Sarcomere length in normal and dystrophic chick muscles.

An electron microscope study of 2- and 8-week-old normal and dystrophic chickens compared sarcomere lengths in relaxed and passively extended Patagialis (PAT) muscles. Sarcomeres were measured in dystrophic muscles only in fibers which exhibited no morphological signs of degeneration. Sarcomere lengths were not different from each other in normal muscles of 2- and 8-week-old chickens. Passive extension of the normal wing increased mean sarcomere length by 44%. Sarcomere lengths in unstretched dystrophic PAT muscles were 22 and 25% longer than unstretched normal sarcomeres at 2 and 8 weeks of age. Passive extension of the wing further increased sarcomere length of 2-week-old dystrophic muscles to the length of stretched sarcomeres in 2-week-old normal muscles. In 8-week-old dystrophic chickens, the wings could be passively extended to only 134 degrees, rather than the normal range of 180 degrees. In this case, passive extension of the wings did not further increase the length of sarcomeres. Increased sarcomere lengths in dystrophic muscles may indicate that dystrophic muscle fibers are being subjected to greater degrees of passive tension than normal muscle fibers during early stages of growth. Passive tension is known to promote fiber hypertrophy, nuclear proliferation, and increased oxidative metabolism in normal muscle. These responses to passive tension are also characteristic of prenecrotic stages of muscular dystrophy in chickens.

Passive stretch of adult chicken muscle produces a myopathy remarkably similar to hereditary muscular dystrophy.

The wings of 10 chickens between 1 and 5 years of age were passively extended. An increase in plasma creatine phosphokinase activity was observed in 30 min, continued to rise for 24 h, and then declined, suggesting mechanically induced damage to muscle fibers. Wing muscles were removed and examined histologically at various times after stretch. Patagialis muscles, but not biceps brachii, showed the development of muscle fiber pathology. The patagialis muscle is less active than the biceps brachii and is stretched to a greater degree by wing extension. Susceptibility of muscles to development of pathology appeared to be correlated with the age of the chickens. Pathology was remarkably similar to that observed in young chickens with hereditary muscular dystrophy. Necrotic fibers exhibiting segmental necrosis, abnormal shapes, enlargement, splitting, vacuolation, and phagocytosis were evident. Of particular interest was the appearance of abnormal clusters of acetylcholinesterase activity along the sarcolemma. These sites were shown to appear on fibers of 2-week-old dystrophic chicks prior to necrosis and increase in plasma creatine phosphokinase activity. It is suggested that aging of inactive muscles may promote adhesions between muscle fibers rendering them susceptible to damage when stretched and that necrosis of dystrophic fibers may be initiated by a similar mechanism. Such could occur if the genetic defect resulted in interfiber adhesions. Support for this hypothesis by other reports in the literature is discussed.

Cimaterol-induced growth in rats: growth pattern and biochemical characteristics.

Sixty Sprague-Dawley female rats weighing 170 g were used to investigate the effect of cimaterol on the growth pattern and biochemical characteristics of skeletal muscles. Cimaterol (CIM) was mixed in a powdered rat chow at 10 ppm. A significant (P less than .01) improvement in the weight gain of CIM group was observed during the first 8 days of the 49-day trial period, followed by no significant improvement thereafter. When CIM was withdrawn from the diet, treated rats lost weight, eventually regressing to the same weight as control rats after 28 days. CIM-fed rats showed 28.6% (P less than .01) and 12.9% (P less than .05) increases in plantaris muscle weight over the control at 8 and 14 d, respectively. DNA concentration significantly (P less than .05) decreased, whereas RNA concentration increased at 3 d (31%) and 8 d (12.7%), but decreased at 14 d compared to the control. The increase in RNA concentration preceded the significant improvement of muscle weight in the CIM group. In the soleus muscle, there was no significant difference in muscle weight or DNA concentration between the control and CIM groups. Cimaterol-feeding significantly (P less than .05) increased the size of both type I and II fibers. The size increase of type II fibers in plantaris was twice that of type I fibers in soleus (34.2% vs 17.5%). This may explain why the plantaris muscle (type II predominant) showed a greater weight increase than the soleus muscle (type I predominant).

