Myotendinous Junction Defects and Reduced Force Transmission in Mice that Lack α7 Integrin and Utrophin
2009; Elsevier BV; Volume: 175; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2009.090052
ISSN1525-2191
AutoresJennifer V. Welser, Jachinta E. Rooney, Nicolette C. Cohen, Praveen B. Gurpur, Cherie A. Singer, Rebecca A. Evans, Bryan A. Haines, Dean J. Burkin,
Tópico(s)Cellular Mechanics and Interactions
ResumoThe α7β1 integrin, dystrophin, and utrophin glycoprotein complexes are the major laminin receptors in skeletal muscle. Loss of dystrophin causes Duchenne muscular dystrophy, a lethal muscle wasting disease. Duchenne muscular dystrophy-affected muscle exhibits increased expression of α7β1 integrin and utrophin, which suggests that these laminin binding complexes may act as surrogates in the absence of dystrophin. Indeed, mice that lack dystrophin and α7 integrin (mdx/α7−/−), or dystrophin and utrophin (mdx/utr−/−), exhibit severe muscle pathology and die prematurely. To explore the contribution of the α7β1 integrin and utrophin to muscle integrity and function, we generated mice lacking both α7 integrin and utrophin. Surprisingly, mice that lack both α7 integrin and utrophin (α7/utr−/−) were viable and fertile. However, these mice had partial embryonic lethality and mild muscle pathology, similar to α7 integrin-deficient mice. Dystrophin levels were increased 1.4-fold in α7/utr−/− skeletal muscle and were enriched at neuromuscular junctions. Ultrastructural analysis revealed abnormal myotendinous junctions, and functional tests showed a ninefold reduction in endurance and 1.6-fold decrease in muscle strength in these mice. The α7/utr−/− mouse, therefore, demonstrates the critical roles of α7 integrin and utrophin in maintaining myotendinous junction structure and enabling force transmission during muscle contraction. Together, these results indicate that the α7β1 integrin, dystrophin, and utrophin complexes act in a concerted manner to maintain the structural and functional integrity of skeletal muscle. The α7β1 integrin, dystrophin, and utrophin glycoprotein complexes are the major laminin receptors in skeletal muscle. Loss of dystrophin causes Duchenne muscular dystrophy, a lethal muscle wasting disease. Duchenne muscular dystrophy-affected muscle exhibits increased expression of α7β1 integrin and utrophin, which suggests that these laminin binding complexes may act as surrogates in the absence of dystrophin. Indeed, mice that lack dystrophin and α7 integrin (mdx/α7−/−), or dystrophin and utrophin (mdx/utr−/−), exhibit severe muscle pathology and die prematurely. To explore the contribution of the α7β1 integrin and utrophin to muscle integrity and function, we generated mice lacking both α7 integrin and utrophin. Surprisingly, mice that lack both α7 integrin and utrophin (α7/utr−/−) were viable and fertile. However, these mice had partial embryonic lethality and mild muscle pathology, similar to α7 integrin-deficient mice. Dystrophin levels were increased 1.4-fold in α7/utr−/− skeletal muscle and were enriched at neuromuscular junctions. Ultrastructural analysis revealed abnormal myotendinous junctions, and functional tests showed a ninefold reduction in endurance and 1.6-fold decrease in muscle strength in these mice. The α7/utr−/− mouse, therefore, demonstrates the critical roles of α7 integrin and utrophin in maintaining myotendinous junction structure and enabling force transmission during muscle contraction. Together, these results indicate that the α7β1 integrin, dystrophin, and utrophin complexes act in a concerted manner to maintain the structural and functional integrity of skeletal muscle. Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disease that affects 1 in every 3500 live male births. 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110: 2873-2881Crossref PubMed Google Scholar Enhanced expression of the α7 integrin in the skeletal muscle of severely dystrophic mice reduced muscle pathology and increased lifespan by threefold.10Burkin DJ Wallace GQ Nicol KJ Kaufman DJ Kaufman SJ Enhanced expression of the alpha 7 beta 1 integrin reduces muscular dystrophy and restores viability in dystrophic mice.J Cell Biol. 2001; 152: 1207-1218Crossref PubMed Scopus (236) Google Scholar, 11Burkin DJ Wallace GQ Milner DJ Chaney EJ Mulligan JA Kaufman SJ Transgenic expression of alpha7beta1 integrin maintains muscle integrity, increases regenerative capacity, promotes hypertrophy, and reduces cardiomyopathy in dystrophic mice.Am J Pathol. 