biomedicines Article Growth Factors Do Not Improve Muscle Function in Young or Adult mdx Mice Tue L. Nielsen 1 , Tessa M. Hornsyld 1 , Tomàs Pinós 2,3 , Camilla Brolin 4 , John Vissing 1 and Thomas O. Krag 1, * 1 Copenhagen Neuromuscular Center, Department of Neurology, Rigshospitalet, University of Copenhagen, DK-2100 Copenhagen, Denmark; tue.leth.nielsen.01@regionh.dk (T.L.N.); tmuho@dtu.dk (T.M.H.); john.vissing@regionh.dk (J.V.) 2 Mitochondrial and Neuromuscular Disorders Unit, Vall d’Hebron Institut de Recerca, Universitat Autònoma de Barcelona, 08035 Barcelona, Spain; tomas.pinos@vhir.org 3 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 28029 Madrid, Spain 4 Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark; cbh@sund.ku.dk * Correspondence: thomas.krag@regionh.dk; Tel.: +45-3545-7614; Fax: +45-3545-6138 Abstract: Muscular dystrophies constitute a broad group of genetic disorders leading to muscle wasting. We have previously demonstrated that treating a muscular atrophy mouse model with growth factors resulted in increased muscle mass. In the present study, we treated the Duchenne mouse model mdx for 12 weeks with myogenic growth factors peri- and post-onset of muscular degeneration to explore the effects in the oxidative muscle soleus and the glycolytic muscle extensor digitorum longus (EDL). We found no overall beneficial effect in the peri-onset group at the conclusion of the study. In the post-onset group, the functional improvement by means of electrophysiological examinations ex vivo was mostly confined to the soleus. EDL benefitted from the treatment on a molecular level but did not improve functionally. Histopathology revealed signs of inflammation  at the end of treatment. In conclusion, the growth factor cocktail failed to improve the mdx on a  functional level. Citation: Nielsen, T.L.; Hornsyld, T.M.; Pinós, T.; Brolin, C.; Vissing, J.; Keywords: mdx; DMD; muscular regeneration; hepatocyte growth factor; leukemia inhibitory factor Krag, T.O. Growth Factors Do Not Improve Muscle Function in Young or Adult mdx Mice. Biomedicines 2022, 10, 304. https://doi.org/10.3390/ biomedicines10020304 1. Introduction Duchenne muscular dystrophy (OMIM, 300377: DMD) is a fatal, X-linked disease, Academic Editor: Marco Segatto which affects nearly 1 in 5000 newborn boys [1]. The disease is caused by absence of Received: 6 January 2022 dystrophin, which in the healthy muscle fiber functions as a link between the sarcolemma- Accepted: 27 January 2022 associated dystrophin-associated glycoprotein complex and the actin cytoskeleton ap- Published: 28 January 2022 paratus of the skeletal muscle fiber, thereby connecting the contractile apparatus to the Publisher’s Note: MDPI stays neutral membrane [2]. Disturbances of this relationship lead to loss of sarcolemmal integrity, ele- with regard to jurisdictional claims in vated levels of plasma creatine kinase (CK), muscular wasting, and fibro-fatty replacement published maps and institutional affil- of the muscle tissue [3,4]. While therapeutic strategies to rescue muscle function in DMD iations. have been numerous, including stem cell therapy, utrophin replacement, growth factors, antisense-mediated exon-skipping, and viral reintroduction of mini/micro-dystrophins, a cure has yet to emerge. We previously treated a hypoxia-induced muscular atrophy mouse model with growth Copyright: © 2022 by the authors. factors (hepatocyte growth factor (HGF) and leukemia inhibitory factor (LIF)) to activate Licensee MDPI, Basel, Switzerland. satellite cells and increase myoblast proliferation and differentiation, thus boosting what is This article is an open access article known as the regenerative response to muscle degeneration [5]. In the muscular atrophy distributed under the terms and model, this led to a significant increase in muscle mass compared to untreated animals. conditions of the Creative Commons This result would make the growth factor cocktail, which should be considered a potential Attribution (CC BY) license (https:// general treatment to muscle wasting, a candidate treatment for the mdx mouse model of creativecommons.org/licenses/by/ DMD, which–as patients–lacks dystrophin. Unlike other treatments that are meant to result 4.0/). Biomedicines 2022, 10, 304. https://doi.org/10.3390/biomedicines10020304 https://www.mdpi.com/journal/biomedicines Biomedicines 2022, 10, 304 2 of 16 in larger muscles, the concept of the growth factor treatment was to address both muscle wasting by increasing muscle mass and reinforce the sarcolemma, as membrane-associated proteins are upregulated as part of the maturation process of the regenerating/treated muscle fiber, by boosting the existing regenerative process. In this study, we tested if the findings from the hypoxia-induced atrophy