A study of the fine structure of the accessory muscle of the horseshoe crab,Tachypleus gigas

The accessory muscle of the walking leg of the horseshoe crab, Tachypleus gigas, was examined electron microscopically. The muscle fibers vary in size but are small in diameter, when compared with other arthropod skeletal muscles. They are striated with A, I, Z and poorly defined H bands. The sarcomere length ranges from 3-10 μm with most sarcomeres in the range of about 6 μm. The myofilaments are arranged in lamellae in larger fibers and less well organized in the smaller ones. Each thick filament is surrounded by 9-12 thin filaments which overlap. The SR is sparse but well organized to form a fenestrated collar around the fibrils. Individual SR tubules are also seen among the myofibrils. Long transverse tubules extend inward from the sarcolemma to form dyads or triads with the SR at the A-I junction. Both dyads and triads coexist in a single muscle fiber, a feature believed to have evolutionary significance. The neuromuscular relationship is unique. In the region of synaptic contact, the sarcolemma is usually elevated to form a large club-shaped structure containing no myofilaments and few other organelles. The axons or axon terminals and glial elements penetrate deep into the club-shaped sarcoplasm and form synapses with the fiber. As many as 13 terminals have been observed within a single section. Synaptic vesicles of two types are found in the axon terminals.     

FINE STRUCTURE OF RAT INTRAFUSAL MUSCLE FIBERS : The Equatorial Region

An ultrastructural study has been undertaken on the equatorial (sensory) region of the rat muscle spindle. Two kinds of intrafusal muscle fibers, a nuclear bag fiber and a nuclear chain fiber, have been identified in this region on the basis of fiber diameter, nuclear disposition, and M-band appearance. The large-diameter nuclear bag fiber contains an aggregation of tightly packed vesicular nuclei, while the small-diameter nuclear chain fiber contains a single row of elongated, well-separated nuclei. Both muscle fibers contain an attenuated peripheral cylinder of myofilaments surrounding a central core of sarcoplasm. Elements of the sarcotubular system, dilatations of the sarcoplasmic reticulum, and the presence of other sarcoplasmic organelles and inclusions are considerably more abundant in the nuclear chain fiber than in the nuclear bag fiber. Leptomeric organelles and membrane-bounded sarcoplasmic granules are present in both intrafusal fiber types and may be situated between the myofibrils or in intimate association with the sarcolemma. The functional significance of some of these structural findings is discussed.     

Distribution of sarcoplasmic reticulum Ca-ATPase and of calsequestrin at the polar regions of rat, rabbit and cat intrafusal fibers

Sarcoplasmic reticulum (SR) Ca2+-pumping ATPase (Ca-ATPase) and calsequestrin (CaS) were visualized by indirect immunofluorescence at the polar regions of adult rat, rabbit and cat intrafusal fibers. The immunohistochemical reaction products were regarded as histochemical markers of the SR and as valid indicators of the distribution of the two Ca2+-sequestering proteins. Static nuclear bag2 fibers displayed lower levels of both Ca-ATPase and CaS than the other two intrafusal fiber types. Nuclear chain fibers presented the highest Ca-ATPase levels and, together with dynamic nuclear bag1 fibers, they also exhibited relatively high amounts of CaS. The level of Ca-ATPase was lower in bag 1 fibers than in nuclear chain fibers, but not as low as in bag2 fibers. The comparatively high levels of Ca-ATPase and CaS seen in nuclear chain fibers coincided with their reported faster contractile speeds compared to nuclear bag fibers.     

