Introduction

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The absence of dystrophin is responsible for the life-threatening progressive skeletal muscle degeneration in Duchenne muscular dystrophy (DMD) (Hoffman and Dressman, 2001). No treatment is available; in fact, gene therapy is not feasible yet, and beneficial glucocorticoids have severe side effects (Hoffman and Dressman, 2001; Dubowitz, 2002). The identification of prompt-to-use drugs to ameliorate DMD is delayed by the partial understanding of the pathological cascade leading to death of dystrophin-deficient fibers (Hoffman and Dressman, 2001; Blake et al., 2002). Also, the mdx mouse, the most used model of DMD, has a limited usefulness for preclinical drug studies, because a successful muscle regeneration leads to a mild progression of the pathology (Hoffman and Dressman, 2001; Blake et al., 2002).

Dystrophin is a subsarcolemmal component of the dystrophin-glycoprotein complex linking the contractile machinery to the extracellular matrix and likely confers mechanical reinforcement to sarcolemma. Its absence may facilitate contraction-induced focal membrane damage with increases in cytosolic Ca²⁺ level and consequent activation of Ca²⁺-dependent proteases and necrosis (Alderton and Steinhardt, 2000; Blake et al., 2002). As a mark of altered calcium homeostasis, the excitation-contraction coupling mechanism of mdx muscle fibers is affected, the voltage for contraction being more negative versus that of control (De Luca et al., 2001). The mechanical role of dystrophin is supported by the susceptibility of dystrophin-deficient fibers to the workload the muscle withstand. The diaphragm of mdx mice, continuously active throughout life, progressively degenerates similarly to DMD muscles (Petrof et al., 1993; Dupont-Versteegden et al., 1994; Blake et al., 2002). Low-intensity swimming as well as immobilization of hind limb muscle seem to ameliorate muscle performance (Hayes and Williams, 1998; Mokhatarian et al., 1999), whereas forced treadmill running, especially with episodes of eccentric contractions, induces severe muscle damage with even fatal consequences (Sandri et al., 1997; Vilquin et al., 1998). The treadmill-exercised mdx mice have been used by Granchelli et al. (2000) for a large drug screening, considering drug effectiveness the ability to prevent the loss of forelimb muscle strength occurring after 4 to 5 weeks of exercise. However, the use of in vivo muscle strength as the sole endpoint to state both muscle function and drug effectiveness is rather elusive, because this parameter is also influenced by nervous reflexes and cognitive function. A cellular parameter useful to functionally assess the degeneration and regeneration events occurring in dystrophic fibers is the resting conductance to chloride ions (gCl), the parameter that controls the electrical stability of sarcolemma (Klocke et al., 1994; De Luca et al., 1997). gCl is significantly impaired in progressively degenerating mdx diaphragm (DIA), whereas in the hind limb extensor digitorum longus (EDL) muscle, gCl rises over the control values between the 8th and the 12th week of age, the period of mouse life in which the spontaneous regeneration is still ongoing (De Luca et al., 1997, 1999; McIntosh et al., 1998). The change in gCl may work as a fine-tuning of the mechanical-stress by modulating sarcolemma excitability (De Luca et al., 1997). In turn, gCl can be a sensitive mark of the dystrophic progression due to increased workload such as exercise. We presently used the exercised mdx mice for preclinical drug screening. Three- to four-week-old mdx animals were made running on a treadmill twice a week for 4 to 8 weeks and the effect of either exercise or exercise plus drug treatment, were evaluated both in vivo on forelimb strength and in vitro, at the end of the protocol, on gCl of EDL and DIA muscles, and on mechanical threshold (MT), the calcium-sensitive index of excitation-contraction (e-c) coupling (De Luca et al., 1996, 2001). The drugs used were creatine, taurine, and insulin-like growth factor-1 (IGF-1). Taurine and creatine levels, normally high in striated fibers, fluctuate in mdx muscle in relation to pathology progression (McIntosh et al., 1998). Creatine stimulates muscle metabolism in vivo, whereas either compounds seem to control calcium-handling mechanisms in vitro, with potential ability to counteract calcium-induced degeneration (Pulido et al., 1998; De Luca et al., 2001). IGF-1 stimulates muscle regeneration, a failing process in DMD patients (Hoffman and Dressman, 2001). In support of its therapeutic potential, we found higher levels of IGF-1 in hind limb muscle and plasma during regeneration period of mdx mouse, and recently transgenic mdx mice overexpressing IGF-1 showed an amelioration in force and muscle mass (De Luca et al., 1999; Barton et al., 2002). Steroids are beneficial in dystrophic patients (Anderson et al., 2000; Dubowitz, 2002); thus, a treatment with -methyl-prednisolone (PDN) was also used as positive control.

