Novel and optimized strategies for inducing fibrosis in vivo: focus on Duchenne Muscular Dystrophy
© Pessina et al.; licensee BioMed Central Ltd. 2014
Received: 29 October 2013
Accepted: 20 January 2014
Published: 25 August 2014
Fibrosis, an excessive collagen accumulation, results in scar formation, impairing function of vital organs and tissues. Fibrosis is a hallmark of muscular dystrophies, including the lethal Duchenne muscular dystrophy (DMD), which remains incurable. Substitution of muscle by fibrotic tissue also complicates gene/cell therapies for DMD. Yet, no optimal models to study muscle fibrosis are available. In the widely used mdx mouse model for DMD, extensive fibrosis develops in the diaphragm only at advanced adulthood, and at about two years of age in the ‘easy-to-access’ limb muscles, thus precluding fibrosis research and the testing of novel therapies.
We developed distinct experimental strategies, ranging from chronic exercise to increasing muscle damage on limb muscles of young mdx mice, by myotoxin injection, surgically induced trauma (laceration or denervation) or intramuscular delivery of profibrotic growth factors (such as TGFβ). We also extended these approaches to muscle of normal non-dystrophic mice.
These strategies resulted in advanced and enhanced muscle fibrosis in young mdx mice, which persisted over time, and correlated with reduced muscle force, thus mimicking the severe DMD phenotype. Furthermore, increased fibrosis was also obtained by combining these procedures in muscles of normal mice, mirroring aberrant repair after severe trauma.
We have developed new and improved experimental strategies to accelerate and enhance muscle fibrosis in vivo. These strategies will allow rapidly assessing fibrosis in the easily accessible limb muscles of young mdx mice, without necessarily having to use old animals. The extension of these fibrogenic regimes to the muscle of non-dystrophic wild-type mice will allow fibrosis assessment in a wide array of pre-existing transgenic mouse lines, which in turn will facilitate understanding the mechanisms of fibrogenesis. These strategies should improve our ability to combat fibrosis-driven dystrophy progression and aberrant regeneration.
In skeletal muscle, accumulation of collagens (fibrosis) in the extracellular matrix (ECM) is most often associated with the muscular dystrophies, characterized by muscle wasting, leading to loss of patient mobility. Duchenne muscular dystrophy (DMD) is one of the severest of the dystrophies and is caused by loss of the dystrophin protein due to genetic mutations. As a result, the sarcolemma becomes fragile and susceptible to contraction-induced damage . Skeletal muscle stem cells (satellite cells) mediate the repair process, but in the absence of dystrophin, the muscle undergoes continuous cycles of degeneration and regeneration, eventually leading to satellite cell depletion and myofiber loss [2–4]. The severity of this childhood-associated pathology may also be exacerbated by the growth of myofibers that occurs in boys with DMD over many years . Affected children eventually succumb to muscle wasting, with muscle progressively being replaced by fat and fibrotic tissue, leading to premature death in the late teens or early twenties from respiratory and cardiac failure . There are currently only palliative treatments for DMD patients. Importantly, no effective clinical treatments are available yet to combat or attenuate fibrosis in patients with DMD. Halting or diminishing the development of fibrosis could not only ameliorate DMD progression, but could also increase the success of new cell- and gene-based therapies [7, 8]. The mdx mouse strain, the most widely used animal model for studying human DMD, has a nonsense mutation in dystrophin exon 23 leading to dystrophin protein absence . Although mdx mice and DMD patients share many genetic, biochemical and histological similarities, the clinical manifestations are generally less severe in mdx mice . While DMD individuals have a high degree of muscle fibrosis, mdx mice present extensive fibrosis exclusively in the diaphragm muscle. In the limb muscles of mdx mice, however, fibrosis only becomes apparent around 20 months of age . Therefore, despite our awareness of the importance of fibrosis in DMD, there is a lack of appropriate mouse models for studying dystrophic skeletal muscle fibrosis in accessible muscles, such as limb muscles, without requiring nearly two years for fibrosis to appear. Thus, there is a genuine need to develop mouse models that present fibrosis at early stages in life and that more closely mimic human DMD.
In this manuscript, we present several experimental strategies to simply and effectively advance and enhance muscle fibrosis in young mdx mice. We use both physiological exercise, as well as more direct tissue-damaging procedures or delivery of profibrotic growth factors to limb muscle of young dystrophic mice, and demonstrate sustained collagen deposition reminiscent of aged mdx diaphragm muscle, and muscle of human DMD patients. Notably, we could extend these strategies to induce fibrosis in muscles of normal, non-dystrophic mice, which will facilitate studying fibrosis in a wide array of genetically modified mouse lines, and this will in turn increase our understanding of the cells and molecules involved in fibrosis development. Thus, we offer for the first time, to the best of our knowledge, a comparative and quantitative set of new and improved strategies for inducing muscle tissue fibrosis, which will greatly foster our ability to combat fibrosis-dependent dystrophy progression.
