Relationship of the sarcoplasmic reticulum to fibril and triadic junction development in skeletal muscle fibers of fetal monkeys and humans.

Examinations of stages of fibril development in muscle fibers of seven Rhesus monkey and six human fetuses reveal SR tubules encircling the Z lines at all stages of fibril development. The encircling SR tubules are continuous with the SR network of tubules which is found surrounding fibrils at all stages of development observed. The SR tubules encircling the Z lines show connections (electron-opaque strands) with the Z lines. The developing triadic junction shows a progressive increase in complexity of structures within the junction. First, membranes of T and SR become apposed with no visible structure between them- Second, tenuous connections are found traversing the space between apposed membranes. Third, well developed bridges are seen traversing the space. And finally, an intermediate density midway between the apposed membranes and parallel to them is found in favorable sections. Junctions between T tubule membranes were also observed and the structures in these junctions are somewhat similar to those found in junctions between T and SR membranes. The change in orientation of triads from predominantly longitudinal to predominantly transverse is complete in the 18-week monkey fetus and incomplete in the latest stage (28-week) of fetal development observed in humans.     

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.     

Transport of the alpha subunit of the voltage gated L‐type calcium channel through the sarcoplasmic reticulum occurs prior to localization to triads and requires the beta subunit but not Stac3 in skeletal muscles

Contraction of skeletal muscle is initiated by excitation‐contraction (EC) coupling during which membrane voltage is transduced to intracellular Ca²⁺ release. EC coupling requires L‐type voltage gated Ca₂₊ channels (the dihydropyridine receptor or DHPR) located at triads, which are junctions between the transverse (T) tubule and sarcoplasmic reticulum (SR) membranes, that sense membrane depolarization in the T tubule membrane. Reduced EC coupling is associated with ageing, and disruptions of EC coupling result in congenital myopathies for which there are few therapies. The precise localization of DHPRs to triads is critical for EC coupling, yet trafficking of the DHPR to triads is not well understood. Using dynamic imaging of zebrafish muscle fibers, we find that DHPR is transported along the longitudinal SR in a microtubule‐independent mechanism. Furthermore, transport of DHPR in the SR membrane is differentially affected in null mutants of Stac3 or DHPRβ, two essential components of EC coupling. These findings reveal previously unappreciated features of DHPR motility within the SR prior to assembly at triads.      

Abnormal formation of sarcoplasmic reticulum networks and triads during early development of skeletal muscle cells in mitsugumin29-deficient mice

Recently, we detected a novel membrane protein, mitsugumin29 (MG29), in the triads in rabbit skeletal muscle cells and suggested important roles for this membrane protein in the formation of the sarcoplasmic reticulum (SR) networks and triads in muscle cells. In the present study, we examined the development of skeletal muscle cells in MG29-deficient mice to try to determine the roles played by MG29 in the formation of the SR networks and triads. Ultrastructural observations revealed some morphological abnormalities in these mice, such as incomplete formation of the SR networks, an irregular running of the transverse tubule and a partial defect in the triads at the A-I junctional region. These ultrastructural abnormalities occurred during early myogenesis and were preserved until the adult stage. The possible roles for MG29 in the formation of SR networks and triads in skeletal muscle cells are discussed in the light of these observations.     

THE SARCOPLASMIC RETICULUM OF THE BAT CRICOTHYROID MUSCLE

The bat cricothyroid muscle is believed to participate in the production of the short bursts of frequency modulated ultrasound which these animals use as an echolocation device. The evidence seems to indicate that this muscle must be extremely fast acting. It possesses a very well developed sarcoplasmic reticulum, consisting of intercommunicating longitudinal and transverse tubular elements. The transverse elements, situated at the level of the junction between the A and the I bands, are tripartite complexes of tubules called triads, and these are sometimes replaced by more complex structures, the pentads. The intermediate element of the triad appears as a slender continuous tubule, which can be shown to come into close contact with the sarcolemma and also to share with it certain common staining properties. The longitudinal components of the reticulum consist of very numerous tubules which link successive triads to each other and anastomose to form multiple layers of close-meshed reticula in the interfibrillar sarcoplasm. Both the longitudinal and the transverse elements of the sarcoplasmic reticulum form a continuous network across the muscle fiber. It is suggested that the extraordinary development of the sarcoplasmic reticulum in the bat cricothyroid is related to the unusual physiological properties of this muscle.     

