https://orcid.org/0000-0002-6050-4556
Figures
Abstract
Purpose
Greater muscle fragility is thought to cause the exhaustion of the muscle stem cells during successive degeneration/repair cycles, leading to muscle wasting and weakness in Duchenne muscular dystrophy. Chronic voluntary exercise can partially reduce the susceptibility to contraction induced-muscle damage, i.e., muscle fragility, as shown by a reduced immediate maximal force drop following lengthening contractions, in the dystrophic mdx mice. Here, we studied the effect of Prospero-related homeobox factor 1 gene (Prox1) transfer (overexpression) using an AAV on fragility in chronically exercised mdx mice, because Prox1 promotes slower type fibres in healthy mice and slower fibres are less fragile in mdx muscle.
Methods
Both tibialis anterior muscles of the same mdx mouse received the transfer of Prox1 and PBS and the mice performed voluntary running into a wheel during 1 month. We also performed Prox1 transfer in sedentary mdx mice. In situ maximal force production of the muscle in response to nerve stimulation was assessed before, during and after 10 lengthening contractions. Molecular muscle parameters were also evaluated.
Results
Interestingly, Prox1 transfer reduced the isometric force drop following lengthening contractions in exercised mdx mice (p < 0.05 to 0.01), but not in sedentary mdx mice. It also increased the muscle expression of Myh7 (p < 0.001), MHC-2x (p < 0.01) and Trpc1 (p < 0.01), whereas it reduced that one of Myh4 (p < 0.001) and MHC-2b (p < 0.01) in exercised mdx mice. Moreover, Prox1 transfer decreased the absolute maximal isometric force (p < 0.01), but not the specific maximal isometric force, before lengthening contraction in exercised (p < 0.01) and sedentary mdx mice.
Conclusion
Our results indicate that Prox1 transfer increased the beneficial effect of chronic exercise on muscle fragility in mdx mice, but reduced absolute maximal force. Thus, the potential clinical benefit of the transfer of Prox1 into exercised dystrophic muscle can merit further investigation.
Citation: Monceau A, Delacroix C, Lemaitre M, Revet G, Furling D, Agbulut O, et al. (2022) The beneficial effect of chronic muscular exercise on muscle fragility is increased by Prox1 gene transfer in dystrophic mdx muscle. PLoS ONE 17(4): e0254274. https://doi.org/10.1371/journal.pone.0254274
Editor: Atsushi Asakura, University of Minnesota Medical School, UNITED STATES
Received: June 9, 2021; Accepted: April 5, 2022; Published: April 18, 2022
Copyright: © 2022 Monceau et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Duchenne muscular dystrophy (DMD), the most common X-linked inherited muscular disease, is caused by mutations in the DMD gene, leading to dystrophin deficiency that results in skeletal muscle fibre injury and progressive muscle wasting and weakness. Dystrophin is a costameric protein that plays a role in force transmission and sarcolemma stability in skeletal muscle [1]. In line, muscle of dystrophin-deficient mdx mouse, the “classic” animal model for DMD, exhibits two important functional dystrophic features. First, muscular weakness that is the decrease of the specific maximal force (the absolute maximal force generated relatively to muscle cross-sectional area or weight) with an unmodified/maintained absolute maximal force due to the muscle hypertrophy [2]. A second robust phenotype is muscle fragility that is revealed by the high susceptibility of the fast and low oxidative mdx muscle for damage caused by the lengthening (eccentric) contractions, leading in particular to an immediate marked force drop following lengthening contractions [2–4]. This force drop is proportional to both the length of the stretch and the absolute maximal lengthening force produced during the first contraction in fast mdx muscle [3, 4]. It was also found no muscle histological structural change immediately following lengthening contractions in mdx mice [5], as well as no reduction in maximal force of permeabilized muscle fiber [5, 6]. The greater fragility in mdx mice is associated to reduced muscle excitability [5, 7–9], and several genes coding ion membrane channels interacting with dystrophin are involved in muscle excitability, such as Scn4a, Cacna1s, Slc8a1, Trpc1 and chrna1 [10, 11]. Increased fragility is also related to NADPH oxidase 2 (NOX2) activity [12–14] and aggravated by inactivation of Utrn and Des coding utrophin and desmin respectively in mdx mice [15, 16].
