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.     

A combined histochemical and immunohistochemical study on the dynamics of fast-to-slow fiber transformation in chronically stimulated rabbit muscle

Summary Chronically stimulated fast-twitch muscles of the rabbit were histochemically and immunohistochemically analyzed in serial cross sections (1) for percentages of fiber types, and (2) for the presence of myosin heavy chain isoforms during fast-to-slow transformation. By four weeks of stimulation the number of type-I fibers had increased more than fourfold, while only about 6% of the original IIB fibers remained. Type-IC and -IIC fibers transiently rose to 20% of the total fiber population. After 16 weeks, the number of type-I fibers had increased to 42%. With prolonged stimulation fewer fibers reacted with antibodies against embryonic and neonatal myosins and more with the antibody against slow myosin. The reaction for embryonic myosin was most often detected in the C fibers (IC, IIC). Immunohistochemical subtypes were observed for each fiber type in the stimulated muscles. The greatest number was seen in type-IIC fibers, which, in addition to their reaction for fast/neonatal and slow myosins, might also react with the antibodies against neonatal/embryonic and embryonic myosins. These findings indicated that the transforming fibers temporarily expressed myosin heavy chain isoforms normally not detectable in adult skeletal muscle. Myotubes reacted strongly with the antibodies against fast/neonatal and embryonic myosins, and some of them also with the antibody against slow myosin. Thus, it appears that under the influence of the low frequency stimulus pattern some of the newly formed myotubes developed into type-I fibers.     

Regenerating adult chicken skeletal muscle and satellite cell cultures express embryonic patterns of myosin and tropomyosin isoforms

Regenerating areas of adult chicken fast muscle (pectoralis major) and slow muscle (anterior latissimus dorsi) were examined in order to determine synthesis patterns of myosin light chains, heavy chains and tropomyosin. In addition, these patterns were also examined in muscle cultures derived from satellite cells of adult fast and slow muscle. One week after cold-injury the regenerating fast muscle showed a pattern of synthesis that was predominately embryonic. These muscles synthesized the embryonic myosin heavy chain, beta-tropomyosin and reduced amounts of myosin fast light chain-3 which are characteristic of embryonic fast muscle but synthesized very little myosin slow light chains. The regenerating slow muscle, however, showed a nearly complete array of embryonic peptides including embryonic myosin heavy chain, fast and slow myosin light chains and both alpha-fast and slow tropomyosins. Peptide map analysis of the embryonic myosin heavy chains synthesized by regenerating fast and slow muscles showed them to be identical. Thus, in both muscles there is a return to embryonic patterns during regeneration but this return appears to be incomplete in the pectoralis major. By 4 weeks postinjury both regenerating fast and slow muscles had stopped synthesizing embryonic isoforms of myosin and tropomyosin and had returned to a normal adult pattern of synthesis. Adult fast and slow muscles yielded a satellite cell population that formed muscle fibers in culture. Fibers derived from either population synthesized the embryonic myosin heavy chain in addition to alpha-fast and beta-tropomyosin. Thus, muscle fibers derived in culture from satellite cells of fast and slow muscles synthesized a predominately embryonic pattern of myosin heavy chains and tropomyosin. In addition, however, the satellite cell-derived myotubes from fast muscle synthesized only fast myosin light chains while the myotubes derived from slow muscle satellite cells synthesized both fast and slow myosin light chains. Thus, while both kinds of satellite cells produced embryonic type myotubes in culture the overall patterns were not identical. Satellite cells of fast and slow muscle appear therefore to have diverged from each other in their commitment during maturation in vivo.     

Expression of myosin isoforms during notexin-induced regeneration of rat soleus muscles

Myosin isozymes and their fiber distribution were studied during regeneration of the soleus muscle of young adult (4-6 week old) rats. Muscle degeneration and regeneration were induced by a single subcutaneous injection of a snake toxin, notexin. If reinnervation of the regenerating muscle was allowed to occur (functional innervation nearly complete by 7 days), then fiber diameters continued to increase and by 28 days after toxin treatment they attained the same values as fibers in the contralateral soleus. If the muscles were denervated at the time of toxin injection, the early phases of regeneration still took place but the fibers failed to continue to increase in size. Electrophoresis of native myosin showed multiple bands between 3 and 21 days of regeneration which could be interpreted as indicating the presence of embryonic, neonatal, fast and slow myosins in the innervated muscles. Adult slow myosin became the exclusive from in innervated regenerates. In contrast, adult fast myosin became the predominant form in denervated regenerating muscles. Immunocytochemical localization of myosin isozymes demonstrated that in innervated muscles the slow form began to appear in a heterogeneous fashion at about 7 days, and became the major form in all fibers by 21-28 days. Thus, the regenerated muscle was almost entirely composed of slow fibers, in clear contrast to the contralateral muscle which was still substantially mixed. In denervated regenerating muscles, slow myosin was not detected biochemically or immunocytochemically whereas fast myosin was detected in all denervated fibers by 21-28 days. The regenerating soleus muscle therefore is clearly different from the developing soleus muscle in that the former is composed of a uniform fiber population with respect to myosin transitions. Moreover the satellite cells which account for the regeneration process in the soleus muscle do not appear to be predetermined with respect to myosin heavy chain expression, since the fibers they form can express either slow or fast isoforms. The induction of the slow myosin phenotype is entirely dependent on a positive, extrinsic influence of the nerve.     