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.

Conversion of muscle fiber types in regenerating chicken muscles following cross-reinnervation.

Slow-tonic anterior latissimus dorsi (ALD) and fast-twitch posterior latissimus dorsi (PLD) muscles of 7 to 10-day-old White Leghorn chickens were crushed and allowed to be reinnervated by their own nerve, or crushed and transplanted to the other side and allowed to be reinnervated by the nerve of the side to which they were transplanted. Following transplantation, changes in the weight of the muscle, fiber-type composition and innervation pattern during regeneration were investigated. Normal growth rate of PLD was about twice that of ALD. Regenerating PLD, however, atrophied rapidly after crushing and denervation whether innervated by its own nerve or the other nerve type, whereas ALD reinnervated by its own nerve showed marked hypertrophy. PLD fibers transformed rapidly to fast-twitch alpha or slow-tonic (ST) fibers when they were reinnervated by PLD or ALD nerve, respectively. When ALD fibers were reinnervated by their own nerve, they differentiated into ST fibers that were surrounded by smaller immature fibers. ALD fibers were, however, resistant to complete control by fast-twitch PLD nerve and contained a large number of slow fibers (ST and beta) long after transplantation. Slow fibers in regenerates were initially multiply innervated, but later transformed into fast-twitch alpha fibers that were focally innervated. The mode of differentiation and innervation pattern of different muscle fiber types in regenerating muscles are discussed.

Stretch-induced growth in chicken wing muscles: role of soluble growth-promoting factors.

The involvement of soluble growth-promoting factors in stretch-induced hypertrophy of the Patagialis muscle (PAT) in the chicken wing was investigated. Soluble extracts were prepared from young chicken PAT muscles made hypertrophic by passive stretch and from unstretched contralateral controls. Extracts were tested for their ability to stimulate cell proliferation and creatine phosphokinase (CPK) activity in primary monolayer cultures of chick embryo muscle cells. Factors were present in muscle extracts which showed a dose-dependent stimulation of cell proliferation and CPK activity in vitro. Passive stretch for 5 days produced a rapid hypertrophy of the PAT which was accompanied by a dramatic increase in the activity of the growth factor(s). Release of stretch resulted in an arrest of growth and an immediate fall in growth factor activity. The difference in growth-stimulating activity between control and stretched PAT extracts could be demonstrated in chicken transferrin-sensitive chick myoblast cultures. Stretch thus induces an increase in a class-specific growth factor, possibly Transferrin, in the PAT. Stretched PAT extracts stimulated: (a) chick myoblast proliferation to a greater extent than an optimum concentration of chick embryo extract, and (b) CPK activity in vitro to a greater extent than excess Transferrin. Both control and stretched PAT extracts supported the growth of rat myoblasts. We conclude that PAT muscle extracts also contain unknown growth factor(s) which are different from Transferrin.

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.

Effects of stretch and denervation on protease activities of normal and dystrophic chicken muscle.

Changes of protease activities that follow passive stretch, denervation, and denervation plus stretch were followed in the patagialis muscle of normal and dystrophic chicks between 6 and 7 weeks of age. The baseline activities of cathepsin C, cathepsin D, and leucine aminopeptidase in dystrophic muscle were 2 to 3.5 times higher than in normal muscle. Passive stretch and denervation induced increases in protease activities by 40 to 120% in normal muscle, whereas the same treatments did not significantly affect the activities of the enzymes in dystrophic muscle. We conclude that the level of protease activity in dystrophic chicken muscle at 6 weeks of age had already attained a maximum limit and could not be increased even by denervation. In spite of protease activities, which were not different from control dystrophic muscle, denervated dystrophic muscles lost muscle weight rapidly whether they were stretched or not. They weighed 60% less than the innervated control muscle after 7 days. Inherently high protease activities in dystrophic muscle do not vary at this age regardless of whether or not the muscle is gaining or losing weight.

