Beneficial effects of resistance training on both mild and severe mouse dystrophic muscle function as a preclinical option for Duchenne muscular dystrophy

Medhi Hassani; Dylan Moutachi; Mégane Lemaitre; Alexis Boulinguiez; Denis Furling; Onnik Agbulut; Arnaud Ferry

doi:10.1371/journal.pone.0295700

. 2024 Mar 8;19(3):e0295700. doi: 10.1371/journal.pone.0295700

Beneficial effects of resistance training on both mild and severe mouse dystrophic muscle function as a preclinical option for Duchenne muscular dystrophy

Medhi Hassani ¹, Dylan Moutachi ², Mégane Lemaitre ³, Alexis Boulinguiez ⁴, Denis Furling ², Onnik Agbulut ¹, Arnaud Ferry ^2,^5,^*

Editor: Aldrin V Gomes⁶

PMCID: PMC10923407 PMID: 38457407

Abstract

Mechanical overloading (OVL) resulting from the ablation of muscle agonists, a supra-physiological model of resistance training, reduces skeletal muscle fragility, i.e. the immediate maximal force drop following lengthening contractions, and increases maximal force production, in mdx mice, a murine model of Duchene muscular dystrophy (DMD). Here, we further analyzed these beneficial effects of OVL by determining whether they were blocked by cyclosporin, an inhibitor of the calcineurin pathway, and whether there were also observed in the D2-mdx mice, a more severe murine DMD model. We found that cyclosporin did not block the beneficial effect of 1-month OVL on plantaris muscle fragility in mdx mice, nor did it limit the increases in maximal force and muscle weight (an index of hypertrophy). Fragility and maximal force were also ameliorated by OVL in the plantaris muscle of D2-mdx mice. In addition, OVL increased the expression of utrophin, cytoplamic γ-actin, MyoD, and p-Akt in the D2-mdx mice, proteins playing an important role in fragility, maximal force gain and muscle growth. In conclusion, OVL reduced fragility and increased maximal force in the more frequently used mild mdx model but also in D2-mdx mice, a severe model of DMD, closer to human physiopathology. Moreover, these beneficial effects of OVL did not seem to be related to the activation of the calcineurin pathway. Thus, this preclinical study suggests that resistance training could have a potential benefit in the improvement of the quality of life of DMD patients.

Introduction

Lengthening contractions, for example when the muscle slows down the movement, are known to cause immediate and prolonged reduction in skeletal muscle function and delayed muscle soreness, even in healthy muscle. The skeletal muscle in murine Duchenne muscular dystrophy (DMD) models, with dystrophin deficiency, is more susceptible to lengthening contraction-induced loss of function compared to healthy muscle, i.e., more fragile. More precisely, it is the fast and low oxidative muscle that is more susceptible to lengthening contraction-induced loss of function in the mild mdx murine DMD model, in contrast to the slow and more oxidative muscle which is not more fragile [1, 2]. Certain proteins, such as utrophin, cytoplamic γ-actin as well as desmin, involved in muscle structure, are also known to significantly modulate this increased fragility when dystrophin is absent. Indeed, the overexpression of utrophin [3] or cytoplamic γ-actin [4] markedly reduces the fragility of mdx mice, while the absence of desmin increases it [5]. Genetic activation of the calcineurin and Akt pathways can also protect the murine dystrophic muscle from susceptibility to lengthening contraction-induced loss of function whereas genetic inactivation of the mTOR pathway increases it in healthy muscle [6–9]. It was also recently reported that overexpression of the myogenic transcription factor MyoD also aggravates muscle fragility [10]. These pathways are also known to regulate fiber-type specification and growth, and play a role in the adaptations induced by chronic muscular exercise in healthy muscle [11–15].

Interestingly, chronic muscular exercise can decrease the susceptibility to lengthening contraction-induced loss of function in mdx mice. Chronic voluntary running and low-frequency electrical stimulation, likely animal models of endurance training, reduce fragility in mdx mouse fast muscle [16–18], whereas physical inactivity aggravates it [16]. Recently, it was found that the voluntary running-induced improvement of mdx mice muscle fragility was related to the activation of the calcineurin pathway, and associated with changes in the program of genes involved in slower contractile phenotype of the muscle fibre [17]. In this latest study, these beneficial effects of calcineurin were blocked by administration of cyclosporin (CsA), a calcineurin inhibitor [17]. In addition, it has been shown that murine models of resistance training also reduce fragility in the mdx mouse [19, 20]. In fact, 6 resistance training sessions [19] and chronic mechanical overloading (OVL) of the plantaris muscle [20], a supra-physiological model of resistance training [21], reduce the force drop following lengthening contractions in the lower leg muscles of mdx mice [19, 20]. However, it is not yet known whether CsA blocks the beneficial effect of resistance training on fragility in dystrophic muscle. Moreover, since murine models of resistance training also increase muscle weight (an index of hypertrophy) and maximal force in mdx mice [19, 20, 22], it would be interesting to know whether these maximal force and weight gains are blocked by CsA, suggesting or not a role of the calcineurin pathway in these beneficial effects. CsA has been reported to decrease the hypertrophy in OVL healthy muscle [23, 24], although this is not found by all [11]. To our knowledge, the contribution of the calcineurin pathway to the strength gain induced by resistance training is not yet known either in healthy or dystrophic muscles.

The previous studies investigating chronic resistance-like muscular exercise did not use a severe murine DMD model, with a major functional deficit closer to that of the DMD patient. A recently developed murine model for DMD is the D2-mdx mice that exhibits a more marked weakness (reduced absolute maximal force) compared with mdx mice since there is no muscle hypertrophy in D2-mdx mice contrary to mdx mice [25–27]. Thus, it would be relevant to determine whether a model of resistance training also improves the fragility in the D2-mdx mice, and likewise the absolute maximal force.

The purpose of the present study was to further analyze the beneficial effect of OVL on muscle fragility and absolute maximal force in dystrophin deficient mice, two important functional dystrophic features. In particular, we wanted to know if the administration of CsA, known to inhibit the calcineurin signaling pathway, blocked the beneficial effect of OVL on fragility of mdx dystrophic muscle, as it did in chronic running exercise [17]. We also wanted to determine whether CsA blocked the effect of OVL on maximal force gain in dystrophic muscle since CsA has been reported to inhibit hypertrophy at least in healthy muscle and possibly the maximal force gain [23, 24]. Another important specific aim was to determine if OVL also exerted these beneficial effects in a more severe murine model of DMD, the D2- mdx mice.