Tenotomy of the avian anterior latissimus dorsi muscle. I. Effect of age on fiber-type transformation and regeneration from the stump in chicks

The chick's anterior latissimus dorsi muscle (ALD) was tenotomized at its origin at either 1 day or 4 weeks of age, and investigated histochemically and ultrastructurally at intervals thereafter to determine whether muscle fiber-type transformation from a slow to a twitch type is greater in young birds than older birds. No transformation of fiber type occurred in either procedure, but a new muscular connection regenerated between the scar tissue at the end of the original tenotomized stump and the former origin. This regenerated muscle had a mosaic pattern of fiber types, as demonstrated by myofibrillar ATPase activity, and contained predominantly fast fibers, as contrasted with controls or the tenotomized portion, which contained predominantly slow tonic muscle fibers. The regenerated portion contained muscle spindles. The original portion of the tenotomized muscle was indistinguishable from the control muscle. These responses of the chick ALD to tenotomy are quite different from those in the pigeon, which are reported in the following study.     

Analysis of fiber types in the pigeon's metapatagialis muscle. I. Histochemistry, end plates and ultrastructure

The pigeon's metapatagialis muscle has a pale slip and two pink slips containing slow and fast fibers respectively. The fast fibers in the pink slip are of three types: large white fibers, small red fibers with a fibrillar pattern throughout, and small red fibers with a fibrillar pattern only in the L-band. The large fibers are intermediate in ATPase activity, low in SDH activity, the small fibers are high in ATPase and SDH activity. The slow fibers are intermediate in size and SDH staining, and low in ATPase activity. Cholinesterase staining for end plates corroborate the fast and slow identities. These results reinforce the idea that gross color should not be used in classifying a muscle's contractile or morphological parameters.     

Regeneration after cardiotoxin injury of innervated and denervated slow and fast muscles of mammals. Myosin isoform analysis

The regeneration of adult rat and mouse slow (soleus) and fast (sternomastoid) muscles was examined after the degeneration of myofibers had been achieved by a snake venom cardiotoxin, under experimental conditions devised to spare as far as possible the satellite cells, the nerves, and the blood vessels of the muscles. Three days after the injury, no myosin was detectable in selected portions of the muscles. New myosins of embryonic, neonatal, and adult types started to be synthesized during the following two days. Adult myosins thus appeared more precociously than in development, which implies that the synthesis of myosin isoforms during regeneration does not entirely 'recapitulate' the sequence of myosin transitions observed during normal development. Two weeks after the injury, the isomyosin electrophoretic pattern displayed by regenerated muscles was already the same as that of control muscles; the normal adult pattern was therefore expressed more rapidly in regenerating than in developing muscles. Except for the synthesis of the slow isoform which was generally inhibited in denervated muscles, the same types of myosins were expressed during the early stages of regeneration in denervated as in innervated muscles; long-term denervation prevented however the qualitative and quantitative recovery of the normal myosin pattern.     

