Myosin light chain phosphorylation during contraction of chicken fast and slow skeletal muscles

A modified automatic freezing apparatus (K. M. Kretzschmar and D. R. Wilkie, 1962, J. Physiol. (London), 202, 66–67) was used for studying light chain phosphorylation during the early phase of contraction of the fast, posterior latissimus dorsi, and slow, anterior latissimus dorsi, muscles of chicken at 37 °C. The frozen muscles were worked up under conditions which avoid artifacts in quantitating the level of light chain phosphorylation in contracting and resting muscles. The posterior latissimus dorsi muscle reached 80% of its maximal isometric tension at 0.1 s of tetanic stimulation. At the same time, light chain phosphorylation increased by 60% of its maximal extent. The peak tension of the posterior muscle at 0.2 s of stimulation was accompanied by maximal light chain phosphorylation. In case of the slow anterior latissimus dorsi muscle, maximal tetanic tension was developed in 2.5 – 5 s and light chain phosphorylation also proceeded at a much slower rate than in the fast posterior muscle. When contralateral posterior latissimus dorsi muscles were stimulated for 0.2 s and one muscle was frozen at the height of tetanus while the other muscle was allowed to relax and frozen 0.4 s after terminating the stimulation, both contracted and relaxed muscles exhibited maximal light chain phosphorylation. However, when the muscle was allowed to relax for 0.8 s before freezing, half of the phosphorylated light chain became dephosphorylated. The resting level of phosphate content of the light chain was restored in both the posterior and anterior muscles during a longer time after relaxation.     

An autoradiographic study of the role of satellite cells and myonuclei during myogenesis in vitro

Satellite cells and myonuclei of neonatal rat muscles were differentially labeled with 3H-thymidine according to the procedure of Moss and Leblond (1971). Minced muscles fragments containing either labeled satellite cells or labeled myonuclei were cultured until multinucleated myotubes grew out from the explants. Reutilzation of isotope released from degenerating nuclei was competitively inhibited by using a culture medium containing excess (0.32-0.41 mM) cold thymidine. after an 8-10 day growth period, the explants were fixed and prepared for autoradiographic (ARG) examination to determine whether labeled satellite cells or myonuclei had contributed to the myonuclear population of the developing myotubes. Counts were made of the number of labeled myotubes in the explants and compared with the number of labeled satellite cells and myonuclei in samples of the original muscle tissues fixed at the time of explantation. The original muscles showed a mean satellite cell labeling index of 51.7% and gave rise to myotubes with a mean labeling incidence of 40%. In contrast, myonuclear labeling in the original muscle tissues showed no correlation with subsequent myotube labeling. Only 3.4% myotube labeling was found in explants in which over 30% of the original tissue myonuclei had been labeled. Under conditions controlled for isotope reutilization, these observations confirm results of in vivo ARG studies indicating that satellite cells are the only significant source of regenerating myoblasts in injured muscle tissue.     

There is selective accumulation of a growth factor in chicken skeletal muscle. II. Transferrin accumulation in dystrophic fast muscle

Transferrin or a transferrin-like protein, with ability to stimulate myogenesis and terminal differentiation in vitro, is found in fast chicken muscle during embryonic development. After hatching, however, transferrin is no longer accumulated or is only weakly accumulated by fast muscles like the pectoralis major and the posterior latissimus dorsi but continues to be accumulated by slow muscles like the anterior latissimus dorsi. In congenic lines of chickens bearing the gene for muscular dystrophy, however, adult fast muscles do not lose the ability to accumulate transferrin. While transferrin is found selectively in adult normal and dystrophic muscle it does not appear to be synthesized by muscle cells. Immunocytochemical localization shows that transferrin is accumulated not so much by muscle fibers as it is by single cells in the muscle interstitial space. The relationship between transferrin presence and growth patterns in adult skeletal muscle is not currently understood but evidence suggests that transferrin stimulation of myogenesis observed in vitro may be mediated in vivo by non-muscle cells dwelling within the muscle interstitial space. These cells may act as transferrin-uptake sources for subsequent satellite cell stimulation.     