Stretch-induced growth in chicken wing muscles: nerve-muscle interaction in muscular dystrophy.

Skeletal muscle growth following denervation and denervation plus passive stretch was characterized in the patagialis muscle of normal and dystrophic chicks until 8 wk of age. In both genotypes, muscles denervated at 1 wk of age grew at reduced rates compared with contralateral control muscles whether or not they were passively stretched. Histograms of fiber size distributions as well as morphological criteria showed that passive stretch of denervated dystropic muscles substantially delayed the development of pathology. Denervation alone provided less protection. There was no evidence of fiber necrosis in any denervated dystrophic muscle, although many fibers did exhibit extreme hypertrophy and abnormal morphology. When denervated dystrophic muscles were allowed to reinnervate, growth and development of pathology was rapid. Because denervation, denervation with passive stretch, or passive stretch alone retards, but does not prevent, the development of pathology, it is concluded that dystrophy in the chick is a myogenic defect that is exacerbated by neurally mediated contractile activity.

Effects of graded duration of stretch on normal and dystrophic skeletal muscle.

Daily passive stretch for six weeks ranging from 30 minutes to 24 hours per day was studied in the patagialis (PAT) muscle of normal and dystrophic chickens. Significant increases in wet weight, cross-sectional area, and mean fiber cross-sectional area occurred in both normal and dystrophic PAT in response to stretch of all daily durations tested. More than 50% of the growth occurring in response to continuous stretch was elicited by as little as 30 minutes of stretch per day. Oxidative enzyme capacity increased proportionately with increasing durations of stretch in the normal PAT. Similarly, increasing duration of stretch progressively retarded the onset of histopathological signs in the dystrophic PAT. We conclude that daily stretching for as little as 30 minutes per day is a powerful inducer of growth in normal and dystrophic muscle and that the progress of the histopathology in dystrophic muscle is delayed in proportion to the daily duration of stretch.

Regeneration and reinnervation of the dystrophic mouse soleus muscle. A light- and electron-microscopic study.

The regeneration and reinnervation of the dystrophic mouse soleus muscle was investigated in response to a double crush-lesion, which causes degeneration of muscle fibres leaving the innervation intact. In normal and dystrophic muscles, injury produced degeneration of muscle fibres, proliferation and fusion of muscle satellite cells, and growth and reinnervation of regenerating fibres. Four, 6 and 21 days after injury, regenerating dystrophic fibres were 50% smaller in cross-sectional area than regenerating normal fibres and showed several pathological changes. Nerve terminal morphology was initially unaffected by the crush, and nerve terminals were associated with degenerating muscle fibres 2 days after injury and with regenerating muscle fibres 6-28 days after crushing. In intact muscles dystrophic endplates were longer and showed increased ultraterminal sprouting compared to normal endplates. At 28 days after crushing normal nerve terminal sprouting was significantly increased compared to the contralateral control. The extent of nerve terminal sprouting and endplate length in dystrophic muscles was not affected by the degeneration and subsequent regeneration of the muscle fibres. We conclude that a proportion of dystrophic mouse soleus muscle fibres can regenerate after a crush when the innervation is left intact.

Stretch-induced growth in chicken wing muscles: effects on hereditary muscular dystrophy.