The fragility of the dystrophic muscle is thought to cause the exhaustion of the muscle stem cells during successive degeneration/repair cycles [17]. Thus, attempt to reduce this fragility is very important because it has the potential to slow the progression of the dystrophic disease. Interestingly, chronic muscular exercise can improve (reduced) the fragility in mdx mice [9, 18–20]. In particular, voluntary running decreases fragility, i.e., reduces the force drop following lengthening contractions, in mdx mouse fast muscle [9, 19], whereas physical inactivity aggravates it [19]. Recently, it was found that the reduced fragility induced by voluntary running in mdx mice was related to calcineurin pathway activation, and changes in the program of genes involved in slower contractile features of muscle fibre and genes coding membrane ions channels involved in muscle excitability [9]. However, voluntary running only partly reduced the susceptibility to exercise-induced muscle damage [9, 19], so it would be interesting to combined the effects of exercise with those of another treatment.
While voluntary exercise offers potential therapeutic benefit, additional adjunct therapies could further improve functional dystrophic features. In the recent years, genetic or pharmacological treatments promoting slower and more oxidative fibres are been shown to be beneficial in the mdx mice. In fact, several studies support the idea that activation of the AMPK, calcineurin, E2F1, ERRγ, IGF1, SIRT1 and PGC1 signalling pathways alleviates some of the dystrophic features in mdx muscle [21–30]. For example, genetic activation of calcineurin pathway improves fragility in fast muscle of the mdx mouse, but decreases maximal force production, thus, aggravating weakness [28]. Recently, in healthy fast muscle, it was demonstrated that the loss of Prospero-related homeobox factor 1 (Prox1), a transcription factor essential for the development of several organs like lymphatic vessels and highly conserved among vertebrates, represses the expression of slow contractile genes, whereas its overexpression via Prox1 transfer has the opposite effect and down-regulates the fast contractile genes, [31, 32]. In particular, the inactivation of Prox1 reduces the expression of the slowest myosin heavy chain Myh7 in fast healthy muscle, without affecting oxidative capacity (succinate dehydrogenase staining) and absolute maximal force [32]. Prox1, that is more expressed in slow fibres, is involved in the activation of the NFAT/calcineurin pathway, and promotes the slow contractile gene program in healthy muscle [31].
The principal purpose of the present study was to determine whether Prox1 transfer using an adeno-associated vectors (AAV9) carrying the Prox1 construct reduced muscle fragility in voluntary exercised mdx mice, with fragility being defined as the immediate loss of muscle function (i.e., maximal force drop) following lengthening contractions. The study including physiological outcome measurement of fragility was complemented by molecular analyses. Because we found that voluntary running and Prox1 transfer have additive beneficial effects on fragility, a second set of experiment was performed to compare the effect of Prox1 transfer on fragility between voluntary exercised and sedentary mdx mice. Interestingly, Prox1 transfer did not reduced fragility in sedentary mdx mice.
Materials and methods
Animal groups and voluntary running
All procedures were performed in accordance with national and European legislations and were approved by our institutional Ethics Committee “Charles Darwin” (Project # 01362.02). Male mice with exon 23 mutation in the dmd gene encoding dystrophin (Mdx mice) were used (hybrid background C57Bl/6 x C57Bl/10). Mice (2–3 months of age) were randomly divided into different control and experimental groups (Fig 1). In the first set of experiment, Mdx mice were placed (Mdx+W) in separate cages containing a wheel and were allowed to run 1-month ad libitum. The muscles of Mdx mouse runners received (Mdx+W+P) or not (Mdx+W) Prox1 transfer into the muscle 3 days before the initiation of voluntary exercise. The running distances were collected and daily running distance was 4.2 ± 0.1 km/day. A group of sedentary Mdx mice was also studied (Mdx). At the end of the experiment, the body weight of the Mdx+W+P/Mdx+W mice and Mdx mice was 29.9 ± 0.3 g and 31.4 ± 0.2 g respectively (p = 0.019). The first set of experiment was performed to study the effect of Prox1 transfer in exercised Mdx mice. Because we found an effect of Prox1 transfer on fragility in Mdx+W+P muscle, we then performed a second set of experiment to compare the effect of Prox1 transfer on fragility between voluntary exercised muscle and sedentary muscle. In the second set of experiment, the muscles of sedentary Mdx mice received (Mdx+P) or not (Mdx) Prox1 transfer. The muscles were measured and collected 4 weeks after Prox1 transfer.
Two sets of experiments were performed. In the first set of experiment, we want to determine the effect of Prox1 transfer on fragility in voluntary exercised mdx mice. AAV-Prox1 and PBS were injected in TA muscles of the same mdx mice before voluntary exercise. The aim of the second set of experiment was to compare the effect of Prox1 transfer between voluntary exercised and sedentary mdx mice.
