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dy2J mouse with merosin deficient congenital muscular dystrophy treated with Glatiramer acetate
Neuromuscular Disorders 20 (2010) 267–272
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Improved muscle strength and mobility in the dy2J/dy2J mouse with merosin deficient congenital muscular dystrophy treated with Glatiramer acetate Oshrat Dadush a,1, Shlomit Aga-Mizrachi a,1, Keren Ettinger a, Rinat Tabakman a, Moran Elbaz a, Yakov Fellig b, Nurit Yanay a, Yoram Nevo a,* a b
Pediatric Neuromuscular Laboratory and Neuropediatric Unit, Hadassah – Hebrew University Medical Center, Mount Scopus, P.O.B. 24035, Jerusalem 91240, Israel Department of Pathology, Hadassah – Hebrew University Medical Center, Jerusalem, Israel
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Article history: Received 27 August 2009 Received in revised form 29 January 2010 Accepted 1 February 2010
Keywords: dy2J/dy2J mice Congenital muscular dystrophy Glatiramer acetate Regeneration Fibrosis Anti-inflammatory agent
a b s t r a c t The therapeutic effect of Glatiramer acetate, an immune modulating agent, was evaluated in the dy2J/dy2J mouse with merosin deficient congenital muscular dystrophy, which is a milder variant of the dy/dy mouse. The treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter and in motor performance quantified by video detection software. Glatiramer acetate treatment was associated with significantly increased expression of regeneration transcription factors MyoD and myogenin, and attenuation of the fibrosis markers vimentin and fibronectin. No effective treatment is currently available in congenital muscular dystrophy and Glatiramer acetate may present a new potential treatment for this disorder. Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction Congenital muscular dystrophy with merosin deficiency (MDC1A, OMIM # 607855) is an autosomal recessive disorder caused by mutations in the LAMA2 gene on chromosome 6q22– q23 encoding the a2 chain of the laminin-2 protein. Laminin-2, a heterotrimer comprised of a2, b1 and c1 chains, is mainly expressed in the basal lamina of skeletal and cardiac muscle cells, trophoblasts and peripheral nerve Schwann cells. Partial or complete deficiency of laminin-2 impairs linking of the muscle cell outer membrane to extra cellular components [1,2]. Children affected with MDC1A suffer from severe early onset muscle weakness with significant motor impairment. They often do not achieve independent ambulation and die in childhood or early adulthood [3,4]. Skeletal muscle pathology shows dystrophic changes with replacement of the normal muscle by fibrous tissue and fat [5]. The Lama2dy-2J (dy2J/dy2J) is a mouse model for MDC1A. It has a spontaneous G to A mutation in the donor splice site of exon 2 of the mouse LAMA2 gene which results in exon skipping. This abnormal splicing causes a deletion without shifting of the reading frame in domain VI of the a2-polypeptide leading to a truncated and par* Corresponding author. Tel.: +972 2 5844751; fax: +972 2 532 8963. E-mail address: [email protected] (Y. Nevo). 1 Equal contribution to this study. 0960-8966/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2010.02.002
tially functional a2 chain of the laminin-2 protein [6–8]. Histopathological analysis reveals progressive dystrophic muscle changes including muscle fiber necrosis, regeneration, and fibrosis [7,9]. The clinical course of the homozygous dy2J/dy2J mouse involves early onset progressive muscle weakness and motor deterioration; however, it is less severe than the allelic form (dy/dy). Comparable to merosin deficient congenital muscular dystrophy children, dy2J/dy2J mice demonstrate a peripheral neuropathy in addition to the muscular dystrophy [2,10,11] . Inflammation plays an important role following muscle degeneration by removing tissue debris and preparing for regeneration. Invasion of inflammatory cells including macrophages, CD4+ and CD8+ T-cells, and occasionally eosinophils accompany muscular dystrophy [12–14]. However, abundant activation of inflammatory pathways has been shown to be involved in enhancement of muscle fibrosis, thus aggravating pathology [12,15–17]. Anti-inflammatory and anti-fibrotic agents have been suggested as potential therapies in these disorders [18–21]. Glatiramer acetate (GA) is a commercially available drug for the treatment of multiple sclerosis that slows the progression of disability and reduces relapse rate with a very high safety profile. It is composed of the amino acids L-alanine, L-lysine, L-glutamic acid, and L-tyrosine in a molar ratio of 4.2:3.4:1.4:1.0 [22]. GA was initially designed to simulate myelin basic protein, but is also involved in immune modulation in different pathways inside and outside the nervous system [23–26].
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The aim of the current study was to evaluate the therapeutic effect of GA, an immunomodulating and anti-fibrotic agent, in the dy2J/dy2J mouse model of MDC1A. 2. Materials and methods 2.1. Mice C57BL/6 J Lama2dy-2J (dy2J/dy2J) heterozygote mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and were bred at the Hebrew University SPF animal housing facility. The experiments were approved by the Hebrew University Animal Care and Use Committee. Mice were maintained under standard conditions, 23 ± 1 °C, 12 h light cycle (7 a.m.–7 p.m.) with ad libitum access to food and drink. No animal had paralysis with inability to reach food or water or dramatic weight loss (more than 10% weight loss between two weighings, or 20% or more from initial weight) or any other severe stress signs requiring withdrawal from the study. No other side effects were noted in the treatment group. Delineation between the dy2J/dy2J affected mice, heterozygous for the LAMA2 gene mutation and wild type C57BL/6 J (WT) mice was detected by PCR reaction with the following primers: forward 50 -TCCTGCTGTCCTGAATCTTG30 and reverse 50 -CTCTATTACTGAACTTTGGATG-30 . The digestion of the PCR products with the NdeI restriction enzyme (recognition sequence: CATATG) resulted in characteristic product size for each of the mice genotypes [7]. Following preliminary studies in our laboratory, WT and dy2J/dy2J mice were injected intra-peritoneally three times a week with 200 lg GA (Copaxone, Teva Pharmaceutical Industries, Petah–Tikva, Israel) or with saline as control, for 12 weeks from the age of 6 weeks (n = 5/group, each group consisted of 3 males and 2 female mice). At the end of the study the mice were sacrificed with ketamine-xylazine. Both hind limb muscles were dissected. Part of the muscle sample was frozen in liquid nitrogen and stored at 80 °C for biochemical analysis. Quadriceps femoris muscle was rapidly frozen in isopentane pre-chilled by liquid nitrogen for cryostat sections and histology. 2.2. Muscle strength Total peak force (in gram force /gram body weight) was determined once a week using an electronic Grip Strength Meter, Columbus Instruments (Columbus, OH, USA). Each week muscle strength measurements of both fore and hind limbs were performed according to Tanase et al. [27] with five measurements done on each fore and hind limb from each animal. The three highest measurements were averaged to give the strength score. The mice were allowed to rest for 10 min between fore and hind limb measurements. All measurements were performed by the same examiner.