Myofibrillar gene expression in differentiating lobster claw muscles

Lobster claw muscles undergo a process of fiber switching during development, where isomorphic muscles containing a mixture of both fast and slow fibers, become specialized into predominantly fast, or exclusively slow, muscles. Although this process has been described using histochemical methods, we lack an understanding of the shifts in gene expression that take place. In this study, we used several complementary techniques to follow changes in the expression of a number of myofibrillar genes in differentiating juvenile lobster claw muscles. RNA probes complementary to fast and slow myosin heavy chain (MHC) mRNA were used to label sections of 7th stage (approximately 3 months old) juvenile claw muscles from different stages of the molt cycle. Recently molted animals (1-5 days postmolt) had muscles with distinct regions of fast and slow gene expression, whereas muscles from later in the molt cycle (7-37 days postmolt) had regions of fast and slow MHC expression that were co-mingled and indistinct. Real-time PCR was used to quantify several myofibrillar genes in 9th and 10th stages (approximately 6 months old) juvenile claws and showed that these genes were expressed at significantly higher levels in the postmolt claws, as compared with the intermolt and premolt claws. Finally, Western blot analyses of muscle fibers from juvenile lobsters approximately 3 to 30 months in age showed a shift in troponin-I (TnI) isoform expression as the fibers differentiated into the adult phenotypes, with expression of the adult fast fiber TnI pattern lagging behind the adult slow fiber TnI pattern. Collectively, these data show that juvenile and adult fibers differ both qualitatively and quantitative in the expression of myofibrillar proteins and it may take as much as 2 years for juvenile fibers to achieve the adult phenotype.     

Fiber subtype distribution of pharyngeal dilator muscles and diaphragm in the cat

In previous studies differences were frequently found between the pharyngeal dilator muscles and the thoracic respiratory muscles in their patterns of electrical and mechanical activity during the respiratory cycle, with both resting and stimulated breathing. However, little is known about the intrinsic properties of the pharyngeal muscles and how they relate to the intrinsic properties of the diaphragm. In the present study, the fiber subtype distributions of two pharyngeal dilator muscles, the geniohyoid and the sternohyoid, were ascertained histochemically in the cat. The geniohyoid and the sternohyoid muscles had a preponderance of fast glycolytic (FG) fibers (mean 48 and 55%, respectively), a smaller number of fast oxidative-glycolytic (FOG) fibers (mean 36 and 31%, respectively), and few slow oxidative (SO) fibers (mean 16 and 14%, respectively). The percentages of SO fibers of both hyoid muscles were significantly (P less than 0.01) lower than that of the costal diaphragm, and the percentages of FOG and FG fibers were significantly higher than that of the diaphragm. In conclusion, the geniohyoid and sternohyoid muscles have histochemical characteristics usually associated with fast contraction and intermediate endurance properties.     

Differentiation of original and regenerated skeletal muscle fibres in mdx dystrophic muscles

The differentiation of both original muscle fibres and the regenerated muscle fibres following necrosis in mdx muscles was investigated using immunoblotting and immunocytochemical procedures. Before the onset of necrosis, postnatal skeletal muscles in mdx mouse differentiated well with only a slight delay in differentiation indicated by the level of developmental isoforms of troponin T. Prior to the onset of apparent myopathic change, both fast and slow skeletal muscle fibre types in mdx leg muscles also differentiated well when investigated by analysis of specific myosin heavy chain expression pattern. While the original muscle fibres in mdx leg muscles developed well, the differentiation of regenerated myotubes into both slow and distinct fast muscle fibre types, however, was markedly delayed or inhibited as indicated by several clusters of homogeneously staining fibres even at 14 weeks of age. The number of slow myosin heavy chain-positive myotubes amongst the regenerated muscle clusters was quite small even in soleus. This study thus established that while muscle fibres initially develop normally with only a slight delay in the differentiation process, the differentiation of regenerated myotubes in mdx muscles is markedly compromised and consequently delayed.     

Contractile proteins of fast and slow fibers during differentiation of lobster claw muscle

Contractile protein populations were determined, using gel electrophoresis, during development of the claw closer muscles of the lobster Homarus americanus. In the adult the paired claw closer muscles are asymmetric, consisting of a crusher muscle with all slow fibers and a cutter muscle with a majority of fast and a few slow fibers. The electrophoretic banding pattern of these adult fast and slow fibers shows a similarity in the major proteins including myosin, actin, and tropomyosin which are common to both fiber types. Paramyosin is slightly heavier in fast fibers than in slow. However, fast fibers have three proteins and slow fibers have four proteins which are unique to themselves. Several of these unique proteins belong to the regulatory troponin complexes. In juvenile 4th stage lobster, where the paired closer muscles are undifferentiated, the banding pattern reveals the presence of proteins common to both fiber types including myosin, actin, and tropomysin but the conspicuous absence of all unique fast fiber proteins as well as one unique slow fiber protein. By the juvenile 10th stage most of these unique proteins are present except for one unique slow fiber protein. Thus lobster fast and slow fiber differentiation entails coordinate gene activation to add unique contractile proteins.     

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.     