The effect of reduced activity on the enzymatic development of phasic and tonic muscles in the chicken

The effects of reduced activity (immobilisation) on the development of the contractile enzyme, Mg2+-activated myofibrillar ATPase was studied in a tonic muscle, the anterior latissimus dorsi and in a phasic muscle, the posterior latissimus dorsi of the chicken. Mg2+-activated myofibrillar ATPase activity showed a decreased and delayed activity peak in both the immobilised muscles. Large differences between the two muscles were observed using this marker enzyme. These data indicate that the activity of Mg2+-activated myofibrillar ATPase and the associated differential gene expression involved in fibre type differentiation are influenced by the early activity pattern of the muscles.     

Some characteristics of myotubes cultured from slow and fast chick muscles

Explant cultures were prepared from the slow anterior latissimus dorsi muscle and the fast posterior latissimus dorsi muscle of 15 day chick embryos. The morphology and growth pattern of myotubes from the two types of muscle were very similar. Intracellular microelectrode studies did not reveal consistent differences between the myotube types in regard to resting potential, input resistance, input time constant, or ability to produce active electrogenic responses. It is suggested that specific differentiation of the two muscles is determined by their innervation.     

Turnover of muscle protein in the fowl (Gallus domesticus). Rates of protein synthesis in fast and slow skeletal, cardiac and smooth muscle of the adult fowl.

Rates of protein synthesis in skeletal, cardiac and smooth muscle of fully grown fowl (Gallus domesticus) were determined in vivo by means of the constant infusion method using [14C]proline. In the anterior latissimus dorsi muscle, containing predominantly slow fibres, the average synthesis rate of non-collagen muscle proteins was 17.0 +/- 3.1% per day, a value higher than that obtained for cardiac muscle (13.8 +/- 1.3% per day) and for smooth muscle of the gizzard (12.0 +/- 1.9% per day). In the posterior latissimus dorsi muscle, containing predominantly fast fibres, synthesis rates were much lower (6.9 +/- 1.8% per day). In each case these average rates for the non-collagen protein were similar to the average rate for the sarcoplasmic and myofibrillar protein fractions. The RNA concentration of these four muscles showed that relative rates of protein synthesis were determined mainly by the relative RNA concentrations. The rate of protein synthesis per unit of DNA (the DNA activity) was similar in the two skeletal muscles, but somewhat lower in cardiac muscle and gizzard, possibly reflecting the larger proportion of less active cell types in these two muscles. These quantitative aspects of protein turnover in the two skeletal muscles are discussed in terms of the determination of ultimate size of the DNA unit, and in relation to muscle ultrastructure.     

Differentiation of slow and fast muscles in chickens

1. The development of the characteristic histochemical appearance of the slow anterior latissimus dorsi (ALD) and fast posterior latissimus dorsi (PLD) was studied in chickens during embryonic development as well as during regeneration of minced muscle. 2. During embryonic development the activity of the oxidative enzyme succinic dehydrogenase (SDH) is higher in the slow ALD muscle already at 16 days of incubation. At this time the fast PLD has a higher activity of the glycolytic enzyme, phosphorylase. Although the histochemical appearance of the two types of muscle is already different at 16 days, their contractile speeds are still similar. No difference in myosin ATP-ase was found in the two muscles in young embryos but in 20-day old embryos the two muscles became distinctly different when stained for this enzyme. 3. When PLD muscles in hatched chickens redeveloped during regeneration in place of ALD the histochemical characteristics of the regenerated muscle resembled ALD, and when ALD regenerated in place of PLD it resembled PLD. 4. It is concluded that the histochemical characteristics of slow and fast muscles become determined during early development, even before any difference in contractile properties can be detected and that they are determined by the nerve.     

