Slow-tonic Myosin Heavy Chain Research Articles

Changing muscle function with sustained glial derived neurotrophic factor treatment of rabbit extraocular muscle

Recent microarray and RNAseq experiments provided evidence that glial derived neurotrophic factor (GDNF) levels were decreased in extraocular muscles from human strabismic subjects compared to age-matched controls. We assessed the effect of sustained GDNF treatment of the superior rectus muscles of rabbits on their physiological and morphological characteristics, and these were compared to naïve control muscles. Superior rectus muscles of rabbits were implanted with a sustained release pellet of GDNF to deliver 2μg/day, with the contralateral side receiving a placebo pellet. After one month, the muscles were assessed using in vitro physiological methods. The muscles were examined histologically for alteration in fiber size, myosin expression patterns, neuromuscular junction size, and stem cell numbers and compared to age-matched naïve control muscles. GDNF resulted in decreased force generation, which was also seen on the untreated contralateral superior rectus muscles. Muscle relaxation times were increased in the GDNF treated muscles. Myofiber mean cross-sectional areas were increased after the GDNF treatment, but there was a compensatory increase in expression of developmental, neonatal, and slow tonic myosin heavy chain isoforms. In addition, in the GDNF treated muscles there was a large increase in Pitx2-positive myogenic precursor cells. One month of GDNF resulted in significant extraocular muscle adaptation. These changes are interesting relative to the decreased levels of GDNF in the muscles from subjects with strabismus and preliminary data in infant non-human primates where sustained GDNF treatment produced a strabismus. These data support the view that GDNF has the potential for improving eye alignment in subjects with strabismus.

Changes in Myosin Heavy Chain Isoforms Along the Length of Orbital Fibers in Rabbit Extraocular Muscle.

Extraocular muscles express 10 myosin heavy chain (MyHC) isoforms that cater for a wide range of contractile speeds. We aim to characterize the variations in MyHC expression along the length of singly (SIFs) and multiply innervated fibers (MIFs) in the orbital layer of rabbit superior rectus muscle. Monospecific antibodies to nine MyHCs, including an anti-slow-tonic antibody characterized here were used to immunohistochemically map variations in MyHC distribution in serial sections along the muscle's full length. The fastest MyHC, EO, is expressed at the endplate zone (EPZ) of SIFs, flanked proximally and distally by segments expressing the slower 2A, with or without embryonic MyHC. MIFs with constant diameter express α-cardiac MyHC at the EPZ, flanked by segments co-expressing α-cardiac/embryonic and possibly slow-tonic MyHCs. MIFs with varying diameter also express α-cardiac MyHC at the EPZ in their thin, central region, flanked by thin segments co-expressing α-cardiac/embryonic MyHCs, with long proximal and distal extensions of larger diameter that co-express embryonic/slow-tonic and α-cardiac or β/slow MyHCs. Orbital fiber types express multiple MyHCs, with faster ones in SIFs, slower ones in MIFs, but all have fast EPZs and slower end segments. We hypothesize that these unique MyHC distributions enable these fibers to relax in two kinetically distinct phases while acting in an antagonistic manner during a saccade: the fast phases facilitate acceleration of eyeball rotation during agonist contraction, while the slow phases help its deceleration toward the visual target, thereby linearizing the saccade. These properties also facilitate pulley movements to implement Listing's law.

Cloning of Skeletal Myosin Heavy Chain Gene Family From Adult Pleopod Muscle and Whole Larvae of Shrimps

