ORIGINAL ARTICLE Journal of Cachexia, Sarcopenia and Muscle 2022; 13: 1373–1384 Published online 7 February 2022 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/jcsm.12923 RhoA/ROCK2 signalling is enhanced by PDGF-AA in fibro-adipogenic progenitor cells: implications for Duchenne muscular dystrophy Esther Fernández-Simón1,5 , Xavier Suárez-Calvet1,2, Ana Carrasco-Rozas1, Patricia Piñol-Jurado1,5, Susana López-Fernández3, Gemma Pons3, Joan Josep Bech Serra4, Carolina de la Torre4, Noemí de Luna1,2, Eduard Gallardo1,2 & Jordi Díaz-Manera1,2,5* 1 Neuromuscular Diseases Unit, Neurology Department, Hospital de la Santa Creu i Sant Pau and Biomedical Research Institute Sant Pau (IIB Sant Pau), Barcelona, Spain; 2 Centro de Investigaciones Biomédicas en Red en Enfermedades Raras (CIBERER)Madrid, Spain; 3Plastic Surgery Department, Hospital de la Santa Creu i Sant Pau, Autonomous University of Barcelona, Barcelona, Spain; 4Proteomics Unit, Josep Carreras Leukaemia Research Institute, Barcelona, Spain; 5John Walton Muscular Dystrophy Research Center, University of Newcastle, Newcastle upon Tyne, UK Abstract Background The lack of dystrophin expression in Duchenne muscular dystrophy (DMD) induces muscle fibre and re- placement by fibro-adipose tissue. Although the role of some growth factors in the process of fibrogenesis has been studied, pathways activated by PDGF-AA have not been described so far. Our aim was to study the molecular role of PDGF-AA in the fibrotic process of DMD. Methods Skeletal muscle fibro-adipogenic progenitor cells (FAPs) from three DMD treated with PDGF-AA at 50 ng/mL were analysed by quantitative mass spectrometry-based proteomics. Western-blot, immunofluorescence, and G-LISA were used to confirm the mass spectrometry results. We evaluated the effects of PDGF-AA on the activation of RhoA pathway using two inhibitors, C3-exoenzyme and fasudil. Cell proliferation and migration were determined by BrdU and migration assay. Actin reorganization and collagen synthesis were measured by phalloidin staining and Sircol assay, respectively. In an in vivo proof of concept study, we treated dba/2J-mdx mice with fasudil for 6 weeks. Muscle strength was assessed with the grip strength. Immunofluorescence and flow cytometry analyses were used to study fi- brotic and inflammatory markers in muscle tissue. Results Mass spectrometry revealed that RhoA pathway proteins were up-regulated in treated compared with non- treated DMD FAPs (n = 3, mean age = 8 ± 1.15 years old). Validation of proteomic data showed that Arhgef2 expres- sion was significantly increased in DMD muscles compared with healthy controls by a 7.7-fold increase (n = 2, mean age = 8 ± 1.14 years old). In vitro studies showed that RhoA/ROCK2 pathway was significantly activated by PDGF- AA (n = 3, 1.88-fold increase, P < 0.01) and both C3-exoenzyme and fasudil blocked that activation (n = 3, P < 0.05 and P < 0.001, respectively). The activation of RhoA pathway by PDGF-AA promoted a significant increase in proliferation and migration of FAPs (n = 3, P < 0.001), while C3-exoenzyme and fasudil inhibited FAPs proliferation at 72 h and migration at 48 and 72 h (n = 3, P < 0.001). In vivo studies showed that fasudil improved muscle function (n = 5 non-treated dba/2J-mdx and n = 6 treated dba/2J-mdx, 1.76-fold increase, P < 0.013), and histological studies demonstrated a 23% reduction of collagen-I expression area (n = 5 non-treated dba/2J-mdx and n = 6 treated dba/2J- mdx, P < 0.01). Conclusions Our results suggest that PDGF-AA promotes the activation of RhoA pathway in FAPs from DMD patients. This pathway could be involved in FAPs activation promoting its proliferation, migration, and actin reorganization, which represents the beginning of the fibrotic process. The inhibition of RhoA pathway could be considered as a poten- tial therapeutic target for muscle fibrosis in patients with muscular dystrophies. Keywords Duchenne muscular dystrophy; Fibro-adipogenic precursor cells; Platelet-derived growth factor; Muscular dystrophies; Fibrosis © 2022 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. 1374 E. Fernández-Simón et al. Received: 19 May 2021; Revised: 21 December 2021; Accepted: 30 December 2021 *Correspondence to: Jordi-Díaz-Manera, John Walton Muscular Dystrophy Research Center, University of Newcastle, Central Parkway, Newcastle upon Tyne NE1 3, UK. Tel: +34 609413007. Email: jordi.diaz-manera@newcastle.ac.uk Introduction signalling pathways such as Ras-MAPK, PI3K, or PLC-ƴ, which are involved in different cellular responses including prolifera- Duchenne muscle dystrophy (DMD) is a genetic disease pro- tion, cell differentiation, apoptosis inhibition, mobilization of duced by mutations in the DMD gene that leads to a fragile intracellular calcium, or cell motility.18,19 muscle membrane susceptible to be damaged during muscle Although the role of PDGF-AA has been widely studied in contraction.1,2 Muscle membrane fragility is the responsible fibrotic processes, its role in driving muscle fibrosis in muscu- of chronic injury resulting on continuous cycles of necrosis lar diseases is not completely known. We have previously and regeneration of muscle fibres, persistent inflammation demonstrated that PFGF-AA activates fibroblast proliferation and eventually loss of muscle fibres and their replacement and migration and increases the release of components of by fat and fibrous tissue leading to permanent weakness the extracellular