SB431542

Denervation-induced Skeletal Muscle Fibrosis is mediated by CTGF/CCN2 independently of TGF

Daniela L. Rebolledo1,4, David González1,2, Jennifer Faundez-Contreras1,2, Osvaldo Contreras1,2, Carlos P. Vio1,3, 1
 
ABSTRACT

Muscular fibrosis is caused by excessive accumulation of extracellular matrix (ECM) that replaces functional tissue, and it is a feature of several myopathies and neuropathies. Knowledge of the biology and regulation of pro-fibrotic factors is critical for the development of new therapeutic strategies. Upon unilateral sciatic nerve transection, we observed accumulation of ECM proteins such as collagen and fibronectin in the denervated hindlimb, together with increased levels of the profibrotic factors transforming growth factor type (TGF-β) and connective tissue growth factor (CTGF/CCN2). In mice hemizygous for CTGF/CCN2 or in mice treated with a blocking antibody against CTGF/CCN2, we observed reduced accumulation of ECM proteins after denervation as compared to control mice, with no changes in fibro/adipogenic progenitors (FAPs), suggesting a direct role of CTGF/CCN2 on denervation-induced fibrosis. During time course experiments, we observed that ECM proteins and CTGF/CCN2 levels are increased early after denervation (2-4 days), while TGF-β signaling shows a delayed kinetics of appearance (1-2 weeks). Furthermore, blockade of TGF-β signaling does not decrease fibronectin or CTGF levels after 4 days of denervation. These results suggest that in our model CTGF/CCN2 is not up-regulated by canonical TGF-β signaling early after denervation and that other factors are likely involved in the early fibrotic response following skeletal muscle denervation.
 
INTRODUCTION

After acute damage, most tissues undergo a series of events that remove damaged structures and replace them with new functional cells. Under chronic damage, the affected tissue cannot be completely repaired, producing an imbalance that leads to excessive production and accumulation of extracellular matrix (ECM) proteins such as proteoglycans, fibronectin and different types of collagens, which surround the cells of the affected tissue [1-3]. This phenomenon, called fibrosis, is a hallmark of several chronic pathologies affecting almost every organ [4], including myopathies as muscular dystrophies, where scar-like tissue replaces functional cells and creates a physical barrier that inhibits regeneration, neovascularization, and the possibility of successful cell therapy [5-8].
Skeletal muscle denervation is the interruption of synaptic transmission from motoneurons to their skeletal muscle targets. It can occur as a consequence of trauma or pathological conditions: transection of the nerve, death of motoneurons as in neurodegenerative diseases like Amyotrophic Lateral Sclerosis (ALS) [9], loss of acetylcholine receptors (AChRs) in diseases such as Myasthenia gravis [10], and during aging [11]. Transection of the sciatic nerve and blockade of nerve transmission have been largely used as experimental approaches [1214]. Skeletal muscle alterations after denervation are numerous, including atrophy and increased autophagy [15, 16], inflammation [17], AChR degradation [18], changes in gene expression profile [19], alterations in mitochondrial function [20] and delocalization of neuronal nitric oxide synthase μ from the sarcolemma [21]. Increases in ECM components and fibrotic markers have also been described after denervation [12, 14, 22-25]. Furthermore, fibro/adipogenic progenitors (FAPs), skeletal muscle resident cells that differentiate to myofibroblasts producing ECM and are involved in the fibrotic response, are also augmented after denervation [26] and in a murine ALS model [27, 28].
Connective tissue growth factor (CTGF/CCN2) is a matricellular protein that is expressed at very low levels in adulthood but its levels increase in challenged tissues, for example under pathological conditions [29, 30]. CTGF/CCN2 is increased in muscular dystrophies [31] and in the mdx mouse, a murine model for Duchenne Muscular Dystrophy [32, 33], as well in the tg-hSODG93A mouse model for ALS [27, 34]. CTGF/CCN2 is a strong profibrotic factor that colocalizes with fibrotic and necrotic/regenerative foci in damaged muscle [35, 36] and promotes fibroblast proliferation and ECM production by several different cell types, both in vitro and in vivo [29, 37-40]. Through experiments of gain and loss of CTGF/CCN2 function, our laboratory showed that CTGF/CCN2 plays a critical role in promoting fibrosis: overexpression of CTGF/CCN2 in healthy wild type skeletal muscle is sufficient to induce a fibrotic phenotype with a reduction of skeletal muscle strength [39], whereas decreased CTGF/CCN2 levels or activity reduces muscle damage, fibrosis, muscle weakness and can improve cell therapy [32, 34].
Transforming growth factor type β (TGF-β) has been extensively studied and linked to fibrosis associated with different pathologies [24, 41-43] including muscular dystrophies [44, 45]. The canonical TGF-β or Smaddependent pathway involves phosphorylation of Smad2/3 directly by the TGF-β receptor I kinase (TGF-βRI), binding to Smad4 and translocation of this complex into the nucleus to drive the expression of target genes that contribute to the fibrotic process, including induction of CTGF/CCN2 [29, 40, 41, 46-48]. Increased TGF-β signaling after denervation or inhibition of electric activity has been reported previously [13, 23, 26]. Furthermore, it has been shown increased TGF-β signaling and CTGF/CCN2 levels in an ALS model during symptomatic stages, when muscle innervation is already compromised [27, 34, 49, 50]. However, whether TGF-β induces CTGF/CCN2 after denervation and if CTGF/CCN2 has a role in denervation-induced fibrosis, are questions that have not yet been explored.

