Force and power output of diaphragm muscle strips from mdx and control mice after clenbuterol treatment

Based on its anabolic properties, treatment with the beta(2)-adrenoceptor agonist, clenbuterol, has been proposed as a strategy for ameliorating the symptoms of muscular dystrophy. In the dystrophic mdx mouse, only the diaphragm muscle exhibits progressive and severe degeneration in muscle structure and function similar to that observed in Duchenne muscular dystrophy. We tested the hypothesis that 20 weeks of clenbuterol treatment ( approximately 1.5-2 mg kg(-1)day(-1)) would increase the force and power output of diaphragm muscle strips of 6-month-old mdx and control mice. At this age, the diaphragm muscles of mdx mice show extensive degeneration and impaired contractility compared with control mice. Clenbuterol treatment did not increase the normalized force or power output of diaphragm strips from either mdx or control mice. The degeneration and necrosis within the diaphragm muscle of mdx mice was also not ameliorated by clenbuterol treatment. The results indicate that clenbuterol treatment does not improve the structure or function of diaphragm muscles from mdx mice.     

Administration of insulin-like growth factor-I improves fatigue resistance of skeletal muscles from dystrophic mdx mice

Muscle fatigue occurs in many neuromuscular diseases, including the muscular dystrophies, and it contributes to a loss of functional capacity and reduced quality of life for affected patients. An improvement in fatigue resistance has been observed in diaphragm muscles of mdx mice following insulin-like growth factor-I (IGF-I) administration. Whether similar treatment can improve locomotor muscle function in mdx mice is not known. We examined the efficacy of IGF-I administration (1 mg/kg daily s.c. for 8 weeks) on structural, metabolic, and functional properties of extensor digitorum longus (EDL) and soleus muscles of mdx mice, and tested the hypothesis that IGF-I treatment would improve function in these muscles. After treatment, muscles were more resistant to fatigue during repeated maximal contractions than muscles from untreated mice, an improvement associated with increased muscle fiber succinate dehydrogenase activity in the absence of changes in cellular (single-fiber) contractile activation characteristics. The findings have important clinical implications, not just for the dystrophinopathies, but for all neuromuscular pathologies where fatigue of locomotor muscles limits functional capacity and decreases quality of life.     

Differential expression of muscular dystrophy in diaphragm versus hindlimb muscles of mdx mice.

Contractile properties of diaphragm (DIA) from mdx and control mice were compared with those of hindlimb muscles [soleus (SOL) and extensor digitorum longus (EDL)] in vitro. Mice ranged in age from 2 weeks to 1.5 years. Muscles were directly stimulated and properties measured were: contraction time, half-relaxation time, active tension per unit area, fatigue index, and maximal velocity of shortening (Vmax). Active tension decreased significantly with age in mdx DIA but not in control DIA. SOL and EDL active tensions were less in mdx than control over the whole age range and did not decrease with age. Vmax was decreased in mdx DIA, but not in mdx SOL or EDL. These results demonstrate that DIA is more affected by muscular dystrophy than hindlimb muscles. Since many Duchenne patients exhibit respiratory distress, this differential expression of dystrophy in diaphragm, as compared to limb muscles, may have important clinical implications.     

Clenbuterol reduces degeneration of exercised or aged dystrophic (mdx) muscle

Evidence of dystrophic muscle degeneration in the hind limb muscles of young (20-week-old) treadmill-exercised or aged (87-week-old) sedentary mdx mice was greatly reduced by treatment with clenbuterol, a beta(2)-adrenoceptor agonist. Daily treadmill exercise for 10 weeks increased the size of regions within the mdx plantaris but not the soleus or gastrocnemius muscles, in which necrotic muscle fibers or the absence of fibers was observed. Clenbuterol reduced the size of these abnormal regions from 21% of total muscle cross-sectional area to levels (4%) found in sedentary mdx mice. In addition, the muscles obtained from aged clenbuterol-treated mdx or wild-type mice did not display the extensive fibrosis or fiber loss observed in untreated mdx mice. These observations are consistent with a mechanism of dystrophic muscle degeneration caused by work load-induced injury that is cumulative with aging and is opposed by beta(2)-adrenoceptor activation. Optimization of beta(2)-agonist treatment of muscular dystrophy in mdx mice may lead to a useful therapeutic modality for human forms of the disease.     