matrix.20 Treatment of murine models of and disability.3,4 DMD with tyrosine kinase inhibitors blocking the PDGFRα, Fibro-adipogenic precursor cells (FAP) are mesenchymal such as imatinib, crenolanib, and nintedanib, decreased mus- muscle-resident stem cells characterized by the expression of cle fibrosis and improved muscle function.20–22 Moreover, platelet-derived growth factor receptor alpha (PDGFRα). FAPs previous studies have shown that PDGF-AA expression is are the main responsible of the fibrotic and fatty tissue expan- higher in dystrophic compared with healthy muscles23 and sion in muscular dystrophies.5–7 FAPs are activated upon acute that PDGF-AA serum levels are increased in DMD patients cor- injury, proliferate, and release components of the extracellular relating with several muscle function tests.24 However, the matrix (ECM) to serve as scaffold where the new regenerated pathways activated by PDGF-AA in FAPs in muscular dystro- myofibres are displayed. After muscle regeneration, excessive phies have not been described so far. This knowledge could FAPs are cleared by apoptosis mediated by tumour necrosis help us to identify new targets for therapies aiming to reduce factor alpha (TNF-α).8 However, in dystrophic muscles, contin- fibrosis in DMD and slow down the on-going degenerative uous release of growth factors by M2 macrophages perma- process. In this study, we have used mass spectrometry to nently activates FAPs proliferation, that release collagen-I identify Rho-kinase as one of the pathways activated by among other components of the ECM leading to an expansion PDGF-AA in FAPs isolated from human skeletal muscles. These of the fibrotic tissue.7,9 The molecular pathways driving FAPs results lead us to test fasudil, a pan-Rho-kinase inhibitor, in a fate in muscular dystrophies are just started to be understood. murine model of DMD observing a reduction of muscle fibro- It is well-known that TGF-β promotes FAP proliferation, sis and consequently an improvement on muscle strength. inhibits TNF-α mediated FAP apoptosis, and drives FAPs to differentiate into fibroblasts.10,11 Apart from TGF-β, the family of platelet-derived growth fac- tors (PDGFs) has been involved in the regeneration and degen- Material and methods eration process. On the one hand, PDGF-BB has been shown to activate satellite cell proliferation and chemotaxis and PDGFRβ Cell culture is highly expressed in dystrophic muscle compared with healthy muscle probably influencing muscle regeneration.12,13 We used muscle biopsies from three DMD patients seen at On the other hand, PDGF-AA has been involved in the fibrotic Hospital de la Santa Creu i Sant Pau (HSCSP) in Barcelona process of several tissues including the skin, lungs, or liver.14,15 (Table 1). The Ethics Committee of HSCSP approved the PDGF-AA binds to PDGFRα, a tyrosin kinase receptor, inducing study, and all participants signed an informed consent form. receptor dimerization and autophosphorylation of the intra- All study procedures were performed in accordance with cellular domain. 16,17 Autophosphorylation of PDGFRα triggers Spanish regulations. Table 1 Clinical information of DMD patients DMD patient 1 DMD patient 2 DMD patient 3 Age (years) 9 9 7 DMD mutations 48–52 deletion 50–52 deletion 52 deletion Muscle biopsied Biceps Biceps Biceps Clinical status Ambulant Ambulant Ambulant Steroid treatment Yes Yes Yes Journal of Cachexia, Sarcopenia and Muscle 2022; 13: 1373–1384 DOI: 10.1002/jcsm.12923 Platelet-Derived Growth Factor AA enhances RhoA/ROCK2 signalling 1375 Muscle explants were cultured and FAPs were isolated as with inserts (Ibdi, Munich, Germany) at 48 and 72 h described in the supporting information. FAPs were treated (see supporting information). with PDGF-AA at 50 ng/mL each day for 4 days (Figure S1E). To test the activation of the RhoA pathway with PDGF-AA, the pathway was first inhibited with fasudil 50 μM (Bio- Western blot Techne, Minnesota, USA) or C3-exoenzyme 2 μg/mL (Cyto- skeleton, Denver, USA) for 15 h and then activated with Cells corresponding to each condition or muscle tissue were PDGF-AA at 50 ng/mL for 20 min and proceed to the G-LISA lysed in RIPA buffer (Sigma-Aldrich) with protease and or IF assay of myosin light chain phosphorylation (p-MLC). phosphatase inhibitors (Roche, Basel, Switzerland), and the To test the functional effect of the PDGF-AA pathway and specific bands corresponding to the proteins of interest were its inhibition, C3-exoenzyme at 2 μg/mL or fasudil at 50 μM visualized with the Odyssey infrared detection system (Li-Cor) was first added to the culture and then PDGF-AA was added together with the Image Studio software (Li-Cor). Total pro- 4 h later at 50 ng/mL. This inhibition/induction cycle was tein bands were measured with the Revert 700 kit (Li-Cor) repeated for as many days as the test lasted. (Figure S1F). The primary and secondary antibodies used in the study are listed in Table 1 (see supporting information). Quantitative proteomics Histology and immunofluorescence The protein samples were extracted and analysed using a Lumos Orbitrap mass spectrometer (Thermo Fisher Scientific, Cultured FAPs were fixed with ethanol and incubated with San Jose, CA, USA) coupled to an EASY-nLC 1000 [Thermo blocking solution (Santa Cruz Biotech, Dallas, TX, USA). Frozen Fisher Scientific (Proxeon), Odense, Denmark]. The samples muscle sections were obtained with a cryostat (Leica were analysed with the MaxQuant