Correspondence to: Stephen J. Kaufman, Department of Cell and Structural Biology, University of Illinois, B107 Chemical and Life Sciences Laboratory, Urbana, IL 61801. Tel:(217) 333-3521 Fax:(217) 244-1648 E-mail:stephenk{at}uiuc.edu.
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Abstract |
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Muscle fibers attach to laminin in the basal lamina using two distinct mechanisms: the dystrophin glycoprotein complex and the 7ß1 integrin. Defects in these linkage systems result in Duchenne muscular dystrophy (DMD),
2 laminin congenital muscular dystrophy, sarcoglycan-related muscular dystrophy, and
7 integrin congenital muscular dystrophy. Therefore, the molecular continuity between the extracellular matrix and cell cytoskeleton is essential for the structural and functional integrity of skeletal muscle. To test whether the
7ß1 integrin can compensate for the absence of dystrophin, we expressed the rat
7 chain in mdx/utr-/- mice that lack both dystrophin and utrophin. These mice develop a severe muscular dystrophy highly akin to that in DMD, and they also die prematurely. Using the muscle creatine kinase promoter, expression of the
7BX2 integrin chain was increased 2.02.3-fold in mdx/utr-/- mice. Concomitant with the increase in the
7 chain, its heterodimeric partner, ß1D, was also increased in the transgenic animals. Transgenic expression of the
7BX2 chain in the mdx/utr-/- mice extended their longevity by threefold, reduced kyphosis and the development of muscle disease, and maintained mobility and the structure of the neuromuscular junction. Thus, bolstering
7ß1 integrinmediated association of muscle cells with the extracellular matrix alleviates many of the symptoms of disease observed in mdx/utr-/- mice and compensates for the absence of the dystrophin- and utrophin-mediated linkage systems. This suggests that enhanced expression of the
7ß1 integrin may provide a novel approach to treat DMD and other muscle diseases that arise due to defects in the dystrophin glycoprotein complex. A video that contrasts kyphosis, gait, joint contractures, and mobility in mdx/utr-/- and
7BX2-mdx/utr-/- mice can be accessed at http://www.jcb.org/cgi/content/full/152/6/1207.
Key Words:
7ß1 integrin, muscular dystrophy, dystrophin, utrophin, neuromuscular junction
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Introduction |
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The defective association of skeletal and cardiac muscle with their surrounding basal lamina underlies the pathologies associated with a variety of muscular dystrophies and cardiomyopathies (
Mutations in the dystrophin gene result in a lack of dystrophin, a 427-kD protein localized at the cytoplasmic side of the plasma membrane of skeletal and cardiac muscle cells (
The integrins are ß heterodimeric receptors that bind extracellular matrix proteins and interact with the cell cytoskeleton (
7ß1 integrin is a laminin receptor on skeletal and cardiac muscle (
7 and ß1 chains are generated by developmentally regulated RNA splicing, resulting in a family of receptors with diverse structures and functions (for reviews see
The 7 integrin chain is encoded by a single autosomal gene on human chromosome 12q13 (
7A,
7B, and
7C) and two extracellular domain variants (X1 and X2) of the protein have been identified (
The ß1 chain cytoplasmic domain also undergoes developmentally regulated alternative splicing. ß1A is the most common isoform of the ß1 chain and is expressed in a wide variety of tissues including replicating myoblasts. The alternative ß1D form is generated upon differentiation of myoblasts to myofibers (
Mutations in the genes that encode the many components of the dystrophin glycoprotein complex cause a variety of muscular dystrophies. Mutations in the 7 gene also cause congenital myopathies (
7 transcript and protein in DMD patients and mdx mice (the mouse model that has a mutation in its dystrophin gene) (
Utrophin, a protein homologous to dystrophin, is also increased in DMD patients and mdx mice (
Although DMD patients (7 integrin chain (
To explore the hypothesis that enhanced expression of the 7ß1 integrin may compensate for the absence of the dystrophin glycoprotein complex and reduce the development of severe muscle disease, transgenic mice were made that express the rat
7 chain. We report that mdx/utr-/- mice with enhanced expression of the
7BX2 chain isoform show greatly improved longevity and mobility compared with nontransgenic mdx/utr-/- mice. Transgenic mice maintained weight and had reduced spinal curvature (kyphosis) and joint contractures. Transgenic expression of the
7BX2 chain also reduced the degree of mononuclear cell infiltration and expression of fetal myosin heavy chain (fMyHC) in muscle fibers. Together these results show that enhanced expression of
7BX2ß1D integrin significantly reduces the development of muscular dystrophy.