First evaluation of the potential effectiveness in muscular dystrophy of a novel chimeric compound, BN 82270, acting as calpain-inhibitor and anti-oxidant

BN 82270 is a membrane-permeable prodrug of a chimeric compound (BN 82204) dually acting as calpain inhibitor and anti-oxidant. Acute in vivo injection of dystrophic mdx mice (30 mg/kg, s.c.) fully counteracted calpain overactivity in diaphragm. A chronic 4-6 weeks administration significantly prevented in vivo the fore limb force drop occurring in mdx mice exercised on treadmill. Ex vivo electrophysiological recordings showed that BN 82270 treatment contrasted the decrease in chloride channel function (gCl) in diaphragm, an index of spontaneous degeneration, while it was less effective on both exercise-impaired gCl and calcium-dependent mechanical threshold of the hind limb extensor digitorum longus (EDL) muscle fibres. The BN 82270 treated mdx mice showed a marked reduction of plasma creatine kinase and of the pro-fibrotic cytokine TGF-beta1 in both hind limb muscles and diaphragm; however, the histopathological profile of gastrocnemious muscle was poorly ameliorated. In hind limb muscles of treated mice, the active form was detected by HPLC in the low therapeutic concentration range. In vitro exposure to 100 microM BN 82270 led to higher active form in diaphragm than in EDL muscle. This is the first demonstration that this class of chimeric compounds, dually targeting pathology-related events, exerts beneficial effects in muscular dystrophy. The drug/prodrug system may require posology adjustment to produce wider beneficial effects on all muscle types.     

IGF-I treatment improves the functional properties of fast- and slow-twitch skeletal muscles from dystrophic mice

Although insulin-like growth factor-I (IGF-I) has been proposed for use by patients suffering from muscle wasting conditions, few studies have investigated the functional properties of dystrophic skeletal muscle following IGF-I treatment. 129P1 ReJ-Lama2(dy) (129 ReJ dy/dy) dystrophic mice suffer from a deficiency in the structural protein, laminin, and exhibit severe muscle wasting and weakness. We tested the hypothesis that 4 weeks of IGF-I treatment ( approximately 2 mg/kg body mass, 50 g/h via mini-osmotic pump, subcutaneously) would increase the mass and force producing capacity of skeletal muscles from dystrophic mice. IGF-I treatment increased the mass of the extensor digitorum longus (EDL) and soleus muscles of dystrophic mice by 20 and 29%, respectively, compared with untreated dystrophic mice (administered saline-vehicle only). Absolute maximum force (P(o)) of the EDL and soleus muscle was increased by 40 and 32%, respectively, following IGF-I treatment. Specific P(o) (sP(o)) was increased by 23% in the EDL muscles of treated compared with untreated mice, but in the soleus muscle sP(o) was unchanged. IGF-I treatment increased the proportion of type IIB and type IIA fibres and decreased the proportion of type I fibres in the EDL muscles of dystrophic mice. In the soleus muscles of dystrophic mice, IGF-I treatment increased the proportion of type IIA fibres and decreased the proportion of type I fibres. Average fibre cross-sectional area was increased in the EDL and soleus muscles of treated compared with untreated mice. We conclude that IGF-I treatment ameliorates muscle wasting and improves the functional properties of skeletal muscles of dystrophic mice. The findings have important implications for the role of IGF-I in ameliorating muscle wasting associated with the muscular dystrophies.     

Analysis of dystrophin in fast- and slow-twitch skeletal muscles from mdx and dy2J mice at different ages

Muscles from mdx, control, and dy2J/dy2J mice at different ages were analyzed for dystrophin in an attempt to relate the chronology of the protein expression with the final phenotypes in regenerated, normal, and dystrophic muscle, respectively. Immunostaining and gold staining of electrophoresis gels were carried out in the investigation. At 5, 25, and 219 days of age, control muscles exhibited dystrophin bands in both the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus (SOL) muscles. Muscles from the mdx mice at comparable ages (8, 28, and 217 days) never exhibited bands for dystrophin, although titin, nebulin, myosin, and other protein bands were present at intensities comparable to those in control muscles. The dystrophin band was present in both the EDL and SOL from dy2J/dy2J dystrophic mice. As indicated by the present study, the dystrophin deficiency from mdx tissue is not transient. This suggests that dystrophin is not necessary for the success of mdx muscle regeneration.     

