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.     

Calcium-mediated myopathy at neuromuscular junctions of normal and dystrophic muscle

Previous studies showed that a myopathy which can be produced in vertebrate muscle by inactivating esterases is mimicked by exposure to carbamylcholine and requires agonist-receptor interaction and extracellular calcium. A most consistent aspect of the myopathy is dissolution of Z-disks in myofibrils near the postsynaptic membrane. Using mouse extensor digitorum longus (EDL) muscles in vitro, we found that leupeptin partially protects the Z-disks from dissolution. Chloroquine had much less, if any, effect. These data are compatible with the suggestion that prolonged agonist action at the neuromuscular junction results in the activation of the calcium-activated protease known to destroy the Z-disk protein. Because dystrophic mouse muscles reportedly have increased activities of calcium-activated proteases, we compared the response of normal and dystrophic EDL muscle. These muscles showed no significant difference after 3 h in Krebs baths, but when carbachol was added, there was a significantly greater amount of Z-disk damage in dystrophic muscles than in muscles from wild types (129 ReJ) or from albino mice. As in normal muscle, the agonist-induced myopathy in dystrophic muscle is both calcium- and protease dependent.     

Functional properties of muscles transplanted between normal and dystrophic mice

Tibialis anterior muscles were transplanted between 12-week-old normal and dystrophic mice with intact or polydimethyl silicone-capped peroneal nerve. After 150 days the transplants were removed and their isometric twitch contraction properties were studied in vitro at 20 C. Intact normal and dystrophic muscles of equivalent age were used as controls. Dystrophic muscles developed lower twitch and tetanus tension than normal muscles and showed prolonged half relaxation time. The contraction time and twitch/tetanus ratio of both types of muscle were similar. Of all transplantations performed, only those in normal mice with intact nerve responded upon stimulation. Both normal and dystrophic transplants in normal hosts showed similar isometric properties. Although intact dystrophic muscles and viable dystrophic transplants in normal hosts were similar in weight, the transplants developed about three to four times more tension. In addition, dystrophic transplants showed relaxation times similar to normal muscles. It is suggested that the dystrophic lesion in mice may have a neural origin.     

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.     

Effect of neonatal denervation-reinnervation on the functional capacity of a 129ReJ dy/dy murine dystrophic muscle

The sciatic nerves of 14-day-old 129 ReJ normal (++) and dystrophic (dy/dy) mice were transected in the mid-thigh region. The cut ends of the nerves were approximated to facilitate regeneration. One hundred days after denervation, contractile properties of denervated-reinnervated, normal and dystrophic extensor digitorum longus (EDL) muscles were compared to age-matched normal and dystrophic muscles. In dystrophic muscle, in vitro twitch and tetanic tensions were reduced, compared to those of normal muscle. The denervation-reinnervation procedure resulted in an increase in these parameters as compared to unoperated dy muscle. These data correlated with increases in total myofiber cross-sectional areas. Twitch contraction time was not significantly affected by the dystrophic condition or by the denervation-reinnervation protocol. Whereas dystrophic muscle had a longer half-relaxation time than normal muscle, denervation-reinnervation of the dystrophic EDL resulted in a significantly faster half-relaxation time. While fatigue resistance was greater in dystrophic muscles than in normal muscle, there was a significant decrease in fatigue resistance in the denervated-reinnervated dystrophic muscle. Transient neonatal denervation results in modification of both the morphological and physiological characteristics of murine dystrophy.     

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.     

Abnormal distribution of skeletal muscle acetylcholinesterase molecular forms in dystrophic mice

Acetylcholinesterase (AChE) activities and molecular forms, as separated by density gradient centrifugation, were studied in dystrophic and clinically normal mouse muscle. Dystrophic hemidiaphragms exhibited normal AChE activity, but there was little or no 10 S enzyme, a form that constitutes 27% of control tissue AChE. The 10 S-AChE abnormality was similarly present in dystrophic extensor digitorum longus (EDL) muscle, but this muscle exhibited significantly reduced AChE activity. The EDL muscles also had reduced 16 S-AChE but normal 4 S enzyme activity. Chronic denervation of EDL muscles resulted in proportionally similar reductions of weight, total AChE, and 16 S enzyme in dystrophic and control muscles. We conclude that murine dystrophy involves some alterations that resemble denervation, but that there are major qualitative and quantitative differences in AChE that cannot be explained by a denervation-like effect.     

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.     

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.     

Observations on the efficiency of dystrophic muscle in vitro

A study of muscles of the dystrophic mouse has failed to substantiate earlier claims that these muscles were especially resistant to fatigue in vitro or that fast muscles are preferentially damaged. It has been found that the fast muscle selected for previous studies is very often unable to withstand isolation in an organ bath if it is working, and both the difficulty in removing the normal gastrocnemius muscle intact and the need to trim it surgically contribute independently toward its deterioration in vitro. The smaller dystrophic gastrocnemius muscle is less liable to excision damage, is able to satisfy its resting metabolic needs in nutrient solution, and requires no damaging dissection, but is nevertheless unable to recover normally from fatigue. Using EDL and soleus muscles which are small enough to withstand isolation in vitro, no differences are found between fatigue patterns of normal and dystrophic specimens. Responses to rest, KCl, and 2 mM caffeine are also quite similar, and the only distinguishing biomechanical characteristic we have found in dystrophic mouse muscle is a weaker contraction and a longer total twitch time.     