粘虫蛾飞行肌超微结构的研究

应用电子显微镜对粘虫雌蛾Mythimna separata(Walker)飞行(背纵)肌的研究结果表明,其肌原纤维由500-700根肌球蛋白丝(粗丝)组成,每根粗丝由6根肌动蛋白丝(细丝)环绕排列成六角形,每根细丝精确地位于两根粗丝间1/2处,从而使粗丝和细丝的比为1：3。肌节较短,长度约2.2-2.6μm。肌原纤维之间充满着线粒体和横管。每个肌节约有线粒体三个,横管二根。线粒体约占肌纤维体积的40%,而横管为7%。每根横管准确地位于肌节的1/4、3/4处,或Z线和中膈的中央,并与肌质网交接形成二位体(dyads)或三位体(triads)。肌质网相当不发达,约占肌纤维体积的2.5%。但其分布很有特色,即除了紧贴于肌原纤维周围的由单层液泡组成的肌质网以外,在中膈处还有一层横穿于肌原纤维的肌质网。和其它同步飞行肌的结构和功能分析比较的结果还表明,粘虫蛾飞行肌具有较善于飞行的结构。     

SARCOPLASMIC RETICULUM AND T TUBULES IN DIFFERENTIATING RAT SKELETAL MUSCLE

An electron microscope study has been made of the distribution of membrane couplings between the sarcoplasmic reticulum (SR) and either the plasmalemma or the T tubules in fetal and neonatal rat intercostal muscle. Within primitive muscle cells at 12 days of gestation, the SR forms both simple and specialized membrane junctions with the plasmalemma; caveolae are very few, and T tubules are not detected. Undifferentiated cells neighbor muscle cells. Occasionally these cells contain subsurface couplings between the endoplasmic reticulum and plasmalemmae. Possible relationships between these couplings and the peripheral couplings of muscle cells are discussed. By 15–18 days of gestation, caveolae and beaded T tubules, comparable to those of cultured muscle, develop; T tubules lie along-side myofibrils and are rarely transverse. SR couples both to T tubules and to plasmalemmae during this period. T tubules with lineal profiles appear after further development and their orientation transverse to A–I junctions becomes increasingly evident. Membrane couplings between SR and T tubules also increase in number, whereas the incidence of peripheral coupling declines rapidly Evidence suggests that peripheral couplings are swept into myotubes as caveolae proliferate and T tubules form. SR thus appears to initially couple with the plasmalemma and then to await T tubular growth. This contrasts with the developmental pattern described in cultured chick muscle in which peripheral couplings are not reported and T tubules with diads and triads occur at very primitive stages of muscle differentiation.     

Simultaneous maturation of transverse tubules and sarcoplasmic reticulum during muscle differentiation in the mouse

The development and maturation of transverse (T) tubules and sarcoplasmic reticulum (SR) have been studied in pre- and postnatal mouse muscle, using selective "staining" of these membrane systems. As previously reported in the literature, orderly transverse orientation of the T tubules occurs late in development and early T-SR junctions (triads and dyads) are located at random along the T tubules in a predominantly longitudinal orientation. We find that initial appearance of transverse tubules occurs fairly abruptly, and that all early T tubules have a longitudinal orientation. Transverse orientation of the T tubule network, location of triads at the A-I junction, and development of differentiated regions of the SR are coordinated events which occur gradually over a period of about 3 weeks for leg muscle.s The timing of triad development coincides with that reported for the increase in slow calcium current and dihydropyridine binding. Differences in T tubule patterns between muscle fibers of EDL and soleus are apparent only at relatively late stages.     

The fine structure of the drum muscles of the Tigerfish,Therapon jarbua,as compared with the trunk musculature

Summary The fibers of drum and trunk muscles of the Tigerfish, Therapon jarbua, differ greatly in diameter. The myofibrils of the trunk muscles are irregularly oriented, while those of the drum muscles are rolled into spiral or concentric bands. Both muscle types possess the sarcomere structure typical of cross-striated musculature. However, the myofibrils of the drum muscles differ greatly in sarcomere length and width from those in the trunk musculature. The trunk muscles contain few mitochondria, whereas in the drum muscles mitochondria are abundant. The sarcoplasmic reticulum (SR) of the drum muscles takes the form of elongated tubes in both the A and the I region; that of the trunk musculature consists of small vesicles. Of the two muscle types, the drum muscle contains more SR. With respect to the form of the T system, the trunk musculature is of the Z type and the drum muscles of the A-I type. The drum muscle displays a considerably greater number of motor endplates; these lack typical junctional folds and have mitochondria with very few cristae. No fat could be demonstrated in either the drum or the trunk muscles. However, the concentration of glycogen is higher in the drum muscle than in the musculature of the trunk.This work was accomplished with support from the Deutsche Forschungsgemeinschaft and is gratefully dedicated to Prof. R. Danneel on the occasion of his 75th birthday.     