	Materials and Methods

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Animal Groups and Drug Treatment. Fifty-four mdx and 28 wild-type (C57/BL10) male mice 3 to 4 weeks old (IFFA Credo, Lyon, France; The Jackson Laboratories, Bar Harbor, Maine) were used. Initially, all the animals were weighed and forelimb force measured by means of grip strength meter (see below). Each group was then subdivided into two further groups: exercised and sedentary. For drug treatments, a total of 45 exercised mdx mice were used, subdivided into three groups as follows: group 1, five untreated, six creatine-treated, and six taurine-treated; group 2, five untreated and six PDN-treated; and group 3, five untreated, six treated with IGF-1 at 50 µg/kg, and six treated with IGF-1 at 500 µg/kg. No significant differences were found between the untreated exercised mdx mice of the three groups. For this reason, and for giving a global view of the effect of exercise between the two strains (control and mdx), the data regarding the untreated exercised mdx mice have been pooled from the three groups. However, to have a more strict statistical evaluation of the drug effectiveness, each group of drug-treated mice has been compared with its own related untreated mdx group. All treatments started a few days before the beginning of the exercise protocol, and continued until the day of sacrifice. Creatine and taurine were given every day with amino acid-enriched chow (10% in weight). The daily amount of food eaten by each animal ranged between 2 and 4 g. The chow enriched with PDN (0.00125% in weight) was given 6 days a week up to the daily amount for the dose of 1 mg/kg. Then, the mice received ad libitum the standard drug-free chow to avoid PDN overdose. Both standard and drug-enriched chow made under request were purchased from Eberle Nafag AG (Gossau, Switzerland). IGF-1 (recombinant human; Chiron, Emeryville, CA) at 50 and 500 µg/kg was administered s.c. and untreated mdx mice belonging to the same group received daily the same amount of solvent. The nine mdx mice, taken as sedentary, were left free to move in the cage, without additional exercise. The sedentary mice were monitored for in vivo and in vitro studies at the same time points of exercised counterparts, as needed.

Exercise Protocol and in Vivo Studies. The wild-type and mdx (both untreated and treated) mice belonging to the exercised groups underwent a 30-min run on a horizontal treadmill (Columbus Instruments, Columbus, OH) at 12 m/min, twice a week, for 4 to 8 weeks (Granchelli et al., 2000). The training protocol started at the mouse age of 3 to 4 weeks. About half of the mdx mice showed an avoidance behavior with respect to exercise, with a higher tendency to fatigue, and had to be gently stimulated, or left resting, to complete the 30-min running session. This behavior, never observed in wild-type animals, was not modified by either exercise or drugs. Every week all the exercised mice were monitored for body weight and compared with related sedentary counterparts. The force for exercised mice (both controls and mdx) was evaluated before each training section by means of a grip strength meter (Columbus Instruments). For this measurement, the mice were allowed to grasp a triangular ring connected to a force transducer and then gently pulled away until the grip was broken. The transducer saved the force value at this point, which was a measure of the maximal resistance the animal can use with its forelimbs. Five measurements were taken from each animal within 2 min, and the maximum values were used for statistical analysis. The sedentary mice, both wild type and mdx, were monitored for muscle strength every week. At the end of the 4th week of exercise, the electrophysiological in vitro experiments were started. The animals continued to be exercised until the day of sacrifice.

In Vitro Electrophysiological Studies. Electrophysiological studies were performed in vitro on sedentary control and mdx mice as well as on exercised control and untreated or treated mdx mice. The age of the animals at the time of experiment was 8 to 12 weeks. The animals were anesthetized with 1.2 g/kg urethane; EDL muscle of one hind limb and diaphragm was removed and rapidly placed in the recording chamber at 30 ± 1°C and superfused with normal and chloride-free physiological solutions. The normal physiological solution had the following composition: 148 mM NaCl, 4.5 mM KCl, 2.0 mM CaCl₂, 1.0 mM MgCl₂, 12.0 mM NaHCO₃, 0.44 mM NaH₂PO₄, and 5.55 mM glucose. The chloride-free solution was made by equimolar substitution of methyl sulfate salts for NaCl and KCl and nitrate salts for CaCl₂ and MgCl₂. The solutions were continuously gassed with 95% O₂ and 5% CO₂ (pH 7.2-7.4) (De Luca et al., 1997). For evaluation of in vitro effect of IGF-1 (Chiron) on the electrical parameter described below, the final concentration was obtained with adequate dilutions of microliter amounts of concentrated stock solutions with normal or chloride-free physiological solution according to the experimental need (De Luca et al., 1997, 1998).