Mice handling and sample collection
All experiments were approved by the Ethics Committee of the Pompeu Fabra University (UPF) and performed according to Spanish and European legislation. Mice were housed in standard cages under 12-hour light–dark cycles and fed ad libitum with a standard chow diet. Three-month-old normal C57Bl/6 J mice (the classic standard laboratory mouse strain, hereafter referred to as WT) and dystrophic C57Bl/10scsn-mdx (mdx) male mice were used in experiments: the background strain for mdx mice is similar to, but not identical with, the C57Bl/6 J strain. All operations were performed after injection intraperitoneal (i.p.) of ketamine/metedomidine anesthesia (50 mg/kg and 1 mg/kg body weight). Atipamezol (1.0 mg/kg body weight) by subcutaneous injection was used to reverse the effects of anesthesia. Mice were sacrificed at the indicated ages and the tissues were immediately processed to avoid artifacts, either by direct freezing in liquid nitrogen for protein and RNA extraction or in 2-methylbutane cooled with liquid nitrogen for histological analysis, as described below.
Skeletal muscle fibrogenic treatments
Chronic exercise: Mdx mice were exercised three times per week on a treadmill for 30 minutes at a speed of 12 meters per minute, with a rest of 5 minutes every 10 minutes of exercise. Mdx mice of three, four and five months of age were exercised for three, two and one month, respectively and were sacrificed at the age of six months, together with age-matched unexercised control mdx mice. At the end of the training period muscles were collected and processed for further analyses.
Single treatments in WT and mdx mice:
Cardiotoxin injury: Tibialis anterior (TA) muscles of three-month-old WT or mdx mice were injected with 50 μl of 10–5 M cardiotoxin (CTX; Latoxan, Rosans, France). Muscles were collected at the indicated times on each set of experiments, which was usually two weeks after myotoxin injection. Muscle samples were also obtained at one month post-injection (in WT mice) and two months post-injection (in mdx mice).
Barium chloride injury: TA muscle of three-month-old WT mice was injected with 50 μl of 0.2% barium chloride (BaCl2) and was isolated after two weeks. For repeated BaCl2 injuries, BaCl2 injections were made in the same muscle, one per week for six weeks, and muscles were isolated and collected for analysis two weeks after the last injection.
Laceration: TA muscles of three-month-old WT or mdx mice were subjected to laceration (LAC) as previously described [11, 12]. Briefly, the skin was carefully cut and separated from the underlying tissue, then the TA muscle of one leg was cut horizontally at its middle of the length by making a lesion through 75% of their width and 50% of the muscle thickness with a scalpel. Contralateral control muscles were sham-operated. In WT mice, muscles were collected at two weeks and one month post-surgery. In mdx mice, muscles were obtained at two weeks and two months post-surgery.
Denervation: Muscle denervation (DEN) was performed as previously described [13, 14]. In brief, a 5 mm segment of the sciatic nerve was surgically removed down to the gluteus maximum from the right legs. In three-month-old WT mice, TA muscles were isolated for analysis at two weeks and one month post-surgery. In three-month-old mdx mice, TA muscles were collected at two weeks and two months post-surgery. Contralateral muscles of sham-operated mice were used as controls on every single treatment (CTX, BaCl2, LAC and DEN).
Profibrotic growth factor treatments:
Transforming growth factor beta treatment: 50 ng of transforming growth factor beta 1 (TGFβ1) (recombinant human TGFβ1; R&D Systems, Minneapolis, MN, USA) were injected in the TA muscle in a volume of 50 μl of phosphate-buffered saline (PBS, vehicle). Two injections (one per week) were made in TA muscle of three-month-old mdx mice, and muscles were collected for analysis two weeks or two months after the first injection.
Connective tissue growth factor delivery: TA muscles of three-month-old mdx mice were injected with 1 × 1011 viral particles of Ad-m connective tissue growth factor (CTGF) or Ad-GFP or with PBS (vehicle)  as a control in a total volume of 50 μl. Muscles were collected two weeks after injury.
Combined treatments in WT mice:
Cardiotoxin injury combined with denervation: TA muscles of three-month-old WT mice were injected with 50 μl of 10–5 M CTX immediately after DEN, as indicated above. Muscles were collected at two weeks and one month after the treatments. Contralateral muscles of non-denervated left legs were used as controls.
Cardiotoxin injury combined with TGFβ/CTGF treatment: TA muscles of three-month-old WT mice were injected with 50 μl of 10–5 M CTX. TGFβ1 was injected intramuscularly twice at day 7 and 10 after cardiotoxin injection. Muscles were collected at two weeks and one month after the cardiotoxin injection. Contralateral muscles of sham-operated legs were used as controls. When indicated, CTGF adenoviral delivery was performed immediately after cardiotoxin injection.
Specific information about starting and sampling ages of mice after the different experimental protocols is included in Table S1 in Additional file 1.
Dystrophic patients study
Human samples were provided from Dr. J. Colomer (Hospital Sant Joan de Deu, Barcelona, Spain). DMD diagnosis was established on a total absence of dystrophin by immunohistochemistry and Western blotting. Muscle samples were obtained by a standard quadriceps muscle biopsy from six DMD patients (ranging from five to eleven years of age) and five healthy male human controls of similar age (seven to fourteen years). Quantification of fibrosis was carried out by color image segmentation and automatic measurement using Fiji image analysis software . The ratio of the total area of fibrosis to the total biopsy area was used to estimate the extent of fibrosis (fibrosis index). Histological analysis was performed similarly to mouse samples, as explained in the next section.