RESULTS

Denervation-induced skeletal muscle fibrosis is accompanied by increased TGF-β and CTGF/CCN2 levels

To evaluate the role of TGF-β and CTGF/CCN2 in the fibrotic process induced after denervation, we performed unilateral sciatic nerve transection in 6-8-month old C57BL10 wild type mice and compared changes between the denervated hindlimb and the non-denervated (contralateral) internal control. Two weeks after denervation we observed an evident increment in the interstitial space as well as the already described muscle atrophy [15] (Figure 1A). We used sirius red staining and indirect immunofluorescence (IFI) to evaluate levels of ECM proteins. When compared to contralateral non-denervated gastrocnemius (GT), denervated muscles showed augmented deposition of total collagen, collagen I and fibronectin (Figure 1A), indicating an increase in skeletal muscle fibrosis as a consequence of denervation, which confirmed previous reports [12, 24, 26]. These changes were also observed in other muscles such as tibialis anterior (TA) and flexor digitorius brevis (data not shown).
Because the role of TGF-β signaling in skeletal muscle fibrosis is well described [45], we evaluated whether this signaling pathway also participates in denervation-induced fibrosis. We observed that after 2 weeks of sciatic nerve section TGF-β1 mRNA levels (Figure 1B) and the total number of positive nuclei for effector pSmad3 were increased in denervated GT (Figure 1C-D), indicating the activation of canonical TGF-β signaling in denervated muscles. Given the high diversity of skeletal muscle cell types, we aimed to determine which cells were activating Smad-dependent TGF-β signaling. Then, we used laminin immune staining to outline muscle fibers, and Pdgfrαtm11(EGFP)Sor mice to discriminate PDGFRα expressing FAPs by localizing nuclear EGFP fluorescence [51]. Interestingly, we observed that the increment in pSmad3 positive nuclei after denervation correspond mostly to nuclei of myofibers and not from interstitial cells (Figure 1E) and do not colocalize with EGFP expressing FAPs (Figure 1F).
Since the expression of CTGF/CCN2 is induced by TGF-β, we aimed to determine whether CTGF/CCN2 was upregulated after denervation. We observed that CTGF/CCN2 levels were increased in denervated muscles after 2 weeks as compared to contralateral controls (Figure 1G-J) when evaluated by western blot (WB) and immunohistochemistry (IHC), suggesting a role for CTGF/CCN2 in developing denervation-induced fibrosis. These results indicate that after denervation of skeletal muscle there is an important increase in pro-fibrotic factors and ECM proteins.

Mice with decreased CTGF/CCN2 levels or activity show less skeletal muscle ECM accumulation after 2 weeks of denervation.