Albumin is a major protein component of transverse tubule vesicles isolated from skeletal muscle

Despite multiple procedures used to isolate transverse tubule vesicles from rabbit skeletal muscle, few proteins have been identified and shown to be specific to transverse tubule vesicles. Markers for purified transverse tubules have included high affinity dihydropyridine binding, cholesterol content, Mg2+-ATPase activity, (Na+,K+)-ATPase activity, and [3H] ouabain binding. Despite these markers, few proteins from purified transverse tubules can be unequivocally identified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In this report we have biochemically and immunologically identified rabbit albumin as a major component of purified transverse tubule membranes from rabbit skeletal muscle. Albumin composed between 5.1 and 9.8% (n = 4) of the total protein in purified transverse tubules based on scans of SDS-PAGE. Furthermore, albumin and other serum proteins are present in preparations of transverse tubules and triads but not in light sarcoplasmic reticulum. Extraction of triads with low concentrations of saponin or sodium dodecyl sulfate completely removes albumin without removing intrinsic membrane proteins. Our results suggest that albumin and other serum proteins are present in the lumen of preparations of transverse tubules and albumin may be used as a marker for the transverse tubules when analyzed on SDS gels.     

T-system formation in cultured rat skeletal tissue

By using a lanthanum-staining technique which enhances the visualization of the plasma membrane and its derivatives we have studied the formation of the T system in rat muscle cells differentiating in vitro. We have found that: (1) T-system formation normally occurs after myoblast fusion and is especially extensive in mature myotubes; myoblasts grown in calcium-deficient medium to prevent fusion show increased number of sarcolemmal caveolae but rare, short T tubules. (2) T-system formation in vitro differs from that displayed by rat muscle cells in vivo in that it precedes and is independent of junctional SR differentiation; the uncoordinated development of T tubules and junctional SR in vitro leads to the formation of ‘inverted’ triads and labyrinthine T-system networks. (3) Coated vesicles are frequently found either free in the cytoplasm or associated with growing T tubules in rat muscle cells differentiating in vitro. A role of coated vesicles in T-system formation is proposed.     

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.     

A transverse tubule NADPH oxidase activity stimulates calcium release from isolated triads via ryanodine receptor type 1 S -glutathionylation

We report here the presence of an NADPH oxidase (NOX) activity both in intact and in isolated transverse tubules and in triads isolated from mammalian skeletal muscle, as established by immunochemical, enzymatic, and pharmacological criteria. Immunohistochemical determinations with NOX antibodies showed that the gp91(phox) membrane subunit and the cytoplasmic regulatory p47(phox) subunit co-localized in transverse tubules of adult mice fibers with the alpha1s subunit of dihydropyridine receptors. Western blot analysis revealed that isolated triads contained the integral membrane subunits gp91(phox) and p22(phox), which were markedly enriched in isolated transverse tubules but absent from junctional sarcoplasmic reticulum vesicles. Isolated triads and transverse tubules, but not junctional sarcoplasmic reticulum, also contained varying amounts of the cytoplasmic NOX regulatory subunits p47(phox) and p67(phox). NADPH or NADH elicited superoxide anion and hydrogen peroxide generation by isolated triads; both activities were inhibited by NOX inhibitors but not by rotenone. NADH diminished the total thiol content of triads by one-third; catalase or apocynin, a NOX inhibitor, prevented this effect. NADPH enhanced the activity of ryanodine receptor type 1 (RyR1) in triads, measured through [3H]ryanodine binding and calcium release kinetics, and increased significantly RyR1 S-glutathionylation over basal levels. Preincubation with reducing agents or NOX inhibitors abolished the enhancement of RyR1 activity produced by NADPH and prevented NADPH-induced RyR1 S-glutathionylation. We propose that reactive oxygen species generated by the transverse tubule NOX activate via redox modification the neighboring RyR1 Ca2+ release channels. Possible implications of this putative mechanism for skeletal muscle function are discussed.     