2005; 166: 253-263Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar In contrast, loss of both dystrophin and α7 integrin in mice results in severe muscular dystrophy and premature death by 4 weeks of age.28Rooney JE Welser JV Dechert MA Flintoff-Dye NL Kaufman SJ Burkin DJ Severe muscular dystrophy in mice that lack dystrophin and alpha7 integrin.J Cell Sci. 2006; 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The utrophin glycoprotein complex is a third major laminin receptor in skeletal muscle. 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To understand the functional overlap between the α7β1 integrin and utrophin in skeletal muscle, we produced mice that lack both α7 integrin and utrophin (α7/utr−/−). Since both complexes are highly enriched at the MTJ and NMJ, we hypothesized that α7/utr−/− mice may have severe abnormalities at these critical junctional sites. Our study demonstrates α7/utr−/− mice exhibit partial embryonic lethality comparable with that observed in α7−/− mice. Dystrophin is increased in these animals and enriched at the NMJ but not the MTJ. α7/utr−/− mice display ultrastructural defects in their MTJ and compromised force transmission. Together, these results indicate that the α7β1 integrin, dystrophin and utrophin laminin binding complexes provide continuity between laminin in the extracellular matrix and the cell cytoskeleton, which are necessary for the normal structural and functional properties of skeletal muscle. Male α7−/− mice (C57BL6 × 129 strain) generated in the Nevada Transgenic Center26Flintoff-Dye NL Welser J Rooney J Scowen P Tamowski S Hatton W Burkin DJ Role for the alpha7beta1 integrin in vascular development and integrity.Dev Dyn. 2005; 234: 11-21Crossref PubMed Scopus (73) Google Scholar were mated to female utr−/− mice (C57BL6 × 129 strain and a kind gift from Dr. Joshua Sanes, Harvard University, MA). The resulting male offspring were backcrossed to utr−/− females. To generate α7/utr−/− mice, F2 generation mice heterozygous at the α7 integrin locus were mated. Wild-type mice (C57BL6 × 129) were used as controls for this study. Age-matched male mice were used for all experiments. Mice were genotyped using genomic DNA isolated from tail clips. Tail clips were incubated in TNES buffer (200 mmol/L TrisHCl pH 8.0, 5 mmol/L EDTA pH8.0, 400 mmol/L NaCl, and 1% SDS) and 100 μg proteinase K overnight at 55°C. 5 M NaCl was added to the samples and DNA precipitated using ice-cold 100% ethanol. The DNA pellet was washed with 70% ethanol and resuspended in TE buffer (10 mM Tris-HC1, 1 mM EDTA pH 8.0). The wild-type and mutant α7 integrin alleles were detected using a multiplex PCR as previously described.28Rooney JE Welser JV Dechert MA Flintoff-Dye NL Kaufman SJ Burkin DJ Severe muscular dystrophy in mice that lack dystrophin and alpha7 integrin.J Cell Sci. 2006; 119: 2185-2195Crossref PubMed Scopus (126) Google Scholar A multiplex PCR was done for utrophin using the following primers: 553 (5′-TTGCAGTGTCTCCCAATAAGG TATGAAC-3′), 554 (5′-CTGAGTCAAACAGCTTGGAAGCCTCC-3′), 22803 (5′-TGCCAAGTTCTAATTCCA TCAGAAGCTG-3′). The PCR conditions were: 95°C for 2 minutes, 40 cycles of 95°C for 1 minute, 62°C for 1 minute, and 72°C for 1 minute. The utrophin wild-type band produced a 640 bp and the utrophin targeted allele produced a 450 bp band. Five week-old wild-type, α7−/−, utr−/−, and α7/utr−/− mice were euthanized according to a protocol approved by the University of Nevada, Reno, Animal Care and Use Committee. Gastrocnemius, triceps, and tibialis anterior muscles were dissected from mice and flash frozen in liquid nitrogen cooled isopentane. To detect α7 integrin, protein was extracted from gastrocnemius muscle as previously reported.28Rooney JE Welser JV Dechert MA Flintoff-Dye NL Kaufman SJ Burkin DJ Severe muscular dystrophy in mice that lack dystrophin and alpha7 integrin.J Cell Sci. 2006; 119: 2185-2195Crossref PubMed Scopus (126) Google Scholar The anti-α7A integrin (A2 345) and anti-α7B integrin (B2 347) rabbit polyclonal antibodies (a gift from