Histological analysis and immunohistochemistry
Cryosections (10 μm thickness) were stained with hematoxylin/eosin (H&E) or Sirius red (Sigma-Aldrich, St Louis, MO, USA). Quantification of collagen content in muscle was performed according to Ardite et al. . Briefly, 10 cryosections were collected in a tube and were sequentially incubated with a solution containing 0.1% Fast green in saturated picric acid, washed with distillated water, incubated with 0.1% Fast green and 0.1% Sirius red in saturated picric acid, washed with distillated water, and gently resuspended in a solution of 0.1 M NaOH in absolute methanol (1 vol:1 vol). Absorbance was measured in a spectrophotometer at 540 and 605 nm wavelengths and used to calculate total protein and collagen.
Immunohistochemistry on frozen sections was performed using the following primary antibodies: rabbit polyclonal collagen I (Coll 1) (Millipore, Billerica, MA, USA), rabbit polyclonal fibronectin (FN) (Abcam, Cambridge, MA, USA) and rabbit polyclonal phosphorylated-Smad2/3 (P-Smad2/3) (Abcam). For immunoperoxidase staining, labeling of sections was performed using the peroxidase staining kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. For immunofluorescence, secondary antibodies were coupled to Alexa Fluor 488 or 568 fluorochromes (Invitrogen, Carlsbad, CA, USA). Stained sections were photographed on a Leica DM6000B microscope (Leica Microsystems, Wetzler, Germany).
RNA isolation, reverse transcription (RT) and real-time quantitative PCR
Total RNA was isolated from muscle tissue using Trizol (Invitrogen). cDNA was synthesized from 1 μg of total RNA using the First Strand cDNA Synthesis kit and random priming according to the manufacturer’s instructions (Promega, Madison, WI, USA). RT-PCR was performed on a LightCycler 480 System using LightCycler 480 SYBR Green I Master Mix (Roche, Basel, Switzerland) with 10 μM each primer and normalized to L7 ribosomal RNA as a housekeeping gene: mL7 5′-GAAGCTCATCTATGAGAAGGC–3′ and 5′–AAGACGAAGGAGCTGCAGAAC-3′; mCollagen I, 5′-GGTATGCTTGATCTGTATCTGC-3′ and 5′-AGTCCAGTTCTTCATTGCATT-3′; mCTGF, 5′-CAGGCTGGAGAAGCAGAGTCGT-3′ and 5′-CTGGTGCAGCCAGAAAGCTCAA–3′; mTIMP-1 5′-TTCCAGTAAGGCCTGTAGC-3′ and 5′-TTATGACCAGGTCCGAGTT-3′; mTGFβ 5′-TATGACCAGGTCCGAGTT-3′ and 5′-CTGGTGCAGCCAGAAAGCTCAA-3′; hFibronectin: 5′- GGATGACAAGGAAAATAGCCCTG-3′ and 5′-GAACATCGGTCACTTGCATCT-3′; hTIMP-1 5′-CTTCTGCAATTCCGACCTCGT-3′ and 5′-CCCTAAGGCTTGGAACCCTTT-3′; hTGFβ 5′-CCTAA GGCCAGATCCTGTCCAAGC-3′ and 5′- GTGGGTTTCCACCATTAGCAC-3′; hCTGF 5′- CAAGGGCCTCTTCTGTGACT-3′ and 5′-ACGTGCACTGGTACTTGCAG-3′.
Quantification of TGFβ protein
The protein concentration of active and total (active plus latent) TGFβ1 levels in dystrophic muscle was quantified by ELISA (Promega), following the manufacturer’s instructions.
Muscle force measurement
Muscle strength was determined as described previously . Briefly, after the indicated days of treatment, mice were sacrificed and the TA was rapidly excised into a dish containing oxygenated Krebs-Ringer solution. The optimum muscle length (Lo) was determined from micromanipulations of muscle length to produce the maximum isometric twitch force. Maximum isometric-specific tetanic force was determined from the plateau of the curve of the relationship between specific isometric force with a stimulation frequency (Hz) ranging from 1 to 200 Hz for 450 ms, with 2 minutes of rest between stimuli. The force was normalized per total muscle fiber cross-sectional area (CSA), to calculate the specific net force (mN/mm2).
Comparison between groups was done using the nonparametric Mann–Whitney U test for independent samples, with a confidence level of 95% being considered statistically significant. One-way or two-way analysis of variance (ANOVA) was used for comparisons between multiple groups as appropriate, and post hoc analysis was performed using Tukey’s test. All statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). The number of samples analyzed per group is detailed on each figure. Differences were considered to be statistically significant at P <0.05.