Histology. Tibialis anterior muscle was rapidly rinsed in normal physiological solution and immediately frozen in isopentane cooled in liquid nitrogen. The samples were stored at 80°C until used for histological determination. Frozen muscle was cut into 10-mm-thick section with a cryostat taken from the midpoint of the muscle body and stained with hematoxylin-eosin. A semiquantitative approach has been used to evaluate histopathological indexes and to allow statistical analysis. In particular, within a comparable number of fibers per section taken for each condition, it has been calculated the number of cell showing necrosis, centronucleation, and/or belonging to inflammatory infiltrates.

Statistics. All data are expressed as the mean ± S.E.M. The S.E. estimate for gCl was obtained as described previously (De Luca et al., 1997, 1998). Statistical analysis for direct comparison between two groups of data means was performed by unpaired Student's t test, whereas multiple statistical comparison between groups (either strain/exercise or untreated/treated as independent factors) was performed by one-way ANOVA, with Bonferroni's t test post hoc correction for allowing a better evaluation of intra- and intergroup variability and avoiding false positive. The MT values are expressed as both the mean ± S.E.M. for the absolute values of voltage threshold at each pulse duration and fitted rheobase (R) and rate constant (1/) parameters ± S.E. These latter parameters were determined from the variance-covariance matrix in the nonlinear least-squares fitting algorithm (De Luca et al., 1996, 2001). For these fitted parameters, the statistical significance between groups was estimated by using the above-mentioned tests using these standard errors and a number of degrees of freedom equal to the total number of threshold values determining the curves minus the number of means minus two for the free parameters (De Luca et al., 1996, 2001).

	Results

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Effect of Exercise on mdx Mice

Effect of Exercise on Body Weight Gain and Forelimb Strength. At the beginning of the training protocol 3- to 4-week-old mdx mice were significantly lighter (16.4 ± 0.57 g; n = 24) than age-matched wild-type animals (18 ± 0.47 g; n = 23) (by Student's t test; p < 0.05). The two groups were subdivided each into two further groups, exercised and sedentary, of similar body weight. An age-dependent increase in body weight was observed with no significant differences between groups and strain. In fact, after 4 weeks of exercise protocol, the mean of body weight gain in exercised mdx mice was 4.93 ± 0.68 g (n = 15) versus 4.79 ± 0.79 g (n = 9) of sedentary ones. Similarly, the weight gain was 4.28 ± 0.47 g (n = 13) and 4.10 ± 0.48 g (n = 10) in exercised and sedentary wild-type mice, respectively. At the beginning of the training period the mdx mice were significantly weaker with respect to age-matched wild-type, the forelimb strength being 0.069 ± 0.004 kg (n = 24) and 0.084 ± 0.002 kg (n = 23), respectively. Figure 1 shows the effect of 4 weeks of exercise on the increment of forelimb strength in the two strains. A significant difference between groups was found by ANOVA test on either absolute increment (Fig. 1A; F = 5.85; p < 0.005) or on strength increment normalized for body weight (Fig. 1B; F = 3.66; p < 0.05). In fact, a similar, not significant, forelimb muscle strength increment was observed in both sedentary and exercised wild-type animals as well as in sedentary mdx mice, whereas the exercised mdx mice differed significantly from each of any other groups, showing a marked impairment of such a force increment (0.0005 < p < 0.029 by Bonferroni's t test).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effects of exercise and drug treatments on in vivo forelimb muscle strength of mdx mice. Each column is the mean ± S.E.M. from 5 to 15 animals. A, effect of exercise and drug treatment on the increment in forelimb strength calculated as the difference in force at the end of 4 weeks of exercise (time 4) with respect to the force measured at the beginning (time 0) of the training section. B, for each mouse, the force at time 0 and the force at time 4 have been normalized with respect to the relative body weights at related time points. The difference has been used for statistical calculation of the normalized force increment. For A and B, a statistical difference was found by ANOVA comparison between sedentary and exercised mice (see Effect of Exercise on Body Weight Gain and Forelimb Strength) as well as between untreated and treated groups (9.3 > F > 3.33; p < 0.05). Bonferroni's t test data are as follows: *, Significantly different with respect to both wild-type groups (both sedentary and exercised WT) and the group of sedentary mdx (Sed MDX) (0.0097 < p < 0.035); and **, significantly different with respect to untreated exercised mdx mice (Exer MDX) (0.000012 < p < 0.033). ANOVA between drug treated group was not significant (p > 0.05).

