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.     

Acetylcholinesterase molecular forms in C57BL/6J dystrophic mice

Acetylcholinesterase (AChE) was extracted from normal and dystrophic C57BL/6J mouse hindlimb muscles and its molecular forms fractionated by sucrose density gradient ultracentrifugation. In the soleus muscles from 6- to 7-week-old mice an increase in the 3 Svedberg unit (S) and a decrease in the 16S AChE molecular forms was observed in dystrophic animals compared to controls. At 12-13 weeks of age, no major significant differences in the relative proportions of AChE molecular forms were noted. In the extensor digitorum longus (EDL) muscles of 6- to 7-week-old dystrophic mice a significant decrease in the proportion of the 10S AChE molecular form and an increase in the 3S and 5S forms was observed. At 12-13 weeks, the dystrophic EDL muscles again displayed a decrease in the 10S form; however, the increase in the 3S and 5S AChE forms, while still apparent, was not significant. These results provide evidence for a biochemical abnormality in the distribution of specific AChE molecular forms, and a differential expression of this abnormality in the soleus and EDL muscles.     

Role of glucocorticoids in the response of rat leg muscles to reduced activity

Adrenalectomy did not prevent atrophy of rat soleus muscle during 6 days of tail cast suspension. Cortisol treatment enhanced this atrophy and caused atrophy of the weight-bearing soleus and both extensor digitorum longus (EDL) muscles. Unloading led to increased sarcoplasmic protein concentration in the soleus, but cortisol administration increased the myofibrillar (+stromal) protein concentration in both muscles. Suspension of hindlimbs of adrenalectomized animals led to faster protein degradation, slower sarcoplasmic protein synthesis, and faster myofibrillar protein synthesis in the isolated soleus, whereas with cortisol-treated animals, the difference in synthesis of myofibrillar proteins was enhanced and that of sarcoplasmic proteins was abolished. Both soleus and EDL of suspended, cortisol-treated animals showed faster protein degradation. It is unlikely that any elevation in circulating glucocorticoids was solely responsible for atrophy of the soleus in this model, but catabolic amounts of glucocorticoids could alter the response of muscle to unloading.     

3H]Ouabain binding in normal and dystrophic mouse skeletal muscles and the effect of age

The numbers of Na+-K+ ATPase sites in skeletal muscles of normal and dystrophic mice between 3 and 17 months of age have been estimated using [3H]ouabain binding assays. In normal mice, at all ages, slow twitch muscle, soleus (SOL), bound significantly more [3H]ouabain than fast-twitch muscle, extensor digitorum longus (EDL). [3H]Ouabain binding did not alter in either SOL or EDL from normal mice over the age range studied. The numbers of Na+-K+ ATPase sites did alter in muscles taken from dystrophic mice (C57BL/6J dy2J/dy2J). In EDL there was an increase and in SOL a decrease in [3H]ouabain binding. This may be related to a change in muscle fibre metabolism from glycolytic to oxidative or to an altered activity pattern. Increasing age resulted in a progressive reduction in [3H]ouabain binding of both SOL and EDL from dystrophic mice. Part of this reduction may be only apparent and due to an increase in connective tissue composition of dystrophic muscles. A limited study of muscles from neonate dystrophic mice indicated that abnormal [3H]ouabain binding was not present in EDL before two weeks of age.     

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.     

Effect of hind-limb suspension on young and adult skeletal muscle. II. Dystrophic mice

Disuse atrophy induced by limb immobilization reportedly protects dystrophic mouse muscle from histopathological changes. This study was conducted to determine whether disuse atrophy induced by hind-limb suspension (HS) limits the histopathology and contractile abnormalities typically observed in the dystrophic mouse. Two weeks of hind-limb suspension were applied to dystrophic mice (line 129B6F1) at two ages, 4 weeks (6 mice) and 12 weeks (8 mice). Thirty-one untreated dystrophics served as controls. In general, HS exaggerated the dystrophic signs, especially in the younger mice; it reduced animal weight, muscle weight, maximum tetanic and twitch tensions, and rates of tetanic and twitch tension development. HS further slowed the contractile properties of soleus (SOL) and extensor digitorum longus (EDL) muscles, and increased their fatigue resistance. HS reduced the size of type I and IIA fibers in the 6-week SOL and EDL, but not in the 14-week muscles. HS produced a preferential atrophy of SOL type I fibers, with a parallel increase in type IIA fibers. However, it did not alleviate the fiber size variability, degree of necrosis, central nucleation, inflammation, or muscle fibrosis in dystrophic muscles. These data demonstrate that disuse by hind-limb suspension does not prevent the histopathological deterioration or loss of muscle function in 6- and 14-week dystrophic mice.     

