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.     

Phosphorylation in vivo of the P light chain of myosin in rabbit fast and slow skeletal muscles.

The P light chain of myosin is partially phosphorylated in resting slow and fast twitch skeletal muscles of the rabbit in vivo. The extent of P light-chain phosphorylation increases in both muscles on stimulation. Rabbit slow-twitch muscles contain two forms of the P light chain that migrate with the same electrophoretic mobilities as the two forms of P light chain in rabbit ventricular muscle. The rate of phosphorylation of the P light chain in slow-twitch muscle is slower than its rate of phosphorylation in fast-twitch muscles during tetanus. The rate of P light-chain dephosphorylation is slow after tetanic contraction of fast-twitch muscles in vivo. The time course of dephosphorylation does not correlate with the decline of post-tetanic potentiation of peak twitch tension in rabbit fast-twitch muscles. The frequency of stimulation is an important factor in determining the extent of P light-chain phosphorylation in fast- and slow-twitch muscles.     

Properties of standard avian slow muscle grafts following long-term regeneration

The tonic anterior latissimus dorsi (ALD) of adult pigeons was orthotopically homografted and evaluated after 11 months of regeneration for histological, histochemical, electromyographic (EMG), and mechanical properties. The resting EMG activity of the grafts was lower in amplitude than that of the controls, but showed the tonic pattern typical for these tonic muscles. The control and grafted muscles had a histochemically homogeneous population of fibers with moderate myofibrillar adenosine triphosphatase activity. Succinic dehydrogenase activity was moderate for the control muscles, but low for the grafts. The regenerated muscles had fewer and smaller fibers and had much larger intersynaptic distances. Both the regenerated and the contralateral control muscles were slow contracting and maintained tetanic tension for prolonged periods with direct electrical stimulation. The relaxation was slower in the grafted muscle than in the control. The grafts produced 40% of the maximum tension of the control muscles, but the rate of tension development was similar between the two groups. The results indicate that the tonic properties were regenerated, but the innervation pattern was altered and the grafted muscles did not have normal mature fibers even after long-term regeneration.     

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.     

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.     

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.     

Phosphorylation of rabbit skeletal muscle myosin in situ

Myosin light chain (P light chain) is phosphorylated by Ca2+ X calmodulin-dependent myosin light chain kinase. Based on studies with rat skeletal muscles, it has been shown that P light chain phosphorylation correlated to the extent of potentiation of isometric twitch tension. It is not clear whether this correlation exists in rabbit skeletal muscle, which has been the primary source of contractile proteins for biochemical studies. Therefore, phosphorylation of myosin P light chain in rabbit slow-twitch soleus and fast-twitch plantaris muscles in situ was examined. Electrical stimulation (5 Hz, 20 seconds) of plantaris muscle produced an increase in the phosphate content of P light chain from 0.17 to 0.45 mol phosphate/mol P light chain. This increase in phosphate content was accompanied by a 58% increase in maximal isometric twitch tension. Tetanic stimulation (100 Hz, 15 seconds) of rabbit soleus muscle resulted in only a small increase in P light chain phosphate content from 0.02 to 0.10 mol phosphate/mol P light chain, and posttetanic twitch tension did not increase significantly. The correlation between potentiated isometric twitch tension and P light chain phosphorylation in rabbit fast-twitch muscle is similar to that observed in rat skeletal muscle. These results were consistent with the hypothesis that phosphorylation of rabbit skeletal muscle myosin, which results in an increase in actin-activated ATPase activity, may be related to isometric twitch potentiation.     

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.     

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.     

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.     

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.     

Effect of curarization on sensitivity of chicken latissimus dorsi to electric current]

(1.) To test the sensitivity to electric current of the latissimus dorsi and, therefore, its ability to yield action potential, we have studied the effect of curarisation on the amplitude of the isometric tetanus during "massive" stimulations by alternative current. (2.) Curarisation causes a considerable but reversible reduction of the tetanic tension of the latissimus dorsi anterior (L.D.A.) but has no effect on the tetanus of the latissimus dorsi posterior (L.D.P.). It is concluded that a part of the L.D.A. is not sensitive to electric current and is thus unable to yield action potentials, unlike the opinion of some authors. (3.) Rising the temperature has an inverse effect on the speed of the tetanus ascending phase, depending on whether the L.D.A. has been curarised or not. This show the existence of two types of excitation processes of very different nature in this muscle. (4.) Direct current is much less efficient than alternative current on the curarised L.D.A.     

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.     

A histochemical study of twitch and tonus fibers

The distribution of glycogen, lipids and succinic dehydrogenase (SDH) in twitch and tonus fibers of several amphibians and birds is described, and the correlation of histochemical properties with fiber structure and function is discussed. Twitch and tonus fibers were identified histologically by the presence of Fibrillenstruktur and Felderstruktur respectively. The rectus abdominis, sartorius and semitendinosus were studied in Rana pipiens, Xenopus laevis and Necturus maculosus; the pectoralis major, pectoralis minor, anterior latissimus dorsi and posterior latissimus dorsi were investigated in Gallus gallus and Passer domesticus. Periodic acid-Schiff was used to stain for glycogen, Sudan Black B for lipids and Nitro BT for localization of SDH activity. In amphibian muscles, fibers with Fibrillenstruktur and Felderstruktur constitute the rectus abdominis. Except in one case, only Fibrillenstruktur fibers were seen in the sartorius and semitendinosus. In the avian muscles, fibers with Fibrillenstruktur comprise the pectoralis major, pectoralis minor and posterior latissimus dorsi, while fibers with Felderstruktur constitute the anterior latissimus dorsi. These types of muscle fibers showed no consistent pattern in the distribution of glycogen, lipids and SDH. The evidence precludes the use of such data alone for distinguishing twitch (Fibrillenstruktur) and tonus (Felderstruktur) fibers.     

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.     

Selected muscle and nerve extracts contain an activity which stimulates myoblast proliferation and which is distinct from transferrin

Extracts from normal chicken anterior latissimus dorsi and dystrophic pectoralis major muscles and from normal chicken sciatic nerves induce a growth stimulation in chicken and rat myogenic cell cultures. Transferrin is only partially responsible for the observed stimulation since the addition of the extracts to transferrin-saturated cultures induces a further growth response and extracts from which transferrin has been removed by immunoabsorption still retain a substantial portion of their stimulation activity. The active fractions of muscle and nerve extracts display heat, acid, and organic solvent inactivation. Gel filtration of ammonium sulfate fractionated activity from the anterior latissimus dorsi muscle suggests the presence of a growth factor in the molecular weight range of 10,000 to 30,000.     

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.     

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.     

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.