Effect of Exercise on Component Ionic Conductances on DIA and EDL Muscle Fibers. In agreement with previous observations (De Luca et al., 1997), the DIA muscle fibers of 8- to 12-week-old mdx animals were characterized by a significantly higher value of Rm with respect to age-matched wild types (Table 1). This high Rm value was due to the 30% decrease of gCl, the gK being almost unchanged. Four to 8 weeks of exercise produced a slight increase of Rm of mdx DIA fibers, due to a further, albeit not significant, 10% lowering of gCl, whereas gK showed no significant increase. Interestingly, the exercise produced similar effects on DIA fibers of wild-type animals, significantly decreasing gCl (Table 1), suggesting a particular susceptibility of DIA to the increase of workload.

Effect of Exercise on Mechanical Threshold of EDL Muscle Fibers. EDL muscle fibers of mdx mice, in spite of the active regeneration, maintains an alteration in e-c coupling mechanism, likely in relation to the proposed increase in cytosolic calcium levels, the MT being typically shifted toward more negative potentials irrespective of mouse age (De Luca et al., 2001). This alteration of MT was also observed in the present study; in fact, at any pulse duration, the voltage threshold for contraction was significantly more negative in the mdx versus wild-type animals (Table 2), with rheobase values of 72.2 ± 0.8 and 66.4 ± 0.4 mV, respectively (p < 0.001; Student's t test). The mdx EDL muscle fibers also showed a significantly smaller rate constant (1/) with respect to wild type (0.114 ± 0.006 versus 0.143 ± 0.006 s¹; p < 0.001; Student's t test), accounting for a longer time constant to reach the rheobase. The chronic exercise produced significant changes when multiple comparison between groups was performed at level of both voltage threshold values (Table 2) and fitted rheobase voltages (F = 13.6; p < 0.0001). Exercise produced a further shift of MT of mdx EDL muscle toward more negative potentials, the fitted parameters being 73 ± 0.7 mV for the rheobase and 0.110 ± 0.008 s¹ for 1/. Interestingly, the exercise caused a significant 4.5 mV shift of the MT of wild-type muscle fibers toward more negative potentials (70.3 ± 0.7 mV), along with a small reduction of 1/ (0.123 ± 0.008 s¹), suggesting that e-c coupling mechanism of normal muscle fibers is sensitive to the increase in muscle workload (Table 2). However, from a statistical point of view, the voltage threshold parameters of exercised wild-type muscles did not reach the severity of mdx ones. Also, preliminary histological evaluation allowed to exclude any damaging effect of exercise in hind limb muscles of wild-type animals (see below).

View this table: [in this window] [in a new window]   TABLE 2 Effect of exercise on the voltage threshold for contraction of extensor digitorum longus muscle fibers of wild-type and dystrophic mdx mouse The columns from left to right are as follows. Experimental conditions, the fibers sampled are from extensor digitorum longus muscle from sedentary and exercised wild-type and mdx mice of 8 to 12 weeks of age. For each experimental condition are shown the threshold membrane potential values obtained with depolarizing command pulse of duration ranging from 5 up to 500 ms. The values are expressed as mean ± S.E.M. from the number of fibers shown in parantheses below each value from 5 to 10 preparations. The ANOVA multiple comparison showed high significant differences between voltage threshold values at each pulse duration (81.7 > F > 11.5; p < 6.3 × 101).