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Materials and Methods |
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Muscle Creatine Kinase-7BX2 Integrin Construct
The cDNA encoding the rat 7BX2 integrin isoform was cloned into pBK-RSV vector (Stratagene) downstream of the 3.3-kb mouse muscle creatine kinase (MCK) promoter and the mouse
7 integrin cell surface localization signal sequence using the restriction sites AatII and KpnI. The MCK promoter was provided by Dr. Stephen Hauschka (University of Washington, Seattle, WA). The construct was verified by DNA sequencing. Previous studies have shown that the MCK promoter is only active in heart and skeletal muscle (
7BX2 integrin construct were verified by transfecting C2C12 myoblasts (
Production of Transgenic mdx/utr-/- Mice
The MCK-7BX2 construct was gel purified. mdx/utr+/- female mice were superovulated, mated to mdx/utr+/- male mice, and fertilized oocytes were collected. The MCK-
7BX2 construct was microinjected into male pronuclei, and injected oocytes were placed into pseudopregnant mice at the University of Illinois Transgenic Animal Facility. Resulting pups were weaned at 3 wk of age, and genomic DNA was isolated from 0.5-cm tail clips using a DNA isolation kit (Promega). Primers (MCK1, 5'-caagctgcacgcctgggtcc-3'; and AATII, 5'-ggcacccatgacgtccagattgaag-3'), used to amplify between the MCK promoter and the
7 integrin cDNA, produced a 455-bp fragment only in transgenic mice. Transgenic mdx/utr+/- male mice were bred with mdx/utr+/- female mice to produce transgenic
7BX2-mdx/utr-/- offspring. The mdx/utr+/- mice (
The mdx mutation was screened by the amplification-resistant mutation system described by
Antibodies and Reagents
For immunofluorescence analysis, the mouse monoclonal antibody O26 was used to detect the transgenic rat 7 chain. Polyclonal anti-
7CDA (345) and anti-
7CDB (347) were used in Western blots to detect the
7A and
7B cytoplasmic domains, respectively (
7 chain cytoplasmic domains are identical, thus these antisera will detect both species with equal affinity. Peptides used to make these polyclonal antibodies were used as blocking controls. Rabbit polyclonal antibodies to the cytoplasmic domains of the ß1A and ß1D integrin chains were provided by Dr. W.K. Song (K-JIST, Kwang-ju, Korea;
-bungarotoxin (Molecular Probes). FITC-labeled donkey antimouse and antirabbit antibodies were purchased from Jackson ImmunoResearch Laboratories.
Western Blot Analysis
Samples of muscle tissue were extracted in 200 mM octyl-ß-D-glucopyranoside or 1% NP-40 in 50 mM Tris-HCl, pH 7.4, 2 mM PMSF, 1:200 dilution of Protease Cocktail Set III (Calbiochem), 1 mM CaCl2, and 1 mM MgCl2 at 4°C for 1 h. The supernatants were collected and protein concentrations were determined using Bradford assays. Equal amounts of extracted muscle proteins were separated on 8% polyacrylamide-SDS gels at 40 mA for 50 min, and the protein was transferred to nitrocellulose filters. Blocked filters were incubated with 1:500 dilutions of either polyclonal anti-7CDA (345), anti-
7CDB (347), or anti-ß1D antibody. HRP-linked antirabbit secondary antibody (Jackson ImmunoResearch Laboratories) was used to detect primary antibodies. Immunoreactive protein bands were detected using an ECL kit (Amersham Pharmacia Biotech). Specificity of the bands was determined using the blocking peptides used to raise the antibodies. Blots were reprobed with an anti-creatine kinase antibody. The intensities of the
7 bands were compared with creatine kinase and to total protein stained with Ponceau S using ImageQuant software. Comparable results were obtained by both methods.
