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Angiotensin II type 1 receptor antagonists alleviate muscle pathology in the mouse model for laminin-α2-deficient congenital muscular dystrophy (MDC1A)

Angiotensin II type 1 receptor antagonists alleviate muscle pathology in the mouse model for... Background: Laminin-α2-deficient congenital muscular dystrophy (MDC1A) is a severe muscle-wasting disease for which no curative treatment is available. Antagonists of the angiotensin II receptor type 1 (AT1), including the anti-hypertensive drug losartan, have been shown to block also the profibrotic action of transforming growth factor (TGF)-β and thereby ameliorate disease progression in mouse models of Marfan syndrome. Because fibrosis and failure of muscle regeneration are the main reasons for the severe disease course of MDC1A, we tested whether W W L-158809, an analog derivative of losartan, could ameliorate the dystrophy in dy /dy mice, the best-characterized model of MDC1A. W W Methods: L-158809 was given in food to dy /dy mice at the age of 3 weeks, and the mice were analyzed at the age of 6 to 7 weeks. We examined the effect of L-158809 on muscle histology and on muscle regeneration after injury as well as the locomotor activity and muscle strength of the mice. W W Results: We found that TGF-β signaling in the muscles of the dy /dy mice was strongly increased, and that L-158809 treatment suppressed this signaling. Consequently, L-158809 reduced fibrosis and inflammation in skeletal W W muscle of dy /dy mice, and largely restored muscle regeneration after toxin-induced injury. Mice showed improvement in their locomotor activity and grip strength, and their body weight was significantly increased. W W Conclusion: These data provide evidence that AT1 antagonists ameliorate several hallmarks of MDC1A in dy /dy mice, the best-characterized mouse model for this disease. Because AT1 antagonists are well tolerated in humans and widely used in clinical practice, these results suggest that losartan may offer a potential future treatment of patients with MDC1A. Keywords: TGF-β, Smad2/3, Notexin, Skeletal muscle, Fibrosis, Muscle regeneration, losartan, Angiotensin II Background which has been shown to be a key regulator of TGF-β Losartan (Cozaar ; Merck Sharpe & Dohme, White- activation [1,2]. Therefore, AT1 inhibition and subse- house Station, NJ, USA), is widely used in clinics to treat quent reduction of TSP-1 production has been shown to hypertension, cardiomyopathy and chronic renal disease. block TGF-β activation [1]. It is very well tolerated by all age groups. Losartan is a TGF-β is a cytokine whose activity is known to inhibit potent inhibitor of angiotensin II receptor type 1 (AT1) myoblast differentiation, promote fibrosis [3], and impair and thus lowers the blood pressure by directly causing regeneration capacity in muscle [4]. TGF-β signals via vasodilation, and by reducing secretion of vasopressin phosphorylation of Smad2/Smad3, which then form a and aldosterone. Activation of AT1 by angiotensin II complex with Smad4 that translocates into the nucleus, results in the production of thrombospondin (TSP)-1, where it modulates transcription [5]. TGF-β also acti- vates the mitogen-activated protein kinases (MAPKs), * Correspondence: markus-a.ruegg@unibas.ch including extracellular signal-regulated kinase (ERK)1/2, Biozentrum, University of Basel, Basel, Switzerland © 2012 Meinen et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Meinen et al. Skeletal Muscle 2012, 2:18 Page 2 of 13 http://www.skeletalmusclejournal.com/content/2/1/18 Jun kinase (JNK) and p38, which then can regulate Smad remains difficult. Hence, several pharmacological proteins or transcription factors [6,7]. Raised TGF-β approaches have been tested, which would eventually levels and activity were shown to contribute strongly to allow clinical treatment options. These include inhibition W W the phenotype of several diseases [8-10]. For example, of apoptosis in dy /dy mice [29-32] and interference Marfan syndrome (MFS), which is caused by mutations with proteasomal and autophagy-mediated degradation in the gene encoding fibrillin-1 [11], is characterized by of proteins [33,34], Halofuginone, an analog of a plant increased fibrosis and impaired muscle regeneration [1]. alkaloid that blocks TGF-β-mediated collagen synthesis, 2J 2J Beside structural functions, fibrillin-1 negatively regu- was tested in dy /dy mice, which represent a much lates TGF-β signaling [9,10]. Consequently, mutations in milder form of MDC1A that is caused by the partial loss fibrillin-1 lead to increased TGF-β levels in muscles of of laminin-211 [35]. In these mice, halofuginone was patients with MFS and in mouse models of MFS shown to inhibit Smad3 phosphorylation downstream of (Fbn1C1039G/+) [1,12]. In addition, increased TGF-β TGF-β activation and to prevent progression of fibrosis, levels were found in muscles of Duchenne muscular dys- resulting in an amelioration of the dystrophic phenotype 2J 2J trophy patients and mdx mice [8,10], and in old mice [36]. Likewise, in dy /dy mice, losartan was shown to suffering from sarcopenia [13]. Importantly, when inhibit TGF-β signaling, improve grip strength, and re- Fbn1C1039G/+ and mdx mice were treated with losar- duce fibrosis [37]. Besides the mouse data, there is evi- tan, AT1-mediated TGF-β signaling was inhibited, dence that TGF-β levels are increased in muscles of decreased fibrosis, normalized muscle architecture, and patients with MDC1A [38]. improved muscle function and regeneration [1,14,15]. In Therefore, we aimed to test the effect of the AT1 in- mice with sarcopenia, losartan improved muscle remod- hibitor L-158809, a potent derivative of losartan, in the W W eling after injury, and protected muscle from disuse- severe dy /dy mouse model for MDC1A. We found induced atrophy [13]. that AT1-mediated TGF-β signaling contributes to the Laminin-α2-deficient congenital muscular dystrophy pathology in MDC1A, and that L-158809 treatment (MDC1A) is a severe muscle-wasting disease that leads reduces TGF-β levels. Fibrosis was reduced and several to death in early childhood [16]. MDC1A is caused by histological hallmarks of disease were improved. Import- mutations in the gene encoding the laminin-α2 chain, antly, L-158809 supported successful regeneration in W W which is needed to form the heterotrimeric laminin-211, dy /dy muscles, and improved body weight, grip the main laminin isoform in the basement membranes strength, and locomotor activity. Taking into consider- of muscle and peripheral nerve [17]. In MDC1A, ab- ation the fact that losartan is already in clinical use and sence of laminin-211 disrupts the linkage of the base- is well tolerated in all age groups, this treatment could ment membrane to the underlying cell layer, and proceed to clinical testing quickly and, might be a sup- interrupts intracellular signaling. Consequently, muscle portive treatment for patients with MDC1A in the near fibers degenerate upon contraction as a result of the future. poor mechanical stability, fail to regenerate properly [18,19], and often undergo apoptosis [18,20]. The mus- Methods cles of patients with MDC1A and of mouse models of Ethics approval MDC1A are characterized by extensive fibrosis, marked All procedures were approved by the veterinary commis- variation in muscle fiber size, and a greatly impaired sion of the Canton Basel-Stadt, and were performed in ability of muscle to regenerate [19-25]. accordance with the Swiss regulations for animal Over the last 10 years, various studies have been car- experimentation. ried out on MDC1A mouse models to test potential W W treatment options. To date, transgenic expression of Treatment of dy /dy mice with the angiotensin II type 1 laminin-α1, a homolog of laminin-α2, in laminin-α2-de- receptor antagonist L-158809 3K 3K W W ficient dy /dy mice has shown the highest efficacy in Dy /dy mice served as the mouse model for MDC1A, restoring muscle function [26,27]. Similarly, a very pro- and were genotyped as previously described [24]. Age- found restoration of muscle is achieved by transgenic ex- matched wild-type (WT) mice served as the control W W pression of mini-agrin, a miniaturized form of the group. To ensure optimal access of dy /dy mice to 3K 3K basement membrane component agrin in dy /dy [21] water and food, cages were supplied with long-necked W W and dy /dy mice [19,25]. Interestingly, expression of water bottles, and wet food was placed inside the cage. mini-agrin by systemic delivery of recombinant adeno- Mice were treated with L-158809 (5,7-dimethyI-2- associated virus (AAV) has also been shown to have a ethyI-3-[[2'-(−1 H-tetrazol-5yI)[1,1]-bi-phenyl-4-yl]-me- W W strong ameliorating effect in dy /dy mice [28]. thyl]-3 H-imidazo[4,5-b]pyridine: generously provided Although these genetic therapies are interesting, the by Merck Sharp & Dohme Research Laboratories, West translation of such approaches into clinical practice Point, PA, USA). L-158809 is a potent orally bioavailable Meinen et al. Skeletal Muscle 2012, 2:18 Page 3 of 13 http://www.skeletalmusclejournal.com/content/2/1/18 angiotensin II type 1 receptor blocker, and constitutes a Histological quantifications more potent, chemically modified derivative of losartan The triceps brachii and diaphragm muscles were chosen (DuP-553; Merck) [39]. L-158809 was solubilized in 15% for histological analysis to exclude muscles that are NaHCO , then 0.6 g/L of L-158809 and 4% sucrose was affected by the secondary atrophic effect resulting from added to the drinking water. This solution was given hind-limb paralysis, which is caused by demyelination of as drinking water and was used for the preparation of the peripheral nerve [41]. Mid-belly cross-sections were the wet food. L-158809 treatment started at the age analyzed. Nuclear accumulation of pSmad2/3 was counted of 3 weeks and was continued until the animals were in four corresponding squares on cross-sections stained killed by CO2 asphyxiation at the age of 6 or 7 weeks. with phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) L-158809 treatment did not influence the body weight, and DAPI. Fibrosis was quantified by measuring the muscle function, or muscle architecture of WT mice collagenous area on entire cross-sections stained with (data not shown). Masson trichrome, and was normalized to muscle area. F4/80 staining allowed counting of macrophages in four Masson trichrome and immunostaining corresponding squares in the triceps and in the entire The triceps brachii and diaphragm muscles were cross-section of the diaphragm. The muscle fiber-size immersed in 7% gum Tragacanth (Sigma-Aldrich, St. distribution was quantified on entire WGA-stained cross- Louis, MO, USA) and rapidly frozen in liquid nitrogen- sections using the minimum distance of parallel tangents cooled isopentane at −150°C). Cross-sections, 12 μmin at opposing particle borders (minimal Feret’s diameter) as thickness, were cut using a cryostat (Leica CM 1950; described previously [42]. The variance coefficient of the Leica Biosystems, Nussloch, Germany) and collected on muscle fiber