Phenotype plasticity in postural muscles of the crayfish Orconectes limosus Raf.: correlation of myofibrillar ATPase-based fiber typing with electrophysiological fiber properties and the effect of chronic nerve stimulation

The characteristics of the medial and lateral superficial extensor muscles (sem and sel) in the crayfish Orconectes limosus abdomen and their developmental and activity-dependent plasticity were studied. It was shown that both muscles are innervated by at least five excitatory and one inhibitory motor neuron in a nonuniform pattern. The muscles are composed of at least three different mATPase histochemistry-based fiber types that are all different from a fourth type in the uniform deep extensor muscles. sem and sel are composed of different ratios of these fiber types but do not show a constant fiber type pattern between segments and even between hemisegments. The three histochemically defined superficial extensor-fiber types have characteristic electrophysiological properties. The fiber types were shown to develop successively during the first postembryonic stages of development without a change in the number of muscle fibers. Based on histochemical ATPase staining after 21 days of chronic stimulation by means of an implantable, double-hook electrode, we show preliminary evidence that the fiber composition in the sem can switch from the presumably fast fiber type III to an intermediate type II. Repeated axotomy up to 53 days had no effect on the fiber type composition of the muscles.     

Changes in myosin heavy chain isoforms during chronic low-frequency stimulation of rat fast hindlimb muscles. A single-fiber study

Fast-twitch rat muscles contain three fast myosin heavy chains (HC) which can be separated by density gradient gel electrophoresis. Their mobility increases in the order of HCIIa less than HCIId less than HCIIb. In contrast to the rabbit, where chronic low-frequency nerve stimulation induces a fast-to-slow conversion, stimulation for up to 56 days does not lead to appreciable increases in the relative concentration of the slow myosin heavy chain HCI in rat fast-twitch muscles. However, chronic stimulation of rat fast-twitch muscle does evoke a rearrangement of the fast myosin heavy chain isoform pattern with a progressive decrease in HCIIb and progressive increases in HCIIa and HCIId. As judged from the time course and extent of these transitions, it appears that HCIId is an intermediate form between HCIIb and HCIIa. Single-fiber analyses of normal muscles make it possible to assign these heavy chain isoforms to histochemically defined fiber types IIB, IID, and IIA. The stimulation-induced fiber transformations produce numerous hybrid fibers displaying more than one myosin heavy chain isoform. Some transforming fibers contain up to four different myosin heavy chain isoforms.     

Physiological and developmental implications of motor unit anatomy

There is increasing evidence that the architectural design and arrangement of the fibers within a motor unit have important physiological and developmental ramifications. Limited data, however, are available to directly address this issue. In the present study the physiological properties of one motor unit in each of seven cat tibialis anterior (TA) muscles were determined. Each of these units then was repetitively stimulated to deplete the glycogen in all muscle fibers within the unit. Subsequently, the length, type of ending, and spatial distribution of fibers sampled from these physiologically and histochemically typed motor units were determined. Four fast fatigable (FF), one fast, fatigue resistant (FR), and two slow (S) motor units (MU) were studied. The samples consisted of all those glycogen-depleted fibers (9-27) contained within a single fascicle or a circumscribed area of each of the motor unit territories. The mean fiber lengths for the two slow motor units were 35.9 and 45.5 mm. The mean fiber lengths for the fast motor unit samples ranged from 8.8 to 48.5 mm. Some fibers of both the fast and slow units reached lengths of 58 mm. Most of the fibers in the slow units extended the entire distance between the proximal and distal musculotendinous planes, had relatively constant cross-sectional areas, and terminated at the tendon as blunt endings. In contrast, the majority of the fibers in the fast units terminated intrafascicularly at one end, and the cross-sectional area decreased progressively along their lengths, that is, showed a tapering pattern for a significant proportion of their lengths. Therefore, the force generated by units that end midfascicularly would appear to be transmitted to connective tissue elements and/or adjacent fibers. All fibers of a fast unit within a fascicle were located at approximately the same proximo-distal location. Thus, developmentally the selection of muscle fibers by a motoneuron would seem to be influenced by their spatial distribution. The architectural complexities of motor units also have clear implications for the mechanical interactions of active and inactive motor units. For example, the tension capabilities of a motor unit may be influenced not only by the spatial arrangement of its own fibers, but also by the level of activation of neighboring motor units.     

Lack of desmin results in abortive muscle regeneration and modifications in synaptic structure.

