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First published online 13 July 2004
doi: 10.1242/jcs.01234

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Genetic compensation for sarcoglycan loss by integrin 7ß1 in muscle

¹ Department of Medicine, The University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637, USA
² Department of Human Genetics, The University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637, USA
³ Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester, M13 9PT, UK

View larger version (29K): [in a new window]   Fig. 1. Upregulation of integrin 7 where sarcoglycan level is reduced. Immunoblotting of microsomal membrane fractions purified from skeletal muscle from mice lacking sarcoglycan subunits or dystrophin. The antibodies used are specific to the integrin 7 splice forms (Itg7A and Itg7B) representing splice variants that alter the cytoplasmic domains of integrin 7 (Mayer et al., 1997). A Coomassie blue-stained loading control is shown in the lower panel of each blot. (A) Upregulation of integrin 7 in muscle null for -sarcoglycan (dsg–/–). (B) Upregulation of integrin 7 in muscle null for -sarcoglycan (gsg–/–) and in dystrophin-null mdx muscle. mdx mice have a secondary reduction of sarcoglycan at the plasma membrane (Ohlendieck and Campbell, 1991). (C) Graphical representation of integrin 7 expression from blots shown in A and B. Upregulation of integrin 7 was seen in all three mutant models but was greatest in muscle lacking -sarcoglycan (dsg–/–) where sarcoglycan loss is greatest (Hack et al., 2000b).

View larger version (45K): [in a new window]   Fig. 2. Muscle wasting, kyphosis and enhanced lethality in mice lacking both integrin 7 and -sarcoglycan (gxi). (A) Littermate wild-type (WT) and gxi mice at 3 weeks of age. WT mice are twice the size of gxi mice by 21 days. gxi mice display reduced movement and a hopping gait with limb stiffness. (B) Radiographic images of wild-type and gxi mice showing kyphosis affecting the cervicothoracic spine in the double mutant (arrows). (C) Survival curve showing relative life span of wild-type, gsg–/– and gxi mice. gxi mice have markedly reduced survival rates (n=10 for gsg–/– and gxi mice, n=3 for control and Itg7–/– mice). Integrin 7 mutants do not have reduced lethality less than 6 months of age (Mayer et al., 1997).

View larger version (162K): [in a new window]   Fig. 3. Severe muscle degeneration in gxi double-mutant mice. Masson trichrome staining shows that muscle from integrin 7 mutants (Itg7–/–) appears indistinguishable from the wild type in young mice (aged 3 weeks). Muscle from -sarcoglycan null mice (gsg–/–) displays focal degeneration, seen as blue areas. This regional fibrosis is often immediately adjacent to normal appearing regions of muscle (surrounding red). In contrast, double-mutant mouse muscle (gxi) has widespread degeneration affecting all parts of the muscle. At this low magnification view, fibrosis is seen interspersed throughout gxi muscle (note blue areas in lower right panel). Bar, 200 µm.

View larger version (91K): [in a new window]   Fig. 4. Evans blue dye (EBD) uptake is increased in gxi double-mutant mice. (A) EBD is a vital tracer and mutations in the dystrophin and sarcoglycan genes lead to enhanced muscle uptake of EBD. Following injection with EBD, muscles from wild-type (WT), integrin 7 mutant (Itg7–/–), -sarcoglycan mutant (gsg–/–) and double mutant (gxi) mice were examined grossly and microscopically where EBD staining appears red. Counterstaining with an anti-dystrophin antibody outlines myofibers (green). Bar, 100 µm. (B) Percentage of EBD-positive fibers in wild-type and mutant mice. EBD uptake is increased in both gxi and gsg–/– muscle compared to control muscle (P<0.001 and P<0.01, respectively). EBD uptake is increased gxi and gsg–/– muscle compared to Itg7–/– muscle (P<0.001 and P<0.05, respectively). EBD uptake is also increased in gxi muscle compared to gsg–/– muscle (P<0.05).

View larger version (61K): [in a new window]   Fig. 5. Upregulation of laminin 2 and ß-dystroglycan in gxi mouse muscle. (A) Immunoblot with antibodies specific to laminin 2, ß-dystroglycan or sarcomeric actin in single (Itg7–/– and gsg–/–) and double-mutant (gxi) mice. Upregulation of laminin-2 may be effective through the integrin 7ß1 complex that is upregulated in gsg–/– muscle. An increase of ß-dystroglycan is seen in Itg7–/– mutant muscle and this upregulation may be significant as an intact sarcoglycan-dystroglycan complex is present. In contrast, upregulation of ß-dystroglycan is ineffective in gxi muscle where the major laminin binding complexes, integrin and sarcoglycan-dystroglycan, are disrupted. (B,C) Photomicrographs showing muscle membrane staining for laminin 2 (B) and ß-dystroglycan (C) in each genotype. Scattered fibers with reduced laminin 2 staining can be seen in gxi muscle (B, lower right panel*). Bars in B and C, 50 µm.