Materials and methods

Ethical approval and animal models

Animal were housed in the departmental animal facility, with free access to water and rodent laboratory chow. The animal facility is specific pathogen free, with a 12h-light/12h-dark cycle, and mice were 3–5 per cage. All procedures were performed in accordance with national and European legislations and were approved by the French Ministere de l’Enseignement Supérieur de la Recherche et de l’Innovation (APAFIS #21554–2019071912051421). Male mdx mice (with hybrid background C57Bl/6 x C57Bl/10) and D2- mdx (DBA2/J background) mice were bred locally and were used at 3–6 months of age.

Experimental design

Mice were randomly divided into different age-matched control and experimental groups. We performed 3 separate experiments (Fig 1) to determine: 1) whether the administration of CsA blocked the effects of OVL on plantaris muscle in mdx mice, with the ablation of soleus and the major portion of lateral and medial gastrocnemius muscles, 2) the effects of OVL on lateral gastrocnemius muscle in mdx mice, with the ablation of a smaller weight of agonist muscles, a less severe OVL, and 3) the effects of OVL on plantaris muscle in D2-mdx mice, a much severe murine DMD model compared to mdx mice. In the second experiment, we wanted to induce a lower mechanical overload compared to that imposed on the plantaris muscle since the OVL of the plantaris muscle is supra-physiological, and leads to strength and mass gains approaching 100%, rarely seen in humans [21, 28]. Muscles were studied 1 month after the initiation of OVL.

Mechanical overloading

For mechanical overloading (OVL) in the 3 experiments, dystrophic mice were anaesthetized with isoflurane. The plantaris muscles (Experiments 1 and 3) of both legs were mechanically overloaded (Mdx+OVL, Mdx+OVL+CsA, and D2-mdx+OVL mice) for 1 month by surgical removal of soleus muscles as well as a major portion of the lateral and medial gastrocnemius muscles as described [20, 22] (Fig 1). The lateral gastrocnemius muscles (Experiment 2) of both legs were mechanically overloaded (Mdx+OVL) for 1 month by surgical removal of soleus, plantaris muscles as well as a major portion of the medial gastrocnemius muscles (Fig 1). Muscles were measured and collected 1 month after the initiation of OVL.

Cyclosporin A (CsA) treatment

In experiment 1, mice were treated every day during 1 month with the calcineurin pathway inhibitor cyclosporine A, CsA (25 mg/kg, ip, daily)(Mdx+OVL+CsA mice)[17, 23, 24] or saline (Mdx+OVL and Mdx mice) (Fig 1), the day after muscle ablation.

Muscle weakness and fragility

In the 3 experiments, maximal force and fragility (susceptibility to contraction-induced functional loss) were evaluated by measuring the in situ muscle contraction in response to nerve stimulation, as described previously [20]. 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 measured the absolute maximal force that was generated during isometric contractions in response to electrical stimulation (frequency of 75–150 Hz, train of stimulation of 500 ms). Absolute maximal force was determined at L0 (length at which maximal tension was obtained during the tetanus). L0 was measured with a caliper (distal tendon length was not included). The ability to rapidly generate muscle force was also assessed in Experiment 1. The rate of force development (RFD)(g/s) was measured when the force increased from 0 to 25% (RFD0-25%), 25% to 50% (RFD25-50%) and 50% to 75% (RFD50-75%) of P0 because the mechanisms responsible for RFD vary over time [29].

Fragility, i.e., susceptibility to contraction-induced functional loss in mice was estimated from the immediate force drop resulting from lengthening contractions. The sciatic nerve was stimulated for 700 ms (frequency of 150 Hz). A maximal isometric contraction of the muscle was initiated during the first 500 ms. Then, muscle lengthening (10% L0) at a velocity of 5.5 mm/s was imposed during the last 200 ms. All isometric contractions were made at an initial length L0. Nine lengthening contractions of the plantaris muscles were performed in mice, each separated by a 60-s rest period. Maximal isometric force was measured 1 min after each lengthening contraction and expressed as a percentage of the initial maximal force. After contractile measurements, the animals were killed with cervical dislocation.

Protein levels

Muscles from the Experiment 3 were snap-frozen in liquid nitrogen immediately after dissection. Frozen muscles were homogenized by Ultra Turax into an ice-cold in Newcastle Buffer [30] containing: 75 mM Tris pH 6.8, 3.8% SDS, 4 M urea, 2 M thiourea, 20% glycerol and 1% of protease inhibitor cocktail (Thermo Scientific) for immunoblotting. Samples were, incubated 30 min in ice and then centrifuged at 4°C. Protein concentration was measured using the DC Protein ASSAY (BioRAD) method with bovine serum albumin as a standard. Equal amounts of protein extracts (30 μg) were distributed and separated in Gel 5/15% or Gel 4/7.5% (Mini-Protean TGX Precast Gels, BioRAD) before electrophoretic transfer onto a 0.2 μm nitrocellulose blotting membrane (Amersham Protran). Western-blot analysis was conducted using anti-Utrophin (anti-Mouse; DSHB), anti-Desmin [Y66] (anti-Rabbit; Abcam), anti-fast myosin heavy chain isoform, MHC2a [SC-71] (anti-Mouse; DSHB), anti-AKT (anti-Rabbit; Cell Signaling), anti-P-AKT (Ser473) (anti-Rabbit; Cell Signaling), anti-cytoplamic γ-actin (anti-Mouse; SIGMA), anti-MyoD (anti-Mouse; Santa Cruz), anti-oxidative phosphorylation complex cocktail (OXPHOS) (anti-Mouse; Abcam), and anti-HSP-60 (anti-Goat, Santa Cruz) antibodies. Proteins bound to primary antibodies were visualized with secondary antibodies IRDye® 680LT Donkey anti-Goat or IRDye® 800LT Donkey anti-Goat (LI-COR), or HRP-conjugated anti-Mouse or HRP-conjugated anti-Rabbit secondary antibodies (Biorad). The signals were detected using ECL reagent (Cyanagen) then revealed with Chemidoc. Bands were quantified by Image Studio Lite software. The ratios of utrophin, desmin, cytoplamic γ-actin, MHC-2a, oxidative phosphorylation complexes, Akt, p-Akt, MyoD, to HSP-60 were calculated and then normalized to Mdx values.