The ultrastructure of the striated muscle of the tail of Cercaria chackai Nadakal et al., 1969

The electron microscopic study of the tail of Cercaria chackai reveals that it contains four sets of striated muscle bundles located central to the nonstriated circular and longitudinal muscles. The striated muscle consists of longitudinally oriented lamellar myofibres. Each myofibre contains a single "U" shaped myofibril. The banding pattern is analogous to that of vertebrate striated muscle. The sarcolemma is a simple surface membrane. There are no transverse tubular extensions of sarcolemma. The sarcoplasmic reticulum (SR) is very well developed with cisternae, tubules, and vesicles. SR cisternae form dyadic couplings with the sarcolemma. There is a set of flattened tubules of SR origin traversing the myofibril exactly at the Z region. These tubules are unique to the striated muscle of the cercarian tail and may have functional significance. A diagrammatic reconstruction of the myofibre is presented.     

Coordinated development of myofibrils, sarcoplasmic reticulum and transverse tubules in normal and dysgenic mouse skeletal muscle, in vivo and in vitro.

We studied the development of transverse (T)-tubules and sarcoplasmic reticulum (SR) in relationship to myofibrillogenesis in normal and dysgenic (mdg/mdg) mouse skeletal muscle by immunofluorescent labeling of specific membrane and myofibrillar proteins. At E16 the development of the myofibrils and membranes in dysgenic and normal diaphragm was indistinguishable, including well developed myofibrils, a delicate network of T-tubules, and a prominent SR which was not yet cross-striated. In diaphragms of E18 dysgenic mice, both the number and size of muscle fibers and myofibrillar organization were deficient in comparison to normal diaphragms, as previously reported. T-tubule labeling was abnormal, showing only scattered tubules and fragments. However, many muscle fibers displayed cross striation of sarcomeric proteins and SR comparable to normal muscle. In cultured myotubes, cross-striated organization of sarcomeric proteins proceeded essentially in two stages: first around the Z-line and later in the A-band. Sarcomeric organization of the SR coincided with the first stage, while the appearance of T-tubules in the mature transverse orientation occurred infrequently, only after A-band maturation. In culture, myofibrillar and membrane organization was equivalent in normal and dysgenic muscle at the earlier stage of development, but half as many dysgenic myotubes reached the later stage as compared to normal. We conclude that the mdg mutation has little effect on the initial stage of membrane and myofibril development and that the deficiencies often seen at later stages result indirectly from the previously described absence of dihydropyridine receptor function in the mutant.     

A functional identification of cardiac junctional sarcoplasmic reticulum

Junctional sarcoplasmic reticulum (SR) has been identified in microsomes from canine ventricular muscle by the presence of calsequestrin and ryanodine-sensitive Ca2+ release channels. These properties, however, are not common to cardiac cells from all species. Seiler et al (1) have recently described a high Mr polypeptide in canine junctional SR similar to the spanning protein subunits of skeletal muscle triads. We now report the existence of a polypeptide with the same mobility in SR from rabbit ventricular muscle and show that those cardiac membranes can associate with transverse (T-) tubules from rabbit skeletal muscle in K cacodylate medium. We propose that this polypeptide and the reaction with T-tubules be considered as criteria for the identification of cardiac junctional SR.     