Southern Blot Analysis
Mouse genomic DNA was isolated from whole blood or liver using a genomic DNA isolation kit (Promega). DNA was cleaved with EcoRI and KpnI at 3 U/µg of DNA for 16 h. DNA fragments were separated on 0.8% agarose gels and alkaline transferred to Hybond-XL nylon membranes (Amersham Pharmacia Biotech) (7 3'-untranslated domain was isolated. The probe was directly labeled with HRP using a North2South nonradioactive kit (Pierce Chemical Co.). The hybridized blots were washed following the manufacturer's instructions. Probes were detected using an ECL substrate. Blots were exposed to x-ray film for 130 min.
Immunofluorescence
Quadriceps muscles from 10-wk-old wild-type male mdx, mdx/utr-/-, and 7BX2-mdx/utr-/- mice were embedded in OCT compound (Tissue-Tek) and frozen in liquid nitrogencooled isopentane. Using a Leica CM1900 series cryostat, 10-µm sections were cut and placed on microscope slides coated with 1% gelatin, 0.05% chromium potassium sulfate. Sections were fixed in -20°C acetone for 1 min, rehydrated in 1x PBS for 10 min, and blocked in PBS containing 10% horse serum for 15 min. The rat
7 chain was detected using 5 µg/ml of purified O26 monoclonal antibody directly labeled with Alexa 488 (Molecular Probes). The anti-ß1D antibody was used at a 1:100 dilution in 1% horse serum in PBS. The antidystrophin antibody was used at a 1:100 dilution, whereas antiutrophin and anti-fMyHC antibodies were diluted 1:2 in 1% horse serum in PBS. Rhodamine-labeled
-bungarotoxin was used at a 1:3,000 dilution to detect neuromuscular junctions (NMJs).
Endogeneous mouse immunoglobulin was blocked before the addition of monoclonal antibodies using 60 µg/ml goat antimouse monovalent Fabs (Jackson ImmunoResearch Laboratories) in 1% horse serum in PBS for 30 min at room temperature. Slides were then washed three times for 5 min in 1% horse serum in PBS. Primary antibodies were added for 1 h at room temperature. Slides were washed three times for 5 min in 1% horse serum in PBS. Primary antibodies were detected with a 1:100 dilution of FITC-labeled donkey antimouse or antirabbit antibody in 1% horse serum in PBS. Slides were mounted using Vectorshield mountant (Vector Laboratories). Localization of the antibody was observed with a ZEISS Photomicroscope III. Images were acquired with a Sony DXC9000 color video CCD camera using SiteCam software and a ZEISS Axiocam digital camera (see Fig 10).
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Histology
10-µm cryosections from the quadriceps muscles of 5-, 8-, and 10-wk-old wild-type, mdx, mdx/utr-/-, and transgenic mdx/utr-/- mice were placed on uncoated slides and stained with hematoxylin and eosin. The occurrence of central nuclei was scored in 1,000 fibers in two mice from each line. The extent of mononuclear cell infiltration was estimated as the percentage of fields (40x objective) in which two or more clusters (>10 cells) of mononuclear cells were detected. At least 80 fields in four sections from each of the duplicate mice were scored.
Electron Microscopy
Sternomastoid muscles from 5-wk-old animals were fixed in 2% glutaraldehyde and 2.5% paraformaldehyde in 0.2 M Sorenson's phosphate buffer, embedded in LX-112 epoxy (Ladd Research Industries), sectioned at 0.1 µm using a Reichert Ultracut E Ultramicrotome, stained with uranyl acetate and lead citrate, and viewed with a Hitachi H-600 transmission electron microscope at a 20,000x magnification.
X-Ray and Magnetic Resonance Imaging
Spinal curvature (kyphosis) in 10-wk-old mdx, mdx/utr, and transgenic 7BX2-mdx/utr-/- mice was visualized by x-ray imaging using a Siemens Heliodent 70 x-ray machine (model D3104). X-rays were taken at 70 kVp and 7 mA. At least two mice of each genotype were analyzed.
