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.     

Ultrastructural transformation of fast chicken muscle fibres induced by nerve cross-union

Summary The fast posterior latissimus dorsi (PLD) muscle of newly hatched chickens was transposed and cross-innervated by the slow-type nerve originally innervating the anterior latissimus dorsi (ALD) muscle. The innervation and the ultrastructure of the cross-innervated posterior latissimus dorsi (PLD-X) muscle was investigated from one week up to 18 months after the operation and compared with that of the control fast (PLD-C) and control slow (ALD-C) muscles. All nerve terminals in the PLD-X muscle were of the slow type. Yet the degree of ultrastructural transformation differed from fibre to fibre. Only about 30% of PLD-X fibres had transformed ultrastructure closely resembling the control slow fibres. In this group of maximally altered fibres, the myofibrils had large diameters, wide Z lines and indistinct M lines as the control slow fibres. The amount of mitochondria was increased to levels found in control slow fibres. The mean percentage of triads was also comparable to that of control slow fibres, being approximately by two thirds lower than in control fast fibres.The differences in the degree of ultrastructural transformation are presumably due to different plasticity of muscle cells at the time of cross-innervation. In the transposed PLD-X muscles large areas undergo degeneration and regeneration. It is suggested that an almost complete changeover of the fibre type is only brought about after cross-innervation of newly differentiating muscle cells, whereas partial alteration occurs after reinnervation of young myofibres.The skillful technical assistance of Dr. Z. Liková, Mrs. M. Sobotková, Ing. M. Doubek and Mr. H. Kunz is gratefully acknowledged.     

EFFECTS OF NEUROMUSCULAR BLOCKING AGENTS ON THE DIFFERENTIATION OF NERVE-MUSCLE CONNECTIONS IN SLOW AND FAST CHICK MUSCLES

The effects of neuromuscular blocking drugs on the development of slow and fast muscle fibres and their neuromuscular junctions was studied in chick embryos.
Treatment of embryos with the depolarizing neuromuscular blocking agent suxamethonium affected the development of muscle fibres of the slow anterior latissimus dorsi (ALD) muscle more than that of muscle fibres of the posterior latissimus dorsi (PLD). The differentiation of the presynaptic elements of the neuromuscular junction was delayed and this was particularly obvious in PLD. Normally the number of axon profiles at individual endplates is reduced by 18 days of incubation, but in suxamethonium treated embryos this reduction took place only at 21 days. During earlier stages of development the axon profiles from treated embryos were small with sparse synaptic vesicles. Nevertheless the subsynaptic site of endplates on ALD and PLD muscle fibres became specialized earlier than normal and to a greater extent. Treatment with hemicholinium (HC-3), a drug that reduces the synthesis of acetylcholine (ACh) in nerve terminals affected the development of PLD muscle fibres more than ALD muscle fibres. Although in HC-3 treated embryos nerve-muscle contacts were formed, the axon terminals look immature and remain small even in 18-day old embryos at both ALD and PLD muscle fibres. The reduction of the number of axon profiles normally seen at 18 days failed to take place in treated embryos. At 18 days of incubation many endplates on PLD muscle fibres showed little sign of postsynaptic specilization and resembled endplates usually seen at this stage on ALD muscle fibres.
It is concluded that while neuromuscular activity may be important for the reduction of the number of axon profiles at individual endplates, the specialization of the subsynaptic membrane is brought about by depolarizing effect of ACh.     

