Twenty-one days of low-intensity eccentric training improve morphological characteristics and function of soleus muscles of mdx mice

Duchene muscular dystrophy (DMD) is caused by the absence of the protein dystrophin, which leads to muscle weakness, progressive degeneration, and eventually death due to respiratory failure. Low-intensity eccentric training (LIET) has been used as a rehabilitation method in skeletal muscles after disuse. Recently, LIET has also been used for rehabilitating dystrophic muscles, but its effects are still unclear. The purpose of this study was to investigate the effects of 21 days of LIET in dystrophic soleus muscle. Thirty-six male mdx mice were randomized into six groups (n = 6/each): mdx sedentary group; mdx training group-3 days; mdx training group-21 days; wild-type sedentary group; wild-type training group-3 days and wild-type training group-21 days. After the training sessions, animals were euthanized, and fragments of soleus muscles were removed for immunofluorescence and histological analyses, and measurements of active force and Ca2+ sensitivity of the contractile apparatus. Muscles of the mdx training group-21 days showed an improvement in morphological characteristics and an increase of active force when compared to the sedentary mdx group. The results show that LIET can improve the functionality of dystrophic soleus muscle in mice.


Results
Morphological alteration. Immunofluorescence analysis confirmed the absence of dystrophin in mdx mice muscle. Figure 1 shows that dystrophin is present in the sarcolemma of soleus fibers in wild-type mice (green color- Fig. 1A), but is absent in mdx mice (Fig. 1B).
The morphological examination of sections from muscles stained by Hematoxylin and Eosin (HE) revealed a homogeneous pattern of muscle fibers in wild-type mice. Photomicrography shows polyhedral fiber and nuclei in the periphery of the fiber ( Fig. 2A). The dystrophic muscles from the mdxSED group (Fig. 2B) showed a large variation in fiber size, nuclear centralization, basophilic fibers and necrosis. Although we have not quantified the amount of connective tissue in these muscles, which may limit a definitive conclusion, qualitative analysis showed an increase in connective tissue in dystrophic muscles, a result on line with previous studies 29 .
After eccentric low-intensity training during 3-days (mdxTr 3 ) the dystrophic muscles showed a dramatic increase in the degree of morphological abnormalities (Fig. 2C). These alterations include an increase in necrosis and basophilic fibers, variation in fiber size and an increase of connective tissue when compared to animals of mdxSED group. Notably, the muscles from dystrophic animals trained during 21 days (mdxTr 21 ) showed an improvement in their morphological characteristics. This group presented moderate abnormality when compared with mdxTr 3 . There is a significant decrease of basophilic fibers, degree of necrosis and connective tissue (Fig. 2D). These muscles of mdxTr 21 group still showed a high-centralized nucleus and with a more homogenous trophism of the fibers, leading to a small variation in fiber sizes (Fig. 2D).
Minimal Feret's diameter of the different fiber types. The absence of dystrophin protein caused a significant alteration in the trophism in muscle fibers of mdx mice. The muscle of mdxSED group showed a significant increase in the diameter of unmixed (FTI, FTIIA and FTIID) and hybrids fibers (FTIC and FTIIAD) when compared to animals of the wild-type group (mdxSED vs. wtSED; p < 0.05). The results of the minimal Feret's diameter of the different fiber types are shown in Table 1.
The effects of LIET on the trophism of all conditions investigated in this study are summarized in Table 1. LIET was effective to improve the trophism of muscle fibers in the mdx mice. The 3-days of training already caused a reduction in the diameter of the dystrophic muscles, while causing only a reduction in the FTI fibers the  Proportion of the different fiber types. Morphometrics alterations were observed through analysis of the slides processed by immunofluorescence for different MHC isoforms. Figure 3A,B show slides from wtSED and mdxSED, respectively. Figure 3A shows a homogeneous distribution between FTI (blue color) and FTIIA (green color) fibers while Fig. 3B shows grouping of fiber in the fascicles, i.e., typing group. Figure 4 illustrates the distribution of fiber types in all groups tested in this study. As expected, the absence of dystrophin protein implied a significant reduction in the number of FTIIA fibers with a concomitant increased of FTIIC fibers in sedentary animals. The short-term training applied to mdx mice (mdxTR 3 ) did not cause a significant alteration in the proportion of different type fibers. Although it the mdxTR 3 showed a small increase of the number in FTIIA fibers and a reduction in FTI fibers (Fig. 4), the difference was not statistically significant. Figure 3C illustrates a predominance of glycolytic FTIIA (green color) in the mdxTR 3 group.
Training of mdx mice for 21 days caused a reduction of oxidative fibers (FTI) compared to the mdx sedentary group (mdxSED vs. mdxTR 21 ; p < 0.05). There was not a difference between the proportions of FTI in both trained groups (mdxTR 3 vs. mdxTR 21 ; p > 0.05), but there was a slight increase in these fibers (mdxTR 3 vs mdxTR 21 ; p < 0.05). Figure 3D shows a large homogeneity between FTI and FTIIA fibers after a long period Total force of the single cell. Figure 5 shows the force traces collected during contractions developed by singles fibers dissected from mice from wtSED, mdxSED and mdxTR 21 . The isometric contractions were recorded during experiments in which fibers were maximally activated (pCa 2+ 4.5) at an initial sarcomere length of 2.5 μm. The forces produced during maximal activation were lower in mdx fibers than wild-type fiber (black trace). After 21 days of low-intensity eccentric training the total force increased significantly in the mdx fibers, but was still lower than the force produced by wild-type fibers (Fig. 5). The force values were significantly different among the wtSED (241.5 ± 33.8), mdxSED (mean: 67.20 ± 17.24) and mdxTR 21 (106.5 ± 8.82) groups.
We also tested the force responses to different levels of Ca 2+ activation, to construct a force-pCa curve and evaluate the Ca 2+ sensitivity of the contractile apparatus (Fig. 6). A difference in Ca 2+ sensitivity (measured by the pCa 50 ) was not observed among groups, i.e., Ca 2+ sensitivity was not impaired in the mdx fibers investigated in this study (Fig. 6).