The physiological and biological properties of skeletal muscle in crustacea have not been well understood compared with those of vertebrates. The present study focused on myosin, the major protein in skeletal muscle, from shrimps. In our previous study, two full-length genes encoding myosin heavy chain (MHC), a large subunit of the myosin molecule, were cloned from abdominal fast skeletal muscle of kuruma Marsupenaeus japonicus, black tiger Penaeus monodon and Pacific white Penaeus vannamei shrimps, and named as MHCa and MHCb. In this study, we renamed these as MHC1 and MHC2, respectively, due to the presence of various isoforms newly identified. Partial MHC sequences were identified from pleopod muscle of these shrimps. Two MHCs, named MHC3 and MHC4, were identified from pleopod muscle of kuruma shrimp, whereas two MHCs, named MHC4 and MHC5, were cloned from Pacific white shrimp pleopod. MHC3 was cloned only from black tiger shrimp pleopod. Partial MHC sequences from zoea, mysis, and postlarvae of black tiger and Pacific white shrimps were also determined. The phylogenetic tree demonstrated that most MHCs from pleopod muscle and larval MHCs formed clades with MHC1 and MHC2, respectively. These MHCs were considered to be of fast type, since MHC1 and MHC2 are fast-type MHCs according to our previous study. MHC5 obtained from pleopod muscle of Pacific white shrimp in this study was monophyletic with American lobster Homarus americanus S2 slow tonic MHC previously reported, indicating that MHC5 from Pacific white shrimp is of slow type.

Sarcomeric Myosin Expression in the Tongue Body of Humans, Macaques and Rats

Expression of developmental and unconventional myosin heavy chain (MHC) isoforms in some adult head and neck muscles is thought to reflect specific contractile demands of muscle fibers active during kinematically complex movements. Mammalian tongue muscles are active during oromotor behaviors that encompass a wide range of tongue movement speeds and tongue shape changes (e.g. respiration, oral transport, swallowing, rejection), but the extent to which tongue muscles express developmental and unconventional MHC is not known. Quantitative PCR was used to determine the mRNA content of conventional MHC-beta, MHC-2a, MHC-2b and MHC-2x, the developmental isoforms embryonic MHC and neonatal MHC and the unconventional isoforms atrial/cardiac-α MHC (MHC-alpha), extraocular MHC, masseter MHC and slow tonic MHC in tongue body muscles of the rat, macaque and human. In all species, conventional MHC isoforms predominate. MHC-2b and MHC-2x account for 98% of total MHC mRNA in the rat. MHC-2a, MHC-2x and MHC-beta account for 94% of total MHC mRNA in humans and 96% of total MHC mRNA in macaque. With the exception of MHC-alpha in humans (5%), developmental and unconventional MHC mRNA represents less than 0.3% of total MHC mRNA. We conclude that in these species, there is limited expression of developmental and unconventional MHC and that diversity of tongue body muscle fiber contractile properties is achieved primarily by MHC-beta, MHC-2a, MHC-2x and MHC-2b. Whether expression of MHC-alpha mRNA in tongue is unique to humans or present in other hominoids awaits further investigation.

Limited expression of slow tonic myosin heavy chain in human cranial muscles.

Recent reports of slow tonic myosin heavy chain (MHCst) in human masticatory and laryngeal muscles suggest that MHCst may have a wider distribution in humans than previously thought. Because of the novelty of this finding, we sought to confirm the presence of MHCst in human masticatory and laryngeal muscles by reacting tissue from these muscles and controls from extraocular, intrafusal, cardiac, appendicular, and developmental muscle with antibodies (Abs) ALD-58 and S46, considered highly specific for MHCst. At Ab dilutions producing minimal reaction to muscle fibers positive for MHCI, only extraocular, intrafusal, and fetal tongue tissue reacted with Ab S46 had strong immunoreaction in an appreciable number of muscle fibers. In immunoblots, Ab S46, but not Ab ALD-58, labeled adult extraocular muscles; no other muscles were labeled with either Ab. We conclude that, in humans, Ab S46 has greater specificity for MHCst than does Ab ALD-58. We suggest that reports of MHCst in human masticatory and laryngeal muscles reflect false-positive identification of MHCst due to cross-reactivity of Ab ALD-58 with another MHC isoform.