Immunohistochemical demonstration of embryonic myosin heavy chains in adult mammalian intrafusal fibers

Serial cross sections of rat, rabbit and cat intrafusal fibers from muscle spindles of normal adult hindlimb muscles were incubated with a monoclonal antibody against embryonic myosin heavy chains. Intrafusal fiber types were identified by noting their staining patterns in adjacent sections incubated for myofibrillar ATPase after acid or alkaline preincubation. In rat and rabbit muscle spindles dynamic nuclear bag1 fibers reacted strongly at the polar and juxtaequatorial regions. Static nuclear bag2 fibers reacted weakly or not at all at the polar region, but showed a moderate amount of activity at the juxtaequator. At the equatorial region both types of nuclear bag fibers displayed a rim of fluorescence surrounding the nuclear bags, while the areas occupied by the nuclear bags themselves were negative. Nuclear chain fibers in rat and rabbit muscle spindles were unreactive with the specific antibody over their entire length. In cat muscle spindles both types of nuclear bag fibers presented profiles which resembled those of the nuclear bag fibers in the other two species, but unlike in rat and rabbit spindles, cat nuclear chain fibers reacted as strongly as dynamic nuclear bag1 fibers.     

Immunohistochemical demonstration of embryonic myosin heavy chains in adult mammalian intrafusal fibers

Summary Serial cross sections of rat, rabbit and cat intrafusal fibers from muscle spindles of normal adult hindlimb muscles were incubated with a monoclonal antibody against embryonic myosin heavy chains. Intrafusal fiber types were identified by noting their staining patterns in adjacent sections incubated for myofibrillar ATPase after acid or alkaline preincubation. In rat and rabbit muscle spindles dynamic nuclear bag₁ fibers reacted strongly at the polar and juxtaequatorial regions. Static nuclear bag₂ fibers reacted weakly or not at all at the polar region, but showed a moderate amount of activity at the juxtaequator. At the equatorial region both types of nuclear bag fibers displayed a rim of fluorescence surrounding the nuclear bags, while the areas occupied by the nuclear bags themselves were negative. Nuclear chain fibers in rat and rabbit muscle spindles were unreactive with the specific antibody over their entire length. In cat muscle spindles both types of nuclear bag fibers presented profiles which resembled those of the nuclear bag fibers in the other two species, but unlike in rat and rabbit spindles, cat nuclear chain fibers reacted as strongly as dynamic nuclear bag₁ fibers.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Muscle fibers from dysgenic mouse in vivo lack a surface component of peripheral couplings.

We have studied the structure of developing normal and dysgenic (mdg/mdg) mouse muscle fibers in vivo, with special attention to the components of the junctions between the sarcoplasmic reticulum and either the surface membrane or the transverse tubules. Triads and dyads are rare in dysgenic muscle fibers, but have apparently normal disposition of feet and calsequestrin. Peripheral couplings in normal developing muscle fibers have junctional tetrads in their surface membrane in association with the junctional feet. Muscle fibers in dysgenic mice lack junctional tetrads. This provides indirect evidence for the identification of the components of junctional tetrads with dihydropyridine receptors, which are known to be absent in dysgenic muscle fibers.     

