Myosin expression in hypertrophied fast twitch and slow tonic muscles of normal and dystrophic chickens.

Disruption of the development program of myosin gene expression has been reported in chicken muscular dystrophy. In the present report, the relationship between muscular dystrophy and the ability of muscle to respond to an increased work load with a transition in the myosin phenotype has been investigated. Hypertrophy of slow tonic anterior latissimus dorsi (ALD) and fast twitch patagialis (PAT) muscles was induced by overloading for 35 days and myosin expression was analyzed by electrophoresis and immunocytochemistry. Normal and dystrophic chicken ALD muscles have nearly identical proportions of SM-1 and SM-2 isomyosins and both exhibit an age-related repression of the SM-1 isomyosin which is enhanced and accelerated by overloading. Immunocytochemistry with anti-myosin heavy chain (MHC) antibodies demonstrates the appearance of nascent myofibers in overloaded ALD muscles from both normal and dystrophic chickens. A minor fast twitch fiber population is also identified which doubles in number with overloading in normal ALD muscles. There are only half as many fast twitch fibers in control dystrophic ALD muscles and this number does not increase with overloading. In contrast to ALD muscles, the isomyosin profile of normal and dystrophic PAT muscles is quite different. There is significantly more FM-3 and significantly less FM-1 isomyosin in the dystrophic PAT muscle. However, both normal and dystrophic PAT muscles exhibit an overload-induced accumulation of the FM-3 isomyosin. Immunocytochemistry reveals that, unlike the normal PAT muscle, the dystrophic PAT muscle contains a population of myofibers which express slow MHCs. As in the ALD muscle, overload-induced hypertrophy is associated with a repression of the SM-1 MHC in these fibers. Nascent myofiber formation does not occur in either normal or dystrophic overloaded PAT muscles.     

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.     

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.     

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.     

Fatigability of normal and dystrophic chicken muscle in vivo

Changes in the indirectly elicited isometric twitch tension, tetanic tension, twitch-to-tetanus ratio, and post-tetanic potentiation in response to fatigue were examined in the posterior latissimus dorsi muscles of normal and genetically dystrophic New Hampshire chickens. Contractile parameters were studied during 3-h fatigue stimulations and during continuous infusion of potassium chloride (0.24 m equiv/min) initiated at the conclusion of the fatigue period. Both twitch and tetanic tension of dystrophic muscles showed a relative resistance to fatigue; no significant changes in either twitch-to-retanus ratio or post-tetanic potentiation occurred during the fatigue period. In contrast, twitch and tetanic tension of normal muscles decreased more rapidly and to a greater extent in response to fatigue. The twitch-to-tetanus ratio decreased and post-tetanic potentiation increased such that after 30 min they were not significantly different from values seen in dystrophic muscles. Potassium chloride infusion produced a significant recovery (two- to ninefold improvement) of the fatigued twitch response to dystrophic muscle but did not have a significant effect on fatigued normal muscles. A comparison of directly and indirectly elicited twitch contractions indicated that part of the decrement of contractile response in dystrophic muscle was due to synaptic failure at the neuromuscular junction and that potassium chloride infusion resulted in restoration of neuromuscular transmission. It is suggested that the difference in fatigue pattern observed between normal and dystrophic muscle was a function of an altered distribution in the physiological types of motor units present in the diseased muscle.     

Nonequivalence of surgical and natural denervation in dystrophic mouse muscles

The electrical “cable” properties of fibers in the soleus muscle of normal and dystrophic mice were measured in vitro at 37C with intracellular microelectrodes. The motor nerve of the dystrophic muscle was stimulated to determine whether or not the fibers chosen for measurement were functionally innervated. Normal and dystrophic muscles which had been surgically denervated were also studied. Specific membrane resistance was higher in normal than in dystrophic muscle fibers; the respective mean values were 1516 and 636 ohm cm². Dystrophic fibers which were not functionally innervated had significantly higher resistance than those which were innervated, but in neither case did values approach the normal. In both normal and dystrophic muscles, previous sectioning of the motor nerve led to an increase in muscle fiber input resistance. Surgically denervated dystrophic fibers had considerably higher resistance than measured in fibers which had become functionally denervated during the course of the disease. The results suggest that denervation per se cannot account for the differences between normal and dystrophic muscle fibers.     

New muscle transplant method produces normal twitch tension in dystrophic muscle.

Grafting newborn muscle is an innovative method of muscle transplant. This method overcomes hypoxia in the deeper fibers and facilitates reinnervation and revascularization of the grafted muscle fibers, thus promoting the survival and development of the characteristics of the donor muscle. The result achieved is superior to that obtained from mature muscle grafts or from minced muscle transplants. When an intact soleus from a 1-day-old normal mouse was grafted into a recipient soleus of a 20-day-old dystrophic C57BL/6J-dy2J mouse, the actively developing normal graft helped to improve the structure and function of the dystrophic muscle. When compared to the intact dystrophic solei, the test dystrophic muscles five to six months after operation showed increases in cross-sectional area, in wet weight, in twitch and tetanic tension, and in the number of muscle fibers with high resting membrane potentials. This is the first procedure to have raised the muscle twitch tension in an adult dystrophic mouse to the normal level.     

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.     