Magnetic resonance imaging (MRI) of 10-wk-old wild-type, mdx, mdx/utr-/-, and 7BX2-mdx/utr-/- mice was used to visualize soft tissues. Mice were imaged at 1-mm thickness using a 4.7 T/31 cm Surrey Medical Imaging Spectrophotometer. At least two mice of each genotype were analyzed.
Statistical Analysis
Survival data from 84 mdx/utr-/- mice and 43 transgenic 7BX2-mdx/utr-/- mice were analyzed using the Kaplan-Meier method (
Online Supplemental Material
A video that contrasts kyphosis, gait, joint contractures, and mobility in mdx/utr-/- and 7BX2-mdx/utr-/- mice can be viewed at http://www.jcb.org/cgi/content/full/152/6/1207.
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Results |
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Production of Transgenic Mice Expressing the Rat 7BX2 Chain
To test the hypothesis that the 7ß1 integrin linkage system can alleviate severe muscle disease, transgenic mice were produced that express the rat
7 chain. DNA encoding the rat
7 integrin
7BX2 isoform, under the transcriptional control of the mouse MCK promoter, was cloned and shown to have biological activity in vitro (
7BX2, was used to express the rat integrin in mdx/utr-/- mice. Due to the mortality of the double knockout mice, the rat transgene was initially introduced into a heterozygous (mdx/utr+/-) background, and these animals were then bred to produce double knockout transgenic offspring. The ratio of offspring followed expected Mendelian genetics, indicating that the transgenic expression of the rat
7 integrin did not have an obvious effect on embryonic development. Two independent lines of transgenic mice were produced:
7BX2#2 and
7BX2#9. In this report, the data from the
7BX2#2 line are presented, but similar results were obtained with both.
The presence of the rat 7 transgene was detected by both PCR and Southern blot analyses. Using MCKI and AATII primers, a 455-bp product was amplified only in transgenic mice (Fig 1 A). Southern blot analysis produced a strong 7.1-kb band only in transgenic mice. This is the expected size of the EcoRI and KpnI digested MCK-
7BX2 construct (Fig 1 B). A weak 14.2-kb band was also detected by Southern blot analysis, suggesting that a portion of the constructs had lost one of these restriction sites.
The status of the utrophin gene was analyzed by PCR using the primers 553, 554, and 22803 described previously (
The status of the dystrophin gene was determined by the amplification-resistant mutation detection system (
Protein expression from the rat 7 chain transgene was determined by immunofluorescence analysis of cryosections using the rat-specific
7 monoclonal antibody O26 (Fig 2). The rat
7 chain was only detected by immunofluorescence in the muscle of transgenic mice (Fig 2). Immunofluorescence also showed the absence of dystrophin in muscle fibers and the absence of utrophin at NMJs in both transgenic and nontransgenic mdx/utr-/- mice (Fig 2). The fluorescent specks seen in Fig 2 upon staining mdx/utr-/- muscle with mouse antidystrophin, antiutrophin, and anti-
7 integrin antibodies are visible in the absence of primary antibody and are due to residual staining with secondary antimouse antibody.
The alternative spliced form of the ß1 integrin chain, ß1D, is expressed in differentiated skeletal and cardiac muscle (7BX2-mdx/utr-/- mice had normal levels of ß1A integrin. Immunofluorescence and Western blot analysis showed that mdx and mdx/utr-/- mice have more cell surface ß1D chain than wild-type mice. This increase in ß1D coincided with an increase in endogenous
7 chain in nontransgenic mdx and mdx/utr-/- mice as well as total
7 in
7BX2-mdx/utr-/- mice. The
7BX2-mdx/utr-/- mice also had an additional 1.5-fold more ß1D compared with mdx/utr-/- mice (Fig 3 and Fig 4 C). Thus, an increase in the
7BX2ß1D integrin is promoted by increased expression of the
7 transgene.
As reported previously, mdx mice express approximately twofold more 7 integrin mRNA than wild-type controls (
7 protein was detected in the mdx/utr-/- animals. The amount of
7BX2 protein in the
7BX2-mdx/utr-/- mouse hindlimb detected by Western blots was 2.02.3-fold greater than the endogenous
7BX2 chain in mdx/utr-/- mice (Fig 4A and Fig B). As expected, the levels of
7AX2 were equivalent in the transgenic and nontransgenic mice (Fig 4A and Fig B).