COORDINATED DEVELOPMENT OF THE SARCOPLASMIC RETICULUM AND T SYSTEM DURING POSTNATAL DIFFERENTIATION OF RAT SKELETAL MUSCLE

An electron microscope study has been carried out on rat psoas muscle, during the early postnatal stages of development. Among the several subcellular components, the sarcotubular system undergoes the most striking modifications during this period. In muscle fibers of the newborn rat, junctional contacts between the T system and the SR are sparse and are, mostly, longitudinally or obliquely oriented. The T tubules do not penetrate deeply into the muscle cell, as indicated by the predominantly peripheral location of the triads and the persistence, at these stages of development, of a highly branched subsarcolemmal system of tubules. Diadic associations of junctional SR elements with the plasma membrane are also occasionally observed. The early SR elaborations incompletely delineate the myofibrils, at both the A- and I-band level. Longitudinal sections show irregularly oriented SR tubules, running continuously over successive sarcomeres. Flattened junctional cisterns filled with granular material are sparse and laterally interconnected, at circumscribed sites, with the SR tubules. Between 1 and 2 wk postpartum, transversal triadic contacts are extensively established, at the A-I band level, and the SR network differentiates into two portions in register with the A and I band, respectively. At 10–15 days after birth, the SR provides a transversely continuous double sheet around the myofibrils at the I-band level, whereas it forms a single discontinuous layer at the A-band level. The relationship that these morphological modifications of the sarcotubular system may bear to previously described biochemical and physiological changes of rat muscle fibers after birth is discussed.     

Distribution of sarcoplasmic reticulum Ca-ATPase and of calsequestrin at the polar regions of rat,rabbit and cat intrafusal fibers

Summary Sarcoplasmic reticulum (SR) Ca²⁺-pumping ATPase (Ca-ATPase) and calsequestrin (CaS) were visualized by indirect immunofluorescence at the polar regions of adult rat, rabbit and cat intrafusal fibers. The immunohistochemical reaction products were regarded as histochemical markers of the SR and as valid indicators of the distribution of the two Ca²⁺sequestering proteins. Static nuclear bag₂ fibers displayed lower levels of both Ca-ATPase and CaS than the other two intrafusal fiber types. Nuclear chain fibers presented the highest Ca-ATPase levels and, together with dynamic nuclear bag₁ fibers, they also exhibited relatively high amounts of CaS. The level of Ca-ATPase was lower in bag₁ fibers than in nuclear chain fibers, but not as low as in bag₂ fibers. The comparatively high levels of Ca-ATPase and CaS seen in nuclear chain fibers coincided with their reported faster contractile speeds compared to nuclear bag fibers.Dedicated to Professor Dr. T.H.Schiebler on the occasion of his 65th birthday     

Fiber content and myosin heavy chain composition of muscle spindles in aged human biceps brachii.

The present study investigated potential age-related changes in human muscle spindles with respect to the intrafusal fiber-type content and myosin heavy chain (MyHC) composition in biceps brachii muscle. The total number of intrafusal fibers per spindle decreased significantly with aging, due to a significant reduction in the number of nuclear chain fibers. Nuclear chain fibers in old spindles were short and some showed novel expression of MyHC alpha-cardiac. The expression of MyHC alpha-cardiac in bag1 and bag2 fibers was greatly decreased in the A region. The expression of slow MyHC was increased in nuclear bag1 fibers and that of fetal MyHC decreased in bag2 fibers whereas the patterns of distribution of the remaining MyHC isoforms were generally not affected by aging. We conclude that aging appears to have an important impact on muscle spindle composition. These changes in muscle spindle phenotype may reflect an age-related deterioration in sensory and motor innervation and are likely to have an impact in motor control in the elderly.     

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.     

A comparative electron microscope study of visceral muscle fibers in Cambarus,Drosophila and Lumbricus

Summary The arrangement of myofilaments in the striated visceral muscle fibers of two arthropods (crayfish and fruitfly) and in the unstriated visceral fibers of one annelid (earthworm) was studied comparatively. Transverse sections through the A bands of arthropod visceral fibers indicate that each thick myofilament is surrounded by approximately 12 thin filaments. The myofilaments are less organized in the visceral fibers of the earthworm than in muscle fibers of the crayfish and fruitfly. The thick myofilaments of the earthworm are composed of subunits, 20–30 Å in diameter. The presence of two distinct sets of myofilaments in these slowly contracting striated and unstriated visceral muscle fibers suggests that contraction is accomplished via a sliding filament mechanism.In crayfish visceral fibers the sarcolemma invaginates at irregular intervals to form a long and unbranched tubular system at any level in the sarcomere. Dyads formed by the apposition of T and SR membranes are observed frequently. The distribution of the T and SR systems in the visceral fibers of the fruitfly and the earthworm is markedly reduced and dyads are infrequently observed. The reduced T and SR systems may be related to the slow contraction of these fibers. Transport of specific substances across the sarcolemma could initiate contraction or relaxation in these fibers.This study was supported by a training grant GM-00582-06 from the U.S. Public Health Service.     