size was defined as follows: variance slides (SuperFrost Plus; Thermo Fisher Scientific Inc., coefficient = (standard deviation of the muscle fiber size Rockford, IL, US). Masson's trichrome staining [40] was ÷ mean muscle fiber size) × 1000. Normalization of the performed to visualize collagenous tissue, using a com- number of fibers in each Feret class of 5 μm was based on mercial kit (HT-15; Trichrome Stain Kit; Sigma-Aldrich). the total fiber number per muscle. Fibers with centrally The antibodies used for stainings were purchased located nuclei (centrally nucleated fibers; CNF) were from commercial sources as follows. Monoclonal rabbit counted in the entire WGA/DAPI-stained cross-sections. anti-mouse TGF-β (56E4, CST #3709), monoclonal Regenerating dMyHC-positive fibers were quantified on rabbit anti-human phospho-Smad2(Ser465/467)/Smad3 entire dMyHC/laminin-γ1/DAPI-stained cross-sections. (Ser423/425) antibody (CST #9510) (both Cell Signaling Only dMyHC-positive fibers that appeared to be healthy Technology, Beverly, MA, USA) polyclonal rabbit anti- were included. Because the antibody used to detect human TSP 1 (LS-C26356; Lifespan Biosciences, Inc., dMyHC was raised in mice, the mean number of Seattle, WA, USA), polyclonal rabbit anti-human perios- dMyHC-positive fibers represents the mean number of tin (RD181045050; BioVendor LLC, Candler, NC, USA), dMyHC-positive fibers minus the mean number of fibers monoclonal rat anti-mouse F4/80 (ab6640; Abcam, that were stained with the secondary antibody alone (that Cambridge, MA, USA), monoclonal mouse anti-rat de- is, 2.8 muscle fibers/cross-section in dy -L158 mice and W W velopmental myosin heavy chain (dMyHC) (NCL- 9 fibers/cross-section in dy /dy mice). In all histological MHCd; Novocastra, Norwell, MA, USA), monoclonal quantification experiments, at least four mice from each rat anti-mouse laminin-γ1 chain (MAB1914; Chemicon group were analyzed. (now EMD Millipore, Billerica, MA, USA)). Membrane- bound and extracellular epitopes were visualized with Western blotting analysis Alexa-488-conjugated wheatgerm agglutinin (WGA) Triceps brachii and diaphragm muscles were homoge- (Molecular Probes, Eugene, OR, USA). Depending on nized in radio-immunoprecipitation assay (RIPA) protein the source of the primary antibody, the appropriate Cy3- extraction buffer (Abcam). A commercial kit (23227; conjugated (Jackson ImmunoResearch Laboratories, BCA Protein Assay Kit; Pierce Biotechnology Inc., Rock- West Grove, PA, USA) or Alexa Fluor 488-conjugated ford, IL, USA) was used to determine protein concentra- (Molecular Probes) secondary antibodies or tetramethyl tions. Equal amounts of protein (20 μg) were separated rhodamine isothiocyanate (TRITC)-labeled streptavidin in 8% (periostin; 75 to 90 kDa) or 20% (transforming were used for visualization. DAPI (4',6'-diamidino-2- growth factor (TGF)-β; 12 kDa in reducing conditions) phenylindole hydrochloride) was used to stain nuclei. SDS–PAGE and transferred to nitrocellulose membrane. Pictures of stained cross-sections were taken using a The membrane was incubated with antibodies to perios- fluorescence microscope (DM5000B; Leica, Heerbrugg, tin (RD181045050; BioVendor) or TGF-β (PAB11274; Switzerland), a digital camera (F-View), and analySIS Abnova Corp., Taipei, Taiwan). An antibody to β-actin software (both Soft Imaging Systems Corp, Lakewood, (#4970; Cell Signaling Technology) was used as loading CO, USA). control. For detection, the appropriate horseradish Meinen et al. Skeletal Muscle 2012, 2:18 Page 4 of 13 http://www.skeletalmusclejournal.com/content/2/1/18 peroxidase-conjugated antibodies were used, and immu- 180 seconds. In all tests, at least 12 animals of each noreactivity was visualized using the enhanced chemilu- genotype were analyzed, and values were normalized to minescence detection method (32106; Pierce). those obtained from WT animals. Quantitative real-time PCR Statistical analysis The total RNA of the triceps brachii and diaphragm To compare the different genotypes, P-values were muscles was extracted (#Z3105; SV Total RNA Isolation calculated using the one-way ANOVA test. System; Promega, Corp., Madison, QI, USA). Samples were calibrated to equal amounts for cDNA synthesis using Results a commercial kit (#170–8891; iScript cDNA Synthesis Kit; Angiotensin II receptor type 1 (AT1) signaling and TGF-β W W Bio-Rad Laboratories, Inc., Hercules, CA, USA). SYBR levels are increased in dy /dy mice Green (4367659; Power SYBR Green PCR Master Mix) was In the first set of experiments, we tested whether TGF-β used to perform quantitative PCR in a PCR system levels were increased in muscles of laminin-α2-deficient W W (4376600; StepOnePlus™ Real-Time PCR System; both Ap- dy /dy mice. We found that TGF-β (Figure 1, green plied Biosystems, Foster City, CA, USA) with the following staining) was present in the perimysium and endomy- 0 W W primers for TGF-β1(sense: 5 GACTCTCCACCTGCAA sium of triceps muscle from dy /dy mice, but was ab- 0 0 GACCAT 3 and anti-sense: 5 GGGACTGGCGAGCCT sent in WT mice (Figure 1A). Accordingly, expression of 0 0 TAGTT 3)and on β-actin (sense: 5 CAGCTTCTTTG periostin and nuclear accumulation of phosphorylated 0 0 CAGCTCCTT 3 and anti-sense: 5 GCAGCGATATCGT Smad2/3 complexes (pSmad2/3), both downstream med- 0 Ct W W CATCCA 3 ) for normalization. The ΔΔ method was iators of TGF-β signaling, were seen in dy /dy but not used to analyze changes in TGF-β1 mRNA expression rela- in WT triceps (Figure 1A). Western blotting analysis tive to WT levels. confirmed the presence of TGF-β and periostin in dy / dy muscles (Figure 1B, first panel). Hydroxyproline assay To test whether L-158809 decreased TGF-β levels, we W W Fibrosis in the triceps brachii muscles was measured by treated dy /dy mice with L-158809 for 3 weeks. Stains assaying for the exclusive collagen-specific modified and immunoblots of triceps muscles from the treated W W W amino acid hydroxyproline. Tendons were carefully dy /dy mice (dy -L158) showed a strong reduction in removed before muscles were speed-dried under vac- TGF-β and periostin (right panels, Figure 1A and uum and sent for amino acid analysis (Analytical Figure 1B). Correspondingly, nuclear accumulation of W W Research Services; Bern, Switzerland) as described pre- pSmad2/3 complexes was 25 times higher in dy /dy viously [19]. There, muscles were hydrolyzed, then than in WT muscles, but was reduced to almost WT evaporated to dryness, and resuspended in 0.1% tri- levels after L-158809 treatment (Figure 1A, right panel; fluoroacetic acid. Amino acids were determined by a Figure 1C). The suppression of TGF-β by L-158809 was routine method [43] using high-performance liquid also seen for mRNA levels, which were less than 50% of W W chromatography (HPLC) to identify and quantify the those in dy /dy mice (Figure 1D). Importantly, TSP-1, amino acid hydroxyproline. The relative hydroxyproline which is increased by AT1 signaling [44], and has been amount was assessed with reference to the total amount shown to mediate activation of TGF-β in cardiac muscle of amino acids. and kidney [45], was present in the extracellular matrix of W W dy /dy mice, but was strongly reduced in L-158809- W W Notexin-induced muscle damage treated dy /dy triceps and diaphragm muscle (Figure 1E). The tibialis anterior (TA) muscle of 5 week-old mice These results indicate that AT1-mediated TGF-β signaling was injured by injection of 15 μl notexin (50 μg/ml; is increased in MDC1A , and that L-158809 dampens this Sigma-Aldrich) as described previously [21]. Mice were pathway. killed 5 days after injection, and muscles were isolated and processed as described above. Angiotensin II type 1 receptor antagonism improves fibrosis, inflammation, and overall histology Body weight, locomotion, and grip strength To test for a beneficial effect of L-158809, we treated W W Body weight was measured at the age of 7 weeks before dy /dy mice starting at the age of 3 weeks and con- mice were sacrificed. Locomotive behavior was recorded tinuing until the age of 7 weeks. First, we analyzed by placing the mice into a new cage and measuring the potential of L-158809 to reduce fibrosis and inflam- W W motor activity (walking, digging, stand-ups on hindlegs) mation in triceps and diaphragm muscle of dy /dy for 10 minutes [25]. Grip strength was evaluated by pla- mice. Masson trichrome staining showed a more prom- cing the animals onto a vertical grid, and measuring inent replacement of muscle tissue with non-muscle W W the time until they fell down; the cut-off time was cells in untreated dy /dy mice than in mice treated Meinen et al. Skeletal Muscle 2012, 2:18 Page 5 of 13 http://www.skeletalmusclejournal.com/content/2/1/18 Figure 1 L-158809 lowered angiotensin II receptor type 1 (AT1) -mediated transforming growth factor (TGF)-β levels in the W W muscles of dy /dy mice. (A) AT1-mediated TGF-β levels were W W increased in muscles of dy /dy mice and decreased after L-158809 W W administration. Triceps cross-sections of dy /dy mice showed expression of TGF-β (green) and periostin (green), and nuclear accumulation of pSmad2/3 (green), which is a downstream target of W W W AT1. Oral L-158809 treatment of dy /dy mice (dy -L158) reduced expression of all three proteins. Nuclei were visualized with 4',6'- diamidino-2-phenylindole hydrochloride (DAPI) (blue). (B) Western blotting analysis of triceps muscles confirmed increased protein expression levels of periostin (75 to 85 kDa) and TGF-β (12 kDa, W W reduced) in dy /dy mice as well as the reduction after L-158809 treatment. β-actin was used as a loading control. (C) The number of pSmad2/3-positive nuclei was more than 25-fold increased in cross- W W sections of dy /dy muscle compared with wild-type (WT) muscles, and was significantly reduced by L-158809 to levels 2-fold (triceps) and 2.6-fold (diaphragm) those of WT mice, which was not significant (n ≥ 4). (D) Quantitative real-time PCR showed an increase of approximately 3.5-fold in TGF-β1 mRNA levels in both W W dy /dy triceps and diaphragm muscles, which was reduced to nearly WT levels by L-158809 (n ≥ 4). (E) Thrombospondin-1 (green) W W was increased in both triceps and diaphragm muscles of dy /dy mice, and was minimized by L-158809 administration. All values represent the mean ± SEM. One-way ANOVA: **P ≤ 0.001; * P ≤ 0.05; n.s. (non-significant) P > 0.05. Scale bar = 50 μm. with L-158809 (Figure 2A). The blue color, indicative of collagen, suggests that the non-muscle cells found in un- treated muscles represent mainly fibrotic tissue (Figure 2A). The fibrotic area measured in cross-sections W W of triceps and diaphragm muscle of dy /dy mice trea- ted with L-158809 was less than 50% of that seen in un- treated mice (Figure 2B). As an independent measure of fibrosis, we also determined the hydroxyproline content in triceps muscles. In agreement with the histological measurements, L-158809 reduced the hydroxyproline W W content in dy /dy muscles (Figure 2C). Because TGF- β plays an important role in regulating skeletal-muscle inflammation [38,46], we also assessed inflammation using the F4/80 antibody that recognizes