde (PFA) for 2 or 5 minutes then rehydrated using PBS. Slides were blocked in 5% BSA in PBS then incubated with antibodies against laminin-α2G or β1D integrin, as previously described (Rooney et al., 2009b). For detection of galectin-1, H00003959-D01P (Abnova) antibody was used. Galectin-3 was visualized using ab53082 (AbCam). Antibody T3413 (Sigma) was used to detect tenascin C. Mmp2 and Timp1 were detected using antibodies ab37150 and ab86482 respectively (AbCam). Slides were then incubated using an appropriate secondary, which was a FITC-conjugated anti-rabbit-IgG antibody in all cases except for tenascin C, where a FITC-conjugated anti-rat-IgG secondary antibody was used. For detection of spectrin, slides were fixed for 1 minute in ice-cold acetone then treated with the M.O.M. kit according to package instructions (FMK-2201, Vector Laboratories). A mouse monocloncal anti-spectrin antibody (Novo Castra NCL-spec2) was then used at 1:100 for 30 minutes followed by a FITC-conjugated anti-mouse-IgG secondary antibody at 1:1000 for 1 hour. Slides were mounted using Vectashield with DAPI and imaged using a Zeiss Axioskop 2 Plus fluorescence microscope. Images were captured using a Zeiss AxioCam HRc digital camera with Axiovision 4.1 software.

Inflammatory cell infiltrate

TA muscle cryosections from 4-week-old mice were fixed in 4% PFA for 5 minutes followed by rehydration with PBS. Slides were incubated with FITC-conjugated rat anti-mouse-CD11b antibody (BD Biosciences) at 1:1000 for 1 hour to detect macrophages in the muscle tissue. Slides were washed with PBS and mounted using Vectashield with DAPI. Muscle sections from five mice of each genotype were analyzed and CD11b-positive cells per 20 fields at 400× magnification were counted. A Zeiss Axioskop 2 Plus fluorescent microscope was used to view the slides and images were captured using a Zeiss AxioCam HRc digital camera with Axiovision 4.1 software.

Confocal microscopy

The TA muscles from 4-week-old male mice from each genotype were sectioned and subjected to immunofluorescence. For detection of α7B integrin, sections were fixed in ice-cold acetone (−20°C) for 1 minute then rehydrated using PBS. Cryosections were blocked in a 5% BSA in PBS solution for 20 minutes followed by incubation with CDB347 (which recognizes the cytoplasmic domain of both mouse and rat α7B integrin) or anti-β1DA2-integrin antibodies for 1 hour. Slides were then washed with 1% BSA in PBS and incubated with FITC-conjugated anti-rabbit-IgG antibody for 1 hour. Slides were again washed with 1% BSA in PBS. To outline the myofibers, sections were incubated with rhodamine-labeled wheat germ agglutinin for 30 minutes. Slides were mounted using Vectashield with DAPI. Images were captured using an Olympus Fluoview confocal scanning system.

Survival and weight gain analysis

Male mice were allowed to age and monitored daily for weight loss and any signs of pain, distress or illness. A weight loss of >10% over a 1-week period was also considered a terminal sign and the animals were humanely euthanized. Weights from animals of each genotype were compared at 3, 8 and 12 weeks of age.

Grip strength and activity assays

The forelimb grip strength of 4- and 8-week-old male wild-type, dy^W−/− and dy^W−/−;itga7+ mice were measured using a SDI grip strength system and a Chatillon DFE digital force gauge (San Diego Instruments) as per a standard protocol. Five consecutive tests were performed for each mouse and the data averaged for each mouse genotype. In order to assess mobility 4- and 8-week-old male wild-type, dy^W−/− and dy^W−/−;itga7+ mice were placed in a clean cage by themselves and monitored for 5 minutes. Periods of moving about the cage, standing up and digging were considered times of activity. Additionally, during this time period the number of times the mouse stood up was recorded. Stand-up testing was only performed on animals who were physically able to stand up. Some mice were excluded from these samples owing to the extent of their peripheral neuropathy.

Hematoxylin and eosin staining

Cryosections from 4-week-old TA and diaphragm muscle were stained using hematoxylin and eosin, as previously described (Rooney et al., 2009a), and were used to determine the percentage of myofibers that contained CLN using a Zeiss Axioskop 2 Plus fluorescence microscope. A minimum of 1000 fibers per animal (five animals per group) were counted and the percentage of myofibers with CLN calculated. Images were captured using a Zeiss AxioCam HRc digital camera and Axiovision 4.1 software.

Myofiber area determination

Cryosections from TA and diaphragm muscles from 4-week-old mice were fixed for 5 minutes in 4% PFA and rehydrated in PBS. Myofibers were outlined with 2 μg/ml Oregon-Green-488-conjugated WGA (Molecular Bioprobes) for 30 minutes. Sections were then washed with PBS for 15 minutes and mounted in Vectashield. A minimum of 1000 fibers per animal with five animals per group were assessed for the TA muscle. For diaphragm muscle, a minimum of 500 fibers per animal with five animals per genotyped were used. Myofiber cross-sectional area was determined with a Zeiss Axioskop 2 Plus fluorescence microscope and images were captured with a Zeiss AxioCam HRc digital camera with Axiovision 4.1 software.

Quantitative real-time PCR analysis

Total RNA was purified from five 4-week-old male wild-type, dy^W−/−, and dy^W−/−;itga7+ mice gastrocnemius muscles using TRIzol (Invitrogen). After the concentration was determined, mRNA was pooled equally by genotype for cDNA production. The cDNA was prepared from 4 μg of pooled total RNA with random hexamers and Superscript III (Invitrogen) using standard procedures. Quantitative real-time PCR was conducted with 50 pg total cDNA using SYBR Green Jumpstart (Sigma–Aldrich). Primer sequences for the mouse extracellular matrix genes are listed in supplementary Table S1 and levels were normalized to that of Gapdh. The fold change over wild-type was calculated using the ΔΔCt method after normalization and the average fold change in transcript (±s.e.m.) were calculated.

Statistics

Data is reported as the mean±s.d. One-way analysis of variance (ANOVA) was used to compare animals across groups. A Kaplan–Meier log-rank test was used to determine significance of lifespan changes. Myofiber cross-sectional area was analyzed using the GLIMMIX statistical analysis package in SAS. P<0.05 was considered significant.