Numbers of myonuclei and satellite cell nuclei in latissimus dorsi muscles of the chicken

Summary In the 3-, 33- and 66-day-old chicken, two muscles, the oxidative slow tonic anterior latissimus dorsi and the glycolytic fast twitch posterior latissimus dorsi were compared by the measurement of muscle fibre diameter and the fraction of total muscle tissue nuclei which were either myonuclei or satellite cell nuclei. Between 3 and 33 days there was a period of rapid growth (more marked in the posterior latissimus dorsi) which coincided with a sharp fall in numerical density of myonuclei and satellite cell nuclei (number per cubic millimetre muscle tissue). The fraction of all nuclei which were satellite cell nuclei declined steadily.The higher levels of myonuclei and satellite cell nuclei in the anterior latissimus dorsi were thought to be a reflection of its oxidative metabolism and the presence of multiple endplates.The volume of sarcoplasm occupied by single myonuclei in anterior and posterior latissimus dorsi muscles was shown to be considerably greater than that occupied by nuclei in other cell systems.     

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.     

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.     

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.     

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.     

Effects of Electrical Stimulation on Molecular Forms of Butyrylcholinesterase in Denervated Fast and Slow Latissimus Dorsi Muscles of Newly Hatched Chicken

The effects of denervation and direct electrical stimulation upon the activity and the molecular form distribution of butyrylcholinesterase (BuChE) were studied in fast-twitch posterior latissimus dorsi (PLD) and in slow-tonic anterior latissimus dorsi (ALD) muscles of newly hatched chicken. In PLD muscle, denervation performed at day 2 substantially reduced the rate of rapid decrease of BuChE specific activity which takes place during normal development, whereas in the case of ALD muscle little change was observed. Moreover, the asymmetric forms which were dramatically reduced in denervated PLD muscle were virtually absent in denervated ALD muscle at day 14. Denervated PLD and ALD muscles were stimulated from day 4 to day 14 of age. Two patterns of stimulation were applied, either 5-Hz frequency (slow rhythm) or 40-Hz frequency (fast rhythm). Both patterns of stimulation provided the same number of impulses per day (about 61,000). In PLD muscle, electrical stimulation almost totally prevented the postdenervation loss in asymmetric forms and led to a decrease in BuChE specific activity. In ALD muscle, electrical stimulation partially prevented the asymmetric form loss which occurs after denervation. This study emphasizes the role of evoked muscle activity in the regulation of BuChE asymmetric forms in the fast PLD muscle and the differential response of denervated slow and fast muscles to electrical stimulation.     

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.     

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.     

In vitro differentiation in the absence of nerve of avian myoblasts derived from slow and fast muscle rudiments

Slow anterior latissimus dorsi (ALD) and fast posterior latissimus dorsi (PLD) muscles of 9-day-old quail embryos were cultured in vitro without neurons for 1 to 12 weeks. Several differences could be observed between ALD- and PLD-derived cells. PLD cultures proliferated less rapidly than ALD cultures. ALD-derived muscle fibres exhibited wide Z lines, numerous mitochondria, and a poorly developed sarcotubular system, while PLD-derived muscle fibres exhibited narrow Z lines, few mitochondria, and an abundant sarcotubular system. Staining for myofibrillar ATPase revealed that all well-differentiated ALD-derived muscle fibres were of the beta' type, while PLD-derived fibres were of beta and beta R types. These results show that myoblasts from slow and fast muscle rudiments can express in vitro some of the characteristic features of slow and fast muscle fibres, independently of motor innervation.     

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.     

Posthatching changes in levels and molecular forms of acetylcholinesterase in slow and fast muscles of the chicken: Effects of denervation and direct electrical stimulation

The evolution of acetylcholinesterase (AChE) activity and AChE molecular form distribution were studied in slow-tonic anterior latissimus dorsi (ALD) and in fast-twitch posterior latissimus dorsi (PLD) muscles of chickens 2-18 days of age. In ALD as well as in PLD muscles, the AChE-specific activity increased transiently from day 2 to day 4; the activity then decreased more rapidly in PLD muscle. During this period asymmetric AChE forms decreased dramatically in ALD muscle and the globular forms increased. In PLD muscle, the most striking change was the decline in A8 form between days 2 and 18 of development. Denervation performed at day 2 delayed the normal decrease in AChE-specific activity in PLD muscle, whereas little change was observed in ALD muscle. Moreover, A forms in these two muscles were virtually absent 8 days after denervation. Direct electrical stimulation depressed the rise in AChE-specific activity in denervated PLD muscle and prevented the loss of the A forms. Furthermore, the different molecular forms varied according to the stimulus pattern. In ALD muscle, electrical stimulation failed to prevent the effect of denervation. This study emphasizes the differential response of denervated slow and fast muscles to electrical stimulation and stresses the importance of the frequency of stimulation in the regulation of AChE molecular forms in PLD muscle during development.     