Determination of slow-tonic MyHC immunoreactivity is an important step in the evaluation of muscle spindles in porcine extraocular muscles

We have tested our hypothesis suggesting (i) that for the reliable determination and counting of muscle spindles (Msp) at the light microscopy level in extraocular muscles (EOM), analysis of the spindle specific myosin heavy chain (MyHC) immunoreactivity of intrafusal fibers, especially after staining with anti-slow-tonic MyHC antibodies, is the most convenient tool, (ii) that the number of Msp determined by the slow-tonic MyHC immunoreactivity of intrafusal fibers in EOM is much lower than that based on histological examination and (iii) that the previously reported numbers of Msp based on histological examination of EOM could be overestimated. In order to determine the number and distribution of Msp and to analyze the MyHC isoform immunoreactivity of intrafusal fibers in porcine EOM, paraffin sections of three 9-month-old pig medial (MR) and lateral rectus (LR), levator palpebrae (LP) and retractor bulbi (RB) muscles were stained histologically or using specific monoclonal antibodies (mAbs) against MyHC isoforms. Msp in recti and LP muscles studied by immunocytochemistry contained nuclear bag (NB) fiber(s) reacting with mAbs against slow-tonic, slow-twitch, α-cardiac and neonatal MyHCs, but not with the mAb against fast-twitch MyHC, which, on the contrary, stained nuclear chain (NC) fibers. Based on determination of spindle specific slow-tonic MyHC isoform immunoreactivity we have found 72 Msp in the MR and 68 Msp in the LR and 12 Msp in LP muscles, which was only 62, 55 and 32% of the Msp total counts according to histological examination, respectively. In the RB muscle, we have even found only 15 spindle-like-structures composed of encapsulated thin muscle fibers, which possessed only a reaction with anti-fast-twitch MyHC mAb, but lacked slow-tonic, slow-twitch or α-cardiac MyHCs immunoreactivity. Our analysis of porcine EOM confirmed the above suggestions, demonstrating, for the first time in the pig, the presence of “false Msp” mimicking encapsulated muscle fibers on histological sections that lack spindle specific MyHC immunoreactivity. In analogy with other muscles we suggest that “false Msp” are not innervated by sensory axons and therefore do not contribute to the physiological sensation of the muscle length changes. Our results thus show that the reliable identification of functionally effective Msp in EOM must involve immunohistochemical analysis of spindle specific MyHC isoforms of intrafusal fibers, as “false” spindles appearing on histologically stained sections as encapsulated muscle fibers could be regarded as “true” Msp and thus increase the spindle number counts in earlier studies.

Differentiation of cardiac Purkinje fibers requires precise spatiotemporal regulation of Nkx2‐5 expression

Nkx2-5 gene mutations cause cardiac abnormalities, including deficits of function in the atrioventricular conduction system (AVCS). In the chick, Nkx2-5 is elevated in Purkinje fiber AVCS cells relative to working cardiomyocytes. Here, we show that Nkx2-5 expression rises to a peak as Purkinje fibers progressively differentiate. To disrupt this pattern, we overexpressed Nkx2-5 from embryonic day 10, as Purkinje fibers are recruited within developing chick hearts. Overexpression of Nkx2-5 caused inhibition of slow tonic myosin heavy chain protein (sMHC), a late Purkinje fiber marker but did not affect Cx40 levels. Working cardiomyocytes overexpressing Nkx2-5 in these hearts ectopically up-regulated Cx40 but not sMHC. Isolated embryonic cardiomyocytes overexpressing Nkx2-5 also displayed increased Cx40 and suppressed sMHC. By contrast, overexpression of a human NKX2-5 mutant did not effect these markers in vivo or in vitro, suggesting one possible mechanism for clinical phenotypes. We conclude that a prerequisite for normal Purkinje fiber maturation is precise regulation of Nkx2-5 levels.

Laryngeal muscle fibre types

The internal laryngeal muscles have evolved to subserve the highly specialized functions of airways protection, respiration, and phonation. Their contractile properties, histochemistry, biochemical properties, myosin heavy chain (MyHC) expression and their regulation by nerves and hormones are reviewed and compared with limb muscle fibres. Cricothyroid, the vocal cord tensor, is limb-like in MyHC composition and fibre type properties, while the vocal fold abductor and adductors are allotypically different, with capacity for expressing an isoform of MyHC that is kinetically faster than the fastest limb MyHC. In rats and rabbits the faster isoform is the extraocular (EO) MyHC, while in carnivores, it is the IIB MyHC. These adaptations enable the abductor and adductor muscles to remain always faster than the cricothyroid as the latter changes in speed during evolution to match changing metabolic and respiratory rates in relation to scaling with body mass. Such phylogenetic plasticity is vital to the airways protection and respiratory functions of these muscles. The posterior cricoarythenoid, the abductor muscle, is tonically driven during expiration, and consequently has a slower fibre type profile than the principal adductor, the thyroarythenoid. The human thyroarythenoid appears not to express EO or IIB MyHC significantly, but is unique in expressing the slow-tonic MyHC. The concepts of allotype and phylogenetic plasticity help to explain differences in fibre type between limb and laryngeal muscles and between homologous laryngeal muscles in different species. Laryngeal muscle fibres exhibit physiological plasticity as do limb muscles, being subject to neural and hormonal modulation.