software (version Microsystems, Wetzlar, Germany), fixed with acetone and 1.6.1.0) through the human Swissprot database (see incubated with blocking solution (Santa Cruz Biotech). The supporting information). primary and secondary antibodies used in the study are listed in Table 2. Images were obtained with an Olympus BX51 microscope Cell viability coupled to an Olympus DP72 camera. ImageJ software was used to quantify the intensity of individual cells and the area The effect of each treatment in FAPs viability was measured of positive staining in muscle tissue. A minimum of five inde- with the PrestoBlue reagent (Invitrogen) following manufac- pendent fields per staining were quantified.25 turer’s instructions. Fluorescence was measured at wave- lengths 570 nm excitation and 600 nm emission using a microplate reader (INFINITE M1000 PRO, Tecan Trading AG, Switzerland). Mouse model Animal procedures were performed according to the National G-LISA, ROCK activity, Sircol assay, and cytokine Institutes of Health Guidelines for the Care and Use of Labo- array ratory Animals and were approved by the Ethical Committee of the Universitat Autònoma de Barcelona. The animals used The RhoA-GTP protein was analysed by a colorimetric assay in this study were all 7-week-old males. Six dba/2J-mdx males using the G-LISA assay (Cytoskeleton). The enzymatic activity were treated with fasudil (LC Laboratories) that was diluted of ROCK was assessed by a colorimetric method by using a in water and administered at 100 mg/kg/day orally for ROCK activity assay (Abcam) in protein extract from quadri- 6 weeks by gavage, according to previous studies.26–30 Five ceps. Total soluble collagen released by FAPs was measured untreated dba/2J-mdx males were used as controls and six using Sircol kit (Biocolor, UK). Mouse cytokines were deter- dba/2J males were used as healthy controls. mined using the Proteome Profiler Cytokine Array Panel A (R&D Systems, USA) (see supporting information). Grip strength test Proliferation and migration assays Force was measured using a grip strength meter. Mice were initially held by the tail above the grid. Once mice The proliferation assay was carried out following the manu- grasped the grid, they were pulled out until release. The facturer’s instructions (Roche, Indianapolis, IN) at 24 and procedure was repeated 5 times, and the mean of the 72 h. The migration assay was performed using 24-well plates three highest values was used. Mean result was normalized Journal of Cachexia, Sarcopenia and Muscle 2022; 13: 1373–1384 DOI: 10.1002/jcsm.12923 1376 E. Fernández-Simón et al. Table 2 Primary, secondary antibodies, and dyes used in this study Target Host Dilution Application Manufacturer ArHGEF2 Rabbit 1/100 WB Sigma-Aldrich RhoA Rabbit 1/100 WB Cell Signalling TE-7 Mouse 1/100 IF Merck Desmin Mouse 1/50 IF Novocastra p-MLC2 Mouse 1/100 IF Cell Signalling PDGFRα Goat 1/50 IF R&D System LIMK2 Rabbit 1/100 IF ProteinTech PDGFA Rabbit 1/50 IF Merck Millipore MLC2 Rabbit 1/50 IF Cell Signalling F4/80 Rabbit 1/200 IF Cell Signalling CD3 Rabbit 1/50 IF Abcam Ly6g + Ly6c Rat 1/50 IF Abcam CD19 Rabbit 1/50 IF Abcam a Wheatgerm agglutinin-488 N/A 1/100 IF Invitrogen a Hoesch 33342 N/A 1/1000 IF Invitrogen a Rhodamine Phalloidin N/A 1/1000 IF Abcam CD140a-PE Recombinant 1/200 IF Miltenyi Biotec b CD56 Ms N/A MACS Miltenyi Biotec PDGFRα-biot Goat 1/75 FC R&D System CD56-PE Ms 1/20 FC Miltenyi Biotec a LIVE/DEAD N/A 1/1000 FC Thermo Fisher Scientific b F4/80 Rat N/A FC Bio Legend b CD45 Rat N/A FC eBioscience b Integrin-α7 Rat N/A FC Miltenyi Biotec b Ter119 Rat N/A FC eBioscience b CD31 Rat N/A FC Thermo Fisher BD b Sca1 Rat N/A FC BD b CD163 Rat N/A FC eBioscience b CD206 Rat N/A FC Bio Legend b CD80 REA N/A FC Miltenyi Biotec b HLA-DR Rat N/A FC Miltenyi Biotec Secondary antibody Dilution Conjugation Application Manufacturer Donkey anti-goat Goat 1/500 Alexa594 IF Invitrogen anti-mouse IgG Alexa594 Donkey anti-rabbit Goat 1/500 Alexa647 IF Invitrogen anti-mouse IgG Alexa594 Goat anti-mouse 1/500 Alexa488 IF Invitrogen Goat anti-rabbit 1/500 Alexa488 IF Invitrogen Goat anti-rat 1/500 Alexa594 IF Invitrogen Goat anti-rabbit 1/7500 IRDye 800 WB LICOR Goat anti-rabbit biotinilyated 1/2000 N/A WB Invitrogen Streptavidin 800 1/2000 IRDye 800 WB LICOR Streptavidin PE-Cy5 1/200 PE-Cy5 FC Biolegend a It is not an antibody. b Volume per test following manufacturer’s instructions. FC, flow cytometry; IHQ, immunohistochemistry; MACS, Magnetic activated cell sorting; Ms, mouse; N/A, not applicable. to the body weight of the animal and is presented as grip adjusted according to the single stained controls. Total viable force/weight (N/g). FAPS (Viable, Ter119-/CD45/CD31 /integrin-α7 /sca1+) and macrophages (Viable, CD45 + F4/80+) counts were quantified using MACSQuantify software and normalized according to Flow cytometry grams of muscle tissue. FlowJo v10 software was used for data analysis and data elaboration. Single cell suspension from cell culture, quadriceps, and tibial anterior were obtained for flow cytometry. Cultured human muscle derived cells were labelled using anti-PDGFRα Statistical analysis followed by streptavidine-PECy5 and anti-CD56. Cells ob- tained from mice were labelled using anti-sca1, anti-CD45, Results are expressed as mean ± standard error of means anti-F4/80, anti-CD163, and a viability marker (Table 1). Sam- (SEM). Differences among the groups were analysed using ples were acquired with the