Prox1 transfer
To overexpress Prox1, adeno-associated vectors (AAV9) carrying the Prox1 construct (AAV-Prox1) [31] was injected in one of the Tibialis anterior (TA) muscles of the mouse (2.1 x 1011 vector genomes). The other TA muscle (control muscle) of the same mouse was injected with saline solution only (Fig 1). The mouse was anesthetized (3% isoflurane) and TA muscles were injected (30 μl). Briefly, hProx1 cDNA’s were cloned into psub plasmid (promoter CMV) [31]. The plasmid was purified using the PureYield™ endotoxin-free Plasmid Maxiprep System (Promega, Lyon, France) and then verified by restriction enzyme digestion and by sequencing (Eurofins MWF Operon, Ebersberg, Germany). The AAV-Prox1 was produced in human embryonic kidney 293 cells by the triple-transfection method using the calcium phosphate precipitation technique. The virus was then purified by 2 cycles of cesium chloride gradient centrifugation and concentrated by dialysis. The final viral preparations were kept in PBS solution at -80°C. The number of viral genomes was determined by a quantitative PCR. Titer for AAV-Prox1 was 7.1 x 1012 vector genomes (vg).ml-1.
Muscle fragility measurement
Muscle fragility (susceptibility to contraction induced damage) was evaluated by measuring the in situ TA muscle contraction properties in response to nerve stimulation, as described previously [5]. Fragility was estimated from the isometric force drop resulting from lengthening contraction-induced damage. Briefly, mice were anesthetized using pentobarbital (60 mg/kg, ip). Body temperature was maintained at 37°C using radiant heat. The knee and foot were fixed with pins and clamps and the distal tendon of the muscle was attached to a lever arm of a servomotor system (305B, Dual-Mode Lever, Aurora Scientific) using a silk ligature. The sciatic nerve was proximally crushed and distally stimulated by a bipolar silver electrode using supramaximal square wave pulses of 0.1 ms duration. We first determined the optimal length (L0, length at which maximal isometric force was obtained during the tetanus). Once L0 was obtained, a maximal isometric contraction of the TA muscle was initiated during the first 500 ms. Then, muscle lengthening (10% L0) at a velocity of 5.5 mm/s (0.85 fibre length/s) was imposed during the last 200 ms. Nine lengthening contractions of the TA muscles were performed in Mdx mice, each separated by a 60 s rest period. Absolute maximal isometric force was measured 1 min after each lengthening contraction and expressed as a percentage of the initial maximal force (force drop). Absolute maximal isometric force measured before the first lengthening contraction was also normalized to the muscle mass in order to calculate the specific maximal isometric force, an index of muscle weakness. In addition, we measured the absolute maximal lengthening force during the first lengthening contraction, and index of the muscle stress. After contractile measurements, the animals were killed with cervical dislocation.
Real-time quantitative PCR (polymerase chain reaction)
Muscles (TA) were snap frozen in liquid nitrogen and stored at −80°C until use. Total RNA was isolated from TA muscles using Trizol (Invitrogen). Complementary DNA (cDNA) was then synthesized from 1 μg of total RNA using the RevertAid First Strand cDNA Synthesis kit with random hexamers, according to the manufacturer’s instructions (Thermo Scientific). RT-PCR was performed on a LightCycler 480 System at the platform iGenSeq of the Institut du Cerveau et de la Moelle epinière, using LightCycler 480 SYBR Green I Master Mix (Roche, Basel, Switzerland) [5]. The expression of Hmbs was used as reference transcript because it’s expression did not differ between groups. The 2-ΔΔCP method has been used as a relative quantification strategy for quantitative real-time polymerase chain reaction (qPCR) data analysis. All sequences of primers used are presented in Table 1.
SDS-PAGE electrophoresis of MHC isoforms (proteins)
The muscles were extracted on ice for 60 min in four volumes of extracting buffer containing 0.3 M NaCl, 0.1 M NaH2PO4, 0.05 M Na2HPO4, 0.01 M Na4P2O7, 1 mM MgCl2, 10 mM EDTA, and 1.4 mM 2-mercaptoethanol (pH 6.5). Following centrifugation, the supernatants were diluted 1:1 (vol/vol) with glycerol and stored at -20°C. MHC isoforms (proteins) were separated on 8% polyacrylamide gels, which were made in the Bio-Rad mini-Protean II Dual slab cell system. The gels were run for 31 h at a constant voltage of 72 V at 4°C [33]. Following migration, the gels were silver stained. The gels were scanned using a video acquisition system. The relative level of MHC isoforms was determined by densitometric analysis using Image J software.