In order to evaluate whether CTGF/CCN2 has a role in developing fibrosis upon denervation, we used two different strategies to decrease CTGF/CCN2 levels or activity. First, we performed sciatic nerve transection in mice hemizygous for the Ctgf/Ccn2 gene (Ctgf/Ccn2+/-), which produce reduced levels of CTGF/CCN2 protein [32, 52]. As second approach, we used FG-3019 (FibroGen Inc.), a human monoclonal antibody that blocks CTGF/CCN2 activity [32, 34]. This strategy allowed us to neutralize CTGF/CCN2 activity just prior to performing the sciatic nerve transection, whereas reduced levels of CTGF/CCN2 in Ctgf/Ccn2+/- mice were sustained from developmental stages. Thus, wild type C57BL10 mice were systemically treated with IP FG-3019 injections (FibroGen, USA) or control human IgG (h-IgG), for 3 weeks. After 1 week of treatment, mice were unilaterally denervated, and their muscles collected for analysis 2 weeks later. Two weeks after denervation we observed less muscular fibronectin and total collagen deposition in Ctgf/Ccn2+/- mice as compared to Ctgf/Ccn2+/+ littermates (Figure 2A, C). We also observed less accumulation of fibronectin and collagen I upon denervation in muscle sections from mice treated with FG-3019, compared to h-IgG-treated animals (Figure 2B, D). Lower abundance of fibronectin was also observed when we analyzed whole muscle extracts of GT by WB: the amount of fibronectin in denervated muscle from Ctgf/Ccn2+/- mice is lower than in wild type mice (Figure 3A) and in FG3019-treated mice compared to controls (Figure 3B). Denervation increased CTGF/CCN2 levels as showed above, but a lower amount of CTGF/CCN2 is observed in muscles from Ctgf/Ccn2+/- mice compared Ctgf/Ccn2+/+ animals when denervated. In contrast, CTGF/CCN2 levels remain the same in treated and control mice, since the antibody blocks CTGF/CCN2 activity without necessarily decreasing its levels [34]. Nevertheless, TGF-β signaling, determined by total TGF-β1 mRNA levels and percent of pSmad3 positive nuclei, remained increased in denervated GT from both Ctgf/Ccn2 hemizygous mice and FG-3019-treated mice (Figure 3C-H). As control for FG3019 treatment, we used a fluorophore (Alexa-568) labeled anti-human antibody in IFI and were able to detect the presence of treatment antibodies in muscle cryosections, indicating that FG-3019 and control h-IgG reached the target tissue. Also, the FG-3019 anti-CTGF/CCN2 antibodies are more abundant in the tissue only in denervated muscle, where CTGF/CCN2 is elevated (Supplementary Figure 1). Furthermore, skeletal muscle sequential cross-sections from denervated mice treated with FG-3019 shows that anti-CTGF staining colocalizes with FG-3019 accumulation in the tissue, detected by the anti-human antibody, indicating the efficacy of FG3019 in binding CTGF (Figure 3I). Altogether, these results strongly suggest that CTGF/CCN2 has a direct role in denervation-induced fibrosis in skeletal muscle and that blocking CTGF/CCN2 decreases the accumulation of ECM proteins after denervation. Our laboratory has already shown that inhibition of CTGF/CCN2 reduces fibrosis but also enhances skeletal muscle strength in mdx mice [32] and in the hSOD1G93A mouse, a model for ALS [34]. Here, we observed that reduced isometric force in isolated denervated TA muscle and muscle fiber atrophy was not improved in Ctgf/Ccn2+/- or FG-3019-treated mice (Supplementary Figure 2), suggesting that the effects of decreasing CTGF/CCN2 are related to muscle fibrosis and not muscle force and atrophy under full denervation conditions for 2 weeks.
FAPs expressing PDGFR-α and Tcf4 are increased in denervated skeletal muscle [26]. To evaluate if CTGF has a role in regulating the amount of FAPs, we used total homogenates from control and denervated muscles for 2 weeks and evaluated PDGFR-α and Tcf4 levels. As shown before, denervation increases FAPs markers compared to contralateral hindlimbs, however, the increment on PDGFR-α and Tcf4 observed after denervation is not changed by FG-3019 treatment (Figure 3J) or in Ctgf/Ccn2 hemizygous mice (Supplementary Figure 3). These results suggest that the reduction in denervation-induced fibrosis observed in CTGF/CCN2 inhibition models is not related to reduction in FAPs.