Skeletal muscle growth induced by passive stretch was characterized in the Patigialis muscle of chicks with hereditary muscular dystrophy. When the muscle of 6-wk-old chicks was stretched for 1 wk, the effects on muscle growth and on muscle pathology were variable, but in general few differences between stretched and unstretched muscles were observed. However, when the muscle of 1-wk-old chicks was stretched for 6 wk, the effects on muscle growth and on prevention of pathology were dramatic. Similar to results obtained previously when normal chick muscles were stretched [Holly et al., Am. J. Physiol. 238 (Cell Physiol. 7): C62-C71, 1980; Barnett et al., Am. J. Physiol. 239 (Cell Physiol. 8): C39-C46, 1980], stretched dystrophic muscle increased in weight (200%), cross-sectional area (107%), and fiber cross-sectional area (82%). DNA concentration, which is severalfold higher in unstretched dystrophic muscle compared with unstretched normal muscle, fell to values not different from normal values after being stretched. Nuclei per square millimeter also were the same for stretched dystrophic and stretched normal muscle. Histograms indicated that stretching induced a fiber distribution in dystrophic muscle qualitatively similar to that found in stretched normal muscle. Cytochemical observations revealed a dramatic protective effect of stretch against the progressive pathology of dystrophy. It is concluded that stretch of muscle applied to newly hatched dystrophic chicks is a powerful deterrent of symptoms characteristic of hereditary muscular dystrophy. Stretch imposed after the symptoms of dystrophy are apparent provides little, if any, protection.

Prediction of lamb meat quality traits based on muscle biopsy fibre typing.

The aim of the experiment described in this paper was to determine the relationship between muscle fibre type and meat quality. Two indicator muscles, the Stylohyoïdeus and the Scutulo auricularis superficialis accessorius, were biopsied on one-month-old lambs and used as an index to select the animals of the redder and of the whiter metabolic types among the lamb population studied. Evaluation of the organoleptic qualities of meats from two groups was performed on the Longissimus dorsi muscle by a taste panel. The results of the taste panel evaluation suggest, on one hand, that the meat from the redder animals is juicier and has a more intense flavour than that from the whiter. On the other hand, it appears possible to obtain an in vivo prediction of lamb meat quality based on muscle biopsy fibre typing.

Stretch-induced growth in chicken wing muscles: myofibrillar proliferation.

The patigialis muscle (PAT) in the wing of the chicken can be induced to grow rapidly in length and in diameter by passively stretching the muscle with a spring-loaded aluminum bar (Holly et al., Am. J. Physiol. 238 (Cell Physiol. 7): C62-C71, 1980). Rates of DNA, RNA, and protein synthesis are accelerated. Sarcomere length falls from 3.19 micrometers after 1 day of stretch to only 10% above control values at 7 days of stretch. Myofibrils are wavy and misaligned. Electron microscopy of cross-sectioned muscles shows that the fraction of cell volume occupied by myofibrils remains constant throughout the experimental period, even though cross-sectional area of the muscle fibers increases by 55%. The mean diameter of myofibrils in stretched muscle increases by more than 25%. The number of splitting myofibrils increases from 15% before stretching to 45% after 1 wk of stretch. Splits appear to originate in the center of the I band, and then progress to the A band and the periphery of the myofibril. Elements of the sarcotubular system develop quickly at the origin of the fractures. It is concluded that rapid growth of the myofibril is required for initiation of splitting. Neither neurally mediated active tension nor muscle contraction are required.

Stretch-induced growth in chicken wing muscles: biochemical and morphological characterization.

Skeletal muscle growth induced by passive stretch was characterized in chicken wing muscles. Within 24 h after installing the stretch apparatus on the birds, the sarcomere length of the stretched patagialis was 40% greater than control. This was accompanied by increasing muscle wet weight, protein content, DNA and RNA concentrations. Sarcomere length returned to near control values by 3 days, but muscle wet weight and protein content continued to increase through 10 days. DNA and RNA concentrations reached a peak after 5-7 days and began to decline. Histological examination after 7 days revealed no change in the concentration of nuclei inside the basement membrane surrounding the muscle fibers, but the concentration of nuclei outside the basement membrane had increased. Therefore, the ratio of protein to DNA can be a poor index of muscle fiber DNA unit size. Additionally, no evidence of new muscle fiber formation was found. Electron microscopy demonstrated that passive stretch destroyed sarcomere registration between adjacent myofibrils. We concluded that passive stretch is a powerful inducer of muscle growth.