activated macrophages (Figure 2D,E). Macrophages were mainly located in areas where muscle fiber degeneration is tak- W W ing place (Figure 2D). In untreated dy /dy muscles, around 100 macrophages were found per mm , whereas L-158809 reduced this number by six times in triceps and by three times in diaphragm muscle (Figure 2E). These data provide evidence that L-158809 inhibits and thus minimizes TGF-β-induced fibrosis and inflamma- W W tion in muscles of dy /dy mice. We next investigated whether the L-158809-mediated reduction of fibrosis also ameliorated other histological hallmarks of MDC1A. A prominent feature of laminin- α2-deficient muscles is the loss of muscle fibers, which is probably due to muscle degeneration and the difficulty in muscle regeneration [18]. Indeed, the number of W W muscle fibers in dy /dy mice was much lower than in Meinen et al. Skeletal Muscle 2012, 2:18 Page 6 of 13 http://www.skeletalmusclejournal.com/content/2/1/18 Figure 2 (See legend on next page.) Meinen et al. Skeletal Muscle 2012, 2:18 Page 7 of 13 http://www.skeletalmusclejournal.com/content/2/1/18 (See figure on previous page.) W W Figure 2 L-158809 reduces fibrosis and inflammation in the muscles of dy /dy mice. (A) Masson trichrome staining showed higher W W W collagen content (blue) in triceps and diaphragm muscles of dy /dy when compared with dy -L158 mice. (B) Average amount of fibrotic area relative to wild-type (WT) muscles. L-158809 treatment more than halved the fibrotic area in triceps and diaphragm muscles (n ≥ 4). W W (C) Measurement of the amount of hydroxyproline as an indicator of fibrosis. L-158809 treatment of dy /dy mice reduced the hydroxyproline amount in triceps muscle (n = 3). (D) F4/80 staining of macrophages in diaphragm muscles. (E) L-158809 treatment reduced the number of W W macrophages in muscles of dy /dy mice (n ≥ 4). All values represent the mean ± SEM. One-way ANOVA: **P ≤ 0.001; * P ≤ 0.05; n.s. (non-significant) P > 0.05. Scale bar = 50 μm. WT mice, with the triceps and diaphragm, respectively To test the effect of L-158809 on the regenerative cap- losing 46% and 40% of their fibers (Figure 3A). L-158809 acity, the TA muscle was injured by injection of notexin. decreased this fiber loss in triceps muscle, and increased Four days after injury, only a few dMyHC-positive fibers W W the fiber number to 68% of WT. In diaphragm, were found in dy /dy mice, whereas many were L-158809 showed a similar trend, but this was not sig- present after L-158809 treatment or in WT muscle nificant (Figure 3A). Further, L-158809 did not affect (Figure 4D). The number of regenerating fibers increased W W fiber size distribution in dy /dy triceps muscle from 61 ± 14 in untreated to 490 ± 25 in L-158809-treated W W (Figure 3B). In diaphragm, where muscle fiber-size dis- dy /dy TA muscle (Figure 4E). Thus, L-158809-mediated tribution was only slightly shifted to smaller diameters TGF-β inhibition improved the regeneration capacity in W W W W in dy /dy mice, L-158809 shifted fiber-size distribu- muscles of dy /dy mice. Because a similar improve- W W tion only slightly towards the values in WT (Figure 3C). ment in muscle regeneration in dy /dy mice has also Accordingly, the minimal Feret’s variance coefficient [42] been reported for treatments that inhibited apoptosis W W was increased in dy /dy muscles but was not cor- [47], we also tested whether L-158809 would influence rected by L-158809 (Figure 3D). However, because of the number of apoptotic nuclei. However, we could not the higher number of muscle fibers in L-158809-treated detect any effect of this treatment on the number of W W dy /dy mice, the mean cross-section area of the terminal deoxynucleotidyl transferase-mediated dUTP muscle was significantly increased (Figure 3E). Thus, nick end labeling-positive myonuclei (see Additional file 1: L-158809 partially prevents the loss of muscle fibers, but Figure S1), indicating that L-158809 does not affect this W W does not significantly affect the diameter of dy /dy pathway. muscle fibers. W W Effect of L-158809 on overall health of dy /dy mice Finally, we tested whether there was any improvement L-158809 strongly improves spontaneous and injury- in motor function and overall health in L-158809- W W induced muscle regeneration treated dy /dy mice, and found that L-158809 W W W W The muscles of dy /dy mice are strongly impaired in increased the body weight of dy /dy mice, by 2 g muscle regeneration [18,19,21]. In addition, increased (Figure 4F). However, age-matched WT animals still TGF-β activity leads to failure of muscle regeneration weighed more than twice as much. The muscle strength W W [1]. Therefore, L-158809 may improve regeneration of of dy /dy mice, measured as the time the animals W W dy /dy muscle. However, the number of centrally could stay on a vertical grid, was doubled by treatment nucleated fibers, which are indicative of ongoing degen- with L-158809 from 26 seconds to 51 seconds eration/regeneration, were not significantly increased (Figure 4G); however, the WT animals far exceeded the W W after L-158809 administration (Figure 4A). By contrast, time measured with L-158809-treated dy /dy mice, staining of diaphragm muscle with the regeneration normally staying more than 180 seconds on the grid. In marker dMyHC (Figure 4B, green) indicated that more the 10-minute open-field locomotion test L-158809- W W fibers regenerated after L-158809 treatment of dy /dy treated mice were more active and explored the new sur- mice. Muscle fibers expressing dMyHC in the dy -L158 roundings for 5 minutes, whereas untreated MDC1A mice appeared to have a proper basement membrane as mice were active only for 3.5 minutes (Figure 4H). Thus, indicated by laminin-γ1 staining (red), whereas in non- the ameliorating effect of L-158809 on muscle histology W W treated dy /dy mice, dMyHC staining was often punc- is sufficient to improve body and muscle condition of W W tated and the basement membrane appeared disrupted, dy /dy mice. indicating that the fibers fail to complete regeneration (Figure 4B). In both triceps and diaphragm muscle, Discussion L-158809 clearly increased the number of intact Muscular dystrophies are caused by mutations in many dMyHC-positive fibers (Figure 4C), suggesting that different genes but they are often accompanied by a regeneration was improved by L-158809. strong inflammatory and fibrotic response, which might Meinen et al. Skeletal Muscle 2012, 2:18 Page 8 of 13 http://www.skeletalmusclejournal.com/content/2/1/18 W W W Figure 3 L-158809 reduced muscle-fiber loss but did not change fiber-size distribution in dy /dy muscles. (A) L-158809 (dy -L158) W W stabilized the loss of muscle fibers in dy /dy triceps muscle, but only tended to reduce fiber loss in the diaphragm (n = 6). (B,C) L-158809 did W W not normalize the shift of fiber sizes to smaller fibers, either (B) in triceps or (C) in diaphragm muscle of dy /dy mice (n ≥ 4). (D) The minimal Feret`s diameter coefficient was unchanged by L-158809 (n ≥ 4). (E) L-158809 increased the cross-sectional area in both mid-belly triceps (P = 0.0007) and diaphragm (P = 0.0024) muscles (n ≥ 4). All values represent the mean ± SEM. P-values (one-way ANOVA): **P ≤ 0.001; * P ≤ 0.05; n.s. (non-significant) P > 0.05. be triggered by similar signaling pathways. For example, the decreased TGF-β levels and Smad2/3 phosphoryl- 2J 2J TGF-β signaling has been shown to be upregulated in ation seen in dy /dy mice [37]. Previous work in kid- several muscle diseases [8-10], and is known to inhibit ney [44] has shown that the AT1-mediated increase in myoblast differentiation and promote fibrosis, and also TSP-1 involves the MAPK pathway. This pathway may to impair regeneration by inhibiting satellite cell activa- also be active in MDC1A, as the MAPKs ERK1/2, p38 2J 2J tion, proliferation, and differentiation [3,4]. and JNK are all increased in dy /dy mice, and this in- In this study, we found that TGF-β signaling, including crease can be inhibited by losartan [37]. increased levels of TGF-β and of the phosphorylated forms of Smad2 and Smad3, was also enhanced in the Angiotensin II type 1 receptor antagonists have a strong W W W W severe dy /dy mouse model of MDC1A (Figure 1). effect on muscle regeneration in dy /dy mice These data are in good agreement with previous reports Increased TGF-β signaling inhibits activation of satellite that TGF-β levels are also upregulated in the muscles cells [48] and promotes differentiation of myogenic into of human patients with MDC1A [38] and in the mild fibrotic cells in injured skeletal muscles [3], indicating that 2J 2J 2J 2J dy /dy mouse model [37]. As in the study on dy /dy TGF-β-dependent fibrosis is associated with impaired mice, we found an increase in the TGF-β-activating pro- muscle regeneration. In mouse models of MFS and tease TSP-1, indicating that the elevation of TGF-β is Duchenne muscular dystrophy (DMD), losartan treatment probably caused by the activation of AT1, as production restored the muscle-regeneration capacity after cardiotoxin- of TSP-1 precedes TGF-β activation in this pathway induced muscle injury [1]. In mice with sarcopenia, losartan [1,2,44,45]. Indeed, inhibition of AT1 signaling by oral restored the necessary downregulation of Pax7 and MyoD administration of L-158809 for only 3 weeks was suffi- and upregulation of p21 and myogenin, which are required cient to lower the amount of TSP-1 in muscle basement for successful completion of regeneration of injured muscle, membrane and to normalize TGF-β levels and Smad2/3 by modulating TGF-β signaling both via Smad2/Smad3 phosphorylation (Figure 1). These experiments provide phosphorylation and via activation of MAPKs [6,13]. strong evidence that the AT1-TSP-TGF-β axis is also Muscle regeneration is largely impaired in MDC1A, and we activated in MDC1A and they are in accordance with have recently shown that the main effect of anti-apoptosis Meinen et al. Skeletal Muscle 2012, 2:18 Page 9 of 13 http://www.skeletalmusclejournal.com/content/2/1/18 Figure 4 (See legend on next page.) Meinen et al. Skeletal Muscle 2012, 2:18 Page 10 of 13 http://www.skeletalmusclejournal.com/content/2/1/18 (See figure on previous page.) W W Figure 4 L-158809 improved muscle regeneration, body weight, locomotion, and muscle strength in dy /dy mice. (A) L-158809 W W W (dy -L158) did not significantly affect the number of centrally nucleated fibers (CNF) in either triceps or diaphragm muscle of dy /dy mice (n ≥ 4). (B) Staining of diaphragm muscle for the regeneration marker developmental myosin heavy chain (dMyHC; green), the basement membrane marker laminin-γ1 (red), and the nuclear marker 4',6'-diamidino-2-phenylindole hydrochloride (DAPI) (blue). L-158809 enabled W W W W damaged dy /dy muscle fibers to regenerate, whereas in non-treated dy /dy muscle many dMyHC-expressing cells continued degenerating. W W (C) L-158809 application strongly increased the number of intact regenerating muscle fibers in dy /dy mice (n ≥ 4). (D) Tibialis anterior (TA) muscle stained for dMyHC (green), laminin-γ1 (red), and DAPI four days after notexin-induced injury. L-158809 increased