Histology
Transverse serial sections (8 μm) of TA muscles were obtained using a cryostat, in the mid-belly region. For determination of muscle fibre diameter (min ferret), frozen unfixed sections were blocked 1h in phosphate buffer saline plus 2% bovine serum albumin, 2% fetal bovine serum. Sections were then incubated overnight with primary antibodies against laminin (Sigma, France). After washes in PBS, sections were incubated 1 h with secondary antibody (Alexa Fluor, Invitrogen). Slides were finally mounted in Fluoromont (Southern Biotech). Images were captured using a digital camera (Hamamatsu ORCA-AG) attached to a motorized fluorescence microscope (Zeiss AxioImager.Z1), and morphometric analyses were made using the software ImageJ. We attempt to analyze all the fibers of the muscle section, but some were excluded from the analysis for reasons of improper labeling (mean: 1474 fibres measured per muscle).
Statistical analysis
Groups were statistically compared using the Prism software v8 (GraphPad, La Jolla, CA, USA). Data were tested for homogeneity of variance using a Brown-Forsythe test. For the first set of experiment, one-way ANOVA was used to analyze the following variables: mRNA expression, absolute and specific maximal force, absolute maximal lengthening force, the ratio of absolute maximal lengthening force to the absolute maximal lengthening force, and muscle weight. Fragility was analyzed by two-way ANOVA, groupes (Mdx, Mdx+W, Mdx+W+P) by lengthening contractions (0, 3, 6, 9), with the repeated measures on lengthening contractions. Unpaired t-test with Welch’s correction was used to analyze the % of MHC-2x and MHC-2a (electrophoresis) and body weight of the mice. For experiment 2, unpaired t-test with Welch’s correction was used for the following variable: mRNA expression, absolute and specific maximal force, absolute maximal lengthening force, and muscle weight. Fragility was analyzed by two-way ANOVA, groupes (Mdx, Mdx+P) by lengthening contractions (0, 3, 6, 9), with the repeated measures on lengthening contractions. Moreover, when significant main effect (ANOVA) was observed, multiple-comparisons were performed with Tukeys test. Finally, when significant interaction was found (ANOVA), differences were tested with Holm-Sidak test. Values are means ± SEM.
Results
Prox1 transfer in voluntary exercised Mdx muscle promotes slower contractile features
In the first set of experiment, we first determined whether Prox1 transfer increased slower contractile features in voluntary exercised Mdx mice. Prox1 transfer into the TA muscle markedly increased the expression of Prox1 (x 37.0) in voluntary exercised Mdx TA muscle (Mdx+W+P) as compared to voluntary exercised Mdx TA muscle (Mdx+W)(p < 0.0001) (Fig 2A), as assessed by qPCR analysis. We also found that the expression of Myh7 coding for MHC-1 (x 15.1)(p < 0.001) was increased in Mdx+W+P muscle as compared to Mdx+W muscle, whereas that of Myh4 coding for MHC-2b was reduced (x 0.6) (Fig 2B) (p < 0.001). In agreement, using gel electrophoresis technique, we found that the relative amounts (percentage of total) of MHC-2b protein were reduced (x 0.8, p < 0.01) whereas that of MHC-2x protein was increased (x 1.6, p < 0.01), respectively (Fig 2C) in Mdx+W+P muscle as compared to Mdx+W muscle. In contrast, there was no difference between Mdx+W+P and Mdx+W muscles in the expression of a marker of oxidative capacity, Sdha, a gene encoding a complex of the mitochondrial respiratory chain (Fig 2B).
(A) Prox1 expression in Mdx+W+P and Mdx+P muscle. N = 6–8 per group. (B) Expression of genes encoding fibre type specific contractile proteins in Mdx+W+P and Mdx+P muscle. N = 6–8 per group. (C) Relative amounts of MHC-2x and MHC-2b proteins in Mdx+W+P and Mdx+P muscle. N = 3 per group. Mdx+W+P: voluntary exercised mdx muscle that received Prox1 transfer into the muscle. Mdx+W: voluntary exercised mdx muscle. Mdx: mdx muscle. a2, a3, a4: significant different from Mdx, p < 0.01, p < 0.001, p < 0.0001, respectively. b2, b3, b4: significant different from Mdx+W, p < 0.01, p < 0.001, p < 0.0001, respectively.
These data indicate that intramuscular delivery of AAV-Prox1 induced a fast to slow contractile transition in the TA muscle of voluntary exercised Mdx mice.