Fiber type susceptibility in the dystrophic mouse

Slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) muscles from young normal and dystrophic ( ) mice were examined histochemically. Counts were made of total fiber content and of the proportions of various fiber types in whole cross sections. By 3 weeks of age the number of fibers in the SOL of dystrophic mice decreased by 16%. The proportions of both type I and type II_ox fibers decreased approximately equal amounts. Even at this relatively early stage of the disease process, almost 20% of the fibers appeared abnormal and could not be clearly identified as belonging to a specific fiber type. Very few type I fibers with dystrophic characteristics were seen. However, this may simply reflect an alteration in staining characteristics which preceded structural changes in involved fibers. In 6- to 7-week-old EDL muscles the most marked change occurred in the proportion of type II_glyc fibers. The percentage of these fibers in dy^2J mice was 8.4% compared with 57.3% in control animals. At this stage the total number of fibers in dy^2J EDL had decreased by 30%, although the number of type II_ox fibers remained virtually constant. Various explanations for these findings are considered.     

Dystrophic mouse muscles have leaky cell membranes

We examined the spaces occupied by mannitol, sucrose, polyethylene glycol molecular weight 900 (PEG 900), and polyethylene glycol molecular weight 4000 (PEG 4000) in the soleus and extensor digitorum longus (EDL) muscles of normal and dystrophic mice (strain ReG-129 $\text{dy}\text{dy}$). The space occupied by all those tracers was larger in dystrophic than in normal muscles. The enlargement of tracer space was more marked in EDL than in soleus muscles. The smaller tracers (sucrose, mannitol, and PEG 900) equilibrate with nearly all the water in dystrophic EDL whereas there is a water space inaccesible to the larger tracers (inulin and PEG 4000). We suggest that the tracers penetrate into the cell through defects in the cell membrane of the dystrophic muscles.     

Changes in isometric contractile properties of fast-twitch and slow-twitch skeletal muscle of C57BL/6J dy2J/dy2J dystrophic mice during postnatal development

Our primary aim was to determine if there exists a preferential involvement of the fast-twitch or slow-twitch skeletal muscle fibers in the dy2J/dy2J strain of murine dystrophy. The changes in the contractile properties of the slow-twitch soleus (SOL) and the fast-twitch extensor digitorum longus (EDL) muscles of normal and dystrophic mice were studied at 4, 8, 12, and 32 weeks of age. Isometric twitch and tetanus tension were decreased in the 4- and 8-week-old dystrophic EDL compared with controls, this situation being reversed in the older animals. At 12 weeks, the dystrophic EDL generated 15% more tetanic tension than normal EDL and by 32 weeks no significant difference was seen between normal and dystrophic EDL twitch or tetanus tension. By 8 weeks, dystrophic EDL exhibited a prolonged time-to-peak twitch tension (TTP) and half-relaxation time (1/2RT) of the isometric twitch which continued to 32 weeks. For the dystrophic SOL, decreased twitch and tetanus tension was observed from 4 to 32 weeks. At 8 and 12 weeks, TTP and 1/2RT of dystrophic SOL were prolonged. However, by 32 weeks there was no longer a significant difference seen in TTP or 1/2RT between normal and dystrophic SOL. Our results appear to indicate that a loss of the primary control which is determining the fiber composition of the individual muscles is occurring as the dystrophic process advances.     

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.     

Fatiguability and oxidative capacity of forelimb and hind limb muscles of dystrophic mice

Fatigue indices and succinic dehydrogenase (SDH) activities were determined in the extensor digitorum longus (EDL) and soleus muscles of the hind limb and the extensor carpi radialis longus of the forelimb in control and dystrophic mice aged 4 to 26 weeks. A good correlation was found between SDH activities and fatigue indices in muscles from normal mice. In the dystrophic (dy2J) mice, however, this correlation was not present. The EDL muscles from 26-week-old dy2J mice showed a much higher resistance to fatigue than age-matched controls but this was not accompanied by a significant change in SDH. The increased fatigue resistance in dy2J EDL appeared between 8 and 12 weeks of age and was temporally correlated with the onset of fused bursts of spontaneous activity in the hind limb muscles. Nevertheless, there was no conclusive evidence for a link among this spontaneous activity, oxidative enzyme capacity, and fatigue resistance.     

