Research Article 2185 Severe in mice that lack and 7

Jachinta E. Rooney1,*, Jennifer V. Welser1,*, Melissa A. Dechert1, Nichole L. Flintoff-Dye1, Stephen J. Kaufman2 and Dean J. Burkin1,3,‡ 1Department of Pharmacology, University of Nevada, Reno, NV 89557, USA 2Department of Cell and Developmental Biology, University of Illinois, Urbana, IL 61801, USA 3Nevada Transgenic Center, University of Nevada, Reno, NV 89557, USA *These authors contributed equally to this work ‡Author for correspondence (e-mail: [email protected])

Accepted 21 February 2006 Journal of Cell Science 119, 2185-2195 Published by The Company of Biologists 2006 doi:10.1242/jcs.02952

Summary The dystrophin glycoprotein complex links in the mice exhibited extensive loss of membrane integrity, to the cell cytoskeleton. Loss of increased centrally located nuclei and inflammatory cell dystrophin causes Duchenne muscular dystrophy, the most infiltrate, greater necrosis and increased muscle common human X--linked genetic disease. degeneration compared to mdx or 7-integrin null animals. The 71 integrin is a second transmembrane laminin In addition, loss of dystrophin and/or 7 integrin resulted receptor expressed in . Mutations in the 7 in altered expression of laminin-2 chain. These results integrin cause in humans and point to complementary roles for dystrophin and 71 mice. The 71 integrin is increased in the skeletal muscle integrin in maintaining the functional integrity of skeletal of Duchenne muscular dystrophy patients and mdx mice. muscle. This observation has led to the suggestion that dystrophin and 71 integrin have complementary functional and Supplementary material available online at structural roles. To test this hypothesis, we generated mice http://jcs.biologists.org/cgi/content/full/119/11/2185/DC1 lacking both dystrophin and 7 integrin (mdx/7–/–). The mdx/7–/– mice developed early-onset muscular dystrophy Key words: 7 integrin, Dystrophin, Transgenic mice, Double and died at 2-4 weeks of age. Muscle fibers from mdx/7–/– knockout, Skeletal muscle, Muscular dystrophy Journal of Cell Science Introduction In DMD patients, loss of dystrophin results in a disruption Duchenne muscular dystrophy (DMD) is the most common X- of the DGC-extracellular matrix linkage. This leads to the loss chromosome-linked human disease that affects approximately of sarcolemmal integrity, unregulated influx of Ca2+ into 1 in 3,500 males. DMD patients suffer from severe, progressive muscle cells, activation of Ca2+-dependent proteases and muscle wasting with clinical symptoms first detected between muscle degeneration (Gillis, 1996). Muscle degeneration is 2 and 5 years of age. As the disease progresses, patients are accompanied by regeneration through the activation of muscle confined to a wheelchair in their early teens and die in their satellite cells; however, regenerative capacity in DMD patients early twenties from cardiopulmonary failure (Emery, 1993; is exceeded by progressive muscle degeneration. This results Moser, 1984). DMD patients and mdx mice have dystrophin in the replacement of muscle fibers with adipose and gene mutations that result in an absence of the dystrophin connective tissue (O’Brien and Kunkel, 2001). (Bulfield et al., 1984; Campbell, 1995; Matsumura et The mdx mouse is a valuable model for DMD and has been al., 1992; Monaco et al., 1986; Sicinski et al., 1989). Dystrophin used extensively to analyze the progression of muscle disease. is localized to the cytoplasmic face of the plasma membrane Although DMD patients (Monaco et al., 1986) and mdx mice and mediates the interaction between the cell cytoskeleton and (Bulfield et al., 1984; Sicinski et al., 1989) both lack dystrophin, the extracellular matrix (ECM) through a complex of associated the mdx mouse develops less severe muscle pathology glycoproteins called the dystrophin-glycoprotein complex compared with DMD patients, and has a normal life span. (DGC). In skeletal muscle, the DGC is localized along muscle Several factors may explain the reduced pathology in mdx mice fibers and at myotendinous and neuromuscular junctions (MTJs including differences in muscle use between humans and and NMJs, respectively). This complex includes dystroglycans, captive mice. In addition, mdx mice might compensate for the sarcoglycans and syntrophins (Suzuki et al., 1994). Dystrophin loss of dystrophin, possibly by overexpressing one or more interacts with F-actin through its N-terminus (Way et al., 1992). with functional similarity to dystrophin. One candidate The C-terminus interacts with syntrophins and with the protein is utrophin, an autosomal homolog of dystrophin that is cytoplasmic tail of -dystroglycan (Henry and Campbell, expressed at the post-synaptic membrane of the NMJ in adult 1996). -dystroglycan binds to laminin in the extracellular skeletal muscle. Utrophin is upregulated in the muscle of both matrix. Overall, the DGC links the muscle cell cytoskeleton to DMD patients and mdx mice and is localized at extrajunctional the extracellular matrix and maintains sarcolemmal integrity. sites (Ohlendieck et al., 1991; Pons et al., 1991). 2186 Journal of Cell Science 119 (11)

Overexpression of utrophin can rescue the dystrophic phenotype in mdx mice (Deconinck et al., 1997b; Rafael et al., 1998; Squire et al., 2002; Tinsley et al., 1998; Tinsley et al., 1996). Mice that lack utrophin show mild neuromuscular defects (Deconinck et al., 1997a; Grady et al., 1997a). By contrast, mice lacking both utrophin and dystrophin (mdx/utr–/– mice) develop severe muscular dystrophy and cardiomyopathy similar to that of DMD patients, and die between 4-14 weeks of age (Deconinck et al., 1997b; Grady et al., 1997b). The 71 integrin is a second laminin receptor in skeletal muscle (Burkin and Kaufman, 1999). Like the DGC, 71 integrin serves as a structural link between the cell cytoskeleton and laminin in the basal lamina and, therefore, contributes to the overall integrity of the . Mutations in the 7-integrin gene are responsible for human congenital myopathy, in which patients exhibit delayed motor milestones (Hayashi et al., 1998). Mice that lack 7 integrin develop myopathy and demonstrate altered force transmission, compliance and viscoelasticity in diaphragm muscle (Lopez et al., 2005; Mayer et al., 1997). In addition, 71 integrin plays an important role in the development of NMJs (Burkin et al., 1998; Burkin et al., 2000) and MTJs (Mayer et al., 1997; Nawrotzki et al., 2003). These junctional sites are often damaged in diseased muscle (Lyons and Slater, 1991; Nagel et al., 1990; Nawrotzki et al.,