cocktail of protease and phosphatase inhibitors (Sigma–Aldrich, St. Louis, MO, USA). The preparations were incubated for 30 min on ice and centrifuged at 17,400g for 15 min at 4 °C. Forty microgram of total protein were electrophoretically separated on 7.5% or 10% SDS–PAGE gels followed by transfer to an Immobilon-P membrane (Millipore, Bedford, USA). Immunoblotting was performed using anti-myogenin (1:500; Santa Cruz, CA, USA), anti-MyoD (1:500; Santa-Cruz), anti-fibronectin (1:1000; Sigma–Aldrich) and anti-vimentin (1:1000; Sigma–Aldrich) antibodies according to standard procedures [30]. Equal protein loading of blots was confirmed by immunoblotting of a-actin (1:5000; Sigma–Aldrich) or GAPDH (1:1000; Santa Cruz). Densitometry of the bands was obtained by ‘‘ImageJ” software. For each protein (MyoD, myogenin, fibronectin and vimentin) 100% was defined as the densitometry of this protein in the WT mice. 2.5. Statistical analysis All data are expressed as mean ± standard error of the mean (SEM) and all statistical analysis was completed in SPSS (SPSS 15.0 for windows). Statistical analysis for direct comparison between two groups was performed by unpaired Student’s t-test and non parametric Mann–Whitney test. Multiple comparisons between groups were made using repeated-measures ANOVA. Significance was set at p < 0.05 for all comparisons. 3. Results 3.1. Muscle strength Significant difference in hind limb muscle strength was detected between the untreated dy2J/dy2J and the WT mice throughout the study (p < 0.01; Fig. 1). A significant increase in hind limb muscle strength was noted in the GA treated compared to the untreated dy2J/dy2J mice (p < 0.01). During the study period muscle strength increased from 1.91 ± 0.09 to 2.9 ± 0.34 (gram force /gram body weight) in the dy2J/dy2J treated mice, while decreased from 1.84 ± 0.06 to 1.32 ± 0.05 in the untreated dy2J/dy2J group. However, following treatment muscle strength was not completely normalized in the dy2J/dy2J compared to the WT mice (2.9 ± 0.34 vs. 3.63 ± 0.42; p < 0.01). Significant difference in fore (stronger) limb muscle strength was detected between the untreated dy2J/dy2J and the WT mice throughout the study (p < 0.01; data not shown). No difference was detected in the fore limbs between the treated
2.3. Mobility At the end of the study the mice were video recorded for 10 min sessions and analyzed by the Ethovision XT system (version 5, Noldus Information Technology, Wageningen, The Netherlands) [28,29]. The test arena was white, 27 cm wide, 48 cm long and 25 cm high. To reduce body movements which were not associated with mobility, only recordings which resulted in movement of at least 0.5 cm in 0.2 s were included in the calculated data. Total distance and mean velocity as well as maximal distance and velocity in 0.2 s were calculated using the software. 2.4. Western blot analysis Muscles were homogenized on ice in NP-40 buffer [1% Nonidet P-40, 150 mM NaCl and 50 mM Tris–HCl, (pH = 8)] containing a
Fig. 1. Hind limb muscle strength in WT and dy2J/dy2J treated and untreated groups. The data of five mice in each group is expressed as mean ± SEM. ANOVA test for repeated measures showed significant difference in strength in the hind limbs (p < 0.01) between treated and untreated dy2J/dy2J.
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and untreated dy2J/dy2J mice (data not shown), and in the fore and hind limbs of the treated and untreated WT groups. 3.2. Mobility Following 12 weeks of treatment the mice were video recorded and their mobility parameters analyzed by the Ethovision system. Significant differences were found in all following four parameters; total and maximal distance as well as mean and maximal velocity, between the untreated dy2J/dy2J and WT groups (p < 0.001). Total (339.56 ± 128.18 vs. 1535.27 ± 208.14 cm; Fig. 2A) and maximal (1.88 ± 0.17 vs. 4.94 ± 0.3 cm; Fig. 2B) distances were shorter in the dy2J/dy2J mice. Mean (3.95 ± 0.28 vs. 7 ± 0.23 cm/s; Fig. 2C) and maximal (9.43 ± 0.85 vs. 24.75 ± 1.52 cm/s; Fig. 2D) velocities were slower in the untreated dy2J/dy2J compared to the WT mice. Significant improvement in these four parameters was noted in the GA treated compared to untreated dy2J/dy2J mice. Total (898.33 ± 199.41 vs. 339.56 ± 128.18 cm; p = 0.05; Fig. 2A) and maximal (3.07 ± 0.34 vs. 1.88 ± 0.19 cm; p < 0.02; Fig. 2B) distances were longer in the treated group. Mean (5.20 ± 0.32 vs. 3.95 ± 0.28 cm/s; p < 0.03; Fig. 2C) and maximal (15.34 ± 1.59 vs. 9.43 ± 0.85 cm/s; p < 0.02; Fig. 2D) velocities were faster in the treated compared to the untreated dy2J/dy2J mice. Following GA treatment the total distance parameter of the dy2J/dy2J mice was no longer significantly different compared to treated WT mice (898.33 ± 199.41 vs. 1096 ± 196.69 cm; p = 0.50). However, each of the other three mobility parameters; maximal distance, mean and maximal velocity was still significantly sub-optimal in the treated dy2J/dy2J compared to the treated WT group (p < 0.01) (maximal distance; 3.07 ± 0.34 vs. 4.82 ± 0.26 cm, mean velocity; 5.20 ± 0.32 vs. 6.46 ± 0.2 cm/s and maximal velocity; 15.34 ± 1.59 vs. 24.12 ± 1.32 cm/s). There was no difference in these four parameters between treated and untreated WT groups. 3.3. Regeneration and fibrosis markers Immunoblotting of skeletal muscle was carried out at the end of the study using anti-myogenin and anti-MyoD as regeneration markers. Treated and untreated WT mice showed a similar level of MyoD and myogenin expression (Fig. 3). Untreated dy2J/dy2J mice
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showed significantly lower amounts of MyoD and myogenin compared to WT groups (MyoD: 31.25 ± 5.66; p < 0.05 myogenin: 59.88 ± 13.6; p < 0.05 [densitometry, arbitrary units]). GA treatment of dy2J/dy2J mice was associated with a significantly increased MyoD and myogenin expression (MyoD: 89.06 ± 16.94; p < 0.05 myogenin: 101.29 ± 12.93; p < 0.05). Following GA treatment there was no longer a significant difference in the expression of these two regeneration markers between the treated WT and dy2J/dy2J groups. Fibronectin and vimentin were used as fibrosis markers. Treated and untreated WT mice showed very low expression of fibronectin and vimentin (Fig. 4). In contrast, untreated dy2J/dy2J mice showed increased expression of fibronectin and vimentin which were significantly attenuated in the GA treated dy2J/dy2J group (Fibronectin: 2906.16 ± 999.36 vs. 411.76 ± 114.75; p < 0.05. Vimentin: 609.16 ± 46.52 vs. 111.28 ± 13.3; p < 0.001). Following treatment there was no longer a significant difference in expression of these two fibrosis markers between the treated WT and dy2J/dy2J groups. General histology of muscle biopsies obtained at the end of the study showed patchy and variable fibrosis in different areas even within the same muscle and quantitative collagen measurement by the Sircol Collagen Assay did not show a significant decrease in total collagen I–IV content in treated dy2J/dy2J mice (data not shown). 