Differentiation of fiber types in aneural musculature of the prenatal rat hindlimb

The presynaptic neurotoxin, beta-bungarotoxin, was injected into rat fetuses in utero to destroy the innervation of their hindlimb muscles. These injections were made prior to the invasion of motor axons into the muscles and, in some cases, prior to the cleavage of individual muscles. Examination of the lateral motor column of the spinal cord showed a dramatic reduction (greater than 95%) in the number of motoneuron cell bodies. Staining of sections of the hindlimb with silver and with antibodies to neurofilament proteins and to a synaptic vesicle protein indicated that the muscles were aneural. Anti-myosin antibodies applied to sections of the hindlimb revealed that these aneural muscles by the 20th day of gestation had the same types of fibers as were present in normal muscles of the same age. Moreover, fiber types in most muscles showed their characteristic intramuscular distributions. These findings suggest that fiber types can differentiate in the absence of the nervous system. However, some fibers achieved their ultimate fiber type fate without passing through the normal sequence of myosin expressions. Moreover, some slow fibers lost their slow expression, suggesting that the maintenance of the slow differentiation may require innervation. Muscle growth was dramatically affected by the absence of motoneurons; some muscles were decreased in size and others disappeared completely. In muscles which had not degenerated by the time secondary myogenesis normally begins, secondary muscle fibers were generated indicating that the genesis of these fibers is not strictly nerve dependent. Because fiber types differentiate independently of the nervous system, this study suggests that motoneurons selectively innervate fiber types during normal development.     

Development of muscle fiber specialization in the rat hindlimb

The appearance of fast and slow fiber types in the distal hindlimb of the rat was investigated using affinity-purified antibodies specific to adult fast and slow myosins, two-dimensional electrophoresis of myosin light chains, and electron microscope examination of developing muscle cells. As others have noted, muscle histogenesis is not synchronous; rather, a series of muscle fiber generations occurs, each generation forming along the walls of the previous generation. At the onset of myotube formation on the 15th d of gestation, the antimyosin antibodies do not distinguish among fibers. All fibers react strongly with antibody to fast myosin but not with antibody to slow myosin. The initiation of fiber type differentiation can be detected in the 17-d fetus by a gradual increase in the binding of antibody to slow myosin in the primary, but not the secondary, generation myotubes. Moreover, neuromuscular contacts at this crucial time are infrequent, primitive, and restricted predominantly, but not exclusively, to the primary generation cells, the same cells which begin to bind large amounts of antislow myosin at this time. With maturation, the primary generation cells decrease their binding of antifast myosin and become type I fibers. Secondary generation cells are initially all primitive type II fibers. In future fast muscles the secondary generation cells remain type II, while in future slow muscles most of the secondary generation cells eventually change to type I over a prolonged postnatal period. We conclude that the temporal sequence of muscle development is fundamentally important in determining the genetic expression of individual muscle cells.     

Differentiation of muscle fiber types in the chicken hindlimb

The differentiation of myotubes into fiber types was studied by examining the ATPase staining characteristics of chicken embryo thigh muscles. Two distinct fiber types, designated type IEMB and IIEMB, could be distinguished as early as stage 29. Paralysis of the embryo with d-tubocurarine prevented the differentiation of type IEMB but not type IIEMB characteristics. The two embryonic fiber types differed from each other, and mature type I and II fibers, in the acid and alkali labilities of their ATPases. Myotubes which were type IEMB at stage 29 matured into type I fibers, whereas those which were type IIEMB predominantly but not exclusively developed into type II fibers. The process of maturation involved sequential changes in the staining characteristics of the myotubes. Thus, the ultimate fiber type of a myotube can be detected long before it expresses its mature characteristics.     

Conversion of rat muscle fiber types

Summary Rats were used in this study to determine the time course of conversion of muscle fiber types. The right or left gastrocnemius muscle was removed thereby causing an overload on the ipsilateral soleus and plantaris muscles. The contralateral limb served as a control. The type II to type I fiber conversion was followed histochemically in the soleus and plantaris muscles for one to six weeks following surgery. Muscle sections were stained for myofibrillar actomyosin ATPase and NADH tetrazolium reductase. The type I population in the soleus muscle was 99.3% six weeks after synergist removal. The plantaris muscle underwent a two fold increase in the percentage of type I fibers after six weeks. Transitional fibers were prominent in the plantaris muscle and reached their peak at 4% (P<0.05) of the total population, four weeks after surgery.This research was funded in part by grants from The Graduate School at Washington State University, and The Society of the Sigma Xi     

Relationship between muscle fiber types and sizes and muscle architectural properties in the mouse hindlimb