Prox1 transfer in voluntary exercised Mdx muscle further improves muscle fragility
The first set of experiment revealed that the immediate isometric force drop following lengthening contractions in Mdx+W muscle was reduced as compared to Mdx muscle (p < 0.0001) (Fig 3A). Interestingly, Prox1 transfer in voluntary exercised Mdx muscle further reduced the isometric force drop following lengthening contractions (Fig 3A). In fact, the isometric force drops following the 6th (p < 0.05) and 9th (p < 0.01) lengthening contractions were lower in Mdx+W+P muscle as compared to Mdx+W muscle (Fig 3A), indicating that Prox1 transfer improved (reduced) fragility in voluntary exercised Mdx muscle.
(A) Force drop following lengthening contractions (Fragility) in Mdx+W+P and Mdx+P muscle. n = 6–8 per group. (B) Expression of genes encoding ion channels, related to excitability in Mdx+W+P and Mdx+P muscle. N = 6–8 per group. (C) Expression of genes, related to NADPH oxidase 2 (NOX2) in Mdx+W+P and Mdx+P muscle. N = 6–8 per group. (D) Expression of genes encoding utrophin (Utrn) and desmin (Des) in Mdx+W+P and Mdx+P muscle. N = 6–8 per group. Mdx+W+P: voluntary exercised mdx muscle that received Prox1 transfer into the muscle. Mdx+W: voluntary exercised mdx muscle. Mdx: mdx muscle. a1, a4: significant different from Mdx, p < 0.05, p < 0.0001, respectively. b1, b2: significant different from Mdx+W, p < 0.05, p < 0.01, respectively.
The fast to slower contractile conversion described above can explained, at least in part, the improved fragility in Mdx+W+P muscle. Moreover, we tested the possibility that Prox1 transfer also improved fragility via the modifications of the expression of genes coding membrane ions channels. The expression of Trpc1 encoding for transient receptor potential cation channel subfamily C member 1 (x 2.1) was increased in Mdx+W+P muscle as compared to Mdx+W muscle (p < 0.01) (Fig 3B). No difference between Mdx+W+P and Mdx+W muscles was observed concerning the expression of Scn4a, Cacna1s, Slc8a1 and Chrna1 (Fig 3B). Then, we determined whether the reduced isometric force drop following lengthening contractions induced by Prox1 transfer was associated to change (decrease) in NOX2 pathway. We found no change in the expression of PrxII, Gp91phox, P47phox and Rac1 (Fig 3C) in Mdx+W+P muscle as compared to Mdx+W muscle (Fig 3C). We also determined whether Prox1 transfer increased Utrn and Des expression. The expression of Utrn was not increased in Mdx+W+P muscle as compared to Mdx+W muscle, whereas that one of Des increased (x 1.2) in Mdx+W muscle, although not significantly (p = 0.052) (Fig 3D).
Thus, the improved TA muscle fragility induced by Prox1 transfer in voluntary exercised mice was associated with the modification of expression of MHC-2b and MHC-2x proteins and several genes involved in different aspects of muscle function and structure (Myh7, Myh4, Trpc1).
Prox1 transfer in voluntary exercised Mdx muscle reduced absolute isometric maximal force
In addition, the first set of experiment revealed that Prox1 transfer combined to voluntary running and voluntary running alone did not affect specific maximal isometric force before lengthening contractions (Fig 4A). However, absolute maximal isometric force was reduced in Mdx+W+P muscle (x 0.6) as compared to Mdx+W muscle (p < 0.01) (Fig 4B). Similarly, absolute maximal lengthening force was lower (x 0.6) in Mdx+W+P muscle (157.2 g ± 7.5) compared to Mdx+W muscle (240.0 g ± 10.8) muscle (p < 0.01). In addition, the ratio of absolute maximal lengthening force to the absolute maximal isometric force was not different between Mdx+W+P muscle (1.9 ± 0.1) and Mdx+W muscle (1.8 ± 0.1).
(A) Specific maximal force in Mdx+W+P Mdx+W+P and Mdx+P muscle. n = 6–8 per group. (B) Absolute maximal force in Mdx+W+P and Mdx+P muscle. n = 6–8 per group. (C) Muscle weight in Mdx+W+P and Mdx+P muscle. n = 6–8 per group. (D) Fibre diameters (min feret) in Mdx+W+P and Mdx+P muscle. n = 3–4 per group. (E) Representative image of muscle cross-section. Fiber outline was visualized by antilaminin antibody (green). Scale bar = 200μm. (F) Expression of genes related to atrophy in Mdx+W+P and Mdx+P muscle. N = 6–8 per group. Mdx+W+P: voluntary exercised mdx muscle that received Prox1 transfer into the muscle. Mdx+W: voluntary exercised mdx muscle. Mdx: Mdx muscle. a1, a2, a3, a4: significant different from Mdx, p < 0.05, p < 0.01, p < 0.001, p < 0.0001, respectively. b2, b3, b4: significant different from Mdx+W, p < 0.01, p < 0.001, p < 0.0001, respectively.
