Transgenic Overexpression of ADAM12 Suppresses Muscle Regeneration and Aggravates Dystrophy in Aged mdx Mice
2007; Elsevier BV; Volume: 171; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2007.070435
ISSN1525-2191
AutoresLouise Helskov Jørgensen, Charlotte Harken Jensen, Ulla M. Wewer, Henrik Daa Schrøder,
Tópico(s)Knee injuries and reconstruction techniques
ResumoMuscular dystrophies are characterized by insufficient restoration and gradual replacement of the skeletal muscle by fat and connective tissue. ADAM12 has previously been shown to alleviate the pathology of young dystrophin-deficient mdx mice, a model for Duchenne muscular dystrophy. The observed effect of ADAM12 was suggested to be mediated via a membrane-stabilizing up-regulation of utrophin, α7B integrin, and dystroglycans. Ectopic ADAM12 expression in normal mouse skeletal muscle also improved regeneration after freeze injury, presumably by the same mechanism. Hence, it was suggested that ADAM12 could be a candidate for nonreplacement gene therapy of Duchenne muscular dystrophy. We therefore evaluated the long-term effect of ADAM12 overexpression in muscle. Surprisingly, we observed loss of skeletal muscle and accelerated fibrosis and adipogenesis in 1-year-old mdx mice transgenically overexpressing ADAM12 (ADAM12+/mdx mice), even though their utrophin levels were mildly elevated compared with age-matched controls. Thus, membrane stabilization was not sufficient to provide protection during prolonged disease. Consequently, we reinvestigated skeletal muscle regeneration in ADAM12 transgenic mice (ADAM12+) after a knife cut lesion and observed that the regeneration process was significantly impaired. ADAM12 seemed to inhibit the satellite cell response and delay myoblast differentiation. These results discourage long-term therapeutic use of ADAM12. They also point to impaired regeneration as a possible factor in development of muscular dystrophy. Muscular dystrophies are characterized by insufficient restoration and gradual replacement of the skeletal muscle by fat and connective tissue. ADAM12 has previously been shown to alleviate the pathology of young dystrophin-deficient mdx mice, a model for Duchenne muscular dystrophy. The observed effect of ADAM12 was suggested to be mediated via a membrane-stabilizing up-regulation of utrophin, α7B integrin, and dystroglycans. Ectopic ADAM12 expression in normal mouse skeletal muscle also improved regeneration after freeze injury, presumably by the same mechanism. Hence, it was suggested that ADAM12 could be a candidate for nonreplacement gene therapy of Duchenne muscular dystrophy. We therefore evaluated the long-term effect of ADAM12 overexpression in muscle. Surprisingly, we observed loss of skeletal muscle and accelerated fibrosis and adipogenesis in 1-year-old mdx mice transgenically overexpressing ADAM12 (ADAM12+/mdx mice), even though their utrophin levels were mildly elevated compared with age-matched controls. Thus, membrane stabilization was not sufficient to provide protection during prolonged disease. Consequently, we reinvestigated skeletal muscle regeneration in ADAM12 transgenic mice (ADAM12+) after a knife cut lesion and observed that the regeneration process was significantly impaired. ADAM12 seemed to inhibit the satellite cell response and delay myoblast differentiation. These results discourage long-term therapeutic use of ADAM12. They also point to impaired regeneration as a possible factor in development of muscular dystrophy. Skeletal muscle is capable of regeneration after injury, such as in trauma or disease. 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23: 55-61Crossref PubMed Scopus (126) Google Scholar Ectopic expression of ADAM12 in an ADAM12 transgenic (TG) mouse model10Moghadaszadeh B Albrechtsen R Guo LT Zaik M Kawaguchi N Borup RH Kronqvist P Schroder HD Davies KE Voit T Nielsen FC Engvall E Wewer UM Compensation for dystrophin-deficiency: ADAM12 overexpression in skeletal muscle results in increased alpha 7 integrin, utrophin and associated glycoproteins.Hum Mol Genet. 