Fast drum strokes: Novel and convergent features of sonic muscle ultrastructure,innervation, and motor neuron organization in the pyramid butterflyfish (hemitaurichthys polylepis)

Sound production that is mediated by intrinsic or extrinsic swim bladder musculature has evolved multiple times in teleost fishes. Sonic muscles must contract rapidly and synchronously to compress the gas‐filled bladder with sufficient velocity to produce sound. Muscle modifications that may promote rapid contraction include small fiber diameter, elaborate sarcoplasmic reticulum (SR), triads at the A–I boundary, and cores of sarcoplasm. The diversity of innervation patterns indicate that sonic muscles have independently evolved from different trunk muscle precursors. The analysis of sonic motor pathways in distantly related fishes is required to determine the relationships between sonic muscle evolution and function in acoustic signaling. We examined the ultrastructure of sonic and adjacent hypaxial muscle fibers and the distribution of sonic motor neurons in the coral reef Pyramid Butterflyfish (Chaetodontidae: Hemitaurichthys polylepis) that produces sound by contraction of extrinsic sonic muscles near the anterior swim bladder. Relative to adjacent hypaxial fibers, sonic muscle fibers were sparsely arranged among the endomysium, smaller in cross‐section, had longer sarcomeres, a more elaborate SR, wider t‐tubules, and more radially arranged myofibrils. Both sonic and non‐sonic muscle fibers possessed triads at the Z‐line, lacked sarcoplasmic cores, and had mitochondria among the myofibrils and concentrated within the peripheral sarcoplasm. Sonic muscles of this derived eutelost possess features convergent with other distant vocal taxa (other euteleosts and non‐euteleosts): small fiber diameter, a well‐developed SR, and radial myofibrils. In contrast with some sonic fishes, however, Pyramid Butterflyfish sonic muscles lack sarcoplasmic cores and A–I triads. Retrograde nerve label experiments show that sonic muscle is innervated by central and ventrolateral motor neurons associated with spinal nerves 1–3. This restricted distribution of sonic motor neurons in the spinal cord differs from many euteleosts and likely reflects the embryological origin of sonic muscles from hypaxial trunk precursors rather than occipital somites. J. Morphol., 2013. © 2012 Wiley Periodicals, Inc.     

Myosin flares and actin leptomeres as myofibril assembly/disassembly intermediates in sonic muscle fibers

The sonic muscle of type 1 male midshipman fish produces loud and enduring mating calls. Each sonic muscle fiber contains a tubular contractile apparatus with radially arranged myofibrillar plates encased in a desmin-rich cytoskeleton that is anchored to broad Z bands (~1.2 μm wide). Immunomicroscopy has revealed patches of myosin-rich “flares” emanating from the contractile tubes into the peripheral sarcoplasm along the length of the fibers. These flares contain swirls of thick filaments devoid of associated thin filaments. In other regions of the sarcoplasm at the inner surface of the sarcolemma and near Z bands, abundant ladder-like leptomeres occur with rungs every 160 nm. Leptomeres consist of dense arrays of filaments (~4 nm) with a structure that resembles myofibrillar Z band structure. We propose that flares and leptomeres are distinct filamentous arrays representing site-specific processing of myofibrillar components during the assembly and disassembly of the sarcomere. Recent reports that myosin assembles into filamentous aggregates before incorporating into the A band in the skeletal muscles of vertebrates and Caenorhabditis elegans suggest that sonic fibers utilize a similar pathway. Thus, sonic muscle fibers, with their tubular design and abundant sarcoplasmic space, may provide an attractive muscle model to identify myofibrillar intermediates by structural and molecular techniques. This work was supported by the Intramural Research Program of the NIAMS, NIH, HHS (KW).     