MACSQuant Analyzer 10 flow one-way ANOVA Test. When ANOVA revealed significant dif- cytometer (MiltenyiBiotec). Doublet cells were excluded ferences, the Tukey post hoc test was performed. The signif- using Forward scatter area and height. Compensations were icance level was set at P < 0.05. The statistical analyses were Journal of Cachexia, Sarcopenia and Muscle 2022; 13: 1373–1384 DOI: 10.1002/jcsm.12923 Platelet-Derived Growth Factor AA enhances RhoA/ROCK2 signalling 1377 calculated using GraphPad Prism version 7 for Windows non-treated FAPs. We observed that 1890 proteins were dif- (San Diego, CA: GraphPad Software, Inc). ferentially expressed (Volcano plot in Figure 1A). We built a proteomap of the up-regulated proteins according to the functional gene classification [Kyoto Encyclopedia Genes and Genomes (KEGG)] (Figure 1B). We observed an increase Results in the expression of proteins involved in various signalling pathways (MAPK, PI3K-Akt, FoxO, or Hedgehog), cell metabo- PDGF-AA induces activation of Rho-A pathway in lism, and genetic information processing (Figure 1C,D). human muscle FAPs Among the different protein groups that were up-regulated, we were interested in those involved in cell functions related We first confirmed that the number of FAPs was increased in to fibrosis such as cytoskeletal rearrangement, cell adhesion, DMD muscle biopsies (n = 2) compared with healthy controls and rearrangement of actin filaments. Proteins related to (n = 2) (Figure S1A,B). Then, we isolated FAPs from muscle bi- these cellular functions, such as proteins of the Ras homo- opsies of three DMD patients and treated them with 50 ng/ logue gene pathway (Rho), were increased in FAPs treated mL of PDGF-AA for 4 days. To analyse changes in the protein with PDGF-AA as displayed in a heatmap showing the differ- profile, we used quantitative proteomic analysis with mass ences between DMD-FAPs untreated and treated with spectrometry comparing PDGF-AA treated FAPs and PDGF-AA (Figure 1E). Among the different proteins of this Figure 1 Proteome analysis of PDGF-AA treated DMD FAPs. (A) Volcano plot representing the total proteome. Data are presented as the protein abun- dance changes of PDGF-AA treated FAPs relative to untreated FAPs. Significantly differentially expressed proteins (Student’s t-test, P ≤ 0.05) are highlighted in blue. (B–D) Proteomap of up-regulated proteins after PDGF-AA treatment. The proteomic map visualizes the composition of the proteomes in terms of abundance and function of the proteins. Each protein is represented by a polygon, and the area of each polygon reflects the abundance of the proteins (calculated with fold-change). Functionally related proteins appear in adjacent regions. The three panels represent three hierarchy levels, being the first one the individual protein detected (B), the second one shows proteins grouped into pathways (C), and the last one shows the functional category associated to a group of proteins (D). (E) Up-regulated proteins shown in proteomaps were also represented in heatmap showing the results of Reactome database. Journal of Cachexia, Sarcopenia and Muscle 2022; 13: 1373–1384 DOI: 10.1002/jcsm.12923 1378 E. Fernández-Simón et al. family, we focused on the Ras homologue gene family mem- analysed if the addition of PDGF-AA to DMD-FAPs in culture ber A (RhoA) pathway because it has been involved in fibro- induced a significant increase in RhoA bound to guanosine tri- blast activation and fibrosis in several other pathologies.31 phosphate (GTP) (RhoA-GTP) compared with untreated FAPs (c ). To further confirm whether the increase in RhoA-GTP was mediated via activation of RhoA pathway, the effect of C3 exoenzyme and Fasudil treatment block Rho- pretreatment with the exoenzyme C3 (C3) was also analysed. kinase pathway activation mediated by PDGF-AA Exoenzyme C3 is an ADP ribosyl transferase that selectively ribosylates RhoA proteins at asparagine residue 41, rendering We validated mass spectrometry results in two different ex- it inactive. As expected, PDGF-AA treatment induced a signif- periments. First, we studied the expression of guanine nucle- icant 1.88-fold increase in the RhoA-GTP levels (n = 3, otide exchange factor Rho 2 (ArhGEF2), which is the effector SEM = 0.206, P < 0.01), while pretreatment with C3 blocked protein in the Rho-kinase pathway (Figure 2A), in DMD mus- the effect induced by PDGF-AA (n = 3, SEM = 0.07, P < 0.05) cle samples and aged and sex matched controls (loading con- (Figure 2C). Treatment with PDGF-AA, C3, or fasudil did not trol in Figure S1E). We observed a 7.7-fold increase in the affect cell viability at any time tested (Figure S1H). expression of ArhGEF2 in DMD muscles compared with Fasudil is a commercially available vasodilator used to control muscles (n = 2) (Figure 2B). In a second step we treat cerebral vasospasm but also useful for pulmonar hyper- Figure 2 RhoA pathway is up-regulated in DMD and PDGF-AA activates it. (A) Molecular pathway of RhoA/ROCK2 signalling downstream of ArhGEF2 which catalyses the replacement of Rhoa-GDP to RhoA-GTP, controlling the RhoA activation. RhoA bound to GTP activates the rho-associated coiled coil-forming protein kinase (ROCK), a serine/threonine kinase that regulates actin filament remodelling trough light chain myosin (MLC) phosphoryla- tion. (B) Quantification of WB bands of ArhGEF2 relative to total protein detection and representative bands of ArhGEF2 expression in muscle tissue