2003; 12: 2467-2479Crossref PubMed Scopus (61) Google Scholar resulted in a faster and more efficient regeneration after freeze injuries.10Moghadaszadeh B Albrechtsen R Guo LT Zaik M Kawaguchi N Borup RH Kronqvist P Schroder HD Davies KE Voit T Nielsen FC Engvall E Wewer UM Compensation for dystrophin-deficiency: ADAM12 overexpression in skeletal muscle results in increased alpha 7 integrin, utrophin and associated glycoproteins.Hum Mol Genet. 2003; 12: 2467-2479Crossref PubMed Scopus (61) Google Scholar Overexpression of ADAM12 also induced adipogenesis in these TG mice,17Kawaguchi N Xu X Tajima R Kronqvist P Sundberg C Loechel F Albrechtsen R Wewer UM ADAM 12 protease induces adipogenesis in transgenic mice.Am J Pathol. 2002; 160: 1895-1903Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar in accordance with the ADAM12-null mouse study.9Kurisaki T Masuda A Sudo K Sakagami J Higashiyama S Matsuda Y Nagabukuro A Tsuji A Nabeshima Y Asano M Iwakura Y Sehara-Fujisawa A Phenotypic analysis of Meltrin alpha (ADAM12)-deficient mice: involvement of Meltrin alpha in adipogenesis and myogenesis.Mol Cell Biol. 2003; 23: 55-61Crossref PubMed Scopus (126) Google Scholar ADAM12 was shown to alleviate the pathology of the mdx mouse when overexpressed in this model for Duchenne muscular dystrophy.10Moghadaszadeh B Albrechtsen R Guo LT Zaik M Kawaguchi N Borup RH Kronqvist P Schroder HD Davies KE Voit T Nielsen FC Engvall E Wewer UM Compensation for dystrophin-deficiency: ADAM12 overexpression in skeletal muscle results in increased alpha 7 integrin, utrophin and associated glycoproteins.Hum Mol Genet. 2003; 12: 2467-2479Crossref PubMed Scopus (61) Google Scholar, 12Kronqvist P Kawaguchi N Albrechtsen R Xu X Schroder HD Moghadaszadeh B Nielsen FC Frohlich C Engvall E Wewer UM ADAM12 alleviates the skeletal muscle pathology in mdx dystrophic mice.Am J Pathol. 2002; 161: 1535-1540Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Analyses indicated that ADAM12 stabilized the sarcolemma through an up-regulation of utrophin thereby protecting the mdx fibers and thus modulating the effect of lack of dystrophin.12Kronqvist P Kawaguchi N Albrechtsen R Xu X Schroder HD Moghadaszadeh B Nielsen FC Frohlich C Engvall E Wewer UM ADAM12 alleviates the skeletal muscle pathology in mdx dystrophic mice.Am J Pathol. 2002; 161: 1535-1540Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar In accordance with this, the effect of ADAM12 on muscle damage seemed to be specific for the mdx phenotype, because overexpression of ADAM12 in the laminin α2-deficient (dy/dy) muscular dystrophic mice had no effect on the damaged muscle.18Guo LT Shelton GD Wewer UM Engvall E ADAM12 overexpression does not improve outcome in mice with laminin alpha2-deficient muscular dystrophy.Neuromuscul Disord. 2005; 15: 786-789Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar Based on the ability of ADAM12 to boost utrophin gene levels, ADAM12 was suggested as a candidate for therapy of Duchenne muscular dystrophy.7Engvall E Wewer UM The new frontier in muscular dystrophy research: booster genes.FASEB J. 2003; 17: 1579-1584Crossref PubMed Scopus (86) Google Scholar With the aim of further characterizing the therapeutic potential of ADAM12 in substitution or nonreplacement gene therapy in dystophinopathies, we decided to evaluate the long-term effect of ectopic ADAM12 expression. Analysis of skeletal muscle samples obtained from 1-year-old ADAM12+/mdx mice, however, revealed an aggravating effect of the transgene on the mdx muscle pathology, and investigations indicated a reduction of muscle stem cell mobilization response in these old mice. This prompted us to investigate skeletal muscle regeneration after major acute injury in ADAM12+ TG mice, with focus on satellite cell