SPHEROIDAL BODIES IN THE JUNCTIONAL SARCOPLASMIC RETICULUM OF LIZARD MYOCARDIAL CELLS

The sarcoplasmic reticulum (SR) of lizard (Anolis carolinensis) myocardial cells has been examined, with particular attention being paid to the structural details of the peripheral couplings (junctional SR). Spheroidal bodies are present within the opaque core of junctional SR; these can be seen both in sections made en face and in sections cut to show the apposition of the junctional SR with the sarcolemma. Opaque junctional processes extend between the sarcolemma and the peripheral junctional SR. The myocardial cells in addition contain some SR cisternae deep within the cells which also possess opaque cores composed of spheroids. Although the significance of the junctional SR spheroidal bodies is unknown, it is thought that they could act as a matrix on which enzymes such as calcium-specific ATPase may be located.     

CARDIAC MUSCLE : Its Ultrastructure in the Finch and Hummingbird with Special Reference to the Sarcoplasmic Reticulum

Cardiac muscle fibers of the hummingbird and finch have no transverse tubules and are smaller in diameter than those of mammalian hearts. The fibers are connected by intercalated discs which are composed of desmosomes and f. adherentes; small nexuses are often interspersed. As in cardiac muscle of several other animals, the junctional SR of the couplings is highly structured in these two birds but, in addition, and after having lost sarcolemmal contact, the junctional SR continues beyond the coupling to extend deep into the interior of the cells and to form belts around the Z-I regions of the sarcomeres. This portion of the sarcoplasmic reticulum, which we have named "extended junctional SR," and which is so prominent and invariant a feature of cardiac cells of hummingbirds and finches, has not been observed in chicken cardiac cells. The morphological differences between these species of birds may be related to respective differences in heart rates characteristic for these birds.     

Detection and localization of triadin in rat ventricular muscle

Summary Dyads (transverse tubule—junctional sarcoplasmic reticulum complexes) were enriched from rat ventricle microsomes by continuous sucrose gradients. The major vesicle peak at 36% sucrose contained up to 90% of those membranes which possessed dihydropyridine (DHP) binding sites (markers for transverse tubules) and all membranes which possessed ryanodine receptors and the putative junctional foot protein (markers for junctional sarcoplasmic reticulum). In addition, the 36% sucrose peak contained half of the vesicles with muscarine receptors. Vesicles derived from the nonjunctional plasma membrane as defined by a low content of dihydropyridine binding sites per muscarine receptor and from the free sarcoplasmic reticulum as defined by the M_r 102K Ca²⁺ ATPase were associated with a diffuse protein band (22–30% sucrose) in the lighter region of the gradient. These organelles were recovered in low yield. Putative dyads were not broken by French press treatment at 8,000 psi and only partially disrupted at 14,000 psi. The monoclonal antibody GE4.90 against skeletal muscle triadin, a protein which links the DHP receptor to the junctional foot protein in skeletal muscle triad junctions, cross-reacted with a protein in rat dyads of the same M_r as triadin. Western blots of muscle microsomes from preparations which had been treated with 100mm iodoacetamide throughout the isolation procedure showed that cardiac triadin consisted predominantly of a band of Mr 95 kD. Higher molecular weight polymers were detectable but low in content, in contrast with the ladder of oligomeric forms in rat psoas muscle microsomes. Cardiac triadin was not dissolved from the microsomes by hypertonic salt or Triton X-100, indicating that it, as well as skeletal muscle triadin, was an integral protein of the junctional SR. The cardiac epitope was localized to the junctional SR by comparison of its distribution with that of organelle markers in both total microsome and in French press disrupted dyad preparations. Immunofluorescence localization of triadin using mAb GE4.90 revealed that intact rat ventricular muscle tissue was stained following a well-defined pattern of bands every sarcomere. This spacing of bands was consistent with the interpretation that triadin was present in the dyadic junctional regions.