4. Discussion The dy2J/dy2J is a spontaneous mouse model of merosin deficient congenital muscular dystrophy (MDC1A) [6,31]. This milder variant of the dy/dy mouse is a useful model to study pharmacological intervention on mouse strength and mobility. At the age of 6 weeks dy2J/dy2J mice were found to be significantly weaker than the C57BL/6 J wild type mice. In the ensuing 12 weeks muscle strength further decreased in the dy2J/dy2J while increasing in the wild type. In the present study, a beneficial clinical effect of GA on muscle strength and mobility was shown in the dy2J/dy2J mouse. These mice have predominantly hind limb muscle weakness initially. The treated mice showed significant improvement in hind limb muscle strength compared to the untreated group as measured by muscle strength meter. In addition, the treated mice showed significant improvement in motor performance which was obvious
Fig. 2. The effect of GA on mouse mobility. Data is expressed as mean ± SEM of 5 WT; 4 WT + GA; 4 dy2J/dy2J; 5 dy2J/dy2J + GA mice. Student’s t-test and non parametric Mann– Whitney test showed significant difference in total distance (A; *p = 0.05), maximal distance (B; **p < 0.02), mean velocity (C; ***p < 0.03) and maximal velocity (D; **p < 0.02), between the treated and untreated dy2J/dy2J.
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Fig. 3. The effect of GA on muscle regeneration markers. Western blot analysis gels and densitometry graphs of regeneration markers’ expression in the WT and dy2J/dy2J mice. MyoD (A) and myogenin (B) levels were higher in the GA treated compared to the untreated dy2J/dy2J mice. GAPDH and a-actin were used as controls. These results represent 3 independent experiments. Graphs of densitometry measurements are normalized to the protein controls. Each bar represents the mean ± SEM of measurements of four mice (*p < 0.05; Student’s t-test).
Fig. 4. The effect of GA on muscle fibrosis markers. Western blot analysis gels and densitometry graphs of fibrosis markers’ expression in the WT and dy2J/dy2J mice. Fibronectin (A) and vimentin (B) levels markedly decreased in the treated compared to the untreated dy2J/dy2J mice. GAPDH was used as controls. These results represent three independent experiments. Densitometry measurements are normalized to GAPDH. Each bar represents the mean ± SEM of measurements of four mice (*p < 0.05, **p < 0.01; Student’s t-test).
clinically and quantified by the Ethovision video detection software. Both velocity and distance parameters indicated significant improvement in mobility for the treated group. However, both strength and mobility parameters were not completely ‘‘normalized” following treatment and were still sub-optimal in the treated dy2J/dy2J compared to the WT group. The mechanism of the effect of GA in the dy2J/dy2J mouse is unknown. GA effect in this model may be through suppression of inflammatory processes, anti-fibrotic effect, or enhanced regeneration, improving the muscular dystrophy. A neuroprotective effect on the peripheral neuropathy should also be considered. Inflammatory processes have been shown to play a major role in promoting pathology in muscular dystrophy with various inflammatory cell types accompanying the muscle fiber degeneration [12]. In the mdx mouse model of Duchenne muscular dystrophy, T-cells are reported to contribute significantly to skeletal muscle death and progressive fibrogenesis during repeated cycles
of muscle degeneration [12,32]. Macrophage depletion at the peak of the pathology prevents most of the muscle membrane lysis [33], while transplantation of normal thymic tissue into mdx-nu/nu mice replenishes muscle collagen deposition [32]. GA exerts anti-inflammatory activity by modulating the immune system, suppressing pro-inflammatory, and enhancing anti-inflammatory cytokines [34–36]. Induction of GA-reactive Th2 cells by shifting the immune response from Th1 to Th2 is thought to be the main therapeutic mechanism of action for GA [25,37,38]. Additional immunomodulatory activities include effects on antigen presenting cells and secretion of anti-inflammatory cytokines [39]. GA activity is not limited to the central nervous system. In the skin transplantation system, GA significantly prevents skin graft rejection, and in bone marrow transplantation GA alleviates immune rejection and drastically reduces cytotoxic activity toward host targets [22,40]. In future studies evaluation of pro and anti-inflammatory cytokines following GA
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treatment will aid determination of the importance of this mechanism in the dy2J/dy2J mouse. MDC1A is characterized by failure of regeneration and striking proliferation of the connective tissue associated with progressive loss of muscle function [4]. GA has an anti-fibrotic effect which has been shown in the animal model of liver cirrhosis [41]. In muscle the anti-fibrotic effect of GA may be either synergistic or secondary to anti-inflammatory activity [12,15–17]. In this study, we found significant suppression of the increased fibrosis markers fibronectin and vimentin in the GA treated dy2J/dy2J group. Fibronectin is a major extra-cellular matrix glycoprotein essential for types I and III collagen assembly [42,43]. It is up-regulated in dystrophic conditions [44]. Vimentin is an intermediate filament protein detected in fibroblasts and also expressed in myoblasts. It gradually disappears with their maturation into myotubes [45]. Our results of vimentin up-regulation in the dy2J/dy2J mouse are consistent with previous reports of its up-regulation in mdx mice diaphragm, and in DMD and MDC1A human patients [46,47]. However, even though both fibrosis markers decreased in treated dy2J/dy2J, the abundant fibrosis on muscle biopsy and quantitative muscle collagen measurement was not significantly affected by GA. In the same experimental paradigm other anti-fibrotic agents showed a more pronounced decrease in fibrosis and collagen content in dy2J/dy2J mice (Nevo Y, unpublished data). The mechanism of fibrosis in MDC1A is not fully understood. Transforming growth factor beta (TGF-b) is a key mediator of fibrosis and tissue remodeling [48]. TGF-b1 transcript levels are shown to be greatly increased in muscle from dystrophin and LAMA2 mutated muscular dystrophy patients [47,49,50]. The intracellular signaling