Effect of denervation on sarcolemmal proteins and glycoproteins of fast and slow mammalian skeletal muscle

We compared the protein and glycoprotein composition of a sarcolemmal membrane fraction isolated from normal and denervated rat extensor digitorum longus (EDL) and soleus muscles. Membranes from EDL and soleus muscles showed significantly different protein compositions. A relatively small number of glycoproteins, which were all minor proteins, accounted for the majority of concanavalin A (ConA) and Ricinus communis agglutinin (RCA120) binding. These glycoproteins appear to be common to EDL and soleus but bound different relative amounts of lectin in the two muscles. A large proportion of the ConA binding sites in EDL, but not soleus, were cryptic (not accessible by ConA unless the membrane structure was disrupted). Denervation had a differential effect on sarcolemma from the two muscles with EDL exhibiting large changes and soleus changing little if at all. Several major proteins changed their relative concentrations after denervation and the relative amount of RCA120 bound to the major glycoproteins also changed. The major ConA-binding glycoproteins did not change in either membrane but denervation resulted in the exposure of most of the cryptic ConA-binding sites in EDL membranes. Endogenous sialyl- and galactosyl-transferase activities in the membrane fractions significantly increased in EDL, but did not change in soleus, suggesting that the turnover of the glycoproteins is increased in EDL after denervation.     

Low dose formoterol administration improves muscle function in dystrophic mdx mice without increasing fatigue

The beta(2)-adrenoceptor agonist (beta(2)-agonist), formoterol, has been shown to cause muscle hypertrophy in rats even when administered at the micromolar dose of 25 micro g/kg/day. We investigated whether a similar low dose of formoterol could improve muscle function in the dystrophic mdx mouse. Ten-week-old male mdx and wild-type (C57BL/10) mice were administered formoterol (25 micro g/kg/day, i.p.) for 4 weeks. Formoterol treatment increased extensor digitorum longus (EDL) and soleus muscle mass, increased median muscle fibre size in diaphragm, EDL, and soleus muscles, and increased maximum force producing capacity in skeletal muscles of both wild-type and mdx mice. In contrast to other studies where beta(2)-agonists have been administered to mice and rats, generally at higher doses, low dose formoterol treatment did not increase the fatiguability of EDL, soleus or diaphragm muscles. Although others have found formoterol can decrease ubiquitin mRNA and proteasome activity when administered to tumour bearing rats at high doses (2mg/kg/day), in the present study low dose formoterol treatment did not alter ubiquitin or the E1 and E3 ubiquitin ligases in diaphragm muscles of wild-type or mdx mice, but it did reduce the level of ubiquitinated proteins in diaphragm of wild-type mice. The findings indicate that formoterol has considerably more powerful anabolic effects on skeletal muscle than older generation beta(2)-agonists (like clenbuterol and albuterol), and has considerable therapeutic potential for muscular dystrophies and other neuromuscular disorders where muscle wasting is indicated.     

Slowing of fast-twitch muscle in the dystrophic mouse

Isometric twitch tension was measured in fast-twitch and slow-twitch muscles of normal and dystrophic ( ) mice in vivo. In dystrophic mice more than 6 months old the fast-twitch extensor digitorum longus (EDL) showed a prolongation of the time to peak tension as well as the time to relax to one-half peak tension ( ) compared with age-matched controls. In younger dystrophic mice (4 to 6 weeks) the time to peak tension was prolonged but not significantly so. This apparent “slowing” of dystrophic fast-twitch muscle was accompanied by a reduction in both cooling potentiation and post-tetanic potentiation toward values typical of slow-twitch muscle. Slow-twitch soleus muscle (SOL) of old mice was almost unaffected by the dystrophic process with regared to its contractile characteristics. However, there appeared to be a slight, but significant “speeding” of young dystrophic SOL compared with age-matched control muscles. This was apparent in reduced times to peak tension and half-relaxation as well as an enhanced cooling potentiation. We suggest that the altered contractile characteristics result from a change in some intrinsic property of the muscle fibers rather than from extrinsic factors such as the additional perimysial connective tissue seen in these muscles.     

Observations on the effects of low frequency electrical stimulation on fast muscles of dystrophic mice.

The deterioration of tibialis anterior (TA) and extensor digitorum longus (EDL) muscles in dystrophic mice (C 57 BL dy/dy) was compared. The effects of chronic electrical stimulation on various characteristic properties of these muscles were also studied. The results indicate that EDL muscles are less affected by the disease than TA. This "selectivity" is difficult to explain since both muscles have similar fibre type composition. TA and EDL muscles that were stimulated for 10-28 days developed greater tetanic tensions than the contralateral muscles, but this effect was apparent only when the muscles were severely affected by the disease, that is the contralateral TA or EDL muscles developed less than 50% of the tension produced by muscles from normal animals. In all EDL muscles, stimulation increased the fatigue resistance. The time course of contraction and relaxation of dystrophic muscles is usually slower than that of normal muscles. The stimulation reduced this slowing effect, so that the stimulated muscles became similar to homologous muscles from normal littermates.     