Influence of neurons on the occurrence of fibre types and myosin light chains in cultured presumptive slow and fast myoblasts

Myoblasts from 9-day-old quail embryo slow anterior latissimus dorsi (ALD) and fast posterior and latissimus dorsi (PLD) muscles were co-cultured with neurons. The presence of neurons allowed ALD-derived muscle fibres to express characteristic features of a slow muscle (occurrence of alpha' and of beta' fibres and predominance of slow myosin light chains). On the contrary, PLD-derived fibres did not differentiate into normal fast fibres (occurrence of alpha'-like fibres and absence of LC3f). These results are compared with the differentiation of ALD and PLD myoblasts in aneural condition. It is suggested that neurons can modify some phenotypic expression of presumptive slow or fast myoblasts.     

Quantitation of myosin in muscle

The amount of myosin per gram of cardiac and skeletal muscle was determined in sodium dodecyl sulfate-solubilized tissue homogenates by radioimmunoassay and by isotope dilution. In the rabbit ventricle, there was an average of 27 mg myosin/g wet wt of tissue. In chickens, the myosin content of typical "red" (anterior latissimus dorsi) and "white" (posterior latissimus dorsi) skeletal muscles was higher than that of ventricular muscle, averaging 36 and 48 mg/g of tissue, respectively. The stoichiometry of the heavy and light chains in cardiac myosin was also determined from the quantitative binding of 125I-labeled Coomassie blue to each subunit after separation of the subunits by sodium dodecyl sulfate-gel electrophoresis. With this procedure, we found that the combined light-chain subunits contributed 19% of the myosin mass. After adjustment for the light-chain contribution, the myosin heavy-chain content of the rabbit ventricle averaged 22 mg/g wet wt of tissue.     

Detection of the nicotinic acetylcholine receptor alpha-subunit mRNA by in situ hybridization at neuromuscular junctions of 15-day-old chick striated muscles.

In adult vertebrate striated muscle, the nicotinic acetylcholine receptor (AChR) is almost exclusively localized in the postsynaptic membrane of the neuromuscular junction. Using in situ hybridization, we show that, in two different chicken muscles [the slow multi-innervated anterior latissimus dorsi (ALD) and the fast singly innervated posterior latissimus dorsi (PLD)], the AChR alpha-subunit mRNA is detected at discrete regions on myofibres and that these regions co-localize (80% correspondence) with neuromuscular junctions identified by histochemical staining for acetylcholinesterase. Moreover, autoradiographic grains densely accumulate on and around subsynaptic nuclei. In contrast, hybridization with an actin probe results in a strong signal distributed over the entire length of the myofibres. Denervation increases the level of AChR alpha-subunit mRNA both in the PLD and to a lesser extent in the ALD. By in situ hybridization we observe that, although a perinuclear pattern is maintained, the labelled nuclei appear randomly distributed among approximately 10% of the nuclei. These results are discussed in a model of AChR gene expression in vertebrate striated muscle fibres.     

Regional injury and the terminal differentiation of satellite cells in stretched avian slow tonic muscle.

In the avian stretch model, the application of a weight overload to the humerus induces enlargement of the anterior latissimus dorsi (ALD) muscle and an increase in muscle fiber number which is accompanied by satellite cell activation. Myofiber injury may be an important stimulus to muscle fiber hyperplasia; therefore, light and electron microscopic evaluation was undertaken to determine if myofiber injury occurs in the stretch-enlarged ALD muscle of the adult quail. Autoradiographic studies were used to determine the terminal differentiation of labeled myogenic cells. A weight equal to 10% of body mass was attached to one wing of 27 adult quail and 3 birds were euthanized at 9 intervals of stretch, from 1 to 30 days. Birds were injected with tritiated thymidine at intervals ranging from 1 hr to 3 days prior to euthanization. Labeled nuclei were detected by light microscopic examination and identified by electron microscopy of a serial section. Three regions of the muscle were examined for disorganization of contractile elements, presence of cytoplasmic vacuoles, and/or phagocytic cell infiltration. The percentage of fibers exhibiting one or more of these criterion was significantly greater in the stretched ALD by Days 5 and 7 and declined at Day 10, reaching near control values by Day 14. Myofiber necrosis and phagocytic cell infiltration were only observed in the middle and distal regions of the stretched ALD muscle. Traditional signs of regeneration and repair were observed, including clusters of labeled myoblast-like cells and myotube formation within an existing basal lamina. New myotube formation with labeled central nuclei was also noted in the interstitial space, outside of basal lamina of persisting fibers. Labeled myonuclei were observed in the stretched fibers. These results demonstrate that chronic stretch produces regional injury and fiber degeneration and resultant regeneration in the ALD muscle of the adult quail. This may be an important stimulus for new fiber formation in this model.     