Mdx mice reproduce the human DMD fibrotic phenotype in aging diaphragm muscle
Exercise training triggers fibrosis in muscles of young dystrophic mice
Surgical muscle injuries advance and enhance fibrosis in young dystrophic mice
Raising TGFβ levels in dystrophic muscle of young mdx mice accelerates fibrosis and accentuates disease severity
Despite the profibrotic effect of the surgical methods on mdx muscle, each one has particularities and limitations. In the LAC model, the injury is confined to a small area of the muscle and this reduces the amount of tissue available for further studies, whereas, for reasons of animal welfare, DEN can only be performed in one leg of the mouse, affecting only the muscles under the knee. Therefore, based on our observation of the elevated levels of TGFβ in human and mouse dystrophic muscle (Figures 1 and 2), and its correlation with the extent of dystrophy-associated fibrosis, we reasoned that exogenous delivery of TGFβ1 to muscle of young dystrophic mice might increase and accelerate the development of fibrosis. We therefore performed two intramuscular TA injections of TGFβ1 (50 ng of TGFβ1 in 50 μl of PBS per injection), spaced seven days apart, in an attempt to sustain the profibrogenic action of this growth factor. Contralateral control muscles received the same number of injections of PBS. Analysis of the muscles histologically by H&E and Sirius red staining showed that TGFβ1 delivery lead to substantial increase in collagen deposition already at two weeks after the first injection, which persisted for up to two months and this was also confirmed by biochemical quantification of muscle extracts (Figure 4B and C). Of note, local muscle overexpression of the TGFβ1 target gene product CTGF also increased fibrogenesis in limb muscle of young mdx mice (Figure S3A, B in Additional file 4).Overall, comparing the distinct biochemical and functional parameters in all the procedures tested revealed that LAC and TGFβ treatments gave statistically higher quantitative measures of collagen than NI age-matched control mdx muscles. The collagen values for LAC and TGFβ1 treatments were comparable to the values recorded in limb muscles of old mdx mice (see Figure 2D), indicating that either one of these methods advances fibrosis by the equivalent of about fourteen months (that is inducing fibrosis at four months of age instead of eighteen months). DEN also significantly increases muscle collagen content over mdx controls, but to a lesser extent than LAC or TGFβ1 treatment (Figure 4A and C). Interestingly, the levels of endogenous TGFβ1 mRNA were increased in young dystrophic muscle in response to LAC, DEN and exogenous TGFβ1 delivery, but not CTX. Consistent with this, the expression of TGFβ-dependent signaling fibrotic target genes, such as, Coll I, CTGF, TIMP-1, were increased in mdx limb muscle after all three treatments, but not in CTX-damaged muscle (Figure 4D). Finally, immunostaining for FN and Coll I on sections from the different damaged mdx muscles showed greater ECM production than uninjured (or CTX-injured) dystrophic muscle (Figure 4E).Remarkably, at two months after injury, collagen deposition still persisted in TGFβ-treated young dystrophic muscles as it did in lacerated and denervated muscles, as revealed by histological and biochemical analysis (Figure 4F and G). In agreement with this, and demonstrating the deleterious physiological consequences of the increased fibrosis in young mdx muscles, the maximum force of the muscles subjected to the distinct treatments decreased with respect to NI mdx muscles (Figure 4H), therefore better mimicking the severe phenotype of the human condition.
Induction of fibrosis in non-dystrophic, wild-type muscle by combining surgical injury and growth factor delivery
Fibrosis persistence has negative consequences on tissue wound healing. Severe muscle injuries caused by trauma often result in scar formation at the expense of tissue repair. Thus, we designed easy-to-perform profibrotic procedures in non-dystrophic WT muscle, which could ideally be extended to a wide variety of transgenic mouse lines for research or therapeutic purposes.
As stated above, one of the limitations of the LAC procedure is the restricted availability of biopsy material. Trying to induce fibrosis by methods that would render more fibrotic tissue available for analysis, we decided to combine CTX injury, which individually was a poor fibrosis-inducing method, with either DEN or co-injection of TGFβ1 in muscle of WT mice, methods which we previously showed were able to increase fibrosis in young mdx muscle (Figure 4). Both DEN and injection of TGFβ1 failed to induce fibrosis in WT muscles when used alone (Figure 5, and Figure S4C and D in Additional file 5). Accordingly, TA muscles of WT mice were first subjected to CTX injection (50 μl of 10–5 M) and subsequently denervated or injected twice with TGFβ1 (50 ng of TGFβ1 in 50 μl PBS per injection) (at day 7 and 10 after CTX injection) and muscles were collected two and four weeks later. We found that the combination of CTX injury with DEN or TGFβ1 delivery induced fibrosis significantly compared to CTX injury alone, as shown by Sirius red staining (Figure 6A), collagen quantification as well as expression of fibrotic markers by quantitative RT-PCR and immunohistochemistry analyses, after 14 days (Figure 6B-D), correlating with delayed regeneration kinetics (Figure 6A). Of note, a combination of CTX injury and CTGF local overexpression produced similar profibrotic effects as combining CTX injury and TGFβ1 delivery (Figure S3C in Additional file 4), suggesting that part of the TGFβ profibrotic actions are likely to be mediated by CTGF.We next compared the persistence of fibrosis over time and the consequences on muscle function of each of the distinct fibrogenic regimes on WT muscle. Sirius red staining and collagen quantification showed that muscle fibrosis still persisted after four weeks of either laceration or CTX combined with TGFβ1 or DEN, compared to muscle injured with CTX alone or NI muscle (Figure 6E and 6F). The relevance of these results was supported by functional studies of WT muscle after the combined profibrotic treatments (CTX combined with TGFβ1 or DEN). Indeed, dual treatments on muscles exerted a synergistic effect, resulting in increased fibrosis and reduced net force compared to uninjured muscle or muscle injured with CTX alone (Figure 6G). These results suggest that in WT mice, LAC, as well as a combination of CTX injury with either DEN or TGFβ1, proved to be effective fibrosis-inducing models that trigger a rapid accumulation of fibrotic tissue that is sustained for an extended period of time, with negative consequences on muscle function.
