Myosin heavy chain composition of muscle spindles in human biceps brachii.

Data on the myosin heavy chain (MyHC) composition of human muscle spindles are scarce in spite of the well-known correlation between MyHC composition and functional properties of skeletal muscle fibers. The MyHC composition of intrafusal fibers from 36 spindles of human biceps brachii muscle was studied in detail by immunocytochemistry with a large battery of antibodies. The MyHC content of isolated muscle spindles was assessed with SDS-PAGE and immunoblots. Four major MyHC isoforms (MyHCI, IIa, embryonic, and intrafusal) were detected with SDS-PAGE. Immunocytochemistry revealed very complex staining patterns for each intrafusal fiber type. The bag(1) fibers contained slow tonic MyHC along their entire fiber length and MyHCI, alpha-cardiac, embryonic, and fetal isoforms along a variable part of their length. The bag(2) fibers contained MyHC slow tonic, I, alpha-cardiac, embryonic, and fetal isoforms with regional variations. Chain fibers contained MyHCIIa, embryonic, and fetal isoforms throughout the fiber, and MyHCIIx at least in the juxtaequatorial region. Virtually each muscle spindle had a different allotment of numbers of bag(1), bag(2) and chain fibers. Taken together, the complexity in intrafusal fiber content and MyHC composition observed indicate that each muscle spindle in the human biceps has a unique identity.     

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.     

Expression of Myosin heavy chain isoforms in rat soleus muscle spindles after 19 days of hypergravity.

The aim of this study was to determine whether a period of 19 days in hypergravity was long enough to induce changes in the expression of myosin heavy chain (MyHC) isoforms in the muscle spindles. The soleus muscle of 10 male Wistar rats (control: CONT, n=5; hypergravity: HG, n=5) was frozen, cut into serial sections, and labeled with antibodies against MyHCs: I, IIA, IIA + IIX + IIB, slow-tonic, and alpha-cardiac. Forty CONT and 45 HG spindles were analyzed. The results from HG spindles compared to CONT showed that there was no change in the cross-sectional area of intrafusal fibers. However, along the entire length of B1 fibers, the expression of both MyHC I and alpha-cardiac was increased significantly, whereas the labeling against MyHC IIA and MyHC slow-tonic was decreased. In B2 fibers, the labeling against MyHC IIA (region A), slow-tonic (region A), and fast myosins (regions A-C) was statistically decreased. In chain fibers, the labeling against both MyHC IIA and fast MyHC was reduced significantly. We conclude that hypergravity has a real impact on the MyHC content in the muscle spindles and induces some inverse changes of those observed in hypogravity for MyHCs I, alpha-cardiac, and slow-tonic.     

Multiple-bag-fiber muscle spindles in tenuissimus muscles of the cat

Over 300 complete and incomplete cat muscle spindles were examined in serial transverse sections of tenuissimus muscles in search of spindles with more than two nuclear bag intrafusal muscle fibers. Several histochemical and histological stains were used to identify the intrafusal fibers and assess their motor and sensory innervation. About 13% of the spindles contained either three or four bag fibers rather than the usual two. Every multiple-bag-fiber spindle possessed at least one nuclear bag1 and one nuclear bag2 fiber. The supernumerary bag fibers were either another bag1 and/or bag2 fiber, or a mixed bag fiber. The extra bag fibers had the usual morphologic and histochemical properties of cat nuclear bag fibers. All multiple-bag spindles received primary sensory innervation, and most had secondary sensory endings in addition. Their motor pattern was similar in the number, appearance and disposition of intrafusal motor endings to that of the usual two-bag-fiber spindles. Bag fibers of the same kind shared motor nerve supply in three multiple-bag spindles in which tracings of individual motor axons were obtained histologically. It is unclear whether any functional advantage is conveyed to a muscle spindle by its having more than one bag1 and one bag2 fiber.     