Skeletal muscle ECM accumulates and CTGF/CCN2 levels increase early after denervation independently of TGF-β signaling.

To evaluate the kinetics of the denervation-related fibrotic response, we performed time course experiments, to evaluate muscles after 2, 4, 7 and 14 days of unilateral sciatic nerve transection. IFI experiments show that ECM proteins such as fibronectin and collagen I are increased early after denervation, as soon as 2 days post sciatic nerve transection (Figure 4). Increased fibronectin was also confirmed by WB analyses of muscle homogenates obtained at the indicated days (Figure 5A-B). In the same samples, we observed that CTGF/CCN2 protein levels, evaluated by WB, were also elevated since 2 days after denervation (Figure 5A-B). Elevated CTGF/CCN2 protein levels 2 days after denervation were more variable than other time points, then not statistically significant. Then, CTGF/CCN2 levels were also evaluated by IHC in muscle cryosections (Figure 5C-D), ratifying increased protein levels since 2 days after denervation. These results suggest an early role for CTGF/CCN2 in the induction of skeletal muscle fibrosis after denervation. Interestingly, we observed that TGF-β1 mRNA also increases after denervation but at later times and with a slightly delayed expression kinetics compared to increased CTGF/CCN2 levels (Figure 5E), as we previously showed [26]. Furthermore, the number of positive nuclei for the downstream canonical effector pSmad3 remains unchanged 2 and 4 days after denervation and only appears significantly elevated 2 weeks after denervation (Figure 5E), while TGF-α1,2,3 detected by WB increased with time after denervation with no statistical significance due to high variability between samples (Figure 5F). These results suggest that the induction of skeletal muscle CTGF/CCN2 levels upon denervation occurs before the activation of canonical TGF-signaling and might be independent of it.
In order to evaluate whether TGF-β signaling is required for the early induction of CTGF/CCN2, we treated mice with SB525334, an inhibitor of TGF-β receptor I (TGF-β-RI) kinase, and evaluated the levels of CTGF/CCN2 and fibronectin 4 days after denervation. We observed that CTGF/CCN2 and fibronectin levels were increased 4 days after denervation despite TGF-β-RI inhibition, and at similar levels as in vehicle-treated mice (Figure 6A-C). We performed the same experiment with a different TGF-β-RI kinase inhibitor, SB431542, and observed the same response (Supplementary Figure 4). To further reinforce this observation, we used a different approach to block TGF-β signaling using SRI31277, an inhibitor of thrombospondin 1 (TSP1) [53]. TPS1 regulates the release of latent TGF-β from insulted tissue and SRI31277 has been shown to block TGF-β signaling in different models [53-57]. We observed that treatment with SRI31277 was unable to reduce skeletal muscle CTGF/CCN2 and ECM accumulation 4 days after denervation (Figure 6D-F), similar to what we observed when blocking TGF-β-RI kinase activity. This lack of effect was not due to inactivity of these inhibitors in this model as there was a decrease in pSmad3 immunostaining in animal treated with either the TGF-βRI kinase inhibitors or the TSP1 antagonist (Figure 6G-H and Supplementary Figure 4).

