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.     

Similar in vitro fatigue patterns of normal and BIO 40.54 dystrophic hamster extensor digitorum longus muscle.

Fatigue patterns of normal and BIO 40.54 dystrophic hamster extensor digitorum longus (EDL) muscles were studied in vitro (22°C) at 60, 120, 170, and 320 days of age. The diseased muscle showed similar rates of tension decline compared to their normal counterparts when stimulated intermittently (a twitch or tetanus every 90 s) for 3 h or in rapid succession (1-s tetanus every 5 s) until tetanic tension was decreased 50%. Electron microscopic observations revealed a subsarcolemmal accumulation of enlarged mitochondria in dystrophic muscle compared to normal EDL. These results suggest that the availability of adenosine triphosphate for cross-bridge formation may not be impaired in dystrophic hamster muscles.     

Proteolytic enzyme activities and onset of muscular dystrophy in the chick

The relationship between proteolytic enzyme activities, soluble protein profiles, and progression of pathology in dystrophic chick muscle was investigated. Activities of cathepsins C and H, and calcium-activated protease were significantly higher in dystrophic patagialis and pectoralis muscles compared with normal muscles prior to the onset of extensive muscle fiber necrosis. Proteolytic enzyme activity of dystrophic muscle remained constant relative to normal muscle during development while muscle pathology progressed in both patagialis and pectoralis muscles. There were more protein bands (60-80 kDa) in the dystrophic muscle extracts compared with normal at all ages studied. Activities of calcium-activated protease in the dystrophic pectoralis and patagialis were similar although muscle pathology progressed much more rapidly in the dystrophic pectoralis. We conclude there is no causal relationship between the activity of the above proteolytic enzymes and the development of muscle fiber necrosis. The elevated activities of proteolytic enzymes in dystrophic muscle may be due to abnormally accelerated growth.     

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.     

An endogenous inhibitor of calcium-activated neutral protease in UMX 7.1 hamster dystrophy

An endogenous inhibitor for calcium-activated neutral protease (CANP) from skeletal and cardiac muscles of muscular dystrophic hamsters (UMX 7.1) was compared with that from normal control animals at 4 and 10 weeks of age by Western blotting using antibody raised against CANP inhibitor. Fragmented CANP inhibitor was found in dystrophic skeletal muscles in all cases at both ages, while only intact inhibitor was detected in the skeletal muscle of the normal hamsters. A total absence of intact inhibitor was shown in one 10-week-old dystrophic hamster. In contrast, there was little difference in CANP inhibitor from heart between dystrophic and control hamsters at 4 weeks. However, fragmentation similar to that in skeletal muscle was seen in the heart inhibitor in a few of the 10-week-old dystrophic hamsters.     

Increased calcium-activated neutral protease activity in muscles of dystrophic hamsters and mice

A Ca²⁺-activated neutral protease activity was examined in muscles of normal and dystrophic hamsters and mice. Light grey and golden brown strains of normal and B10 14.6 strain of dystrophic hamsters were used. Normal and dystrophic mice were of the Bar Harbor 129 ReJ strain. Enzyme activity was measured in the post myofibrillar fraction (homogenate) and in the 75,000 × g pellet (particulate fraction) and supernatant using purified myofibrils.

In normal and dystrophic hamsters or mice, the Ca²⁺-activated neutral protease was most active in the supernatant followed by the homogenate and particulate fractions. As compared to fractions from normal muscle, enzyme activity was significantly elevated in all 3 fractions from dystrophic muscles of hamsters and mice. Both homogenate and supernatant fractions from muscles of normal hamsters had significantly higher enzyme activity than those of normal mice. Enzyme activity was similar in the particulate fraction. Similarly enzyme activity in the 3 fractions from dystrophic hamster and mouse muscles showed no significant difference.

It is suggested that the Ca²⁺-activated neutral protease may be involved in muscle fibre necrosis in muscular dystrophy.     

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 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.     

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.     

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.     

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.     

Identification of regenerated dystrophic minced muscle transplanted in normal mice

The origin of the reconstituted normal and dystrophic transplants in normal mice of the Bar Harbor 129 ReJ strain was investigated by transplanting [³H]thymidine-labelled minced tibialis anterior muscles into the legs of unlabelled hosts. After 20 days the transplants were processed for autoradiography and histology. At varying time intervals between 0 and 50 days radioactivity counts were made on the transplants and compared with those from the adjacent EDL and contralateral tibialis anterior muscles of the hosts.Both autoradiography and radioactivity counts showed that the transplanted muscles were formed from muscle cells derived from within the donor tissue. Moreover, normal and dystrophic transplants from normal hosts were histologically similar.     