Morphometric studies on tenuissimus muscle spindles in the cat

Muscle spindles were studied histochemically in serial transverse sections of 42 cat tenuissimus muscle specimens. Staining for myofibrillar adenosine triphosphatase was employed to identify nuclear bag 1, nuclear bag 2, and nuclear chain intrafusal muscle fibers. The nuclear chain fibers were further subdivided into three categories according to their polar length and the intensity of their staining for nicotinamide adenine dinucleotide tetrazolium reductase. A total of 430 spindle poles were surveyed. The mean spindle content of bag 1, bag 2, and chain fibers was established. The mean polar length of intrafusal fibers as well as that of the intracapsular and extracapsular spindle regions was determined. A cholinesterase (ChE) staining technique was used to demonstrate the termination sites of motor axons along intrafusal fibers. Two types of circumscribed ChE deposits. The "rim" and the "plate," occurred on the fibers. The nuclear chain fibers usually carried both the ChE rims and plates, while most nuclear fibers displayed only the plates. The ChE plates were assessed in term of their appearance, staining intensity, length, and location along the fibers. The mean number of ChE plates found along the fibers was established for each of the various intrafusal fiber types. These histochemical observations are discussed with regard to the current concepts of cat spindle morphology and motor innervation. The results suggest a degree of predictability in the spindle fiber content and in the distribution of motor nerve terminals along intrafusal muscle fibers, at least in the tenuissimus muscle.     

One-bag-fiber muscle spindles in tenuissimus muscles of the cat

Over 150 complete and 139 incomplete single muscle spindles were examined in serial transverse sections of cat tenuissimus muscles in search for spindles lacking one of the two types of nuclear bag intrafusal fiber. Several histochemical reactions were used to type the intrafusal muscle fibers and assess the spindle motor and sensory innervation. One complete spindle lacked a bag1 fiber, and another spindle lacked a bag2 fiber. Several incomplete spindles also lacked bag1 fibers. In addition, ten double tandem spindles contained one capsular unit each that lacked the bag1 fiber, and one triple tandem spindle had two such capsules. All one-bag-fiber spindles had primary sensory innervation, but none had secondary sensory innervation. Their motor innervation was similar to that of the usual two-bag-fiber spindles in the number and disposition of intrafusal motor endings. It is unclear whether the one-bag fiber spindles, either single or tandem-linked, are products of an aberrant spindle development or represent a true anatomical and functional subcategory of the cat muscle spindle.     

The occurrence of "mixed" nuclear bag intrafusal fibers in the cat muscle spindle

A total of 147 muscle spindles was studied histochemically in serial transverse sections of 42 cat tenuissimus muscle specimens. Nuclear bag1, nuclear bag2 and nuclear chain intrafusal muscle fibers were distinguished by the differential staining resulting from the reactions for myosin adenosine 5'-triphosphatase and nicotinamide adenine dinucleotide tetrazolium reductase. The majority of intrafusal fibers were of the same histochemical type at both fiber poles. However, seven muscle spindles contained one nuclear bag fiber each that presented as a bag1 in one pole and as a bag2 in the other pole. These "mixed" nuclear bag fibers were found in spindles that also contained at least one bag1 and one bag2 fiber of equivalent histochemical presentation in both fiber poles. The "mixed" bag fibers displayed differences of apparent fiber diameter and relative polar length between the two fiber poles. The motor innervation pattern, as revealed by staining for cholinesterase, was also dissimilar between the two poles of "mixed" bag fibers. The study indicates that the spindle equatorial region may in some instances serve as a boundary between two morphologically and histochemically different poles of the same intrafusal fiber.     

Motor innervation of intrafusal fibers in rat muscle spindles: incomplete separation of dynamic and static systems

Distributions of 53 motor axons to different types of intrafusal fibers were reconstructed from serial 1-micron-thick transverse sections of 13 poles of spindles in the rat soleus muscle. The mean number of motor axons that innervated a spindle pole was 4.1. Approximately 60% of motor axons lost their myelination prior to or shortly after entry into the periaxial fluid space of spindles. Motor innervation to the juxtaequatorial portion of nuclear bag fibers (particularly the bag1) consisted of groups of short, synaptic contacts that were terminations of thin, unmyelinated axons. In contrast, motor endings on both the bag1 and bag2 fibers were platelike in the polar intracapsular region. Chain fibers had a single midpolar platelike ending. The ratio of motor axons that innervated the bag1 fiber exclusively to axons that innervated bag2 and/or chain fibers was 1:1. However, one-fourth of motor axons coinnervated the dynamic bag1 fiber in conjunction with static bag2 and/or chain fibers. Thus the complete separation of motor control of the dynamic bag1 and static bag2 intrafusal systems observed in cat tenuissimus spindles is neither representative of the pattern of motor innervation in all other species of mammals nor essential to normal spindle function.     