Expression of unique and developmental myosin heavy chain isoforms in adult human digastric muscle.

Digastric muscle (DGM) is a powerful jaw-opening muscle that participates in chewing, swallowing, breathing, and speech. For better understanding of its contractile properties, five pairs of adult human DGMs were obtained from autopsies and processed with immunocytochemistry and/or immunoblotting. Monoclonal antibodies against alpha-cardiac, slow tonic, neonatal, and embryonic myosin heavy chain (MHC) isoforms were employed to determine whether the DGM fibers contain these MHC isoforms, which have previously been demonstrated in restricted specialized craniocervical skeletal muscles but have not been reported in normal adult human trunk and limb muscles. The results showed expression of all these MHC isoforms in adult human DGMs. About half of the fibers reacted positively to the antibody specific for the alpha-cardiac MHC isoform in DGMs, and the number of these fibers decreased with age. Slow tonic MHC isoform containing fibers accounted for 19% of the total fiber population. Both the alpha-cardiac and slow tonic MHC isoforms were found to coexist mainly with the slow twitch MHC isoform in a fiber. A few DGM fibers expressed the embryonic or neonatal MHC isoform. The findings suggest that human DGM fibers may be specialized to facilitate performance of complex motor behaviors in the upper airway and digestive tract.

Muscle spindles in the deep muscles of the human neck: a morphological and immunocytochemical study.

Muscle spindle density is extremely high in the deep muscles of the human neck. However, there is a paucity of information regarding the morphology and immunoreactivity of these muscle spindles. The objective of this study was to investigate the intrafusal fiber content and to assess the myosin heavy chain (MyHC) composition of muscle spindles from human deep neck muscles. In addition to the conventional spindles containing bag(1), bag(2), and chain fibers (b(1)b(2)c spindle), we observed a number of spindles lacking bag(1) (b(2)c spindle) or bag(2) (b(1)c spindle) fibers. Both bag(1) and bag(2) fibers contained slow tonic MyHCs along their entire fiber length and MyHCI, MyHCIIa, embryonic, and alpha-cardiac MyHC isoforms along a variable length of the fibers. Fetal MyHC was present in bag(2) fibers but not in bag(1) fibers. Nuclear chain fibers contained MyHCIIa, embryonic, and fetal isoforms with regional variations. We also compared the present data with our previous results obtained from muscle spindles in human biceps brachii and the first lumbrical muscles. The allotment of numbers of intrafusal fibers and the MyHC composition showed some muscle-related differences, suggesting functional specialization in the control of movement among different human muscles.

Proportions of slow myosin heavy chain-positive fibers in muscle spindles and adjoining extrafusal fascicles, and the positioning of spindles relative to these fascicles

Chicken leg muscles were examined to calculate the percentages of slow myosin heavy chain (MHC)-positive fibers in spindles and in adjacent extrafusal fascicles, and to clarify how the encapsulated portions of muscle spindles are positioned relative to these fascicles. Unlike mammals, in chicken leg muscles slow-twitch MHC and slow-tonic MHC are expressed in intrafusal fibers and in extrafusal fibers, suggesting a close developmental connection between the two fiber populations. In 8-week-old muscles the proportions of slow MHC-positive extrafusal fibers that ringed muscle spindles ranged from 0-100%. In contrast, proportions of slow MHC-positive intrafusal fibers in spindles ranged from 0-57%. Similar proportions in fiber type composition between intrafusal fibers and surrounding extrafusal fibers were apparent at embryonic days 15 and 16, demonstrating early divergence of extrafusal and intrafusal fibers. Muscle spindles were rarely located within single fascicles. Instead, they were commonly placed where several fascicles converged. The frequent extrafascicular location of spindles suggests migration of intrafusal myoblasts from developing clusters of extrafusal fibers toward the interstitium, perhaps along a neurotrophic gradient established by sensory axons that are advancing in the connective tissue matrix that separates adjoining fascicles.