Skeletal muscle fiber and architectural properties both contribute to the functional behavior of a muscle. This study uses discriminant analysis and mathematical modeling to identify the structurally and functionally significant properties. The architectural properties of fiber length, muscle length, and pennation angle are found to be the most structurally significant parameters, whereas fiber length, muscle length, and fiber type distribution are found to be most functionally determining. Architectural speed and fiber type do not appear to be complimentary (i.e., the architectural determinant of sspeed, fiber length, is not associated with fibers of high intrinsic velocity). However, there does seem to be a synergistic relation between the two property classes and force production. Muscles with large physiological cross sectional areas (PCSAs) tend to contain a greater proportion of larger, faster fibers. Structurally or morphologically significant parameters are not always found to have a large functional effect. Pennation angle, though one of the most structurally significant variables, was found to have very little functional effect. © 1994 Wiley-Liss, Inc.     

Enhancement of hybrid-fiber types in rat soleus muscle after clenbuterol administration during hindlimb unloading

Our objective was to determine the effects of a clenbuterol (CB) treatment orally administered (2 mg per kg) to rats submitted to 14 days of hindlimb unloading (HU). The morphological and the contractile properties as well as the myosin heavy chain isoforms contained in each fiber type were determined in whole soleus muscles. As classically described after HU, a decrease in muscle wet weight and in body mass associated with a loss of muscular force, an evolution of the contractile parameters towards those of a fast muscle type, and the emergence of fast myosin heavy chain isoforms were observed. The CB treatment in the HU rats helped reduce the decrease in 1) muscle and body weights, 2) force and 3) the proportion of slow fibers, without preventing the emergence of fast myosin isoforms. Clenbuterol induced a complex remodelling of the muscle typing promoting the combination of both slow and fast myosin isoforms within one fiber. To conclude, our data demonstrate that CB administration partially counteracts the effects produced by HU, and they allow us to anticipate advances in the treatment of muscular atrophy.     

The origin and selective innervation of early muscle fiber types in the rat

The diversity of muscle fiber types present in adult animals is present also in the fetus. Fibers generated early and late in fetal development undergo a stereotyped sequence of myosin expressions in giving rise to these fiber types. The differentiation of these fetal fiber types does not require innervation. However, evidence obtained from experiments identifying the types of fibers innervated by single motors suggests that the nervous system comes to recognize this diversity, at least during early postnatal life. Reinnervation experiments suggest that this recognition can occur in the absence of the timing cues normally present in the genesis of fiber types. Thus, a selective innervation of muscle fiber types occurs during development. The role of rearrangement of initial synaptic connections in generating this selectivity is discussed.     

Myosin heavy chain isoforms in histochemically defined fiber types of rat muscle

Summary Combined histochemical and biochemical analyses were performed on rat skeletal muscles in order to determine the myosin heavy chain patterns in specific fiber types. Four myosin heavy chain isoforms were separated by gradient polyacrylamide gel electrophoresis of extracts from single fibers and whole muscle homogenates. Their electrophoretic mobility increased in the order HCIIa, HCIIb, and HCI. HCIIa, HCIIb and HCI were present as unique isoforms in histochemically defined fiber types IIA, IIB and I, respectively. The isoforms HCI and HCIIa coexisted at variable ratios in type IC and IIC fibers. An additional fast myosin heavy chain isoform with an electrophoretic mobility between HCIIa and HCIIb was designated as HCIId because of its abundance in fast fibers of large diameter in the diaphragm. With the exception of slight differences in mATPase staining intensity after acid preincubation, these fibers were almost indistinguishable from type IIB fibers. In view of their specific myosin heavy chain composition (HCIId), these fibers were named type IID. In the extensor digitorum longus muscle, type IID fibers were of smaller size than type IIB and differed from the latter by higher NADH tetrazolium reductase activities. Circumstantial evidence suggests that type IID fibers are identical with the 2X fibers, previously described by Schiaffino et al. (1986).     

Conversion of rat muscle fiber types. A time course study

Rats were used in this study to determine the time course of conversion of muscle fiber types. The right or left gastrocnemius muscle was removed thereby causing an overload on the ipsilateral soleus and plantaris muscles. The contralateral limb served as a control. The type II to type I fiber conversion was followed histochemically in the soleus and plantaris muscles for one to six weeks following surgery. Muscle sections were stained for myofibrillar actomyosin ATPase and NADH tetrazolium reductase. The type I population in the soleus muscle was 99.3% six weeks after synergist removal. The plantaris muscle underwent a two fold increase in the percentage of type I fibers after six weeks. Transitional fibers were prominent in the plantaris muscle and reached their peak at 4% (P less than 0.05) of the total population, four weeks after surgery.     