Statistical analysis

Statistical analyses were performed Prism v8.4.0 software (GraphPad, La Jolla, CA, USA). When comparing two groups of data for one variable, T-test, T-test with Welch’s correction (when variance between groups was different) or Mann-Whitney test (when the number of data per group ≤ 5, typically protein levels) was used. When comparing three groups of data for one variable, one-way ANOVA with Tukey test or Brown-Forsythe ANOVA with Dunnet T3 test (when variance between groups was different) was used. Variance difference between group was tested using Brown-Forsythe test. When comparing data groups for more than one variable (typically the force drop following lengthening contractions in the 3 experiments), two-way ANOVA and Sidak test (when there was an interaction between factors) was used. We also calculated the Pearson correlation coefficients. P value less than 0.05 was considered significant. We did not remove any outlier. Individual values are presented in graphs, mean and SEM.

Results

CsA did not block the OVL-reduced fragility and maximal force and weight gains in Mdx mice (Experiment 1)

In situ isometric and lengthening contractions in response to nerve stimulation in the OVL plantaris muscle were performed 1 month after muscle agonist ablation (Experiment 1, Fig 1). As previously observed [20], the isometric maximal force drops following lengthening contractions were reduced in Mdx+OVL mice compared with Mdx mice (Fig 2A), indicating a reduced fragility in Mdx+OVL. In fact, the remaining force following the 9th lengthening contraction was increased in Mdx+OVL mice compared with Mdx mice (55.4% versus 28.2%)(p< 0.0001) (Fig 2A). We also found that absolute maximal force (+120.4%)(p < 0.0001) (Fig 2B) and muscle weight (+ 43.7%)(p < 0.05) (Fig 2C) were increased in Mdx+OVL mice compared to Mdx mice. To determine whether the activation of the calcineurin pathway needs to be involved to have the beneficial effects of OVL, we treated a group of Mdx+OVL mice with CsA (25 mg/kg), an inhibitor of this pathway (Fig 1). We have previously shown that CsA, at this administered dose, blocks the activation of the calcineurin pathway in murine dystrophic muscle [17]. Interestingly, we observed no difference (p > 0.05) between Mdx+OVL+CsA and Mdx+OVL mice concerning the force drop following lengthening contractions (Fig 2A), absolute maximal force (Fig 2B) and muscle weight (Fig 2C). However, CsA did alter the change in the rate of force development induced by OVL (p < 0.05)(Fig 2D), indicating that CsA does have an effect, since this functional parameter was not changed in Mdx+OVL+CsA, contrary to Mdx+OVL. Together these results indicate that the beneficial effects of OVL on fragility and maximal force of the plantaris muscle were minimally affected by CsA in Mdx+OVL mice.

Fig 2 — (A) Force drop following lengthening contractions (Fragility). n = 8–12 per group. (B) Absolute maximal force (P0). n = 9–12 per group. (C) Muscle weight. n = 6–8 per group n = 11–12 per group. (D) Rate of force development. n = 9–12 per group. CsA: cyclosporin A. Mdx+OVL: mechanically overloaded Mdx mice. Mdx+OVL+CsA: mechanically overloaded Mdx mice that received *CsA*. Mdx: non-overloaded Mdx muscle. o1, o3, and o4: significant different from Mdx (p < 0.05), (p < 0.001) and (p < 0.0001) respectively. c1: significant different from Mdx+OVL (p < 0.05).

A lower OVL also generated beneficial effects in Mdx mice (Experiment 2)

Next, we determined the effect of OVL on the lateral gastrocnemius (LG) muscle in mdx mice, a more physiological OVL than in the case of plantaris muscle (Experiment 2, Fig 1). The OVL of the LG muscle was less important that the OVL of the plantaris muscle because the amount of agonist muscle removed was lower in the case of the OVL of the LG muscle (the weight of the plantaris muscle is lower as compared to medial gastrocnemius muscle). In line with the lower OVL, the maximal force (Fig 3A) and weight (Fig 3B) gains in the OVL LG muscle were decreased compared to the OVL plantaris muscle (Fig 2B and 2C). Indeed, we found that LG muscle absolute maximal force (Fig 3A) (p < 0.05) and weight (Fig 3B) (p < 0.001) increased only 22.7% and 26.2%, respectively, in Mdx+OVL mice compared with Mdx mice. The force drops following lengthening contractions were reduced in the LG muscle from Mdx+OVL mice compared to Mdx mice. The remaining force following the 9th lengthening contraction was increased in the LG muscle from Mdx+OVL mice compared with Mdx mice (39.8% versus 25.5%) (p < 0.01) (Fig 3C), but to a lower extent in comparison to the OVL plantaris muscle (Fig 2A).

Fig 3 — (A) Absolute maximal force. n = 14–16 per group. (B) Muscle weight. n = 6–8 per group n = 14–17 per group. (C) Force drop following lengthening contractions (Fragility). n = 14–16 per group. Mdx+OVL: mechanically overloaded Mdx mice. Mdx: non-overloaded Mdx muscle. o1, o2, o3: significant different from Mdx (p < 0.05), (p < 0.01) and (p < 0.001) respectively.

Together, these results show that improvements in fragility, maximal force and muscle weight are also achieved by the OVL of the LG muscle, although to a lesser extent than for plantaris muscle.

OVL was also beneficial in the more severe dystrophic D2-mdx mice (Experiment 3)

We also studied the effect of OVL of the plantaris muscle in D2-mdx mice, a more severe murine DMD model (Experiment 3, Fig 1). We found that the force drops following lengthening contractions were reduced in D2-mdx+OVL mice compared with D2-mdx mice (Fig 4A). For example, the remaining force following the 9th lengthening contraction was increased in D2-mdx+OVL mice compared with D2-mdx mice (p < 0.0001)(66.1% versus 39.1%)(Fig 4A). In addition, absolute maximal force (p < 0.01)(Fig 4B) and muscle weight (p < 0.0001) (Fig 4C) increased by 48% and 49%, respectively, in D2-mdx+OVL mice compared with D2-mdx mice.