mobilization. The results point to ADAM12 as a negative regulator of the satellite cell response during regeneration promoting adipogenic and fibrogenic substitution of muscle tissue. ADAM12+ TG mice and age-matched normal littermate controls (LC), 10 to 12 weeks of age, were used for the knife injury experiment. The generation of TG mice expressing human ADAM12 under the control of the muscle creatine kinase promoter has been described previously.12Kronqvist P Kawaguchi N Albrechtsen R Xu X Schroder HD Moghadaszadeh B Nielsen FC Frohlich C Engvall E Wewer UM ADAM12 alleviates the skeletal muscle pathology in mdx dystrophic mice.Am J Pathol. 2002; 161: 1535-1540Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar ADAM12+/dystrophin− animals were obtained by mating homozygous female mdx mice [C57BL/10ScSn-Dmd(mdx); Jackson Laboratories, Bar Harbor, ME] with male ADAM12+ mice.12Kronqvist P Kawaguchi N Albrechtsen R Xu X Schroder HD Moghadaszadeh B Nielsen FC Frohlich C Engvall E Wewer UM ADAM12 alleviates the skeletal muscle pathology in mdx dystrophic mice.Am J Pathol. 2002; 161: 1535-1540Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar One-year-old individuals were used for analysis along with 1-year-old homozygous mdx mice and normal controls. All animal experiments were performed in accordance with Danish legislation on animal welfare and approved by the Danish Council for Supervision with Experimental Animals. ADAM12+ TG (n = 23) and LC mice (n = 24) (10 to 12 weeks of age) were anesthetized using Hypnorm/Dormicum (fentanyl:fluanisone/midazolam), and experimental regeneration of skeletal muscle was induced by a stab incision in both calf muscles (musculus triceps surae). The mice were sacrificed by cervical dislocation 0.25, 1, 2, 3, 4, 5, 7, 10, and 14 days after injury. The injured muscles were removed and processed for either histological analysis or RNA purification. Uninjured muscle tissue was obtained from three ADAM12+ mice and three control mice. For histological analysis, muscles were fixed in formalin and embedded in paraffin and for RNA analysis kept in RNAlater (Invitrogen, Tåstrup, Denmark) until further processing. From the 1-year-old mice (ADAM12+/mdx, mdx, and control) musculus quadriceps and musculus tibialis muscles were formalin-fixed and embedded in paraffin whereas musculus gastrocnemius and musculus tibialis muscles were snap frozen for cryosectioning. Total RNA from muscle samples kept in RNAlater was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. One μg of pooled total RNA was used to synthesize cDNA using the SuperscriptIII first-strand synthesis system (Invitrogen). The RNA samples from each genotype were pooled (10 μg of each sample) for each time point after surgery. Two μl of cDNA were used as template for each PCR experiment, and primers were added at a concentration of 5 pmol/20 μl each. Primer sequences used: Pax7 sense: 5′-GCTACCAGTACAGCCAGTATG-3′, Pax7 antisense: 5′-GTCACTAAGCATGGGTAGATG-3′, MyoD sense: 5′-GCCCGCGCTCCAACTGCTCTGAT-3′, MyoD antisense: 5′-CCTACGGTGGTGCGCCCTCTGC-3′, Myogenin sense: 5′-GGGCCCCTGGAAGAAAAG-3′; Myogenin antisense: 5′-AGGAGGCGCTGTGGGAGT-3′; NCAM sense: 5′-ATTGTCTGC- TCCTCGGTCCATT-3′; NCAM antisense: 5′-GAGGGTAGAGGAGTCGTCATCA-3′; GAPDH sense: 5′-AACGACCCCTTCATTGAC-3′, GAPDH antisense: 5′-TCCACGACATACTCAGCAC-3′, 18s rRNA sense: 5′-GTAACCCGTTGAACCCC- ATT-3′ and 18s rRNA antisense: 5′-CCATCCAATCGGTAGTGACG-3′. All PCR reactions were run as multiplex reactions with GAPDH. 