Journal of Cell Science 2003; Rafael et al., 2000). Fig. 1. mdx/7–/– mice lack dystrophin and 7 integrin. (A) Multiplex PCR and ARMS Increased amounts of 7 integrin are found in DMD patients and mdx mice (Hodges et al., assays were performed on genomic tail DNA to detect wild-type versus 7-integrin- targeted allele and the wild-type versus mdx mutation, respectively. The wild-type 7- 1997). These observations suggest that integrin allele (727 bp) was detected in wild-type and mdx mice, whereas the targeted enhanced 7 integrin expression is a 7-integrin allele (482 bp) was detected in the 7–/– and double-knockout mice. Wild- mechanism by which muscle can compensate type dystrophin (275 bp) was detected in wild-type and 7–/– mice, whereas the mdx for the loss of dystrophin. This hypothesis mutation (275 bp) was present in the mdx and double-knockout mice. Neg, negative was supported by transgenic overexpression control lacking DNA. (B) Western blotting with anti-7A-integrin and 7B-integrin of the 7-integrin chain which alleviated the antibodies in gastrocnemius muscle of 3-week-old mice confirmed the absence of 7- severe muscular dystrophy and extended the integrin protein in 7–/– and mdx/7–/– mice. (C) Immunofluorescence with anti-CA5.5 life-span of mice that lacked dystrophin and antibody, which recognizes 7 integrin, demonstrated loss of the 7 integrin in triceps muscle from 3-week-old 7–/– and mdx/7–/– mice. The absence of dystrophin in mdx utrophin (Burkin et al., 2001; Burkin et al., –/– 2005). These observations suggest potential and mdx/ 7 mice was verified by immunofluorescence with an anti-dystrophin antibody. Bar, 10 m. structural and functional overlap between the 71 integrin and DGC. To explore the structural and functional overlap between structural overlap between DGC and 71 integrin in dystrophin and 71 integrin, we generated mice that lack both maintaining the integrity of skeletal muscle. laminin-binding complexes (mdx/7–/– mice). These mice exhibited more severe muscular dystrophy than either mdx- or Results 7-integrin-deficient animals and died at 2-4 weeks of age. In mdx/7–/– mice exhibit muscle wasting and reduced addition, the muscle of 3-week-old mdx/7–/– mice showed viability inflammation and centrally located nuclei, indicative of severe To determine whether functional overlap exists between muscle damage and increased requirement for muscle repair. dystrophin and 71 integrin, mdx mice were bred with mice Finally, loss of dystrophin and/or 7 integrin resulted in that lack the 7-integrin gene (Flintoff-Dye et al., 2005) to reduced laminin-2-chain expression in skeletal muscle, produce mice that lack both dystrophin and 7 integrin demonstrating regulation of laminin expression by 71 (mdx/7–/–). Mutations in the dystrophin and 7 integrin integrin. Collectively, these studies support functional and were confirmed by genotyping (Fig. 1A). Multiplex PCR Dystrophin and 7-integrin null mice 2187