View larger version (15K): [in a new window]   Fig. 6. Regeneration is present in gxi muscle. The percentage of centrally placed nuclei is increased in gxi and gsg–/– muscle reflecting enhanced regeneration. Centrally placed nuclei develop after myoblasts fuse to regenerate muscle. Quadriceps muscle was studied. There is no significant increase in number of centrally nucleated myofibers in integrin 7 mutant (Itg7–/–) muscle (P>0.05). -sarcoglycan mutant (gsg–/–) and double-mutant gxi muscle both have significantly increased centrally nucleated myofibers compared to the wild type and Itg7–/– (P<0.001 for all comparisons). No difference was detected between the number of centrally nucleated fibers between gsg–/– and gxi mice (P>0.05).

View larger version (70K): [in a new window]   Fig. 7. Upregulation of embryonic myosin heavy chain expression in gxi mouse muscle. Embryonic myosin heavy chain is expressed in response to regeneration, as it is a developmentally expressed isoform of myosin. (A) Embryonic myosin heavy chain (eMyHC) expression is shown in green, with Evans Blue Dye showing in red. DAPI staining alone is shown (in blue) for both wild-type (WT) and integrin 7 mutant (Itg7–/–) sections as no embryonic myosin heavy chain staining was present. The pattern of embryonic myosin heavy chain expression differs between -sarcoglycan mutant (gsg–/–) and gxi double-mutant muscle. In gsg–/– muscle, embryonic myosin is found in discrete regions consistent with the pattern of focal degeneration and regeneration (A, lower left panel). In contrast, gxi muscle shows widespread expression of eMyHC reflecting widespread degeneration and responsive regeneration. (B) TUNEL-positive nuclei are shown in green and are increased near regions of degeneration in gsg–/– and are seen scattered throughout gxi muscle. This is consistent with the widespread degenerative pattern and relatively small increase in TUNEL-positive nuclei compared to wild-type muscle. TUNEL-positive cells were absent in sections from the wild type and integrin 7 mutants, and only DAPI staining is shown (blue). Bars in A and B, 100 µm.

View larger version (65K): [in a new window]   Fig. 8. In vitro myoblast differentiation and fusion to myotubes is normal in gxi mutant mouse. (A) To assess the regenerative capacity of myoblasts, we cultured myoblasts from wild-type (WT), integrin 7 mutant (Itg7–/–), -sarcoglycan mutant (gsg–/–) and double mutant (gxi) neonatal mice. Each culture was differentiated to myotubes, and stained for dystrophin (green) and embryonic myosin heavy chain expression (red). Bar, 50 µm. (B) The fusion index was determined as described in Materials and Methods for each genotype and no significant differences were noted in the timing or degree of myoblast fusion to myotubes (P>0.05 for all comparisons).

View larger version (47K): [in a new window]   Fig. 9. Upregulation of integrin 5 is found in regenerating fibers. (A) Immunoblot and graphical representation of integrin 5 expression in wild-type (normal), -sarcoglycan mutant (dsg–/–), -sarcoglycan mutant (gsg–/–) and dystrophin mutant (mdx) muscle protein. Integrin 5 is a fibronectin receptor in muscle and is upregulated in DGC mutant muscle. ß-actin labelling is shown in the lower panels as a loading control. (B) Immunoblot showing that integrin 5 is upregulated in double-mutant gxi and gsg–/– mutant muscle compared to muscle from integrin 7 mutant (Itg7–/–). ß-actin labelling is again shown in the lower panel as a loading control. (C) Immunostaining with an antibody to integrin 5 shows little to no increase in Itg7–/– muscle compared to wild-type (WT) muscle. (D) Immunostaining shows that integrin 5 is found in regions of regeneration as the same fibers that are positive for integrin 5 also stain with embryonic myosin heavy chain (green). The increased fibrosis in gxi muscle also reacts with the integrin 5 antibodies consistent with its expression outside of muscle. Bar, 50 µm (C,D).