Discussion
The results of this study demonstrate that LIET improved the general histology, trophism, the overall distribution of different fiber types and the contractile force of mdx mice. These findings suggest that a well-controlled LIET is a viable therapeutic model for improving muscle function in DMD.
Physical exercise has been studied as a therapeutic strategy to delay the progression of DMD. However, physical exercise must be carefully planned because it can also accelerate a degenerative process in DMD muscles. High-intensity exercise, for example, can increase muscle damage and degeneration 18,30 . Although our findings showed a worsening of the phenotype of mdx animals trained for 3 days with LIET, this result was reverted with 21 days of training. It has been shown that eccentric exercise when applied for a prolonged period may improve the muscle morphological characteristics 14,31 .
This study confirmed some of the main alterations induced by the absence of dystrophin in soleus muscle: nuclear centralization, splitting, necrosis, increased connective tissue and basophilic cells. The centralization observed in the dystrophic muscle of mdx mice is a result of muscle degeneration and regeneration 32 . During repair of skeletal muscle tissues, satellite cells, which are precursors of myogenesis, are activated and proliferate www.nature.com/scientificreports/ to the site of the lesion where they will merge into the focus of the lesion and subsequently differentiate into myoblasts. These newly repaired cells present the centralized nucleus until the maturation occurs, with subsequent migration of the nucleus to the periphery 33 . Other cytoarchitectural changes such as basophilia and splitting are characteristic of cell membrane damage and Ca 2+ influx into the cytoplasm of the dystrophic cell 34 , which triggers degenerative reactions that increase the inflammation, contributing to chronic damage and degeneration of  . Force-pCa 2+ relation for the wtSED and the mdxSED groups. There was no difference in Ca 2+ sensitivity, as the value for pCa 50  www.nature.com/scientificreports/ dystrophic cells 35 . The degenerative process culminates with increased connective tissue that can lead to fibrosis and impairment of muscle function 32,36,37 .
There was an increase in minimal Feret's diameter of fibers of the mdxSED animals when compared to wtSED animals, as previously observed 14,[38][39][40] . The successive degeneration/regeneration processes that occur in the dystrophic fiber exacerbate the inflammatory process in the cytosol, increasing the cell volume [41][42][43] . After 21 days of training, the soleus muscle cells of the mdx animals reduced their volume compared to the mdx sedentary; in it has been shown that low-intensity eccentric training applied during long period reduces inflammatory processes 44 and reduce the cross-section area (CSA) of mdx muscle fibers 15 .
The wtSED animals presented a homogeneous distribution between type I and type IIA fibers, as previously shown 15,27 . The absence of dystrophin promoted an imbalance in this distribution, as the proportion of FTIIA fibers was reduced while the proportion of FTI fibers was increased in mdxSED animals 27,45,46 . We observed that the LIET applied during a short period of training (3-days) reduced the number of FTI fibers. However, with the maintenance of the training for 21 days, the proportion of these oxidative fibers was increased, concomitant with a reduction in FTIIA fibers, in agreement with previous studies 6, [47][48][49] . It is likely that low-intensity training promotes adaptations in the mitochondria thus increasing the oxidative capacity of the fibers and the resistance to fatigue.
The most striking symptom of DMD is the loss of muscle strength due to progressive degeneration of muscle fibers. In this condition, non-contractile tissues, such as adipose and connective tissue, replace the contractile tissue. Our results from experiments conducted with single fibers showed that DMD reduced the specific force of mdx muscle, confirming previous results 27, [50][51][52][53][54] . The reduction in force in the mdx mice could be explained by a reduction of the sensitivity of the contractile system to Ca 2+ , which would lead to a decrease in force produced at a given level of activation 55,56 . However, our results do not show an altered difference in Ca 2+ sensitivity in fibers dissected from mdx mice, as previously observed [57][58][59] . Therefore, the decrease in force in mdx fibers may be a result of dysfunction in contractile proteins. In this regard, there are conflicting results in the literature. Canepari, et al. 52 observed an impairment of the myosin function in dystrophic muscles that was attributed to post-translational modifications, which could determine a change in its enzymatic or mechanical properties of myosin. On the other hand, Bates, et al. 60 did not observe a significant reduction in myosin activity or crossbridge kinetics in mdx mice. The difference in results is difficult to explain, but it may be associated with differences in experimental protocols, and with the age of the mdx mice. In this study, we used 7-week-old mice, an age in the peak phase of the lesion, presenting many cells in degeneration/regeneration, as discussed earlier.
We are not aware of other studies investigating the effects of LIET on contractile properties of single fibers from dystrophic muscles. However our results are on line with studies showing an improvement in the forelimb and hindlimb strength of mdx mice after long periods of low intensity training on a flat treadmill 11,44,61 . There are studies showing an improvement in fatigue resistance and oxidative capacity after 12 weeks of low-intensity training 47 .