Muscular dystrophies constitute a heterogeneous group of inherited myopathies, characterized by progressive muscular degeneration, of which DMD is one of the severest. Progressive replacement of skeletal muscle by fat and fibrotic tissue not only exacerbates disease progression, but also impairs the efficiency of gene- and stem cell-based therapies [30, 31]. Yet, there is no effective clinical treatment to reverse or attenuate fibrosis in DMD patients, except for promising new agents such as halofuginone . To a great extent, this deficit may derive from the poor understanding of the mechanisms underlying fibrogenesis in muscular dystrophy. Indeed, chronic inflammation and production of collagens by myoblasts are among the few reported causal factors promoting progression to fibrosis in dystrophic muscle [33–38]. The largely unknown etiology of fibrogenesis in DMD in turn may be principally due to the lack of adequate animal models of muscle fibrosis. Here we report the application of simple models of tissue damage that are able to significantly enhance the fibrotic response in skeletal muscle and which may be useful for investigating therapeutic strategies for DMD.
Studies using mdx mice, the most common mouse model of DMD, may not be translated directly to dystrophic patients due to the mild phenotype they display. In particular, limb muscles of mdx mice show a relatively efficient regeneration and no significantly aberrant deposition of ECM proteins until very old age. Progressive endomysial fibrosis only develops in diaphragm muscle, but is still not significantly advanced until well into adulthood . To try to accelerate or exacerbate this phenotype, other mouse models have been generated such as mdx mice lacking arginase-2, PAI-1 (plasminogen activator inhibitor-1) or Cmah (cytidine monophosphate-sialic acid hydroxylase) [11, 40, 41] and previously the mdx/utrn+/- mouse line (mdx mice with haploinsufficiency of utrophin) . However, mdx/utrn+/- mice show early mortality and the manipulation of the line requires time and resources in genotyping and breeding. Moreover, the genetics of these mouse models do not adequately reflect human DMD patients. Therefore, the need for fibrotic models that do not require waiting for the natural physiological onset of fibrosis in the hindlimb of old mice, and that recapitulate the human DMD phenotype becomes increasingly more important. One recent attempt to address this problem came from Desguerre and colleagues (2012) who described a model of mechanical muscle injury by daily repeated micro-punctures in mdx hindlimb muscle . Induction of endomysial fibrosis in dystrophic muscle through this method is ascribed to a small fibrotic area and requires daily animal manipulation during two weeks. In addition, this procedure does not seem to induce fibrosis in WT mice .
The strategies we propose here are valid alternatives to both hasten the appearance and prolong the duration of fibrosis in hindlimb muscles of young mdx mice, with very limited (non-daily) animal manipulation, which notably are also able to induce relatively sustained fibrosis in WT muscle. Therefore, these methods would be applicable to other genetically modified mice, and this will help further delineating the cellular and genetic basis of muscle fibrosis. Exercise training of young mdx mice induced endomysial fibrosis, resembling the phenotype of old hindlimb dystrophic muscles; however, although this method can be considered more physiological, it still requires a lengthy time period to obtain a fibrotic muscle tissue, in addition to significant amount of effort and time, since exercise protocols need to be applied several times a week for ideally three months. At variance, the methods based on muscle growth factor delivery and surgical injuries that we present here offer a faster and less labor-intensive alternative. The rationale for the proposed profibrotic growth factor-based methods relies on the observation that, in fibrotic muscles of human DMD patients and old mdx mice, TGFβ1 (and its downstream target CTGF) is present at high levels [22, 44], correlating with the increased activation of Smad2/3 transcriptional mediators (see Figure S5 in Additional file 6). Of the surgical methods tested, muscle laceration proved to be the most effective for inducing sustained fibrosis; however, this method has the disadvantage that the affected area is relatively small (and only one muscle per mouse can be lesioned due to the severity of the procedure) thereby limiting the amount of material available for downstream processing. Subsequent cellular analysis of fibrotic muscle by techniques such as fluorescence-activated cell sorting (FACS) may not be possible in this type of model without vast improvements of sensitivity or without increasing the number of animals used, which has extra cost and ethical implications. Sciatic nerve denervation of mdx mice generates increased collagen deposition, as a possible mechanism to replace the tissue volume lost due to myofiber atrophy. All of these fibrogenesis-inducing methods persist with time, since at two months after injury muscles still displays a fibrotic phenotype. Moreover, consistent with the idea that fibrosis aggravates muscle dysfunction in DMD, we showed that maximal muscle force was also reduced in young mdx mice after fibrosis induction through the different protocols.