Slow tonic muscle fibers in the thyroarytenoid muscles of human vocal folds; a possible specialization for speech.

Most of the sounds of human speech are produced by vibration of the vocal folds, yet the biomechanics and control of these vibrations are poorly understood. In this study the muscle within the vocal fold, the thyroarytenoid muscle (TA), was examined for the presence and distribution of slow tonic muscle fibers (STF), a rare muscle fiber type with unique contraction properties. Nine human TAs were frozen and serially sectioned in the frontal plane. The presence and distribution pattern of STF in each TA were examined by immunofluorescence microscopy using the monoclonal antibodies (mAb) ALD-19 and ALD-58 which react with the slow tonic myosin heavy chain (MyHC) isoform. In addition, TA muscle samples from adjacent frozen sections were also examined for slow tonic MyHC isoform by electrophoretic immunoblotting. STF were detected in all nine TAs and the presence of slow tonic MyHC isoform was confirmed in the immunoblots. The STF were distributed predominantly in the medial aspect of the TA, a distinct muscle compartment called the vocalis which is the vibrating part of the vocal fold. STF do not contract with a twitch like most muscle fibers, instead, their contractions are prolonged, stable, precisely controlled, and fatigue resistant. The human voice is characterized by a stable sound with a wide frequency spectrum that can be precisely modulated and the STF may contribute to this ability. At present, the evidence suggests that STF are not presented in the vocal folds of other mammals (including other primates), therefore STF may be a unique human specialization for speech.

Expression of myosin heavy chain isoforms and myogenesis of intrafusal fibres in rat muscle spindles.

This review concerns the pattern of expression and regulation of myosin heavy chain (MHC) isoforms in intrafusal fibres of rat muscle spindles detected by immunocytochemistry. The three types of intrafusal fibres--nuclear bag1, nuclear bag2, and nuclear chain fibres--are unique in co-expressing several MHCs including special isoforms such as slow tonic and alpha cardiac-like MHC and isoforms typical of muscle development, such as embryonic and neonatal MHC. The distinct intrafusal fibre types appear sequentially during rat hind limb development, the nuclear bag2 precursors being first identifiable at 17-18 days in utero as the only primary myotubes expressing slow tonic MHC. Sensory innervation is required for the expression of "spindle-specific" MHC isoforms. Motor innervation contributes to the diversity in distribution of the different MHCs along the length of the nuclear bag fibres. It is suggested that unique populations of myoblasts are destined to become intrafusal fibres during development in the rat hind limb muscles and that the regional heterogeneity in MHC expression is related both to sensory and motor innervation and to the properties of the myoblast lineages. These distinct features make intrafusal fibres an attractive in situ model for investigating myogenesis, myofibrillogenesis, and the mechanisms regulating MHC expression.

Type and regional diversity in the distribution of myosin heavy chains in chicken intrafusal muscle fibers

Chicken intrafusal fibers were classified on the basis of their myosin heavy chain (MHC) composition, which was compared to that of mammalian nuclear bag and nuclear chain types. Immunoreactivities of intrafusal fibers from leg muscles of 8-week-old chickens were evaluated in serial cross-sections after incubation with monoclonal antibodies against slow-twitch, slow-tonic, or fast-twitch MHC and fast muscle C-protein. Four categories of slow intrafusal fiber could be distinguished on the basis of differential expression of slow-twitch and slow-tonic MHC. Segregation into types was most evident at the motor axon supplied pole, followed by the sensory region of the equator. Fiber types were least distinct at the juxtaequator where sensory and motor axons meet. Intrafusal fibers negative for slow myosins reacted with anti-fast myosins. Fast fibers were best viewed as a single group without subdivisions. Immunostaining for fast muscle C-protein paralleled in large part reactivities for neonatal/fast MHC, indicating that proteins other than MHC are useful fiber type markers. Despite regional changes along the length of intrafusal fibers and some variation within fiber types, the concept of separate MHC-based fiber types was valid as long as typing of fibers was restricted to the proximal polar region. Comparisons of MHC profiles revealed similarities between chicken fast intrafusal fibers and mammalian nuclear chain fibers and between some chicken slow intrafusal fibers and mammalian nuclear bag fibers.