Conclusion
LIET improved the morphological and functional characteristics of the dystrophic soleus muscle of mice by reducing cell degeneration, improving trophism and distribution of different types of fibers and increasing the contractile force of muscle fibers. LIET may be an important method to be used for rehabilitation of dystrophic muscles, with potentially far-reaching implications for the treatment of the disease.

Methods
Ethics statement. This study was conducted in accordance with the Ethical Principles in Animal Research, adopted by the National Council for the Control of Animal Experimentation (CONCEA, Brazil) and was approved by the Ethics Committee on Animal Use in Ribeirão Preto Medical School (Brazil) (protocol number 173/2013).
The mice were maintained in cages on a light-dark cycle (12 h/12 h) at 22ºC and supplied with water and food ad libitum. The mdx mice were randomized into three groups (n = 6 for each group): Sedentary group (mdxSED), Training group-3 days (mdxTR 3 ) and Training group-21 days (mdxTR 21 ). Wild-type mice were similarly randomized into three groups (n = 6 for each group): wtSED; wtTR 3 and wtTR 21 .
Training started when the animals were 6-weeks old, because at this age severe morphological alterations and signals of degeneration and/or regeneration are detected in dystrophic muscles 62 . The exercise protocol was performed during three days per week (Monday, Wednesday and Friday) for 3 days (1 week) or 21 days (7 weeks) of training. Therefore, at the end of training the animals of mdxTR 3 and wtTR 3 groups were 7-week-old and the animals of mdxTR 21  Contractile measurements of single fiber. The muscles were dissected, tie to wood sticks, and chemically permeabilized following standard procedures [64][65][66] . Briefly, the muscle samples were incubated in rigor solution (50 mM Tris, 100 mM NaCl, 2 mM KCl, 2 mM MgCl 2 , and 10 mM EGTA pH7.0, Sigma Aldrich, San Luis, Missouri, USA) for 4 h, after which they were transferred to a rigor-glycerol (50:50) solution for 15 h. Then, the samples were placed in a fresh rigor-glycerol (50:50) solution with a cocktail of protease inhibitors (Roche Diagnostics, Basel, Switzerland) and stored in a freezer −20 ºC for at least 7 days.
Single cell force measurements. On the day of experiment, single fibers were carefully dissected in relaxing solution, and gripped at their ends with T-shaped clips made of aluminum foil. The fiber was transferred to a temperature-controlled chamber (802 D, Aurora Scientific, Aurora, Ontario, Canada) where it was attached between a force transducer (403A, Aurora Scientific, Aurora, Ontario, Canada) and a length controller (322C, Aurora Scientific, Aurora, Ontario, Canada).
Before the start of each experiment, the average sarcomere length (SL) was measured in relaxing solution (100 mM KCl, 2 mM EGTA, 20 mM imidazole, 4 mM ATP, and 7 mM MgCl2, Sigma-Aldrich, San Luis, Missouri, USA) using a high-speed video system (HVSL, Aurora Scientific 901B, Aurora, Ontario, Canada). Images from a selected region of the fibers were used to calculate the SL by fast Fourier transform (FFT) analysis based on the striation spacing produced by dark and light bands of myosin and actin filaments. The fiber diameter and length were measured using a CCD camera (Go-3, QImaging; pixel size: 3.2 μm × 3.2 μm, Aurora Scientific, Aurora, Ontario, Canada), and the cross-sectional area was estimated assuming a circular geometry. Fibers were activated with different Ca 2+ concentration (pCa 4.5; 5.5; 6.0; 6.5; 9.0) (20 mM imidazole, 14.5 mM creatine phosphate, 7 mM EGTA, 4 mM MgATP, 1 mM free Mg 2+ , and free Ca 2+ ranging from 1 nM (pCa 2+ 9.0) to 32 M (pCa 2+ 4.5) (Sigma-Aldrich, San Luis, Missouri, USA) at an intitial SL of 2.5 μm. The active force produced during the experiments was measured after force stabilized during the contractions. Statistical analysis. All contractile data were compared among groups using a two-way analysis of variance (ANOVA) for repeated factors. When significant changes were observed, post hoc analyses were performed with Newman-Keuls tests. Data for the minimal Feret's diameter and the proportion of different type fibers were analyzed using mixed-effects linear models, and multiple comparisons were performed using diagonal contrasts. All statistical analyses were performed using the SAS software version 9.4, with the level of significance set at p < 0.05. The results are shown as means and standard error of the mean (SEM).