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.     

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.     

Abnormal distribution of proteins in the soleus and extensor digitorum longus of dystrophic mice

Proteins of the whole muscle homogenates of the slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) of normal and dystrophic C57BL/6J mice at 4, 8, 12, and 32 weeks of age were resolved on polyacrylamide isoelectric focusing gels. Gels of the normal SOL proteins at all ages contained two bands specific to SOL and not represented in EDL. Gels of normal EDL contained three bands highly amplified in EDL but barely detectable in SOL. The distribution of proteins in dystrophic SOL was abnormal at all age groups studied due, in part, to a decrease in the proportion of SOL-specific proteins relative to other proteins in the muscle. The distribution of proteins in dystrophic EDL appeared abnormal first at 12 weeks due to a decrease in the relative proportion of EDL-amplified proteins. Due to these and other changes, at 32 weeks the dystrophic SOL and EDL were almost indistinguishable on the basis of their proteins' distributions.     

Isometric contractile properties of fast- and slow-twitch muscles in normal and spastic mice

Isometric contractile properties of the fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles of 24 male C57 mice were studied in vitro at 35°C. Ten animals exhibited the hereditary movement disorder known as spasticity. EDL muscles of spastic and normal mice developed similar specific tensions and they had similar twitch tension:tetanic tension ratios. The time-to-peak tension and the time to half relaxation in the twitch of spastic EDL muscles were, on average, slightly longer. SOL muscles of spastic and normal mice also developed similar specific tensions and their twitch contractions had similar times to half-relaxation. The time-to-peak tension in the twitch and the twitch tension: tetanic tension ratio of spastic SOL muscles were, on average, higher than in normal SOL. The significance of our findings remains somewhat uncertain.     

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.     

Potassium and caffeine contractures in limb muscles of normal and dystrophic (C57 BL/6J dy2J/dy2J) mice

The strength of contractures, produced by 15 to 146 mM [K]0 (as L-glutamate), was measured in isolated small bundles of muscle fibers from the fast-twitch extensor digitorum longus and from the slow-twitch soleus of normal and dystrophic (C57 BL/6J dy2J/dy2J) mice. The analysis of the relation between the maximal amplitude of the contracture vs the membrane potential and the time constant of relaxation of the K-contractures has shown that dystrophy induced an attenuation of the differences between fast- and slow-twitch muscles. The repriming of K-contractures was more affected by changes in [Ca]0 in normal soleus than in normal extensor digitorum longus and this difference was unaffected by dystrophy. For both types of muscles, the ability of caffeine to produce contractures was reduced in dystrophic muscle and this modification was not related to a change in the fiber typing.     

Contractility of mdx skeletal muscle after denervation and devascularization.

To evaluate the regenerative capacity of mdx skeletal muscle, changes in contractile properties of the fast-twitch extensor digitorum longus (EDL) of normal and mdx mice were studied at 7 and 16 weeks of age, following denervation and devascularization (DD) at 4 weeks of age. At 7 weeks, DD EDL of both strains showed significantly decreased isometric twitch and tetanus tensions compared with their non-DD controls. By 16 weeks, normal operated muscle exhibited a recovery of 57% and 58% of absolute tetanus and twitch tensions while the mdx EDL recovered remarkably to 96% and 99% of 7-week values. At 7 weeks, the DD EDL of both strains exhibited significantly slower time-to-peak (TTP) and one-half relaxation time (1/2RT). By 16 weeks, TTP and 1/2RT of the mdx DD EDL no longer differed from non-DD controls, but the normal EDL showed slowed TTP. No differences were found in the maximum velocity of shortening (Vo) or in posttetanic potentiation (PTP). Following DD, there was an increase in resistance to fatigue in both strains at 7 weeks. This resistance persisted at 16 weeks in the normal mouse, but the operated mdx EDL returned to normal. It would appear that following a denervation/devascularization insult, the mdx EDL is able to recover contractile characteristics to a remarkably larger extent than normal EDL.     