DISCUSSION

Fibrosis is the final outcome of most chronic, inflammatory and autoimmune pathologies affecting almost every organ and accounting for approximately 40% of deaths in developed countries [4, 42]. Consequently, knowledge of the physiology and the cellular and molecular basis of the fibrotic process will open possibilities for new therapeutic alternatives to be used in patients suffering fibrotic diseases of diverse etiology. Changes in tissue remodeling and increased accumulation of ECM proteins after denervation have been described by different groups. Despite differences in the animal models used (rat vs mouse, for example), type of damage and muscles evaluated, a common feature is the accumulation of ECM proteins associated with enhanced TGF-β expression and changes in metalloprotease activity [24, 58, 59]. To date, several molecules involved in the skeletal muscle fibrotic response have been studied by us and others. The data shows that the inhibition of pro-fibrotic factors or the activation of anti-fibrotic pathways can reduce fibrosis, resulting in improved structure, perfusion, and tissue function [2, 7, 8, 60, 61]. In this respect, our laboratory has been interested in studying the biology and pro fibrotic effects of CTGF/CCN2 in skeletal muscle fibrotic disease. Here, we report that skeletal muscle fibrosis induced by sciatic denervation associates with increased TGF-β signaling and CTGF/CCN2 expression. Liu et al., previously reported that denervation of the sternocleidomastoid muscle was accompanied by increased TGF-β and CTGF/CCN2 levels 1 week after denervation [58]. Also, increased Smad2/3 proteins after 1 week of denervation has been reported and showed to be required for denervationinduced protein degradation and atrophy [62]. However, earlier times were not evaluated, and neither was the effect of inhibition of either of the profibrotic factors.
In this work, we propose a role for CTGF/CCN2 in denervation-induced fibrosis since we observed a decrease in ECM proteins accumulation when CTGF/CCN2 levels (in hemizygous mice) or activity (using the FG3019 antibody) were reduced. The reduction of skeletal muscle fibrosis as a consequence of CTGF/CCN2 inhibition not only recounts what had been reported on fibrosis related to dystrophic mdx muscle [32], but also to what we observed in the hSOD1G93A transgenic mouse model of ALS [34]. Interestingly, here we show that under conditions where CTGF/CCN2 is inhibited, the augmented number of FAPs after denervation were not affected, suggesting that CTGF/CCN2 is not influencing the increase in FAPs. In the ALS model [34]., fibrosis and skeletal muscle CTGF/CCN2 increased only in the symptomatic stages, when there were already signs of muscle denervation. This observation opened the question of whether muscle fibrosis is due to overexpression of mutant SOD1 or a consequence of muscle denervation. The inhibition of CTGF/CCN2 in the ALS model reduces skeletal muscle fibrosis but also improves muscle strength and locomotory function [34]. This might be explained because in this model, CTGF/CCN2 is also increased in the spinal cord in pre-symptomatic stages, and the inhibition of CTGF/CCN2 preserved nerve and neuromuscular junction integrity, decreasing denervation, and also showing an effect in the central nervous system. Here, we show that under conditions of full denervation, the loss of motoneuron communication increases skeletal muscle CTGF/CCN2 and fibrosis. However, bilateral denervation of the lower limbs is not able to induce CTGF/CCN2 expression in the spinal cord (Supplementary Figure 5), suggesting that in the ALS model increased CTGF/CCN2 in skeletal muscle is a consequence of denervation, but augmented CTGF/CCN2 in the spinal cord is indeed due to overexpression of mutant SOD1 and the concomitant spinal cord degeneration [34].