Control of contractile properties within adaptive ranges by patterns of impulse activity in the rat

These experiments explore the relationship between patterned impulse activity and contractile properties of skeletal muscles. Soleus (SOL) and extensor digitorum longus (EDL) muscles of adult rats were denervated and stimulated directly from 4 to 15 weeks with the same number of pulse trains at different intratrain pulse frequencies (1-500 Hz), with different numbers of pulse trains (864-4,320,000 pulses/d) at the same intratrain pulse frequencies, or with different combinations of pulse trains at 10 and 100 Hz. Chronic stimulation of the denervated SOL resulted in twitch times-to-peak and half-relaxation times that varied in a graded manner between values longer than those in the normal SOL to values as fast as those in the normal EDL, depending upon the pattern used. Increasing pulse frequencies (constant number) resulted in faster twitches, lower twitch/tetanus ratios, increasing post-tetanic potentiations, and larger tetanic tensions. Increasing pulse numbers (constant frequencies) resulted in slower twitches, lower twitch/tetanus ratios, post-tetanic depressions, and higher fatigue indices. The effect of varying the pulse number on twitch parameters was greater at low frequencies (10-20 Hz) than at high frequencies (100 Hz). SOL muscles receiving pulse trains at both 10 and 100 Hz became much faster than muscles receiving pulse trains at 10 Hz only, even in the experiments where the stimulation pattern contained 9 times as many pulses at 10 as at 100 Hz. Chronic stimulation of both the denervated and the innervated EDL with large numbers of pulses at 10 or 15 Hz resulted in twitches that were only half as slow as those induced in the SOL by the same "slow" patterns. In addition, these patterns led to a marked decrease in maximum tetanic tension and a marked increase in twitch/tetanus ratio. During stimulation with a small number of pulses at 150 Hz, on the other hand, twitch speed, twitch/tetanus ratio, and maximum tetanic tension remained normal or almost normal. We conclude that the isometric twitch and related properties of the rat SOL muscle can be graded within wide "adaptive ranges" by varying either the number or the frequency of pulses. In the EDL, the corresponding adaptive ranges appear much narrower, suggesting that the EDL and the SOL contain intrinsically different muscle fibers.     

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.     

Isometric contractile properties of the posterior latissimus dorsi muscle in normal and genetically dystrophic chickens

Various isometric contractile properties of posterior latissimus dorsi muscles from normal and genetically dystrophic New Hampshire chickens were examined. Compared to normal, the dystrophic muscles were lighter, the indirectly elicited twitch was weaker, and the twitch-to-tetanus ratio was reduced. The time course of the twitch contraction was not significantly different from normal. However, fusion frequency was higher in dystrophic muscle and the entire frequency-tension relationship was shifted to higher frequencies. The maximal rate of rise of tension was significantly reduced during both twitch and tetanus in dystrophic muscle. There was no difference in the decrement of the gross electromyogram during high-frequency stimulation. A significantly greater post-tetanic potentiation of the twitch contraction was observed in dystrophic muscle. We suggest that the modification of contractile properties observed in dystrophic chicken muscle represents a shift toward slow muscle characteristics. The paradoxical observation of an unchanged twitch time course in the presence of a reduced maximal rate of rise of tension is discussed in relation to an apparent reduction in duration of the plateau of the active state.     

Comparison of the development of isometric contractile properties of embryonic avian normal and dystrophic skeletal muscle

Embryonic posterior latissimus dorsi (PLD) muscles were isolated at 24-h intervals between days 14 and 20 in ovo from a line of normal chickens (412) and a line afflicted with hereditary muscular dystrophy (413), and their isometric contractile properties were compared. The results demonstrated differences in the isometric contractile responses between normal and dystrophic embryonic PLD muscles. The normalized twitch and tetanic tensions were significantly less for the dystrophic muscle immediately before hatching. Some kinetics of the isometric responses were also different between normal and dystrophic muscles. At embryonic day 16 the times to one-half peak twitch tension, to peak twitch tension, and to one-half peak tetanic tension were significantly longer for the dystrophic muscles. The maximum rate of tetanic force development at days 14, 16, and 18 was lower in the dystrophic muscles. At embryonic day 18 the twitch relaxation of the dystrophic muscle was significantly slower. The results indicated that as early as the final week in ovo, the dystrophic PLD produced less tension and, in some respects, was slower than the normal muscle. Moreover, the differences in the kinetics of the responses were transient, i.e., differences in the kinetics that were observed at day 16 in ovo were not seen closer to hatching.     