Epinephrine-activation of heparin-nonreleasable lipoprotein lipase in 3 skeletal muscle fiber types of the rat

Epinephrine was used to activate the heparin non-releasable lipoprotein lipase (LPL) in the 3 skeletal muscle fiber types of the perfused rat hindlimb. Following a 9 min washout of the capillary-bound lipoprotein lipase, the hindquarter of the rat was perfused with a buffer containing 10 nM of epinephrine. Activity of the residual LPL in soleus, red vastus lateralis, and white vastus lateralis muscles increased 75%, 96%, and 102% respectively, following epinephrine perfusion. These results suggest that skeletal muscle LPL is under hormonal control possibly through protein phosphorylation by cyclic AMP dependent protein kinase.     

Postnatal development and plasticity of specialized muscle fiber characteristics in the hindlimb

Recent progress in defining molecular components of pathways controlling early stages of myogenesis has been substantial, but regulatory factors that govern the striking functional specialization of adult skeletal muscle fibers in vertebrate organisms have not yet been identified. A more detailed understanding of the temporal and spatial patterns by which specialized fiber characteristics arise may provide dues to the identity of the relevant regulatory factors. In this study, we used immunohistochemical, in situ hybridization, and Northern blot analyses to examine the time course and spatial characteristics of expression of myoglobin protein and mRNA during development of the distal hindlimb in the mouse. In adult animals, myoglobin is expressed selectively in oxidative, mitochondria-rich, fatigue-resistant myofibers, and it provides a convenient marker for this particular subset of specialized fibers. We observed only minimal expression of myoglobin in the hindlimb prior to the second day after birth, but a rapid and large (50-fold) induction of this gene in the ensuing neonatal period. Myoglobin expression was limited, however, to fibers located centrally within the limb which coexpress myosin isoforms characteristic of type I, IIA, and IIX fibers. This induction of myoglobin expression within the early postnatal period was accompanied by increased expression of nuclear genes encoding mitochondrial proteins, and exhibited a time course similar to the upregulation of myoglobin and mitochondrial protein expression that can be induced in adult muscle fibers by continuous motor nerve stimulation. This comparison suggests that progressive locomotor activity of neonatal animals may provide signals which trigger the development of the specialized features of oxidative, fatigue-resistant skeletal muscle fibers. © 1996 Wiley-Liss, Inc.     

Catalase-positive microperoxisomes in rat soleus and extensor digitorum longus muscle fiber types

The size, distribution, and content of catalase-reactive microperoxisomes were studied cytochemically in slow-twitch oxidative (SO), fast-twitch oxidative glycolytic (FOG), and fast-twitch glycolytic (FG) fibers of soleus and extensor digitorum longus (EDL) rat muscles. Fiber types were classified on the basis of mitochondrial content and distribution, Z-band widths, and myofibril size and shape. Microperoxisomes were generally located between myofibrils at the I-bands. The absence of crystalloid inclusions prevented positive identification of microperoxisomes in nonreacted and aminotriazole-inhibited muscles. EDL and soleus SO fibers possessed the largest microperoxisomes, whereas FOG and FG fibers of the EDL contained small- to medium-sized microperoxisomes. Comparing either microperoxisome number per muscle fiber area or microperoxisome area per fiber area revealed significant differences between fiber types with this ranking: soleus SO greater than EDL SO greater than EDL FOG greater than EDL FG. The present observations demonstrate that the content of catalase-positive microperoxisomes is greatest in the oxidative muscle fiber types. These cytochemical findings account for the higher catalase activity in homogenates of soleus muscles as compared to that of EDL muscles, because the soleus contains more oxidative fibers than EDL.     

Development of different types of muscle fiber in the postnatal ontogeny of guinea pigs

As demonstrates estimation of myosin ATPase and SDG activity, the guinea pig is already born with differentiated muscle fibers (MF), and the first histochemical differences between them take place in the uterine 10 days before birth. Tonic oxidative fibers of the first type, arranging hexagonally, develop especially quickly at early stages of postnatal ontogenesis. Their relative contents up to the end of the observations (185 days) do not change, and area of their transversal section increases but slightly in comparison to the phasic fibers. The main age changes of the muscle tissue are connected with formation and rearrangement of the phasic fibers. The most intensive reconstructions of the phasic fibers coincide with the period of game activity and sex maturation. In mixed muscles the part of the glycolytic fibers increase during the postnatal ontogenesis. In the process of ontogenesis the soleus muscle fully consists of oxidative fibers. The definitive level of the MF development is established after the guinea pigs have reached their sex maturation. Comparing the results of the given investigation with the previous data on development of MF in rats, it is possible to conclude that term and premature animals have various rates in development of the muscle system, however, main stages of myogenesis coincide, though they are connected with various phases of ontogenesis.     