of TGF-b is mediated via smad proteins [49]. Smad3 has a role in transcriptional regulation of type I collagen gene expression in the development of fibrosis [51,52]. The fibrotic process involves an imbalance of matrix metalloproteinases (MMPs) and their specific tissue inhibitors (TIMPs) [48]. MMP 9 directly activates latent TGF-b, and in turn TGF-b induces MMPs up-regulation of MMPs 2 and 9 [53–55]. Previous in vitro studies showed increased secretion of TGF-b following GA treatment [34]. Modification of contradicting pathways including GA induced TGF-b pro-fibrotic activity may explain the lack of improvement in muscle fibrosis in this study even though fibrotic markers were attenuated. If this is the case, then the combined effect of GA and TGF-b inhibitors may potentially further improve the clinical and histopathological outcome. Significant up-regulation of muscle regeneration transcription factors in parallel with suppression of fibrosis markers following treatment were found in this study. During muscle regeneration the satellite cells undergo self-renewal and proliferate to produce myoblasts that fuse into newly regenerated myofibers. MyoD and myogenin are transcription factors in skeletal myogenesis considered as regeneration markers. MyoD is up-regulated early during activation of the satellite cell population and is required for differentiation of activated myogenic progenitor cells into myoblasts. Later, up-regulation of myogenin induces differentiation of myoblasts into myotubes and myocytes [56–58]. Enhanced expression of MyoD and myogenin were detected at the age of 18 weeks in the treated dy2J/dy2J compared to the untreated group. At this age significant motor impairment is noted in these mice. These preliminary findings suggest that regeneration potential is still available at advanced stages of the disease. These results are in concordance with the Cohn et al. study which showed that inhibition of TGF-b pathways in the mdx mouse was associated with both attenuation of fibrosis and up-regulation of muscle regeneration [18]. A neuroprotective effect of GA should also be considered. In multiple sclerosis the GA mechanism of action is postulated to include neuroprotection in addition to immune modulation [59]. Neuroprotective effect on damaged axons and neuronal cells is
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mediated by modulation of neurotrophic factors such as brain-derived neurotrophic factor (BDNF) secreted by GA-reactive T-cell lines [39]. Congenital muscular dystrophy with merosin deficiency is associated with central and peripheral nervous system dysmyelination. Both children and dy2J/dy2J mice have peripheral neuropathy in addition to progressive muscular dystrophy [2,10]. As neuroprotection and prevention of central nervous system damage may be a primary or secondary component of the beneficial effect of GA in multiple sclerosis, GA also may possibly have a favorable impact on peripheral nerve dysmyelination. This hypothesis was not tested in the current study. In summary, in this study a beneficial effect of GA on muscle strength and mobility was detected in the dy2J/dy2J mouse model of congenital muscular dystrophy. GA is associated with a low side effect profile in adults. However, towards clinical application, GA safety in children, its effect in other animal models of muscular dystrophy and the mechanism of action of GA in muscle should be further explored. Acknowledgments The authors thank Danny Sason for assistance in the Ethovision mobility data analysis, S. Miterani-Rosenbaum and O. El-Peleg for helpful comments and M. Rabie for assistance in English editing of the manuscript. Following the results of this study Hadassit, Hadassah hospital implementation office issued an international PCT patent application No. PCT/IL2008/001289 on GA treatment in muscular dystrophy. References [1] Lisi MT, Cohn RD. Congenital muscular dystrophies: new aspects of an expanding group of disorders. Biochim Biophys Acta 2007;1772:159–72. [2] Miyagoe-Suzuki Y, Nakagawa M, Takeda S. Merosin and congenital muscular dystrophy. Microsc Res Tech 2000;48:181–91. [3] Muntoni F, Voit T. The congenital muscular dystrophies in 2004: a century of exciting progress. Neuromuscul Disord 2004;14:635–49. [4] Jimenez-Mallebrera C, Brown SC, Sewry CA, Muntoni F. Congenital muscular dystrophy: molecular and cellular aspects. Cell Mol Life Sci 2005;62:809–23. [5] Talim B, Kale G, Topaloglu H, et al. Clinical and histopathological study of merosin-deficient and merosin-positive congenital muscular dystrophy. Pediatr Dev Pathol 2000;3:168–76. [6] Xu H, Wu XR, Wewer UM, Engvall E. Murine muscular dystrophy caused by a mutation in the laminin alpha 2 (Lama2) gene. Nat Genet 1994;8:297–302. [7] Vilquin JT, Vignier N, Tremblay JP, Engvall E, Schwartz K, Fiszman M. Identification of homozygous and heterozygous dy2J mice by PCR. Neuromuscul Disord 2000;10:59–62. [8] Colognato H, Yurchenco PD. The laminin alpha2 expressed by dystrophic dy(2J) mice is defective in its ability to form polymers. Curr Biol 1999;9:1327–30. [9] Vainzof M, Ayub-Guerrieri D, Onofre PC, et al. Animal models for genetic neuromuscular diseases. J Mol Neurosci 2008;34:241–8. [10] Shorer Z, Philpot J, Muntoni F, Sewry C, Dubowitz V. Demyelinating peripheral neuropathy in merosin-deficient congenital muscular dystrophy. J Child Neurol 1995;10:472–5. [11] Schessl J, Zou Y, Bonnemann CG. Congenital muscular dystrophies and the extracellular matrix. Semin Pediatr Neurol 2006;13:80–9. [12] Spencer MJ, Tidball JG. Do immune cells promote the pathology of dystrophindeficient myopathies? Neuromuscul Disord 2001;11:556–64. [13] Morrison J, Palmer DB, Cobbold S, Partridge T, Bou-Gharios G. Effects of Tlymphocyte depletion on muscle fibrosis in the mdx mouse. Am J Pathol 2005;166:1701–10. [14] Tidball JG. Inflammatory processes in muscle injury and repair. Am J Physiol Regul Integr Comp Physiol 2005;288:R345–53. [15] Farini A, Meregalli M, Belicchi M, et al. T and B lymphocyte depletion has a marked effect on the fibrosis of dystrophic skeletal muscles in the scid/mdx mouse. J Pathol 2007;213:229–38. [16] Vidal B, Serrano AL, Tjwa M, et al. Fibrinogen drives dystrophic muscle fibrosis via a TGF-b/alternative macrophage activation pathway. Genes Dev 2008;22:1747–52. [17] Butterfield TA, Best TM, Merrick MA. The dual roles of neutrophils and macrophages in inflammation: a critical balance between tissue damage and repair. J Athl Train 2006;41:457–65. [18] Cohn RD, van Erp C, Habashi JP, et al. Angiotensin II type 1 receptor blockade attenuates TGF-b-induced failure of muscle regeneration in multiple myopathic states. Nat Med 2007;13:204–10.
