Loss of Dystrophin and β-Sarcoglycan Significantly Exacerbates the Phenotype of Laminin α2 Chain–Deficient Animals
2014; Elsevier BV; Volume: 184; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2013.11.017
ISSN1525-2191
AutoresKinga I. Gawlik, Johan Holmberg, Madeleine Durbeej,
Tópico(s)Adipose Tissue and Metabolism
ResumoThe adhesion molecule laminin α2 chain interacts with the dystrophin-glycoprotein complex, contributes to normal muscle function, and protects skeletal muscles from damage. Complete loss of the laminin α2 chain in mice results in a severe muscular dystrophy phenotype and death at approximately 3 weeks of age. However, it is not clear if the remaining members of the dystrophin-glycoprotein complex further protect laminin α2 chain–deficient skeletal muscle fibers from degeneration. Hence, we generated mice deficient in laminin α2 chain and dystrophin (dy3K/mdx) and mice devoid of laminin α2 chain and β-sarcoglycan (dy3K/Sgcb). Severe muscular dystrophy and a lack of nourishment inevitably led to massive muscle wasting and death in double-knockout animals. The dy3K/Sgcb mice were generally more severely affected than dy3K/mdx mice. However, both double-knockout strains displayed exacerbated muscle degeneration, inflammation, fibrosis, and reduced life span (5 to 13 days) compared with single-knockout animals. However, neither extraocular nor cardiac muscle was affected in double-knockout animals. Our results suggest that, although laminin α2 chain, dystrophin, and β-sarcoglycan are all part of the same adhesion complex, they have complementary, but nonredundant, roles in maintaining sarcolemmal integrity and protecting skeletal muscle fibers from damage. Moreover, the double-knockout mice could potentially serve as models in which to study extremely aggressive muscle-wasting conditions. The adhesion molecule laminin α2 chain interacts with the dystrophin-glycoprotein complex, contributes to normal muscle function, and protects skeletal muscles from damage. Complete loss of the laminin α2 chain in mice results in a severe muscular dystrophy phenotype and death at approximately 3 weeks of age. However, it is not clear if the remaining members of the dystrophin-glycoprotein complex further protect laminin α2 chain–deficient skeletal muscle fibers from degeneration. Hence, we generated mice deficient in laminin α2 chain and dystrophin (dy3K/mdx) and mice devoid of laminin α2 chain and β-sarcoglycan (dy3K/Sgcb). Severe muscular dystrophy and a lack of nourishment inevitably led to massive muscle wasting and death in double-knockout animals. The dy3K/Sgcb mice were generally more severely affected than dy3K/mdx mice. However, both double-knockout strains displayed exacerbated muscle degeneration, inflammation, fibrosis, and reduced life span (5 to 13 days) compared with single-knockout animals. However, neither extraocular nor cardiac muscle was affected in double-knockout animals. Our results suggest that, although laminin α2 chain, dystrophin, and β-sarcoglycan are all part of the same adhesion complex, they have complementary, but nonredundant, roles in maintaining sarcolemmal integrity and protecting skeletal muscle fibers from damage. Moreover, the double-knockout mice could potentially serve as models in which to study extremely aggressive muscle-wasting conditions. Deficiency of laminin α2 chain leads to a severe form of muscular dystrophy (MDC1A), and patients with MDC1A display severe muscle weakness, joint contractures, respiratory insufficiency, and feeding difficulties.1Helbling-Leclerc A. Zhang X. Topaloglu H. Cruaud C. Tesson F. Weissenbach J. Tomé F.M.S. Schwartz K. Fardeau M. Tryggvason K. Guicheney P. Mutations in the laminin α2 chain gene (LAMA2) cause merosin-deficient muscular dystrophy.Nat Genet. 1995; 11: 216-218Crossref PubMed Scopus (569) Google Scholar, 2Allamand V. Guicheney P. 