The reduced absolute maximal isometric force was related to a lower muscle weight (x 0.7)(p < 0.001) (Fig 4C) and reduced fibre diameters (p < 0.01) (Fig 4D and 4E) in Mdx+W+P muscle as compared to Mdx+W muscle. Numerous genes encoding proteins are involved in muscle atrophy, growth and maintenance [34, 35]. The ubiquitin-proteasome system plays a key role in triggering muscle atrophy when the expressions of Murf1 and Mafbox are increased. Quantitative real-time PCR revealed that the expressions of these genes were not increased in Mdx+W+P muscle as compared to Mdx+W muscle (Fig 4F). We then analyzed another atrophic mechanism, autophagy, which involves a battery of genes including Lc3 which could contribute to the degradation of muscle proteins [36]. We did not find any change in Lc3 expression in Mdx+W+P muscle (Fig 4F). Similarly, Gadd45, Hdac4, Fn14, Redd1, Redd2, Mstn, Fst, Igf1, and Smox genes also did not seem to participate to the atrophic state of Mdx+W+P muscle (Fig 4F). For example, Mstn, the negative regulator of muscle growth, was down-regulated in Mdx+W+P muscle as compared to Mdx+W muscle (p < 0.001) (Fig 4F).
Thus, the reduction in maximal isometric force induced by Prox1 transfer in voluntary exercised Mdx muscle was related to decreased muscle weight and increased expression of Mstn.
Prox1 transfer in sedentary Mdx muscle promotes slower contractile features but does not reduce muscle fragility
A second set of experiment was performed to compare the effect of Prox1 transfer on fragility between voluntary Mdx mice and sedentary Mdx mice. Similarly to voluntary exercised Mdx muscle, Prox1 transfer in sedentary Mdx muscle (Mdx+P muscle) increased the expressions of Prox1 (x 27.3)(p < 0.0001) (Fig 5A), Myh7 (x 6.2)(p < 0.05) (Fig 5B), and reduced that one of Myh4 (x 0.7)(p < 0.05) (Fig 5B) compared to sedentary Mdx muscle. In contrast to voluntary exercised Mdx muscle, Prox1 transfer increased the expression of Tnni1 (x 2.4)(p < 0.01), reduced the expression of Sdha (x 0.8) (Fig 5B) (p < 0.01) and did not change the relative amounts of MHC-2b and MHC-2x proteins (Fig 5C) in Mdx+P muscle as compared to Mdx muscle. Overall, intramuscular delivery of AAV-Prox1 also induced a fast to slow contractile conversion in the TA muscle of sedentary Mdx mice, but without consequence at the MHC protein level.
(A) Prox1 expression in Mdx+P and Mdx muscle. N = 6–11 per group. (B) Expression of genes encoding fibre type specific contractile proteins in Mdx+P and mdx muscles. N = 6–11 per group. (C) Relative amounts of MHC-2x and MHC-2b proteins in Mdx+P and Mdx muscle. N = 3 per group. Mdx+P: Mdx muscle that received Prox1 transfer into the muscle. Mdx: Mdx muscle. a1, a2, a4: significant different from Mdx, p < 0.05, p < 0.01, p < 0.0001, respectively.
In contrast to voluntary exercised Mdx muscle, we found in the second set of experiment that the isometric force drop following lengthening contractions was not significantly reduced by Prox1 transfer in sedentary Mdx muscle because there was no significant difference between Mdx+P muscle and Mdx muscle (Fig 6A). Similarly to voluntary exercised Mdx muscle, Prox1 transfer in Mdx+P muscle increased Trpc1 expression (p < 0.01) (Fig 6B), but to lesser extent (x 1.4), did not alter the expression of PrxII, Gp91phox, P47phox and Rac1 (Fig 6C), and increased not significantly Des expression (p = 0.059) (Fig 6D). In contrast to voluntary exercised Mdx muscle, the expression of Cacn1s and Chrna1 was increased in Mdx+P muscle compared to Mdx muscle (p < 0.01) (Fig 6B).