7BX2-mdx/utr-/- Mice Exhibit Increased Longevity and Mobility
Longevity was significantly extended in the 7BX2-mdx/utr-/- transgenic mice (Fig 5). Kaplan-Meier survival analysis (
7BX2-mdx/utr-/- mouse was killed at 64 wk of age.
Compared with mdx mice that exhibit minimal pathology, mdx/utr-/- mice do not maintain weight. Instead, these mice undergo a crisis period that results in weight loss and premature death at 420 wk of age (7BX2-mdx/utr-/- transgenic mice did not show sudden weight loss. Animal weight stabilized between 20 and 25 g (Fig 6). No significant differences were found in the weights of mdx mice compared with
7BX2-mdx mice 330 wk of age. Thus, extra
7BX2 chain itself does not promote weight gain.
By 8 wk of age, mdx/utr-/- mice exhibited limited mobility and a waddling gait. In contrast, 7BX2-mdx/utr-/- littermates had highly improved mobility compared with mdx mice. A video that contrasts kyphosis, gait, joint contractures, and mobility in mdx/utr-/- and
7BX2-mdx/utr-/- transgenic mice is accessible at http://www.jcb.org/cgi/content/full/152/6/1207.
Enhanced Expression of the 7BX2 Chain Stabilizes Regeneration in mdx/utr-/- Mice
Nuclei are normally localized along the periphery of myofibers, whereas in regenerating muscle nuclei are centrally located (7BX2-mdx/utr-/- mice were stained with hematoxylin and eosin to determine the extent of mononuclear infiltration and centrally located nuclei (Fig 7 and Table 1). Immunofluorescence of fMyHC was also determined. Degeneration and regeneration that are characteristic of muscle disease occur earlier in mdx/utr-/- animals compared with mdx mice (Fig 7 and Table 1). These results are consistent with the earlier onset of necrosis and cell infiltration reported previously in these animals (
7BX2-mdx/utr-/- mice was similar to that in mdx/utr-/- mice, indicating that enhanced expression of the integrin does not prevent early degeneration and regeneration. Likewise, fMyHC expression was most extensive at 5 wk in the mdx/utr-/- and
7BX2-mdx/utr-/- mice. In contrast, mdx mice exhibited very little fMyHC at 5 wk. At 8 wk, fMyHC was elevated in mdx mice, and at 10 wk it was reduced, indicating that a cycle of degeneration and regeneration was followed by stabilization. The shift from the ß1A to ß1D chain supports this conclusion. At all ages examined, the extent of fMyHC expression in the
7BX2-mdx/utr-/- animals was intermediate between that found in the mdx and mdx/utr-/- animals. In the 8- and 10-wk-old transgenic mdx/utr-/- mice, fMyHC expression approached that in mdx mice (Fig 7). This decreased expression of fMyHC in
7BX2-mdx/utr-/- mice paralleled the greater integrity of tissue seen in the 8- and 10-wk-old transgenic animals compared with the mdx/utr (-/-) mice. The extensive mononuclear cell infiltration observed in the mdx/utr-/- mice (8997% positive fields) was also partially reduced in the
7BX2-mdx/utr-/- animals (Fig 7). The percentage of fields with cell infiltration was reduced approximately 17 and 19% in 5- and 8-wk-old transgenic versus nontransgenic mdx/utr-/- mice, respectively. This represents a minimal estimate since the areas of cell infiltration were considerably larger in the nontransgenic animals (Fig 7). Thus, enhanced expression of the
7ß1 integrin does not alter the initial degenerative cycle, but once regeneration has taken place, the additional integrin appears to stabilize muscle integrity, reducing muscle pathology.