Choline acetyltransferase activity in muscles of old rats

The total activity of choline acetyltransferase (ChAc) in the rat extensor digitorum longus (EDL) and soleus muscles increased by 50 and 55%, respectively, between 3 and 9 months of age. In rats 28 to 29 months old, the activity of ChAc in EDL and soleus diminished to 41 and 40%, respectively, of the activity observed in 9-month-old animals. Age changes of ChAc activity in the diaphragm were not significant. The number of muscle fibers in EDL and soleus muscles of rats 28 to 29 months old decreased by 44 and 38% respectively, in comparison with younger animals. Mean muscle fiber diameters did not change between 3 and 9 months of age and decreased by 24, 35 and 9% in the EDL, soleus and diaphragm, respectively, in the 28- to 29-month-old rats. The activity of ChAc expressed in relation to one muscle fiber was about the same in the EDL and soleus muscles. It increased between 3 and 9 months and decreased between 9 and 28 to 29 months of age. The observation that ChAc activity per muscle fiber was identical in the fast EDL and slow soleus muscle suggests that the physiological differences between the two muscles are not caused by a difference in the capacity of their motor nerves to synthesize ACh. In the diaphragm the activity of ChAc per muscle fiber apparently did not diminish in old age. The decrease in the total ChAc activity in the limb muscles of old animals seems due both to a decrease in the number of nerve terminals in the muscles and to a decrease in the amount of enzyme present in individual terminals. We suggest that the maintenance of ChAc activity in the motor nerve terminals in the diaphragm of old rats is due to the continuous activity of this muscle and its motor nerves.     

Beneficial versus adverse effects of long-term use of clenbuterol in mdx mice

Long-term administration of the β₂-adrenergic agonist clenbuterol in mdx mice was used to test the hypothesis that increasing contractile protein content in skeletal muscle will decrease the progression of muscular dystrophy. C57BL/10SNJ (control) and dystrophic (mdx) mice were given clenbuterol (1.0–1.5 mg/kg body weight/day) in the drinking water. Ventilatory function and morphological and functional characteristics of soleus (SOL) and diaphragm (DIA) muscles were evaluated. Clenbuterol administration was associated with increased SOL muscle weight, and SOL muscle weight to body weight ratio in control and mdx mice at both ages. There was a 22% increase in myosin concentration of mdx DIA at 1 year of age, correlating well with increased normalized active tension in mdx DIA at this age. Also, absolute tetanic tension increased in control and mdx SOL with clenbuterol at both ages. Ventilatory function was significantly impaired in mdx mice at both ages and clenbuterol administration did not alleviate this. Clenbuterol treatment was associated with a 30–40% increase in fatigability in DIA and SOL muscles of control and mdx mice at both ages. Furthermore, 1-year-old mdx mice receiving clenbuterol exhibited deformities in hindlimbs and spine. These results suggest that long-term clenbuterol treatment has a positive effect on muscle growth and force generation, but has adverse side effects such as increased muscle fatigability and development of deformities. © 1995 John Wiley & Sons, Inc.     

Muscular dystrophy in the mdx mouse: histopathology of the soleus and extensor digitorum longus muscles

We have used light microscopic histomorphometry to quantify the developmental histopathological changes induced by muscular dystrophy in the soleus and extensor digitorum longus (EDL) muscles of the mdx mouse. We find that this X-linked disease exhibits early fibre necrosis with foci of invasive cells, clustering of affected fibres, hyaline fibres, and, in the mixed soleus muscle, a progressive increase in the proportion of type 1 fibres, the mdx soleus containing 58 +/- 5% type 1 fibres by 26 weeks, compared with 27 +/- 4% in control C57BL/10 ScSn mice. This increase is not due to atrophy or slow axon reinnervation of type 2 fibres. Although only 5% of all original fibres survive by 26 weeks in the EDL, the diseased mdx fibres are continuously and successfully replaced by new fibres with internal nuclei, the affected mice thus avoiding the end-stage histopathology and physical disability characteristic of the X-linked human Duchenne and Emery-Dreifuss muscular dystrophies. Homozygous mdx mice share the life expectancy of normal C57BL/10 mice and appear behaviourly normal. The mdx mouse is therefore an excellent mammalian model in which to study the processes of muscle fibre degeneration and regeneration.     