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McNally E.M. γ-Sarcoglycan deficiency leads to muscle membrane defects and apoptosis independent of dystrophin.J Cell Biol. 1998; 142: 1279-1287Crossref PubMed Scopus (256) Google Scholar, 23Duclos F. Straub V. Moore S.A. Venzke D.P. Hrstka R.F. Crosbie R.H. Durbeej M. Lebakken C.S. Ettinger A.J. van der Meulen J. Holt K.H. Lim L.E. Sanes J.R. Davidson B.L. Faulkner J.A. Williamson R. Campbell K.P. Progressive muscular dystrophy in α-sarcoglycan-deficient mice.J Cell Biol. 1998; 142: 1461-1471Crossref PubMed Scopus (311) Google Scholar, 24Coral-Vazquez R. Cohn R.D. Moore S.A. Hill J.A. Weiss R.M. Davisson R.L. Straub V. Barresi R. Bansal D. Hrstka R.F. Williamson R. Campbell K.P. Disruption of the sarcoglycan-sarcospan complex in vascular smooth muscle: a novel mechanism for cardiomyopathy and muscular dystrophy.Cell. 1999; 98: 465-474Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 25Durbeej M. Cohn R.D. Hrstka R.F. Moore S.A. Allamand V. Davidson B.L. Williamson R.A. Campbell K.P. Disruption of the β-sarcoglycan gene reveals pathogenetic complexity of limb-girdle muscular dystrophy type 2E.Mol Cell. 2000; 5: 141-151Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 34Ohlendieck K. Campbell K.P. Dystrophin-associated proteins are greatly reduced in skeletal muscle from mdx mice.J Cell Biol. 1991; 115: 1685-1694Crossref PubMed Google Scholar However, it still remains unclear if removing DGC components from laminin α2 chain–deficient muscle would exacerbate the already severe dy3K/dy3K phenotype. In addition, it is unclear how beneficial laminin α2 chain expression is in mice lacking various DGC components. Therefore, we generated two new double-knockout strains: one lacking laminin α2 chain and dystrophin (dy3K/mdx mice) and one devoid of laminin α2 chain and β-sarcoglycan (dy3K/Sgcb mice). We show that dy3K/mdx and dy3K/Sgcb mice represent two of the most severely affected mouse models of muscular dystrophy, with grave muscle degeneration, inflammation, fibrosis, and a short life span (5 to 13 days). Yet, neither extraocular nor cardiac muscle was affected in double-knockout animals. Laminin α2 chain–deficient dy3K/dy3K mice were used and previously described.20Miyagoe Y. Hanaoka K. Nonaka I. Hayasaka M. Nabeshima Y. Arahata K. Nabeshima Y. Takeda S. Laminin α2 chain-null mutant mice by targeted disruption of the Lama2 gene: a new model of merosin (laminin 2)-deficient congenital muscular dystrophy.FEBS Lett. 1997; 415: 33-39Crossref PubMed Scopus (220) Google Scholar Mdx (C57BL/10Scsn-mdx/J) and β-sarcoglycan–deficient (Sgcb-null) mice (B6.129-Sgcbtm1Kcam/1J)25Durbeej M. Cohn R.D. Hrstka R.F. Moore S.A. Allamand V. Davidson B.L. Williamson R.A. Campbell K.P. Disruption of the β-sarcoglycan gene reveals pathogenetic complexity of limb-girdle muscular dystrophy type 2E.Mol Cell. 2000; 5: 141-151Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar were obtained from The Jackson Laboratory (Bar Harbor, ME) and bred in our animal facility. The dy3K/+ females were bred with mdx males. The resulting dy3K/+; mdx males were mated with dy3K/+; mdx/+ females to generate mdx/+, mdx, dy3K/dy3K, and dy3K/mdx mice. Also, dy3K/+; mdx males and females were mated. dy3K/+ females were bred with Sgcb-null males. The resulting dy3K/+; Sgcb/+ females and males were mated to generate wild-type, Sgcb-null, dy3K/dy3K, and dy3K/Sgcb mice. Also, dy3K/+; Sgcb-null males and females were mated. Control mice were wild type, dy3K/+, mdx/+, Sgcb/+, or dy3K/+; mdx/+. Mice were maintained in the animal facilities of Biomedical Center (Lund, Sweden), according to the animal care guidelines, and permission was given by the Malmö/Lund (Sweden) ethical committee for animal research. Quadriceps femoris, triceps brachii, diaphragm, heart, masseter, tongue, and extraocular muscles (EOMs) were dissected after euthanasia and frozen in OCT (Tissue Tek; Sakura Finetek, Torrence, CA) in liquid nitrogen. Sections (7 μm thick) were stained with H&E. Briefly, sections were acclimated to room temperature for 45 minutes before beginning the staining procedure and subsequently incubated in hematoxylin (1 minute), water, 0.5% acid alcohol, water, Scott's tap water, water, 80% ethanol, eosin (30 seconds), 95% and 100% ethanol, and xylene, and mounted in Pertex (Histolab, Göteborg, Sweden). Sirius red staining was used to analyze collagen content. Sections were incubated in 4% paraformaldehyde, water, 0.02% Sirius red in picric acid (10 minutes), acidified water, 70%, 95%, and 100% ethanol, and xylene, and mounted in Pertex. Stained sections were scanned using an Aperio Scanscope CS2 (with a Scanscope console, version 8.2.0.1263; Vista, CA). Necropsy analysis was performed at the National Veterinary Institute (Uppsala, Sweden). Sections were processed for immunofluorescence analyses following standard procedures35Gawlik K. Miyagoe-Suzuki Y. Ekblom P. Takeda S. Durbeej M. Laminin α1 chain reduces muscular dystrophy in laminin α2 chain deficient mice.Hum Mol Genet. 