obtained from healthy controls (n = 2) and DMD patients (n = 2). Total protein detection was used as loading control signal (Figure S1D). (C) Optical density at 490 nm indicating RhoA activity relative to total RhoA protein detected by WB. (D) Representative images of p-MLC staining using fluores- cence microscopy. Scale bar: 50 μm. (E) Quantification of p-MLC intensity of each cell after different treatments. (F) Representative images FAPs stained with phalloidin after different treatments. Scale bar: 50 μm. C-: Untreated FAPs. Data are represented as the mean of three replicates ± standard error of the mean. Results were statistically analysed using one-way ANOVA followed by Tukey post hoc test. Statistical signifi- cance was set at P < 0.05. **P < 0.01; ***P < 0.001. Journal of Cachexia, Sarcopenia and Muscle 2022; 13: 1373–1384 DOI: 10.1002/jcsm.12923 Platelet-Derived Growth Factor AA enhances RhoA/ROCK2 signalling 1379 tension. Fasudil inhibits RhoA pathway by blocking the Addition of PDGF-AA to the culture medium significantly in- Rho-associated protein kinase (ROCK) which phosphorylates creased FAPs proliferation at both 48 (n = 3, SEM = 0.02, myosin light chain 2 (p-MLC2). Treatment of DMD-FAPs with P < 0.001) and 72 h (n = 3, SEM = 0.05, P < 0.001) (3.6-fold PDGF-AA induced a statistically significant 2.3-fold increase in and 6-fold increase, respectively) compared with untreated MLC2 phosphorylation (n = 3, SEM = 0.33, P < 0.001), which FAPs. In contrast, treatment of FAPs with C3-exoenzyme was reversed by pretreatment with C3-exoenzyme (n = 3, (n = 3, SEM = 0.01, P < 0.001) and fasudil (n = 3, SEM = 0.11, P < 0.001) and fasudil (n = 3, SEM = 0.09, SEM = 0.02, P < 0.001) prior to stimulation with PDGF-AA P < 0.001), as shown in Figure 2D,E. Last step on the lead to a significant decrease in proliferation rate (3.27-fold Rho-kinase pathway is the polymerization of F-actin filament and 2.6-fold decrease, respectively) compared with FAPs mediated by MLC2 phosphorylation. Treatment of FAPs with treated with PDGF-AA at 72 h (Figure 3A). All treatment PDGF-AA induced polymerization on intracellular actin fila- doses and the different times of cell exposure were tested ments, which was impinged by pretreatment with C3 or for cell viability. PrestoBlue data showed that neither fasudil (Figure 2F). C3-exoenzyme nor fasudil had any effect on cell viability (Figure S1H). Treatment with PDGF-AA significantly enhanced FAPs mi- Fasudil blocks PDGF-AA mediated increase in gration in vitro compared with untreated FAPs at both 48 h proliferation, migration and expression of (n = 3, SEM = 0.51, P < 0.001) and 72 h (n = 3, SEM = 0.66, collagen-I by human FAPs in vitro P < 0.001) (2.48-fold and a 3.37-fold increase, respectively). This effect was significantly blocked when C3-exoenzyme We studied the effect of PDGF-AA, C3-exoenzyme, and fasudil and fasudil were added to the medium (Figure 3B,C) either in proliferation, migration, and collagen-I production in vitro. at 48 or 72 h. C3-exoenzyme reduced migration rate 2.1-fold Figure 3 Effects of C3, fasudil, and PDGF-AA on FAPs from DMD patients. (A) FAP proliferation was analysed after 48 and 72 h. (B) FAP migration was analysed after 48 and 72 h. (C) Representative images of migration assay at both 48 and 72 h. Scale bar: 200 μm. (D) The amount of collagen delivered to the supernatant was analysed after 4 days treatment. C-: Untreated FAPs. Data are represented as the mean of three replicates ± standard error of the mean. Results were statistically analysed using one-way ANOVA followed by Tukey post hoc test. Statistical significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001. Journal of Cachexia, Sarcopenia and Muscle 2022; 13: 1373–1384 DOI: 10.1002/jcsm.12923 1380 E. Fernández-Simón et al. at 48 h (n = 3, SEM = 0.47, P < 0.001) and 3-fold at 72 h SEM = 0.64, P < 0.001) (Figure 4C). Additionally, we observed (n = 3, SEM = 0.25, P < 0.001). Fasudil reduced migration rate a 42% decrease in the PDGFRα area as a measure of the num- 2.43-fold at 48 h (n = 3, SEM = 0.41, P < 0.001) and 4-fold at ber of FAPs present in the quadriceps of treated mice (n = 6, 72 h (n = 3, SEM = 0.22, P < 0.001). SEM = 0.38) compared with non-treated mice (n = 5, PDGF-AA treatment also increased non-significantly the re- SEM = 0.95), although these differences did not reach signifi- lease of collagen-I to the culture medium an effect that was cance (Figure 4E,F). reversed by the addition of C3-exoenzyme and fasudil (Figure 3D). C3-exoenzyme significantly reduced the release of collagen-I 1.7-fold (n = 3, SEM = 0.33, P < 0.05). Effect of fasudil treatment in inflammation Quantification of macrophages was performed by analysing Fasudil treatment improves muscle function and the area of the quadriceps stained with F4/80 maker. Fasudil reduces muscle fibrosis in the dba/2J-mdx murine induced a 74% reduction in treated mice (n = 6, SEM = 0.15) model of DMD when compared with untreated mice (n = 5, SEM = 0.73) (Fig- ure 4F,G). Those results were in accordance with flow cytom- Based in our in vitro results, we decided to test if fasudil had etry analysis which showed a 18.3% decrease in the number an antifibrotic effect in vivo in the dba/2J-mdx murine model of F4/80 positive cells in treated mice (n = 6, SEM = 33.49) of DMD. We first confirmed that PDGF-AA levels were in- compared with non-treated mice (n = 5, SEM = 39.82) (Figure creased in muscle samples of dba/2J-mdx mice