About the T-system in the myofibril-free sarcoplasm of the frog muscle fibre

Previous investigations of the T-system in skeletal muscle fibres described the inter-myofibrillar relationships between T-tubules and the sarcoplasmic reticulum. They disregarded the arrangement of the T-system in the myofibril-free sarcoplasm in the area of muscle fibre nuclei. In the present investigation, the T-system was filled by means of lanthanum incubation and the myofibril-free sarcoplasm was ultrastructural examined by means of thin (< or = 100 nm) as well as thick sections (> 300 nm-1 microm) with the electron microscope. The investigation of thick sections revealed that T-tubules meander through this myofibril-free sarcoplasm and tangle up at the poles of muscle fibre nuclei and in the area of fundamental nuclei of the motor end plate. They are, far from myofibrils, in proximity to these nuclei, the Golgi apparatus and mitochondria. On basis of this proximity and their openings at the muscle fibre surface, a contribution at the drainage of metabolic products and at the local calcium control is discussed.     

The fine structure of pigeon breast muscle

The pectoralis major muscle of the pigeon is composed of two types of muscle fibre. In the Type I fibres, the myofibrils are closely packed and there are few mitochondria. The myofibrils in the Type II fibres are separated by numerous columns of large mitochondria and lipid droplets. The membrane systems of the two types of fibre are similar. The triads occur at the Z-line; the sarcoplasmic reticulum is in the form of large terminal cisternae which are joined by narrow longitudinal tubules to a broad central cisterna. The value of morphological criteria in the classification of muscle fibres is discussed.     

Confocal microscopy study of membrane organelles of the skeletal muscle fiber in the process of Zenker’s (spreading) necrosis

Using laser confocal microscopy and some vital fluorescent dyes (acridine orange, RH 414, DiOC₆(3), rhodamine 123, fluorescein dextran), changes of the T-system and cellular acidic organelles were studied during spreading (Zenker’s) necrosis of isolated frog skeletal muscle fibers. The most characteristic of the initial stages of development of Zenker’s necrosis is the formation of numerous vacuoles as a result of local T-system swellings. The vacuole length can reach tens of micrometers. They are located both near nuclear poles and between myofibrils. Until the moment of contraction knot separation, the vacuoles preserve their connections with normal T-tubules and under certain conditions (glycerol influx to the fiber) are reversible. The vacuoles deform nuclei and cisternae of the sarcoplasmic reticulum. Acidic cell organelles accumulating acridine orange (lysosomes, late endosomes, trans-Golgi cisternae) are located in the immediate vicinity both of normal and of vacuolated T-tubules. In the course of the development of the pathological process, the size and number of acidic organelles increases and they tend to be clustered. Vacuolation of the T-system during necrosis was not accompanied by vacuole content acidification. At late stages of necrosis, alterations of nuclei and sarcoplasmic reticulum were observed. The role of cellular acidic organelles and of the T-system vacuolation in development of various muscle pathologies is discussed.     

Quantitative succinate-dehydrogenase histochemistry. II. A comparison between visual and quantitative msucle fibre typing.

Most muscles exhibit a mosaic pattern of staining intensities of their muscle fibres after the histochemical reaction for succinate dehydrogenase (SDH). Visually these muscle fibres are usually classified into three groups: with low (A-fibres), intermediate (B-fibres), and high (C-fibres) enzymatic activity (staining intensity). Cytospectrophotometric methods were employed to investigate whether discrete groups of muscle fibres could be discerned, comparable to those found after the visual classification. The classifications were based on quantitative parameters of the total absorbance per cell and the distribution of the coloured endproduct over the fibre cross area.     

Ultrastructure of the myocardial cell and its membrane systems in the adult fly Calliphora erythrocephala (Insecta: Diptera)

Summary Calliphora erythrocephala has cross-striated cardiac muscle cells with A, I and Z-bands. The diameters of the myosin and actin filaments are 200–250 Å and 85 Å respectively and the length of the myosin filaments (A-band) is approximately 1.5 . Usually 8–10 actin filaments surround each myosin filament.The myocardial cells show a well-developed membrane system and interior couplings. A perforated sheet of SR envelopes the myofibrils at the A-band, dilates into flattened cisternae at both A-I band levels before it merges into a three-dimensional net-work between the actin filaments of the I-bands and between the dense bodies of the discontinuous Z-discs. The T-system consists of broad flattened tubules running between the myofibrils at the A-I band levels forming dyads with the SR-cisternae. Longitudinal connections between the transverse (T-) tubules often occur.It is suggested that this well-developed SR may be an adaptation to facilitate a rapid contraction/relaxation frequency by an effective Ca²⁺ uptake.     