18s rRNA was run as an additional control gene (not shown). Reaction conditions: initial denaturation 94°C for 5 minutes; denaturation: 94°C for 30 seconds; annealing: specific temperature for 30 seconds; extension: 72°C for 30 seconds; and final extension: 72°C for 7 minutes; Pax7: 56°C, 35 cycles; MyoD: touchdown 67 to 57°C, 35 cycles; Myogenin: 60°C, 35 cycles; NCAM: 57°C, 31 cycles; and 18s rRNA: 55°C, 30 cycles. Both a nontemplate (water) control and a reverse transcriptase control (−RNA) were run as negative controls for all primer pairs. Uninjured muscle was included in the analysis as normal background control. PCR products were visualized on a 2% agarose gel stained with ethidium bromide. DNA sequence analysis confirmed the specificity of the PCR reaction products. Each data point was quantified using the GelDoc 1000 and the software program Quantity One (Bio-Rad, Herlev, Denmark) and normalized to the abundance of GAPDH (glyceraldehyde-3-phophate-dehydrogenase) expressed as log2 ratio followed by calculation of fold expression compared with normal uninjured muscle.19Yan Z Choi S Liu X Zhang M Schageman JJ Lee SY Hart R Lin L Thurmond FA Williams RS Highly coordinated gene regulation in mouse skeletal muscle regeneration.J Biol Chem. 2003; 278: 8826-8836Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar Formalin-fixed and paraffin-embedded muscle tissue was cut in 4-μm sections in a longitudinal direction, to ensure inclusion of the zones of interest in the injured area, and stained with hematoxylin and eosin (H&E) for morphological analysis. A Sirius red staining was performed to detect fibrosis. For immunohistochemistry (IHC), antigen retrieval was done by microwave oven treatment, and sections were subsequently blocked to eliminate endogenous peroxidase and endogenous biotin. The following primary antibodies were used: monoclonal mouse-α-myogenin at 1:200 (F5D; DAKO, Glostrup, Denmark); monoclonal mouse-α-pax7 at 1:10 (Developmental Studies Hybridoma Bank, Iowa City, IA); polyclonal rabbit-α-chicken NCAM (neural cell adhesion molecule) at 1:2000 (AB5032; Chemicon International, Inc., Temecula, CA); rat-α-mouse ki67 at 1:50 (Tec3; DAKO), monoclonal mouse-α-dystrophin at 1:10 (MAB 1692; Chemicon, AH Diagnostics, Arhus, Denmark), polyclonal rat-α-mouse CD45 at 1:50 [30-F11 (Ly 5); BD Pharmingen, Brondby, Denmark], polyclonal rabbit-α-human myostatin 1:200 (Chemicon, AH Diagnostics), monoclonal mouse-α-human transforming growth factor (TGF)-β1 at 1:10 (NCL-TGFB; NovoCastra, Newcastle, UK), monoclonal mouse-α-human follistatin at 1:10 [85918 (MAB669); R&D Systems, TriChem ApS, Skanderborg, Denmark). For detection of mouse monoclonal antibodies, the ARK kit (DAKO) was used. The labeled streptavidin-biotin-system with goat-α-rat (E468 at 1:200; DAKO) and the Envision+ horseradish peroxidase system (K4003, DAKO) were used as detection systems for polyclonal antibodies. DAB+ was used as chromogen. Nuclei were counterstained using Mayer's hemalum with 4.5% chloral hydrate. Controls with exclusion of the primary antibodies were included in all experiments. Crysections of musculus tibialis (n = 6) and musculus gastrocnemius (n = 6) from ADAM12+/mdx and mdx mice were stained with monoclonal utrophin at 1:50 (NCL-DRP2; NovoCastra) and monoclonal β1D integrin at 1:25 [2B1 (MAB1900); Chemicon, AH Diagnostics] using an ARK-immunofluorescence protocol using a fluorescein isothiocyanate-labeled Tyramid (TSA kit, NEL701; Perkin-Elmer, Waltham, MA) procedure. Before staining, sections were blocked to eliminate endogenous biotin and peroxidase. Nuclei were detected with 4,6-diamidino-2-phenylindole mounting medium (Vectashield; Vector Laboratories, Burlingame, CA). The number of cells expressing a particular protein during the course of regeneration was estimated on sections by counting the number of positive nuclei (pax7, ki67, and myogenin) or nuclei surrounded by positive cytoplasm in mononuclear cells (NCAM). Morphometrics was performed using a Leica microscope equipped with a camera connected to a computer and a motorized cross board. By means of the CAST software (2000; Olympus Denmark A/S) systematic random fields for counting were selected. Fields with positive events (positive nuclei or stained cytoplasm) were accepted as lesion area and included. The number of nuclei in cells with positive events