compared 4H). Indeed, we observed a trend towards a reduction in the with healthy WT controls (Figure S2A) as happens in DMD pa- expression of profibrotic M2 markers, such as CD163 and tients. Based on these results, we treated six dba/2J-mdx mice CD206, in macrophages obtained from fasudil treated mice. of 7 weeks old with fasudil at a dose of 100 mg/kg/day orally In detail, we observed a 31% reduction in the expression for 6 weeks. Mice did not experience any change in their be- levels of CD163 marker in macrophages obtained from skele- haviour during the treatment period suggesting that there tal muscles of treated mice (n = 6, SEM = 1.10) although there were not significant adverse effects. However, we observed was only a 3% lower number of CD163 + cells compared with a decrease in weight when compared treated (n = 6, non-treated mice (n = 5, SEM = 2.58). Similarly, we observed a SEM = 0.94) and untreated mice (n = 5, n = 0.60) (Figure 18% reduction in the expression levels of CD206 marker S2B) although these differences did not reach statistical signif- in macrophages obtained from treated mice (n = 6, icance. We confirmed that fasudil inhibited RhoA pathway by SEM = 1.43) although there was only a 10% lower number analysing different downstream effector targets of RhoA, such of CD206 + cells compared with non-treated mice (n = 5, as ROCK2, LIM domain kinase 2 (LIMK2), and Formin Homol- SEM = 5.91) (Figure 4I). We did not observe any difference ei- ogy 2 Domain Containing 1 (FHOD1), being the two later pro- ther in the expression levels or in the number of CD209 cells teins involved in the regulation of actin filament dynamics which also identify M2 macrophages. We did not observe (Figure S3A). We also quantified the enzymatic activity of changes in the percentage of positive cells or in the expres- ROCK, which was decreased in quadriceps in treated mice sion of CD80 and DR, two well-known M1 markers, in the (n = 6, SEM = 0.12) compared with non-treated mice (n = 5, macrophages obtained from skeletal muscles of treated mice SEM = 0.14), although these changes were non-significant compared with non-treated mice (Figure S2C). (Figure S3B). However, the protein expression of LIMK2 and We also studied the population of T cells, B cells, and neu- MLC2 was significantly reduced in treated (n = 6, SEM = 0.45 trophils and observed a trend towards a reduction in neutro- and n = 6, SEM = 0.05, respectively) compared with untreated phils population in treated mice (n = 6, SEM = 0.72) compared mice (n = 5, SEM = 0.97 and n = 5, SEM = 0.64, respectively) with non-treated (n = 5, SEM = 0.74), while there were no (Figure S3C–F). We analysed muscle strength using forelimb changes in the number of T and B cells (Figure S4A–D). Fi- grip-strength after 6 weeks of treatment and compared the nally, we studied if fasudil treatment modified the expression results obtained in non-treated dba/2J-mdx and healthy con- of several cytokines involved in the inflammatory process in trol mice. We observed a significant 1.76-fold increase in fore- muscular dystrophies. As shown in a heatmap in Figure S4E, limb strength in treated mice (n = 6, SEM = 1.63) compared we observed a reduction in TNFα, IL-4, IL-17, CXCL1, CXCL2, with non-treated mice (n = 5, SEM = 2.29, P < 0.01) (Figure CXCL10, CCL2, CCL3, or CCL17 among others in fasudil treated 4A). Interestingly, differences between treated mice and WT mice when compared with non-treated mice. mice did not reach significance. We also analysed the effect of fasudil on quadriceps histology. Fasudil induced a signifi- cant 23% reduction in collagen-I area in the quadriceps of Discussion treated mice (n = 6, SEM = 1.63, P < 0.05) (Figure 4B,D). We detected a significant increase in the myofibre size of treated In this study, we have investigated the molecular pathways mice (n = 6, SEM = 0.60) compared with non-treated (n = 5, activated by PDGF-AA in skeletal muscles of patients with Journal of Cachexia, Sarcopenia and Muscle 2022; 13: 1373–1384 DOI: 10.1002/jcsm.12923 Platelet-Derived Growth Factor AA enhances RhoA/ROCK2 signalling 1381 Figure 4 Effect of fasudil treatment in the dba/2J-mdx mice. (A) Grip strength test after 6 weeks treatment with fasudil in dba-2J WT mice, untreated dba/2J-mdx, and fasudil treated dba/2J-mdx. Results are relative to weight of animals. (B) Quantification of collagen I expression area in quadriceps. Scale bar: 200 μm. (C) Quantification of muscle fibre size mean and frequency in dba-2J WT mice, untreated dba/2J-mdx, and fasudil treated dba/2J- mdx. (D) Representative immunofluorescence images of collagen I expression in muscle samples. Scale bar: 200 μm. (E) Quantification of PDGFRα+ expression area in quadriceps. (F) Representative immunofluorescence images of PDGFRα+ (yellow arrows), F4/80 (white arrows) expression in muscle samples. ECM is stained with wheat germ agglutinin (WGA) in green. Scale bar: 200 μm. (G) Quantification of macrophages expression area in quad- riceps. (H) Quantification of results obtained with flow cytometry to analyse the F4/80 macrophages (Mo) accumulated in quadriceps and tibial ante- rior obtained from mice. Data are represented as counts of F4/80 relative to the weight of quadriceps and tibial anterior. (I) Percentage of total macrophages and mean fluorescence intensity (MFI) of CD163 and CD206 markers were analysed by flow cytometry. Data are represented as the mean of the groups analysed ± standard error of the