Age-related differences in regeneration of dystrophic (mdx) and normal muscle in the mouse

The mdx mouse, a genetic homologue of human Duchenne muscular dystrophy (DMD), has been attributed with a greater regenerative capacity of its skeletal muscles. Here, we have tested the hypothesis that muscles of mdx mice regenerate better than those of nondystrophic animals. We studied muscle regeneration resulting from a denervation-devascularization injury (DD) of extensor digitorum longus muscle (EDL) at 3 weeks and 2 months in mdx and wild-type (C57BL/10) mice. Histological and morphometrical studies of muscle regeneration were made from 3 to 180 days later. When DD was performed in 3-week-old C57BL/10 mice, the percentages of nonperipheral nuclei in regenerated fibers decreased progressively over 3 months. This decrease did not occur in animals where DDs were performed at 2months, suggesting that two different populations of muscle precursor cells are mobilized in muscle regenration in mice at these two ages. Moreover, mdx EDL muscle regenerated similarly to the controls for up to 60 postoperative days, as shown by distribution of mean diameters and percentage of nonperipheral nuclei of muscle fibers. After 60 postoperative days, necrosis/regeneration characteristics of mdx muscles recurred, suggesting that mdx-regenerated muscle fibers remain susceptible to degeneration.© 1995 John Wiley & Sons, Inc.     

Role of tumour necrosis factor alpha, but not of cyclo-oxygenase-2-derived eicosanoids, on functional and morphological indices of dystrophic progression in mdx mice: a pharmacological approach

The role of tumour necrosis factor (TNF)-alpha or cyclo-oxygenase-2 (COX-2) eicosanoids in dystrophinopathies has been evaluated by chronically treating (4-8 weeks) adult dystrophic mdx mice with the anti-TNF-alpha etanercept (0.5 mg/kg) or the COX-2 inhibitor meloxicam (0.2 mg/kg). Throughout the treatment period the mdx mice underwent a protocol of exercise on treadmill in order to worsen the pathology progression; gastrocnemious muscles from exercised mdx mice showed an intense staining for TNF-alpha by immunohistochemistry. In vivo, etanercept, but not meloxicam, contrasted the exercise-induced forelimb force drop. Electrophysiological recordings ex vivo, showed that etanercept counteracted the decrease in chloride channel function (gCl), a functional index of myofibre damage, in both diaphragm and extensor digitorum longus (EDL) muscle, meloxicam being effective only in EDL muscle. None of the drugs ameliorated calcium homeostasis detected by electrophysiology and/or spectrofluorimetry. Etanercept, more than meloxicam, effectively reduced plasma creatine kinase (CK). Etanercept-treated muscles showed a reduction of connective tissue area and of pro-fibrotic cytokine TGF-beta1 vs. untreated ones; however, the histological profile was weakly ameliorated. In order to better evaluate the impact of etanercept treatment on histology, a 4-week treatment was performed on 2-week-old mdx mice, so to match the first spontaneous degeneration cycle. The histology profile of gastrocnemious was significantly improved with a reduction of degenerating area; however, CK levels were only slightly lower. The present results support a key role of TNF-alpha, but not of COX-2 products, in different phases of dystrophic progression. Anti-TNF-alpha drugs may be useful in combined therapies for Duchenne patients.     

Choline acetyltransferase and acetylcholinesterase activities in muscles of aged mice

The activities of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) were assayed in intact diaphragm, extensor digitorum longus (EDL), and soleus muscles or their homogenates of young (2-6 months) and aged (24-34 months) mice. CAT activity (per mg of protein) was significantly higher in diaphragm and soleus of old mice in comparison with the young but the age change in EDL was negligible. On the other hand, AChE activity (per mg of protein) was significantly higher in EDL of old mice but in diaphragm and soleus muscles the enzyme activity did not show any significant change statistically. The diaphragm muscle was divided into two fractions, one being neuromuscular (NM) fraction and the other the remainder of the muscle (M fraction). No appreciable change in the ratio of the enzyme activities of NM fraction to the one of M fraction was obtained between the young and aged preparations. Thus, it seems likely that there is an age-related change in CAT and AChE activities which might be affected by the degree to which muscle activity is maintained.     