detected a 482 bp product representing the targeted 7- integrin allele in both the 7–/– and mdx/7–/– mice, whereas the wild-type allele was 727 bp. Amplification- resistant mutation system (ARMS) PCR yielded a 275 bp product and confirmed the presence of the mdx mutation in mdx and mdx/7–/– mice (Fig. 1A). Loss of 7 protein was confirmed by immunoblotting. Western analysis demonstrated that both 7A-integrin and 7B-integrin isoforms were missing in mdx/7–/– mice (Fig. 1B). The absence of both dystrophin and 7 integrin in the skeletal muscle of mdx/7–/– mice was confirmed by immunofluorescence (Fig. 1C). At 10 days of age, the average weight of male wild- type, mdx, 7–/– or mdx/7–/– pups was 7.9±0.2 g, 6.3±0.9 g, 7.8±0.17 g or 5.6±0.45 g, respectively (Fig. 2B). The weight of mdx and mdx/7–/– littermates was statistically different from wild-type and 7–/– mice (P<0.05), but was not different from each other. By day 18, mdx/7–/– mice exhibited a failure to thrive and appeared smaller compared with mdx littermates (Fig. 2A). By day 21, male mdx/7–/– mice weighed only 5.05±0.51 g (n=8), less than half the weight of wild-type (11.89±0.83 g, n=5), mdx (9.86±0.15 g, n=7) or 7–/– (13.04±0.54 g, n=9) animals, indicating that double- knockout mice failed to gain weight (Fig. 2C). mdx/7–/– animals displayed kyphosis, joint contractures of the limbs, an abnormal waddling gate, tremors and reduced mobility (a movie that contrasts mobility, joint contractures, tremor and gait in mdx and mdx/7–/– mice can be viewed as supplementary material, Movie 1). This debilitating phenotype progressed rapidly, resulting in the death of these mice at day 16-26 (Fig. 2D). Fig. 2. Muscle-wasting and reduced viability in mdx/7–/– mice. (A) Photograph of mdx and mdx/7–/– littermates aged 18 days. Double- Muscle membrane integrity is compromised in knockout mice appear smaller than mdx littermates and show outward –/– Journal of Cell Science mdx/ 7 mice signs of kyphosis. These mice also exhibit joint contractures, reduced To evaluate skeletal muscle integrity, mice were injected mobility and tremors (see supplementary material, Movie 1). (B) The with Evan’s Blue dye (EBD). EBD binds albumin, average weight of 10-day-old male mdx/7–/– mice was indistinguishable –/– fluoresces red and can enter cells with compromised from that of mdx littermates. The weight of both mdx and mdx/7 mice –/– was significantly different from wild-type and 7–/– animals (*P<0.05). plasma membranes. At 3 weeks, wild-type and 7 mice –/– showed no evidence of EBD uptake, signifying that the (C) The average weight of 21-day-old male mdx/ 7 mice was significantly different from wild-type, mdx, and 7–/– animals (*P<0.05), sarcolemma was undamaged in these animals (Fig. 3A). indicating that double-knockout mice failed to gain weight. (D) The By contrast, myofibers from both mdx and dystrophin/ 7 viability of mdx/7–/– mice (n=21) is severely reduced compared with double-knockout mice showed considerable EBD uptake wild-type (n=12), 7–/– (n=148) and mdx (n=19) mice. All mdx/7–/– mice (Fig. 3A). Muscle from mdx and mdx/7–/– mice had died at 16-26 days. 14.6% and 18.9% EBD-positive fibers, respectively; however, these were not significantly different from each other (Fig. 3B). These data indicate that the loss of dystrophin skeletal muscle of 3-week-old mdx/7–/– mice has undergone but not 7 integrin, in the skeletal muscle of 3-week-old mice increased muscle degeneration compared with wild-type, mdx decreases sarcolemmal integrity. or 7-integrin null animals. Earlier studies have implicated inflammation in the mdx/7–/– mice exhibit severe muscle pathology pathogenesis of muscular dystrophy (McDouall et al., 1990; To investigate whether the skeletal muscle from mdx/7–/– Tews and Goebel, 1996). To assess the inflammatory response mice exhibited greater pathology compared with controls, in mice lacking dystrophin and 7 integrin, tissue cryosections muscle sections were stained with hematoxylin and eosin. from the triceps muscle of 3-week-old wild-type, mdx, 7–/– Skeletal muscle from 3-week-old wild-type and 7–/– mice and mdx/7–/– mice were incubated with anti-CD4 antibody. showed no pathology (Fig. 4A). By comparison, skeletal CD4 is a type-I membrane glycoprotein expressed on muscle from 3-week-old mdx mice exhibited small necrotic thymocytes, and to some extent, monocytes and macrophage- lesions (Fig. 4A). By contrast, skeletal muscle from mdx/7–/– related cells. There was on average less than 1 CD4-positive mice exhibited severe pathology including variation in cell per field of view from each of the control genotypes (Fig. myofiber size, large necrotic regions and areas of mononuclear 4B). By contrast, an average of 44 CD4-positive cells per field cell infiltration (Fig. 4A). These data demonstrate that the was observed in the muscle of mdx/7–/– mice. These results 2188 Journal of Cell Science 119 (11)

Fig. 3. Reduced sarcolemmal integrity in muscle from mdx/7–/– mice. (A) Evan’s Blue dye (EBD) uptake was used to assess membrane integrity of the tibialis anterior skeletal muscle. Myofibers were outlined with Oregon Green-488-conjugated WGA (green). EBD uptake (red) was not detected in wild-type and 7–/– myofibers. By contrast, significant EBD uptake was observed in muscle from mdx and mdx/7–/– animals. Bar, 10 m. (B) The number of EBD-positive muscle fibers was assayed. Wild-type and 7–/– muscle had less than 1% EBD-positive fibers. By contrast, 18.9% of fibers were positive for EBD in mdx/7–/– mice, but this was not significantly different from the number of EBD-positive fibers seen in mdx animals.

demonstrate that mdx/7–/– mice experienced an increased muscle showed 2.8% and 0.3% of muscle fibers with centrally inflammatory response in skeletal muscle compared with wild- located nuclei, respectively, which was not statistically type, mdx or 7-integrin null animals at 3 weeks of age. significant from the wild-type controls (Fig. 5B). By contrast, 17.8% of muscle fibers in the tibialis anterior muscle and Increased muscle regeneration in mdx/7–/– mice 40.1% of muscle fibers from the triceps muscle from mdx/7–/– To examine the extent of skeletal muscle regeneration in contained centrally located nuclei (Fig. 5B). These results mdx/7–/– mice, fibers containing centrally located nuclei in suggest an earlier onset of regeneration in response to muscle triceps and tibialis anterior muscles were counted and fiber damage in mdx/7–/– mice. compared with control genotypes. As expected, skeletal muscle Embryonic myosin heavy chain (eMyHC) expression was from wild-type mice had few centrally located nuclei (Fig. 5A- examined as a second measure of muscle regeneration. B). By comparison, the tibialis anterior muscle from 3-week- Consistent with data obtained from the analysis of centrally old mdx and 7–/– mice showed 4.1% and 3.2% fibers with located nuclei in muscle fibers, a significant increase in the centrally located nuclei, respectively (Fig. 5B). Their triceps number of eMyHC-positive fibers was observed in 3-week-old Journal of Cell Science

Fig. 4. Severe muscle pathology in mdx/7–/– mice. (A) Hematoxylin and eosin staining of tibialis anterior muscle from 3-week-old mice shows the characteristically uniform fiber diameters in wild-type mice. Small necrotic regions and variations in myofiber size were observed in muscle of 3-week-old mdx mice. At this age, muscle from 7–/– mice appeared to be similar to wild-type. By contrast, muscle from mdx/7–/– mice exhibited extreme variations myofiber size and large areas of mononuclear cell infiltration (arrow). Bar, 10 m. (B) Inflammation in the muscle of mdx/7–/– mice was quantified by counting the number of CD4-positive immune cells per field of view in triceps muscle of 3-week-old mice. Muscle of wild-type, mdx and 7–/– animals showed less than 1% CD4-positive cells. By contrast, an average of 43.88 CD4-positive immune cells per field was observed in the triceps muscle of mdx/7–/– mice, which was significantly higher when compared with control genotypes (*P<0.05). Dystrophin and 7-integrin null mice 2189