Effect of Exercise on Histopathology. To ascertain the effect of exercise on pathology progression of dystrophin-deficient muscle, tibialis anterior (TA) muscle, removed after the 4 to 8 weeks of exercise protocol from both wild-type and mdx animals was analyzed for histopathological signs in comparison with sedentary counterparts. Figure 2 shows representative sections for each group, along with the main differences found in the muscle histology as a consequence of exercise. The analysis was performed by evaluating morphological differences over comparable number of fibers per section (NF/S) for each experimental condition. In wild-type muscle, the exercise only produced an increase in number of mitochondria, without substantial morphological changes (NF/S: 649.3 ± 68, n = 3 sedentary and 690 ± 124, n = 3). In line with classical description of dystrophin-less muscle (Blake et al., 2002), TA muscles of mdx mice were histologically distinguishable from wild type for the more disorganized structure due to the presence of necrosis, centronucleated fibers, regenerating fibers, and infiltration of mononuclear inflammatory cells. Over 622.8 ± 37 (n = 5) and 680.3 ± 52 (n = 5) NF/S for sedentary and exercised mdx muscles, it was possible to verify that the histopathological signs of mdx TA muscles were worsened by exercise. This was particularly evident for the number of necrotic fibers (F = 46.1; p < 1 × 10⁵) and for the number of infiltrates (F = 68.3; p < 1 × 10⁶), both clear indexes of increased muscle damage and inflammatory reaction in the dystrophic muscle as a consequence of the increased contractile stress. Other parameters were also present but not significantly changed by exercise.

View larger version (86K): [in this window] [in a new window]   Fig. 2.   Effect of exercise on the histological characteristics of tibialis anterior muscle of wild-type and mdx mice. Top, representative sections of tibialis anterior muscles from sedentary (A) and exercised (B) wild-type mice and from sedentary (C) and exercised (D) mdx mice stained with standard hematoxylin-eosin. A disorganization of muscle morphology is evident in both sedentary and exercised dystrophic muscles with clear sign of fibrosis and necrosis and infiltration of inflammatory cells, in contrast with the more uniform structure of wild-type muscles. Bottom, quantification of the number of necrotic and infiltrates cells in each experimental condition. This evaluation has been performed over a comparable number of fibers per section (see text) and showed significant differences by ANOVA between the four groups. Significant difference by Bonferroni's t test with respect to wild-type groups (*) and sedentary mdx mice (**).

Effect of Drug Treatments on Exercised mdx Mice

Effect of Drugs on Body Weight and Forelimb Strength. Taurine treatment almost doubled the body weight gain of exercised mdx mice, whereas all other drugs were ineffective on this parameter. After 4 weeks of treatment, all the compounds were significantly effective in fully counteracting the deleterious effect of exercise on the absolute force increment (Fig. 1A; 9.26 >F > 3.73; p < 0.02). Surprisingly, taurine produced an increase in muscle strength larger than that observed in both sedentary mdx mice and wild-type animals, followed by creatine that also was highly effective. To rule out any possible effect of body weight in the drug-induced increase in strength, for each mouse we normalized the forelimb strength to body weight at the beginning (time 0) and at the end of 4 weeks of exercise (time 4) and we considered the difference as normalized force increment (Fig. 1B). Also by this approach taurine and creatine were similarly highly effective, followed by IGF-1, which was effective in a dose-dependent manner (7.43 > F > 3.33; p < 0.03). Surprisingly, PDN was the less beneficial compound, producing a nonsignificant effect on normalized strength increment (F = 2.62; p = 0.066). Control exercised animals treated with 50 mg/kg IGF-1 showed increments in forelimb muscle strength totally overlapping that of untreated counterpart, allowing to rule out any aspecific anabolic effect as the basis of IGF-1 effectiveness in exercised mdx animals. In fact, the absolute increment of strength in exercised wild types that were IGF-1-treated was 0.040 ± 0.009 kg (n = 5), whereas the normalized increment strength/body weight value was 0.7 ± 0.3.

Effect of Drug Treatments on Component Ionic Conductances of Exercised mdx Mice. The effects of the various drug treatments on gCl and gK of EDL and DIA muscle fibers are illustrated in Figs. 3 and 4. A different effect of the various drugs was observed in the two muscle types. In fact, in the taurine-treated group the value of Rm of EDL muscle fibers was 396 ± 17  · cm² (n = 6/56), which almost overlapped that typically recorded in the sedentary mdx mice. The ANOVA test between untreated wild-type and mdx and taurine-treated mice was significant (F = 11.9; p < 1 × 10⁶). This was due to the ability of taurine to maintain gCl at the high level typical of regenerating EDL muscle of sedentary mdx mice (Fig. 3). Also the gK value in taurine-treated mice was lower than that of untreated exercised mdx EDL muscle fibers. In contrast, in DIA muscle, the taurine treatment did not significantly change the Rm value that was 573 ± 35  · cm² (n = 6/33), with respect to untreated mdx counterparts, being only able to prevent the 10% decrease of gCl due to exercise (Fig. 3). The treatment with creatine was not effective, being unable to significantly ameliorate Rm, gCl, and gK in both muscle types (Fig. 3).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Effects of taurine and creatine treatments on gCl of EDL and DIA muscle fibers of exercised mdx mice. Each column is the mean ± S.E.M. from 20 to 88 fibers from 5 to 10 preparations. ANOVA was 22.8 > F > 2.85 (p < 0.05) for taurine and 22.3 > F > 2.8 (p < 0.05) for creatine. Bonferroni's t test is as follows: Significantly different (p < 0.05) with respect to wild-type (WT) (a), sedentary mdx (Sed mdx) (b), and untreated exercised mdx mice (Exer MDX) (c).