model could be reproduced and lead to improved muscle function in the mdx. HGF stimulates satellite cell activation and differentiation through the cMet receptor [6–8] while LIF enhances myoblast survival and maintains proliferation [9–11]. We also introduce L-arginine, as it has been demonstrated to ameliorate the mdx phenotype in different distinct ways by decreasing inflammation, increasing regeneration and improving membrane integrity by upregulating utrophin [12]. Utrophin is an autosomal homolog of dystrophin which serves as a dystrophin precursor during the first 4–5 weeks of age in mice [12,13] after which it is only found in the mature muscle fiber near the neuromuscular junction [14]. However, it maintains the ability to prevent the development of muscular dystrophy [15]. In general, myofiber regeneration leads to upregulation of several membrane-associated proteins that reinforce the membrane and bind to the extra-cellular membrane. As utrophin expression is extra-synaptic until the mdx is 4 to 5 weeks old, this pro- vides an opportunity to initiate treatment peri- and post-onset of muscular degeneration, which occurs when the animals are 3–4 weeks old. Thus, to elude the possible effects of endogenous utrophin, we initiated our treatment in the mdx at the age of 4 and 8 weeks to determine if peri-onset treatment had a different impact from that of treating the mice post-onset. We were not able to commence treatment earlier than 4 weeks of age due to animal welfare protocols. All animals were treated for 12 weeks for the sake of comparison to previous studies [16–18]. We were looking for a positive effect of the treatment in ex vivo electrophysiological muscle performance-tests, since specific force production and stretch resistance are hallmarks of a translatable improvement in muscle function of the mdx [19]. While our approach was not designed to cure DMD by attempting to re-express dystrophin per se, we hypothesized that the treatment with growth factors would improve muscle function and that peri-onset treatment would improve muscle function more than post- onset treatment and delay or attenuate the compensatory hypertrophy, affecting specific force generation positively. This could make the combined molecular approach attractive to a larger number of myopathies with muscle wasting, where the regeneration mechanism is unaffected by the disease. 2. Experimental Section 2.1. Animals Only male mice were used for this study. Cohorts of mdx (C57BL/10ScSn Dmdmdx /J) were treated and age-matched C57BL/10ScSn (wt) mice were used as the background strain, all were bred at the University of Copenhagen. Animals had access to food and water ad libitum and three to six mice were typically housed together in individually ventilated cages under a 12 h/12 h light/dark cycle at the animal facility of the University of Copenhagen Department of Experimental Medicine. 2.2. Treatment Mdx mice were randomized to receive the HGF/LIF/L-arginine growth factor cocktail (GF) or placebo (PBS). The treatment would commence when mice were either 4 (4W) or 8 weeks (8W) of age and last for 12 weeks, with the entire study consisting of six groups including wild-type mice, each with 12 male mice. Sample size of each group was based on cohort sizes previously used [16]. The treatment was given as weekly intraperitoneal injections of HGF (20 ng/g body mass, 2207HG/CF, R&D Systems, Minneapolis, MN, USA) and L-arginine (200 µg/g body mass, 11009 Sigma-Aldrich, St. Louis, MO, USA) followed by LIF (10 ng/g body mass, AMSBIO, Cambridge, MA, USA) three-to-four days later. Doses were based on previous works [5,12,13]. Body mass was recorded weekly at the time of the HGF/L-arginine injection. Biomedicines 2022, 10, 304 3 of 16 2.3. Physiological Experiments After receiving 12 weeks of treatment, mice were anaesthetized using Hypnorm/ midazolam 0.5 mg/g body mass (Department of Experimental Medicine, University of Copenhagen, Copenhagen, Denmark) and kept on a heating pad for the dissecting proce- dure. Hindlimb muscles tibialis anterior (TA), extensor digitorum longus (EDL), soleus, gastrocnemius, and quadriceps were removed. The mass of all muscles was determined, and length was measured for EDL and soleus. Dissected EDL and soleus were immediately mounted on pins in the organ bath in oxygenated Krebs/Ringer solution at room temper- ature using 4-0 Vicryl surgical suture and left to equilibrate in oxygenated Krebs/Ringer solution at room temperature for 5 min. The electrophysiology setup consisted of an 840MD organ bath setup with force transducers and motors (DMT A/S, Aarhus, Denmark). Muscles were stimulated with 20 V·cm−1 using a DMT CS8 stimulator (DMT A/S). Analog signals were converted using an AD Instruments PowerLab 4/35 A/D converter and recorded