Examination of chronically de-efferented cat muscle spindles for cholinesterase activity

Summary Cat tenuissmus muscles were deprived of motor nerve supply for three months by sectioning of the appropriate ventral spinal roots. Muscle spindles were located in the chronically de-efferented muscles and examined histochemically in serial transverse sections. Staining for nicotinamide adenine dinucleotide tetrazolium reductase showed that the spindle sensory innervation was preserved. The de-efferented intrafusal muscle fibers retained their differential staining with the reaction for myosin adenosine triphosphatase. However, all cholinesterase-active areas that are normally found along nuclear bag and nuclear chain intrafusal fibers demonstrated loss of the enzyme activity in the chronically de-efferented spindles. It is concluded that all histochemically demonstrable cholinesterase activity within the cat muscle spindle is dependent upon the continuous presence of motor innervation.     

Nonselective motor innervation of nuclear bag1 intrafusal muscle fibers in the cat

The motor nerve supply to cat nuclear bag1 intrafusal muscle fibers was reconstructed from light and electron microscopy of serial transverse sections of spindles in the tenuissimus muscle. Twenty-six of thirty poles of bag1 fibers that were examined received motor innervation. Every innervated bag1 pole received at least one (range 1-3) selective motor axon that supplied this fiber type only. Four of the innervated bag1 poles (15%) received additional motor supply from a nonselective motor axon that also innervated one nuclear chain fiber in the same spindle pole. The chain fibers co-innervated with bag1 fibers were among the longest chain fibers although they were shorter than two long chain fibers also present in the spindle poles. In cross-sections stained with toluidine blue they displayed 1-3 equatorial nuclei side by side, and there were fewer intermyofibrillar granules in their polar regions than in most of the other chain fibers. The endings of nonselective motor axons on the bag1 and chain fibers were morphologically and ultrastructurally dissimilar. It is suggested that instances of common innervation of the (dynamic) bag1 fiber and a (static?) chain fiber represent an integral and, presumably, functionally meaningful part of the motor pattern in some cat spindles.     

Slow-tonic MHC expression in paralyzed hindlimbs of fetal rats.

Whether nerve activity and active contraction of myotubes are essential for the assembly and initial differentiation of muscle spindles was investigated by paralyzing fetal rats with tetrodotoxin (TTX) from embryonic day 16 (E16) to E21, prior to and during the period when spindles typically form. TTX-treated soleus muscles were examined by light and electron microscopy for the presence of spindles and expression of myosin heavy chain (MHC) isoforms by the intrafusal fibers. Treatment with TTX did not inhibit the formation of a spindle capsule or the expression of a slow-tonic MHC isoform characteristic of intrafusal fibers, but did retard development of spindles. Spindles of TTX-treated E21 muscles usually consisted of one intrafusal fiber (bag2) only rather than two fibers (bag1 and bag2) typically present in untreated (control) E21 spindles. Intrafusal fibers of TTX-treated spindles also had only one sensory region supplied by multiple afferents, and were devoid of motor innervation. These features are characteristic of spindles in normal E18-E19 muscles. Thus, nerve and/or muscle activity is not essential for the assembly of muscle spindles, formation of a spindle capsule, and transformation of undifferentiated myotubes into the intrafusal fibers containing spindle-specific myosin isoforms. However, activity may promote the maturation of intrafusal bundles, as well as the maturation of afferent and efferent nerve supplies to intrafusal fibers.     

Expression of myosin heavy chain (MyHC) isoforms in rat intrafusal muscle fibres after neonatal deefferentation and subsequent denervation

The analysis of developing intrafusal fibres is not feasible in the absence of primary sensory axons, as neonatal denervation leads to the disintegration of muscle spindles. On the other hand, neonatal deefferentation does not arrest their differentiation and, moreover, it leads to the neomyogenesis of supernumerary intrafusal profiles. If the sciatic nerve was sectioned in 4-week-old rats deefferented at the birth, muscle spindles survived, the neomyogenesis proceeded and the denervated intrafusal fibres expressed the spindle specific slow tonic (STO) MyHC. The expression of MyHC pattern in individual fibres and the differentiation of the fibre type characteristics were, however, less obvious compared to the control or deefferented spindles. The newly formed intrafusal profiles (which differentiated from satellite cells in the absence of innervation) expressed the STO MyHC particularly when they developed in a spatial relation to nuclear bag fibres.     