Fig. 5. Enhanced myofiber regeneration in mdx/7–/– mice. (A) Hematoxylin and eosin staining of tibialis anterior muscle of 3-week- old mice to analyze the localization of nuclei within myofibers. Centrally localized nuclei were observed in the muscle of mdx/7–/– mice. Compared with control genotypes they indicate extensive muscle regeneration, which follows degeneration of diseased myofibers. Central nuclei are indicated by arrows. Bar, 10 m. (B) Quantitation of centrally-located nuclei in triceps and tibialis anterior muscles. As expected, wild-type mice exhibited peripherally located nuclei in both muscle groups. Centrally located nuclei were observed in muscle fibers from mdx and 7–/– mice compared with wild- type (2.8% and 0.32%, respectively, for triceps muscle; 4.1% and 3.2%, respectively, for tibialis anterior muscle). By contrast, mdx/7–/– mice showed 40.1% and 17.5% centrally located nuclei in triceps and tibialis anterior muscle, respectively, which are significantly higher than controls (*P<0.05).

mdx/7–/– muscle. As expected, wild-type muscle showed only also been shown to regulate laminin deposition (Li et al., 2003). 0.3 eMyHC-positive fibers per field. Muscle from 3-week-old Immunoblotting and immunofluorescence were undertaken to mdx and 7–/– mice showed 0.2 and 0.1 eMyHC-positive fibers determine whether the absence of 7, coupled with the loss of per field, respectively. By contrast, 7.5 eMyHC-positive fibers dystrophin, affected the expression and localization of 1D per field of view were counted in muscle from mdx/7–/– mice integrin, utrophin and/or laminin. Wild-type, mdx, 7–/– and (Fig. 6B). Collectively, these data indicate that loss of mdx/7–/– mice exhibited similar amounts of 1D integrin in dystrophin and 7 integrin results in more muscle regeneration skeletal muscle (Fig. 7A), which was confirmed by western –/– Journal of Cell Science in mdx/7 mice at the age of 3 weeks compared with wild- analysis (data not shown). type, mdx or 7 integrin deficient animals. Immunofluorescence revealed an increase in laminin-2- chain expression in mdx mice and downregulation in the Altered expression of laminin-2-chain expression in muscle of 7–/– and mdx/7–/– mice (Fig. 7A). This was 7–/– and mdx/7–/– mice confirmed by quantitative western analysis with laminin-2 In mature skeletal muscle, the 7-integrin chain preferentially bands normalized for protein loading to the constitutively and associates with the 1D subunit and binds to laminin-211 and ubiquitously expressed protein cyclooxygenase-1 (Cox-1). laminin-221 in the extracellular matrix. The 71 integrin has Expression of Cox-1 did not appear to change in wild-type or

Fig. 6. Increased expression of eMyHC in mdx/7–/– mice. (A) Immunofluorescence was used to detect eMyHC expression in tibialis anterior muscle fibers from 3-week- old mice. Muscle from wild-type, mdx and 7–/– mice showed few eMyHC-positive fibers (green). Many muscle fibers from mdx/7–/– mice were positive for eMyHC. Rhodamine-labeled WGA (red) was used to define muscle fibers. Bar, 10 m. (B) Quantitation of eMyHC-positive fibers. Few eMyHC-positive muscle fibers were counted in wild-type, mdx or 7–/– mice. By contrast, an average of 7.46 fibers per field were positive for eMyHC in mdx/7–/– mice, which is significantly different from the controls (*P<0.05). 2190 Journal of Cell Science 119 (11)

Fig. 7. Altered expression of laminin-2 chain in the skeletal muscle of mdx/7–/– mice. (A) Immunofluorescence was used to detect the localization of 1D and laminin-2 chain in triceps muscle of 3- week-old mice. A similar pattern of 1D localization at the myofiber sarcolemma was observed in wild-type, 7–/–, mdx and mdx/7–/– mice. Immunofluorescence revealed a decrease in laminin-2 chain in the muscle of 7–/– and mdx/7–/– mice compared with both wild-type and mdx mice. Bar, 10 m. (B) Laminin-2 expression in the gastrocnemius muscle was quantitated by western analysis. mdx mice showed a significant increase in laminin-2 expression compared with wild-type. 7–/– and mdx/7–/– mice showed a decrease in laminin-2-chain expression compared with wild-type and mdx animals (*P<0.05).