Fig 4 — (A) Force drop following lengthening contractions (Fragility). n = 14–16 per group. (B) Absolute maximal force. n = 15–18 per group. (C) Muscle weight. n = 6–8 per group. n = 16–20 per group. D2-mdx+OVL: mechanically overloaded D2-mdx mice. D2-mdx: non-overloaded D2-mdx muscle. o2, and o4: significant different from (p < 0.01) and (p < 0.0001) respectively.

We also evaluated the protein expression of important modulators of muscle fragility and maximal force/hypertrophy. Using immunoblotting analysis, we found that OVL increased the protein levels of utrophin (p < 0.05) (Fig 5A and 5B) and cytoplamic γ-actin (p < 0.05) (Fig 5C and 5D) in plantaris muscle from D2-mdx+OVL, without affecting that of desmin (Fig 5E and 5F). The phosphorylation of Akt (p-Akt)(p < 0.05) (Fig 6A and 6B) as well as the expression of MyoD (p < 0.05) (Fig 6C and 6D) were also increased in D2-mdx+OVL mice compared to D2-mdx mice. The amount of MHC-2a protein was not significantly changed by OVL (Fig 6C and 6E) neither the components of the oxidative respiratory chain (Fig 6F and 6G).

Fig 5 — (A) Representative image of a immunoblot showing utrophin band. (B) Utrophin protein levels. n = 4–5 per group. (C) Representative image of a immunoblot showing cytoplamic γ-actin band. (D) Cytoplamic γ-actin protein levels. n = 4–5 per group. (E) Representative image of a immunoblot showing desmin band. (F) Desmin protein levels. Immunoreactive bands were normalized to HSP-60 bands. Values are then expressed relative to mdx levels (mdx = 1). n = 4–5 per group. D2-mdx+OVL: mechanically overloaded D2-mdx mice. D2-mdx: non-overloaded D2-mdx muscle. o1: significant different from Mdx (p < 0.05).

Fig 6 — (A) Representative image of a immunoblot showing the bands of Akt and p-Akt. (B) p-Akt levels. n = 4–5 per group. (C) Representative image of a immunoblot showing the bands of MyoD and MHC-2a. (D) MyoD protein levels. n = 4–5 per group. (E) MHC-2a protein levels. n = 4–5 per group. (F) Representative image of a immunoblot showing the bands of the complexes (CIII, CIV and CV) of the respiratory chain. (G) Protein levels of the complexes (CIII, CIV and CV) of the respiratory chain protein levels. n = 4–5 per group. Immunoreactive bands were normalized to HSP-60 bands. Values are then expressed relative to mdx levels (mdx = 1). D2-mdx+OVL: mechanically overloaded D2-mdx mice. D2-mdx: non-overloaded D2-mdx muscle. o1: significant different from Mdx (p < 0.05).

Together, these results indicate that the beneficial effects of OVL on muscle maximal force, weight and fragility were also observed in the plantaris muscle from D2-mdx+OVL mice, concomitantly with increased levels of different proteins known to play a role in fragility and maximal force/hypertrophy (utrophin, cytoplamic γ-actin, p-Akt and MyoD).

Discussion

OVL improved fragility in mdx mice treated or not with CsA and D2- mdx mice

The present study confirms our previous observations [20] showing that 1-month OVL alleviates the fragility, a major dystrophic functional feature, in a similar manner to dystrophin restoration [31]. It was also recently reported an improvement in fragility after 6 training sessions comporting each 10 isometric maximal tetanic contractions per session in the mdx mice [19]. For the first time, we show that the beneficial effect of OVL was exerted in a muscle independent manner, independent of the degree of the OVL and the severity of the murine DMD model. This is very encouraging if we were to consider the prescription of resistance training by the physician for DMD patients since murine models are less affected than patients. Thus, our results suggest that the reduction of fragility can contribute to the improvement of the quality of life of the DMD patients insofar as they would suffer less this loss of function following lengthening contractions, together with having an increased absolute maximal force (see below).

Interestingly, we found that the calcineurin inhibitor CsA did not block the OVL-induced decrease in fragility in mdx mice, suggesting that activation of the calcineurin pathway is not the mechanism involved in this beneficial effect of OVL in murine dystrophic mice. It is possible that the calcineurin pathway was not activated by OVL. One study reports the absence of activation of the calcineurin pathway by OVL in healthy rodents [11], but several other do not [23, 24, 32]. One limitation of our study is that we do not provide a direct measure of calcineurin pathway activation. This deserves to be studied more closely in the future. Whatever, the present and previous results [17] suggest that different types of chronic exercise (voluntary running and OVL) exert the same improvement of fragility in murine dystrophic muscle via different mechanisms, since the beneficial effect of the two different types of chronic muscular exercise was blocked by CsA [17] or not (the present study).

Moreover, the improvement in fragility by OVL does not seem to be explained by an increase in the number of potentially less fragile muscle fibers [1] since our results suggest that the percentage of slower and more oxidative fibers remains unchanged in the D2-mdx (no increase in the amounts of MHC-2a and complexes of the respiratory chain), in contrast to what was observed in mdx mice [22]. However, it would be interesting to confirm the absence of muscle fiber type transition using a histological study and a larger sample size. We also eliminated the possibility that a decrease in MyoD expression in murine dystrophic mice would contribute to the improvement in fragility. It has recently been suggested that blocking the induction of the MyoD-dependent fetal gene program in myofibers improves sarcolemmal membrane stability and fragility [10]. However, we reported the contrary, an increase in the expression of MyoD in D2-mdx+OVL mice.