the regenerative capacity W W W W of damaged dy /dy muscle. (E) The number of dMyHC-expressing fibers in TA muscle of dy /dy mice 5 days after notexin injection was more W W than 8 times higher in L-158809 treated dy /dy mice but was still lower than in controls (n = 3). (F) L-158809 increased the average body W W weight of dy /dy mice from 7.6 g to 9.5 g, although age-matched wild-type (WT) mice weigh more than 20 g. (n ≥ 12). (G). L-158809 doubled W W the time a dy /dy mouse could hold itself on a vertical grid, from 26 seconds to 51 seconds (P = 0.0001) (n ≥ 14). (H) L-158809 increased the W W time dy /dy mice explored an unknown surrounding (P = 0.0004) within 10 minutes (n ≥ 12). All values represent the mean ± SEM (n ≥ 4). One-way ANOVA: **P ≤ 0.001; * P ≤ 0.05; n.s. (non-significant) P > 0.05. Scale bar = 50 μm, if not indicated differently. W W treatment in dy /dy mice is the prevention of cell death hallmarks of the condition, such as fiber loss and muscle during the regeneration process [47]. In addition, this area. In contrast to its effects in mdx mice, L-158809 did previous work also provided evidence that the cell death not correct the shift of the fiber-size distribution to smal- W W prevention by anti-apoptosis treatment had a synergistic ler fibers in dy /dy mice. This phenomenon was due to effect when combined with the re-establishment of the con- the presence of many newly formed regenerating nection between the basement membrane and the muscle (dMyHC-positive) fibers that were not grown to full size. membrane by mini-agrin [47]. Treatment with L-158809 also resulted in a significant In the current study, we found evidence that AT1 block- functional improvement in skeletal muscle, manifesting as ade by L-158809 also ameliorated the muscle regeneration increased locomotor activity and increased grip strength. W W capacity in dy /dy mice. Importantly, we found that Interestingly, an improvement of the hind-limb and fore- 2J 2J L-158809 treatment enabled newly formed muscle fibers limb strength was also seen in the dy /dy mouse model to stay intact and to complete regeneration, whereas of MDC1A [37]. W W non-treated dy /dy fibers started to regenerate, but then turned into fibrotic cells. The beneficial effect of Therapeutic potential of the different treatment options L-158809 on regeneration is superior to that seen in The highest potential to restore function in mouse models W W dy /dy mice treated with an apoptosis inhibitor alone for MDC1A is the body-wide, transgenic expression of [47], and reached almost the same efficacy as the laminin-α1, which largely cures the disease [26,27]. How- combination of apoptosis inhibition and mini-agrin [47]. ever, because the cDNA encoding laminin-α1is morethan Our observation of a relatively strong beneficial effect of 9 kb in size, its use in gene therapy is challenging, and no AT1 antagonists on muscle regeneration is in good successful attempts in creating smaller versions of the accordance with the finding in mice with sarcopenia, in functional protein have been reported. Another very which losartan enabled the regeneration of skeletal muscle effective treatment option is the muscle-specific overex- by fostering satellite cells to transit from a proliferation pression of a miniaturized form of agrin (mini-agrin), stage into the differentiation process [13]. Our finding that specifically designed to structurally reconnect the cell L-158809 treatment lowered the number of centrally surface protein α-dystroglycan to laminins in the basement located myonuclei suggests that AT1 blockade might also membrane [19,21,25]. Finally, overexpression of insulin- prevent necrosis of muscle fibers. like growth factor (IGF)-1 also ameliorates disease and prolongs life span [49], but its efficacy is clearly lower than Effect of Angiotensin II receptor type 1 antagonist on that of mini-agrin. This lower efficacy is probably based on fibrosis, inflammation, and overall muscle function the fact that IGF-1 interferes with rather late events in the Previous work has provided evidence that AT1 antagonists course of the disease, whereas mini-agrin tackles the struc- can ameliorate disease in mouse models of MFS and tural deficits that are the primary cause of the muscular DMD and in mice with sarcopenia [1,13-15]. We found dystrophy. Recombinant AAV-based gene therapy with W W that 4 weeks of L-158809 treatment decreased fibrosis mini-agrin was shown to be successful in dy /dy mice from 19.7% to 8.1% in triceps and from 23.3% to 10% in [28]. However, the use of gene therapy in clinics has been diaphragm muscle, and thereby prevented the characteristic rather slow, and thus delivery of mini-agrin to the muscles inflammatory response. Similarly, 12 weeks of losartan of patients with MDC1A remains difficult. 2J 2J treatment reduced the fibrotic area in dy /dy by This difficulty in translating gene therapy into clinics approximately 4% [37]. In addition, L-158809 treatment in has led to attempts to interfere with disease progression W W dy /dy mice corrected some of the histological using protein therapy and pharmacological compounds. Meinen et al. Skeletal Muscle 2012, 2:18 Page 11 of 13 http://www.skeletalmusclejournal.com/content/2/1/18 An interesting approach that has been shown to be survival in a large cohort of animals and could only rec- W W W efficacious in dy /dy mice is the systemic delivery of ord age at death in four cases of L-158809-treated dy / laminin-111 (heterotrimer of the α1, β1, and γ1 chains). dy mice. Nevertheless, all four mice lived at least W W In those studies, laminin-111 reached the skeletal 5 weeks longer than the untreated dy /dy mice muscles, improved muscle histology, and prolonged life (~10 weeks in average). Thus, as L-158809 or losartan span [50]. Pharmacological approaches include the use of does not act on apoptosis (see Additional file 1: Figure the apoptosis inhibitors omigapil [30] or of doxycycline or S1), it remains to be tested whether a combination of W W minocycline [31] to treat dy /dy mice. In both studies, losartan and omigapil could provide an additive benefit W W there was a clear effect on functional parameters and on in dy /dy mice. The fact that losartan is well tolerated the longevity of the mice; however, the efficacy of either in all age groups and that omigapil has proven to be safe W W treatment was much lower than in dy /dy mice expres- in clinical trials of patients with Parkinson’s disease and sing mini-agrin [47]. those with amyotrophic lateral sclerosis [52,53], makes Another consequence of muscular dystrophies is a such a combination attractive for further clinical trials. severe muscle-wasting. Preservation of muscle mass depends on the proper balance between protein synthe- Conclusion sis and protein degradation [51]. Thus, muscle wasting In this study, we investigated the efficacy of AT1 antago- can be due to a prevalence of protein degradation, and nists in the most widely used mouse model of MDC1A. based on this idea, pharmacological inhibition of the two The benefits of the therapy included decreased fibrosis main systems involved in protein degradation, the pro- and inflammation, improved muscle regeneration (which teasomal and the autophagic pathway, has been tested in leads to preservation of muscle-fiber number), and the preclinical mouse models for MDC1A. Again, inter- improved locomotion and grip strength. The AT1 antag- ference with either of those pathways ameliorated dis- onist losartan is an approved anti-hypertensive drug that ease progression [33,34]. It is, however, questionable if is well tolerated, including in children. And, importantly, such an approach is a good option for the treatment of losartan harbors fewer potential side-effects than any patients with MDC1A, as inhibition of proteosomal and other pharmacological options that have been tested to autophagosomal degradation has a strong likelihood of date in preclinical mouse models of MDC1A. Therefore, causing severe side-effects. AT1 blockade could provide a supportive treatment that In the current study, we provide evidence that AT1 alleviates some of the pathology in patients with W W antagonists also provide benefit to dy /dy mice. Our MDC1A in the near future. results are similar to those of other researchers who tested inhibition of TGF-β-induced fibrosis by intraperi- Additional file 2J 2J toneal injection of halofuginone in the milder dy /dy mouse model of MDC1A [36]. Halofuginone blocked Additional file 1: Figure S1. TGF-β-mediated collagen synthesis, prevented muscle fibrosis, and improved the performance of both muscles Abbreviations AAV: Adeno-associated virus; AT1: Angiotensin II receptor type 1; and animals. In addition, the same group also used losar- DMD: Duchenne muscular dystrophy; dMyHC: Developmental myosin heavy W W W tan to block TGF-β-signaling and this also showed bene- chain; dy /dy : Laminin-α2-deficient MDC1A mouse model; dy -L158: L- 2J 2J W W fit in dy /dy mice [37]. Thus, our work is an important 158809-treated dy /dy mice; Fbn1C1039G/+: MFS mouse model; mdx: DMD mouse model; MDC1A: Laminin-α2-deficient congenital muscular dystrophy; confirmation of efficacy of AT1 antagonists in the more MFS: Marfan syndrome; Mini-agrin: Miniaturized form of agrin specifically severe mouse model of MDC1A. designed to contain laminin and α-dystroglycan binding domains; WT: Wild-type. It is difficult to compare studies that were performed W W 3K Competing interests in dy /dy mice with those using the more severe dy / 3K 2J 2J The authors declare no competing interests dy mouse model or the much milder dy /dy model. W W As our current study used dy /dy mice, it can be com- Authors' contributions SM performed and analyzed the experiments. SL assisted in some of the pared with those using the pharmacological apoptosis experiments, and provided scientific input. SM and MAR conceived and inhibitors omigapil or doxycycline. A detailed analysis of designed the study, and wrote the manuscript. All authors read and those studies indicates that all of the treatments are of approved the final manuscript. comparable benefit to the mice. 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Miller R, Bradley W, Cudkowicz M, Hubble J, Meininger V, Mitsumoto H, Moore D, Pohlmann H, Sauer D, Silani V, et al: Phase II/III randomized trial of TCH346 in patients with ALS. Neurology 2007, 69:776–784. 53. Olanow CW, Schapira AH, LeWitt PA, Kieburtz K, Sauer D, Olivieri G, Pohlmann H, Hubble J: TCH346 as a neuroprotective drug in Parkinson's disease: a double-blind, randomised, controlled trial. Lancet Neurol 2006, 5:1013–1020. doi:10.1186/2044-5040-2-18 Cite this article as: Meinen et al.: Angiotensin II type 1 receptor antagonists alleviate muscle pathology in the mouse model for laminin- α2-deficient congenital muscular dystrophy (MDC1A). Skeletal Muscle 2012 2:18. 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Angiotensin II type 1 receptor antagonists alleviate muscle pathology in the mouse model for laminin-α2-deficient congenital muscular dystrophy (MDC1A)

Skeletal Muscle , Volume 2 (1) – Sep 3, 2012