Interpretation of the cross sectional structure of skeletal muscle fibers. 2. Light and electron microscopic studies of m. rectus abdominis in Rana esculenta]

In both longitudinal and cross sections of rectus abdominis muscle of Rana esculenta three types of muscle fibres are identified by means of light and electron microscopy. A comparison is made between these fibre types in homologous muscles of frog and mammals (rat and mouse). In longitudinal sections of mammalian and frog muscle the Z-line can be used for discrimination of the fibre types A, B and C because that line is of different thickness in each type. The proportions of the thickness in frog and mammalian muscles are relatively the same, but the absolute values are different. In cross sections there are no differences between frog and mammalian muscle fibres concerning the typical form of myofibrils in type A- and B-fibres, whereas in type C-fibres the arrangement of the filaments in the Z- and H-layer is different in the members of both animal classes. The amount of mitochondria and lipid droplets is different as well. In the species examined the distribution of A-, B- and C-fibres changes within the whole muscle. In frog, this pattern depends on the level in which the muscle has been sectioned. This is not true for mammalian muscle. On the other hand both ends of the rectus abdominis muscle in frog, rat and mouse show an accumulation of B- and C-type fibres.     

Morphological and physiological characterization of fiber types in the iliofibular muscle of Rana esculenta]

In both longitudinal and cross sections of the M. iliofibularis of Rana esculenta three types of muscle fibres are identified by means of light and electron microscopy. These fibretypes called A-, B- and C-fibres are according to the fibres of m. rectus abdominis of the frog. They can be compared with the fibres of the m. rectus abdominis of rat and mouse. But there is another distribution of the fibretypes A, B and C in the m. iliofibularis and in the m. rectus abdominis. The m. iliofibularis is divided into two parts called "Tonusbündel" and "nichttonischer Teil" by means of their reaction to acetylcholine. The surface of the "Tonusbündel" consists of A-, B- and C-fibres while its inside is onlyformed by A- and B-fibres. They continue the "Tonusbündel" in the "nichttonischer Teil". This part chiefly consists of A-fibres. In cross sections their myofibrils are larger in their extent than the A-fibres known before. Therefore the A-fibretype has to be distinguished into two A-fibres: A1 and A2. The new one is called A2-fibre. A1-fibre is described in the "Tonusbündel" and in further investigations. The difference between the two fibres can be understood as a greater manifestation of power of the A1-fibre. The surface of the "nichttonischer Teil" of the m. iliofibularis consists of A2-fibres which easily could be found opposite the "Tonusbündel". At this point in contrary to the "Tonusbündel" could be found a defined morphological substrate for physiological investigations. The different reactions of "Tonusbündel" and "nichttonischer Teil" to acetylcholine could only be explained by the sum of reactions of all fibretypes in each bundle in correspondence with the reaction of the fibres in the neighbour bundle. But their different behaviour by summer- and winterfrogs is unknown. Therefore it is to discuss whether it is allowed to refer generally the results to "muscle" or "musclefibre" got from frogs living in cooled rooms. It is known in literature that not all results of physiological investigations can be interpreted with the two fibre- theorie ("twitch" and "slow") of muscle. Those not interpretable physiological results could be associated to the B-fibre can not be explained by morphological methods but must be proofed by physiological investigations. In tables are summerised morphological criteria of the three types and it is tried to associate the physiological qualities known from literature. Besides there is summerised the usual nomenclature with the first citations.     

Molecular organization of transverse tubule/sarcoplasmic reticulum junctions during development of excitation-contraction coupling in skeletal muscle.