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Effects of IGF-1 and PND (Prednisolone) treatments on gCl and gK of EDL and DIA muscle fibers of exercised mdx mice. The effect of IGF-1 treatment has been evaluated at two close to therapeutic doses, 50 µg/kg (IGF-1 50) and 500 µg/kg (IGF-1 500). Each column is the mean ± S.E.M. from 22 to 88 fibers from 5 to 10 preparations. ANOVA test results as follows: 32.1 > F > 25 (p < 1 × 106) for gCl of IGF-1 treated EDL and DIA, whereas gK was not significant. F = 30.2 (EDL muscle) and 27.3 (DIA) (p < 1 × 106) for gCl of PDN-treated group, whereas gK was not significant. Bonferroni's t test is as follows: significantly different (p < 0.05) with respect to wild-type (WT) (a), sedentary mdx (Sed mdx) (b), and untreated exercised MDX mice (Exer MDX) (c).

Specificity of IGF-1 Effects for Dystrophic Muscle. To evaluate the specificity of observed drug effect for dystrophic muscle, we performed a series of experiments with the highly effective IGF-1. First, we evaluated the sensitivity of gCl of EDL and DIA muscles of both untreated and IGF-1-treated exercised mdx mice to the in vitro application of IGF-1. In fact, previous studies showed the ability of IGF-1 to modulate the function of muscle chloride channel through both acute and long-term mechanisms (De Luca et al., 1998, 1999).

Effect of Drug Treatments on Mechanical Threshold of EDL Muscle Fibers of Exercised mdx Mice. As shown in Fig. 5, taurine treatment had a significant effect on MT of exercised EDL mdx muscle fibers, shifting the strength-duration curve toward the more positive potentials typical of sedentary wild-type muscles. In fact, the rheobase value was 67.9 ± 0.6 mV, about 5 mV more positive than the value recorded in both sedentary and exercised mdx EDL muscle and very close to the value recorded in sedentary EDL fibers. The taurine treatment was also effective on 1/ changing it from 0.11 ± 0.01 to 0.13 ± 0.007 s¹, a value similar to that recorded in sedentary wild type. These effects were very similar to those previously observed after in vitro application of taurine on mdx EDL muscle (De Luca et al., 2001). Creatine had no effect. IGF-1 had a little but significant dose effect at 50 and 500 µg/kg, being the curve shifted toward more positive potentials by 2 and 4 mV, respectively, with respect to the related untreated control group of mdx mice, whose rheobase voltage was 74.1 ± 0.4 mV. On the other hand, PDN treatment was almost as effective as taurine, bringing the rheobase voltage to 69 ± 0.5 mV and 1/ to 0.14 ± 0.007 s¹.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Effect of drug treatments on the strength-duration curves of mechanical threshold of EDL muscle fibers of exercised mdx mice. In each panel the experimental data, expressed as the mean ± S.E.M. from 10 to 40 values from 5 to 8 preparations, have been fitted to the equation under Materials and Methods to obtain the strength-duration curves and the fitted parameters of rheobase and 1/, described in the text. A, total lack of effect of creatine treatment, with respect to the high effectiveness of taurine. B, dose-dependent slight amelioration of MT brought about by IGF-1. For comparison, C shows the effect of PDN. In each panel, the effect of drug treatment has been compared with the strength duration curve of untreated exercised mdx mice belonging to the same group. For some data point the standard error bar is not visible being smaller than symbol size. The Bonferroni's post hoc test after ANOVA was as follows. A, taurine-treated fibers had threshold values not significantly different with respect to wild type, whereas they were significantly different with respect to both exercised mdx and creatine-treated (0.00001 < p < 0.05). Creatine treatment was not significantly different with respect to exercised mdx fibers. B, muscle fibers from exercised mdx mice treated with both 50 and 500 µg/kg had threshold values significantly different with respect to those of both wild-type and exercised mdx mice (p < 0.02), whereas they were not significantly different with respect to each others. C, threshold values from PDN-treated fibers were significantly different with respect to both wild-type and exercised untreated mdx mice (p < 0.001).