Prevention of muscle fibrosis and improvement in muscle performance in the mdx mouse by halofuginone

Fibrosis is a known feature of dystrophic muscles, particularly the diaphragm, in the mdx mouse. In this study we evaluated the effect of halofuginone, a collagen synthesis inhibitor, on collagen synthesis in various muscles of young wild-type (C57/BL/6J) and mdx mice. Halofuginone prevented the age-dependent increase in collagen synthesis in the diaphragms of mdx with no effect on wild-type mice (n = 5 for each time point). This was associated with a decrease in the degenerated areas and number of central nuclei. Halofuginone also inhibited collagen synthesis in cardiac muscle. Moreover, enhanced motor coordination, balance and improved cardiac muscle function were observed implying reduced muscle injury. Halofuginone inhibited Smad3 phosphorylation downstream of TGFbeta in the diaphragm and cardiac muscles, in C2 cell line and in primary mouse myoblast cultures representing various muscular dystrophies. We suggest that via its effect on Smad3 phosphorylation, halofuginone inhibits muscle fibrosis and improves cardiac and skeletal muscle functions in mdx mice.     

Peripheral mechanisms of fatigue in muscles of normal and dystrophic mice.

We evaluated the contribution of different processes to fatigue of normal and dystrophic mouse muscles using an in vitro electromyography chamber. Fatigue was induced by repetitive nerve stimulation at 30 Hz for 0.5 s, every 2.5 s until tension decreased by about 50%. We monitored the compound nerve action potential (AP), compound muscle AP, and isometric tension responses to nerve stimulation, and compound muscle AP and tension responses to direct muscle stimulation. In normal mice, about 50% reduction in nerve-evoked tension occurred by 2.4 min in extensor digitorum longus (EDL), 4.8 min in diaphragm, and 9 min in soleus. Analysis of the responses revealed that the fatigue was caused by failure of more than one process in all muscles, and failure of nerve conduction did not contribute to fatigue in any muscle. Failure of neuromuscular transmission, muscle membrane excitation, and excitation-contraction (E-C) coupling and contractility accounted for 55, 45, and 0%, respectively, of the fatigue in EDL, for 21, 74, and 5% of the fatigue in diaphragm, and for 2, 54, and 44% of the fatigue in soleus. In dystrophic mice, while about 50% reduction in nerve-evoked tension occurred by 8.1 min in EDL and 5.6 min in diaphragm, only 29% reduction in tension occurred by 80 min in soleus. Failure of neuromuscular transmission, muscle membrane excitation, E-C coupling and contractility accounted for 22, 63 and 15% of the fatigue in EDL, for 21, 79, and 0% of the fatigue in diaphragm, and for 15, 59, and 26% of the fatigue in soleus. The proportion of slow-twitch oxidative fibers was more than normal in dystrophic EDL, but the same as normal in dystrophic diaphragm and soleus. The slower onset of fatigue was attributable to lesser failure of neuromuscular transmission in dystrophic EDL, and to lesser failure of E-C coupling and contractility in dystrophic soleus.     

Morphometric analysis of mdx diaphragm muscle fibres. Comparison with hindlimb muscles

Histological, histochemical and morphometric characteristics of diaphragm muscles from mdx and control mice were compared with those of hindlimb muscles [extensor digitorum longus (EDL), tibialis anterior (TA) and soleus (SA) muscles]. In contrast to mdx limb muscles, regeneration after muscle necrosis does not restore diaphragm muscle structure. In mdx mice at 270 days of age, 70–80% of fibres in hindlimb muscles had central nuclei, compared with only 35% in diaphragm muscle. At 270 days of age, mdx diaphragm muscle was characterized by perimysial and endomysial fibrosis; this latter feature was absent from mdx hindlimb muscles. Fibre diameter remained smaller than the control in mdx diaphragm muscle. We suggest that the similarity in muscle pathology between the diaphragms of mdx mice and in patients suffering from Duchenne muscular dystrophy (DMD) makes these an appropriate model for DMD, since respiratory failure is the leading cause of death in DMD patients.     