The relationship between the molecular composition and organization of the triad junction and the development of excitation-contraction (E-C) coupling was investigated in cultured skeletal muscle. Action potential-induced calcium transients develop concomitantly with the first expression of the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR), which are colocalized in clusters from the time of their earliest appearance. These DHPR/RyR clusters correspond to junctional domains of the transverse tubules (T-tubules) and sarcoplasmic reticulum (SR), respectively. Thus, at first contact T-tubules and SR form molecularly and structurally specialized membrane domains that support E-C coupling. The earliest T-tubule/SR junctions show structural characteristics of mature triads but are diverse in conformation and typically are formed before the extensive development of myofibrils. Whereas the initial formation of T-tubule/SR junctions is independent of association with myofibrils, the reorganization into proper triads occurs as junctions become associated with the border between the A band and the I band of the sarcomere. This final step in triad formation manifests itself in an increased density and uniformity of junctions in the cytoplasm, which in turn results in increased calcium release and reuptake rates.     

Identification of a constituent of the junctional feet linking terminal cisternae to transverse tubules in skeletal muscle

This study describes the biochemical composition of junctional feet in skeletal muscle utilizing a fraction of isolated triad junctions. [3H]Ouabain entrapment was employed as a specific marker for T-tubules. The integrity of the triad junction was assayed by the isopycnic density of [3H]ouabain activity (24-30% sucrose for free T-tubules, 38- 42% sucrose for intact triads). Trypsin, chymotrypsin, and pronase all caused separation of T-tubules from terminal cisternae, indicating that the junction is composed as least in part of protein. Trypsin and chymotrypsin hydrolyzed four proteins: the Ca2+ pump, a doublet 325,000, 300,000, and an 80,000 Mr protein. T-tubules which had been labeled covalently with 125I were joined to unlabeled terminal cisternae by treatment with K cacodylate. The reformed triads were separated from free T-tubules and then severed by passage through a French press. When terminal cisternae were separated from T-tubules, some 125I label was transferred from the labeled T-tubules to the unlabeled terminal cisternae. Gel electrophoresis showed that, although T-tubules were originally labeled in a large number of different proteins, only a single protein doublet was significantly labeled in the originally unlabeled terminal cisternae. This protein pair had molecular weights of 325,000 and 300,000 daltons. Transfer of label did not occur to a substantial degree without K cacodylate treatment. We propose that the transfer of 125I label from T-tubules to terminal cisternae during reformation and breakage of the triad junction is a property of the protein which spans the gap between T-tubules and terminal cisternae.     

Histochemical study of calcium on T-tubule membranes and in the sarcoplasmic reticulum, in frog twitch muscle fibres at rest and during activity

The trigger calcium hypothesis of signal transmission between T-tubules and terminal cisternae (TC) of the sarcoplasmic reticulum (SR) in twitch muscle fibres implies the presence of calcium along T-tubule membranes at rest and its release upon excitation. To test this hypothesis, calcium was immobilised using a fixing and precipitating solution of glutaraldehyde in phosphate buffer at pH 8.0 and the calcium was substituted for by lead. Simultaneous tension recordings revealed the occurrence of contractions or a burst of twitches upon perfusion with the fixative. Procaine or tetrodotoxin (TTX) was used to inhibit this activity. In fibres without fixative-induced activity, precipitates were observed along T-tubules and in adjoining parts of TC. In activated fibres, tubular and TC precipitates were absent. These results are consistent with the trigger calcium hypothesis. In fibres activated by depolarisation, calcium returned to TC after passing successively through different parts of the SR.     

Histochemical study of calcium on T-tubule membranes and in the sarcoplasmic reticulum,in frog twitch muscle fibres at rest and during activity

Summary The trigger calcium hypothesis of signal transmission between T-tubules and terminal cisternae (TC) of the sarcoplasmic reticulum (SR) in twitch muscle fibres implies the presence of calcium along T-tubule membranes at rest and its release upon excitation. To test this hypothesis, calcium was immobilised using a fixing and precipitating solution of glutaraldehyde in phosphate buffer at pH 8.0 and the calcium was substituted for by lead. Simultancous tension recordings revealed the occurence of contra tions or a burst of twitches upon perfusion with the tixative. Procaine or tetrodotoxin (TTX) was used to inhibit this activity. In fibres without fixative-induced activity, precipitates were observed along T-tubules and in adjoining parts of TC. In activated fibres, tubular and TC precipitates were absent. These results are consistent with the trigger calcium hypothesis. In fibres activated by depolarisation, calcium returned to TC after passing successively through different parts of the SR.     