In vitro regulation of the innervation pattern of quail muscle fibres by quail and mouse neurons

Myoblasts from rudiments of slow and fast muscle, anterior latissimus dorsi (ALD) and posterior latissimus dorsi (PLD) respectively, of 9-day-old quail embryos were cultured in vitro for a period of up to 60 days in order to give rise to well-differentiated muscle fibres. These fibres were innervated by neurons from either quail or mouse embryo spinal cord and their innervation pattern was examined by the visualization of acetylcholine receptors (ACh-R) and of acetylcholinesterase (ACh-E) activity at the neuromuscular contacts. In the culture system used, quail neurons always innervated muscle fibres at several sites and only when a fast-type activity was imposed on these neurons did a reduction in the number of the previously established neuromuscular contacts take place. In contrast, in the muscle fibres innervated by mouse neurons, a spontaneous reduction in the number of the previously established neuromuscular contacts occurred but this spontaneous reduction depended upon the level of differentiation reached by the muscle fibres in vitro. In the cultures of muscle fibres previously innervated by mouse neurons, the addition of quail neurons did not provoke any modification in the initial innervation pattern, and no quail ACh-R cluster was observed. In contrast, in the muscle fibres previously innervated by quail neurons, the mouse neurons contacted these fibres, resulting in a decrease in the number of quail ACh-R clusters. These results emphasize the part played by neurons in the establishment of the innervation pattern when muscle fibres have reached a high level of differentiation. In vitro, the slow and fast characteristics of the muscle fibres do not influence this pattern.     

Developmental changes in myosin isoforms from slow and fast latissimus dorsi muscles in the chicken

In the course of muscle differentiation, changes in fibre-type population and in myosin composition occur. In this work, the expression of native myosin isoforms in developing fast-twitch (posterior latissimus dorsi; PLD) and slow-tonic (anterior latissimus dorsi; ALD) muscles of the chick was examined using electrophoresis under nondissociating conditions. The major isomyosin of 11-day-old embryonic PLD comigrated with the adult fast myosin FM3. Two additional components indistinguishable from adult fast FM2 and FM1 isomyosins appeared successively during the embryonic development. The relative proportion of these latter isoforms increased with age, and the adult pattern was established by the end of the 1st month after hatching. Between day 11 and day 16 of embryonic development, PLD muscle fibres also contained small amounts of slow isomyosins SM1 and SM2. This synthesis of slow isoforms may be related to the presence of slow fibres within the muscle. At all embryonic and posthatch stages, ALD was composed essentially of slow isomyosins that comigrated with the two slow components SM1 and SM2 identified in adult. Several studies have reported that the SM1:SM2 ratio decreases progressively throughout embryonic and posthatching development, SM2 being predominant in the adult. In contrast, we observed a transient increase in SM1:SM2 ratio at the end of embryonic life. This could reflect a transitional neonatal stage in myosin expression. In addition, the presence in trace amounts of fast isomyosins in developing ALD muscle could be related to the presence of a population of fast fibres within this muscle.     

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.     

Effects of electrical stimulation upon post-hatching development of fibre types in normally innervated fast and slow latissimus dorsii muscles of the chicken

In chicken, the main characteristic properties of muscle fibre types in slow anterior (ALD) and fast posterior (PLD) latissimus dorsii are acquired during post-hatching development. At day 4 it becomes possible to distinguish between alpha' and beta' fibre types in ALD muscle. At the same time, mATPase staining and NADH-TR activity permit recognition of alpha w and alpha R fibres within PLD muscle. During further development, muscle fibre typology progressively changes towards the adult slow and fast type. Chronic stimulation at a slow rhythm (5 Hz) of PLD prevents the change in relative proportions of alpha R and alpha W fibres within the muscle that occurs in normal post-hatching development and increases the number of beta R fibres. Moreover, oxidative activity is increased in all muscle fibre types following stimulation. In ALD muscle, chronic stimulation at a fast rhythm (40 Hz) results in a decrease in oxidative activity and inhibits the differentiation of alpha' and beta' muscle fibre types. This study demonstrates that in young chicken, the pattern of activity influences the differenciation of fibre types in slow and fast muscles.     