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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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Abstract

Background: Laminin-α2-deficient congenital muscular dystrophy (MDC1A) is a severe muscle-wasting disease for which no curative treatment is available. Antagonists of the angiotensin II receptor type 1 (AT1), including the anti-hypertensive drug losartan, have been shown to block also the profibrotic action of transforming growth factor (TGF)-β and thereby ameliorate disease progression in mouse models of Marfan syndrome. Because fibrosis and failure of muscle regeneration are the main reasons for the severe disease course of MDC1A, we tested whether W W L-158809, an analog derivative of losartan, could ameliorate the dystrophy in dy /dy mice, the best-characterized model of MDC1A. W W Methods: L-158809 was given in food to dy /dy mice at the age of 3 weeks, and the mice were analyzed at the age of 6 to 7 weeks. We examined the effect of L-158809 on muscle histology and on muscle regeneration after injury as well as the locomotor activity and muscle strength of the mice. W W Results: We found that TGF-β signaling in the muscles of the dy /dy mice was strongly increased, and that L-158809 treatment suppressed this signaling. Consequently, L-158809 reduced fibrosis and inflammation in skeletal W W muscle of dy /dy mice, and largely restored muscle regeneration after toxin-induced injury. Mice showed improvement in their locomotor activity and grip strength, and their body weight was significantly increased. W W Conclusion: These data provide evidence that AT1 antagonists ameliorate several hallmarks of MDC1A in dy /dy mice, the best-characterized mouse model for this disease. Because AT1 antagonists are well tolerated in humans and widely used in clinical practice, these results suggest that losartan may offer a potential future treatment of patients with MDC1A. Keywords: TGF-β, Smad2/3, Notexin, Skeletal muscle, Fibrosis, Muscle regeneration, losartan, Angiotensin II Background which has been shown to be a key regulator of TGF-β Losartan (Cozaar ; Merck Sharpe & Dohme, White- activation [1,2]. Therefore, AT1 inhibition and subse- house Station, NJ, USA), is widely used in clinics to treat quent reduction of TSP-1 production has been shown to hypertension, cardiomyopathy and chronic renal disease. block TGF-β activation [1]. It is very well tolerated by all age groups. Losartan is a TGF-β is a cytokine whose activity is known to inhibit potent inhibitor of angiotensin II receptor type 1 (AT1) myoblast differentiation, promote fibrosis [3], and impair and thus lowers the blood pressure by directly causing regeneration capacity in muscle [4]. TGF-β signals via vasodilation, and by reducing secretion of vasopressin phosphorylation of Smad2/Smad3, which then form a and aldosterone. Activation of AT1 by angiotensin II complex with Smad4 that translocates into the nucleus, results in the production of thrombospondin (TSP)-1, where it modulates transcription [5]. TGF-β also acti- vates the mitogen-activated protein kinases (MAPKs), * Correspondence: markus-a.ruegg@unibas.ch including extracellular signal-regulated kinase (ERK)1/2, Biozentrum, University of Basel, Basel, Switzerland © 2012 Meinen et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Meinen et al. Skeletal Muscle 2012, 2:18 Page 2 of 13 http://www.skeletalmusclejournal.com/content/2/1/18 Jun kinase (JNK) and p38, which then can regulate Smad remains difficult. Hence, several pharmacological proteins or transcription factors [6,7]. Raised TGF-β approaches have been tested, which would eventually levels and activity were shown to contribute strongly to allow clinical treatment options. These include inhibition W W the phenotype of several diseases [8-10]. For example, of apoptosis in dy /dy mice [29-32] and interference Marfan syndrome (MFS), which is caused by mutations with proteasomal and autophagy-mediated degradation in the gene encoding fibrillin-1 [11], is characterized by of proteins [33,34], Halofuginone, an analog of a plant increased fibrosis and impaired muscle regeneration [1]. alkaloid that blocks TGF-β-mediated collagen synthesis, 2J 2J Beside structural functions, fibrillin-1 negatively regu- was tested in dy /dy mice, which represent a much lates TGF-β signaling [9,10]. Consequently, mutations in milder form of MDC1A that is caused by the partial loss fibrillin-1 lead to increased TGF-β levels in muscles of of laminin-211 [35]. In these mice, halofuginone was patients with MFS and in mouse models of MFS shown to inhibit Smad3 phosphorylation downstream of (Fbn1C1039G/+) [1,12]. In addition, increased TGF-β TGF-β activation and to prevent progression of fibrosis, levels were found in muscles of Duchenne muscular dys- resulting in an amelioration of the dystrophic phenotype 2J 2J trophy patients and mdx mice [8,10], and in old mice [36]. Likewise, in dy /dy mice, losartan was shown to suffering from sarcopenia [13]. Importantly, when inhibit TGF-β signaling, improve grip strength, and re- Fbn1C1039G/+ and mdx mice were treated with losar- duce fibrosis [37]. Besides the mouse data, there is evi- tan, AT1-mediated TGF-β signaling was inhibited, dence that TGF-β levels are increased in muscles of decreased fibrosis, normalized muscle architecture, and patients with MDC1A [38]. improved muscle function and regeneration [1,14,15]. In Therefore, we aimed to test the effect of the AT1 in- mice with sarcopenia, losartan improved muscle remod- hibitor L-158809, a potent derivative of losartan, in the W W eling after injury, and protected muscle from disuse- severe dy /dy mouse model for MDC1A. We found induced atrophy [13]. that AT1-mediated TGF-β signaling contributes to the Laminin-α2-deficient congenital muscular dystrophy pathology in MDC1A, and that L-158809 treatment (MDC1A) is a severe muscle-wasting disease that leads reduces TGF-β levels. Fibrosis was reduced and several to death in early childhood [16]. MDC1A is caused by histological hallmarks of disease were improved. Import- mutations in the gene encoding the laminin-α2 chain, antly, L-158809 supported successful regeneration in W W which is needed to form the heterotrimeric laminin-211, dy /dy muscles, and improved body weight, grip the main laminin isoform in the basement membranes strength, and locomotor activity. Taking into consider- of muscle and peripheral nerve [17]. In MDC1A, ab- ation the fact that losartan is already in clinical use and sence of laminin-211 disrupts the linkage of the base- is well tolerated in all age groups, this treatment could ment membrane to the underlying cell layer, and proceed to clinical testing quickly and, might be a sup- interrupts intracellular signaling. Consequently, muscle portive treatment for patients with MDC1A in the near fibers degenerate upon contraction as a result of the future. poor mechanical stability, fail to regenerate properly [18,19], and often undergo apoptosis [18,20]. The mus- Methods cles of patients with MDC1A and of mouse models of Ethics approval MDC1A are characterized by extensive fibrosis, marked All procedures were approved by the veterinary commis- variation in muscle fiber size, and a greatly impaired sion of the Canton Basel-Stadt, and were performed in ability of muscle to regenerate [19-25]. accordance with the Swiss regulations for animal Over the last 10 years, various studies have been car- experimentation. ried out on MDC1A mouse models to test potential W W treatment options. To date, transgenic expression of Treatment of dy /dy mice with the angiotensin II type 1 laminin-α1, a homolog of laminin-α2, in laminin-α2-de- receptor antagonist L-158809 3K 3K W W ficient dy /dy mice has shown the highest efficacy in Dy /dy mice served as the mouse model for MDC1A, restoring muscle function [26,27]. Similarly, a very pro- and were genotyped as previously described [24]. Age- found restoration of muscle is achieved by transgenic ex- matched wild-type (WT) mice served as the control W W pression of mini-agrin, a miniaturized form of the group. To ensure optimal access of dy /dy mice to 3K 3K basement membrane component agrin in dy /dy [21] water and food, cages were supplied with long-necked W W and dy /dy mice [19,25]. Interestingly, expression of water bottles, and wet food was placed inside the cage. mini-agrin by systemic delivery of recombinant adeno- Mice were treated with L-158809 (5,7-dimethyI-2- associated virus (AAV) has also been shown to have a ethyI-3-[[2'-(−1 H-tetrazol-5yI)[1,1]-bi-phenyl-4-yl]-me- W W strong ameliorating effect in dy /dy mice [28]. thyl]-3 H-imidazo[4,5-b]pyridine: generously provided Although these genetic therapies are interesting, the by Merck Sharp & Dohme Research Laboratories, West translation of such approaches into clinical practice Point, PA, USA). L-158809 is a potent orally bioavailable Meinen et al. Skeletal Muscle 2012, 2:18 Page 3 of 13 http://www.skeletalmusclejournal.com/content/2/1/18 angiotensin II type 1 receptor blocker, and constitutes a Histological quantifications more potent, chemically modified derivative of losartan The triceps brachii and diaphragm muscles were chosen (DuP-553; Merck) [39]. L-158809 was solubilized in 15% for histological analysis to exclude muscles that are NaHCO , then 0.6 g/L of L-158809 and 4% sucrose was affected by the secondary atrophic effect resulting from added to the drinking water. This solution was given hind-limb paralysis, which is caused by demyelination of as drinking water and was used for the preparation of the peripheral nerve [41]. Mid-belly cross-sections were the wet food. L-158809 treatment started at the age analyzed. Nuclear accumulation of pSmad2/3 was counted of 3 weeks and was continued until the animals were in four corresponding squares on cross-sections stained killed by CO2 asphyxiation at the age of 6 or 7 weeks. with phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) L-158809 treatment did not influence the body weight, and DAPI. Fibrosis was quantified by measuring the muscle function, or muscle architecture of WT mice collagenous area on entire cross-sections stained with (data not shown). Masson trichrome, and was normalized to muscle area. F4/80 staining allowed counting of macrophages in four Masson trichrome and immunostaining corresponding squares in the triceps and in the entire The triceps brachii and diaphragm muscles were cross-section of the diaphragm. The muscle fiber-size immersed in 7% gum Tragacanth (Sigma-Aldrich, St. distribution was quantified on entire WGA-stained cross- Louis, MO, USA) and rapidly frozen in liquid nitrogen- sections using the minimum distance of parallel tangents cooled isopentane at −150°C). Cross-sections, 12 μmin at opposing particle borders (minimal Feret’s diameter) as thickness, were cut using a cryostat (Leica CM 1950; described previously [42]. The variance coefficient of the Leica Biosystems, Nussloch, Germany) and collected on muscle fiber size was defined as follows: variance slides (SuperFrost Plus; Thermo Fisher Scientific Inc., coefficient = (standard deviation of the muscle