Based on previous studies [6, 8, 33], we next formulated the hypothesis that the improved fragility in murine dystrophic mice was associated to hypertrophy (increased muscle weight) and/or maximal force gain for the following reasons. First, we found that the improvement in fragility was roughly proportional to the hypertrophy and maximal force gain in mdx mice (the improvement in fragility was greater in the OVL plantaris compared to OVL LG gastrocnemius muscles). Consistently, there were correlations between fragility and muscle weight (S1 Table). Second, we found an increase in the Akt phosphorylation in response to OVL in D2-mdx mice, as previously shown in mdx mice [22], suggesting the activation of the Akt pathway. The activation of the Akt/mTOR pathway by OVL is indispensable to hypertrophy (and likely maximal force gain) [11]. Interestingly, we found a correlation between the activation of the Akt/mTOR pathway and hypertrophy in D2-mdx mice (S1 Table). Third, genetic activation of Akt in mdx mice leads to hypertrophy and increased maximal force as well as reduced fragility [6]. Fourth, genetic inactivation of mTOR in healthy muscle results in atrophy, reduced maximal force and increased fragility [8]. In a previous study, we found that OVL increased p-S6 in mdx mice, suggesting the activation of the mTOR pathway [22]. Fifth, the administration of formoterol in mdx mice, that induces hypertrophy and greater maximal force, decreases the fragility [33]. Formoterol is reported to activate the Akt pathway [13]. Sixth, inactivity, achieved by leg immobilization, reduced maximal force and increased fragility in mdx mice [16]. Together these results suggest that there is a link between the improved fragility in OVL dystrophic mice and maximal force and weight gains and the activation of the Akt and mTOR pathways, even if the downstream events have not yet been identified [6]. However, this link remains to be demonstrated in the future. It has also been reported that the Akt and mTOR pathways control the expression of several proteins providing a physical link between the extracellular matrix and the intracellular cytoskeleton, such as dystrophin, utrophin and desmin [6, 8]. These proteins are also known to modulate fragility in dystrophic muscle [2, 3, 5, 34]. However, we demonstrate that utrophin was increased by OVL in D2-mdx mice but not in mdx mice [22] and desmin was not increased by OVL in D2-mdx mice. It is possible however that the increased cytoplamic γ-actin in D2-mdx+OVL mice contributed to the improved fragility since transgenic overexpression of cytoplamic γ-actin reduces fragility in mdx mice [4]. Finally, and in a manner always linked to hypertrophy, it is possible that the reduction in fragility in response to OVL is due to the increase in the ratio of fiber length to muscle length [35]. This increase would have the effect of reducing the percentage of lengthening at the fiber level, since we achieved a stretch of 10% of the length of the muscle.

OVL increased maximal force production in both mild and severe murine DMD models

In addition to the improvement in fragility, OVL markedly increased the absolute maximal force of the mdx mice, independent the lower leg muscle concerned (plantaris or LG gastrocnemius muscles), in line with previous results [20, 22]. A smaller improvement of approximately 12% in absolute maximal force after resistance training was also reported in mdx mice [19]. In the future, it would be interesting to know if the restoration of these parameters is complete (no more difference with healthy muscle). What is new is that this maximal force gain was also observed in the case of a severe murine model of DMD (D2- mdx mice), closer to the human DMD phenotype. Thus, unlike the absence of desmin [36], another protein of the costamer, the loss of dystrophin does not prevent the increase in maximal force induced by OVL. This beneficial effect of OVL is related to hypertrophy (increased muscle weight), increased level of p-Akt and MyoD in D2-mdx (the present study) and mdx mice [22], increased level of p-S6 in mdx mice [22], thus possibly the activation of the Akt/mTOR pathway that is indispensable for the OVL-induced hypertrophy [11]. Moreover, we found that the hypertrophy was not blocked by CsA, suggesting that the calcineurin pathway does not play an important role which in line with a previous study [11] but not for unknown reasons with others studies [23, 24, 32], which have been widely debated [37]. This increase in maximal force in D2-mdx mice after 1-month of OVL was interesting because it has been previously reported that the restoration of dystrophin in response to a gene therapy approach in D2-mdx mice was able or not to increase maximal force [38, 39]. Of note, several other types of fragility-improving preclinical therapies do not increase absolute maximal force in mouse models of DMD, i.e., could not potentially mitigate muscle weakness, and sometimes even increased it [9, 18, 27, 40, 41]. Finally, another aspect of muscular performance that is improved by OVL is the ability to produce force rapidly. We show that the increase in the rate of force development was diminished by CsA, suggesting that fiber type is not the only factor determining this ability [29].

Conclusion

Our results show that OVL reduced the susceptibility to contraction-induced force drop, independent of the severity of the OVL or the type of leg muscle, in both mild and severe murine DMD models. This beneficial effect of OVL on muscle fragility is associated to maximal force gain, hypertrophy, increased expression of p-Akt and cytoplamic γ-actin, and was not inhibited by CsA which is known to block the activation of the calcineurin pathway. Moreover, OVL also induced substantial increase in maximal force related to muscle growth. Thereby, this murine model of resistance training improved two important functional dystrophic features in both mildly and severely affected mice. Collectively, studies using more or less severe murine models of DMD [19, 20, 22] as well as the study in DMD patients [42] support the idea that it would be interesting to determine that resistance training, comprising muscle contractions in small numbers but developing high levels of force, would benefit DMD and Becker patients by allowing notable strength gains.

Supporting information

S1 Table. Correlations.

(DOCX)

pone.0295700.s001.docx^{(12.1KB, docx)}

S1 File. Minimal data.

Plantaris mdx, Force (experiment 1).

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S2 File. Minimal data.

Lat Gast Mdx, force (experiment 2).

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S3 File. Minimal data.

Plantaris D2-mdx, force (experiment 3).

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S4 File. Minimal data.

Plan D2-mdx, Blots values (experiment 3).

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S1 Raw image. Fig 5A UTROPHIN.

Image of blot.

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S2 Raw image. Fig 5C. GAMA ACTIN.

Image of blot.

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S3 Raw image. Fig 5E. DESMIN.

Image of blot.

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S4 Raw image. Fig 6A. P-AKT.

Image of blot.

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S5 Raw image. Fig 6C. MyoD and MHC2a.

Image of blot.

(PDF)

pone.0295700.s010.pdf^{(3.1MB, pdf)}

S6 Raw image. Fig 6F. Respiratory Chain.

Image of blot.