Nonuniform expression of myosin heavy chain isoforms along the length of cat intrafusal muscle fibers

The expression of four myosin heavy chain (MHC) isoforms, avian slow-tonic (ATO) or neonatal-twitch (ANT) and mammalian slow-twitch (MST) or fast-twitch (MFT) in intrafusal fibers was examined by immunocytochemistry of spindles in the tenuissimus muscle of adult cats. The predominant MHCs expressed by nuclear bag fibers were ATO and MST, whereas the MHCs prevalent in nuclear chain fibers were ANT and MFT. The expression of these isoforms of MHC was not uniform along the length of intrafusal fibers. In general, both bag and chain fibers expressed avian MHC in the intracapsular region and mammalian MHC in the extracapsular region. The nonuniform expression of MHCs observed along the length of bag and chain fibers implies that different genes are activated in myonuclei located in the intracapsular and extracapsular regions of the same muscle fiber. Regional differences in gene activation might result from a greater effect of afferents on myonuclei located near the equator of intrafusal fibers then on myonuclei outside the spindle capsule.     

Histological and histochemical comparisons of muscle spindles in three hind limb muscles of the guinea pig.

Guinea pig soleus, medial gastrocnemius and vastus lateralis muscles were compared for spindle density and distribution, number of intrafusal fibers per spindle and histochemical appearance of the axial bundle. A total of 326 spindles was used in the comparisons. Spindle density was over four times greater in the soleus than in either the medial gastrocnemius or vastus lateralis. In the soleus the spindles were distributed at random, but in the other two muscles no spindles were found in those fascicles in which fast-twitch glycolytic extrafusal fibers predominated. The average number of intrafusal fibers per spindle varied by less than 5% between the three kinds of muscles. About 80% of all spindles located had four intrafusal fibers, two of the nuclear bag type and two of the nuclear chain type. The histochemical appearance of the axial bundle was the same in each kind of muscle. Based on intensities of the myofibrillar adenosine triphosphatase reaction product at polar regions nuclear bag fibers were separable into two histochemical groups; nuclear chain fibers were of only one histochemical type.     

Nonuniform expression of myosin heavy chain isoforms along the length of cat intrafusal muscle fibers

Summary The expression of four myosin heavy chain (MHC) isoforms, avian slow-tonic (ATO) or neonatal-twitch (ANT) and mammalian slow-twitch (MST) or fast-twitch (MFT) in intrafusal fibers was examined by immunocytochemistry of spindles in the tenuissimus muscle of adult eats. The predominant MHCs expressed by nuclear bag fibers were ATO and MST, whereas the MHCs prevalent in nuclear chain fibers were ANT and MFT. The expression of these isoforms of MHC was not uniform along the length of intrafusal fibers. In general, both bag and chain fibers expressed avian MHC in the intracapsular region and mammalian MHC in the extracapsular region. The nonuniform expression of MHCs observed along the length of bag and chain fibers implies that different genes are activated in myonuclei located in the intracapsular and extracapsular regions of the same muscle fiber. Regional differences in gene activation might result from a greater effect of afferents on myonuclei located near the equator of intrafusal fibers then on myonuclei outside the spindle capsule.     

A new grouping of intrafusal muscle fibers based on developmental studies of muscle spindles in the cat.

The development of muscle spindles was studied using the tenuissimus muscle of the cat. Observations show that the intrafusal muscle fibers develop as two separate groups: one group represented by a single nuclear bag fiber while the second group comprises the second nuclear bag fiber in association with all the nuclear chain fibers. This grouping is most pronounced in the fetus and is clearly seen in neonatal kittens (i.e., up to 2 weeks of age). As the intrafusal fibers begin to separate from each other, the groupings become less noticeable, although this basic pattern is often retained in the adult. The pattern of intrafusal fiber grouping is most noticeable in the equatorial regions of the spindle and least noticeable in the polar regions. This is not the grouping of fibers which would have been expected from a consideration of existing reports on muscle spindles. The implications for spindle form and function are considered.     

Expression of myosin heavy chain isoforms along intrafusal fibers of rat soleus muscle spindles after 14 days of hindlimb unloading.

Morphological, contractile, histochemical, and electrophoretical characteristics of slow postural muscles are altered after hindlimb unloading (HU). However, very few data on intrafusal fibers (IFs) are available. Our aim was to determine the effects of 14 days of hindlimb unloading on the morphological and immunohistochemical characteristics of IF in rat soleus muscle. Thirty-three control and 32 unloaded spindles were analyzed. The number and distribution of muscle spindles did not appear to be affected after unloading. There was no significant difference in number, cross-sectional area, and histochemical properties of IF between the two groups. However, after unloading, a significant decrease in slow type 1 MHC isoform and a slight increase in slow-tonic MHC expression were observed in the B and C regions of the bag1 fibers. The alpha-cardiac MHC expression was significantly decreased along the entire length of the bag2 fibers and in the B and C regions of the bag1 fibers. In 12 muscle spindles, the chain fibers expressed the slow type 1 and alpha-cardiac MHC isoforms over a short distance of the A region, although these isoforms are not normally expressed. The most striking finding of the study was the relative resistance of muscle spindles to perturbation induced by HU.     