Confocal-microscopic study of skeletal muscle fibre membrane organelles during Zenker's (spreading) necrosis

The changes of T-system and cellular acidic organelles during spreading (Zenker's) necrosis of frog skeletal muscle fibres have been investigated using laser confocal microscopy and several vital fluorescent dyes acridine orange, RH 414, DiOC6(3), rhodamine 123, fluorescein dextran. The formation of numerous vacuoles as a result of local T-system swelling is most characteristic for initial steps of Zenker's necrosis. Vacuoles can attain tens microns in length. They are located both near nuclear poles and between myofibres. Vacuoles maintain connections with the extracellular space up to the moment of contraction knot rejection, and under definite conditions (glycerol influx to fibre) vacuoles are reversible. They deform nuclei and sarcoplasmic reticulum cisternae. Cellular acidic organelles, accumulating acridine orange (lysosomes, late endosomes, Golgi apparatus cisternae) are situated in direct vicinity with normal and vacuolated T-system. The increase in acidic organelles number and size occur during the pathological process development, and tendency to vacuoles clusterization may be seen. Vacuolation of T-system during necrosis is not followed by vacuole content acidification. The role of cellular acidic organelles and of T-system vacuolation in the development of different muscle pathological changes is discussed.     

A morphometric analysis of regional differences in myotomal muscle ultrastructure in the juvenile eel (Anguilla anguilla L.)

Summary Subpopulations of fast and slow fibres within the trunk musculature of elvers were examined using morphometric analysis of electron micrographs. Fibre regions were characterised by their histochemical staining characteristics, and individual fibres located using a coordinate mapping system utilising morphological features as reference points. Percentages of fibre volume occupied by mitochondria, myofibrils, sarcoplasmic reticulum (S.R.), and T-system were determined in each of the fibre groups, along a transect from the skin to the vertebral column (fibres 1–14, respectively).The fine structure of slow (red) fibres (1–2 fibres deep) is relatively homogeneous throughout its range, giving mean values for mitochondria, 21.4%; myofibrils, 61.0%; S.R., 2.10%; T-system, 0.31%. The fibres are relatively small (204 m²) and the mitochondrial cristae poorly developed.In contrast, there is a marked heterogeneity in the ultrastructure of fast (white) fibres, dependent on both position and size. The moderately small (333 m²) superficial fast fibres (3–4 fibres deep) have a significantly higher mitochondrial content (7.6%) than the larger deep fibres (1.2%) (6–12 fibres deep, 775 m²). The mean fractional volumes occupied by myofibrils, S.R., and T-system in the deep fibres are: 80.4%, 5.95%, and 0.38%, respectively. Fibres < 100 m² constitute up to 5% of the fast muscle and have a significantly higher mitochondrial volume (4.3%), more glycogen granules, and a slightly lower volume of S.R. (5.57%) than larger fibres.It is suggested that metabolic subpopulations of fast fibres correspond to different stages of fibre growth. The relatively poorly developed S.R. of eel fast muscle is thought to be correlated with the low frequency, high amplitude nature of the propagated waveform found in anguilliform locomotion.     

Specialized contacts between sarcolemma and sarcoplasmic reticulum at the ends of muscle fibers in the diaphragm of the rat

Summary An electron-microscopic study of the myotendinous portion of the diaphragm in the Wistar rat has shown that at the ends of muscle fibers, longitudinally oriented invaginations and peripheral furrows of the sarcolemma establish specialized contacts with individual sacs of the sarcoplasmic reticulum. The construction of these terminal contacts is similar to that of contacts between sarcolemmic T-tubules and terminal cisternae of the sarcoplasmic reticulum, characterized by formation of triads. The contact zones of the sac membrane are undulated and bound to the adjoining sarcolemma via electron-dense profiles of varying forms. Frequently, the terminal contacts and triads are located at the same level within the muscle fiber, at the borderline between A- and I-bands of the sarcomeres. At the ends of muscle fibers combined contacts between peripheral furrows of the sarcolemma, terminal cisternae of the sarcoplasmic reticulum, and T-tubules of the triads are also disclosed. The implications of the terminal contacts for muscle contraction are discussed.     

Architecture of the T-system in helical paramyosin muscles of an annelid (Branchiobdella)

Tannic acid impregnation has revealed the existence of a T-system in the helical fibers of Branchiobdella pentodonta (Annelida, Clitellata). T-tubules are L-shaped inside the fiber, within the plane of the I-band: after a short horizontal tract they run longitudinally for a long tract keeping contact with many sarcoplasmic reticulum cisternae and forming dyads. The presence of a T-system in this annelid, the only one demonstrated up to now in annelids, is to be ascribed to the thickness of the contractile layer of those fibers.     