were compared with the total number of nuclei in the counted fields to give an estimate of the number of cells expressing the various proteins at the time points investigated. P values were calculated for the comparison of TG animals with LC animals using two-way factorial analysis of variance analysis with an α-value of 0.05. P values <0.05 were considered statistically significant. Having seen the beneficial effect of short-term ADAM12 expression on the mdx phenotype,12Kronqvist P Kawaguchi N Albrechtsen R Xu X Schroder HD Moghadaszadeh B Nielsen FC Frohlich C Engvall E Wewer UM ADAM12 alleviates the skeletal muscle pathology in mdx dystrophic mice.Am J Pathol. 2002; 161: 1535-1540Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar the next step in evaluating the therapeutic potential of ADAM12 was to study its long-term effects. For this purpose musculus quadriceps and musculus tibialis from 1-year-old ADAM12+/mdx mice, mdx mice, and normal mice was analyzed by IHC (Table 1). A Sirius staining performed on muscle in the 1-year-old ADAM12+/mdx mice to detect fibrosis (Figure 1, A–C) demonstrated that the ADAM12/mdx mice had more extensive fibrosis and more adipocytes, thus having a more dystrophic phenotype compared with the mdx mice. A recent study indicated a relationship between myostatin, fibrosis, and TGF-β1 in skeletal muscle.20Zhu J Li Y Shen W Qiao C Ambrosio F Lavasani M Nozaki M Branca MF Huard J Relationships between TGF-beta-1, myostatin, and decorin: implications for skeletal muscle fibrosis.J Biol Chem. 2007; 282: 25852-25863Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar Therefore we decided to evaluate the expression of myostatin, a known inhibitor of myogenesis,21Bogdanovich S Krag TO Barton ER Morris LD Whittemore LA Ahima RS Khurana TS Functional improvement of dystrophic muscle by myostatin blockade.Nature. 2002; 420: 418-421Crossref PubMed Scopus (725) Google Scholar TGF-β1, and follistatin, a myostatin inhibitor,20Zhu J Li Y Shen W Qiao C Ambrosio F Lavasani M Nozaki M Branca MF Huard J Relationships between TGF-beta-1, myostatin, and decorin: implications for skeletal muscle fibrosis.J Biol Chem. 2007; 282: 25852-25863Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar because these factors could participate as co-regulators in the fibrosis reaction observed. TGF-β1 (data not shown) and follistatin (data not shown) were both expressed in the connective tissue of ADAM12+/mdx and mdx mice. However, we did not observe any difference in expression of either TGF-β1 or follistatin in ADAM12+/mdx mice compared with mdx mice. Thus, expression of these two factors seems to have no apparent correlation to the ADAM12+/mdx phenotype observed in this study. However, in contrast to mdx mice, most of the myofibers in the ADAM12+/mdx mice expressed myostatin.Table 1Results from Immunohistochemical Stainings of Young (6 Weeks) and Old (1 Year) mdx and ADAM12+/mdx Mice Analyzed by Light Microscopy According to Number of Stained Cells/Nucleimdx, 6 weeks (n = 2)mdx, 1 year (n = 6)ADAM12+/mdx 6 weeks (n = 3)ADAM12+/mdx, 1 year (n = 6)ki67+++++CD45+++++++++pax7++++(+)myogenin+(+)/+++++NCAM++++++++myostatin++++++++Sirius+++++++Expression of the markers was analyzed according to a semiquantitative scale where zero protein expression was scored as −, weak expression as (+), modest expression as +, marked expression as ++, and pronounced expression as +++. Open table in a new tab Expression of the markers was analyzed according to a semiquantitative scale where zero protein expression was scored as −, weak expression as (+), modest expression as +, marked expression as ++, and pronounced expression as +++. Muscle in ADAM12+/mdx mice also showed more inflammation, seen as an increase in CD45+ve cells. The number of pax7+ve nuclei was the same as in control mice, indicating that the degeneration was not followed by satellite cell activation and proliferation