diseased states. In mdx mice an increase in laminin-2 chain and loss of 7 integrin has been shown to result in defective was detected compared with wild-type muscle. By contrast, a matrix deposition at this site (Mayer et al., 1997; Nawrotzki et 7.4-fold decrease in laminin-2 protein was observed in the al., 2003), we compared the ultrastructure of the MTJs from 3- muscle of 7-integrin-deficient mice and a 3.3-fold decrease week-old wild-type, mdx, 7–/– and mdx/7–/– mice (Fig. 9). –/– Journal of Cell Science was observed in mdx/ 7 mice compared with wild-type Longitudinal sections were prepared from the gastrocemius animals (Fig. 7B). These results indicate that loss of 7 integrin muscle and examined by electron microscopy. The MTJs of led to reduced deposition of laminin-211 or laminin-221 in the wild-type and mdx mice showed extensive sarcolemmal folding basal lamina of muscle. (Fig. 9, arrows) increasing the contact area between muscle and Utrophin expression was assayed with two independent anti- tendon; also, a highly organized, continuous and dense utrophin antibodies (MANCHO3 and MANCHO7). Utrophin extracellular matrix was observed. By contrast, the MTJs of is normally localized at MTJs and NMJs in adult mice (Law et 7–/– mice showed few sarcolemmal folds (Fig. 9, arrows) al., 1994). Utrophin was localized at the NMJs of 3-week-old indicating loss of structural integrity at this site. The wild-type mice. As previously reported, utrophin expression extracellular matrix appeared less organized and was not as was increased and localized at neuromuscular and electron-dense as wild-type or mdx tissue confirming previous extrajunctional sites in 3-week-old mdx mice (Law et al., observations (Fig. 9, arrowheads) (Mayer et al., 1997; 1994). In 7–/– and mdx/7–/– mice, utrophin appeared Nawrotzki et al., 2003). Like those of 7-integrin-deficient localized at junctional and extrajunctional sites (Fig. 8A). mice, the MTJs of mdx/7–/– mice showed few sarcolemmal Western blot analysis revealed a significant increase in utrophin folds (arrow) and altered deposition of extracellular matrix expression in mdx mice (P<0.05, n=3 mice per genotype) (Fig. 9, arrowheads). In addition to these structural changes, compared with wild-type animals (Fig. 8B), which confirms mdx/7–/– mice also showed mononuclear cell infiltrate at this previous reports. Utrophin levels in the gastrocnemius muscle junctional site. from 7 and mdx/7–/– mice were similar to those observed in wild-type animals (Fig. 8). These results indicate that the loss Discussion of dystrophin alone resulted in increased utrophin. However, The dystrophin glycoprotein complex and 71 integrin are the additional loss of the 7 integrin did not result in an transmembrane receptors in skeletal muscle that provide increase in utrophin expression in the skeletal muscle of molecular continuity between laminin in the extracellular mdx/7–/– mice. matrix and the cell cytoskeleton. Disruption of either receptor leads to neuromuscular disease in both humans and mice Structural changes in the MTJs of 7 and mdx/7–/– reflecting the importance of both complexes in maintaining mice muscle integrity (Emery, 1993; Hayashi et al., 1998; Mayer et Since the 71 integrin and dystrophin are localized at MTJs al., 1997; Monaco et al., 1986; Moser, 1984; Sicinski et al., Dystrophin and 7-integrin null mice 2191

Fig. 8. Increased utrophin expression in mdx but not mdx/ 7–/– mice. (A) Utrophin localization was examined by immunofluorescence in the gastrocnemius muscle of 3-week-old wild-type, mdx, 7–/– and mdx/ 7–/– mice. As expected, utrophin was localized at the NMJs of muscle from wild-type mice and at neuromuscular and extrajunctional sites of muscle from mdx animals, confirming previous studies. Utrophin localization appeared at both junctional and extrajunctional sites in 7–/– or mdx/7–/– mice. Bar, 10 m. (B) Utrophin expression in the gastrocnemius muscle was quantitated by western analysis. mdx mice showed a statistically significant increase in utrophin expression compared with wild-type (*P<0.05). 7–/– and mdx/7–/– mice showed no increase in utrophin compared with wild- type animals.

1989). Levels of the 7-integrin chain are increased twofold in the muscle of DMD patients and mdx mice (Hodges et al., 1997). Transgenic overexpression of 7 integrin in the skeletal muscle of severely dystrophic mdx/utr–/– mice can partially

Journal of Cell Science rescue the diseased phenotype and extend the lifespan of these animals without restoring the expression of DGC components (Burkin et al., 2001; Burkin et al., 2005). Together, these results suggest the DGC and 71 integrin have overlapping and compensatory functions. The severe muscle phenotype and premature death of the mdx/7–/– mice reported in this study further extends these studies, and supports the hypothesis that dystrophin and the 71 integrin have overlapping roles in maintaining the structural and functional integrity of skeletal muscle. We observed only mild muscle pathology in 3-week- old 7–/– and mdx mice, which is consistent with previously reported studies (Grady et al., 1997b; Mayer et al., 1997). By contrast, the absence of both proteins in mdx/7–/– mice results in severe muscle degeneration, variation in myofiber size and inflammatory cell infiltration. The muscle pathology of mice that lack dystrophin and utrophin is similar to that seen in DMD patients (Deconinck et al., 1997b; Grady et al., 1997b). Utrophin is an autosomal homolog of dystrophin localized to junctional sites in adult Fig. 9. Ultrastructure of MTJs in muscle from 3-week-old wild- skeletal muscle (Blake et al., 1996). In mdx mice, utrophin is –/– –/– increased and is localized at extrajunctional sites were it type, mdx, 7 and mdx/ 7 mice. The sarcolemma of the MTJs of wild-type and mdx mice is highly folded, increasing the surface replaces dystrophin and associates with an otherwise normal area between muscle and tendon (arrows); the extracellular matrix dystrophin-associated protein complex (Matsumura et al., at this site is highly organized and electron-dense. By contrast, 1992). Mice that lack dystrophin and utrophin have a mean life MTJs of 7–/– and mdx/7–/– mice show little or no sarcolemmal span of 14 weeks (Burkin et al., 2001; Deconinck et al., 1997b; folding (arrows) and the extracellular matrix appeared less dense Grady et al., 1997b), whereas mice that lack dystrophin and 7 and organized compared with those of wild-type and mdx mice integrin all die before the age of 4 weeks. These results indicate (arrowheads). Bar, 1 m. 2192 Journal of Cell Science 119 (11)