The three-dimensional structure of motor endplates in different fiber types of rat intercostal muscle

Summary The three-dimensional organization of the motor end plates in the red, white and intermediate striated muscle fibers of the rat intercostal muscle was observed under a field-emission type scanning electron microscope after removal of connective tissue components by HCl hydrolysis.The motor endplate of the white fiber had terminal branches (or axon terminals), which were large, long and thin, and small but numerous nerve swellings (or terminal boutons). The motor endplate of the red fiber had terminal branches, which were small, short and thick, and had large but fewer nerve swellings. The motor endplate of the intermediate fiber was intermediate in size and structure between these two. In detached nerve-ending preparations, primary synaptic grooves with slit-like openings of the junctional folds appeared on the surface of the muscle fibers. The primary synaptic grooves were more developed in the white fiber than in the red fiber, and they were intermediate in the intermediate fiber. The numerical ratio of slit-like openings was 11.83.5 in the red, intermediate and white fiber, respectively.The Schwann cells and their processes were observed on the surface of the motor endplate, with the processes covering the upper orifices of the primary synaptic grooves and sealing the terminal branches. The number of Schwann cells was usually three in the white fiber, two in the intermediate fiber and one in the red fiber.     

Disorganization of honey-comb immunoreactive pattern of desmin and plectin in rat atrophic soleus muscle fiber induced by hindlimb suspension

Immunoreactivity for desmin, plectin and alpha-actinin was investigated in rat atrophic soleus muscle fibers induced by hindlimb suspension between 1 and 4 weeks (hindlimb suspension group, HSG), and compared with that of the control group (CG). Some of the HSG for 4 weeks were allowed unrestricted cage activity for 2 weeks as the recovery group (RG). In the cross-sectioned muscle fibers of the CG, desmin and plectin showed honey-comb immunoreactive patterns extending throughout the sarcoplasm. Superimposed images by double immunofluorescence labeling showed overlapping of both immunoreactivities. In the longitudinally sectioned profiles, superimposed images of alpha-actinin and desmin were overlapped at the level of Z-discs. The focal disorganization of the above honeycomb immunoreactive patterns, followed by the reduction of the cross-sectional area (CSA) of atrophic soleus muscle fibers and the appearance of Z-streaming, uniquely arose in the HSG from the first week and extended throughout the sarcoplasm in proportion to the suspension period. Such honey-comb patterns of both desmin and plectin were already restored in the RG at 2 weeks, followed by the disappearance of Z-streaming, prior to the recovery of the CSA. These findings indicate that the disorganization of topological and structural relationships of desmin and plectin with Z-discs surrounding individual myofibrils is primarily evoked, which leads to Z-streaming of atrophic soleus muscle fibers, and that the restoration of the muscle activity results in an early arrangement recovery of desmin and plectin around myofibrils.     

Quantitative distribution of muscle fiber types in the scup Stenotomus chrysops

Because the mass-specific power generated by myotomal muscle during swimming varies along the length of the fish, a realistic assessment of total power generation by the musculature requires integrating the product of mass-specific power and muscle mass at each position over the length of the fish. As a first step toward this goal, we examined the distribution of red, pink, and white muscle along the length of Stenotomus chrysops (scup) using histochemical and image analysis techniques. The largest cross-sectional area of red fibers occurs at 60% of total fish length and declines both anteriorly and posteriorly. By contrast, white fibers have the largest cross-sectional area in the anterior and decline dramatically moving posteriorly. The proportion of the fishes' cross-section occupied by red fibers increases from 1.37% to 8.42% moving posteriorly along the length of the fish. In contrast, the proportion of cross-sectional area occupied by pink fibers is constant (1.19%), while the proportional cross-sectional area of white fibers falls from 82.5% to 66.3%. The red, pink, and white fibers comprise 2.09, 0.73, and 51.1%, respectively, of total fish weight. We also compared the distribution of muscle in 10°C-and 200°C-acclimated animals. The value for red fiber volume, though slightly higher (13%) in cold-acclimated fish, is not statistically different. No difference was found in pink or white fibers. Finally, the finding that most of the red muscle is in the posterior half of the fish further supports the notion that most power for steady swimming at moderate speeds comes from posterior rather than anterior musculature. © 1996 Wiley-Liss, Inc.