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.     

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.     

Rate and extent of functional reinnervation in fast-twitch and slow-twitch muscles of the dystrophic mouse (C57Bl6Jdy2jdy2j)

The extent of functional reinnervation of fast-twitch extensor digitorum longus muscle in dystrophic and normal mice was determined at various times after nerve transection. Functional reinnervation was assessed by measuring the twitch tension evoked by stimulation of the nerve central to the site of transection. In control mice aged 4 to 6 weeks at the time of denervation, complete reinnervation was observed after 6 weeks. In dystrophic mice of the same age reinnervation was clearly impaired. The ratio of functional innervation of the operated leg to that of the contralateral unoperated leg was only 0.62 after 6 weeks. In older dystrophic mice (4 to 6 months at the time of nerve transection) the reinnervation ratio was even lower, 0.43 after 12 weeks. Reinnervation of slow-twitch soleus muscle was assessed 8 weeks after denervation and was also found to be reduced in the older dystrophic animals. The extent of reinnervation was reflected in the measured values of muscle weight, twitch tension per unit wet weight, and twitch time course. The impairment of reinnervation of dystrophic muscle is consistent with, but not proof of, a neurogenic defect in murine muscular dystrophy.     

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.     

Changes in the cholinergic system of rat sciatic nerve and skeletal muscle following suspension-induced disuse

Muscle disuse-induced changes in the cholinergic system of sciatic nerve, slow-twitch soleus (SOL), and fast-twitch extensor digitorum longus (EDL) muscles were studied in rats. Rats with hind limbs suspended for 2 to 3 weeks showed marked elevation in the activity of choline acetyltransferase in sciatic nerve (38%), in the SOL (108%), and in the EDL (67%). Acetylcholinesterase (AChE) activity in the SOL increased 163% without changing the molecular forms pattern of 4S, 10S, 12S, and 16S. No significant (P greater than 0.05) changes in the activity and molecular forms pattern of AChE were seen in the EDL or in AChE activity of sciatic nerve. Nicotinic receptor binding of [3H]acetylcholine was increased in both muscles. When measured after 3 weeks of hind limb suspension the normal distribution of type I fibers in the SOL (87%) was reduced (to 58%) and a corresponding increase in types IIa and IIb fibers occurred. In the EDL no significant change in fiber proportion was observed. Muscle activity, such as loadbearing, appeared to have a greater controlling influence on the characteristics of the slow-twitch SOL muscle than on the fast-twitch EDL muscle.     

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.     

Oxidative Enzyme Activity and Soma Size in Motoneurons Innervating the Rat Slow-Twitch and Fast-Twitch Muscles After Chronic Activity

The effects of chronic activity induced by running training on the activity of the mitochondrial enzyme succinate dehydrogenase (SDH) and soma size in motoneurons innervating the slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) muscles were studied in rats using the retrograde neuronal tracer Nuclear Yellow. Rats were assigned to control and trained groups that were subjected to treadmill running for 10 weeks (2 h/day, 30 m/min, 5 days/week). After training, both SOL and EDL muscles showed clear adaptations (citrate synthase activity in the SOL muscle, and the fast-twitch oxidative-glycolytic fiber area of the EDL muscle increased significantly after training). The SDH activity of the motoneurons innervating both SOL and EDL muscles was unchanged by training. However, SOL motoneurons of trained rats had a significantly larger soma size and a significantly higher total SDH activity (SDH activity × soma size) than those of control. Total SDH activity was calculated to examine the absolute SDH protein content of the motoneurons. On the other hand, there was no difference in both soma size and total SDH activity of EDL motoneurons between the two groups. These data demonstrate that chronic activity has a considerably stronger impact on soma size and total oxidative enzyme activity of motoneurons innervating slow-twitch rather than fast-twitch muscles.     