	Discussion

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In the present study, we found various compounds that were able to counteract, with different potency and specificity, the in vivo and in vitro signs of the deleterious effect of exercise in mdx mouse, allowing to envisage new drugs for the therapy of DMD.

Apart from the impairment of muscle strength in vivo described previously (Granchelli et al., 2000), the novel finding was that chronic exercise could selectively overwhelm the effect of regeneration in hind limb muscle. Other than by histological analysis, this was detected by the state of gCl, a sensitive index of degeneration and regeneration events occurring in different muscle types of mdx phenotype as well as in other pathophysiological conditions of skeletal muscle (De Luca et al., 1997, 1998). Chronic exercise seriously counteracted the regeneration-induced increase in gCl observed in mdx EDL muscle fibers, whereas it slightly affected the already low gCl typical of degenerating DIA (De Luca et al., 1997, 1999). The drugs used were all effective in preserving muscle strength in vivo, but had different effects on gCl in the two muscle types, suggesting that the lowering of this parameter is triggered by diverse molecular mechanisms of the pathological cascade. A long-term mechanism can ensue in DIA muscle for the continuous mechanical stress caused by the respiratory activity. For an adaptive phenomenon, in mdx DIA an age-dependent higher expression of fatigue-resistant fiber I and IIa versus the fast-glycolytic IIb does occur (Petrof et al., 1993). The phenotype transition can account for the decrease in gCl, because slow fiber types are characterized by lower expression of ClC-1, the muscle chloride channel, and, consequently, by lower gCl values (Klocke et al., 1994; Pierno et al., 2002). In this condition, only drugs such as PDN or IGF-1, able to stimulate regeneration and to act on myogenic programs activating specific transcription factors, may have positive functional effects on gCl by restoring channel expression (De Luca et al., 1999; Semsarian et al., 1999; Anderson et al., 2000). Recent findings support the usefulness of IGF-1 as a long-term therapeutic countermeasure. In fact, muscle-specific overexpression of IGF-1 in mdx mouse significantly enhanced muscle strength, muscle mass, and other signaling pathways associated with muscle regeneration, significantly decreasing fibrosis and necrosis (Barton et al., 2002).

In parallel, an acute effect of exercise can blunt regeneration and account for the marked decrease in gCl in the EDL muscle and for the further lowering of gCl in DIA. The short-term decrease in gCl can represent an early event that, through an increase of membrane excitability (De Luca et al., 1997), contributes to the establishment of a chronic state leading to either necrosis and apoptosis or phenotype transition. In support of this view, in myotonic mouse muscles, in which membrane hyperexcitability is triggered by a loss-of-function mutation of ClC-1 channel, a fast-to-slow transition results from activity and calcium-dependent activation of muscle-specific transcription factors, such as MEF-2 (Wu and Olson, 2002). Interestingly, these pathways, also involved in regeneration and/or apoptosis, are activated by exercise in heart of mdx animals (Nakamura et al., 2002). Thus, the understanding of the molecular mechanisms underlying the acute drop of gCl can help the identification of early therapies. A possible starting disease-related event disclosed by exercise is a functional ischemia, already demonstrated in contracting mdx and DMD muscles, due to a reduced activity of nitric-oxide synthase and nitric oxide production, for the disassembling of the dystrophin-glycoprotein complex (Sander et al., 2000; Hoffman and Dressman, 2001; Blake et al., 2002). The ischemia, along with the increased calcium, can trigger an inflammatory reaction; this latter seems a secondary mechanism playing a key role in the pathogenesis of muscular dystrophy (Porreca et al., 1999; Spencer et al., 2000; Hoffman and Dressman, 2001), and a recent DNA microarray study described a significant overexpression of cytokines, chemokines, and relative receptors in mdx muscle (Porter et al., 2002). The consequent switch-on of inflammation-related transducing signals can acutely modulate various cellular targets, among which muscle chloride channels.