Functional transformation of fast posterior latissimus dorsi muscle by "slow" nerve implanted in newly hatched chickens.

1. Contraction properties and the activity of Ca2+ - ATPase were investigated 2 and 5 to 6 1/2 months after transposition of the fast posterior latissimus dorsi muscle (PLD) to the other side in newly hatched chickens. At the same time the muscle was cross-innervated by the nerve originally supplying the slow anterior latissimus dorsi muscle (ALD). 2. The mean isometric twitch contraction time of these transposed, cross-innervated PLD muscles in the 2-month-old and 5 to 6 1/2-month-old groups was 61.6 +/- 4.2 msec and 72.5 +/- 10.8 msec respectively. When compared with values obtained in control PLD and ALD muscles (21.9 +/- 0.6 msec and 107.7 +/- 5.6 msec), contraction time was significantly prolonged by this procedure. 3. Ca2+ - ATPase activity was also found to change towards the slow muscle (activity in control PLD was 0.600 micronmoles Pi/mg myosin/min, in the transposed, cross-innervated PLD 0.462 and in control ALD muscle 0.156 respectively). 4. Foreign innervation may thus induce changes in functional and biochemical properties even in muscles considerably different in structure and function, if transformation is allowed to take place at a sufficiently early stage of development. The muscle transposition itself, by introducing the element of muscle dedifferentiation and regeneration, probably assists the transformation process by making the muscle more plastic to the neural influences.     

Synthesis and assembly of native myosin on muscle polyribosomes

We have used the overload-induced growth of avian muscles to study the assembly of the newly synthesized myosins which were separated by non-denaturing pyrophosphate-polyacrylamide gel electrophoresis. Using this model, we have observed the appearance of fast-like isomyosins in polyribosomes prepared from slow anterior latissimus dorsi muscle after 72 h of overload. These new isoforms comigrating with native myosin from fast posterior latissimus dorsi muscle were not yet present in cellular extracts from the same muscle. The in vitro translation system utilizing muscle specific polyribosomes directs the synthesis of the corresponding myosin isoforms. Under denaturing conditions, myosin heavy chains and light chains dissociate to the expected subunit composition of each specific isoform. The synthesis and assembly of native myosin on polyribosomes indicate that myosin exists as a single mature protein prior to the incorporation in the thick filament.     

Acetylcholinesterase in singly and multiply innervated muscles of normal and dystrophic chickens. II. Effects of denervation.

Neural regulation of mature normal fast twitch muscle of the chicken suppresses high activity, extrajunctional localization, and isozyme forms of acetylcholinesterase (AChE) characteristic of embryonic, denervated and dystrophic muscle. Normal adult slow tonic muscle ofthe chicken retains intermediate levels of activity and embryonic isozyme forms but not extrajunctional activity; it is not affected by muscular dystrophy. The hypothesis that neural regulation of the AChE system is lacking in slow tonic muscle and thus not affected by dystrophy was tested by denervating the fast twitch posterior latissimus dorsi and slow tonic anterior latissimus dorsi muscles of normal and dystrophic chickens. Extrajunctional AChE activity and embryonic isozyme forms increased, then declined, in both muscles. The results suggest that ocntrol of AChE is qualitatively similar in slow tonic and fast twitch muscle of the chicken.     