Finally, to be able to investigate fibrosis development and therapeutic options in muscle of non-dystrophic models, we sought to apply these methods to WT mice. To date, studies on muscle damage in non-dystrophic models have been performed classically with a single injection of myotoxins (for example, CTX or BaCl2). Despite the general use, we have shown in this study that these standard single-injury methods are not appropriate fibrosis-inducing models, as the resolution of the damage occurs rapidly and collagen deposition is very mild and only transient. On the contrary, muscle laceration of WT muscle induces a massive collagen deposition that is relatively stable over long periods of time, despite affecting only a localized small tissue area. However, the combination of regimes showed an improved capacity to generate fibrosis in WT muscle for a sustained period of time, correlating with reduction in muscle force, indicating that they mimic in WT animals pathophysiological situations of severe muscle trauma that result in aberrant regeneration, scar deposition and functional impairment. We propose that this variety of fibrosis-inducing methodologies will enable fibrosis to be studied in a vast array of transgenic mouse lines (with no apparent underlying muscle pathology) or after crossing them with dystrophic strains such as mdx mice.
Collectively, through this study, we propose novel and/or optimized experimental strategies to accelerate, anticipate and boost muscle fibrosis in young dystrophic mice or to drive de novo fibrosis onset in WT mice. We think that our findings provide very useful methodologies that will facilitate research in the emerging field of skeletal muscle fibrosis. In particular, these rapid and feasible procedures for most laboratories will help getting deeper insight into the mechanisms underlying muscle fibrosis, as well as developing therapeutic strategies aimed to reduce its magnitude in dystrophic diseases and to ameliorate dystrophy progression. Since fibrosis is also a main obstacle for stem cell engraftment, availability of appropriate fibrosis models will be a determinant factor in the research toward successful gene/cell therapy-based strategies in muscular dystrophy.
- Coll I:
connective tissue growth factor
Duchenne muscular dystrophy
hematoxylin and eosin
transforming growth factor beta1
tissue inhibitor of metalloproteinases 1
We are indebted to E. Perdiguero, V. Lukesova, L. Correa, A. Vasquez, C. Mann and M. Raya for their continuous help and advice. We also thank previous members of our laboratories, especially E. Ardite and B. Vidal, for setting up the basis of this study, and J. Martín-Caballero for assistance in the PRBB animal facility. The authors acknowledge funding from MINECO-Spain (SAF2012-38547, FIS-PS09/01267, FIS-PI13/02512, PLE2009-0124), AFM, E-Rare, Fundació-MaratóTV3, Duchenne PP-NL, EU-FP7 (Myoage, Optistem and Endostem), MDA, CARE PFB12/2007 and FONDECYT 1110426.
- Emery AE: The muscular dystrophies. Lancet. 2002, 359: 687-695.View ArticlePubMedGoogle Scholar
- Briggs D, Morgan JE: Recent progress in satellite cell/myoblast engraftment - relevance for therapy. FEBS J. 2013, 280: 4281-4293.PubMed CentralView ArticlePubMedGoogle Scholar
- Serrano AL, Munoz-Canoves P: Regulation and dysregulation of fibrosis in skeletal muscle. Exp Cell Res. 2010, 316: 3050-3058.View ArticlePubMedGoogle Scholar
- Yablonka-Reuveni Z, Anderson JE: Satellite cells from dystrophic (mdx) mice display accelerated differentiation in primary cultures and in isolated myofibers. Dev Dynamics. 2006, 235: 203-212.View ArticleGoogle Scholar
- Grounds MD, Shavlakadze T: Growing muscle has different sarcolemmal properties from adult muscle: a proposal with scientific and clinical implications: reasons to reassess skeletal muscle molecular dynamics, cellular responses and suitability of experimental models of muscle disorders. Bio Essays. 2011, 33: 458-468.Google Scholar
- Muntoni F: Cardiac complications of childhood myopathies. J Child Neurol. 2003, 18: 191-202.View ArticlePubMedGoogle Scholar
- Benedetti S, Hoshiya H, Tedesco FS: Repair or replace? Exploiting novel gene and cell therapy strategies for muscular dystrophies. FEBS J. 2013, 280: 4263-4280.View ArticlePubMedGoogle Scholar
- Tedesco FS, Hoshiya H, D’Antona G, Gerli MF, Messina G, Antonini S, Tonlorenzi R, Benedetti S, Berghella L, Torrente Y, Kazuki Y, Bottinelli R, Oshimura M, Cossu G: Stem cell-mediated transfer of a human artificial chromosome ameliorates muscular dystrophy. Sci Trans Med. 2011, 3: 96ra78-View ArticleGoogle Scholar