One-bag-fiber muscle spindles in tenuissimus muscles of the cat

Summary Over 150 complete and 139 incomplete single muscle spindles were examined in serial transverse sections of cat tenuissimus muscles in search for spindles lacking one of the two types of nuclear bag intrafusal fiber. Several histochemical reactions were used to type the intrafusal muscle fibers and assess the spindle motor and sensory innervation. One complete spindle lacked a bag₁ fiber, and another spindle lacked a bag₂ fiber. Several incomplete spindles also lacked bag₁ fibers. In addition, ten double tandem spindles contained one capsular unit each that lacked the bag₁ fiber, and one triple tandem spindle had two such capsules. All one-bag-fiber spindles had primary sensory innervation, but none had secondary sensory innervation. Their motor innervation was similar to that of the usual two-bag-fiber spindles in the number and disposition of intrafusal motor endings. It is unclear whether the one-bag fiber spindles, either single or tandem-linked, are products of an aberrant spindle development or represent a true anatomical and functional subcategory of the cat muscle spindle.     

Histochemistry of rat intrafusal muscle fibers and their motor innervation.

Muscle spindles were followed in serial transverse sections of freshly frozen rat soleus muscles. Adenosine triphosphatase (ATPase) histochemical staining reaction was used to identify nuclear bag1, nuclear bag2 and nuclear chain intrafusal muscle fibers. Regional differences in ATPase staining occurred along bag1 and bag2 fibers but not along chain fibers. Bag1 fibers displayed ultrastructural heterogenity when their intra- and extracapsular regions were compared. Simple "diffuse" and more elaborate "plate" motor nerve terminals were demonstrated histochemically along the poles of bag1 and bag2 fibers by staining for cholinesterase. One motor terminal of the "plate" appearance was present on a chain fiber pole. There was no consistent spatial correlation between the intensity of regional ATPase staining along the nuclear bag fibers and the location, number and type of motor endings. Other factors, such as intrafusal fiber sensory innervation and regional differences in active and passive functional recruitment of nuclear bag fibers during muscle activity, may contribute to the ATPase staining variability along the intrafusal fibers.     

Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles

The composition of adult rat soleus muscle spindles, with respect to myosin heavy chain isoforms and M-band proteins, was studied by light-microscope immunohistochemistry. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal myosin isoforms as well as against myomesin, M-protein and the MM form of creatine kinase. Intrafusal fiber types were distinguished according to the pattern of ATPase activity following acid and alkaline preincubations. Nuclear bag1 fibers were always strongly stained throughout with anti-slow tonic myosin, were positive for anti-slow twitch myosin towards and in the C-region but were unstained with anti-fast twitch and anti-neonatal myosins. The staining of nuclear bag2 fibers was in general highly variable. However, they were most often strongly stained by anti-slow tonic myosin in the A-region and gradually lost this reactivity towards the poles, whereas a positive reaction with anti-slow twitch myosins was found along the whole fiber. Regional staining variability with anti-neonatal and anti-fast myosins was apparent, often with decreasing intensity towards the polar regions. Nuclear chain fibers showed strong transient reactivity with anti-slow tonic myosin in the equatorial region, did not react with anti-slow twitch and were always evenly stained by anti-fast twitch and anti-neonatal myosins. All three intrafusal fiber types were stained with anti-myomesin. Nuclear bag1 fibers lacked staining for M-protein, whereas bag2 fibers displayed intermediate staining, with regional variability, often increasing in reactivity towards the polar regions. Chain fibers were always strongly stained by anti-M-protein. The MM form of creatine kinase was present in all three fiber types, but bag1 fibers were less reactive and clear striations were not observed, in contrast to bag2 and chain fibers. Out of 38 cross sectioned spindles two were found to have an atypical fiber composition (lack of chain fibers) and a rather diverse staining pattern for the different antibodies tested. Taken together, the data show that in adult rat soleus, slow tonic and neonatal myosin heavy chain isoforms are only expressed in the muscle spindle fibers and that each intrafusal fiber type has a unique, although variable, composition of myosin heavy chain isoforms and M-band proteins. We propose that both motor and sensory innervation might be the determining factors regulating the variable expression of myosin heavy chain isoforms and M-band proteins in intrafusal fibers of rat muscle spindles.