Increased autophagy in chloroquine-treated tonic and phasic muscles: An alternative view

Ultrastructural and cytochemical techniques were used to investigate autophagy in the tonic anterior (ALD) and phasic posterior (PLD) latissimus dorsi muscles of the chicken following chloroquine administration. Autophagic vacuoles were seen in the ALD after 1 day of chloroquine administration while no change was seen in the PLD until 3 days. In both muscles, autophagic vacuoles and myeloid bodies were found at the level of the I band. Myeloid bodies usually were found in the longitudinal rows of mitochondria in the ALD muscle. Some, but not all, of the autophagic vacuoles and myeloid bodies were cytochemically acid phosphatase positive, while the portion of the sarcoplasmic reticulum of both muscles which is normally acid phosphatase positive was devoid of activity following chloroquine administration. These observations are discussed in regard to accepted mechanisms of autophagy and the possible inhibition of autophagy in skeletal muscle tissue by chloroquine.     

Can angiogenesis induced by chronic electrical stimulation enhance latissimus dorsi muscle flap survival for application in cardiomyoplasty?

In cardiomyoplasty, the latissimus dorsi muscle is lifted on its primary neurovascular pedicle and wrapped around a failing heart. After 2 weeks, it is trained for 6 weeks using chronic electrical stimulation, which transforms the latissimus dorsi muscle into a fatigue-resistant muscle that can contract in synchrony with the beating heart without tiring. In over 600 cardiomyoplasty procedures performed clinically to date, the outcomes have varied. Given the data obtained in animal experiments, the authors believe these variable outcomes are attributable to distal latissimus dorsi muscle flap necrosis. The aim of the present study was to investigate whether the chronic electrical stimulation training used to transform the latissimus dorsi muscle into fatigue-resistant muscle could also be used to induce angiogenesis, increase perfusion, and thus protect the latissimus dorsi muscle flap from distal necrosis. After 14 days of chronic electrical stimulation (10 Hz, 330 microsec, 4 to 6 V continuous, 8 hours/day) of the right or left latissimus dorsi muscle (randomly selected) in 11 rats, both latissimus dorsi muscles were lifted on their thoracodorsal pedicles and returned to their anatomical beds. Four days later, the resulting amount of distal flap necrosis was measured. Also, at predetermined time intervals throughout the experiment, muscle surface blood perfusion was measured using scanning laser Doppler flowmetry. Finally, latissimus dorsi muscles were excised in four additional stimulated rats, to measure angiogenesis (capillary-to-fiber ratio), fiber type (oxidative or glycolytic), and fiber size using histologic specimens. The authors found that chronic electrical stimulation (1) significantly (p < 0.05) increased angiogenesis (mean capillary-to-fiber ratio) by 82 percent and blood perfusion by 36 percent; (2) did not reduce the amount of distal flap necrosis compared with nonchronic electrical stimulation controls (29 +/- 5.3 percent versus 26.6 +/- 5.1 percent); (3) completely transformed the normally mixed (oxidative and glycolytic) fiber type distribution into all oxidative fibers; and (4) reduced fiber size in the proximal and middle but not in the distal segments of the flap. Despite the significant increase in angiogenesis and blood perfusion, distal latissimus dorsi muscle flap necrosis did not decrease. This might be because of three reasons: first, the change in muscle metabolism from anaerobic to aerobic may have rendered the muscle fibers more susceptible to ischemia. Second, because of the larger diameter of the distal fibers in normal and stimulated latissimus dorsi muscle, the diffusion distance for oxygen to the center of the distal fibers is increased, making fiber survival more difficult. Third, even though angiogenesis was significantly increased in the flap, cutting all but the single vascular pedicle resulted in the newly formed capillaries not receiving enough blood to provide nourishment to the distal latissimus dorsi muscle. The authors' findings indicate that chronic electrical stimulation as tested in these experiments could not be used to prevent distal latissimus dorsi muscle flap ischemia and necrosis in cardiomyoplasty.     