Characterization of the C-protein from posterior latissimus dorsi muscle of the adult chicken: heterogeneity within a single sarcomere

Specific isoforms of myofibrillar proteins are expressed in different muscles and in various fiber types within a single muscle. We have isolated and characterized monoclonal antibodies against C-proteins from slow tonic (anterior latissimus dorsi, ALD) and fast twitch (pectoralis major) muscles of the chicken. Although the antibody against "fast" C-protein (MF-1) did not bind to the "slow" isoform and the antibody to the "slow" C-protein (ALD-66) did not bind to the "fast" isoform, we observed that both antibodies bound C-protein from the posterior latissimus dorsi (PLD) muscle. Here we demonstrate that in the PLD muscle the binding sites of these two antibodies reside in two different C-protein isoforms which have different molecular weights and can be separated by hydroxylapatite column chromatography. Since we have shown previously that both these antibodies stain all myofibers and myofibrils derived from PLD muscle, we conclude that all myofibers in this muscle contain both isoforms with all sarcomeres.     

Immunological cross-reactivity between chicken slow skeletal and ventricular muscle myosin

Antibodies elicited in rabbits against chicken slow skeletal anterior latissimus dorsi and ventricular myosin were analyzed by double immunodiffusion for their ability to react with homologous and heterologous antigen at different stages of immunization (1--12 months). Each anti myosin antiserum formed a single, strong precipitin line with its immunogen after short time of immunization. This reaction was specific for myosin heavy chains as determined by GEDELISA (gel electrophoresis derived enzyme lined immunosorbent assay) test. In rabbits injected with ventricular myosin after long time of immunization a second, fainter precipitin line has generally been observed. The antigenic determinants responsible for this precipitin line have been localized on the light myosin subunits. By comparing the two types of anti myosin antisera with heterologous antigen we have obtained evidence for partial immunological cross-reactivity between slow skeletal and ventricular muscle myosins. In particular, all anti ventricular myosin antisera displayed a marked immunological reactivity with anterior latissimus dorsi myosin whereas most of anti anterior latissimus dorsi myosin antisera showed absence of reciprocity. By means of immunofluorescence and immunoabsorption techniques both common and unique slow skeletal and ventricular antigenic determinants have been demonstrated.     

Comparative analysis of chicken atrial and ventricular myosins.

1. Structural and enzymic properties of myosins from atrial and ventricular cardiac muscle of the chicken were investigated and compared with myosins from the fast skeletal pectoralis and the slow skeletal anterior latissimus dorsi muscle. 2. The Ca2+-ATPase activity, both in function of pH and [K+], of atrial myosin closely resembled that of the fast pectoralis myosin, whereas the enzymic properties of ventricular myosin were similar to those of slow skeletal myosin. 3. By sodium dodecyl sulphate polyacrylamide gel electrophoresis on gradient gel and two-dimensional electrophoresis, involving isoelectric focusing in the first dimension and SDS gel electrophoresis in the second dimension, no difference could be demonstrated in the light-chain pattern of atrial and ventricular myosin. Complete identity was also found between anterior latissimus dorsi and cardiac light chains. 4. Electrophoretic analysis of soluble peptides released by tryptic digestion of myosin and electron microscopic study of light meromyosin paracrystals showed significant differences between the heavy chains of atrial and ventricular myosins, as well as between the heavy chains of cardiac and skeletal myosins. 5. The results confirm previous immunochemical findings and provide direct biochemical evidence for the existence of a new, unique type of myosin in the chicken atrial tissue.     

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.     

Acetylcholinesterase in chick embryo latissimus dorsii muscles: effects of curarization and electrical stimulation

The accumulation of acetylcholinesterase (AChE), the changes in AChE-specific activity and in AChE molecular form distribution were studied in slow-tonic anterior latissimus dorsi (ALD) and in fast-twitch posterior latissimus dorsi (PLD) muscles of the chick embryo. From stage 36 (day 11) to stage 42 (day 17) of Hamburger and Hamilton, the AChE-specific activity decreased, while the relative proportion of asymmetric A 12 and A 8 forms increased. Repetitive injection of curare resulted at stage 42 (day 17) in a decrease in AChE-specific activity, in the accumulation of the synaptic AChE and in the expression of AChE asymmetric forms. Electrical stimulation at a relatively high frequency (40 Hz) of curarized ALD and PLD muscles resulted in a normal increase in AChE asymmetric forms, whereas a lower frequency (5 Hz) resulted in a dominance of globular forms. Both patterns of stimulation partly prevented the loss in synaptic AChE accumulations. These results suggest that in chick embryo muscles, muscle activity and its rhythms are involved in the normal evolution of AChE.     