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Neuromuscular Disorders journal homepage: www.elsevier.com/locate/nmd
Improved muscle strength and mobility in the dy2J/dy2J mouse with merosin deficient congenital muscular dystrophy treated with Glatiramer acetate Oshrat Dadush a,1, Shlomit Aga-Mizrachi a,1, Keren Ettinger a, Rinat Tabakman a, Moran Elbaz a, Yakov Fellig b, Nurit Yanay a, Yoram Nevo a,* a b
Pediatric Neuromuscular Laboratory and Neuropediatric Unit, Hadassah – Hebrew University Medical Center, Mount Scopus, P.O.B. 24035, Jerusalem 91240, Israel Department of Pathology, Hadassah – Hebrew University Medical Center, Jerusalem, Israel
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Article history: Received 27 August 2009 Received in revised form 29 January 2010 Accepted 1 February 2010
Keywords: dy2J/dy2J mice Congenital muscular dystrophy Glatiramer acetate Regeneration Fibrosis Anti-inflammatory agent
a b s t r a c t The therapeutic effect of Glatiramer acetate, an immune modulating agent, was evaluated in the dy2J/dy2J mouse with merosin deficient congenital muscular dystrophy, which is a milder variant of the dy/dy mouse. The treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter and in motor performance quantified by video detection software. Glatiramer acetate treatment was associated with significantly increased expression of regeneration transcription factors MyoD and myogenin, and attenuation of the fibrosis markers vimentin and fibronectin. No effective treatment is currently available in congenital muscular dystrophy and Glatiramer acetate may present a new potential treatment for this disorder. Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction Congenital muscular dystrophy with merosin deficiency (MDC1A, OMIM # 607855) is an autosomal recessive disorder caused by mutations in the LAMA2 gene on chromosome 6q22– q23 encoding the a2 chain of the laminin-2 protein. Laminin-2, a heterotrimer comprised of a2, b1 and c1 chains, is mainly expressed in the basal lamina of skeletal and cardiac muscle cells, trophoblasts and peripheral nerve Schwann cells. Partial or complete deficiency of laminin-2 impairs linking of the muscle cell outer membrane to extra cellular components [1,2]. Children affected with MDC1A suffer from severe early onset muscle weakness with significant motor impairment. They often do not achieve independent ambulation and die in childhood or early adulthood [3,4]. Skeletal muscle pathology shows dystrophic changes with replacement of the normal muscle by fibrous tissue and fat [5]. The Lama2dy-2J (dy2J/dy2J) is a mouse model for MDC1A. It has a spontaneous G to A mutation in the donor splice site of exon 2 of the mouse LAMA2 gene which results in exon skipping. This abnormal splicing causes a deletion without shifting of the reading frame in domain VI of the a2-polypeptide leading to a truncated and par* Corresponding author. Tel.: +972 2 5844751; fax: +972 2 532 8963. E-mail address: [email protected] (Y. Nevo). 1 Equal contribution to this study. 0960-8966/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2010.02.002
tially functional a2 chain of the laminin-2 protein [6–8]. Histopathological analysis reveals progressive dystrophic muscle changes including muscle fiber necrosis, regeneration, and fibrosis [7,9]. The clinical course of the homozygous dy2J/dy2J mouse involves early onset progressive muscle weakness and motor deterioration; however, it is less severe than the allelic form (dy/dy). Comparable to merosin deficient congenital muscular dystrophy children, dy2J/dy2J mice demonstrate a peripheral neuropathy in addition to the muscular dystrophy [2,10,11] . Inflammation plays an important role following muscle degeneration by removing tissue debris and preparing for regeneration. Invasion of inflammatory cells including macrophages, CD4+ and CD8+ T-cells, and occasionally eosinophils accompany muscular dystrophy [12–14]. However, abundant activation of inflammatory pathways has been shown to be involved in enhancement of muscle fibrosis, thus aggravating pathology [12,15–17]. Anti-inflammatory and anti-fibrotic agents have been suggested as potential therapies in these disorders [18–21]. Glatiramer acetate (GA) is a commercially available drug for the treatment of multiple sclerosis that slows the progression of disability and reduces relapse rate with a very high safety profile. It is composed of the amino acids L-alanine, L-lysine, L-glutamic acid, and L-tyrosine in a molar ratio of 4.2:3.4:1.4:1.0 [22]. GA was initially designed to simulate myelin basic protein, but is also involved in immune modulation in different pathways inside and outside the nervous system [23–26].
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The aim of the current study was to evaluate the therapeutic effect of GA, an immunomodulating and anti-fibrotic agent, in the dy2J/dy2J mouse model of MDC1A. 2. Materials and methods 2.1. Mice C57BL/6 J Lama2dy-2J (dy2J/dy2J) heterozygote mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and were bred at the Hebrew University SPF animal housing facility. The experiments were approved by the Hebrew University Animal Care and Use Committee. Mice were maintained under standard conditions, 23 ± 1 °C, 12 h light cycle (7 a.m.–7 p.m.) with ad libitum access to food and drink. No animal had paralysis with inability to reach food or water or dramatic weight loss (more than 10% weight loss between two weighings, or 20% or more from initial weight) or any other severe stress signs requiring withdrawal from the study. No other side effects were noted in the treatment group. Delineation between the dy2J/dy2J affected mice, heterozygous for the LAMA2 gene mutation and wild type C57BL/6 J (WT) mice was detected by PCR reaction with the following primers: forward 50 -TCCTGCTGTCCTGAATCTTG30 and reverse 50 -CTCTATTACTGAACTTTGGATG-30 . The digestion of the PCR products with the NdeI restriction enzyme (recognition sequence: CATATG) resulted in characteristic product size for each of the mice genotypes [7]. Following preliminary studies in our laboratory, WT and dy2J/dy2J mice were injected intra-peritoneally three times a week with 200 lg GA (Copaxone, Teva Pharmaceutical Industries, Petah–Tikva, Israel) or with saline as control, for 12 weeks from the age of 6 weeks (n = 5/group, each group consisted of 3 males and 2 female mice). At the end of the study the mice were sacrificed with ketamine-xylazine. Both hind limb muscles were dissected. Part of the muscle sample was frozen in liquid nitrogen and stored at 80 °C for biochemical analysis. Quadriceps femoris muscle was rapidly frozen in isopentane pre-chilled by liquid nitrogen for cryostat sections and histology. 2.2. Muscle strength Total peak force (in gram force /gram body weight) was determined once a week using an electronic Grip Strength Meter, Columbus Instruments (Columbus, OH, USA). Each week muscle strength measurements of both fore and hind limbs were performed according to Tanase et al. [27] with five measurements done on each fore and hind limb from each animal. The three highest measurements were averaged to give the strength score. The mice were allowed to rest for 10 min between fore and hind limb measurements. All measurements were performed by the same examiner.