(PDF)

pone.0295700.s011.pdf^{(872.4KB, pdf)}

Acknowledgments

We thank Gillian Butler-Browne for her invaluable help.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

References

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PLoS One. doi: 10.1371/journal.pone.0295700.r001

Decision Letter 0

Aldrin V Gomes

3 Sep 2023

PONE-D-23-21038Beneficial effects of resistance training on both mild and severe mouse dystrophic muscle function as a preclinical option for Duchenne muscular dystrophyPLOS ONE

Dear Dr. Ferry,

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Reviewer #2: No

Reviewer #3: Yes

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Reviewer #1: The manuscript by Hassani et. al. investigates the beneficial impact of muscle activity in mouse models of muscular dystrophy. Specifically, the authors show that synergistic ablation induces muscle hypertrophy, maximal force production, and resistance to eccentric damage. While demonstrated previously, this has been expanded to be present even with lower levels of hypertrophy, in a more severely affected mouse model and to not be dependent on calcineurin signaling. The manuscript is generally well written. The influence of exercise and muscle loading in DMD is of importance to the field with questions that this paper can help to address. However, there are substantial concerns that dampen enthusiasm listed below.

Minor comments:

Methods: It is stated that a velocity of 5.5 mm/s was used that is equivalent to a 0.85 fiber length/s velocity. This would assume a constant fiber length of ~6.5 mm. How is this determined, especially in light of potentially changing Lo?

Methods: The methods state that lengthening contractions were induced in the TA, however that is not consistent with the rest of the manuscript.

Fig 2C: Weight is misspelled on the axis.

Fig 2D: The rationale for separating out the different phases of force development is not clear. It may be easier to just show one value (perhaps 0-50%).

Fig 2D: The rate of force development is slowed with CsA, but this would seem counter to expectations of CaN promoting slower fibers (thus blocking it would promote faster fibers). A comment in the discussion could be warranted.

Fig 3C: It isn’t clear if error bars are present in this figure.

Fig 5: The lane labels are not very clear.

Discussion: The statement that improvement in fragility cannot be explained by an increase in less fragile fibers could e overly strong. While a significant change in MHC2a was not observed there is a shift in the mean and with low sample sizes here this appears under powered.

Discussion: Research is cited that gene therapy does not increase muscle force in the D2 model. However, there is data showing substantial increases in muscle force with microdystrophin gene therapy (Cernisova et. al. Int J Mol Sci 2023).

Reviewer #2: The study describes the benefits of resistance training in two mouse DMD models, offering preclinical data that might be relevant to human disease. Results are solid and well presented, yet the study is purely descriptive and the manuscript needs revision before it might be acceptable for publication.

Main concerns with this study are that it offers little mechanistic insight and no direct link to applicability to human medicine. The title should be improved to better phrase the study.

The manuscript would benefit from comparison with other studies given in a comprehensive table. Also, relevance to human medicine should be better explored. What could be a recommended (improved) training strategy based upon the study’s findings? What is the added value for the clinic? Are there any significant differences between mdx and D2-mdx that aid in comparison of severity? Becker MD is not mentioned anywhere, give this some thought and add to the discussion. In view of human DMD being severe, how can resistance training be implement in the window of ambulation? Authors should more thoroughly discuss the translational value of the study.

There are several method concerns. Describe how the CsA dose and administration was determined. Was this based on literature or on own preliminary tests? Give concentrations of antibodies used for western blots in µg/ml IgG and not as a dilution factor. Why was gamma-actin chosen as a loading control, while most use alpha- or beta-? Also, the use of HSP60 as a mitochondrial marker needs to be explained (as well as its preference to VDAC). Legends to fig 5 and 6 do not mention HSP60, nor if the graphs of protein levels are ratio’s to HSP60. Explain rationale of normalization to mitochondrial content. Full western blots need to be provided as a supplement. Figure legends need to specify precise numbers of mice per group, and not ranges.

Minor remarks:

Line 280 benefic

Line 293 cytoplamic

Reviewer #3: I think this research paper is very detailed and thorough. It is technically sound, the data supports the conclusions and the statistical analysis is very rigorous. It just needs some small revisions.

I have one main area of confusion. For the first sub -header of the discussion section, I was very confused by the wording. "OVL improved fragility in both mdx and D2-mdx mice, treated or not with CsA" makes it sound like both the mdx and the D2-mdx mice were treated with CsA. I had to go back to the methods section to double-check that the D2-mdx mice were never treated with CsA.

In addition, I was wondering why you all did not include a CsA group in the D2-mdx mice?

Lastly, I believe almost every time the cytoplasmic gamma-actin are mentioned in the paper, cytoplasmic is spelled incorrectly. I saw "cytoplamic" multiple times throughout the paper.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PLoS One. 2024 Mar 8;19(3):e0295700. doi: 10.1371/journal.pone.0295700.r002

Author response to Decision Letter 0

7 Nov 2023

Academic Editor:

The referees all found that the influence of exercise and muscle loading in DMD is of importance to the field. However, there are concerns that need to be addressed. Improved description of the experimental design, addressing the methods concerns, comparison with other studies and directly testing for significant relationships between one of the parameters such as hypertrophy and force loss or pAKT levels and muscle mass is needed. AUTHORS' RESPONSE: This was revised.

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Reviewers' comments:

Reviewer #1:

The decrease fragility could simply be more sarcomeres in series leading to less sarcomeric strain or strain rate with the eccentric contraction. The description of how lengths are recorded is incomplete. It appears Lo was set to where maximum force was achieved, but how was Lo then measured in situ? Where calipers used and was any tendon length included? This is important given that the lengthening contractions are set to 10% of Lo and Lo is expected to change with overload. AUTHORS' RESPONSE: We thank the reviewer for these excellent remarks. It is possible that the longitudinal growth of the fibers is responsible for the improvement in fragility. We now discussed the possible increase in the ratio of fiber length to muscle length in response to OVL (Jorgenson et al 2020) could be the cause of the improvement in fragility (Discussion). Muscle length (L0) was measured from proximal extremity (knee) to myotendinous junction (distal extremity) using caliper. Distal tendon length was not included. These informations are now included (Materials and Methods).

