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Clinical Science (1995) 89, 447-452 (printed in Great Britain) 447 Effects of treadmill exercise and high-fat feeding on muscle degeneration in mdx mice at the time of weaning Asghar MOKHTARIANI, Jean Pascal LEFAUCHEUR2, Patrick C. EVEN' and Alain SEBILLP 'Laboratoire de Neurobiologie des Regulations, CNRS URA 1860, College de France, and Laboratoire de Physiologie, DRED EA 278, Faculte de Medecine Saint-Antoine, Paris, France (Received 31 October 1994/15 May 1995; accepted I June 1995) 1. Dystrophin-deficient hindlimb muscles of mdx mice undergo necrosis at the time of weaning when the motor activity of the mice greatly increases and muscle energy metabolism becomes more dependent on insulin and carbohydrates. 2. We have attempted to determine if the onset of myofibre necrosis in mdx mice at the time of weaning is related to the development of motor activity and/or the change in diet. 3. Fourteen-day-old mdx mice were divided into two groups after weaning. One group was trained to run on a treadmill and the other group was kept on a high-fat diet. Muscle necrosis was assessed histologically in the soleus and extensor digitorum longus muscles of mice in both experiments. 4. Keeping mice on a high-fat milk diet from the time of weaning up to 42 days of age did not influence the occurrence of necrosis in the soleus and extensor digitorum longus muscles of the mdx pups. In contrast, treadmill exercise greatly increased necrosis in both muscles. 5. We conclude that an increase in motor activity exacerbates the degeneration of hindlimb muscles of mdx mice at the time of weaning. INTRODUCTION The mdx mouse, a mutant of the C57BL/10 strain [1], presents a genetic deficiency of dystrophin [2], like patients with Duchenne muscular dystrophy (DMD) [3, 4]. As a result of this lack of dystrophin, a large subsarcolemmal protein of muscle cells [5, 6], mdx muscles undergo necrosis. In contrast to DMD muscle, in which an extensive fibrosis develops early, necrosis in mdx muscles is followed by a successful regeneration [7-10]. The onset of myofibre necrosis occurs in the hindlimb muscles of mdx mice at approximately 3 weeks of age [7, 10, 11], i.e. at the time during which two simultaneous events take place: (1) the development of exploratory behaviour that greatly increases motor activity, and therefore mechanical as well as metabolic stress to skeletal muscles; (2) a weaning-induced switch from a high-fat (milk) to a low-fat (cube diet) food regimen, associated with the progressive dependence of muscle metabolism on glucose and insulin [12]. Dystrophin is thought to stabilize myofibre membranes [13, 14], and may minimize the damage induced by contractile activity in normal muscles. The increased sarcolemmal permeability of dystrophindeficient muscle [15] could lead to an excessive influx of calcium [16], and thus activation of various proteases [17] that contribute to myofibre degeneration. Consequently, mdx muscle may suffer from an increased susceptibility to exercise-induced injury [18-20], even if the basis for this hypothesis is still not well established [21, 22]. On the other hand, the intracellular free calcium accumulation could also be due to alterations in intracellular organization of enzyme complexes [23, 24], thus affecting the control of mitochondrial function and energy metabolism, and resulting in a reduction in active ion transport. This hypothesis is supported by a reduced metabolic rate observed in myofibres of adult mdx mice [25], a high glycogen concentration [26, 27] and a decreased oxidation of glucose, free fatty acids and various intermediates of the tricarboxylic acid cycle [23]. In this study, we have attempted to determine if the onset of myofibre necrosis in mdx mice at the time of weaning is related to the enhancement of motor activity or to the switch from a high-fat (milk) to a low-fat (cube diet) food regimen. METHODS Animals Mdx mice from the inbred colony of the Faculte de Medecine Saint-Antoine (Paris, France) were housed in large plastic cages (20cm x 27 em) in a room kept at constant temperature (21°C) with a natural night-day light cycle. They were fed a commercial cube diet (A03 DAR) and water was available ad libitum, except when indicated. Control Key words: diet, dystrophin, eccentric contraction, mdx, myofibre type, necrosis, regeneration. .. . . Abbreviations: OMO, Ouchenne muscular dystrophy; EOL, extensor digitorum longus; SOL, soleus; TA, tlblalls anterior. Correspondence: Dr Asghar Mokhtarian, Laboratoire de Neurobiologie des Regulations, CNRS URA 1860, College de France, II place M. Berthelot, 75231 Paris cedex OS, France. 