- Sicinski P, Geng Y, Ryder-Cook AS, Barnard EA, Darlison MG, Barnard PJ: The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science. 1989, 244: 1578-1580.View ArticlePubMedGoogle Scholar
- Carnwath JW, Shotton DM: Muscular dystrophy in the mdx mouse: histopathology of the soleus and extensor digitorum longus muscles. J Neuro Sci. 1987, 80: 39-54.View ArticleGoogle Scholar
- Ardite E, Perdiguero E, Vidal B, Gutarra S, Serrano AL, Munoz-Canoves P: PAI-1-regulated miR-21 defines a novel age-associated fibrogenic pathway in muscular dystrophy. J Cell Biol. 2012, 196: 163-175.PubMed CentralView ArticlePubMedGoogle Scholar
- Menetrey J, Kasemkijwattana C, Fu FH, Moreland MS, Huard J: Suturing versus immobilization of a muscle laceration. A morphological and functional study in a mouse model. Am J Sports Med. 1999, 27: 222-229.PubMedGoogle Scholar
- Serrano AL, Murgia M, Pallafacchina G, Calabria E, Coniglio P, Lomo T, Schiaffino S: Calcineurin controls nerve activity-dependent specification of slow skeletal muscle fibers but not muscle growth. Proc Natl Acad Sci USA. 2001, 98: 13108-13113.PubMed CentralView ArticlePubMedGoogle Scholar
- Glass DJ: Skeletal muscle hypertrophy and atrophy signaling pathways. Int J Biochem Cell Biol. 2005, 37: 1974-1984.View ArticlePubMedGoogle Scholar
- Morales MG, Cabello-Verrugio C, Santander C, Cabrera D, Goldschmeding R, Brandan E: CTGF/CCN-2 over-expression can directly induce features of skeletal muscle dystrophy. J Pathol. 2011, 225: 490-501.View ArticlePubMedGoogle Scholar
- Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A: Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012, 9: 676-682.View ArticlePubMedGoogle Scholar
- Cabello-Verrugio C, Morales MG, Cabrera D, Vio CP, Brandan E: Angiotensin II receptor type 1 blockade decreases CTGF/CCN2-mediated damage and fibrosis in normal and dystrophic skeletal muscles. J Cellular Mol Med. 2012, 16: 752-764.View ArticleGoogle Scholar
- Biernacka A, Frangogiannis NG: Aging and cardiac fibrosis. Aging Dis. 2011, 2: 158-173.PubMed CentralPubMedGoogle Scholar
- Brandan E, Gutierrez J: Role of proteoglycans in the regulation of the skeletal muscle fibrotic response. FEBS J. 2013, 280: 4109-4117.View ArticlePubMedGoogle Scholar
- MacDonald EM, Cohn RD: TGFbeta signaling: its role in fibrosis formation and myopathies. Cur Opinion Rheumatol. 2012, 24: 628-634.View ArticleGoogle Scholar
- Mann CJ, Perdiguero E, Kharraz Y, Aguilar S, Pessina P, Serrano AL, Munoz-Canoves P: Aberrant repair and fibrosis development in skeletal muscle. Skelet Muscle. 2011, 1: 21-PubMed CentralView ArticlePubMedGoogle Scholar
- Morales MG, Cabrera D, Cespedes C, Vio CP, Vazquez Y, Brandan E, Cabello-Verrugio C: Inhibition of the angiotensin-converting enzyme decreases skeletal muscle fibrosis in dystrophic mice by a diminution in the expression and activity of connective tissue growth factor (CTGF/CCN-2). Cell Tissue Res. 2013, 353: 173-187.View ArticlePubMedGoogle Scholar
- Dangain J, Vrbova G: Muscle development in mdx mutant mice. Muscle Nerve. 1984, 7: 700-704.View ArticlePubMedGoogle Scholar
- De Luca A, Pierno S, Liantonio A, Cetrone M, Camerino C, Fraysse B, Mirabella M, Servidei S, Ruegg UT, Conte Camerino D: Enhanced dystrophic progression in mdx mice by exercise and beneficial effects of taurine and insulin-like growth factor-1. J Pharm Exp Thera. 2003, 304: 453-463.View ArticleGoogle Scholar
- Morales MG, Gutierrez J, Cabello-Verrugio C, Cabrera D, Lipson KE, Goldschmeding R, Brandan E: Reducing CTGF/CCN2 slows down mdx muscle dystrophy and improves cell therapy. Hum Mol Gen. 2013, 22: 4938-4951.View ArticlePubMedGoogle Scholar
- Perdiguero E, Sousa-Victor P, Ruiz-Bonilla V, Jardi M, Caelles C, Serrano AL, Munoz-Canoves P: p38/MKP-1-regulated AKT coordinates macrophage transitions and resolution of inflammation during tissue repair. J Cell Biol. 2011, 195: 307-322.PubMed CentralView ArticlePubMedGoogle Scholar
- Suelves M, Lopez-Alemany R, Lluis F, Aniorte G, Serrano E, Parra M, Carmeliet P, Munoz-Canoves P: Plasmin activity is required for myogenesis in vitro and skeletal muscle regeneration in vivo. Blood. 2002, 99: 2835-2844.View ArticlePubMedGoogle Scholar