that compensation by 7 integrin allows mdx/utr–/– mice to live integrin and the laminin-2 chain. This mechanism might also longer. This hypothesis is supported by the partial rescue of the be involved in the rescue of severely dystrophic mice by the dystrophic phenotype in mdx/utr–/– mice by transgenic transgenic overexpression of 7 integrin (Burkin et al., 2001). expression of 7 integrin, which extends the life span of these Interestingly, utrophin and laminin expression are upregulated animals approximately threefold (Burkin et al., 2001; Burkin in mdx mice, but not mdx/7–/– mice, suggesting the possibility et al., 2005). By contrast, endogenous expression of utrophin that utrophin expression is regulated by laminin. in mdx/7–/– mice appears to be unable to compensate to the The inflammatory response to chronic muscle-fiber damage same extent for the loss of 7 integrin and dystrophin. is believed to be a major factor contributing to the progression Interestingly, the loss of dystrophin and 7 integrin does not of pathology observed in DMD patients (Gorospe et al., 1994; result in increased utrophin expression as seen in the skeletal Gosselin and McCormick, 2004). Muscle of mdx mice shows muscle of mdx mice. changes in the expression of genes involved in the The mdx/7–/– mice analyzed in this study exhibit a inflammatory response (Porter et al., 2002). A significant phenotype similar to mice deficient in 7-integrin and - inflammatory response was observed in the skeletal muscle and sarcoglycan (Allikian et al., 2004). The phenotypic similarity MTJs of mdx/7–/– mice compared with mdx or 7-integrin- between these mice suggests that functional overlap exists deficient animals at 3 weeks of age. These observations suggest between -sarcoglycan in the dystrophin complex and 71 that loss of dystrophin and integrin protein complexes lead to integrin. Alternatively, loss of -sarcoglycan might affect the decreased extracellular matrix-to-muscle-cell contacts within stability of the dystrophin-glycoprotein complex, resulting in muscle and at critical junctional sites, resulting in increased the observed phenotypic similarity (Hack et al., 2000b; Hack muscle-fiber damage and inflammation. et al., 2000a; Zhu et al., 2001). The production and analysis of Although our data support the model of overlapping double-knockout mice lacking 7 integrin and other structural roles for dystrophin and 7 integrin in maintaining sarcoglycan subunits may elucidate further the functional the integrity of the sarcolemma, it is also possible that these overlap between members of the sarcoglycan complex and 7 proteins have functionally intersecting cell-signaling pathways integrin. that regulate muscle survival because both mdx and laminin- EBD uptake in the muscle of mdx/7–/– mice was similar to deficient mice show increased apoptosis (Ruegg et al., 2002; that observed in mdx animals, implying that loss of dystrophin Tidball et al., 1995; Tombes et al., 1995). exhibit is responsible for the observed membrane damage in both mechanotransduction ability, and the binding of extracellular groups of animals. Prior investigations have shown that the ligands or integrin-clustering can initiate cell signaling through height of muscle degeneration and its ensuing regeneration in proteins such as focal adhesion kinase or phosphoinisitol-3- mdx mice occurs at 4-6 weeks of age (Grady et al., 1997b; kinase (Schlaepfer et al., 1999; Schwartz, 2001). Interestingly, McGeachie et al., 1993). By conducting the present studies in signaling through FAK and the adaptor molecule growth- 3-week-old mice, only the onset of changes in membrane factor-receptor-binding protein 2 (Grb2) can lead to activation permeability in mdx muscle fibers was observed. This concept of the mitogen-activated protein kinase (MAPK) pathway of age-dependency was further supported by the extensive (Schlaepfer et al., 1994), which promotes cell survival (Bonni

Journal of Cell Science muscle damage and centrally located nuclei observed in et al., 1999). mdx/7–/– mice compared with the single-knockout Evidence that dystrophin and other members of the DCG phenotypes. The absence of both dystrophin and 7 integrin have cell-signaling capacities that regulate cell survival and resulted in significantly more centrally located nuclei and growth is beginning to emerge. Dystrophin itself can become eMyHC expression in skeletal muscle fibers compared with phosphorylated, which can alter its affinity for actin (Senter et control genotypes. Therefore, our data indicate that mdx/7–/– al., 1993). Calmodulin binding sites are present on both mice have a requirement for increased muscle regeneration dystrophin and syntrophin (Madhavan et al., 1992), which link compared with mdx or 7-integrin null animals. Compromised the DGC to Ca2+/calmodulin-dependent protein kinase II contractile function or too little muscle regeneration might be (CaMKII)-mediated signaling (Rando, 2001). Neuronal nitric mechanisms underlying the severe muscle damage observed in oxide synthase (nNOS), which regulates many signaling the mdx/7–/– mice. pathways, is also associated with DGC (Abdelmoity et al., The severe muscle pathology observed in mdx/7–/– mice is 2000). Finally, Grb2 can bind to -dystroglycan (Yang et al., similar to that reported in laminin-2-deficient mice (Miyagoe- 1995) and may therefore be a point of convergence between Suzuki et al., 2000). The skeletal muscle of laminin-2 null dystrophin and integrins. mice show reduced expression of 7 integrin (Hodges et al., Recently, several genes have been identified that appear to 1997). Our data showed increased endogenous expression of compensate for the loss of dystrophin. Using a complementary both 7-integrin and laminin-2 proteins in the muscle of mdx gene therapy approach, these ‘booster’ genes have been mice. Although 7 deficient mice exhibit decreased levels of overexpressed in skeletal muscle of dystrophic mouse models laminin-2 in skeletal muscle, these mice live beyond 20 and were shown to rescue the diseased phenotype (Engvall and weeks of age. These observations suggest that levels of Wewer, 2003). These complementary proteins include laminin, dystrophin and utrophin in 7-integrin-deficient mice utrophin, 7 integrin, GalNac, nNOS and Adam12 (Burkin et are sufficient to maintain muscle integrity. A further reduction al., 2001; Moghadaszadeh et al., 2003; Nguyen et al., 2002; in laminin-2-chain expression was not observed in mdx/7–/– Tidball and Wehling-Henricks, 2004; Tinsley et al., 1998). By mice compared with 7–/– animals, indicating that the loss of taking advantage of single- and double-knockout mice, this the laminin-2 chain is primarily due to the loss of 7 integrin. study has identified areas of structural and functional Together, these data support the idea that, in skeletal muscle, convergence and independence between the 71 integrin and a regulatory mechanism exists between the expression of 7 dystrophin protein complexes. Understanding the functional Dystrophin and 7-integrin null mice 2193