using Chart 7.0 (AD Instruments, Oxford, UK). Stimulation with 5 ms square pulses for isometric twitches were used to determine the optimal base tension and base tension length, L0 . After this, maximum isometric tetani were determined by stimulating the muscle with 5 ms pulses at 100 Hz for EDL and 80 Hz for soleus for 695 ms (EDL) or 705 ms (soleus) with 60 s between five trains. Muscles were then stimulated at a 50% duty cycle using 5 ms pulses for EDL and 6.5 ms pulses for soleus. Following a resting period of 5 min, the muscle was subjected to a stretch protocol, with stimulation for 700 ms of 100 Hz (EDL) or 80 Hz (soleus). After 500 ms of isometric contraction, stretch was applied to induce an eccentric contraction for the remaining 200 ms by stretching the muscle 10% of the base tension length, L0 . The stretch protocol was repeated four times with a 60 s resting period in between, as previously described [16,17,20]. The maximal contraction of the five performed was used in the results. The person operating and recording muscular function of mdx mice was blinded to treatment. Muscles were flash-frozen in isopentane cooled by liquid nitrogen and stored at −80 ◦ C. Physiological cross-sectional area (CSA) was determined by dividing the mass of the muscle (EDL and soleus) with the density of mammalian muscle (1.056 g·cm−3 ) and resting length, lo . Specific force was obtained by dividing the measured absolute force with the CSA. If both had been recorded, muscle mass as well as CSA for an individual animal was recorded as the average of both right and left side muscles. All results are presented as mean with standard deviations unless otherwise stated. The primary outcome of the treatment was change in specific force and stretch resistance whereas secondary outcomes such as changes in body mass, absolute force, muscle mass and CSA, and molecular markers of muscular regeneration were to support the primary findings. 2.4. Western Blot Analyses TA, EDL, and soleus muscles from animals were sectioned on a cryostat and dis- solved in lysis buffer (10 mM Tris, pH7.4, 0.1% Triton-X 100, 0.5% sodium deoxycholate, 0.07 U/mL aprotinin, 20 µM leupeptin, 20 µM pepstatin, 1 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol- bis(β-aminoethyl ether)-N,N,N0 ,N0 -tetraacetic acid (EGTA), 1 mM dithiothreitol (DTT), 5 mM β-glycerophosphate, 1 mM sodium fluoride, 1.15 mM sodium molybdate, 2 mM sodium pyrophosphate decahydrate, 1 mM sodium orthovanadate, 4 mM sodium tartrate, 2 mM imidazole, 10 nM calyculin, and 5 µM cantharidin, (all Sigma-Aldrich) using a Bullet Blender tissue homogenizer at 4 ◦ C (Next Advance Inc., Averill, NY, USA). Samples were mixed with 4× sample buffer (12.5% glycerol, 0.2 g/mL sodium dodecyl sulfate, 0.08 M bromophenol-blue, and 20% β-mercaptoethanol). Equal amounts of extracted muscle proteins were separated on 4–15% and 7.5% TGX polyacrylamide gels (Bio-Rad Laboratories Inc., Hercules, CA, USA) at 200V for 60 min (4–15% gels) and 80 min (7% gels). Proteins were blotted to polyvinylidene difluoride membranes (Bio-Rad Laboratories Inc.) at 2.5 A for 7 min using a Trans-Blot Turbo (Bio-Rad Laboratories Inc.), and 18 h at 0.6 A using a Criterion tank blotting chamber, cooled at 4 ◦ C. Membranes were then Biomedicines 2022, 10, 304 4 of 16 blocked using Baileys Irish Cream (Baileys, Dublin, Ireland). After washing excess Bailey’s with TBS-T, membranes were incubated overnight in 0.5% skimmed milk with antibodies against vinculin at 1:2000 (ab73412) from Abcam (Cambridge, UK), myogenin at 1:500 (F5D) from Developmental Studies Hybridoma Bank (Iowa City, IA, USA), and myoD1 at 1:500 (clone 5.8A, MA1-41017) from Vector Laboratories (Burlingame, CA, USA). Secondary antibodies (goat anti-rabbit and goat anti-mouse) coupled with horseradish peroxidase from DAKO (Glostrup, Denmark) were diluted at 1:10,000 and used to detect primary antibodies. Immuno-reactive bands were detected using SuperSignal West Dura Extended Duration Substrate kit (Thermo Scientific, Rockford, IL, USA) and Clarity Max Western ECL substrate (Bio-Rad Laboratories Inc.). Quantification took place using a ChemiDoc™ MP Imaging system and Image Lab™-software (Bio-Rad Laboratories Inc.) was used to measure the intensities of immune-reactive bands on 16-bit digital photos. All immuno- reactive band intensities were normalized to the intensity of the vinculin bands for each subject to correct for differences in total muscle protein loaded on the gel. Subsequently, all normalized values from the group of a treatment regime were again normalized to the average of soleus or PBS in order to compare the groups and muscles, separately. Results are presented as the fold change of exercised animals from the average of soleus or PBS. All results are presented with standard deviations. 