mean. Results were statistically analysed using one-way ANOVA followed by Tukey post hoc test. Sta- tistical significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001. DMD driving to muscle fibrosis. We have observed that that reduces FAPs activation in vitro. We performed a PDGF-AA activates RhoA/ROCK2 pathway which can be effec- proof-of-concept preclinical study to validate these findings tively blocked by fasudil, a well-known Rho-kinase inhibitor observing a reduction in muscle fibrosis in the murine model Journal of Cachexia, Sarcopenia and Muscle 2022; 13: 1373–1384 DOI: 10.1002/jcsm.12923 1382 E. Fernández-Simón et al. of DMD. Our results expand the molecular pathways involved was only observed at 72 h. The lack of proliferation inhibition in the process of muscle fibrosis in DMD and support further at 48 h after C3-exoenzyme or fasudil treatment could be due investigations in the efficacy of Rho-kinase inhibitors as a to other signalling pathways activated after PDGF-AA treat- treatment of DMD in particular and muscular dystrophies in ment. PDGF-AA is a mitogen growth factor that not only general. activates RhoA pathway but also triggers other signalling Absence of dystrophin in DMD produces sarcolemma in- pathways, such as MAPK, PLC, or PI3K.16,17 While these mo- stability leading to expansion of fibrotic and fat tissue.32,7 lecular pathways are mainly involved in cell proliferation, Several growth factors have been related to the fibrotic ex- RhoA/ROCK2 is involved in actin remodelling. pansion; however, the role of PDGF-AA has not been fully Because the Rho/ROCK-mediated pathway interacts with studied. Although previous studies found that the signalling other signalling pathways known to contribute to fibrosis,41–43 through PDGFRα is persistent in DMD leading to fibrosis we tested whether fasudil reduces fibrosis in the dba/2J-mdx and hindering repair,21 the molecular consequences of persis- mice. Fasudil is a selective inhibitor of ROCK1 and ROCK2 iso- tent exposure of FAPs to PDGF-AA have not been addressed. forms that has been used in clinic as a first-generation selec- To study the molecular changes that occurs in DMD, we tive Rho/ROCK inhibitor.44 Fasudil has been tested in treated FAPs from DMD patients with PDGF-AA and observed different diseases such as pulmonary hypertension, amyotro- an up-regulation of several proteins belonging to the RhoA/ phic lateral sclerosis, or cardiovascular disease.45,46 We ob- ROCK2 pathway. The RhoA/ROCK2 signalling pathway is well served that dba/2J-mdx mice treated with fasudil had better known for regulating actin cytoskeleton organization and cel- performance on grip strength test than non-treated mice. lular dynamics in several cell types. RhoA acts through two These results were associated to a decrease in the amount of molecular conformations: it is inactive when bound to a gua- collagen-I and to a tendency towards a decreased number of nosine diphosphate (GDP) and active when bound to GTP. FAP cells in the skeletal muscles of the treated animals com- ROCK2, the Rho downstream effector molecule, is a serine/ pared with the non-treated ones. These results are in accor- threonine kinase protein that regulates actin filament remod- dance with previous studies using fasudil as an antifibrotic elling by phosphorylating numerous downstream target pro- agent as it has been shown that fasudil reduces collagen area, teins, including the myosin binding subunit of myosin light number of infiltrating macrophages and production of TGF-β1 chain 2 (MLC2) phosphatase.33,34 Actin reorganization trig- and CTGF in renal interstitial fibrosis induced by unilateral ure- gered by RhoA/ROCK2 signalling pathway drive fibroblasts ac- teral obstruction and in bleomycin-induced pulmonary fibrosis tivation in different pathologic conditions such as cardiac in mice.46,47 fibrogenesis, tumour-cell growth31,35,36 or pulmonary artery Because the inflammatory response is another characteris- hypertension.37,38 Actin polymerization regulates cell polari- tic feature of DMD muscles, we also analysed inflammatory zation, organization of adhesion structures, and the genera- cells profile after treatment. Fasudil induced a trend towards tion of the force essential for cell migration.39 These events a reduction in the number of neutrophils and macrophages, allow cells to migrate into the injured site where they prolif- particularly in the number of CD163 and CD206 positive mac- erate and release components of the ECM to regenerate the rophages without modifying M1 population. Additionally, damaged tissue. However, an excessive activation of fibro- fasudil reduced the expression of several inflammatory cyto- blasts increases the ECM deposition leading to accumulated kines; some of them directly involved in the fibrotic process fibrogenesis. Although the rearrangement of actin produced such as IL4, IL17, or CCL17.48–51 Although previous studies in fibroblast activation has not been characterized in FAPs, have shown that RhoA can be expressed in macrophages, we previous studies suggest that fibroblasts and FAPs are pheno- just observed a non-significant reduction in their number typically and biochemically equivalent supporting that when blocking this pathway with fasudil.52 The changes that Rho-kinase activation could have a similar effect in both we have observed in the number of infiltrating inflammatory cells.40 We demonstrated that RhoA/ROCK2 pathway is in- cells and