overlap between each of the complementary proteins and (HRP)-labeled goat anti-mouse secondary antibody. The 395 kDa utrophin band was dystrophin might identify new pathways that can be targeted detected by chemiluminescence and normalized for protein loading by probing the same blot with anti-Cox-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). to treat muscular dystrophy. Band intensities were quantified by using ImageQuant TL software (Amersham Biosciences, Piscataway, NJ). Materials and Methods Generation of mdx/7–/– mice Immunofluorescence 7–/– mice (C57BL6-7gal strain) produced in the Nevada Transgenic Center Triceps muscles were embedded in Tissue-TEK Optimal Cutting Temperature (Flintoff-Dye et al., 2005) were bred with mdx (C57BL10ScSn-Dmd strain) female compound (Sakura Finetek USA Inc., Torrance, CA). Ten-m sections were cut animals. The resulting F1 males, which were heterozygous at the 7 locus, were using a Leica CM1850 cryostat and placed onto Surgipath microscope slides backcrossed with mdx (C57BL10ScSn-Dmd strain) females. Males and females (Surgipath Medical Industries, Richmond, IL). The 7 integrin was detected with produced in the F2 generation that were heterozygous at the 7 locus were bred to a 1:1000 dilution of anti-CA5.5 rat monoclonal antibody (Sierra Biosource, Morgan generate mdx/ double-knockout mice. The wild-type control strain used in this Hill, CA) followed by a 1:1000 dilution of FITC-conjugated anti-rat secondary study was C57BL10ScSn. Male littermates or age-matched mice were used for antibody. The 1D integrin was detected with a 1:500 dilution of rabbit polyclonal analysis. antibody followed by a 1:500 dilution of FITC-conjugated anti-rabbit antibody. To genotype mice, genomic DNA was isolated from tail clips using a Wizard SV Laminin-2 chain was detected with a 1:500 dilution of rabbit anti-2G polyclonal DNA purification system (Promega, Madison, WI) following the manufacturer’s antibody (a kind gift from Peter Yurchenco, Robert Wood Johnson Medical School, instructions. Mice were genotyped by multiplex PCR to detect the wild-type and Department of Pathology, Piscataway, NJ). Dystrophin was detected with the mouse targeted 7-integrin alleles with the following primers: 7PF10 (5-TGAAG- monoclonal anti-Dys2 antibody (Novacastra Laboratories, Ltd, Newcastle upon GAATGAGTGCACAGTGC-3), 7exon1R1 (5-AGATGCCTGTGGGCAGAG- Tyne, UK) and utrophin was detected with MANCHO7 7F3 monoclonal antibody TAC-3) and galR2 (5-GACCTGCAGGCATGCAAGC-3). PCR conditions were against utrophin (a kind gift from Glenn Morris, Center for Inherited Neuromuscular as follows: 95°C for 4 minutes then 34 cycles at 95°C for 1 minute, 62°C for 1 Disease, Shropshire, UK) at a dilution of 1:200. Anti-CD4 monoclonal antibody (a minute and 72°C for 1 minute. A wild-type band was 727 bp, whereas the 7- kind gift from Dorothy Hudig, University of Nevada, Reno) was used to assay integrin-targeted allele produced a 482 bp band. muscle inflammation at a dilution of 1:200. The mouse monoclonal antibodies were The mutation in the dystrophin gene was detected by a modified ARMS assay used in conjunction with a mouse-on-mouse (MOM) immunodetection kit (Vector (Amalfitano and Chamberlain, 1996; Burkin et al., 2001). The following primers Laboratories, Burlingame, CA) to block mouse immunoglobulin and a 1:500 were used: p259E (5-GTCACTCAGATAGTTGAAGCCATTTAA-3), p260E (5- dilution of FITC-conjugated anti-mouse secondary antibody. Acetylcholine GTCACTCAGATAGTTGAAGCCATTTAG-3) and p306F (5-CATAGTTATT- receptors at NMJs were detected with Rhodamine-labeled -bungarotoxin at 1:1000 AATGCATAGATATTCAG-3). PCR conditions were as follows: 95°C for 4 (Molecular Probes). Fluorescence was observed with a Zeiss Axioskop 2 Plus minutes then 34 cycles at 95°C for 1 minute, 55°C for 1 minute and 72°C for 1 fluorescent microscope and images were captured with a Zeiss AxioCam HRc minute. Primer set p259 and p306 produced a 275 bp wild-type dystrophin allele. digital camera and Axiovision 4.1 software. Primer set p260 and p306 detects the mdx point mutation and produced a 275 bp product. To genotype the dystrophin gene, separate PCR reactions were performed Embryonic myosin heavy chain because the product sizes are identical in wild-type and mdx mice. Embryonic myosin heavy chain (eMyHC) was used in immunofluorescence experiments to measure muscle regeneration. Immunofluorescence was performed Isolation of skeletal muscle on 10-m sections from tibialis anterior muscle from 3-week-old wild-type, mdx, –/– –/– Three-week-old wild-type, mdx, 7–/– and mdx/7–/– male mice were euthanized by 7 and mdx/7 mice with a 1:500 dilution of anti-eMyHC antibody (F1.652, Developmental Studies Hybridoma Bank, University of Iowa, IA). The primary CO2 inhalation in accordance with a protocol approved by the University of Nevada, Reno Animal Care and Use Committee. The gluteus, gastrocnemius, triceps and antibody was detected with a 1:500 dilution of FITC-conjugated anti-mouse tibialis anterior muscles from these mice were dissected, flash-frozen in liquid secondary antibody. A 1 g/ml concentration of tetramethylrhodamine-conjugated nitrogen and stored at –80°C. wheat-germ agglutinin (WGA) (Molecular Probes, Eugene, OR) was used to define muscle fibers. Multiple adjacent sections were analyzed with ten random, non- Immunoblotting overlapping microscopic fields that were counted per animal at 630 magnification. The gastrocnemius muscle from 3-week-old male mice was ground in liquid Data was reported as the number of eMyHC-positive fibers per field. Journal of Cell Science nitrogen. Protein was extracted in 200 mM octyl--D-glucopyranoside (Sigma Aldrich, St Louis, MO), 