An insulin receptor defect in murine muscular dystrophy

A study has been made of the I¹²⁵ insulin binding and postbinding effects on excised soleus muscles from the 129 ReJ strain of dystrophic mice. Results are compared with those in sex- and weight-matched controls. The data suggest that, in the range of physiological hormone concentrations, the affinity of insulin receptors on dystrophic muscles is less than normal and that the insulin-dependent uptake of both 2-deoxyglucose (2-DG) and aminoisobutyric acid (AIB) is impaired. These findings are taken to indicate that many of the biochemical and electrophysiological abnormalities observed in murine dystrophy could arise from some genetic defect in the receptor proteins controlling uptake of raw materials.     

Evidence for the identities of five proteins decreased in skeletal muscle of dystrophic mice

In a previous study, protein distributions in normal and dystrophic soleus and EDL were examined using polyacrylamide isoelectric focusing. Whereas normally protein distributions in these two muscles were characteristically different, in dystrophic muscles they were strikingly similar. The soleus-specific proteins were identified as the myosin light chains LCls and LC2s. The EDL-amplified proteins were the myosin light chains LC1f, LC2f, the phosphorylated form of LC2f (LC2f-P), and LC3f. In addition, evidence is presented that one protein, which had not been identified in two-dimensional gels, is parvalbumin. The decreased proportion of parvalbumin, LC2f-P, and LC3f in the dystrophic EDL was also shown in other fast-twitch muscles of the mouse. We suggest that these changes in protein distributions in dystrophic muscles reflect a loss of their acquired state of differentiation.     

Intracellular sodium-activity at rest and after tetanic stimulation in muscles of normal and dystrophic (dy2J/dy2J) C57BL/6J mice

Intrafiber Na+-activity, (aNa)i, was measured in vivo in single muscle fibers in the gastrocnemius and soleus muscles of normal and dystrophic (dy2J/dy2J) C57BL/6J mice by means of double-barrel Na+-selective microelectrodes. Values of (aNa)i in normal gastrocnemius and soleus muscles were 9.8 +/- 1.0 mM (means +/- SE; N = 15) and 13.7 +/- 1 mM (N = 19), respectively. Values of (aNa)i in dystrophic gastrocnemius and soleus muscles were 18.7 +/- 1.3 mM (N = 10) and 26.2 +/- 3.3 mM (N = 15), respectively. The soleus muscle of both normal and dystrophic mice was "Na+ loaded" by an intermittent tetanus delivered via the sciatic nerve. Muscles from both normal and dystrophic mice could extrude the Na+ load at similar rates. The results suggest that in the dystrophic mice the elevated (aNa)i is not due to inability of the Na+ pump to respond to a Na+ load; rather, it may be due to an alteration in the affinity of the pump for Na+, resulting in a different "set point" for (aNa)i.     

Pre- and postnatal growth and protein turnover in four muscles of the rat

Developmental growth and associated changes in protein turnover and nucleic acid concentrations have been studied in four individual skeletal muscles. They have also been related to changes in the whole animal. The growth rates of both fast and slow muscle types progressively diminished from the fetus to old age. Similarly, the fractional rates of protein synthesis (measured in vivo) and breakdown in each muscle type declined with age; the changes in the former correlating with decreases in the ribosomal capacities of the muscles. Throughout, fast muscles possessed lower turnover rates. The mean half-lives of mixed proteins were 12.0, 14.4, 13.5, and 7.2 days in the extensor digitorum longus (EDL), gastrocnemius, diaphragm, and soleus muscles, respectively, 310 days postpartum. Muscle atrophy was found at 735 days, at which stage the decreased protein synthetic rate in the soleus was due to a fall in the ribosomal capacity, while that in the EDL was attributable to a decreased synthetic rate per ribosome.     

Phenytoin application in murine muscular dystrophy: Behavioral improvement with no change in the abnormal intracellular Na : K ratio in skeletal muscles

Dystrophic mice injected with phenytoin showed behavioral improvement (improved motor control of hind limbs). An electromyographic assay of the myotonic-like activity (associated with dystrophy in mice) demonstrated the drug's ability to reduce this activity within 15 min of a single injection. The abnormal intracellular Na and K values of skeletal muscle fibers in the gastrocnemius, plantaris, and soleus muscles of the dystrophic mouse were unchanged after 21 days of phenytoin administration. The data suggest that phenytoin is not acting directly on the muscle membrane determinants of the abnormal (dystrophic) ion concentrations, and that the drug's inhibition of myotonic-like electrical activity, probably at the neural level, is responsible for the behavioral improvement in the dystrophic animals.