Immunochemical analysis of protein isoforms in thick myofilaments of regenerating skeletal muscle

The expression of myosin heavy chain (MHC) and C-protein isoforms has been examined immunocytochemically in regenerating skeletal muscles of adult chickens. Two, five, and eight days after focal freeze injury to the anterior latissimus dorsi (ALD) and posterior latissimus dorsi (PLD) muscles, cryostat sections of injured and control tissues were reacted with a series of monoclonal antibodies previously shown to specifically bind MHC or C-protein isoforms in adult or embryonic muscles. We observed that during the course of regeneration in each of these muscles there was a reproducible sequence of antigenic changes consistent with differential isoform expression for these two proteins. These isoform switches appear to be tissue specific; i.e., the isoforms of MHC and C-protein which are expressed during the regeneration of a "slow" muscle (ALD) differ from those which are synthesized in a regenerating "fast" muscle (PLD). Evidence has been obtained for the transient expression of a "fast-type" MHC and C-protein during ALD regeneration. Furthermore, during early stages of PLD regeneration this muscle contains MHCs which antigenically resemble those found in the pectoralis muscle at embryonic and early posthatch stages of development. Both regenerating muscles express an isoform of C-protein which appears immunochemically identical to that normally expressed in embryonic and adult cardiac muscle. These results support the concept that isoform transitions in regenerating skeletal muscles qualitatively resemble those found in developing muscles but differences may exist in temporal and tissue-specific patterns of gene expression.     

Regionalized adaptations and muscle fiber proliferation in stretch-induced enlargement

The relative contribution of increases in fiber area to stretch-induced muscle enlargement was evaluated in the slow tonic fibers of the anterior latissimus dorsi of adult Japanese quails. A weight corresponding to 10% of the bird's body mass was attached to one wing. Thirty days of stretch in 34 birds averaged 171.8 +/- 13.5% increase in muscle mass and 23.5 +/- 0.8% increase in muscle fiber length. The volume density of noncontractile tissue increased in middle and distal regions of stretch-enlarged muscles. Mean fiber cross-sectional area increased 56.7 +/- 12.3% in the midregion of stretched muscles. Further analysis indicated slow beta-fiber hypertrophy occurred in proximal, middle, and distal regions; however, fast alpha-type fiber hypertrophy was limited to middle regions of stretched muscles. Stretched muscles had a significant increase in the frequency of slow beta-fibers that were less than 500 microns 2 in all regions and fast alpha-type fibers in middle and distal regions. Total fiber number was determined after nitric acid digestion of connective tissue in 10 birds. Fiber number increased 51.8 +/- 19.4% in stretched muscle. These results are the first to clearly show that muscle fiber proliferation contributes substantially to adult skeletal muscle stretch-induced enlargement, although we do not know whether the responses of the slow tonic anterior latissimus dorsi might be similar or different from mammalian twitch muscle.     

Quantification and origin of spindles in orthotopic and heterotopic grafts of avian muscles

Summary Regeneration of muscle spindles was quantified in a series of orthotopically and heterotopically autografted muscles of pigeons. Significantly fewer spindles relative to numbers of extrafusal fibers were present in grafts than in normal muscles. These results are in marked contrast to observations of free-grafted muscles of rats. A majority of grafts of the metapatagialis, a muscle devoid of spindles, into the site of the anterior latissimus dorsi contained spindles. A few spindles were present in grafts of the extensor digitorum communis, which normally contains many spindles, into the site formerly occupied by the metapatagialis whereas muscle spindles were absent in orthotopic grafts of the metapatagialis muscle. These observations suggest that the spindle-like structures observed in the extensor digitorum communis muscles, which regenerated in the sites of the metapatagialis, were derived from spindles of the donor muscle. Thus muscle spindles in transplanted avian muscle can form by two distinct developmental processes.This investigation was supported in part by research grants 1RO1AM26992 from the Public Health Service and PCM 79-16540 from the National Science Foundation