Fibrosis was evaluated by an increase in total collagen, collagen type I and fibronectin. Previously, we have shown that proteoglycans are augmented in skeletal muscle after denervation [12, 14] and in muscular dystrophies [63, 64]. Several types of proteoglycans have been involved in skeletal muscle pathophysiology, either at the neuromuscular junction [65-67] or in the skeletal muscle surface [68]. Particularly attractive is decorin, a chondroitin/dermatan sulfate proteoglycan [69] that increases its expression after denervation [14]. Decorin has the ability to regulate the activity of TGF-β modulating skeletal muscle formation [68, 70-72]. Its role during skeletal muscular diseases has been demonstrated [1]. Interestingly, decorin can inhibit TGF-β mediated action in response to skeletal muscle injury [73, 74] and also interacts with CTGF/CCN2 inhibiting its action and regulating its biological activity [75]. Future experiments are required to understand the role of decorin or other proteoglycans modulating the activity of these pro-fibrotic factors and others in neuromuscular diseases.
Our results indicate that the increase in fibrosis, as consequence of denervation, seems to be at least dependent of two components, CTGF/CCN2 and TGF- mediated signaling, acting at different times after nerve transection. Time course experiments showed that accumulation of CTGF/CCN2 and ECM proteins such as fibronectin and collagen I in the skeletal muscle rise very early after denervation, as soon as 2 days after sciatic nerve transection, while the activation of canonical TGF-β seems to be a later event. The kinetics of the fibrotic response after denervation, together with downregulation of TGF-β signaling (by inhibition of TGF-β-RI kinase or TGF-β release) suggest that in our model skeletal muscle CTGF/CCN2 is not up-regulated by canonical TGF-β signaling early after denervation (Figure 8).
TGF-β is a strong CTGF/CCN2 inducer, but there are other factors that also regulate CTGF/CCN2 expression. In other fibrotic models such as skin scleroderma, renal and lung fibrosis, and systemic sclerosis, cell response to low oxygen pressure or hypoxia has been associated with up-regulation of CTGF/CCN2 and ECM proteins via mechanisms that can be independent of TGF-β [76-79]. Endothelin-1, a vasoconstrictor involved in cardiovascular disease, and its receptors have also been reported to induce CTGF/CCN2 and a subsequent fibrotic response in lung and skin fibrosis, systemic sclerosis and cardiac myocytes [80-83]. Angiotensin II, a vasoactive peptide of the renin-angiotensin system (RAS), can induce CTGF/CCN2 levels via mechanisms that can be dependent or independent of TGF-β [84, 85]. Also, the action of bioactive lipids, such as lysophosphatidic acid (LPA), has been linked to fibrosis of different etiology [86-88] and our laboratory demonstrated that LPA is able to induce CTGF/CCN2 in muscle cells in vitro [75, 89, 90]. Inflammatory cytokines are known triggers of the fibrotic response. Although there is no significant infiltration of immune cells in denervated tissue, small inflammatory responses are observed with increases of IL-1β, IL-15 and TNF-α [91]. Furthermore, there is persistently activated STAT3-IL-6 signaling in denervated FAPs that can contribute to fibrosis and muscle atrophy after denervation [28]. Also, in mechanically stressed fibroblasts, the expression of CTGF/CCN2 precedes that of TGF-β [92]. Therefore, these and other factors that might be modulating the fibrotic response immediately after skeletal muscle denervation need to be studied in detail. The exploration of these factors might open new therapeutic alternatives for skeletal muscle wasting and fibrosis due to full denervation (e.g. from traumatic nerve injury) or pathological conditions in which progressive denervation occurs.
 