Muscular transverse relaxation time measurement by magnetic resonance imaging at 4 Tesla in normal and dystrophic dy/dy and dy(2j)/dy(2j) mice

Muscular transverse relaxation times values were measured in vivo in normal mice (strain C57BL6/J, n=14) and in murine models of human congenital muscular dystrophy (dy/dy, n=9; dy(2j)/dy(2j), n=8). A single-slice multi-echo sequence was used. Gastrocnemius/soleus complex, thigh and buttock muscles were studied. Muscular transverse relaxation times values were compared between different muscle groups in each type of animal and between animal groups. Differences were observed between normal and dy(2j)/dy(2j) mice from 3 to 12 weeks of age, and between normal and dy/dy mice at 6 weeks. In specific age ranges, the values of muscular transverse relaxation times in two dystrophic models are different from those in normal mice, and could thus be used as an index of modifications in dystrophic muscle to evaluate therapies.     

Muscle-specific beta-enolase concentrations after cross- and random innervation of soleus and extensor digitorum longus in rats

The concentration of beta-enolase, a highly specific marker of the skeletal muscle of rats, was determined in a slow-twitch muscle, the soleus (SOL) and a fast-twitch muscle, the extensor digitorum longus (EDL) after cross-innervation, random reinnervation, or denervation. The beta-enolase concentration is normally high in EDL and low in SOL. When the nerves entering into these muscles were cross-sutured, the beta-enolase concentration in EDL decreased and that in SOL increased to reach an almost equal value in 20 weeks and by the 35th week the SOL ultimately had a higher beta-enolase concentration than the EDL. When the sciatic nerve trunk was completely transected and sutured immediately, the beta-enolase concentration in EDL decreased and that of SOL increased; in 20 weeks SOL had a beta-enolase concentration similar to that of the EDL. When these muscles were denervated by cutting the sciatic nerve trunk, their beta-enolase concentrations were markedly lowered, but EDL still retained on the 12th week a beta-enolase value comparable to the normal SOL. Possible mechanisms behind the observed changes in beta-enolase concentration are discussed.     

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.     

Influence of temperature on isometric tension development in mouse fast- and slow-twitch skeletal muscles

The influence of temperature (range 35 to 20°C) on the isometric contractile properties of fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles of the male C57 mice was studied in vitro, with direct stimulation. Cooling from 35 to 25°C resulted in an average twitch tension potentiation of 42% in EDL and 13% in SOL muscles. Further cooling to 20°C did not produce further potentiation in EDL muscles. The time-to-peak tension and the time to half-relaxation increased 2.4 to 3.2 times for a 10°C cooling in both muscles. The maximum tetanic tension was little changed in cooling from 35 to 30°C, but was depressed 16 to 19% in cooling to 20°C in both muscles. These results from mouse fast and slow muscles were compared with previously published data from muscles of the rat and the cat. The behaviors of the slow SOL muscles in the three species were found to be significantly different, but they could be explained on the basis of their muscle fiber-type composition.     

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.     

Contractile and fatigue properties of thyrotoxic rat skeletal muscle

The effects of thyrotoxicosis on the contractile properties and development of muscle fatigue in the slow soleus (SOL) and fast extensor digitorum longus (EDL) muscles were examined in rats given 3 mg of L-thyroxine and 1 mg of L-triiodothyronine per kilogram of diet for 6 weeks. The hormone treatment produced significant decreases in the contraction time, one-half relaxation time, and twitch tension in the SOL, while the peak rate of tension development (+ dP/dt) and decline (? dP/dt) in this muscle were elevated. Additionally, the forcefrequency curve was shifted to the right and, thus, resembled the curve of a normal fast-twitch muscle. In contrast, the contractile properties of the fast EDL were relatively unaltered by the hormone administration. Thyrotoxicosis also changed the SOL response to contractile activity as twitch tension, + dP/dt, and ? dP/dt remained high, and a faster decline in muscle glycogen and an increase in lactate occurred compared to control muscles. These results clearly demonstrate a preferential effect of thyroid hormone on slow compared to fast skeletal muscle.