- Suelves M, Vidal B, Serrano AL, Tjwa M, Roma J, Lopez-Alemany R, Luttun A, de Lagran MM, Diaz-Ramos A, Jardi M, Roig M, Dierssen M, Dewerchin M, Carmeliet P, Muñoz-Cánoves P: uPA deficiency exacerbates muscular dystrophy in MDX mice. J Cell Biol. 2007, 178: 1039-1051.PubMed CentralView ArticlePubMedGoogle Scholar
- Carlson BM, Billington L, Faulkner J: Studies on the regenerative recovery of long-term denervated muscle in rats. Rest Neurol Neurosci. 1996, 10: 77-84.Google Scholar
- Gargioli C, Coletta M, De Grandis F, Cannata SM, Cossu G: PlGF-MMP-9-expressing cells restore microcirculation and efficacy of cell therapy in aged dystrophic muscle. Nat Med. 2008, 14: 973-978.View ArticlePubMedGoogle Scholar
- Muir LA, Chamberlain JS: Emerging strategies for cell and gene therapy of the muscular dystrophies. Expert Rev Mol Med. 2009, 11: e18-View ArticlePubMedGoogle Scholar
- Turgeman T, Hagai Y, Huebner K, Jassal DS, Anderson JE, Genin O, Nagler A, Halevy O, Pines M: Prevention of muscle fibrosis and improvement in muscle performance in the mdx mouse by halofuginone. Neuro Dis. 2008, 18: 857-868.View ArticleGoogle Scholar
- Alexakis C, Partridge T, Bou-Gharios G: Implication of the satellite cell in dystrophic muscle fibrosis: a self-perpetuating mechanism of collagen overproduction. Am J Physiol Cell Physiol. 2007, 293: C661-C669.View ArticlePubMedGoogle Scholar
- Morrison J, Palmer DB, Cobbold S, Partridge T, Bou-Gharios G: Effects of T-lymphocyte depletion on muscle fibrosis in the mdx mouse. Am J Pathol. 2005, 166: 1701-1710.PubMed CentralView ArticlePubMedGoogle Scholar
- Vidal B, Serrano AL, Tjwa M, Suelves M, Ardite E, De Mori R, Baeza-Raja B, Martinez de Lagran M, Lafuste P, Ruiz-Bonilla V, Jardí M, Gherardi R, Christov C, Dierssen M, Carmeliet P, Degen JL, Dewerchin M, Muñoz-Cánoves P: Fibrinogen drives dystrophic muscle fibrosis via a TGFbeta/alternative macrophage activation pathway. Genes Dev. 2008, 22: 1747-1752.PubMed CentralView ArticlePubMedGoogle Scholar
- Vidal B, Ardite E, Suelves M, Ruiz-Bonilla V, Janue A, Flick MJ, Degen JL, Serrano AL, Munoz-Canoves P: Amelioration of Duchenne muscular dystrophy in mdx mice by elimination of matrix-associated fibrin-driven inflammation coupled to the alphaMbeta2 leukocyte integrin receptor. Hum Mol Gen. 2012, 21: 1989-2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Villalta SA, Nguyen HX, Deng B, Gotoh T, Tidball JG: Shifts in macrophage phenotypes and macrophage competition for arginine metabolism affect the severity of muscle pathology in muscular dystrophy. Hum Mol Gen. 2009, 18: 482-496.PubMed CentralView ArticlePubMedGoogle Scholar
- Kharraz Y, Guerra J, Mann CJ, Serrano AL, Munoz-Canoves P: Macrophage plasticity and the role of inflammation in skeletal muscle repair. Mediators Inflam. 2013, 2013: 491497-View ArticleGoogle Scholar
- Stedman HH, Sweeney HL, Shrager JB, Maguire HC, Panettieri RA, Petrof B, Narusawa M, Leferovich JM, Sladky JT, Kelly AM: The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy. Nature. 1991, 352: 536-539.View ArticlePubMedGoogle Scholar
- Chandrasekharan K, Yoon JH, Xu Y, deVries S, Camboni M, Janssen PM, Varki A, Martin PT: A human-specific deletion in mouse Cmah increases disease severity in the mdx model of Duchenne muscular dystrophy. Sci Trans Med. 2010, 2: 42ra54-View ArticleGoogle Scholar
- Wehling-Henricks M, Jordan MC, Gotoh T, Grody WW, Roos KP, Tidball JG: Arginine metabolism by macrophages promotes cardiac and muscle fibrosis in mdx muscular dystrophy. PloS one. 2010, 5: e10763-PubMed CentralView ArticlePubMedGoogle Scholar
- Zhou L, Rafael-Fortney JA, Huang P, Zhao XS, Cheng G, Zhou X, Kaminski HJ, Liu L, Ransohoff RM: Haploinsufficiency of utrophin gene worsens skeletal muscle inflammation and fibrosis in mdx mice. J Neurol Sci. 2008, 264: 106-111.PubMed CentralView ArticlePubMedGoogle Scholar
- Desguerre I, Arnold L, Vignaud A, Cuvellier S, Yacoub-Youssef H, Gherardi RK, Chelly J, Chretien F, Mounier R, Ferry A, Chazaud B: A new model of experimental fibrosis in hindlimb skeletal muscle of adult mdx mouse mimicking muscular dystrophy. Muscle Nerve. 2012, 45: 803-814.View ArticlePubMedGoogle Scholar
- Bernasconi P, Di Blasi C, Mora M, Morandi L, Galbiati S, Confalonieri P, Cornelio F, Mantegazza R: Transforming growth factor-beta1 and fibrosis in congenital muscular dystrophies. Neuromusc Dis. 1999, 9: 28-33.View ArticlePubMedGoogle Scholar
This article is published under license to 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. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.