Chemical sympathectomy further increases muscle protein degradation of acutely diabetic rats

The present work investigated the role of the sympathetic nervous system (SNS) in the control of protein degradation in skeletal muscles from rats with streptozotocin (STZ)-induced diabetes. Diabetes (1, 3, and 5 days after STZ) induced a significant increase in the norepinephrine content of soleus and EDL muscles, but it did not affect plasma catecholamine levels. Chemical sympathectomy induced by guanethidine (100 mg/kg body weight, for 1 or 2 days) reduced muscle norepinephrine content to negligible levels (less than 5%), decreased plasma epinephrine concentration, and further increased the high rate of protein degradation in muscles from acutely diabetic rats. The rise in the rate of proteolysis (nmol.mg wet wt(-1).2h(-1)) in soleus from 1-day diabetic sympathectomized rats was associated with increased activities of lysosomal (0.127 +/- 0.008 vs. 0.086 +/- 0.013 in diabetic control) and ubiquitin (Ub)-proteasome-dependent proteolytic pathways (0.154 +/- 0.007 vs. 0.121 +/- 0.006 in diabetic control). Increases in Ca2+-dependent (0.180 +/- 0.007 vs. 0.121 +/- 0.011 in diabetic control) and Ub-proteasome-dependent proteolytic systems (0.092 +/- 0.003 vs. 0.060 +/- 0.002 in diabetic control) were observed in EDL from 1-day diabetic sympathectomized rats. The lower phosphorylation levels of AKT and Foxo3a in EDL muscles from 3-day diabetic rats were further decreased by sympathectomy. The data suggest that the SNS exerts acute inhibitory control of skeletal muscle proteolysis during the early stages of diabetes in rats, probably involving the AKT/Foxo signaling pathway.     

Effects of long-term administration of ambenonium chloride on motor end-plate fine structure and acetylcholine receptor in rat

Ambenonium chloride was administered orally in a dosage of 6 mg/kg/day to rats for 14–360 days.Motor end-plate fine structure and junctional AChR were quantitatively analyzed in red (soleus) and white (EDL) muscle fibers. In treated animals, degeneration and simplification of postsynaptic folds and widening of synaptic clefts were often observed in soleus end-plates, but infrequently in EDL end-plates. On the other hand, the postsynaptic AChR was reduced markedly in both soleus and EDL end-plates. No presynaptic changes were observed.These results show that long-term administration of Anti-ChE agents in myasthenia gravis may have an adverse effect on neuromuscular transmission.     

Failure of neuromuscular transmission and contractility during muscle fatigue

In previous studies of muscle fatigue, tension was monitored from whole muscle, while action potentials were recorded from a few muscle fibers. To compare more accurately changes in these responses, an in vitro fluid electrode technique was employed to record the action potential of whole muscle simultaneously with tension during fatigue induced by nerve stimulation in the rat extensor digitorum longus (EDL), soleus, and diaphragm muscles. In each muscle, tension declined from the start of stimulation, while action potential amplitude initially increased slightly and then declined most rapidly in EDL, more slowly in diaphragm, and most slowly in soleus. Direct stimulation of the fatigued muscle produced the greatest increase in tension in EDL, next in diaphragm, and least in soleus. These results indicate that while failure of excitation-contraction coupling or of the contractile mechanism is the initial cause of fatigue in all the muscles studied, and remains the predominant cause throughout in the soleus muscle, failure of neuromuscular transmission plays an important role in fatigue after the first 15 seconds in EDL, and to a lesser extent, after the first 90 seconds in diaphragm.     

Regeneration after free muscle grafting in normal and dystrophic (mdx) mice.

Soleus muscles from C57BL/10 and mdx mice were isotransplanted to induce a cycle of degeneration/regeneration. Sixty days post-surgery, transplanted and contralateral soleus muscles were removed for mechanical and biochemical analyses. The regeneration which occurs after transplantation, induces in both mdx and C57BL/10 soleus muscles a decrease in maximal isometric force, together with an increase of the velocity of contraction. This increase in velocity is accompanied by the expression of typically fast-type myosin heavy chains. Thus degeneration/regeneration of both mdx and normal mice are very similar, causing a shift towards physiologically 'faster' muscle. Previous physiological and biochemical studies of mdx muscles have shown that mdx muscle is shifted towards 'slower' muscle compared to normal mice. One explanation of these findings was that the degeneration/regeneration cycles inherent in dystrophin-deficient mdx muscle causes a shift towards 'slow'. Our results argue against this hypothesis: degeneration/regeneration in both normal and mdx mice causes a shift towards 'fast'.