According to the idea of short-term modulation of channel function, the impairment of gCl in untreated exercised EDL muscle was acutely counteracted by in vitro application of IGF-1, an effect that could not be detected in muscles from IGF-1-treated animals. This result, along with the lack of any effect of in vivo IGF-1 treatment in control animals, also suggests that the benefit of IGF-1 is addressed, through both short- and long-term actions, to specific steps of the pathological cascade (Pons and Torres-Aleman, 2000). Accordingly, part of the beneficial effect of PDN treatment can also be attributable to its well known anti-inflammatory activity. The effectiveness of taurine on gCl of exercised mdx EDL muscle may be related to its antioxidant activity able to protect from ischemic injury; however, recent data support a specific anti-inflammatory action of the amino acid, mediated by both the reduction of cytokine production and the inhibition of cytokine-dependent activation of nuclear factor-B (Kontny et al., 1999; Kanayama et al., 2002).

The positive effect of drugs could also be linked to a Ca²⁺-related mechanism and evaluated on MT. In line with evidence from other laboratories (for review, see Alderton and Steinhardt, 2000; Blake et al., 2002), our preliminary results with Fura-2 imaging are in favor of higher cytosolic Ca²⁺ level also in adult native muscle fibers of mdx mice (data not shown), and this correlates with the negative rheobase voltage of mdx EDL muscle (De Luca et al., 2001). The modification of MT by exercise cannot be taken by itself as an index of worsening of dystrophic condition, because the function of sarcoplasmic reticulum easily adapts in response to exercise and cytosolic calcium (Booth and Thomason, 1991). In fact, a remarkable negative shift of MT was observed in exercised wild-type mice, in the absence of any sign of degeneration by histological analysis, whereas a minor effect of exercise on MT occurs in mdx muscle, likely in relation to adaptive changes to functionally buffer the excess of cytosolic Ca²⁺. However, the negative MT is a typical feature of dystrophin-deficient muscle fibers and its modulation by drugs can provide additional information about drug efficacy and mechanism of action. The taurine treatment restored MT of exercised dystrophic muscle, in line its high effectiveness when applied in vitro and likely in relation to a direct stimulation of the Ca²⁺ ATPase pump of sarcoplasmic reticulum (Huxtable, 1992; De Luca et al., 2001). Conditions of muscle taurine depletion are in fact characterized by negative MT and a difficulty of dystrophic muscle to retain adequate amounts of the amino acid has been documented and can be even worsened by exercise (De Luca et al., 1996, 2001; McIntosh et al., 1998; Matsuzaki et al., 2002). Similarly, PDN was very effective on MT, corresponding to its ability in vitro to decrease cytosolic Ca²⁺ levels of dystrophic myotubes (Passaquin et al., 1998). Interestingly, IGF-1, whose cellular action seems rather to involve an increase in Ca²⁺ mobilization (Semsarian et al., 1999), was not as effective as taurine and PDN on MT.

Surprisingly, creatine was ineffective on both gCl of DIA and EDL muscle fibers as well as on MT, in contrast with its high efficacy on muscle force in vivo. Creatine, being converted into phosphocreatine, is widely used as reservoir for quick energy source and clinical trials are currently ongoing in DMD patients and in other neuromuscular disorders (Klivenyi et al., 1999; Tarnopolsky and Beal, 2001). Even by assuming that its positive effect on muscle strength is mediated by the amelioration of nervous system function (Klivenyi et al., 1999), other discrepancies are still present. In vitro creatine supplementation to mdx myotubes counteracts the increase in cytosolic calcium by stimulating the activity of calcium ATPase pump due to the larger availability of ATP (Pulido et al., 1998). Thus, we should have observed an effect of creatine on MT. A possible explanation is that the mice had an overdose of creatine with the drug-enriched food, because creatine toxicity seems to involve muscle and cardiac tissue (Klivenyi et al., 1999; Tarnopolsky and Beal, 2001). In fact, Passaquin et al. (2002), who recently described the ability of creatine to counteract the first degeneration cycle occurring postnatally in mdx mice, performed the creatine feeding to the mothers, suggestive of a much lower amount of drug taken up by pups with the milk.

In conclusion, we found a wide effectiveness of IGF-1, both on the progressive dystrophic degeneration, likely due to its ability to stimulate muscle regeneration, and on the exercise-induced damage, through an acute mechanism that deserves to be better investigated. Also, the results showed the efficacy of taurine, a natural component of skeletal muscle, already used as food supplement and almost free of side effects (Huxtable, 1992), in contrast with the deleterious effect of exercise. In light of this finding, we propose that taurine could be rapidly considered for clinical trials in DMD alone or in combination with other drugs.