EXPERIMENTAL PROCEDURES

Animals, denervation protocol and tissue collection. All protocols were conducted in strict accordance and with formal approval of the Animal Ethics Committee of the Pontificia Universidad Católica de Chile. We used 6-7month-old C57/BL10 wild type and CTGF/CCN2+/- males (in the same genetic background). The latter was kindly donated by Professor Roel Goldschmeding (UMC, The Netherlands). For detection of FAPs, 6-7-month-old Pdgfratm11(EGFP)Sor mice were used (JAX stock #007669) [51]. For denervation experiments, animals were anesthetized with 3.0% isoflurane gas in pure oxygen and a small incision (≤ 0.5 mm) is made in the left tight skin. Muscle packages from gluteal and biceps femoris muscles were carefully separated by cutting the facia. The sciatic nerve was exposed with the help of a small surgical hook and then cut before the separation of sciatic nerve branches [19, 26]. A small (2-5 mm) section was removed to prevent reinnervation. Denervation was performed unilaterally, using the contralateral hindlimb as a control. 2, 4, 7 or 14 days after denervation surgery, mice were anesthetized with isoflurane and euthanized by cervical dislocation. GT muscles were dissected from denervated and contralateral hindlimbs. Muscle samples for cryosectioning were frozen in liquid nitrogen cooled-isopentane (Merck, Darmstadt, Germany) and stored at -80°C until processing.
Treatment with the FG-3019 neutralizing antibody. Human monoclonal IgG antibody against CTGF/CCN2 (FG3019) and non-specific h-IgG were obtained from FibroGen, Inc. 6-7-month-old wild type male mice were treated via intraperitoneal injection (IP). A dose of 25 mg/Kg was administered three times per week [32, 34]. Denervation was performed 1 week after the treatment started. 4 days or 2 weeks after denervation, mice were euthanized as described above.
Treatment with TGF-βR kinase inhibitors: SB525334 and SB431542 were obtained from Sigma. A 10 mg/Kg dose was IP administered daily to 6-7-month-old wild type male mice. Denervation was performed 2 days after the first injection, and muscles were collected 4 days after denervation.
Treatment with SRI31277: SRI31277 peptide was synthesized by Biomatik USA (Wilmington, DE) . Administration to 6-7-month-old wild type male mice was performed implanting a subcutaneous micro-osmotic pump (Alzet, model 1007D) delivering a 30 mg/Kg/day [53] dose during 7 days. The osmotic pump was implanted 3 days prior to denervation, and muscles were collected 4 days after denervation.
Isometric force measurement. Isolated TA muscle was submerged in a bath with a buffer containing oxygenated Krebs-Ringer solution. The knee bone and tendon were used to attach the muscle-to-muscle force transducer and the bottom of the bath respectively. The isometric force of isolated TA muscle was measured at optimum muscle length (Lo). Stimulation voltage was determined from the voltage necessary to produce a maximum isometric twitch force. Maximum isometric force was determined by stimulating from 1 to 200Hz for 450 ms with 2 min rest between the stimuli. After measurements, the muscles were removed from the bath, trimmed of their tendons and of any non-muscle tissue and weighed. Specific force (force normalized with respect to muscle fiber cross-sectional area; mN/mm2) was calculated from muscle mass and Lo [32, 44].
Hematoxylin & eosin staining. GT muscle cryosections (7 µm) were placed onto glass slides. Hematoxylin and eosin (H&E) staining was performed to assess muscle architecture and histology. Briefly, tissue sections were incubated for 10 minutes in formalin (10% v/v), then washed with water, incubated for 5 minutes with diluted H&E (Merck, Darmstadt, Germany; 25% v/v in H2O) and washed with water. Eosin was added for 30 seconds and then dehydration with ethanol was performed. Finally, Entellan (Merck, Darmstadt, Germany) was added to the slices. Sections were imaged using bright field microscopy on a Nikon Eclipse E600 [34].
Immunohistochemistry (IHC): 7μm muscle cryosections were fixed in cold ethanol, rinsed in 0.05M TBS buffer, pH 7.6, and incubated overnight with primary antibodies against CTGF/CCN2/CCN2 (sc-14939, Santa Cruz Biotechnology, Santa Cruz, CA, USA; (1:100 dilution in TCT buffer (TBS, carrageenan 0.7%, Triton X-100 0.25%)). Sections were placed at RT°, washed 3 times for 5 min in TBS and then incubated with secondary antibodies (1:100) for 30min followed by three 5 min TBS washes. The sections were then incubated with peroxidase-antiperoxidase (PAP) complex (1:200) (MP Biomedicals, Aurora, OH, USA) for 30min, then washed in TBS 3 times for 5min. The immunoperoxidase reaction was visualized after incubation of sections in 0.1% diaminobenzidine and 0.03% hydrogen peroxide for 2 min [36]. Sections were washed with tap water and counterstained with H&E, dehydrated in an ethanol gradient and cleared with xylene.