Dr. Stephen Kaufman, University of Illinois) were used to detect the α7A and α7B integrin isoforms at a dilution of 1:500 and 1:2000 respectively. Utrophin was detected using the MANCHO3 8A4 anti-utrophin mouse monoclonal antibody (a kind gift of Glenn Morris, Center for Inherited Neuromuscular Disease, Shropshire, UK) as previously described.28Rooney JE Welser JV Dechert MA Flintoff-Dye NL Kaufman SJ Burkin DJ Severe muscular dystrophy in mice that lack dystrophin and alpha7 integrin.J Cell Sci. 2006; 119: 2185-2195Crossref PubMed Scopus (126) Google Scholar For dystrophin immunoblots, protein was extracted from gastrocnemius using a 10% SDS extraction buffer (100 mmol/L Tris-HCl pH 8.0, 10% SDS, 10 mmol/L EDTA, 10% glycerol). Samples were boiled for 10 minutes and centrifuged for 20 minutes. 10 μg of protein was loaded on 5% polyacrylamide gels (Bio-Rad, Hercules, CA) and transferred to nitrocellulose membranes. The mouse monoclonal antibody Dys-2 (Novocastra Laboratories Ltd, Newcastle Upon Tyne, UK) was used at a dilution of 1:350 to detect dystrophin. Blots were incubated with Alexa Fluor 680-coupled goat anti-rabbit IgG (Molecular Probes, Eugene, OR) to detect the primary antibody. The Odyssey Imaging System (LiCor Biosciences, Lincoln, NE) was used to quantify band intensities. Myosin heavy chain was used as the loading control and analysis performed in the linear range for the detection of these antibodies. For acetylcholine receptor (AChR)-β subunit immunoblots, protein was extracted from gastrocnemius using RIPA buffer (50 mmol/L Hepes pH 7.4, 150 mmol/L NaCl, 1 mmol/L Na3VO4, 10 mmol/L NaF, 0.5% Triton X-100, 0.5% NP40, 10% glycerol, 2 mmol/L phenylmethylsulfonyl fluoride, and 1:200 Protease Inhibitor Cocktail Set III [Calbiochem, EMD Biosciences, San Diego, CA]). 50 μg of protein was loaded on 7.5% Tris-HCL gels (Bio-Rad, Hercules, CA). To detect the AChR-β, the rabbit polyclonal antibody AChR-β (Epitomics, Burlingame, CA) was used at a dilution of 1:2000. Primary antibodies were detected, and blots were normalized for protein loading, as previously published.28Rooney JE Welser JV Dechert MA Flintoff-Dye NL Kaufman SJ Burkin DJ Severe muscular dystrophy in mice that lack dystrophin and alpha7 integrin.J Cell Sci. 2006; 119: 2185-2195Crossref PubMed Scopus (126) Google Scholar Ten-micron-thick cryosections from triceps or tibialis anterior muscles were placed on Surgipath slides (Surgipath Medical Supplies, Richmond, IL). Sections were fixed with methanol, acetone, or 4% paraformaldehyde for 2 minutes. α7 integrin and utrophin were detected as reported28Rooney JE Welser JV Dechert MA Flintoff-Dye NL Kaufman SJ Burkin DJ Severe muscular dystrophy in mice that lack dystrophin and alpha7 integrin.J Cell Sci. 2006; 119: 2185-2195Crossref PubMed Scopus (126) Google Scholar using the rat monoclonal antibody CA5.5 (Sierra Biosource, Morgan Hill, CA) and the mouse monoclonal antibody MANCHO7 7F3. Dystrophin was detected using the mouse monoclonal antibody MANDRA1 (Sigma Aldrich, St. Louis, MO) (1:100) after using the M.O.M. kit (Vector Laboratories, Burlingame, CA) to block endogenous mouse immunoglobulin. Tenascin C was detected using a rat monoclonal anti-tenascin C antibody (Sigma Aldrich, St. Louis, MO) at 1:200 on unfixed TA muscle. Fluorescein isothiocyanate-conjugated secondary antibodies were used to detect primary antibodies at a dilution of 1:500. Rhodamine-labeled α-bungarotoxin (Molecular Probes, Eugene, OR) was used at a 1:1000 dilution to identify AChRs at the NMJ. Images were captured with Zeiss Axioskop 2 Plus fluorescent microscope, AxioCam HRc digital camera and Axiovision 5.0 software. Ten-micron-thick cryosections from triceps muscle were placed on glass slides and fixed in ice-cold 95% ethanol for 2 minutes, placed in 70% ethanol for 2 minutes, and rinsed in running water for 5 minutes. Slides were immersed in Gill’s hematoxylin (Fisher Scientific, Fair Lawn, NJ) for 3 minutes and rinsed in running water for 5 minutes. Slides were then incubated in Scott’s solution for 3 minutes and rinsed in running water for 5 minutes. Slides were immersed in 1× eosin for 2 minutes and progressively dehydrated in ice cold 70% ethanol for 30 seconds, 95% ethanol for 30 seconds, and 100% ethanol for 2 minutes. Slides were