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Kyphosis and Joint Contractures Are Alleviated in 7BX2-mdx/utr-/- Mice
Severe curvature of the spine (kyphosis) in DMD patients and mdx/utr-/- mice is due to a failure of the muscles that would normally support the spinal column (7BX2-mdx/utr-/- mice compared with mdx/utr-/- littermates (Fig 8). This was confirmed by whole body MRI which visualized not only the tissues surrounding the spinal column, but bundles of muscle fibers, the heart, lung, and other soft tissues. The reduction in kyphosis promoted by the enhanced expression of integrin in the
7BX2-mdx/utr-/- animals is likely a major factor in their survival. Kyphosis results in the diaphragm being pushed forward, compromising lung capacity and diaphragm function, and thereby contributing to cardiopulmonary failure. A partial reduction of kyphosis may therefore have dramatic effects on survival.
A hallmark of diseased musculature is the failure to extend limb muscles, resulting in joint contractures. Hindlimb joint contractures are conspicuous in mdx/utr-/- mice but are markedly reduced in the 7BX2-mdx/utr-/- mice (Fig 9). The reduction in hindlimb joint contractures allows the mice to have greatly improved mobility.
Structural Changes in the NMJs of 7BX2-mdx/utr-/- Mice
The NMJs in utr-/- mice exhibit a significant reduction in the number of synaptic folds and density of AChRs (
Since the 7ß1 integrin is normally found at NMJs (
7BX2-mdx/utr-/- mice (Fig 10). Longitudinal sections of hindlimb muscles were stained with rhodamine-labeled
-bungarotoxin, and images of en face sections of the postsynaptic membrane were analyzed. Fluorescence staining of the NMJs of mdx/utr-/- mice appeared less intense compared with wild-type mice and showed discrete boutons. In contrast, most NMJs from
7BX2-mdx/utr-/- mice appeared more continuous. The postsynaptic membrane in longitudinal sections prepared from sternomastoid muscles was examined by EM. The normal folded morphology of the postsynaptic membrane is markedly reduced in the mdx/utr-/- animals, as reported previously (
7BX2-mdx/utr-/- mice. Thus, enhanced levels of the
7ß1 integrin help maintain the normal structure of the NMJ.
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Discussion |
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Our results demonstrate for the first time that enhanced expression of the 7ß1 integrin can alleviate the development of muscular dystrophy and significantly extend longevity. Mice lacking both dystrophin and utrophin were used in this study because in the absence of both proteins direct substitution of dystrophin with utrophin is precluded. This results in the development of severe muscular dystrophy and premature death, symptoms that closely resemble those seen in DMD (
The 7BX2-mdx/utr-/- mice reported here have
2.02.3-fold more
7BX2 chain than their nontransgenic littermates. The ß1D chain, partner to
7, is also increased in the
7BX2 transgenic mice. The increased levels of
7ß1 integrin led to a threefold extension in median survival time, markedly improved mobility, and reduced kyphosis and joint contractures in the transgenic mdx/utr-/- mice. Kaplan-Meier survival analysis of the transgenic and nontransgenic mdx/utr-/- mice shows that the extension of longevity due to expression of the transgene is statistically significant and is evident early and throughout the life of the animals.
The survival times of the mdx/utr-/- mice in these experiments differ slightly from those reported previously. The original reported longevity of the mdx/utr-/- mice bred from the mdx/utr+/- line used to produce the animals in our experiments was 414 wk (7BX2-mdx/utr-/- mice are clearly distinct in longevity, mobility, and histology from nontransgenic littermates. The median life span of the
7BX2-mdx/utr-/- mice was 38 wk, whereas the median life span for those not receiving the transgene was 12 wk.
Although the mechanism by which enhanced expression of the 7ß1 integrin alleviates the development of the dystrophic phenotype is not currently understood, multiple effects that result from additional
and ß integrin chains are possible.
Expression of the ß1D chain is restricted to differentiated skeletal and cardiac muscle (7ß1 for its ligand and the association of the
7ß1 with the cell cytoskeleton (
7BX2 transgenic mice may increase the interaction between the extracellular matrix, sarcolemma, and the cell cytoskeleton, stabilizing muscle integrity. Moreover, ß1A, characteristic of nonmuscle cells and undifferentiated muscle, is increased in mdx/utr-/- and decreased in the transgenic mdx/utr-/- animals. The shift from ß1A and increased ß1D reflects fewer mononuclear cell infiltrates and increased stability of muscle fibers in the rescued mice.