fiber size Rockford, IL, US). Masson's trichrome staining [40] was ÷ mean muscle fiber size) × 1000. Normalization of the performed to visualize collagenous tissue, using a com- number of fibers in each Feret class of 5 μm was based on mercial kit (HT-15; Trichrome Stain Kit; Sigma-Aldrich). the total fiber number per muscle. Fibers with centrally The antibodies used for stainings were purchased located nuclei (centrally nucleated fibers; CNF) were from commercial sources as follows. Monoclonal rabbit counted in the entire WGA/DAPI-stained cross-sections. anti-mouse TGF-β (56E4, CST #3709), monoclonal Regenerating dMyHC-positive fibers were quantified on rabbit anti-human phospho-Smad2(Ser465/467)/Smad3 entire dMyHC/laminin-γ1/DAPI-stained cross-sections. (Ser423/425) antibody (CST #9510) (both Cell Signaling Only dMyHC-positive fibers that appeared to be healthy Technology, Beverly, MA, USA) polyclonal rabbit anti- were included. Because the antibody used to detect human TSP 1 (LS-C26356; Lifespan Biosciences, Inc., dMyHC was raised in mice, the mean number of Seattle, WA, USA), polyclonal rabbit anti-human perios- dMyHC-positive fibers represents the mean number of tin (RD181045050; BioVendor LLC, Candler, NC, USA), dMyHC-positive fibers minus the mean number of fibers monoclonal rat anti-mouse F4/80 (ab6640; Abcam, that were stained with the secondary antibody alone (that Cambridge, MA, USA), monoclonal mouse anti-rat de- is, 2.8 muscle fibers/cross-section in dy -L158 mice and W W velopmental myosin heavy chain (dMyHC) (NCL- 9 fibers/cross-section in dy /dy mice). In all histological MHCd; Novocastra, Norwell, MA, USA), monoclonal quantification experiments, at least four mice from each rat anti-mouse laminin-γ1 chain (MAB1914; Chemicon group were analyzed. (now EMD Millipore, Billerica, MA, USA)). Membrane- bound and extracellular epitopes were visualized with Western blotting analysis Alexa-488-conjugated wheatgerm agglutinin (WGA) Triceps brachii and diaphragm muscles were homoge- (Molecular Probes, Eugene, OR, USA). Depending on nized in radio-immunoprecipitation assay (RIPA) protein the source of the primary antibody, the appropriate Cy3- extraction buffer (Abcam). A commercial kit (23227; conjugated (Jackson ImmunoResearch Laboratories, BCA Protein Assay Kit; Pierce Biotechnology Inc., Rock- West Grove, PA, USA) or Alexa Fluor 488-conjugated ford, IL, USA) was used to determine protein concentra- (Molecular Probes) secondary antibodies or tetramethyl tions. Equal amounts of protein (20 μg) were separated rhodamine isothiocyanate (TRITC)-labeled streptavidin in 8% (periostin; 75 to 90 kDa) or 20% (transforming were used for visualization. DAPI (4',6'-diamidino-2- growth factor (TGF)-β; 12 kDa in reducing conditions) phenylindole hydrochloride) was used to stain nuclei. SDS–PAGE and transferred to nitrocellulose membrane. Pictures of stained cross-sections were taken using a The membrane was incubated with antibodies to perios- fluorescence microscope (DM5000B; Leica, Heerbrugg, tin (RD181045050; BioVendor) or TGF-β (PAB11274; Switzerland), a digital camera (F-View), and analySIS Abnova Corp., Taipei, Taiwan). An antibody to β-actin software (both Soft Imaging Systems Corp, Lakewood, (#4970; Cell Signaling Technology) was used as loading CO, USA). control. For detection, the appropriate horseradish Meinen et al. Skeletal Muscle 2012, 2:18 Page 4 of 13 http://www.skeletalmusclejournal.com/content/2/1/18 peroxidase-conjugated antibodies were used, and immu- 180 seconds. In all tests, at least 12 animals of each noreactivity was visualized using the enhanced chemilu- genotype were analyzed, and values were normalized to minescence detection method (32106; Pierce). those obtained from WT animals. Quantitative real-time PCR Statistical analysis The total RNA of the triceps brachii and diaphragm To compare the different genotypes, P-values were muscles was extracted (#Z3105; SV Total RNA Isolation calculated using the one-way ANOVA test. System; Promega, Corp., Madison, QI, USA). Samples were calibrated to equal amounts for cDNA synthesis using Results a commercial kit (#170–8891; iScript cDNA Synthesis Kit; Angiotensin II receptor type 1 (AT1) signaling and TGF-β W W Bio-Rad Laboratories, Inc., Hercules, CA, USA). SYBR levels are increased in dy /dy mice Green (4367659; Power SYBR Green PCR Master Mix) was In the first set of experiments, we tested whether TGF-β used to perform quantitative PCR in a PCR system levels were increased in muscles of laminin-α2-deficient W W (4376600; StepOnePlus™ Real-Time PCR System; both Ap- dy /dy mice. We found that TGF-β (Figure 1, green plied Biosystems, Foster City, CA, USA) with the following staining) was present in the perimysium and endomy- 0 W W primers for TGF-β1(sense: 5 GACTCTCCACCTGCAA sium of triceps muscle from dy /dy mice, but was ab- 0 0 GACCAT 3 and anti-sense: 5 GGGACTGGCGAGCCT sent in WT mice (Figure 1A). Accordingly, expression of 0 0 TAGTT 3)and on β-actin (sense: 5 CAGCTTCTTTG periostin and nuclear accumulation of phosphorylated 0 0 CAGCTCCTT 3 and anti-sense: 5 GCAGCGATATCGT Smad2/3 complexes (pSmad2/3), both downstream med- 0 Ct W W CATCCA 3 ) for normalization. The ΔΔ method was iators of TGF-β signaling, were seen in dy /dy but not used to analyze changes in TGF-β1 mRNA expression rela- in WT triceps (Figure 1A). Western blotting analysis tive to WT levels. confirmed the presence of TGF-β and periostin in dy / dy muscles (Figure 1B, first panel). Hydroxyproline assay To test whether L-158809 decreased TGF-β levels, we W W Fibrosis in the triceps brachii muscles was measured by treated dy /dy mice with L-158809 for 3 weeks. Stains assaying for the exclusive collagen-specific modified and immunoblots of triceps muscles from the treated W W W amino acid hydroxyproline. Tendons were carefully dy /dy mice (dy -L158) showed a strong reduction in removed before muscles were speed-dried under vac- TGF-β and periostin (right panels, Figure 1A and uum and sent for amino acid analysis (Analytical Figure 1B). Correspondingly, nuclear accumulation of W W Research Services; Bern, Switzerland) as described pre- pSmad2/3 complexes was 25 times higher in dy /dy viously [19]. There, muscles were hydrolyzed, then than in WT muscles, but was reduced to almost WT evaporated to dryness, and resuspended in 0.1% tri- levels after L-158809 treatment (Figure 1A, right panel; fluoroacetic acid. Amino acids were determined by a Figure 1C). The suppression of TGF-β by L-158809 was routine method [43] using high-performance liquid also seen for mRNA levels, which were less than 50% of W W chromatography (HPLC) to identify and quantify the those in dy /dy mice (Figure 1D). Importantly, TSP-1, amino acid hydroxyproline. The relative hydroxyproline which is increased by AT1 signaling [44], and has been amount was assessed with reference to the total amount shown to mediate activation of TGF-β in cardiac muscle of amino acids. and kidney [45], was present in the extracellular matrix of W W dy /dy mice, but was strongly reduced in L-158809- W W Notexin-induced muscle damage treated dy /dy triceps and diaphragm muscle (Figure 1E). The tibialis anterior (TA) muscle of 5 week-old mice These results indicate that AT1-mediated TGF-β signaling was injured by injection of 15 μl notexin (50 μg/ml; is increased in MDC1A , and that L-158809 dampens this Sigma-Aldrich) as described previously [21]. Mice were pathway. killed 5 days after injection, and muscles were isolated and processed as described above. Angiotensin II type 1 receptor antagonism improves fibrosis, inflammation, and overall histology Body weight, locomotion, and grip strength To test for a beneficial effect of L-158809, we treated W W Body weight was measured at the age of 7 weeks before dy /dy mice starting at the age of 3 weeks and con- mice were sacrificed. Locomotive behavior was recorded tinuing until the age of 7 weeks. First, we analyzed by placing the mice into a new cage and measuring the potential of L-158809 to reduce fibrosis and inflam- W W motor activity (walking, digging, stand-ups on hindlegs) mation in triceps and diaphragm muscle of dy /dy for 10 minutes [25]. Grip strength was evaluated by pla- mice. Masson trichrome staining showed a more prom- cing the animals onto a vertical grid, and measuring inent replacement of muscle tissue with non-muscle W W the time until they fell down; the cut-off time was cells in untreated dy /dy mice than in mice treated Meinen et al. Skeletal Muscle 2012, 2:18 Page 5 of 13 http://www.skeletalmusclejournal.com/content/2/1/18 Figure 1 L-158809 lowered angiotensin II receptor type 1 (AT1) -mediated transforming growth factor (TGF)-β levels in the W W muscles of dy /dy mice. (A) AT1-mediated TGF-β levels were W W increased in muscles of dy /dy mice and decreased after L-158809 W W administration. Triceps cross-sections of dy /dy mice showed expression of TGF-β (green) and periostin (green), and nuclear accumulation of pSmad2/3 (green), which is a downstream target of W W W AT1. Oral L-158809 treatment of dy /dy mice (dy -L158) reduced expression of all three proteins. Nuclei were visualized with 4',6'- diamidino-2-phenylindole hydrochloride (DAPI) (blue). (B) Western blotting analysis of triceps muscles confirmed increased protein expression levels of periostin (75 to 85 kDa) and TGF-β (12 kDa, W W reduced) in dy /dy mice as well as the reduction after L-158809 treatment. β-actin was used as a loading control. (C) The number of pSmad2/3-positive nuclei was more than 25-fold increased in cross- W W sections of dy /dy muscle compared with wild-type (WT) muscles, and was significantly reduced by L-158809 to levels 2-fold (triceps) and 2.6-fold (diaphragm) those of WT mice, which was not significant (n ≥ 4). (D) Quantitative real-time PCR showed an increase of approximately 3.5-fold in TGF-β1 mRNA levels in both W W dy /dy triceps and diaphragm muscles, which was reduced to nearly WT levels by L-158809 (n ≥ 4). (E) Thrombospondin-1 (green) W W was increased in both triceps and diaphragm muscles of dy /dy mice, and was minimized by L-158809 administration. All values represent the mean ± SEM. One-way ANOVA: **P ≤ 0.001; * P ≤ 0.05; n.s. (non-significant) P > 0.05. Scale bar = 50 μm. with L-158809 (Figure 2A). The blue color, indicative of collagen, suggests that the non-muscle cells found in un- treated muscles represent mainly fibrotic tissue (Figure 2A). The fibrotic area measured in cross-sections W W of triceps and diaphragm muscle of dy /dy mice trea- ted with L-158809 was less than 50% of that seen in un- treated mice (Figure 2B). As an independent measure of fibrosis, we also determined the hydroxyproline content in triceps muscles. In agreement with the histological measurements, L-158809 reduced the hydroxyproline W W content in dy /dy muscles (Figure 2C). Because TGF- β plays an important role in regulating skeletal-muscle inflammation [38,46], we also assessed inflammation using the F4/80 antibody that recognizes activated macrophages (Figure 2D,E). Macrophages were mainly located in areas where muscle fiber degeneration is tak- W W ing place (Figure 2D). In