448 A. Mokhtarian et al. Table I. Surface area (mean ±SEM) occupied by necrotic and regenerating myofibres, expressed as a percentage of whole crosssectional area, in soleus (SOL) and extensor digitorum longus (EDL) muscles of 25-day~ld sedentaryand exercised mdx miceand age-matched C57BL/IO mice. Significant difference between sedentary and exercised mdx groups: ***p < 0.0002. mice were from an inbred colony of the wild strain C57BL/IO. Treadmill running The treadmill runnmg experiments were conducted on three groups of 14-day-old mice: (1) a group of four sedentary mdx mice placed on a motionless treadmill for 30 min every day, (2) a group of four exercised mdx mice and (3) a group of four exercised C57BL/1O mice. The exercised mice ran for 30 min every day (11.00 to 11.30 hours) for 7 days [28] on an uphill home-made motorized treadmill (speed 4 m/min, slope 15/~). The pups were always handled with disposable gloves, and the separation was strictly limited to the duration of the exercise to prevent dismissal of the pups by the parents. During the first 2 days, the pups were encouraged to run by gently pushing them with the finger. Thereafter, all the pups ran spontaneously. Electric shocks were never used. SOL EDL Sedentary mdx Exercised mdx Exercised CS7BL/10 20.8±ll 4.1 ± 1.0 67.5 ± 6.4'** 28.4 ± 2.8*** 0.6±0.2 0.3±0.1 The high-fat diet consisted of 'first-age' human synthetic milk (Enfalac, Mead-Johnson). The highfat feeding experiment was paired with a control experiment and conducted on two litters of four mdx pups. The first litter was given, up to 42 days of age, only human synthetic milk [weight (g) per 100g: carbohydrates, 57.5; lipids, 30.4; proteins, 12.1]. The second litter was given for the same time period the standard low-fat laboratory cube diet [weight (g) per 100 g: carbohydrates, 63.6; lipids, 6.4; proteins, 30]. independent observer, using manual planimetry. The difference between these two measurements gave the surface occupied by necrotic and regenerating myofibres and was expressed as a percentage of whole cross-section area. To determine the prevalence of necrosis in a specific type of myofibre, cross-sections were also processed for myosin-A'TPase activity [30]. The pH of the preincubation medium was either 4.53 or 10.40, and all final incubations with ATP were performed at pH 9.4 [31]. The number of type I-lIB, IIA and IIC myofibres in both EDL muscles of each mouse was assessed after preincubation at pH 4.53; type I (slow oxidative) and lIB (fast glycolytic) myofibres remained unstained, type IIA (fast oxidative and glycolytic) myofibres were darkly stained and type IIC (intermediate) myofibres exhibited distinct pale staining. The number of type I and II myofibres in both SOL muscles of each mouse was assessed after preincubation at pH 10.40; type I myofibres remained unstained, and type IIA and lIB myofibres were darkly stained. The data are reported as means ± SEM. Statistical differences were assessed using the Mann-Whitney test. Histological studies RESULTS The soleus (SOL) and the extensor digitorum longus (EDL) muscles were removed bilaterally under 3.5% chloral hydrate anaesthesia (0.35 ml per animal intraperitoneally). In the high-fat feeding experiments the muscles were removed when the pups were 42 days of age. In the treadmill experiments they were removed when the mice were 25 days old, i.e. 4 days after the end of exercise, in order to allow for optimal exercise-induced myofibre necrosis [29]. The excised muscles were mounted in a piece of cork with tragacanth gum, and frozen in isopentane chilled by liquid nitrogen. Muscles were cut on a cryostat all along their length. To ensure the greatest cross-sectional area, quantification was performed on whole-muscle cross-sections (10 /lm thick) taken from the mid-point of the muscle body [18]. The whole cross-sectional area and the total surface area occupied by surviving normal myofibres (identified by the presence of peripheral nuclei) were measured on microphotographs of haernatoxylin-eosin-stained cross-sections by an Effect of treadmill running High-fat diet Necrotic and regenerating myofibres were very rare in SOL and EDL muscles of 25-day-old exercised control (C57BL/IO) pups (Table 1). On the other hand, areas of necrotic and regenerating myofibres were found in SOL and to a lesser extent in EDL muscles of 25-day-old sedentary mdx pups (Table 1). Treadmill running enhanced mdx muscle necrosis and resulted in a significant extension of regenerating areas (P < 0.0002) in both SOLand EDL muscles of 25-day-old exercised mdx mice (Table 1). This extension may have been minimized by the presence of early regenerating myofibres (basophilic fibres with central nuclei), which are small in diameter and were particularly observed in foci of degeneration in exercised mdx EDL muscles. Histological features in muscles of sedentary and exercised mdx mice are presented in Fig. 1. Foci of inflammatory cells were seen in SOL but not in EDL muscles. We saw no localization in muscle damage along the length of exercised muscles (Fig. Exercise and high-fat feeding in mdx mice 449 Fig. I. CrosHection of soleus (SOL: a, b) and extensor digitorum longus (EDL: c, tI) muscles in 25-day-old sedentary (a, c) and treadmill-exercised (b, tI) mdx mice. Haematoxylin-eosin staining ( x 250). 