Expression of myosin heavy chain isoforms in developing human muscle spindles.

We studied serial sections of human fetal limb muscles (10-25 weeks of gestation) by light microscopic (LM) immunocytochemistry, using specific antibodies against slow-tonic, slow-twitch, fetal, embryonic, and alpha-cardiac myosin heavy chain (MHC) isoforms, neurofilament protein, laminin, and myomesin. One set of the first-generation myotubes expressed slow-tonic MHCs, and slow-twitch, fetal, and embryonic MHCs from the tenth week of gestation. These primary myotubes were identified as developing nuclear bag fibers. Second-generation myotubes in close apposition to the primary nuclear bag myotubes initially expressed only fetal and embryonic MHCs. One or more of these secondary myotubes acquired expression of slow-tonic and slow-twitch MHCs and gave rise to nuclear bag fibers. Most of the nuclear bag precursors expressed alpha-cardiac MHC. The secondary myotubes that expressed fetal and embryonic MHC but not slow-tonic, slow-twitch, or alpha-cardiac MHCs gave rise to the nuclear chain fibers. This study shows that different populations of fiber precursors, each with a unique sequence of MHC expression, gave rise to the nuclear bag and chain fibers, despite the presence of a common afferent nerve, from the early stages.

Immunohistochemical characterization of human masseter muscle spindles

An enzyme- and immunohistochemical study has been performed on human masseter muscle spindles. Antibodies selective for different myosin heavy chain (MHC) isoforms and M-band proteins (M-protein, myomesin, and MM-CK) were used. The expression of these proteins was determined in the different intrafusal fiber types. Nuclear bag1 and nuclear bag2 fibers expressed predominantly slow-twitch and slow-tonic MHCs. The bag2 fibers in addition contained fetal MHC. Nuclear chain fibers coexpressed embryonic, fetal, and fast-twitch MHCs. The bag2 and chain fibers contained all three M-band proteins, whereas the bag1 fibers contained only myomesin. In general the MHC expression in the human masseter intrafusal fiber types was similar to that previously reported for limb muscles in man as well as for limb and masseter muscles in other species. However, the number of intrafusal fibers per spindle was unusually high (up to 36). This reinforces the idea that masseter muscle spindles have a strong proprioceptive impact during the control of jaw movements.

Transient expression of a slow-tonic MHC isoform by extrafusal fibers in the developing rat.

ALD 19, a monoclonal antibody that recognizes the slow-tonic myosin heavy chain (MHC) isoform, has been used extensively as a marker for nuclear bag intrafusal fibers of muscle spindles in developing and adult rats. Extrafusal fibers of adult rat hindlimb muscles do not express slow-tonic MHC. However, while using ALD 19 to trace the fate of intrafusal fibers following neonatal denervation, we noted that some extrafusal fibers of neonates also bound this antibody. The immunolabeled extrafusal fibers were a subset of slow fibers located in the deep axial regions of crural muscles. The same fiber subset transiently displayed a weak affinity for ALD 19 during the first postnatal week in normal muscles. Denervation at birth increased the intensity of ALD 19 immunolabelling by these extrafusal fibers and extended the duration of the slow-tonic immunoreactivity into the 2nd postnatal week, after which expression diminished or ceased. Demonstration that some developing extrafusal fibers have a nerve-independent capacity for transiently expressing slow-tonic MHC, an MHC previously though to be expressed only by intrafusal fibers, raises the possibility that both types of fiber originate from a subset of bipotential slow primary myotubes in rat hindlimbs.