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM CaCl2, 1 Hematoxylin and eosin staining Tibialis anterior and triceps muscles were cryosectioned and 10-m sections were mM MgCl2, 2 mM PMSF and a 1:200 dilution of Protease Inhibitor Cocktail Set III (Calbiochem, EMD Biosciences, San Diego, CA) for the detection of the 7 and placed on Surgipath microscope slides. Tissue sections were fixed in ice-cold 95% 1 integrins. Protein was quantified by Bradford assay and 80 g of total protein ethanol for 2 minutes followed by 70% ethanol for 2 minutes and then re-hydrated were separated on 8% SDS-PAGE gels under non-reduced conditions, and in running water for 5 minutes. The tissue sections were then stained with Gill’s transferred to nitrocellulose membranes. Membranes were blocked in Odyssey hematoxylin (Fisher Scientific, Fair Lawn, NJ) and rinsed in water for 5 minutes. Blocking Buffer (LiCor Biosciences, Lincoln, NE) that was diluted 1:1 in Tissue sections were placed in Scott’s solution (0.024 M NaHCO3, 0.17 M MgSO4) phosphate-buffered saline (PBS). The 7 integrin was detected with a 1:500 dilution for 3 minutes and rinsed in water for 5 minutes. Tissue sections were stained in of rabbit anti-7A (A2 345) and rabbit anti-7B (B2 347) polyclonal antibodies. eosin solution (Sigma-Aldrich, St Louis, MO) for 2 minutes. Tissue sections were 1D integrin was detected with a 1:500 dilution of rabbit anti-1D polyclonal then dehydrated in ice-cold 70% and 95% ethanol for 30 seconds each, followed by antibody (a kind gift of Woo Keun Song, Gwanju Institute for Science and 100% ethanol for 2 minutes. Tissue sections were then cleared in xylene for 5 Technology, South Korea). Blots were incubated with a 1:5000 dilution of Alexa minutes prior to mounting with DePeX mounting medium (Electron Microscopy Fluor 680-coupled goat anti-rabbit IgG (Molecular Probes, Eugene, OR) to detect Sciences, Washington, PA). Centrally located nuclei were counted at 630 primary antibodies. magnification by bright-field microscopy. The number of central nuclei per muscle To analyze laminin-2, protein was extracted from gastrocnemius muscle with a fiber was determined by counting 1000 muscle fibers for triceps muscle and 700 buffer containing 2% NP40 and 2% Triton X-100. Protein was quantified by a muscle fibers for tibialis anterior muscle per animal. At least five animals from each –/– –/– Bradford assay. 60 g of protein were loaded per well, separated on a 7.5% genotype (wild-type, mdx, 7 and mdx/7 ) were analyzed. polyacrylamide gel and transferred for 1 hour to nitrocellulose. Laminin-2 was detected with an anti-laminin-2 antibody (sc-20142, Santa Cruz Biotechnology, Evan’s Blue dye uptake Santa Cruz, CA) at 1:200 followed by a 1:5000 dilution of Alexa Fluor 680-labeled Mice were injected intraperitoneally with 50 l per 10 g of body weight sterile goat anti-rabbit IgG. The 300 kDa laminin-2 band was normalized for protein Evans Blue dye solution (10 mg/ml). After 3 hours, the gastrocnemius muscle was loading by re-probing the blot with goat anti-Cox-1 polyclonal antibody (Santa Cruz harvested and flash-frozen in liquid nitrogen. 10-m cryosections were placed on Biotechnology, Santa Cruz, CA). Blots were visualized with the Odyssey Imaging microscope slides and fixed in 4% paraformaldehyde. To outline muscle fibers, System and band intensities determined by using Odyssey Imaging software. tissue sections were incubated with 2 g/ml Oregon Green-488-conjugated WGA To examine utrophin expression, protein was extracted from the gastrocnemius (Molecular Probes, Eugene, OR). Muscle fibers positive for Evans Blue dye were muscle with RIPA buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 1 mM Na3VO4, counted in a minimum of 1000 fibers per animal, and at least three animals from 10 mM NaF, 0.5% Triton X-100, 0.5% NP40, 10% glycerol, 2 mM PMSF and a each phenotype were analyzed. Counting was conducted and images captured at 1:200 dilution of Protease Inhibitor Cocktail Set III) and quantified by a Bradford 630 magnification. Assay (BioRad Laboratories Inc., Hercules, CA). 80 g of total protein were separated on a 7.5% SDS-PAGE gel and transferred to nitrocellulose. The blot was Electron microscopy incubated with a 1:200 dilution of anti-utrophin mouse monoclonal antibody The gastrocnemius muscle and tendon were dissected from 3-week-old animals and (MANCHO3 8A4, a kind gift of Glenn Morris, Center for Inherited Neuromuscular fixed in 2% glutaraldehyde and 2.5% paraformaldehyde in 0.2% Sorenson’s Disease, Shropshire, UK) followed by a 1:50,000 dilution of horseradish peroxidase phosphate buffer. Tissue was embedded in LX-112 epoxy (Ladd Research 2194 Journal of Cell Science 119 (11)

Industries), sectioned at 0.1 m wirth a Reichert Ultracut E Ultramicrotome, stained Gosselin, L. E. and McCormick, K. M. (2004). Targeting the immune system to improve with uranyl actetate and lead citrate and viewed with a Hitachi H-600 transmission ventilatory function in muscular dystrophy. Med. Sci. Sports Exerc. 36, 44-51. electron microscope at 5000 magnification. Grady, R. M., Merlie, J. P. and Sanes, J. R. (1997a). Subtle neuromuscular defects in utrophin-deficient mice. J. Cell Biol. 136, 871-882. Statistical analysis Grady, R. M., Teng, H., Nichol, M. C., Cunningham, J. C., Wilkinson, R. S. and All averaged data are reported as the mean ± s.d. Comparisons between multiple Sanes, J. R. (1997b). Skeletal and cardiac myopathies in mice lacking utrophin and groups were performed by one-way-analysis of variance (ANOVA) for parametric dystrophin: a model for Duchenne muscular dystrophy. Cell 90, 729-738. Hack, A. A., Groh, M. E. and McNally, E. M. (2000a). 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