Effect of denervation and tenotomy on slow and fast muscles of the chicken

Summary Changes of muscle weights, fiber diameters and ultrastructure were studied in the slow anterior latissimus dorsi (ALD) and in the fast posterior latissimus dorsi (PLD) of the chick three weeks after denervation and tenotomy, and after combined denervation and tenotomy of the two muscles.The slow ALD muscle becomes hypertrophic after denervation (Feng, Jung and Wu, 1962). Three weeks after nerve section, wet weights of ALD muscles are increased by 60% and fiber diameters become by 30% larger than those of contralateral control muscles. In spite of this hypertrophy, degenerative changes are seen in the ultrastructure, similar to those described in denervated atrophic muscles. Areas of dedifferentiation with autophagic vacuoles and aggregates of tubules are found in superficial layers of some fibers. Disintegration of Z lines and filaments along one or two sarcomeres occurs in a number of myofibrils, especially in muscles of young animals.In contrast to denervation alone, simultaneous denervation and tenotomy of the ALD muscles results in atrophy. Decrease of muscle weights and reduction of fiber diameters are similar as after tenotomy; in both cases muscle fibers waste by degeneration and atrophy of myofibrils.The fast PLD muscles underwent extensive atrophy in all three series of experiments. Corresponding atrophic and degenerative changes of ultrastructure were found in all instances.The authors wish to acknowledge gratefully the skillful technical assistance of Mrs. M. Sobotková and Ing. M. Doubek, and editorial assistance of Miss Virginia Hamilton.     

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.     

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.     

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.     

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.     

Developmental modulation of myosin expression by thyroid hormone in avian skeletal muscle.

It is well established that a rise in circulating thyroid hormone during the second half of chick embryo development significantly influences muscle weight gain and bone growth. We studied thyroid influence on differentiation in slow anterior latissimus dorsi (ALD) and fast posterior latissimus dorsi (PLD) muscles of embryos rendered hypothyroid by hypophysectomy or administration of an anti-thyroid drug. The expression of native myosins and myosin light chains (MLCs) was studied by electrophoretic analysis, and the myosin heavy chain (MHC) was characterized by immunohistochemistry. The first effects of hypothyroid status were observed at day 21 of embryonic development (stage 46 according to Hamburger and Hamilton). Analysis of myosin isoform expression in PLD muscles of hypothyroid embryos showed persistence of slow migrating native myosins and slow MLCs as well as inhibition of neonatal fast MHC expression, indicating retarded differentiation of this muscle. In ALD muscle, hypothyroidism maintained fast embryonic MHC and induced noticeable amounts of fast MLCs, thus delaying slow muscle differentiation. Our results suggest that thyroid hormones play a role in modulating the appearance of neonatal fast MHC and the disappearance of isomyosins transiently present during embryogenesis. However, T3 supplemental treatment would seem to compensate in part for the effects of hypothyroidism induced by hypophysectomy, suggesting that thyroid hormone might interfere with other factors also accounting for the observed effects.     

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.     

Change in motor neurone activity modifies the differentiation of a slow muscle in chick embryo

Slow-tonic anterior latissimus dorsi (ALD) muscle properties were studied following chronic spinal cord stimulation in chick embryo. Stimulation at a fast rhythm was applied from day 7, 8 or 10 of development until the end of embryonic life. When stimulation was applied from day 7 up to day 18 of development, ALD muscle exhibited at day 18 a large decrease in half time to peak of tetanic contraction, a large proportion of fast type II fibres and an increase in fast myosin light chain content as compared to control muscle. When stimulation started at day 8 of development, changes in properties of ALD muscle were reduced when compared to the previous experimental series. Indeed, no fast type II fibres were observed within the muscle, even when stimulation was prolongated until the 20th day of embryonic development. In addition, chronic stimulation at a fast rhythm initiated at day 10 of development did not modify ALD muscle differentiation. The present results indicate that a fast pattern of motor neurone activity can induce some slow-to-fast transformations of ALD muscle fibres. However, after the first week of embryonic life, ALD myotubes appeared refractory to these transformations. The possible mechanisms responsible for the transformation of slow myotubes and for their further loss of plasticity are discussed.     