cocktail of protease and phosphatase inhibitors (Sigma–Aldrich, St. Louis, MO, USA). The preparations were incubated for 30 min on ice and centrifuged at 17,400g for 15 min at 4 °C. Forty microgram of total protein were electrophoretically separated on 7.5% or 10% SDS–PAGE gels followed by transfer to an Immobilon-P membrane (Millipore, Bedford, USA). Immunoblotting was performed using anti-myogenin (1:500; Santa Cruz, CA, USA), anti-MyoD (1:500; Santa-Cruz), anti-fibronectin (1:1000; Sigma–Aldrich) and anti-vimentin (1:1000; Sigma–Aldrich) antibodies according to standard procedures [30]. Equal protein loading of blots was confirmed by immunoblotting of a-actin (1:5000; Sigma–Aldrich) or GAPDH (1:1000; Santa Cruz). Densitometry of the bands was obtained by ‘‘ImageJ” software. For each protein (MyoD, myogenin, fibronectin and vimentin) 100% was defined as the densitometry of this protein in the WT mice. 2.5. Statistical analysis All data are expressed as mean ± standard error of the mean (SEM) and all statistical analysis was completed in SPSS (SPSS 15.0 for windows). Statistical analysis for direct comparison between two groups was performed by unpaired Student’s t-test and non parametric Mann–Whitney test. Multiple comparisons between groups were made using repeated-measures ANOVA. Significance was set at p < 0.05 for all comparisons. 3. Results 3.1. Muscle strength Significant difference in hind limb muscle strength was detected between the untreated dy2J/dy2J and the WT mice throughout the study (p < 0.01; Fig. 1). A significant increase in hind limb muscle strength was noted in the GA treated compared to the untreated dy2J/dy2J mice (p < 0.01). During the study period muscle strength increased from 1.91 ± 0.09 to 2.9 ± 0.34 (gram force /gram body weight) in the dy2J/dy2J treated mice, while decreased from 1.84 ± 0.06 to 1.32 ± 0.05 in the untreated dy2J/dy2J group. However, following treatment muscle strength was not completely normalized in the dy2J/dy2J compared to the WT mice (2.9 ± 0.34 vs. 3.63 ± 0.42; p < 0.01). Significant difference in fore (stronger) limb muscle strength was detected between the untreated dy2J/dy2J and the WT mice throughout the study (p < 0.01; data not shown). No difference was detected in the fore limbs between the treated
2.3. Mobility At the end of the study the mice were video recorded for 10 min sessions and analyzed by the Ethovision XT system (version 5, Noldus Information Technology, Wageningen, The Netherlands) [28,29]. The test arena was white, 27 cm wide, 48 cm long and 25 cm high. To reduce body movements which were not associated with mobility, only recordings which resulted in movement of at least 0.5 cm in 0.2 s were included in the calculated data. Total distance and mean velocity as well as maximal distance and velocity in 0.2 s were calculated using the software. 2.4. Western blot analysis Muscles were homogenized on ice in NP-40 buffer [1% Nonidet P-40, 150 mM NaCl and 50 mM Tris–HCl, (pH = 8)] containing a
Fig. 1. Hind limb muscle strength in WT and dy2J/dy2J treated and untreated groups. The data of five mice in each group is expressed as mean ± SEM. ANOVA test for repeated measures showed significant difference in strength in the hind limbs (p < 0.01) between treated and untreated dy2J/dy2J.
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and untreated dy2J/dy2J mice (data not shown), and in the fore and hind limbs of the treated and untreated WT groups. 3.2. Mobility Following 12 weeks of treatment the mice were video recorded and their mobility parameters analyzed by the Ethovision system. Significant differences were found in all following four parameters; total and maximal distance as well as mean and maximal velocity, between the untreated dy2J/dy2J and WT groups (p < 0.001). Total (339.56 ± 128.18 vs. 1535.27 ± 208.14 cm; Fig. 2A) and maximal (1.88 ± 0.17 vs. 4.94 ± 0.3 cm; Fig. 2B) distances were shorter in the dy2J/dy2J mice. Mean (3.95 ± 0.28 vs. 7 ± 0.23 cm/s; Fig. 2C) and maximal (9.43 ± 0.85 vs. 24.75 ± 1.52 cm/s; Fig. 2D) velocities were slower in the untreated dy2J/dy2J compared to the WT mice. Significant improvement in these four parameters was noted in the GA treated compared to untreated dy2J/dy2J mice. Total (898.33 ± 199.41 vs. 339.56 ± 128.18 cm; p = 0.05; Fig. 2A) and maximal (3.07 ± 0.34 vs. 1.88 ± 0.19 cm; p < 0.02; Fig. 2B) distances were longer in the treated group. Mean (5.20 ± 0.32 vs. 3.95 ± 0.28 cm/s; p < 0.03; Fig. 2C) and maximal (15.34 ± 1.59 vs. 9.43 ± 0.85 cm/s; p < 0.02; Fig. 2D) velocities were faster in the treated compared to the untreated dy2J/dy2J mice. Following GA treatment the total distance parameter of the dy2J/dy2J mice was no longer significantly different compared to treated WT mice (898.33 ± 199.41 vs. 1096 ± 196.69 cm; p = 0.50). However, each of the other three mobility parameters; maximal distance, mean and maximal velocity was still significantly sub-optimal in the treated dy2J/dy2J compared to the treated WT group (p < 0.01) (maximal distance; 3.07 ± 0.34 vs. 4.82 ± 0.26 cm, mean velocity; 5.20 ± 0.32 vs. 6.46 ± 0.2 cm/s and maximal velocity; 15.34 ± 1.59 vs. 24.12 ± 1.32 cm/s). There was no difference in these four parameters between treated and untreated WT groups. 3.3. Regeneration and fibrosis markers Immunoblotting of skeletal muscle was carried out at the end of the study using anti-myogenin and anti-MyoD as regeneration markers. Treated and untreated WT mice showed a similar level of MyoD and myogenin expression (Fig. 3). Untreated dy2J/dy2J mice
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showed significantly lower amounts of MyoD and myogenin compared to WT groups (MyoD: 31.25 ± 5.66; p < 0.05 myogenin: 59.88 ± 13.6; p < 0.05 [densitometry, arbitrary units]). GA treatment of dy2J/dy2J mice was associated with a significantly increased MyoD and myogenin expression (MyoD: 89.06 ± 16.94; p < 0.05 myogenin: 101.29 ± 12.93; p < 0.05). Following GA treatment there was no longer a significant difference in the expression of these two regeneration markers between the treated WT and dy2J/dy2J groups. Fibronectin and vimentin were used as fibrosis markers. Treated and untreated WT mice showed very low expression of fibronectin and vimentin (Fig. 4). In contrast, untreated dy2J/dy2J mice showed increased expression of fibronectin and vimentin which were significantly attenuated in the GA treated dy2J/dy2J group (Fibronectin: 2906.16 ± 999.36 vs. 411.76 ± 114.75; p < 0.05. Vimentin: 609.16 ± 46.52 vs. 111.28 ± 13.3; p < 0.001). Following treatment there was no longer a significant difference in expression of these two fibrosis markers between the treated WT and dy2J/dy2J groups. General histology of muscle biopsies obtained at the end of the study showed patchy and variable fibrosis in different areas even within the same muscle and quantitative collagen measurement by the Sircol Collagen Assay did not show a significant decrease in total collagen I–IV content in treated dy2J/dy2J mice (data not shown). 