untreated dy /dy muscles, around 100 macrophages were found per mm , whereas L-158809 reduced this number by six times in triceps and by three times in diaphragm muscle (Figure 2E). These data provide evidence that L-158809 inhibits and thus minimizes TGF-β-induced fibrosis and inflamma- W W tion in muscles of dy /dy mice. We next investigated whether the L-158809-mediated reduction of fibrosis also ameliorated other histological hallmarks of MDC1A. A prominent feature of laminin- α2-deficient muscles is the loss of muscle fibers, which is probably due to muscle degeneration and the difficulty in muscle regeneration [18]. Indeed, the number of W W muscle fibers in dy /dy mice was much lower than in Meinen et al. Skeletal Muscle 2012, 2:18 Page 6 of 13 http://www.skeletalmusclejournal.com/content/2/1/18 Figure 2 (See legend on next page.) Meinen et al. Skeletal Muscle 2012, 2:18 Page 7 of 13 http://www.skeletalmusclejournal.com/content/2/1/18 (See figure on previous page.) W W Figure 2 L-158809 reduces fibrosis and inflammation in the muscles of dy /dy mice. (A) Masson trichrome staining showed higher W W W collagen content (blue) in triceps and diaphragm muscles of dy /dy when compared with dy -L158 mice. (B) Average amount of fibrotic area relative to wild-type (WT) muscles. L-158809 treatment more than halved the fibrotic area in triceps and diaphragm muscles (n ≥ 4). W W (C) Measurement of the amount of hydroxyproline as an indicator of fibrosis. L-158809 treatment of dy /dy mice reduced the hydroxyproline amount in triceps muscle (n = 3). (D) F4/80 staining of macrophages in diaphragm muscles. (E) L-158809 treatment reduced the number of W W macrophages in muscles of dy /dy mice (n ≥ 4). All values represent the mean ± SEM. One-way ANOVA: **P ≤ 0.001; * P ≤ 0.05; n.s. (non-significant) P > 0.05. Scale bar = 50 μm. WT mice, with the triceps and diaphragm, respectively To test the effect of L-158809 on the regenerative cap- losing 46% and 40% of their fibers (Figure 3A). L-158809 acity, the TA muscle was injured by injection of notexin. decreased this fiber loss in triceps muscle, and increased Four days after injury, only a few dMyHC-positive fibers W W the fiber number to 68% of WT. In diaphragm, were found in dy /dy mice, whereas many were L-158809 showed a similar trend, but this was not sig- present after L-158809 treatment or in WT muscle nificant (Figure 3A). Further, L-158809 did not affect (Figure 4D). The number of regenerating fibers increased W W fiber size distribution in dy /dy triceps muscle from 61 ± 14 in untreated to 490 ± 25 in L-158809-treated W W (Figure 3B). In diaphragm, where muscle fiber-size dis- dy /dy TA muscle (Figure 4E). Thus, L-158809-mediated tribution was only slightly shifted to smaller diameters TGF-β inhibition improved the regeneration capacity in W W W W in dy /dy mice, L-158809 shifted fiber-size distribu- muscles of dy /dy mice. Because a similar improve- W W tion only slightly towards the values in WT (Figure 3C). ment in muscle regeneration in dy /dy mice has also Accordingly, the minimal Feret’s variance coefficient [42] been reported for treatments that inhibited apoptosis W W was increased in dy /dy muscles but was not cor- [47], we also tested whether L-158809 would influence rected by L-158809 (Figure 3D). However, because of the number of apoptotic nuclei. However, we could not the higher number of muscle fibers in L-158809-treated detect any effect of this treatment on the number of W W dy /dy mice, the mean cross-section area of the terminal deoxynucleotidyl transferase-mediated dUTP muscle was significantly increased (Figure 3E). Thus, nick end labeling-positive myonuclei (see Additional file 1: L-158809 partially prevents the loss of muscle fibers, but Figure S1), indicating that L-158809 does not affect this W W does not significantly affect the diameter of dy /dy pathway. muscle fibers. W W Effect of L-158809 on overall health of dy /dy mice Finally, we tested whether there was any improvement L-158809 strongly improves spontaneous and injury- in motor function and overall health in L-158809- W W induced muscle regeneration treated dy /dy mice, and found that L-158809 W W W W The muscles of dy /dy mice are strongly impaired in increased the body weight of dy /dy mice, by 2 g muscle regeneration [18,19,21]. In addition, increased (Figure 4F). However, age-matched WT animals still TGF-β activity leads to failure of muscle regeneration weighed more than twice as much. The muscle strength W W [1]. Therefore, L-158809 may improve regeneration of of dy /dy mice, measured as the time the animals W W dy /dy muscle. However, the number of centrally could stay on a vertical grid, was doubled by treatment nucleated fibers, which are indicative of ongoing degen- with L-158809 from 26 seconds to 51 seconds eration/regeneration, were not significantly increased (Figure 4G); however, the WT animals far exceeded the W W after L-158809 administration (Figure 4A). By contrast, time measured with L-158809-treated dy /dy mice, staining of diaphragm muscle with the regeneration normally staying more than 180 seconds on the grid. In marker dMyHC (Figure 4B, green) indicated that more the 10-minute open-field locomotion test L-158809- W W fibers regenerated after L-158809 treatment of dy /dy treated mice were more active and explored the new sur- mice. Muscle fibers expressing dMyHC in the dy -L158 roundings for 5 minutes, whereas untreated MDC1A mice appeared to have a proper basement membrane as mice were active only for 3.5 minutes (Figure 4H). Thus, indicated by laminin-γ1 staining (red), whereas in non- the ameliorating effect of L-158809 on muscle histology W W treated dy /dy mice, dMyHC staining was often punc- is sufficient to improve body and muscle condition of W W tated and the basement membrane appeared disrupted, dy /dy mice. indicating that the fibers fail to complete regeneration (Figure 4B). In both triceps and diaphragm muscle, Discussion L-158809 clearly increased the number of intact Muscular dystrophies are caused by mutations in many dMyHC-positive fibers (Figure 4C), suggesting that different genes but they are often accompanied by a regeneration was improved by L-158809. strong inflammatory and fibrotic response, which might Meinen et al. Skeletal Muscle 2012, 2:18 Page 8 of 13 http://www.skeletalmusclejournal.com/content/2/1/18 W W W Figure 3 L-158809 reduced muscle-fiber loss but did not change fiber-size distribution in dy /dy muscles. (A) L-158809 (dy -L158) W W stabilized the loss of muscle fibers in dy /dy triceps muscle, but only tended to reduce fiber loss in the diaphragm (n = 6). (B,C) L-158809 did W W not normalize the shift of fiber sizes to smaller fibers, either (B) in triceps or (C) in diaphragm muscle of dy /dy mice (n ≥ 4). (D) The minimal Feret`s diameter coefficient was unchanged by L-158809 (n ≥ 4). (E) L-158809 increased the cross-sectional area in both mid-belly triceps (P = 0.0007) and diaphragm (P = 0.0024) muscles (n ≥ 4). All values represent the mean ± SEM. P-values (one-way ANOVA): **P ≤ 0.001; * P ≤ 0.05; n.s. (non-significant) P > 0.05. be triggered by similar signaling pathways. For example, the decreased TGF-β levels and Smad2/3 phosphoryl- 2J 2J TGF-β signaling has been shown to be upregulated in ation seen in dy /dy mice [37]. Previous work in kid- several muscle diseases [8-10], and is known to inhibit ney [44] has shown that the AT1-mediated increase in myoblast differentiation and promote fibrosis, and also TSP-1 involves the MAPK pathway. This pathway may to impair regeneration by inhibiting satellite cell activa- also be active in MDC1A, as the MAPKs ERK1/2, p38 2J 2J tion, proliferation, and differentiation [3,4]. and JNK are all increased in dy /dy mice, and this in- In this study, we found that TGF-β signaling, including crease can be inhibited by losartan [37]. increased levels of TGF-β and of the phosphorylated forms of Smad2 and Smad3, was also enhanced in the Angiotensin II type 1 receptor antagonists have a strong W W W W severe dy /dy mouse model of MDC1A (Figure 1). effect on muscle regeneration in dy /dy mice These data are in good agreement with previous reports Increased TGF-β signaling inhibits activation of satellite that TGF-β levels are also upregulated in the muscles cells [48] and promotes differentiation of myogenic into of human patients with MDC1A [38] and in the mild fibrotic cells in injured skeletal muscles [3], indicating that 2J 2J 2J 2J dy /dy mouse model [37]. As in the study on dy /dy TGF-β-dependent fibrosis is associated with impaired mice, we found an increase in the TGF-β-activating pro- muscle regeneration. In mouse models of MFS and tease TSP-1, indicating that the elevation of TGF-β is Duchenne muscular dystrophy (DMD), losartan treatment probably caused by the activation of AT1, as production restored the muscle-regeneration capacity after cardiotoxin- of TSP-1 precedes TGF-β activation in this pathway induced muscle injury [1]. In mice with sarcopenia, losartan [1,2,44,45]. Indeed, inhibition of AT1 signaling by oral restored the necessary downregulation of Pax7 and MyoD administration of L-158809 for only 3 weeks was suffi- and upregulation of p21 and myogenin, which are required cient to lower the amount of TSP-1 in muscle basement for successful completion of regeneration of injured muscle, membrane and to normalize TGF-β levels and Smad2/3 by modulating TGF-β signaling both via Smad2/Smad3 phosphorylation (Figure 1). These experiments provide phosphorylation and via activation of MAPKs [6,13]. strong evidence that the AT1-TSP-TGF-β axis is also Muscle regeneration is largely impaired in MDC1A, and we activated in MDC1A and they are in accordance with have recently shown that the main effect of anti-apoptosis Meinen et al. Skeletal Muscle 2012, 2:18 Page 9 of 13 http://www.skeletalmusclejournal.com/content/2/1/18 Figure 4 (See legend on next page.) Meinen et al. Skeletal Muscle 2012, 2:18 Page 10 of 13 http://www.skeletalmusclejournal.com/content/2/1/18 (See figure on previous page.) W W Figure 4 L-158809 improved muscle regeneration, body weight, locomotion, and muscle strength in dy /dy mice. (A) L-158809 W W W (dy -L158) did not significantly affect the number of centrally nucleated fibers (CNF) in either triceps or diaphragm muscle of dy /dy mice (n ≥ 4). (B) Staining of diaphragm muscle for the regeneration marker developmental myosin heavy chain (dMyHC; green), the basement membrane marker laminin-γ1 (red), and the nuclear marker 4',6'-diamidino-2-phenylindole hydrochloride (DAPI) (blue). L-158809 enabled W W W W damaged dy /dy muscle fibers to regenerate, whereas in non-treated dy /dy muscle many dMyHC-expressing cells continued degenerating. W W (C) L-158809 application strongly increased the number of intact regenerating muscle fibers in dy /dy mice (n ≥ 4). (D) Tibialis anterior (TA) muscle stained for dMyHC (green), laminin-γ1 (red), and DAPI four days after notexin-induced injury. L-158809 increased the regenerative capacity W W W W of damaged dy /dy muscle. (E) The number of dMyHC-expressing fibers in TA muscle of dy /dy mice 5 days after