in ADAM12+/mdx mice at this age. A few myogenin+ve nuclei could, however, be detected suggesting a slight regenerative activity. Compared with the mdx mice, ADAM12+/mdx mice had lower numbers of NCAM+ve mononuclear cells and ki67+ve proliferating cells, indicating a more active regeneration in the mdx mice than in ADAM12+/mdx mice. These results implied that long-term expression of ADAM12 in mdx dystrophic mice compromised muscle restoration seen as reduced stem cell function, increased inflammation, fibrosis, and adipogenesis. The mechanisms underlying this could be exhaustion of the stem cell population; however, the expression patterns seen, in particular the myostatin expression, could suggest that the ADAM12+/mdx mice had an overall suppressed regeneration. Membrane stabilization, rather than an increase in regeneration capacity, was proposed to be the mechanism behind early alleviation of the dystrophic phenotype in the ADAM12+/mdx mice.10Moghadaszadeh B Albrechtsen R Guo LT Zaik M Kawaguchi N Borup RH Kronqvist P Schroder HD Davies KE Voit T Nielsen FC Engvall E Wewer UM Compensation for dystrophin-deficiency: ADAM12 overexpression in skeletal muscle results in increased alpha 7 integrin, utrophin and associated glycoproteins.Hum Mol Genet. 2003; 12: 2467-2479Crossref PubMed Scopus (61) Google Scholar We therefore analyzed the expression of utrophin (Figure 2) and β1D integrin (data not shown) in the membranes and found that expression of utrophin seemed mildly elevated in the aged ADAM12+/mdx mice compared with age-matched controls. Expression of β1D integrin reflected the fibrosis reaction observed in the Sirius stain (data not shown). Thus a down-regulation of these compounds could not account for the late dystrophic phenotype. To re-evaluate the regenerative capacity, satellite cell activation and subsequent proliferation were analyzed in young ADAM12+/mdx and mdx mice with focus on the same markers used for the old mice (Table 1). In brief, we found few pax7+ve but many myogenin+ve nuclei in 6-week-old ADAM12+/mdx compared with age-matched mdx mice. Identical staining patterns were observed for NCAM and myostatin in fibers from both genotypes. Although the presence of many CD45+ve nuclei indicated pronounced inflammation in mdx mice, a Sirius staining revealed more fibrosis in the young ADAM12+/mdx mice than in the mdx mice. These results altogether suggested an altered regeneration in the young ADAM12+/mdx mice, too. To address the question about stem cell function in an ADAM12-enriched milieu, we studied the regenerative response after a stab lesion in 10- to 12-week-old ADAM12+ mice. Regeneration after freeze injury has previously been reported for ADAM12+ mice10Moghadaszadeh B Albrechtsen R Guo LT Zaik M Kawaguchi N Borup RH Kronqvist P Schroder HD Davies KE Voit T Nielsen FC Engvall E Wewer UM Compensation for dystrophin-deficiency: ADAM12 overexpression in skeletal muscle results in increased alpha 7 integrin, utrophin and associated glycoproteins.Hum Mol Genet. 2003; 12: 2467-2479Crossref PubMed Scopus (61) Google Scholar but not with specific focus on myogenic cell parameters. The knife cut injury resulted in a total loss of architecture of the injured muscle, but on day 5, the injured area in the control mice had regained most of its original structure (Figure 3A), and on day 10 (Figure 3B) the regeneration was almost complete in these animals. The only indication of a previous injury was the centrally located nuclei in the repaired fiber segments (Figure 3B). By contrast, no obvious regeneration had occurred in the ADAM12+ mice on day 5 (Figure 3C), and on day 10 their muscle structure was still incomplete, although some regenerative activity could be observed (Figure 3D). These findings strongly indicated an impaired regenerative capacity in the ADAM12+ mice compared with their LCs. To analyze th
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