in the reduction of the cytokines levels could be ex- creased in muscles of DMD patients when compared with plained either by a direct effect of fasudil on inflammatory age and sex matched healthy controls subjects. Therefore, cells or by an indirect effect of the reduced fibrosis content we decided to analyse the effect of PDGF-AA on RhoA/ROCK2 of the muscle. It is well known that continuous muscle damage signalling pathway in FAPs isolated from DMD patients. First, perpetuates the inflammatory response in muscular dystro- we confirmed that RhoA/ROCK2 pathway can be modulated phies, and therefore, treatments improving the general archi- by PDGF-AA because C3-exoenzyme and fasudil significantly tecture of the muscles reducing muscle degeneration can have reduced the signalling pathway activation. Our results show an indirect effect reducing inflammation.2 For example, the at- that PDGF-AA activated RhoA/ROCK2 pathway resulting in tenuation of the fibrotic process in the muscle may improve an increase of actin filament polymerization, proliferation, muscle regeneration in treated animals reducing the number and migration and that both C3 and fasudil attenuated these of degenerating fibres and preventing macrophages and neu- effects. Although PDGF-AA induced a higher proliferation rate trophils to infiltrate the injured muscle. Indeed, the increased at 48 and 72 h, the blocking effect of RhoA/ROCK2 inhibitors fibre size observed in treated mice could be due to a better re- Journal of Cachexia, Sarcopenia and Muscle 2022; 13: 1373–1384 DOI: 10.1002/jcsm.12923 Platelet-Derived Growth Factor AA enhances RhoA/ROCK2 signalling 1383 generation of the muscle together with a less affected muscle Acknowledgements architecture. In summary, treatment with fasudil on dba-2J mdx mice improved grip-strength test, reduced expression of This study has been funded by Instituto de Salud Carlos collagen-I and resulted in a trend towards a decreased number III-FEDER through the project ‘PI18/01525’ (Co-funded by of PDGFRα+ cells on skeletal muscles, confirming our results European Union ERDF), by AFM (France) through the in vitro. Our results suggest that these effects are mainly ex- project 22525, by Fundación Isabel Gemio, by Academy of plained by the effect that RhoA/ROCK2 inhibition has on fibro- Medical Sciences Professorship Scheme to JDM (APR4\1007) sis deposition. and by a MRC grant to JDM (MR/W019086/1). Although ROCK is expressed in different cell types, side ef- fects produced by fasudil does not include major safety concerns.44 Fasudil targets the ATP-dependent kinase domain of either ROCK1 and ROCK2 with equal potency and without Conflict of interest selective effects. It is worth noting that ROCK2 is the isoform Esther Fernández-Simón, Xavier Suárez-Calvet, Ana Carrasco- detected in our proteomic analysis, and previous studies have Rozas, Patricia Piñol-Jurado, Susana López-Fernández, Joan shown that ROCK2 is the predominant isoform in skeletal Josep Bech Serra, Carolina de la Torre, Noemí de Luna, muscle.53 Eduard Gallardo, and Jordi-Díaz-Manera declare that they Muscle fibrosis is an irreversible process that takes place in have no competing interests. muscular dystrophies and should be prevented before the ac- cumulation of ECM disturbs muscle architecture and func- tion. Because FAP activation and ECM remodelling start early during skeletal muscle degeneration, inhibiting that Ethical guidelines statement the initial cellular changes that occur on FAPs could prevent the increase of ECM deposition. Therapies focused on The study was approved by The Ethics Committee of Hospital targeting actin rearrangement to prevent FAP migration into de la Santa Creu i Sant Pau (HSCSP) in Barcelona. Patient and the injury site may help to slow down the progression of fi- controls signed an informed consent to use their muscle bi- brosis. Coupling anti-fibrotic therapies with cell therapy or opsy for research purposes. Animal procedures were per- gene therapy could also lead to a better outcome of these ex- formed according to the ethical standards laid down in the perimental therapies. Then, by inhibiting the ROCK2 signal- 1964 Declaration of Helsinki, National Institutes of Health ling in FAPs, we are targeting the first stages of fibrosis Guidelines for the Care, and Use of Laboratory Animals and which could be further studied as a potential treatment to re- were approved by the Ethical Committee of the Universitat duced muscle fibrosis in DMD. Autònoma de Barcelona. The authors of this manuscript cer- In summary, this study demonstrates that PDGF-AA in- tify that they comply with the ethical guidelines for author- duces RhoA/ROCK2 pathway signalling in DMD-FAPs leading ship and publishing in the Journal of Cachexia, Sarcopenia to their proliferation, migration, and actin reorganization. and Muscle.54 Treatment with fasudil, a well-known ROCK inhibitor, blocks the effect of PDGF-AA on cells in vitro and reduces muscle fi- brosis increasing muscle strength in a DMD murine model. Online supplementary material Our results open the door to new studies further characteriz- ing the efficacy of more specific ROCK2 inhibitors as potential Additional supporting information may be found online in the new treatments for DMD. Supporting Information section at the end of the article. References 1. Birnkrant J, Bushby K, Bann CM, Alman and management of Duchenne muscular 6. 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