2), therefore, as others [18], we analysed a single section taken from the mid-point of each muscle. All types of muscle fibres seemed to be identically affected by necrosis after treadmill running as characterized by ATPase reactions (Fig. 3). Although the ratio of type I to type II fibres in SOL muscles after pH lOA preincubation was decreased in exercised mdx pups (- 25°0)' this difference did not reach significance (Table 2). In EDL muscles, the ratio of type IIA to type lIB fibres following pH 4.53 preincubation was not significantly modified (Table 2). Type IIC fibres were also discernible in EDL muscles owing to their pale staining. In sedentary mdx muscles, these fibres exhibited peripheral nuclei and were probably immature, growing post-natal myofibres. After treadmill running, type IIC fibres exhibited central nuclei and were regenerated myofibres. In both groups, their ratio to the total number of myofibres was similar (0.37 ±0.03 in exercised mice versus 0.35 ± 0.03 in sedentary mice). Effect of high-fat feeding Maintenance of mdx pups on a high-fat milk diet from weaning up to 42 days of age did not influence muscle weight gain or the occurrence of necrosis in SOL and EDL muscles of the pups. The extent of necrotic and regenerating areas was similar (high-fat diet, 43.2%; cube diet, 42.7%, whatever the diet. III both muscles) DISCUSSION In this study, the onset of mdx muscle necrosis was not modified by keeping mdx pups on a highfat diet after weaning. In contrast, muscle necrosis was enhanced in mdx mice by treadmill exercise at the time of weaning. In mdx mice, hindlimb muscle necrosis occurs at the time of weaning, i.e. at 3 weeks of age. By this time, the pups exhibit an increase in motor activity (e.g. exploratory behaviour, seeking food) and a switch from a high-fat (maternal milk) to a low-fat (cube diet) food regimen. Weaning therefore increases muscle dependence on carbohydrate, and exercise increases cell energy requirements. Both processes could result in a metabolic stress for mdx muscle cells, which are known to suffer from a defective energy metabolism [25]. Muscle necrosis could also result from an increased influx of calcium into mdx myofibre sarcoplasm, due to either an altered ion channel function [32, 33] or membrane breakings resulting from the absence of dystrophin [15, 17]. The lack of beneficial effects of a food regimen of higher fat content than the standard cube diet (30% versus 6%) suggests that a defective adaptation of muscle energy metabolism to the low- 450 A. Mokhtarian et al. Fig. 2. Cross-section taken in the middle part of a soleus muscle in 25-day-old treadmill-exercised mdx mice. Distance between each section = 200/1 m. Total distance between sections (a) to (e) = BOO 11m. Haematoxylin-eosin staining (x 100). fat cube diet is not the leading cause of muscle cell necrosis at the time of weaning. However. the human synthetic milk used in this study contained the same proportion of carbohydrate as the standard cube diet. To determine then the exact role played by the insulin-dependent metabolic processes at the time of weaning, further experiments using diets of higher fat content and lower carbohydrate content are planned. The paradigm of mdx muscle susceptibility to exercise-induced damage only results from studies on adult mdx regenerated muscles. Most studies on isolated adult muscles in a recording chamber have reported a susceptibility to eccentric activity of mdx muscles. An increased percentage of damaged myo- fibres resulting from eccentric, isometric contractions, or passive lengthenings, was shown in diaphragm and EDL muscles of 90- to l l O-day-old mdx mice [14]. Furthermore, an increased force drop was induced by contractions with stretch in EDL and SOL muscles of mdx mice of unknown age [20]. However, the maximal tetanic force after lengthening contractions during tetanic contraction was altered in EDL but not in SOL muscles of 180to 400-day-old mdx mice [34]. The effects of increased motor activity in mdx mice are more controversial. Long-term overload by removing the synergist tibialis anterior (T A) led to a progressive EDL weakness in 60- to 240-day-old mdx mice [19]. Susceptibility to necrosis was increased in the T A Exercise and high-fat feeding in mdx mice 451 Fig. 3. Cross-section of soleus (SOL: a, b) and extensor digitorum longus (EDL: c, d) muscles in 