Fibre types in locomotory muscles of the cartilaginous fish Chimaera monstrosa

The cartilaginous fish Chimaera monstrosa swims slowly by means of pectoral fin movements, and fast by undulations of the tail. In order to compare the fibres in the corresponding muscles, they were studied by histochemistry and electron microscopy. Three fibre types were identified by microphotometry and morphometry. Most of the axial muscles are white fibres, containing little mitochondria and glycogen. Red fibres, with glycogen and about 5 % mitochondria constitute a thin sheet in the axial muscles, composed of one fibre layer only. Pink fibres, with intermediate amounts of glycogen and mitochondria are situated between these two types, but are often not covered by red fibres. Pectoral muscles contain numerous red and intermediate fibres, partially mixed, superficially, and white fibres deeper. Pectoral muscle red fibres contain about 25 % mitochondria, half of which are situated in subsarcolemmal accummulations. The sarcotubular system has T-tubules at the Z discs, and the terminal cisternae are partially divided by regularly spaced clefts.     

Quantitative analyses of ultrastructure and vascularization of the slow muscle fibres of the anchovy

A quantitative study has been made of the ultrastructure and vascularization of slow fibres in the lateral muscles of the European anchovy (Engraulis encrasicolus). Mitochondria and myofibrils occupy 45.5 and 44.3% of total fibre volume respectively. More than 95% of all myofibrils are adjacent to mitchondria. A total of 51 % of the sarcolemma is in direct contact with capillaries with a mean of 12.9 capillaries per fibre. In transverse sections anchovy slow fibr es are considerably flattened (long to short axis 12:1) such that the surface to volume ratio is more than twice that of a cylindrical fibre of the same area (1115 μm²). The capillary surface required to supply l μm³ of mitochondria is 0.18 μm² and the maximum distance between any capillary and mitochondrion 8 μm. T-system and sarcoplasmic reticulum occupy 0.43 and 2.7% of fibre volume respectively. Adaptations for increasing the capacity of skeletal muscle for aerobic work are discussed.     

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。     

Atypical ‘fibrillar’ flight muscle in strepsiptera

The fine structure of the principal and ancillary metathoracic flight muscle fibres in the adult male of a strepsipteran, Elenchus tenuicornis, is described. Power-producing dorsal longitudinal and dorso-ventral flight muscles show features consistent with myoneural asynchrony: myofibrils are large and discrete and are separated by large closely packed mitochondria; the sarcoplasmic reticulum is very reduced but engages with T-system membranes in dyads at the mid-sarcomere H-band level. With respect to other asynchronous insect flight muscles, the fibres of Elenchus are anomalous (i) in the small fibre diameter, (ii) in the variable contour of the myofibrils and (iii) in the absence of tracheolar invagination. The functional significance of these structural features is discussed. Ancillary metathoracic muscles are structurally comparable with other synchronous fibres in possessing an extensive SR compartment. Structural evidence for asynchrony in the flight mechanism of Strepsiptera is considered in the context of the evolution of this mechanism throughout the insect Orders.     

The effect of starvation on the ultrastructure of the red and white myotomal muscles of the crucian carp (Carassius carassius)

Summary The effect of 16 weeks total starvation on the ultrastructure of the red and white myotomal muscles of the crucian carp (Carassius Carassius) has been investigated. In the white fibres the amount of myofibrillar material fell from 89.6% to 70.7% of the total fibre volume whilst in the red fibres the fall was from 72.2% to 70.3%. The sarcoplasmic reticulum appeared to have become swollen during starvation in both fibre types. In the white fibres the terminal cisternae of some triads seem to have fused. The volume of the red fibres occupied by mitochondria was reduced from 16.2 % to 5.9 %. The concentration of mitochondria in the white fibres was too low to detect any quantitative changes. A marked reduction in the amount of euchromatin material was observed in most white fibre nuclei and many red fibre nuclei. Many of the ultrastructural changes noted in the present study can be correlated with biochemical changes known to occur in the red and white myotomal muscles of fish during starvation. This work was supported by a grant from the Natural Environmental Research Council.