(A) Force drop following lengthening contractions in Mdx+P and Mdx muscle. n = 5–8 per group. (B) Expression of genes encoding ion channels, related to excitability in Mdx+P and Mdx muscle. N = 6–11 per group. (C) Expression of genes, related to NADPH oxidase 2 (NOX2) in Mdx+P and Mdx muscle. N = 6–11 per group. (D) Expression of genes encoding utrophin (Utrn) and desmin (Des) in Mdx+P and Mdx muscle. N = 6–11 per group. (E) Specific maximal force in Mdx+P and Mdx muscle. n = 5–8 per group. (F) Absolute maximal force in Mdx+P and Mdx muscle. n = 5–8 per group. (G) Muscle weight in Mdx+P and Mdx muscle. n = 5–9 per group. (H) Expression of genes related to atrophy in Mdx+P and Mdx muscle. n = 6–11 per group. (I) Fibre diameters (min feret) in Mdx+P and Mdx muscle. n = 3 per group. a1, a2, a3, a4: significantly different from Mdx, p < 0.05, p < 0.01, p < 0.001, p < 0.0001, respectively.
Thus, Prox1 transfer in sedentary Mdx muscle does not reduced fragility, did not change the expression of MHC-2b and MHC-2x, whereas it altered the expression of several genes involved in different aspects of muscle function (Myh7, Myh4, Tnni1, Sdha, Trpc1, Cacn1s and Chrna1).
Prox1 transfer in sedentary Mdx muscle also reduced absolute isometric maximal force
Similarly to voluntary exercised Mdx muscle, we found in the second set of experiment that Prox1 transfer in Mdx+P muscle did not change specific maximal isometric force (Fig 6E), reduced absolute maximal isometric force (x 0.7)(p < 0.05) (Fig 6F), reduced muscle weight (x 0.8) although not significantly (p = 0.08) (Fig 6G), and decreased the expression of Mstn (p < 0.0001) (Fig 6H). Moreover, it decreased absolute maximal lengthening force in Mdx+P muscle (205.9 g ± 18.8) as compared to Mdx muscle (257.0 g ± 15.3), although not significantly (p = 0.07). In contrast to voluntary exercised Mdx muscle, Prox1 transfer did not change the fibre diameter (Fig 6I). Furthermore, it decreased the expression of Mafbox (p < 0.001), Reed2 (p < 0.05) and Smox (p < 0.01), whereas it increased the expression Gadd45 (p < 0.01), Fn14 (p < 0.001), and Fst (p < 0.05) in Mdx+P muscle (Fig 6H).
Discussion
Prox1 transfer improved fragility in voluntary exercised Mdx mice
The present study confirms previous studies [9, 19] showing that voluntary exercise alleviates the great susceptibility to lengthening contraction-induced force drop, a major dystrophic feature, in fast anterior crural muscles (TA and extensor digitorum longus) of mdx mice, such as Dmd based preclinical therapy [5]. For the first time, we demonstrate that Prox1 transfer further improves fragility in voluntary exercised mdx mice. Importantly, the muscle was protected from damaging lengthening muscle contractions by Prox1 transfer only when mdx mice performed voluntary exercise. This improved fragility observed in exercised mdx mice treated with Prox1 transfer might be very interesting if it is assumed that fragility causes the exhaustion of the muscle stem cells during successive degeneration/repair cycles [17]. Prox1 transfer might reduce the progressive muscle wasting in exercised dystrophic muscle because of the promotion of less fragile fibres.
This beneficial effect of Prox1 transfer in exercised mdx mice could be explained by a lower work and stress during lengthening [3]. However, we found that absolute maximal lengthening force (presumably work) is reduced in exercised mdx mice, in proportion to the absolute maximal isometric force. We previously observed no strong association between fragility and lengthening force in mdx mice, when muscle is maximally activated and for a constant stretch [19]. Indeed, fragility was increased by inactivity and reduced by voluntary exercise in mdx mice whereas absolute maximal lengthening force was respectively reduced and unchanged [19].
The reduced fragility induced by Prox1 transfer in exercised mdx mice is associated with the promotion of slower contractile features (increased and reduced expression of Myh7 and Myh4 respectively, reduced and increased relative amounts of MHC-2b and MHC-2x proteins respectively). This relation between improved fragility and slower contractile features is in line with the 2 following points. First, slow muscle appears less fragile than fast muscle in mdx mice [4, 37]. Second, exercise and pharmacological or genetic activation of signaling pathways, such as calcineurin, PPAR-β, PGC1-α, and AMPK, that promote a slower and more oxidative gene program, improve fragility in mdx mice [9, 18, 19, 23, 27–29, 38]. It was previously demonstrated that Prox1 promotes slower features, and activates the NFAT-calcineurin pathway [31], a signaling pathway known to play an important role in fibre type specification [39].