4. Discussion The dy2J/dy2J is a spontaneous mouse model of merosin deficient congenital muscular dystrophy (MDC1A) [6,31]. This milder variant of the dy/dy mouse is a useful model to study pharmacological intervention on mouse strength and mobility. At the age of 6 weeks dy2J/dy2J mice were found to be significantly weaker than the C57BL/6 J wild type mice. In the ensuing 12 weeks muscle strength further decreased in the dy2J/dy2J while increasing in the wild type. In the present study, a beneficial clinical effect of GA on muscle strength and mobility was shown in the dy2J/dy2J mouse. These mice have predominantly hind limb muscle weakness initially. The treated mice showed significant improvement in hind limb muscle strength compared to the untreated group as measured by muscle strength meter. In addition, the treated mice showed significant improvement in motor performance which was obvious
Fig. 2. The effect of GA on mouse mobility. Data is expressed as mean ± SEM of 5 WT; 4 WT + GA; 4 dy2J/dy2J; 5 dy2J/dy2J + GA mice. Student’s t-test and non parametric Mann– Whitney test showed significant difference in total distance (A; *p = 0.05), maximal distance (B; **p < 0.02), mean velocity (C; ***p < 0.03) and maximal velocity (D; **p < 0.02), between the treated and untreated dy2J/dy2J.
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Fig. 3. The effect of GA on muscle regeneration markers. Western blot analysis gels and densitometry graphs of regeneration markers’ expression in the WT and dy2J/dy2J mice. MyoD (A) and myogenin (B) levels were higher in the GA treated compared to the untreated dy2J/dy2J mice. GAPDH and a-actin were used as controls. These results represent 3 independent experiments. Graphs of densitometry measurements are normalized to the protein controls. Each bar represents the mean ± SEM of measurements of four mice (*p < 0.05; Student’s t-test).
Fig. 4. The effect of GA on muscle fibrosis markers. Western blot analysis gels and densitometry graphs of fibrosis markers’ expression in the WT and dy2J/dy2J mice. Fibronectin (A) and vimentin (B) levels markedly decreased in the treated compared to the untreated dy2J/dy2J mice. GAPDH was used as controls. These results represent three independent experiments. Densitometry measurements are normalized to GAPDH. Each bar represents the mean ± SEM of measurements of four mice (*p < 0.05, **p < 0.01; Student’s t-test).
clinically and quantified by the Ethovision video detection software. Both velocity and distance parameters indicated significant improvement in mobility for the treated group. However, both strength and mobility parameters were not completely ‘‘normalized” following treatment and were still sub-optimal in the treated dy2J/dy2J compared to the WT group. The mechanism of the effect of GA in the dy2J/dy2J mouse is unknown. GA effect in this model may be through suppression of inflammatory processes, anti-fibrotic effect, or enhanced regeneration, improving the muscular dystrophy. A neuroprotective effect on the peripheral neuropathy should also be considered. Inflammatory processes have been shown to play a major role in promoting pathology in muscular dystrophy with various inflammatory cell types accompanying the muscle fiber degeneration [12]. In the mdx mouse model of Duchenne muscular dystrophy, T-cells are reported to contribute significantly to skeletal muscle death and progressive fibrogenesis during repeated cycles
of muscle degeneration [12,32]. Macrophage depletion at the peak of the pathology prevents most of the muscle membrane lysis [33], while transplantation of normal thymic tissue into mdx-nu/nu mice replenishes muscle collagen deposition [32]. GA exerts anti-inflammatory activity by modulating the immune system, suppressing pro-inflammatory, and enhancing anti-inflammatory cytokines [34–36]. Induction of GA-reactive Th2 cells by shifting the immune response from Th1 to Th2 is thought to be the main therapeutic mechanism of action for GA [25,37,38]. Additional immunomodulatory activities include effects on antigen presenting cells and secretion of anti-inflammatory cytokines [39]. GA activity is not limited to the central nervous system. In the skin transplantation system, GA significantly prevents skin graft rejection, and in bone marrow transplantation GA alleviates immune rejection and drastically reduces cytotoxic activity toward host targets [22,40]. In future studies evaluation of pro and anti-inflammatory cytokines following GA
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treatment will aid determination of the importance of this mechanism in the dy2J/dy2J mouse. MDC1A is characterized by failure of regeneration and striking proliferation of the connective tissue associated with progressive loss of muscle function [4]. GA has an anti-fibrotic effect which has been shown in the animal model of liver cirrhosis [41]. In muscle the anti-fibrotic effect of GA may be either synergistic or secondary to anti-inflammatory activity [12,15–17]. In this study, we found significant suppression of the increased fibrosis markers fibronectin and vimentin in the GA treated dy2J/dy2J group. Fibronectin is a major extra-cellular matrix glycoprotein essential for types I and III collagen assembly [42,43]. It is up-regulated in dystrophic conditions [44]. Vimentin is an intermediate filament protein detected in fibroblasts and also expressed in myoblasts. It gradually disappears with their maturation into myotubes [45]. Our results of vimentin up-regulation in the dy2J/dy2J mouse are consistent with previous reports of its up-regulation in mdx mice diaphragm, and in DMD and MDC1A human patients [46,47]. However, even though both fibrosis markers decreased in treated dy2J/dy2J, the abundant fibrosis on muscle biopsy and quantitative muscle collagen measurement was not significantly affected by GA. In the same experimental paradigm other anti-fibrotic agents showed a more pronounced decrease in fibrosis and collagen content in dy2J/dy2J mice (Nevo Y, unpublished data). The mechanism of fibrosis in MDC1A is not fully understood. Transforming growth factor beta (TGF-b) is a key mediator of fibrosis and tissue remodeling [48]. TGF-b1 transcript levels are shown to be greatly increased in muscle from dystrophin and LAMA2 mutated muscular dystrophy patients [47,49,50]. The intracellular signaling of TGF-b is mediated via smad