Temperature effects on the kinetics of force generation in normal and dystrophic mouse muscles

The kinetics of isolated extensor digitorum longus and soleus muscles from normal and genetically dystrophic (129/ReJ dy/dy) mice were studied at temperatures from 8 to 38 degrees C. The rate constants for the exponential rise of tetanic force and for the exponential decay of force during an isometric twitch or short tetanus were similar in normal and dystrophic soleus muscles, but the decay rates were significantly reduced in dystrophic extensor digitorum longus muscles. The temperature dependence for several rate constants for isometric twitches and tetani was similar in all muscles studied, suggesting that the same rate limiting processes apply to fast and slow, normal and dystrophic muscles. Thus, the contractile proteins and those in the sarcoplasmic reticulum of dystrophic muscle are probably normal. The slower relaxation phase in dystrophic extensor digitorum longus muscles is compatible with a reduction in Ca2+-pumping sites in the sarcoplasmic reticulum, perhaps secondary to a change in motor unit composition. Some changes in the temperature dependence for measured times, toward those of soleus muscles, is consistent with the increased proportion of slow twitch motor units in dystrophic extensor digitorum longus 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.     

The effect of stretch removal on muscle weight and proteolytic enzyme activity in normal and dystrophic chicken muscles

Hypertrophy was induced in the patagialis (PAT) muscle of 6-week-old normal and dystrophic chicks by passive stretch for 1 week. Stretch was then removed and muscle weights and activities of the proteolytic enzymes cathepsin C, cathepsin D, and leucine aminopeptidase (LAPase) were measured after 3, 5, 7, 10, and 14 days. In both genotypes, weights of stretch-released muscles dropped progressively for 7 days relative to control muscles, after which they were not significantly different. At the time of stretch release, proteolytic enzyme activities were approximately twice as high in stretched normal muscles as in normal control muscles. In dystrophic chicks there was no difference in activities between stretched and control muscles. However, the activities of the enzymes in dystrophic muscles were already about 4 times higher than in normal control muscles. After stretch release, the enzyme activities in normal muscle progressively fell for 10 days, after which they were not different from normal control muscles. In dystrophic muscles the enzyme activities remained elevated and were not different from dystrophic control muscle activities at any time. We conclude that degradative enzyme activities in normal muscle closely parallel changes in muscle weight, whereas in dystrophic muscle proteolytic enzymes remain elevated and constant whether the muscle is gaining or losing weight.     

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.     

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 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.     

Recovery and loss of muscle force of rat plantaris after partial denervation

The isometric contractile characteristics of rat plantaris muscles were assessed 1, 3, 6, 9, and 12 months after resection of the L4 radicular nerve. After 1 month of partial denervation, twitch and tetanus were significantly lower (26.1 and 22.1%, respectively) than those of sham-operated rats. Plantaris muscle weight was reduced (22.5%), but twitch-to-tetanus ratio (TW/TT), time-to-peak, and one-half relaxation time were not changed significantly. At 3 months, average twitch force was normal but average tetanic force was significantly lower (27.1%) than control value. Muscle weight was reduced (28.9%), but TW/TT was increased by 31%. After 6 months, twitch, tetanus, and all other variables were similar to those of control rats. Normal twitch at 3 months indicates that all muscle fibers have been reinnervated by sprouting from L5 motor axons; however, the new synaptic contacts may not support the tetanic response. At 9 months, muscle force was again reduced and remained at about the same level at the 12-month interval. These results are consistent with the recovery and loss of function seen in poliomyelitis and the postpolio syndrome.     

Beneficial effects of transplanting normal limb-bud mesenchyme into dystrophic mouse muscles

A new technique is being developed to remedy muscle weakness of hereditary myopathies. Mesenchymal cells dissected from limb-buds of day-12 normal mouse embryos were transplanted into the right solei of 20-day-old normal or dystrophic C56BL/6J-dy^2J mice. Host and donors were immunocompatible. Unoperated left solei served as controls. Sham control solei receiving similar surgical treatment but no mesenchyme transplant did not differ from contralateral, unoperated solei. Six to seven months postoperatively the test solei (8 normal and 15 dystrophic) exhibited greater cross-sectional area, total fiber number, and twitch and tetanus tensions than their contralateral controls. Test dystrophic solei contained more normal-appearing and less abnormal-appearing fibers than their controls. Their mean fiber resting potential was intermediate between those of normal and dystrophic controls. There is no difference in twitch time course between test and control solei. The results indicate that such transplantation improves the structure and function of the dystrophic muscles.     

Neural regulation of muscle activation

The effects of temperature and repetitive stimulation on isometric twitch contractions of normal, self-innervated and cross-innervated fast extensor digitorum longus and slow soleus muscles of the rat were studied in situ. Normal and self-innervated extensor digitorum longus showed a 2-fold increase in twitch tension following either a train of 200 stimuli at 35 C (post-tetanic potentiation) or lowering of temperature from 35 C to 20 C (cooling potentiation), while under these conditions the twitch tensions of normal and self-innervated slow soleus fell by about 15%. Post-tetanic and cooling potentiation were virtually abolished in cross-innervated extensor digitorum longus but appeared in cross-innervated soleus. The degree of potentiation in these and control muscles was inversely proportional to contraction time. These experiments suggest that mammalian motor nerves exert a controlling influence on the degree of activation of skeletal muscle during a twitch. An hypothesis of the molecular mechanism of post-titanic and cooling potentiation is proposed. According to this hypothesis, neural regulation of muscle activation results from the transformation of myosin after nerve cross-union.     

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.