Indirect immunofluorescence (IFI): For GT immunofluorescence, cryosections (7 µm) were fixed in 4% paraformaldehyde (Merck, Darmstadt, Germany), blocked for 1 hour in blocking buffer (1% BSA, BM-0150, Winkler, Santiago, CL; 1% gelatin from cold water fish skin, G7765, Sigma, St. Louis, MO, USA; 0,01% Triton X100, X100-1L, Sigma, MO, St. Louis, USA) in PBS and incubated overnight at 4°C with the following antibodies: anti-fibronectin (Sigma-Aldrich, St. Louis, MO, USA), anti-collagen-I (Abcam, Cambridge, UK), anti-p-Smad3 (Cell Signaling, Danvers, MA, USA, anti-laminin (Sigma-Aldrich, St. Louis, MO, USA).The corresponding Alexa Fluor 568 or 488-conjugated anti-IgGs (Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies. Alexa Fluor 594 conjugated-wheat germ agglutinin (WGA) (Thermo Fisher, Waltham, MA, USA) was used to stain cell surface. For nuclear staining, sections were incubated with 1µg/mL Hoechst 33258 during the incubation with secondary antibodies. Slices were then washed in water and mounted in fluorescent mounting medium (DAKO, Santa Clara, CA, USA). For visualization of ECM proteins and pSmad3, cross-sections were visualized on a Nikon Eclipse E600 epifluorescence microscope with 20X or 40X objectives, using NIS-Elements software v4.20, 32 bit [26, 27]. For evaluation of fiber versus interstitial nuclei for pSmad3 staining, cross-sections were imaged on a Nikon Eclipse C2 confocal spectral microscope using NIS-Elements AR software 4.00.00 (build 764) LO, 64 bit. The objective used was 40x Oil Plan Apo NA 1.0 WD 0.16 DIC H. a, b, x, y, images were zoomed 2.5x optically from the original picture.
Determination of occupied area and fiber diameter. For quantifying the area occupied by CTGF, collagen I or fibronectin, immunostained microphotographs from transversal muscle cryosections were selected by adjusting color threshold and measuring the area fraction with the ImageJ software (NIH, USA). Values were expressed as a percentage of area occupied or fold-change respect to contralateral muscle. For determination of fiber diameter, muscle cryosections stained for laminin or with fluorescent wheat germ agglutinin (WGA) were selected by adjusting color threshold and using the ROI manager plugin also from the ImageJ software and measuring the minimal Feret´s diameter of each fiber [32]. Fiber size was calculated using individual or reconstructed images of the GT muscle. Quantifications were performed using 3-7 20x images per muscle, and 2-3 muscles per condition.
Immunoblot analysis. Skeletal muscles were homogenized in 10 volumes of Tris-EDTA buffer pH 7.4 with 1mM phenylmethylsulfonyl fluoride (PMSF) using an Ultraturrax T25 (Labortechnik). Then, the same volume of buffer containing 2% glycerol, 4% SDS and 0.125M Tris pH 6.8 was added to the homogenates and mixed. Muscle homogenates were incubated at 50°C for 20 min and centrifuged for 10 min at 14,000 rpm to pellet insoluble material. Protein concentration in the supernatant was determined using the BCA Assay kit (Pierce, Rockford, IL, USA) with BSA as the standard. Aliquots (40-50µg) were subjected to SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked in 5% nonfat milk in TBS (50 mM Tris-Cl, pH 7.6; 150 mM NaCl) and probed with the following antibodies at 4°C overnight: anti-fibronectin (Sigma-Aldrich, St. Louis, MO, USA), anti-pSmad3 (Cell Signaling, Danvers, MA, USA), anti-PDGFRα (R&D Systems, Minneapolis, MN, USA), anti-CTGF/CCN2 (Santa Cruz, USA), anti-GAPDH (Millipore, Billerica, MA, USA) and anti-α-tubulin (Sigma-Aldrich, St. Louis, MO, USA). Following incubation for 1 hour at room temperature, primary antibodies were detected with horseradish-peroxidase-conjugated secondary antibodies. All immunoreactions were visualized by enhanced chemiluminescence (Pierce, Rockford, IL, USA). Densitometric analysis and quantification were performed using the ImageJ software (NIH, USA) [26, 27].
RNA isolation, reverse transcription, and quantitative real-time PCR: Total RNA was isolated from GT muscle using TRIzol (Invitrogen) according to the manufacturer’s recommendations. Total RNA (2 mg) was reverse transcribed into cDNA using random primers and M-MLV reverse transcriptase (Invitrogen). TaqMan quantitative real-time PCR reactions were performed in duplicate on an Eco Real-Time PCR System (Illumina, San Diego, CA, USA) using predesigned primer sets for mouse-TGFb1 gene (Mm01178820_m1) and the housekeeping gene GAPDH (Mm99999915_g1; TaqMan Assays-on-Demand, Applied Biosystems, Foster City, CA, USA). Alternatively, we used primer sets for mouse TGF-β1 (Fwd: 5’-CTCCACCTGCAAGACCAT-3’; Rev: 5’-CTCCACCTGCAAGACCAT-3’) CACCACCACCCACGGAATCG-3’). mRNA expression was quantified with the comparative ΔCt method (2-ΔΔCt), using GAPDH as reference gene. The mRNA levels were expressed relative to the mean expression in control mice [26, 27].
 
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