Immunocytochemical localization of aldolase in normal, denervated, and dystrophic chicken muscles

To investigate whether immunocytochemical localization of muscle-specific aldolase can be used for fiber phenotype determination, we produced specific antibodies against the enzyme and studied its distribution in adult chicken skeletal muscles by indirect immunofluorescence microscopy. Monoclonal antibodies against the myosin heavy chains of fast-twitch (MF-14) and slow-tonic (ALD-58) muscle fibers were also used to correlate aldolase levels with the fiber phenotype. The goat anti-aldolase antibody was found to be specific for the A form of aldolase, as evidenced by sodium dodecyl sulfate gel electrophoresis, immunotitration experiments, and immunoblot analysis. The antibody reacted strongly with the fast-twitch myofibers of normal pectoralis and posterior latissimus dorsi muscles; the phenotype of these muscle fibers was confirmed by a positive immunofluorescent reaction after incubation with MF-14 antibody. By contrast, the slow-tonic myofibers of normal anterior latissimus dorsi, which react positively with ALD-58 antibody, reacted weakly with anti-aldolase antibodies. In denervated chicken muscles, reaction to anti-aldolase antibodies was markedly reduced in fast-twitch fibers, although reaction to MF-14 was not diminished. By contrast, in dystrophic muscle, fast-twitch fibers showed reduced reactivity to anti-aldolase and marked to moderate reduction in MF-14 reactivity. Our results show that: (a) in normal muscles, reactivity to anti-aldolase matches the phenotype obtained by using anti-fast or anti-slow myosin heavy chain antibodies, and therefore can serve to identify mature fibers as fast or slow; and (b) in denervated or dystrophic muscles, the intracellular expressions of aldolase and fast-twitch myosin heavy chains are regulated independently.     

Isoforms of C-protein in adult chicken skeletal muscle: detection with monoclonal antibodies

Monoclonal antibodies (McAbs) specific for the C-proteins of chicken pectoralis major and anterior latissimus dorsi (ALD) muscles have been produced and characterized. Antibody specificity was demonstrated by solid phase radioimmunoassay (RIA), immunoblots, and immunofluorescence cytochemistry. Both McAbs MF-1 (or MF-21) and ALD-66 bound to myofibrillar proteins of approximately 150,000 daltons; the former antibody reacted with pectoralis but not ALD myofibrils, whereas the latter recognized ALD but not pectoralis myofibrils. Chromatographic elution of the antigens from DEAE-Sephadex, and their distribution in the A-band, support the conclusion that both of these antibodies recognize variant isoforms of C-protein. Since both McAbs react with a protein of similar molecular weight in the A-band of all myofibrils of the posterior latissimus dorsi (PLD) muscle, we suggest that either another isoform of C-protein exists in the PLD muscle or both pectoralis and ALD-like isoforms coexist in the A-bands of PLD muscle.     

Embryonic-like myosin heavy chains in regenerating chicken muscle

An antibody to chicken ventricular myosin was found to cross-react by enzyme immunoassay with myosin heavy chains from embryonic chicken pectorials, but not with adult skeletal myosins. This antibody, which was previously shown to label cultured muscle cells from embryonic pectoralis (Cantini et al., J cell biol 85 (1981) 903), was used to investigate by indirect immunofluorescence the reactivity of chicken skeletal muscle cells differentiating in vivo during embryonic development and muscle regeneration. Muscle fibers in 11-day old chick embryonic pectoralis and anterior latissimus dorsi muscles showed a differential reactivity with this antibody. Labelled fibers progressively decreasgd in number during subsequent stages and disappeared completely around hatching. Only rare small muscle fibers, some of which had the shape and location typical of satellite elements, were labelled in adult chicken muscle. A cold injury was produced with dry ice in the fast pectoralis and the slow anterior latissimys dorsi muscles of young chickens. Two days after injury a number of labelled cells was first seen in the intermediate region between the outer necrotic area and the underlying uninjured muscle. These muscle cells rapidly increased in number and size, thin myotubes were seen after 3 days and by 4–5 days a superficial layer of brightly stained newly formed muscle fibers was observed at the site of the injury. Between one and two weeks after the lesion the intensity of staining of regenerated fibers progressively decreased as their size further increased. These findings indicate that an embryonic type of myosin heavy chain is transitorily expressed during muscle regeneration.