A possible mechanism of phenotypic expression of normal and dystrophic genomes on succinic dehydrogenase activity and fiber size within a single myofiber of muscle transplants.

Muscle transplantation was used to evaluate the ability of normal and dystrophic chickens to support regeneration of both normal and dystrophic muscle fragments. Pectoralis muscles were grafted into the site of the biceps muscle of host chickens. Identification of dystrophic characteristics of intact and regenerating muscle fibers was made by cytochemical analysis of mitochondrial succinic dehydrogenase (SDH) and by fiber size. In the biceps muscle of dystrophic chicks at 40 days ex ovo, the mean size of muscle fibers with low activity of SDH and fibers with high SDH activity was 29.0 +/- 5.9 micrometers and 42.0 +/- 10.4 micrometers, respectively. The mean size of normal muscle fibers was notably smaller than in dystrophic muscle and was 17.8 + 3.1 micrometers. The hypertrophy of fibers coupled with elevation of SDH activity tended to increase with age. Transplants were examined at 56 days postoperatively. The results of cross-transplantation between normal and dystrophic genotypes were similar to unoperated muscles in the correlation between SDH activity and fiber size. Donor muscles determined the type of myofibers regenerated in transplants regardless of whether the host was normal or dystrophic. In addition, combined transplantation was attempted to produce a single hybrid myofiber in which normal and dystrophic pectoralis muscle were mixed in equal volume. The mixtures were then allowed to regenerate in host chicks. A number of mosaic myofibers appeared in transplants and had regional differences in SDH activity along their length. It was concluded that: (1) The characteristics of high SDH activity and fiber hypertrophy are an expression of dystrophic nuclei, (2) combined transplantation of both normal and dystrophic muscle fragments can produce mosaic myofibers in SDH reaction; and (3) the local control of SDH activity and fiber size within nuclear territories in mosaic myofibers seems likely to be due to phenotypic expression of either normal or dystrophic genomes.

Stretch-induced growth in chicken wing muscles: a new model of stretch hypertrophy.

A new model of stretch-induced growth is evaluated in four chicken wing muscles stretched to different extents by a spring-loaded tubular assembly. Muscles grew in length and cross section in proportion to the extent to which they were stretched. Longitudinal growth was essentially completed within 1 wk, while muscles grew in cross section through at least 5 wk of stretch. The muscles were neither denervated nor immobilized, and muscle activity as measured by EMG was not increased. Oxidative enzyme activities increased substantially with stretch in the patagialis (PAT), a twitch muscle, but were relatively unchanged in the slow-tonic anterior latissimus dorsi (ALD). Stretch altered mitochondrial enzyme proportions in the PAT, but had little effect in the ALD. Capillary density was unchanged with stretch in the PAT, but decreased in the ALD. Capillary density was unchanged with stretch in the PAT, but decreased in the ALD. Capillary-to-fiber ratio, however, increased in both muscles. We conclude that muscles grow and adapt enzymatically due to stretch, but that these responses are dissimilar in twitch and tonic muscles.

Scoliosis in chickens.

Scoliosis developed in 55 per cent of sexually mature birds (68 per cent of male and 46 per cent of female birds) in a highly inbred line of chickens originally produced from white Leghorns. The curve could first be detected at five to six weeks of age and progressed until spontaneous fusion of the thoracic vertebrae occurred. Studies of these chickens indicated that abnormalities of growth and development of the spine are not the primary cause of the scoliosis. Preliminary studies of the paravertebral musculature also indicated that simple muscle imbalance is not responsible for the curve. Initial studies of collagen extracted from the scoliotic line of chickens showed it to be more soluble than similar collagen extracted from white Leghorn controls.