Differentiation of supernumerary fibres in neonatally deefferented rat muscle spindles

Abstract The myofibrillar ATPase (mATPase) activity and the pattern of expression of several myosin heavy chain (MHC) isoforms and of M-protein (Mr 165000) were studied in serial cross sections of neonatally deefferented 5- to 8-week-old rat hindlimb muscle spindles with supernumerary intrafusal fibres. In a sample of 5- to 6-week-old neonatally deefferented muscle spindles cut through the A region, the average number of intrafusal fibres per spindle was 8.4 in comparison to 4.2 in control spindles. Parent fibres extended throughout the whole encapsulated portion of the spindle, whereas supernumerary fibres were found only in the A region. The diameters of the supernumerary intrafusal fibres varied from less than 1 μ up to 10 μ approximately. On the basis of the mATPase activity and the pattern of expression of MHC isoforms and of M-protein, the vast majority of the supernumerary fibres could be classified as nuclear bag 2, bag 1 or chain fibres. However, some supernumerary fibres with small diameters exhibited features that did not fit any of the three known intrafusal fibre types. Two major processes, namely fibre splitting versus activation and fusion of satellite cells, might account for the formation of supernumerary fibres. The data presented suggest the existence of at least two types of intrafusal satellite cells. One type of satellite cell is related to the nuclear bag fibres and gives rise to myotubes which, if they have sensory innervation, can express slow tonic MHC and, therefore, differentiate into a phenotype similar to that seen in nuclear bag fibres. The other type of satellite cells form myotubes which attain a fast phenotype similar to that seen in nuclear chain fibres irrespective of the presence or absence of sensory innervation.

Differential effects of neonatal denervation on intrafusal muscle fibers in the rat.

The response of developing muscle spindles to denervation was studied by sectioning the nerve to the medial gastrocnemius muscle of rats at birth. The denervated spindles were examined daily throughout the first postnatal week for changes in ultrastructure and expression of several isoforms of myosin heavy chain (MHC). Each of the three different types of intrafusal muscle fiber exhibited a different response to denervation. Within 5 days after the nerve section nuclear bag2 fibers degenerated completely; nuclear bag1 fibers persisted, but ceased to express the 'spindle-specific' slow-tonic MHC isoform and thereby could not be differentiated from extrafusal fibers; nuclear chain fibers did not form. The capsules of spindles disassembled, hence spindles or their remnants could no longer be identified 1 week after denervation. Neonatal deefferentation has little effect on these features of developing spindles, so removal of afferent innervation is presumably the factor that induces the loss of spindles in denervated muscles. Degeneration of the bag2 fiber, but not bag1 or extrafusal fibers, reflects a greater dependence of the bag2 fiber than the bag1 fiber on afferent innervation for maintenance of its structural integrity. This difference in response of the two types of immature bag fiber to denervation might reflect an origin of the bag2 fibers from a lineage of myogenic cells distinct from that giving rise to bag1 or extrafusal fibers, or a difference in the length of contact with afferents between the two types of bag fiber prior to nerve section.

Expression of type-specific MHC isoforms in rat intrafusal muscle fibers.

Myosin heavy chain (MHC) expression by intrafusal fibers was studied by immunocytochemistry to determine how closely it parallels MHC expression by extrafusal fibers in the soleus and tibialis anterior muscles of the rat. Among the MHC isoforms expressed in extrafusal fibers, only the slow-twitch MHC of Type 1 extrafusal fibers was expressed along much of the fibers. Monoclonal antibodies (MAb) specific for this MHC bound to the entire length of bag2 fibers and the extracapsular region of bag1 fibers. The fast-twitch MHC isoform strongly expressed by bag2 and chain fibers had an epitope not recognized by MAb to the MHC isoforms characteristic of developing muscle fibers or the three subtypes (2A, 2B, 2X) of Type 2 extrafusal fibers. Therefore, intrafusal fibers may express a fast-twitch MHC that is not expressed by extrafusal fibers. Unlike extrafusal fibers, all three intrafusal fiber types bound MAb generated against mammalian heart and chicken limb muscles. The similarity of the fast-twitch MHC of bag2 and chain fibers and the slow-tonic MHC of bag1 and bag2 fibers to the MHC isoforms expressed in avian extrafusal fibers suggests that phylogenetically primitive MHCs might persist in intrafusal fibers. Data are discussed relative to the origin and regional regulation of MHC isoforms in intrafusal and extrafusal fibers of rat hindlimb muscles.