Desmin, a muscle-specific intermediate filament protein, is expressed in all muscle tissues. Its absence leads to a multisystemic disorder involving cardiac, skeletal, and smooth muscles. In skeletal muscle, structural abnormalities include lack of alignment of myofibrils, Z disk streaming, and focal muscle degeneration. In this study, we have examined the consequences of an absence of desmin on the mechanisms of regeneration and the integrity of the neuromuscular junction. The muscles of desmin knock-out and wild-type mice were made to regenerate by injecting cardiotoxin and were examined 7 to 42 days following the injection. The absence of desmin resulted in a delayed and modified regeneration and an accumulation of adipocytes. This was associated with a persistence of small diameter muscle fibers containing both N-CAM and developmental myosin isoforms. The amount of the slow myosin was increased, whereas there was a decrease in the fast isoform in the regenerated muscles of desmin knock-out mice. Both regeneration and aging led to the appearance of elongated neuromuscular junctions with diffuse acetylcholinesterase staining and a decrease in the overall acetylcholinesterase activity in the muscles of these mice. The neuromuscular junctions were markedly disorganised and in some cases postjunctional folds were absent. We conclude that desmin is essential for terminal muscle regeneration, maturation of muscle fibers, and maintaining the complex folded structure of the postsynaptic apparatus of the neuromuscular junctions.     

Morphology and histochemistry of the ambiens muscle of the red-eared turtle (Pseudemys scripta)

Six fiber types have been described in the ambiens muscle of red-eared turtles. These include one slow oxidative type, two fast oxidative types, two fast oxidative and glycolytic types, and one fast glycolytic type. Fiber types are non-randomly distributed throughout cross sections of the muscle. There is a decreasing gradient of oxidative staining and an increasing gradient of glycolytic staining along an axis from the superficial to deep regions of the muscle. The slow oxidative fibers are predominantly located within one or two fascicles of the superficial surface of the muscle. The fast glycolytic fibers are predominant in deep fascicles. In contrast to previous reports of histochemically monotypic intrafusal fibers in turtle muscle, ambiens muscle spindles have been observed containing one to eleven intrafusal fibers, including two fiber types. Fiber diameter and area are consistently smaller than observed in most extrafusal fibers. Spindles are predominantly located in superficial and cranial fascicles of the ambiens muscle and are located in regions characterized by extrafusal fibers with high oxidative activity.     

Myosin types in human skeletal muscle fibers

By combining enzyme histochemistry for fiber typing with immunohistochemistry for slow and fast myosin a correlation between fiber type and myosin type was sought in human skeletal muscle. Fiber typing was done by staining for myofibrillar ATPases after preincubation at discriminating pH values. Myosin types were discriminated using type specific anti-rabbit myosin antibodies shown to cross-react with human myosin and were visualized by a protein A-peroxidase method. Type I fibers were shown to contain slow myosin only, type IIA and IIB fibers fast myosin only, and type IIC fibers both myosins in various proportions. When muscle biopsies from well-trained athletes were investigated essentially the same staining pattern was observed. However, rarely occurring type I fibers with high glycolytic activity were detected containing additional small amounts of fast myosin and occasional type IIA fibers had small amounts of slow myosin. Based on the observation of various fiber types in which slow and fast myosin coexist we propose a dynamic continuum of fibers encompassing all fiber types.     

Myosin types in human skeletal muscle fibers

Summary By combining enzyme histochemistry for fiber typing with immunohistochemistry for slow and fast myosin a correlation between fiber type and myosin type was sought in human skeletal muscle. Fiber typing was done by staining for myofibrillar ATPases after preincubation at discriminating pH values. Myosin types were discriminated using type specific anti-rabbit myosin antibodies shown to cross-react with human myosin and were visualized by a protein A-peroxidase method. Type I fibers were shown to contain slow myosin only, type IIA and IIB fibers fast myosin only, and type IIC fibers both myosins in various proportions. When muscle biopsies from well-trained athletes were investigated essentially the same staining pattern was observed. However, rarely occurring type I fibers with high glycolytic activity were detected containing additional small amounts of fast myosin and occasional type IIA fibers had small amounts of slow myosin. Based on the observation of various fiber types in which slow and fast myosin coexist we propose a dynamic continuum of fibers encompassing all fiber types.     

Muscle fiber selectivity of Trichinella spiralis and Trichinella pseudospiralis (1983)

The biceps, semimembranosus, biceps femoris, and soleus muscles of female Rockland Wistar mice infected with either 1,000 Trichinella spiralis or 1,000 Trichinella pseudospiralis larvae were removed on days 12, 14, 16, and 18 post-infection (PI), sectioned and stained histochemically for their myosin ATPase activity. Light microscopic examination of the sections revealed that larvae of T. spiralis invade only the slow twitch muscle fibers, and those of T. pseudospiralis invade both the fast twitch and the slow twitch fibers. In sections obtained from mice infected with either parasite and killed on days 16 and 18 PI, identification of the majority of the infected fibers as fast twitch or slow twitch was not possible due to pathological modification of infected fibers.