25-day-old sedentary (a, b) and treadmill-exercised (b, d) mdx mice. ATPase activity staining following preincubation at pH 10.40 (a, b) and at pH 4.53 (c, d) ( x 250). Table 2. Ratios (mean± SEM) of type I to type II myofibres in soleus (SOL) muscle and of type IIA to type liB myofibres in extensor digitorum longus (EDL) muscles of 25-day-old sedentary and exercised mdx mice. Statistical comparisons between groups are not significant. SOL EDL Sedentary mdx Exercised mdx 0,34±0.08 1.72±0.23 0.25 ±0.05 1.61±O.16 muscle following lengthening (eccentric) contractions induced by stimulation of the sciatic nerve in 100day-old mdx mice [18]. However, force loss in the TA muscle after lengthening contractions induced by stimulation of the peroneal nerve was similar in 110- to 180-day-old mdx mice and age-matched C57BL/10 mice [21]. In addition, endurance swimming has been claimed to have beneficial effects on the contractile properties of EDL and SOL muscles in 35- to 140-day-old mdx mice [22]. Thus, there is some discrepancy regarding the effects of exercise on mdx muscle, even concerning eccentric activity [18, 21], but our study was in some respects different from these previous studies. First, we assessed the extent of myofibre necrosis in young mdx mice at the first round of muscle degeneration, not in adult mdx mice in which muscle regeneration had already occurred. Second, our exercise protocol did not investigate the effects of a specific type of contraction (eccentric or concentric, as with electrical stimulation procedures [18,21]), and is different from non-weight-bearing endurance training with few eccentric contractions, such as swimming [22]. To our knowledge, unweaned 15-day-old mdx pups have never been exercised using an uphill treadmill. Since no significant necrosis was found in exercised C57BL/1O pups, our exercise protocol was adapted to young animals which run spontaneously without collapsing. Compared with sedentary mdx muscles, treadmill running increased the mean surface area occupied by necrotic (SOL) or regenerating (EDL) myofibres. The prejudicial effect of exercise mimics or exacerbates the spontaneous degeneration, which is more evolved in SOL muscles than in EDL muscles in sedentary young mdx mice. Muscle regeneration was allowed to take place in EDL but not in SOL muscles, which exhibited constantly necrotic processes. This predominant involvement in SOL, which is a postural muscle, could be due either to the permanent contractile activity of this muscle or to the presence of some type I myofibres. In our study, the ratio of type I to type II myofibres in exercised mdx SOL muscles was decreased. Type II (fast glycolytic) myofibres were previously shown to have an increased susceptibility to damage induced by eccentric contraction compared with type I (slow oxidative) myofibres [34]. During 452 A. Mokhtarian et al. uphill exercise, SOL muscles probably undergo fewer eccentric contractions than during lengthening or downhill running, but are subjected to more intense motor activity than EDL muscles. Therefore, in young mdx mice, the importance of the exerciseinduced injury may be related to the increase in motor activity rather than to the type of exercise. In EDL muscle of sedentary 25-day-old mdx mice, we observed 37% of myofibres to be of intermediate type (type IIC). These myofibres were mostly immature, growing and peripherally nucleated [35]. In exercised mdx EDL muscles, the percentage of type IIC myofibres was similar, but these cells were regenerating centronucleated myofibres. Thus, immature, growing intermediate myofibres disappeared from EDL muscles after exercise as a result of either an increased susceptibility of these fibres to necrosis or an accelerated maturation in type II myofibres. In conclusion, we suggest that the onset of necrosis in the hindlimb muscles of mdx mice at the time of weaning is mainly related to or exacerbated by the increase in motor activity. Although we should be careful in drawing conclusions regarding DMD pathophysiology on the basis of such experiments in small quadrupedal animals, these results suggest that dystrophin-deficient muscle necrosis could be partially controlled by restraining intensive muscle exercise. However, contraction-induced muscle damage cannot be avoided in incessantly working muscles. ACKNOWLEDGMENTS We thank Dr S. Thornton for his help with the English, Mrs M. J. Meile and J. Chandellier for technical assistance and Mrs N. Ouvrard and Mr P. Casanovas for animal care. This work was supported by the Association Francoise contre fes Myopathies (A.M., P.C.E., A.S.) and the Ministere de fa Recherche et de fa Technofogie, Paris, France (J.-P.L.). REFERENCES I. Bulfield G, Siller WG, Wight PAL. Moore KJ. 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