notexin injection was more W W than 8 times higher in L-158809 treated dy /dy mice but was still lower than in controls (n = 3). (F) L-158809 increased the average body W W weight of dy /dy mice from 7.6 g to 9.5 g, although age-matched wild-type (WT) mice weigh more than 20 g. (n ≥ 12). (G). L-158809 doubled W W the time a dy /dy mouse could hold itself on a vertical grid, from 26 seconds to 51 seconds (P = 0.0001) (n ≥ 14). (H) L-158809 increased the W W time dy /dy mice explored an unknown surrounding (P = 0.0004) within 10 minutes (n ≥ 12). All values represent the mean ± SEM (n ≥ 4). One-way ANOVA: **P ≤ 0.001; * P ≤ 0.05; n.s. (non-significant) P > 0.05. Scale bar = 50 μm, if not indicated differently. W W treatment in dy /dy mice is the prevention of cell death hallmarks of the condition, such as fiber loss and muscle during the regeneration process [47]. In addition, this area. In contrast to its effects in mdx mice, L-158809 did previous work also provided evidence that the cell death not correct the shift of the fiber-size distribution to smal- W W prevention by anti-apoptosis treatment had a synergistic ler fibers in dy /dy mice. This phenomenon was due to effect when combined with the re-establishment of the con- the presence of many newly formed regenerating nection between the basement membrane and the muscle (dMyHC-positive) fibers that were not grown to full size. membrane by mini-agrin [47]. Treatment with L-158809 also resulted in a significant In the current study, we found evidence that AT1 block- functional improvement in skeletal muscle, manifesting as ade by L-158809 also ameliorated the muscle regeneration increased locomotor activity and increased grip strength. W W capacity in dy /dy mice. Importantly, we found that Interestingly, an improvement of the hind-limb and fore- 2J 2J L-158809 treatment enabled newly formed muscle fibers limb strength was also seen in the dy /dy mouse model to stay intact and to complete regeneration, whereas of MDC1A [37]. W W non-treated dy /dy fibers started to regenerate, but then turned into fibrotic cells. The beneficial effect of Therapeutic potential of the different treatment options L-158809 on regeneration is superior to that seen in The highest potential to restore function in mouse models W W dy /dy mice treated with an apoptosis inhibitor alone for MDC1A is the body-wide, transgenic expression of [47], and reached almost the same efficacy as the laminin-α1, which largely cures the disease [26,27]. How- combination of apoptosis inhibition and mini-agrin [47]. ever, because the cDNA encoding laminin-α1is morethan Our observation of a relatively strong beneficial effect of 9 kb in size, its use in gene therapy is challenging, and no AT1 antagonists on muscle regeneration is in good successful attempts in creating smaller versions of the accordance with the finding in mice with sarcopenia, in functional protein have been reported. Another very which losartan enabled the regeneration of skeletal muscle effective treatment option is the muscle-specific overex- by fostering satellite cells to transit from a proliferation pression of a miniaturized form of agrin (mini-agrin), stage into the differentiation process [13]. Our finding that specifically designed to structurally reconnect the cell L-158809 treatment lowered the number of centrally surface protein α-dystroglycan to laminins in the basement located myonuclei suggests that AT1 blockade might also membrane [19,21,25]. Finally, overexpression of insulin- prevent necrosis of muscle fibers. like growth factor (IGF)-1 also ameliorates disease and prolongs life span [49], but its efficacy is clearly lower than Effect of Angiotensin II receptor type 1 antagonist on that of mini-agrin. This lower efficacy is probably based on fibrosis, inflammation, and overall muscle function the fact that IGF-1 interferes with rather late events in the Previous work has provided evidence that AT1 antagonists course of the disease, whereas mini-agrin tackles the struc- can ameliorate disease in mouse models of MFS and tural deficits that are the primary cause of the muscular DMD and in mice with sarcopenia [1,13-15]. We found dystrophy. Recombinant AAV-based gene therapy with W W that 4 weeks of L-158809 treatment decreased fibrosis mini-agrin was shown to be successful in dy /dy mice from 19.7% to 8.1% in triceps and from 23.3% to 10% in [28]. However, the use of gene therapy in clinics has been diaphragm muscle, and thereby prevented the characteristic rather slow, and thus delivery of mini-agrin to the muscles inflammatory response. Similarly, 12 weeks of losartan of patients with MDC1A remains difficult. 2J 2J treatment reduced the fibrotic area in dy /dy by This difficulty in translating gene therapy into clinics approximately 4% [37]. In addition, L-158809 treatment in has led to attempts to interfere with disease progression W W dy /dy mice corrected some of the histological using protein therapy and pharmacological compounds. Meinen et al. Skeletal Muscle 2012, 2:18 Page 11 of 13 http://www.skeletalmusclejournal.com/content/2/1/18 An interesting approach that has been shown to be survival in a large cohort of animals and could only rec- W W W efficacious in dy /dy mice is the systemic delivery of ord age at death in four cases of L-158809-treated dy / laminin-111 (heterotrimer of the α1, β1, and γ1 chains). dy mice. Nevertheless, all four mice lived at least W W In those studies, laminin-111 reached the skeletal 5 weeks longer than the untreated dy /dy mice muscles, improved muscle histology, and prolonged life (~10 weeks in average). Thus, as L-158809 or losartan span [50]. Pharmacological approaches include the use of does not act on apoptosis (see Additional file 1: Figure the apoptosis inhibitors omigapil [30] or of doxycycline or S1), it remains to be tested whether a combination of W W minocycline [31] to treat dy /dy mice. In both studies, losartan and omigapil could provide an additive benefit W W there was a clear effect on functional parameters and on in dy /dy mice. The fact that losartan is well tolerated the longevity of the mice; however, the efficacy of either in all age groups and that omigapil has proven to be safe W W treatment was much lower than in dy /dy mice expres- in clinical trials of patients with Parkinson’s disease and sing mini-agrin [47]. those with amyotrophic lateral sclerosis [52,53], makes Another consequence of muscular dystrophies is a such a combination attractive for further clinical trials. severe muscle-wasting. Preservation of muscle mass depends on the proper balance between protein synthe- Conclusion sis and protein degradation [51]. Thus, muscle wasting In this study, we investigated the efficacy of AT1 antago- can be due to a prevalence of protein degradation, and nists in the most widely used mouse model of MDC1A. based on this idea, pharmacological inhibition of the two The benefits of the therapy included decreased fibrosis main systems involved in protein degradation, the pro- and inflammation, improved muscle regeneration (which teasomal and the autophagic pathway, has been tested in leads to preservation of muscle-fiber number), and the preclinical mouse models for MDC1A. Again, inter- improved locomotion and grip strength. The AT1 antag- ference with either of those pathways ameliorated dis- onist losartan is an approved anti-hypertensive drug that ease progression [33,34]. It is, however, questionable if is well tolerated, including in children. And, importantly, such an approach is a good option for the treatment of losartan harbors fewer potential side-effects than any patients with MDC1A, as inhibition of proteosomal and other pharmacological options that have been tested to autophagosomal degradation has a strong likelihood of date in preclinical mouse models of MDC1A. Therefore, causing severe side-effects. AT1 blockade could provide a supportive treatment that In the current study, we provide evidence that AT1 alleviates some of the pathology in patients with W W antagonists also provide benefit to dy /dy mice. Our MDC1A in the near future. results are similar to those of other researchers who tested inhibition of TGF-β-induced fibrosis by intraperi- Additional file 2J 2J toneal injection of halofuginone in the milder dy /dy mouse model of MDC1A [36]. Halofuginone blocked Additional file 1: Figure S1. TGF-β-mediated collagen synthesis, prevented muscle fibrosis, and improved the performance of both muscles Abbreviations AAV: Adeno-associated virus; AT1: Angiotensin II receptor type 1; and animals. In addition, the same group also used losar- DMD: Duchenne muscular dystrophy; dMyHC: Developmental myosin heavy W W W tan to block TGF-β-signaling and this also showed bene- chain; dy /dy : Laminin-α2-deficient MDC1A mouse model; dy -L158: L- 2J 2J W W fit in dy /dy mice [37]. Thus, our work is an important 158809-treated dy /dy mice; Fbn1C1039G/+: MFS mouse model; mdx: DMD mouse model; MDC1A: Laminin-α2-deficient congenital muscular dystrophy; confirmation of efficacy of AT1 antagonists in the more MFS: Marfan syndrome; Mini-agrin: Miniaturized form of agrin specifically severe mouse model of MDC1A. designed to contain laminin and α-dystroglycan binding domains; WT: Wild-type. It is difficult to compare studies that were performed W W 3K Competing interests in dy /dy mice with those using the more severe dy / 3K 2J 2J The authors declare no competing interests dy mouse model or the much milder dy /dy model. W W As our current study used dy /dy mice, it can be com- Authors' contributions SM performed and analyzed the experiments. SL assisted in some of the pared with those using the pharmacological apoptosis experiments, and provided scientific input. SM and MAR conceived and inhibitors omigapil or doxycycline. A detailed analysis of designed the study, and wrote the manuscript. All authors read and those studies indicates that all of the treatments are of approved the final manuscript. comparable benefit to the mice. For example, they all Acknowledgments increased body weight by approximately 2 g and in the We thank Merck for generously providing us with L-158809, a highly potent open-field locomotion test, both L-158809 and omigapil AT1 antagonist and derivative of losartan. This work was supported by the increased activity time by 1.5 minutes. Treatment with Neuromuscular Research Association Basel (NeRAB) and the The Swiss Foundation for Research on Muscle Diseases. omigapil and doxycycline approximately doubled the W W lifespan of dy /dy mice. Because of changes in the Received: 16 May 2012 Accepted: 31 July 2012 laws regarding animal testing, we could not study the Published: 3 September 2012 Meinen et al. Skeletal Muscle 2012, 2:18 Page 12 of 13 http://www.skeletalmusclejournal.com/content/2/1/18 References laminin alpha 2-chain gene (LAMA2) cause merosin-deficient congenital 1. Cohn RD, van Erp C, Habashi JP, Soleimani AA, Klein EC, Lisi MT, Gamradt M, muscular dystrophy. 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Skeletal MuscleSpringer Journals

Published: Sep 3, 2012

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