2004; 13: 1775-1784Crossref PubMed Scopus (110) Google Scholar with antibodies against dystrophin (rabbit polyclonal, 1:100; Abcam, Cambridge, UK), laminin α2 chain (rat monoclonal 4H8-2, 1:100; Alexis Biochemicals, Farmingdale, NY), CD68 (rat monoclonal FA-11, 1:100; AbD Serotec, Kidlington, UK), CD11b (rat monoclonal M1/70, 1:250; BD Pharmingen, Franklin Lakes, NJ), embryonic myosin heavy chain (mouse monoclonal F1.652, 1:10; Developmental Studies Hybridoma Bank, Iowa City, IA), caspase-3 (mouse monoclonal 46, 1:100; BD Transduction Laboratories, Franklin Lakes, NJ), laminin γ1 chain [rabbit polyclonal 1083+E1, 1:200; kindly provided by Dr. Takako Sasaki (University of Erlangen-Nürnberg, Germany)], collagen III (goat polyclonal, 1:100; Southern Biotech, Birmingham, AL), fibronectin [rabbit polyclonal, 1:1000; Abcam), fibrinogen (rabbit polyclonal fluorescein isothiocyanate conjugated, 1:50; Dako, Glostrup, Denmark), tenascin-C (rat monoclonal MTn15, undiluted),35Gawlik K. Miyagoe-Suzuki Y. Ekblom P. Takeda S. Durbeej M. Laminin α1 chain reduces muscular dystrophy in laminin α2 chain deficient mice.Hum Mol Genet. 2004; 13: 1775-1784Crossref PubMed Scopus (110) Google Scholar α-dystroglycan (mouse monoclonal IIH6, 1:50; Upstate Biotechnology, Billerica, MA), β-dystroglycan (rabbit polyclonal Vimsa, 1:50),35Gawlik K. Miyagoe-Suzuki Y. Ekblom P. Takeda S. Durbeej M. Laminin α1 chain reduces muscular dystrophy in laminin α2 chain deficient mice.Hum Mol Genet. 2004; 13: 1775-1784Crossref PubMed Scopus (110) Google Scholar α-sarcoglycan (mouse monoclonal IVD31A9, undiluted; Developmental Studies Hybridoma Bank), β-sarcoglycan (mouse monoclonal 5B1, 1:50; Novocastra, Wetzlar, Germany), utrophin (mouse monoclonal DRP3/20C5, 1:3; Novocastra), and integrin α7B [rabbit polyclonal U31, 1:300; kindly provided by Dr. Ulrike Mayer (University of East Anglia, Norfolk, UK)].31Cohn R.D. Mayer U. Saher G. Herrmann R. van der Flier A. Sonnenberg A. Sorokin L. Voit T. Secondary reduction of α7B integrin in laminin α2 deficient congenital muscular dystrophy supports an additional transmembrane link in skeletal muscle.J Neurol Sci. 1999; 163: 140-152Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar Primary antibodies were detected with appropriate secondary antibodies (Molecular Probes, Carlsbad, CA). Sections were analyzed and images were captured using a Zeiss Axioplan fluorescence microscope (Zeiss, Oberkochen, Germany), using an ORCA 1394 ER digital camera (Hamamatsu Photonics, Hamamatsu City, Japan) and Openlab software version 3 (Improvision, Coventry, UK). The area corresponding to CD68, collagen III, and fibronectin labeling was quantified in relation to the entire area of the quadriceps cross section, using ImageJ software version 1.43u (NIH, Bethesda, MD). Briefly, low-magnification (×2.5) Tiff images of whole muscles (collagen III and fibronectin staining) or ×10 magnification multiple images covering the whole muscle (CD68 staining) were converted to 8-bit mode images. Measurements were set to the threshold, which was manually adjusted for every individual image (the total muscle area versus stained area; measured in square pixels). Twelve-day-old mice (control, mdx, dy3K/dy3K, and dy3K/mdx) were injected i.p. (50 μL/10 g body weight) with Evans Blue dye (EBD; Sigma-Aldrich, St. Louis, MO) dissolved in sterile saline (10 mg EBD/mL saline). After approximately 16 hours, muscles (quadriceps femoris, triceps brachii, diaphragm, masseter, and tongue) were collected and quickly frozen in liquid nitrogen. Cryosections of the muscles were fixed in ice-cold acetone at −20°C for 10 minutes and then stained with laminin γ1 antibody (green) to visualize muscle fibers. Total muscle area and area of EBD-positive fibers (red staining) in each muscle were quantified using ImageJ software version 1.43u (as previously