placed in xylene for 5 minutes and mounted in DePeX mounting medium (Electron Microscopy Sciences, Washington, PA). Using H&E-stained sections from the triceps muscle, the number of centrally located nuclei in 1000 fibers was counted per mouse (N = 6). The results were expressed as the percentage of fibers containing centrally located nuclei. Wild-type, α7−/−, utr−/−, and α7/utr−/− mice at 5 weeks of age were injected intraperitoneally with 50 μl sterile Evan’s blue dye (500 μg/50 μl PBS) per 10g of body weight. After 3 hours, tissues were frozen in liquid nitrogen cooled isopentane, cryosectioned (10 μm), and placed on slides. Tissue sections were fixed in 4% paraformaldehyde. To define muscle fibers, wheat-germ agglutinin was used at a concentration of 1 μg/ml. A Zeiss Axioscope II fluorescence microscope was used to visualize sections. Fibers positive for Evan’s blue dye in 1000 random fibers per mouse were counted at a magnification of ×630 (N = 5). Five-week-old wild-type, α7−/−, utr−/−, and α7/utr−/− mice were allowed to grasp wire cages and remain suspended. The recorded time was terminated when the mice released their grip. Every mouse was subjected to two tests and results from both tests were averaged. Muscle endurance was determined using the ratio of time suspended over mouse weight. The forelimb grip strength was measured using a SDI Grip Strength System and a Chatillon DFE Digital Force Gauge (San Diego Instruments, Inc., San Diego, CA). Five-week-old wild-type, α7−/−, utr−/−, and α7/utr−/− mice were allowed to grasp a horizontal platform with their forelimbs and then pulled backwards. The peak tension (grams of force) was recorded just before the mice released their grip. Five consecutive tests were performed for each mouse and the data were averaged. At least nine mice per group were analyzed. Tibialis anterior muscles from 5-week-old wild-type and α7/utr−/− mice were fixed in 2% gluteraldehyde and 2.5% paraformaldehyde in a 0.2 M/L phosphate buffer overnight. Tissues were rinsed with PBS and postfixed with 1% osmium tetroxide in phosphate buffer for one hour. Samples were successively dehydrated in 70%, 95%, and 100% ethanol, incubated in propylene oxide for 30 minutes, in 1:1 propylene oxide and LX-112 epoxy embedding resin for 12 hours. 0.1 μm sections were cut on a Reichert Ultracut E Ultramicrotome. Sections were stained with uranyl acetate and lead citrate. The MTJ were viewed on a Hitachi H-600 transmission electron microscope at ×20,000 magnification. All averaged data are reported as the mean ± SD unless otherwise stated. To compare multiple groups, one-way and two-way analysis of variance was performed using SigmaStat 1.0 software (Jandel Corporation, San Rafael, CA). Chi-squared analysis was used to determine statistical significance for the Mendelian inheritance pattern. P < 0.05 was considered statistically significant unless otherwise stated. To determine the contributions of the α7 integrin and utrophin as modifiers of DMD disease progression, α7 integrin null mice were bred with utrophin-deficient animals to produce mice that lack both the α7 integrin and utrophin (α7/utr−/−). Genotyping was used to confirm mutant alleles for α7 integrin and utrophin (Figure 1A). PCR was used to identify the mutant α7 integrin allele at 482 bp while the wild-type allele was detected at 727 bp (Figure 1A). PCR identified the targeted utrophin allele at 450 bp, whereas the wild-type allele produced a 640 bp product (Figure 1A). Loss of both α7 integrin and utrophin proteins was confirmed by immunoblotting and immunofluorescence (Figure 1, B and C). To determine whether loss of both α7 integrin and utrophin results in partial embryonic lethality, the Mendelian inheritance patterns of wild-type, α7−/−, utr−/−, and α7/utr−/− mice were analyzed. Matings of α7+/−/utr+/− mice to α7−/−/utr+/− animals showed that 7.6% of α7−/−/utr−/− mice were born instead of the expected 12.5% (Table 1), indicating that 40% of the α7/utr−/− animals died in utero. This embryonic lethality is similar to that observed in α7 integrin null mice in which about 50% of the mice die before birth (Table 1).26Flintoff-Dye NL Welser J Rooney J Scowen P Tamowski S Hatton W Burkin DJ Role for the a
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