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.     

Phosphorylase kinase activities in damaged mouse skeletal muscles

In the course of work in which the phosphorylase kinase (PhK)-deficient mouse was used as a model of a defined inherited myopathy, we measured the PhK activity in regenerated autografts of normal whole extensor digitorum longus (EDL) muscles. Initially, no PhK activity was found for up to 71 days after grafting. A more sensitive assay technique revealed PhK activity in regenerated normal grafts from 43 days after grafting, but the levels never reached those found in ungrafted normal muscle. PhK activity was also reduced in normal EDL muscles following either: denervation, or tenotomy, or denervation and devascularisation, or denervation, devascularisation and tenotomy, but the reduction was never as great as that observed in grafted muscle of equivalent age. PhK activity was also reduced in the tibialis anterior (TA) muscles of the myopathic C57B1/10 mdx strain of mouse, in which the skeletal muscles undergo persistent bouts of degeneration and regeneration, whilst retaining their vascular and nervous connections. It was concluded that the loss of PhK activity in grafted muscle is due to a combination of the effects of denervation, tenotomy and regeneration which occur on grafting.     

Axoplasmic flow of protein in the sciatic nerve of mice with experimentally induced myopathy.

An experimental myopathy was induced in hind-limb muscles of the mouse by ligating the common iliac artery and subsequently administering serotonin. A marked muscle weakness developed after the serotonin injection, and histological examination showed large areas of fiber degeneration interspersed with regenerating fibers. Axoplasmic flow of ¹⁴C-labeled proteins along the sciatic nerve was measured in these mice and compared with the flow patterns found previously in dystrophic mice. In the dystrophic mouse fast flow of protein was increased but no corresponding increase was found in mice with experimental myopathy. This result is consistent with the proposal that changes in flow patterns in the dystrophic mice are primarily of neural origin.     

A maturational defect in passive membrane properties of dystrophic mouse muscle

Electrical properties, component ionic conductances, and histochemical characteristics of normal and dystrophic (dy^2J/dy^2J) mouse extensor digitorum longus (EDL) and soleus (SOL) muscles were studied between 1 and 6 months of age. Normal EDL and SOL membrane electrical parameters were indistinguishable at 1 month. Between 1 and 3 months, membrane resistance (R_m) in normal EDL decreased progressively to a mature value one-half that found in SOL muscles. Measurements of component conductances indicated that this decrease in R_m was due to a specific increase in membrane chloride conductance (G_C1) in EDL fibers. During the same time membrane capacitance increased slightly in both EDL and SOL. Dystrophic EDL failed to develop fully the changes in membrane resistance seen in controls, showing significant deviation at all time points beyond 1 month. An abnormal high-resistance subpopulation was evident in R_m histograms of mature dystrophic EDL. Average membrane properties of dystrophic SOL appeared normal during the 6-month interval studied but significant changes in R_m histograms were found. Histochemical analysis of normal and dystrophic EDL showed that both contained almost exclusively type II fibers (> 95%); normal and dystrophic SOL contained a nearly equal mixture of type I and type II fibers. Progressive degenerative changes were seen in both dystrophic muscles with age but were more severe in the SOL. The depressed average G_C1 in the dystrophic EDL was due to a subpopulation of fibers (30% of total) having a high membrane resistance. Denervation was ruled out as a cause for this subpopulation by a lack of correlation between membrane resistance and resting potential. Histochemical evidence eliminated the possibility that this subpopulation was composed of slow-twitch fibers. Data are presented indicating that the subpopulation represents a group of dystrophic EDL fibers which fail to undergo normal maturation.     

Calcium-activated protease activity in tenotomized muscle

The purpose of this study was to investigate the possible role of calcium-activated neutral protease in the disorganization and dissolution of the myofibrils of the rat soleus that occurs following tenotomy. Rats were killed 3, 5, 7, 14, 21, and 42 days after tenotomy of the soleus, and the muscles were removed and assayed for calcium-activated protease activity. Maximal protease activity occurred 1 week after tenotomy, at the time when myofibril organization is completely disrupted. Activity was still high 2 and 3 weeks after the operation, but returned to normal levels by 6 weeks, when muscle histology had returned to normal. The time course of the calcium-activated protease activity corresponded closely to the time course of the morphological changes. Thus, calcium-activated neutral protease may play a major role in myofibrillar proteolysis following tenotomy and in making the myofibril susceptible to proteolytic attack by other, less specific proteases.     

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.     

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.