The 7BX2 integrin chain is normally concentrated at neuromuscular and myotendinous junctions (
7 integrin protein is increased, and the
7BX2 isoform is also found extrajunctionally (
7ß1 integrin in dystrophic mice is also seen with utrophin that is normally confined to NMJs (
7 transgene detected with antirat
7 antibodies shows that the rat
7 protein is also distributed more globally in the
7BX2-mdx/utr-/- animals. Enhanced expression of the integrin therefore contributes to the mechanical integration and stability between muscle fibers and at their junctional sites. Other possible mechanisms may also underlie how the
7ß1 integrin rescues mdx/utr-/- mice.
Whereas the MCK promoter drives transcription in skeletal and cardiac muscle (7ß1 integrin in the heart may also contribute to the rescue of these animals. However, expression of utrophin in skeletal muscle, but not cardiac muscle, of mdx/utr-/- mice increased survival and reduced pathology (
The role of the 7ß1 integrin in the formation of the postsynaptic membrane (
7BX2 chain appears to maintain the normal structure of the postsynaptic membrane in mdx/utr-/- mice.
In the absence of dystrophin, there is an increase in total muscle calcium (7ß1 integrin (
7BX2 chain may regulate the activity of calcium channels, stabilizing [Ca2+]i levels in mdx/utr-/- myofibers and reducing Ca2+-dependent proteolysis and muscle degeneration.
Enhanced expression of the 7 integrin may contribute to additional changes in the expression of other proteins, both within the cell and in the extracellular matrix. For example, matrix stability or modeling may potentiate both mechanical and signal transduction capacities of muscle (
7-/- mice. The myotendinous junctions of fast fibers are compromised in
7-deficient mice (
7 chain leads to increased ß1D.
A broad phenotype is seen in children with congenital muscular dystrophies that arise from mutations in the 7 gene (
7ß1 integrin in the formation and stability of the postsynaptic membrane, myotendinous junctions, and overall stability of muscle integrity.
Since enhanced expression of the 7ß1 integrin can alleviate many of the symptoms of severe muscular dystrophy in mdx/utr-/- mice, it appears that the integrin- and dystrophin-mediated linkage systems between myofibers and the extracellular matrix are in many ways functionally complementary mechanisms. As such, the enhanced expression of the
7ß1 integrin may be a novel approach to alleviate DMD and treat
7 integrin congenital muscular dystrophies. Moreover, increasing integrin levels may prove effective in reducing the development of other muscular dystrophies and cardiomyopathies that arise from compromised expression of other components of the dystrophin glycoprotein complex.
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Footnotes |
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The online version of this article contains supplemental material.
1 Abbreviations used in this paper: AChR, acetylcholine receptor; DMD, Duchenne muscular dystrophy; fMyHC, fetal myosin heavy chain; MCK, muscle creatine kinase, MRI, magnetic resonance imaging; NMJ, neuromuscular junction.
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Acknowledgements |
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This paper is dedicated to the memory of Dr. Kiichi Arahata, a distinguished scientist who taught us much about muscle and its diseases.
We gratefully acknowledge the generous gift of the mdx/utr+/- mice provided by Dr. Joshua Sanes (Washington University) and the MCK promoter and advice kindly provided by Drs. Stephen Hauschka and Jean Buskin (University of Washington). We thank Dr. Jonathan Henry (University of Illinois) for his helpful discussions of the X-ray and MRI analyses and Mr. Eric Chaney for his skillful assistance with the EM. Mice were generated at the University of Illinois Transgenic Animal Facility under the direction of Dr. Matt Wheeler, with the expert technical assistance of Ms. Melissa Hentges. MRI data were obtained at the University of Illinois Medical Resonance Imaging Center under the direction of Dr. Paul Lauterbur, with the technical assistance of Ms. Sherrie Frydenger.
Supported by the Muscular Dystrophy Association and the National Institutes of Health.
Submitted: 30 October 2000
Revised: 17 January 2001
Accepted: 22 January 2001
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References |
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