The isometric responses of fast and slow twitch muscles from normal and genetically dystrophic hamsters

There has been a need for some time to examine the effects of the dystrophic process upon the mechanical properties of the limb muscles of the dystrophic hamster. This paper reports the findings of such a study by using an in vivo technique to record the isometric twitch characteristics of the extensor digitorum longus and soleus muscles. Normal and dystrophic animals have been examined at various ages as it has been reported that the disease is progressive. Normal muscle characteristics fall in line with those already published for small mammals. Dystrophic muscles show little difference from normal muscles with respect to their twitch characteristics. Only at the 60 day age point was the time to half relaxation of dystrophic muscles longer than its normal counterpart. Differences were found between normal and dystrophic muscle weights and between the ratio of muscle weight to body weight. It is proposed that these result probably from increased hydration of the muscles caused secondarily to the cardiomyopathy.     

Core myofibers and related alterations induced in rats' soleus muscle by immobilization in shortened position

Experimental induction of core myofibers by tenotomy or local tetanus suggests that mechanical factors such as muscle tension loss, shortening or immobilization may play a role in core fiber formation. To test this hypothesis, we investigated the morphologic alterations induced in soleus (SOL) and extensor digitorum longus (EDL) muscles following immobilization of rats' hindlimb in various positions. The SOL and EDL muscles were immobilized in either shortened or lengthened state by applying wire-meshed plaster cast for 1, 2 and 3 weeks. The muscles were dissected out, measured, weighed and examined by histochemistry and electron microscopy. Gross atrophy was noted in all muscles but was greatest in shortened SOL. The SOL atrophy was diffuse and associated with relative increase in type 2 fibers. In EDL, the atrophy selectively involved fibers with low oxidative enzyme activity. Core myofibers were seen mainly in shortened SOL and consisted of myofibrillar derangement, loss of myofilaments and streaming of Z bands. The preferential involvement of shortened SOL (tonic, fatigue-resistant, slow-twitch muscle) suggests that the functional length, loss of tension subsequent to shortening and intrinsic biochemical properties of the muscle are important in core fiber formation.     

Effects of insulin on protein synthesis in muscles from normal and dystrophic mice

Protein synthesis in soleus and extensor digitorum longus (EDL) muscles was measured in vitro to test the hypothesis that the lack of muscle protein accumulation in dystrophic conditions could be caused by a reduced sensitivity to insulin. We demonstrate that physiological insulin concentrations stimulate protein synthesis in soleus muscles from normal mice but not from muscles obtained from dystrophic (dy) animals. The difference is lost at very high insulin concentrations (1 microM) and could not be shown at any concentration with EDL muscles. These results, together with the reported reduced inhibitory effect of insulin on protein synthesis in dystrophic hamsters and on protein breakdown in dystrophic mice, suggest that protein metabolism in certain muscles from dystrophic animals may be less responsive to the anabolic effects of insulin.     

Effect of low Ca2+ solution on muscle contraction of developing, preclinical dystrophic (dy2j) mice.

EDL muscles from normal and dystrophic (dy2j) mice aged 7 to 21 days of postnatal life were examined. Muscles were divided into 2 groups according to age, 7 to 14 days and 16 to 21 days postnatal, so as to assess age- and/or phenotype-related differences in the muscle response to low Ca2+ solution. Tension production was already much impaired in "predystrophic" muscles. At this stage, however, there was essentially no difference in twitch kinetics between normal and dystrophic muscles. Upon exposure to low Ca2+ solution, twitch responses of both normal and dystrophic muscles declined in a similar manner. In the youngest animals studied (7 to 14 days), the tetanic responses of both normal and dystrophic muscles to low Ca2+ solution were also similar. In animals 15 to 21 days old, however, the tetanic tension developed in low Ca2+ solution by dystrophic muscles, was significantly less than that of normal. Moreover, under these conditions (i.e., in low Ca2+ solution), and following tetanic stimulation, the membrane potential of dystrophic muscles in this age group was significantly more depolarized than that of normal muscles. Our results suggest that the ability of the cell to deal with extracellular Ca2+ is normal in predystrophic muscles up to 21 days of postnatal life. The results also clearly point to the fragility of the membrane in these muscles.     

Effect of caffeine and high potassium on normal and dystrophic mouse EDL muscles at various developmental stages

EDL muscles from normal and dystrophic (dy^2j) mice of various ages were examined. Muscles were divided into three groups according to age: 7 to 14 days postnatal, 16 to 21 days postnatal, and 6 months old, to assess age and/or phenotype related differences in the muscle response to caffeine or high K⁺. The response of normal muscles to caffeine decreased with age and reached adult characteristics between the second and third week of postnatal life. Their response to high K⁺ also changed during post-natal development; specifically, the time taken to recover to 50% pretest twitch tension decreased with age, probably reflecting developmental changes in Cl^? conductance. Up to 21 days of age, the sensitivity of dystrophic muscles to both caffeine and high K⁺ was essentially similar to normal, while marked differences were observed in the adult. Taken altogether, our results suggest that while the maturation of a number of systems might be delayed in dystrophic muscles at preclinical stages of the disease, their e–c coupling and SR function (Ca²⁺ release and reuptake) appear to be quite normal. Our results further suggest that the “abnormal” responses of dystrophic muscles at more advanced stages of the disease, when challenged by drugs acting on either of these systems, may be explained in terms of changes in muscle fiber type proportions. © 1993 John Wiley & Sons, Inc.