2.5. Histochemical Analysis Cryo-sections of TA were stained with hematoxylin and eosin (H&E) for general histopathological evaluation. Wheat germ agglutinin (WGA) Alexa Fluor™ 647stain (Ther- moFisher, Waltham, MA, USA) was diluted 1:200 in Hanks Balanced Salt Solution and applied to muscle sections to visualize fibrosis [21]. 2.6. Immunohistochemistry For immunohistochemistry (IHC), sections were fixed in 10% normal phosphate buffered formalin and subsequently blocked in 5% fetal bovine serum in PBS prior to staining. To assess the number of satellite cells undergoing divisions, sections were incu- bated with Pax7 (DSHB) diluted 1:50 and Ki67 (#15580, Abcam, Cambridge, UK) antibodies diluted 1:500. Positive nuclei were confirmed by DAPI nuclear stain (ThermoFisher). Secondary goat anti-mouse and anti-rabbit Alexa Fluor antibodies were used at a 1:500 dilution in PBS buffer (ThermoFisher). For muscle-fiber-type analysis, we used antibodies against MHC type I (MHC I) (clone BA-D5 at 1:50, DSHB), MHC IIA and IIB (clones SC-71 at 1:10 and BF-F3 at 1:50, DSHB), and MHC IIX at 1:100 (SAB2104768, Sigma-Aldrich). Secondary antibodies were Alexa Flour. At least 300 fibers per section were counted man- ually, by random selections of each muscle cross-section. All sections were observed at room temperature using a Nikon 10x Plan Apo or a 20× Plan Apo VC N/A 0.75 mounted on a Nikon Eclipse 80i or a Nikon Ti-E epi-fluorescence microscope (Nikon Instruments, Melville, NY, USA). Images were acquired with a 5M pixel Nikon DS-5Mc (brightfield) and 5Mpixel Andor Neo (epi-fluorescence) cameras (Andor, Belfast, Northern Ireland) using NIS-Elements Basic Research and Advanced Research (BR/AR) software and merged in software (Nikon). 2.7. Statistical Analysis Two-way analysis of variance (ANOVA) was performed on data using XLSTAT (Ad- dionsoft (2020), New York, USA. https://www.xlstat.com, accessed on 2 November 2020) with Tukey’s HSD post-test to show differences between variables. A p-value of <0.05 was considered significant. 3. Results 3.1. Body Mass In order to assess the influence of the different treatment regimens on the growth of the mice, we followed the body mass of the mice during the 12-week period of treatment was considered significant. 3. Results 3.1. Body Mass Biomedicines 2022, 10, 304 In order to assess the influence of the different treatment regimens on the 5 ofgrowth 16 of the mice, we followed the body mass of the mice during the 12-week period of treatment (Figure 1). In the 4W group, we observed an accelerated growth in GF-treated animals, (Figure with 1). In the 4W group, a significantly we observed increased body mass an accelerated comparedgrowth to PBSinmice GF-treated animals, in weeks 4 andwith 5 of treat- a significantly increased body mass compared to PBS mice in weeks 4 and 5 of ment. During the second half of the treatment period, the two curves of GF- and PBS-mice treatment. During the second half of the treatment period, the two curves of GF- and PBS-mice approached each other. In the 8W group, there was no effect on body mass between GF- approached each other. In the 8W group, there was no effect on body mass between GF- and PBS-treated animals. The growth curves of mdx mice from both age-groups flattened and PBS-treated animals. The growth curves of mdx mice from both age-groups flattened out outat at the conclusionofofthe the conclusion thetreatment treatment period. period. WTWTmicemice didincrease did not not increase mass asmass as rapidly rapidly asasany any mdx mdx cohort, corresponding cohort, corresponding to to compensatory compensatory muscular muscular hypertrophy hypertrophy in mdxin[22,23]. mdx [22,23]. After 12 weeks of treatment, there was no significant difference in body mass After 12 weeks of treatment, there was no significant difference in body mass between the between the treated treated and untreatedcohorts and untreated cohortsof of mdxmdx mice. mice. Figure Figure 1. 1. Growth curvesofofthe Growth curves the different different treatment treatment cohorts. cohorts. The growth The growth pattern pattern of the of the six six in groups groups in the the4W 4W cohort (A)and cohort (A) andthe the8W 8W cohort cohort (B).(B). Lack Lack of data of data pointpoint in 4WinPBS 4WisPBS dueistodue to accidental accidental loss of loss of measurements. measurements. GF,GF,growth growth factor; factor; PBS,PBS, phosphate-buffered phosphate-buffered saline; saline; WT, WT, wild-type wild-type strain. strain. Vertical bars Vertical bars are SEM. Two-way ANOVAs were performed with subsequent Tukey HSD post-hoc tests to assess tests to are SEM. Two-way ANOVAs were performed with subsequent Tukey HSD post-hoc assess significance. significance. Symbols Symbols indicate indicate p