proteins [49]. Smad3 has a role in transcriptional regulation of type I collagen gene expression in the development of fibrosis [51,52]. The fibrotic process involves an imbalance of matrix metalloproteinases (MMPs) and their specific tissue inhibitors (TIMPs) [48]. MMP 9 directly activates latent TGF-b, and in turn TGF-b induces MMPs up-regulation of MMPs 2 and 9 [53–55]. Previous in vitro studies showed increased secretion of TGF-b following GA treatment [34]. Modification of contradicting pathways including GA induced TGF-b pro-fibrotic activity may explain the lack of improvement in muscle fibrosis in this study even though fibrotic markers were attenuated. If this is the case, then the combined effect of GA and TGF-b inhibitors may potentially further improve the clinical and histopathological outcome. Significant up-regulation of muscle regeneration transcription factors in parallel with suppression of fibrosis markers following treatment were found in this study. During muscle regeneration the satellite cells undergo self-renewal and proliferate to produce myoblasts that fuse into newly regenerated myofibers. MyoD and myogenin are transcription factors in skeletal myogenesis considered as regeneration markers. MyoD is up-regulated early during activation of the satellite cell population and is required for differentiation of activated myogenic progenitor cells into myoblasts. Later, up-regulation of myogenin induces differentiation of myoblasts into myotubes and myocytes [56–58]. Enhanced expression of MyoD and myogenin were detected at the age of 18 weeks in the treated dy2J/dy2J compared to the untreated group. At this age significant motor impairment is noted in these mice. These preliminary findings suggest that regeneration potential is still available at advanced stages of the disease. These results are in concordance with the Cohn et al. study which showed that inhibition of TGF-b pathways in the mdx mouse was associated with both attenuation of fibrosis and up-regulation of muscle regeneration [18]. A neuroprotective effect of GA should also be considered. In multiple sclerosis the GA mechanism of action is postulated to include neuroprotection in addition to immune modulation [59]. Neuroprotective effect on damaged axons and neuronal cells is
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mediated by modulation of neurotrophic factors such as brain-derived neurotrophic factor (BDNF) secreted by GA-reactive T-cell lines [39]. Congenital muscular dystrophy with merosin deficiency is associated with central and peripheral nervous system dysmyelination. Both children and dy2J/dy2J mice have peripheral neuropathy in addition to progressive muscular dystrophy [2,10]. As neuroprotection and prevention of central nervous system damage may be a primary or secondary component of the beneficial effect of GA in multiple sclerosis, GA also may possibly have a favorable impact on peripheral nerve dysmyelination. This hypothesis was not tested in the current study. In summary, in this study a beneficial effect of GA on muscle strength and mobility was detected in the dy2J/dy2J mouse model of congenital muscular dystrophy. GA is associated with a low side effect profile in adults. However, towards clinical application, GA safety in children, its effect in other animal models of muscular dystrophy and the mechanism of action of GA in muscle should be further explored. Acknowledgments The authors thank Danny Sason for assistance in the Ethovision mobility data analysis, S. Miterani-Rosenbaum and O. El-Peleg for helpful comments and M. Rabie for assistance in English editing of the manuscript. Following the results of this study Hadassit, Hadassah hospital implementation office issued an international PCT patent application No. PCT/IL2008/001289 on GA treatment in muscular dystrophy. References [1] Lisi MT, Cohn RD. Congenital muscular dystrophies: new aspects of an expanding group of disorders. Biochim Biophys Acta 2007;1772:159–72. [2] Miyagoe-Suzuki Y, Nakagawa M, Takeda S. Merosin and congenital muscular dystrophy. Microsc Res Tech 2000;48:181–91. [3] Muntoni F, Voit T. The congenital muscular dystrophies in 2004: a century of exciting progress. Neuromuscul Disord 2004;14:635–49. [4] Jimenez-Mallebrera C, Brown SC, Sewry CA, Muntoni F. Congenital muscular dystrophy: molecular and cellular aspects. Cell Mol Life Sci 2005;62:809–23. [5] Talim B, Kale G, Topaloglu H, et al. Clinical and histopathological study of merosin-deficient and merosin-positive congenital muscular dystrophy. Pediatr Dev Pathol 2000;3:168–76. [6] Xu H, Wu XR, Wewer UM, Engvall E. Murine muscular dystrophy caused by a mutation in the laminin alpha 2 (Lama2) gene. Nat Genet 1994;8:297–302. [7] Vilquin JT, Vignier N, Tremblay JP, Engvall E, Schwartz K, Fiszman M. Identification of homozygous and heterozygous dy2J mice by PCR. Neuromuscul Disord 2000;10:59–62. [8] Colognato H, Yurchenco PD. The laminin alpha2 expressed by dystrophic dy(2J) mice is defective in its ability to form polymers. Curr Biol 1999;9:1327–30. [9] Vainzof M, Ayub-Guerrieri D, Onofre PC, et al. Animal models for genetic neuromuscular diseases. J Mol Neurosci 2008;34:241–8. [10] Shorer Z, Philpot J, Muntoni F, Sewry C, Dubowitz V. Demyelinating peripheral neuropathy in merosin-deficient congenital muscular dystrophy. J Child Neurol 1995;10:472–5. [11] Schessl J, Zou Y, Bonnemann CG. Congenital muscular dystrophies and the extracellular matrix. Semin Pediatr Neurol 2006;13:80–9. [12] Spencer MJ, Tidball JG. Do immune cells promote the pathology of dystrophindeficient myopathies? Neuromuscul Disord 2001;11:556–64. [13] Morrison J, Palmer DB, Cobbold S, Partridge T, Bou-Gharios G. Effects of Tlymphocyte depletion on muscle fibrosis in the mdx mouse. Am J Pathol 2005;166:1701–10. [14] Tidball JG. Inflammatory processes in muscle injury and repair. Am J Physiol Regul Integr Comp Physiol 2005;288:R345–53. [15] Farini A, Meregalli M, Belicchi M, et al. T and B lymphocyte depletion has a marked effect on the fibrosis of dystrophic skeletal muscles in the scid/mdx mouse. J Pathol 2007;213:229–38. [16] Vidal B, Serrano AL, Tjwa M, et al. Fibrinogen drives dystrophic muscle fibrosis via a TGF-b/alternative macrophage activation pathway. Genes Dev 2008;22:1747–52. [17] Butterfield TA, Best TM, Merrick MA. The dual roles of neutrophils and macrophages in inflammation: a critical balance between tissue damage and repair. J Athl Train 2006;41:457–65. [18] Cohn RD, van Erp C, Habashi JP, et al. Angiotensin II type 1 receptor blockade attenuates TGF-b-induced failure of muscle regeneration in multiple myopathic states. Nat Med 2007;13:204–10.
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