Cholinesterase Activity of Myoneural Junctions from Twitch and Tonic Muscles of the Domestic Fowl

THE posterior latissimus dorsi (PLD) muscle of the chick consists of an almost homogeneous population of “twitch” fibres whereas the anterior latissimus dorsi (ALD) consists of “tonic” fibres¹. Twitch and tonic fibres differ essentially in the time course of contraction but there is also evidence of differences in intracellular organization² and in metabolism³.     

Potential phasic and tonic muscles express a common set of fast and slow myosin light chains and fast tropomyosin during early development of chick embryo

We investigated the expression of myosin light chains and tropomyosin subunits during chick embryonic development of the anterior (ALD) and posterior (PLD) parts of the latissimus dorsi muscles. As early as day 8 in ovo, both muscles accumulate a common set of myosin light chains (LC) in similar ratios (LC1F: 55 per cent; LC2S: 25 per cent; LC2F: 12 per cent; LC1S: 8 per cent) and a common set of tropomyosin (TM) subunits (beta 2, beta 1, alpha 2F). Later during development, the slow components of the LC regularly disappear in the PLD and the fast components of the LC and the alpha 2FTM disappear in the ALD, so that the adult pattern is almost established at the time of hatching. Thus, early in development, the two muscles accumulate a common set of fast and slow myosin light chains and fast tropomyosin and some isoforms are repressed at a later stage during development. These data might suggest that during development, the regulatory mechanisms of muscle specific isoform expression differ from one contractile protein to another.     

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.     

Characterization of skeletal muscles by MR imaging and relaxation times

Magnetic resonance (MR) images of three major flight muscles of chicks were obtained with surface coils using a 0.3 Tesla whole body imaging system (FONAR Beta 3000). The two fast muscles, pectoralis major (PM) and posterior latissimus dorsi (PLD), and a slow muscle, anterior latissimus dorsi (ALD), were identified in the axial, coronal, and sagittal images. The signal intensity (SI) of each muscle was electronically measured and its ratio to the background noise (S/N) was determined. Although visually the three muscles showed intermediate SI, the slow and fast muscles could be differentiated on the basis of their S/N values. These values were invariably higher in the slow muscles than in the fast muscles. To understand these differences, the muscles were excised and their mono- and multiexponential MR relaxation times (T1 and T2) were determined at 30 MHz. Multiexponential analysis enhanced the differences between the muscle types. With the sole exception of short T2, all relaxation components of the slow muscles were significantly longer than those of the fast muscles. These results suggest that elevation in the S/N, T1 and T2 values of muscles may not necessarily indicate a pathologic event, but may reflect the preponderance of slow fibers.     

鸡前、后背阔肌去神经后对辣根过氧化物酶(HRP)的胞纳作用

本文报道了用辣根过氧化物酶(HRP)作为大分子标记物,以组织化学和生物化学方法观察了鸡前后背阔肌在去神经后的胞纳现象。结果表明,在去神经后发生肥大的前背阔肌和去神经后发生萎缩的后背阔肌同样都出现胞纳的明显增加。组织化学所观察的结果表明,浸泡在含 HRP 的任氏液中的有完整神经支配的前、后背阔肌只有极少数的肌纤维摄取 HRP,而在去神经的前、后背阔肌中则有不少的肌纤维内部出现 HRP 染色反应。这种反应在有的纤维表现为弥散性染色,有的表现为浓的 HRP 反应颗粒。生物化学的结果显示,去神经后的前、后背阔肌中 HRP 的相对含量分别比有神经支配的对照肌肉明显地增多54%和87%,我们在鸡前背阔肌用组织化学和生物化学所得的实验结果与 Thesleff 等人提出的关于肌肉萎缩机制的假设显然不符。本工作证实了肌肉的胞纳作用的增加并不一定最终导致肌纤维的变性和萎缩。