Although it did not turn out to induce major effects the treatment with CsA is thought to act through inhibiting slow fiber programs through Calcineurin. However, only one myosin heavy chain molecule was examined. It would be informative to look at fiber types more directly to see if that was related at all to the susceptibility to damage as postulated. AUTHORS' RESPONSE: In two previous studies (Ferry et al 2015, Joanne et al 2012), we did not observe any change in the expression of MHC-2b in response to OVL in the plantaris muscle of mdx mice. Additionally, the change in MHC-1 expression was modest. Unfortunately, we did not prepare muscle samples for histological study. Thus, we revised the statement that improvement in fragility cannot be explained by an increase in less fragile fibers. Moreover, we added that it would be interesting to confirm the absence of muscle fiber type transition using a histological study and a larger sample size (Discussion).

The experimental design section is helpful to show what was done. However, the rationale for some decisions could be more fully explained. Particularly, it may be helpful to include wildtype animals so the degree of recovery could be stated for certain parameters. Also, it isn’t clear why a bilateral design is not used to compare the contralateral limb to that of the OVL. AUTHORS' RESPONSE: We agree, it would have been interesting to study WT mice to determine the degree of recovery compared to WT mice. This idea has been added (Discussion). A bilateral experimental design would indeed be more interesting, but we were always afraid that the mouse would not use much of the leg that was operated on. It is difficult/complicated to verify this hypothesis. Therefore, we followed the protocol of Roy's study in 1995.

There are a number of associations that are proposed between parameters studied, such as that between AKT and muscle growth. Given that many of the parameters are investigated within the same muscle it would be helpful to directly test for significant relationships between parameters such as hypertrophy and force loss or pAKT levels and muscle mass. AUTHORS' RESPONSE: We thank you for this remark. We studied these relationships in the revised manuscript (Discussion, S1 Table). We found correlations between fragility (the percentage of force remaining following 9 lengthening contractions) and the muscle weight in the 3 experiments. There were also correlations between the muscle weight and the pAkt/Akt ratio.

Minor comments:

Methods: The methods state that lengthening contractions were induced in the TA, however that is not consistent with the rest of the manuscript. AUTHORS' RESPONSE: This was revised.

Fig 2C: Weight is misspelled on the axis. AUTHORS' RESPONSE: This was revised.

Fig 2D: The rationale for separating out the different phases of force development is not clear. It may be easier to just show one value (perhaps 0-50%). AUTHORS' RESPONSE: The mechanisms responsible for RFD vary over time (Rodrıguez-Rosell et al 2018). This information has been added (Materials and Methods).

Fig 3C: It isn’t clear if error bars are present in this figure. AUTHORS' RESPONSE: SEM are present (the error bars were shorter than the size of the symbol).

Fig 5: The lane labels are not very clear. AUTHORS' RESPONSE: This was revised.

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Reviewer #2:

Main concerns with this study are that it offers little mechanistic insight and no direct link to applicability to human medicine. The title should be improved to better phrase the study.

The underlying mechanisms of benefit have not been investigated in depth. Administering CsA leads the authors to conclude that calcineurin signalling is not involved, and it is speculated that Akt/mTor might be involved. However, no specific data is offered that might corroborate this statement, and it therefore needs to be downplayed. The study appears to me somewhat immature., and authors need to clearly acknowledge limitations and future routes forward. Lines 344-370 need to be reduced in fit with the shortcomings of the study. AUTHORS' RESPONSE: We now clearly acknowledge limitations and future routes forward (Discussion). We found that Akt phosphorylation was increased by OVL. In previous studies (Bodine et al 2001, Blaauw et al 2008), Akt was demonstrated using antibodies specific for phospho-Akt, with Immunoblot analysis of protein extracts from muscle. Some data were also added to support the hypothesis that Akt/mTOR, hypertrophy and force gain might be associated (S1 Table), as suggested by Reviewer #1. We found correlations between fragility and hypertrophy, and hypertrophy and Akt/mTOR activation. In addition, reviewer #1's remarks led us to suggest adding a very interesting explanation related to hypertrophy. Indeed, it is possible that the improvement in fragility could be linked to the longitudinal growth of the fibers, this phenomenon also participating in hypertrophy (Discussion).

The manuscript would benefit from comparison with other studies given in a comprehensive table. AUTHORS' RESPONSE: Only 2 studies (ours) studied the effect of OVL on fragility and maximal force (Joanne et al 2012, Ferry et al 2015). A 3rd study, that of Lindsay et al (2019), determined the effects of isometric resistance training on maximal force and fragility. They reported that OVL and isometric resistance training improve maximal force and fragility in mdx mice (plantaris and EDL muscles). Comparisons are now made (Discussion).

Also, relevance to human medicine should be better explored. What could be a recommended (improved) training strategy based upon the study’s findings? What is the added value for the clinic? Are there any significant differences between mdx and D2-mdx that aid in comparison of severity? Becker MD is not mentioned anywhere, give this some thought and add to the discussion. In view of human DMD being severe, how can resistance training be implement in the window of ambulation? Authors should more thoroughly discuss the translational value of the study. AUTHORS' RESPONSE: We did not find any notable difference between mdx and D2-mdx mice, which is encouraging for the application of this type of training to DMD patients. This idea was already stated in the Discussion. We more thoroughly discuss the translational value of the study, as suggested (Conclusion).

There are several method concerns. Describe how the CsA dose and administration was determined. Was this based on literature or on own preliminary tests? AUTHORS' RESPONSE: The dose of CsA and administration were based on previous studies and our own experience, as already stated in Materials and Method.

Give concentrations of antibodies used for western blots in µg/ml IgG and not as a dilution factor. AUTHORS' RESPONSE: Indeed it is not relevant to mention dilution. This has been fixed.

Why was gamma-actin chosen as a loading control, while most use alpha- or beta-? Also, the use of HSP60 as a mitochondrial marker needs to be explained (as well as its preference to VDAC). Legends to fig 5 and 6 do not mention HSP60, nor if the graphs of protein levels are ratio’s to HSP60. Explain rationale of normalization to mitochondrial content. Full western blots need to be provided as a supplement. AUTHORS' RESPONSE: Gamma-actin was not chosen as a loading control, unlike HSP60. Gamma-actin is one of the proteins of interest, studied among several others. It is correct that protein levels are ratio's to HSP60 and the expressed relative to mdx. This is now mentioned in the revised manuscript (methods and legend figures). The level of HSP60 did not vary between groups.