described). All statistical analyses were performed with GraphPad Prism software version 6 (La Jolla, CA). Averaged data are reported as means ± SEM. Kruskal-Wallis and U-tests were used (unless stated otherwise), and statistical significance was accepted for P < 0.05. To determine whether laminin α2 chain–deficient dy3K/dy3K mice would develop an even more profound phenotype when missing dystrophin or β-sarcoglycan, we crossed heterozygous dy3K/+ mice with mdx and Sgcb-null mice to produce mice lacking laminin α2 chain and dystrophin (dy3K/mdx) and laminin α2 chain and β-sarcoglycan (dy3K/Sgcb), respectively. The genotype of different mouse lines was confirmed by PCR (data not shown) and immunofluorescence (Supplemental Figure S1, A and B). Mice were born at the normal mendelian ratio in both strains (data not shown). As shown in Figure 1, the overall health of dy3K/mdx and dy3K/Sgcb mice was drastically worsened compared with single-knockout animals. The dy3K/mdx and dy3K/Sgcb mice had a median survival of approximately 13 days. However, many of the dy3K/Sgcb mice died at 5 days of age. Thus, the early lethality of both dy3K/mdx and dy3K/Sgcb mice is in sharp contrast to dy3K/dy3K mice, which died at approximately 3 to 4 weeks of age20Miyagoe Y. Hanaoka K. Nonaka I. Hayasaka M. Nabeshima Y. Arahata K. Nabeshima Y. Takeda S. Laminin α2 chain-null mutant mice by targeted disruption of the Lama2 gene: a new model of merosin (laminin 2)-deficient congenital muscular dystrophy.FEBS Lett. 1997; 415: 33-39Crossref PubMed Scopus (220) Google Scholar, 30Carmignac V. Quere R. Durbeej M. Proteasome inhibition improves the muscle of laminin α2 chain deficient mice.Hum Mol Genet. 2011; 20: 541-552Crossref PubMed Scopus (55) Google Scholar (Figure 1B). Mdx mice, on the other hand, have a near normal life span,36Lynch G.S. Hinkle R.T. Chamberlain J.S. Brooks S.V. Faulkner J.A. Force and power output of fast and slow skeletal muscles from mdx mice 6-28 months old.J Physiol. 2001; 535: 591-600Crossref PubMed Scopus (250) Google Scholar and most Sgcb-null mice live for >1 year.25Durbeej M. Cohn R.D. Hrstka R.F. Moore S.A. Allamand V. Davidson B.L. Williamson R.A. Campbell K.P. Disruption of the β-sarcoglycan gene reveals pathogenetic complexity of limb-girdle muscular dystrophy type 2E.Mol Cell. 2000; 5: 141-151Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar Moreover, both dy3K/mdx and dy3K/Sgcb mice weighed significantly less at 12 to 14 days of age, compared with single-knockout animals (Figure 1C). In addition, weight gain was less in dy3K/Sgcb mice compared with dy3K/mdx mice (Figure 1C). Because many of the dy3K/Sgcb mice died before 14 days of age, we continued to analyze these mice predominantly at 6 to 10 days of age; already at that time point, dy3K/Sgcb mice weighed significantly less than Sgcb-null mice (Supplemental Figure S2). The dy3K/mdx and dy3K/Sgcb mice were indistinguishable from other littermates at birth (data not shown). Histologically, laminin α2 chain–deficient muscle is characterized by cycles of myofiber degeneration and regeneration, inflammation, necrosis/apoptosis, and connective tissue infiltration.7Gawlik K.I. Durbeej M. Skeletal muscle laminin and MDC1A: pathogenesis and treatment strategies.Skelet Muscle. 2011; 1: 9Crossref PubMed Scopus (87) Google Scholar, 37Jeudi S. Wardrop K.E. Alessi A. Dominov J.A. Bcl-2 inhibits the innate immune response during early pathogenesis of murine congenital muscular dystrophy.PLoS One. 2011; 6: e22369Crossref PubMed Scopus (11) Google Scholar Therefore, we analyzed the muscle morphological features, the inflammatory response, and the fibrosis deposition in different muscles of all described genotypes at different ages. At 1 day of age, skeletal muscles of each genotype appeared morphologically normal, with no increased inflammation (Supplemental Figure S3A and data not shown). However, already at days 3 and 7, dy3K/Sgcb and dy3K/mdx muscle, respectively, displayed an acute inflammatory response (Supplemental Figure S3B). In dy3K/dy3K muscle, the first signs of inflammation were visible at day 7 (Supplemental Figure S3B). At 12 to 14 days of age, dy3K/mdx muscles were severely devastated. T
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