It is also possible that Prox1 transfer improves fragility in voluntary exercised mdx mice by a preserved excitability, as voluntary exercise and Dmd based therapy [5, 9]. In our experiments, reduced excitability, i.e. plasmalemma electrical dysfunction leading to defective generation and propagation of muscle potential action, largely contributes to the immediate force drop following lengthening contractions in mdx mice [5, 9], in agreement with other studies [7, 8]. Membrane ion channels are likely damaged following by lengthening contractions and Prox1 transfer possibly interferes with this process. It remains to be determined whether the upregulation of the membrane ion stretch-activated channel Trpc1 induced by Prox1 transfer in voluntary exercised mdx mice contributes to this improvement of excitability. However, a higher level of TRPC1 or activity of stretch-activated channels are generally associated with a worst dystrophic phenotype and fragility [40, 41]. In line with the present study, it was previously reported that the improved TA mdx muscle excitability and fragility induced by voluntary exercise and calcineurin pathway activation were also related to the changes in expression of genes encoding membrane ion channels [9].
Previous studies suggest that increased NOX2 activity is related to fragility in mdx mice [13, 14]. However, our results show that Prox1 transfer in exercised mdx muscle does not reduce the expression of Nox2 subunits (Gp91phox, P47phox and Rac1), which are shown to produce an elevated level of ROS in mdx mice [14]. Moreover, we found no increased expression of the gene encoding the antioxidant enzyme PrxII, whose overexpression improves fragility in mdx mice [13]. Finally, we found that the improvement of the fragility in response to Prox1 transfer is not associated with significantly increased expression of Utr and Des in exercised mdx mice, two genes contributing to fragility in mdx mice [15, 16].
Of note, Prox1 transfer alone does not significantly improve fragility in sedentary mdx mice. It is not excluded that the increase in the number of sedentary mdx mice per group change this conclusion but the potential beneficial effect would nevertheless be less important. The difference cannot be attributed to the fact that Prox1 was not highly overexpressed in sedentary mdx mice treated with Prox1 transfer. However, some changes induced by Prox1 transfer are notably different between exercised and sedentary mdx mice: absolute maximal lengthening force (x 0.6 versus none significant change), MHC-2b (x 0.8 versus none), MHC-2x (x 1.6 versus none), Myh7 (x 15.1 versus x 6.2), and Trpc1 (x 2.1 versus x 1.4). Thus, our study interestingly indicates that voluntary exercise potentiates a possible gene-based therapy, at least in the preclinical field.
Prox1 transfer reduces maximal force production in exercised mdx mice
Although Prox1 transfer improves fragility in voluntary exercised mdx muscle, we found that it has a detrimental effect on absolute maximal isometric force (and maximal lengthening force), without change in specific maximal isometric force. The reduced maximal isometric force is related to a reduced muscle weight and fibre diameter and is associated to the downregulation of Mstn, a negative regulator of muscle growth in mdx muscle [42]. The same effects were also observed in sedentary mdx mice, although less marked. In line with the reduced muscle weight induced by Prox1 transfer, several genetic or pharmacological treatments promoting slower and more oxidative fibres has been shown to induce muscle atrophy/reduced weight in sedentary mdx mice [27, 28, 30], for reasons still largely unknown. It remains to be determined whether the injection of the AAV itself also contributes to the muscle weight reduction (independently of the overexpression of Prox1).
Conclusions
Combined to voluntary exercise, Prox1 transfer using an AAV further improves (reduced) the immediate isometric force drop following lengthening contractions. This beneficial effect on fragility in exercised mdx mice is associated to the reduction in maximal lengthening force, the promotion of slower contractile features, and the change in Trpc1 expression. However, Prox1 transfer also reduces absolute maximal isometric force production. Thus, Prox1 transfer combined to chronic exercise has effects, some of which are beneficial for the mdx dystrophic muscle. Is this knowledge could be exploited for therapeutic advantage?
Acknowledgments
We are grateful to Kari Alitalo (Wihuri Research Institute and Translational Cancer Biology Program, University of Helsinki, Finland) for the gift of Prox1 construct, Pierre Joanne (Sorbonne Université) for assistance during the experiments, Laura Julien and Sofia Benkhelifa for AAV-Prox1 production (Sorbonne Université), Delphine Bouteiller for qPCR measurements (Sorbonne Université), and Saline Jabr (Sorbonne Université) for her help in english.
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