Figure legends need to specify precise numbers of mice per group, and not ranges. AUTHORS' RESPONSE: It is possible to know precisely the number of mice in each group by examining the figures (individual values are shown) and supporting information in the revised manuscript.

Minor remarks:

Line 280 benefic AUTHORS' RESPONSE: This has been corrected in the new version of the manuscript.

Line 293 cytoplamic AUTHORS' RESPONSE: This has been corrected in the new version of the manuscript.

*************

Reviewer #3:

In addition, I was wondering why you all did not include a CsA group in the D2-mdx mice? AUTHORS' RESPONSE: As we did not find an effect of CsA in the mdx mice, we had no reason to think that this might be different in the D2-mdx mouse. For the OVL effects that we compared, we did not find any difference overall between the 2 murine DMD models.

Lastly, I believe almost every time the cytoplasmic gamma-actin are mentioned in the paper, cytoplasmic is spelled incorrectly. I saw "cytoplamic" multiple times throughout the paper. AUTHORS' RESPONSE: This has been corrected.

Attachment

Submitted filename: Response to reviewers.docx

pone.0295700.s012.docx^{(23.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0295700.r003

Decision Letter 1

Aldrin V Gomes

28 Nov 2023

Beneficial effects of resistance training on both mild and severe mouse dystrophic muscle function as a preclinical option for Duchenne muscular dystrophy

PONE-D-23-21038R1

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Reviewer #3: All comments have been addressed

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Reviewer #3: Yes

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Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Reviewer #1: The revised manuscript by Hassani et. al. makes notable improvements in the investigation of muscle overload in dystrophic muscles. The added relationships between hypertrophy and eccentric damage and pAKT support the conclusions. Importantly the limitations of the study have been more thoroughly addressed by the authors which prevent more strong conclusions from being drawn. The topic of overloading dystrophic muscle is of interest and the result of decreased susceptibility to damage is clear although the mechanistic insight is limited.

Reviewer #3: The author addressed all comments and concerns made to the manuscript. The manuscript is sound, the data supports the conclusions, and the statistical analysis has been performed appropriately and rigorously.

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PLoS One. doi: 10.1371/journal.pone.0295700.r004

Acceptance letter

Aldrin V Gomes

4 Dec 2023

PONE-D-23-21038R1

Beneficial effects of resistance training on both mild and severe mouse dystrophic muscle function as a preclinical option for Duchenne muscular dystrophy

Dear Dr. Ferry:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Correlations.

(DOCX)

pone.0295700.s001.docx^{(12.1KB, docx)}

S1 File. Minimal data.

Plantaris mdx, Force (experiment 1).

(PDF)

pone.0295700.s002.pdf^{(30.4KB, pdf)}

S2 File. Minimal data.

Lat Gast Mdx, force (experiment 2).

(PDF)

pone.0295700.s003.pdf^{(28.4KB, pdf)}

S3 File. Minimal data.

Plantaris D2-mdx, force (experiment 3).

(PDF)

pone.0295700.s004.pdf^{(44KB, pdf)}

S4 File. Minimal data.

Plan D2-mdx, Blots values (experiment 3).

(PDF)

pone.0295700.s005.pdf^{(24.1KB, pdf)}

S1 Raw image. Fig 5A UTROPHIN.

Image of blot.

(PDF)

pone.0295700.s006.pdf^{(2.2MB, pdf)}

S2 Raw image. Fig 5C. GAMA ACTIN.

Image of blot.

(PDF)

pone.0295700.s007.pdf^{(503.6KB, pdf)}

S3 Raw image. Fig 5E. DESMIN.

Image of blot.

(PDF)

pone.0295700.s008.pdf^{(861.9KB, pdf)}

S4 Raw image. Fig 6A. P-AKT.

Image of blot.

(PDF)

pone.0295700.s009.pdf^{(1.6MB, pdf)}

S5 Raw image. Fig 6C. MyoD and MHC2a.

Image of blot.

(PDF)

pone.0295700.s010.pdf^{(3.1MB, pdf)}

S6 Raw image. Fig 6F. Respiratory Chain.

Image of blot.

(PDF)

pone.0295700.s011.pdf^{(872.4KB, pdf)}

Attachment

Submitted filename: Response to reviewers.docx

pone.0295700.s012.docx^{(23.7KB, docx)}

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

Beneficial effects of resistance training on both mild and severe mouse dystrophic muscle function as a preclinical option for Duchenne muscular dystrophy

Medhi Hassani

Dylan Moutachi

Mégane Lemaitre

Alexis Boulinguiez

Denis Furling

Onnik Agbulut

Arnaud Ferry

Roles

Abstract

Introduction

Materials and methods

Ethical approval and animal models

Experimental design

Fig 1.

Mechanical overloading

Cyclosporin A (CsA) treatment

Muscle weakness and fragility

Protein levels

Statistical analysis

Results

CsA did not block the OVL-reduced fragility and maximal force and weight gains in Mdx mice (Experiment 1)

Fig 2. Effect of CsA on OVL plantaris muscle in Mdx mice.

A lower OVL also generated beneficial effects in Mdx mice (Experiment 2)

Fig 3. Effect of OVL on lateral gastrocnemius muscle in Mdx mice.

OVL was also beneficial in the more severe dystrophic D2-mdx mice (Experiment 3)

Fig 4. Effect of OVL on plantaris muscle in D2-mdx mice: Physiological data.

Fig 5. Effect of OVL on plantaris muscle in D2-mdx mice: Expression of utrophin, cytoplamic γ-actin, and desmin.

Fig 6. Effect of OVL on plantaris muscle in D2-mdx mice: Expression of pAkt, MyoD, MHC-2a and the complexes of the respiratory chain.

Discussion

OVL improved fragility in mdx mice treated or not with CsA and D2- mdx mice

OVL increased maximal force production in both mild and severe murine DMD models

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Aldrin V Gomes

Roles

Author response to Decision Letter 0

Decision Letter 1

Aldrin V Gomes

Roles

Acceptance letter

Aldrin V Gomes

Roles

Associated Data

Supplementary Materials

Data Availability Statement

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