Preparation and morphology of sarcoplasmic reticulum terminal cisternae from rabbit skeletal muscle

We have developed a procedure to isolate, from skeletal muscle, enriched terminal cisternae of sarcoplasmic reticulum (SR), which retain morphologically intact junctional "feet" structures similar to those observed in situ. The fraction is largely devoid of transverse tubule, plasma membrane, mitochondria, triads (transverse tubules junctionally associated with terminal cisternae), and longitudinal cisternae, as shown by thin-section electron microscopy of representative samples. The terminal cisternae vesicles have distinctive morphological characteristics that differ from the isolated longitudinal cisternae (light SR) obtained from the same gradient. The terminal cisternae consist of two distinct types of membranes, i.e., the junctional face membrane and the Ca2+ pump protein-containing membrane, whereas the longitudinal cisternae contain only the Ca2+ pump protein-containing membrane. The junctional face membrane of the terminal cisternae contains feet structures that extend approximately 12 nm from the membrane surface and can be clearly visualized in thin section through using tannic acid enhancement, by negative staining and by freeze-fracture electron microscopy. Sections of the terminal cisternae, cut tangential to and intersecting the plane of the junctional face, reveal a checkerboardlike lattice of alternating, square-shaped feet structures and spaces each 20 nm square. Structures characteristic of the Ca2+ pump protein are not observed between the feet at the junctional face membrane, either in thin section or by negative staining, even though the Ca2+ pump protein is observed in the nonjunctional membrane on the remainder of the same vesicle. Likewise, freeze-fracture replicas reveal regions of the P face containing ropelike strands instead of the high density of the 7-8-nm particles referable to the Ca2+ pump protein. The intravesicular content of the terminal cisternae, mostly Ca2+-binding protein (calsequestrin), is organized in the form of strands, sometimes appearing paracrystalline, and attached to the inner face of the membrane in the vicinity of the junctional feet. The terminal cisternae preparation is distinct from previously described heavy SR fractions in that it contains the highest percentage of junctional face membrane with morphologically well-preserved junctional feet structures.     

DIFFERENTIATION OF THE SARCOPLASMIC RETICULUM AND T SYSTEM IN DEVELOPING CHICK SKELETAL MUSCLE IN VITRO

The electron microscope was used to investigate the first 10 days of differentiation of the SR and the T system in skeletal muscle cultured from the breast muscle of 11-day chick embryos. The T-system tubules could be clearly distinguished from the SR in developing muscle cells fixed with glutaraldehyde and osmium tetroxide. Ferritin diffusion confirmed this finding: the ferritin particles were found only in the tubules identified as T system. The proliferation of both membranous systems seemed to start almost simultaneously at the earliest myotube stage. Observations suggested that the new SR membranes developed from the rough-surfaced ER as tubular projections. The SR tubules connected with one another to form a network around the myofibril. The T-system tubules were formed by invagination of the sarcolemma. The early extension of the T system by branching and budding was seen only in subsarcolemmal regions. Subsequently the T-system tubules could be seen deep within the muscle cells. Immediately after invaginating, the T-system tubule formed, along its course, specialized connections with the SR or ER: triadic structures showing various degrees of differentiation. The simultaneous occurrence of myofibril formation and membrane proliferation is considered to be important in understanding the coordinated events resulting in the differentiated myotube.     

Morphology of isolated triads

The triad is the junctional association of transverse tubule with sarcoplasmic reticulum terminal cisternae. A procedure for the isolation of highly enriched triads from skeletal muscle has been described in the previous paper. In the present study, the structural features of isolated triads have been examined by thin-section, negative-staining, and freeze-fracture electron microscopy. In isolated triads, key features of the structure observed in situ have been retained, including the osmiophilic "feet," junctional structures between the transverse tubule and terminal cisternae. New insight into triad structure is obtained by negative staining, which also enables visualization of feet at the junctional face of the terminal cisternae, whereas smaller surface particles, characteristic of calcium pump protein, are not visualized there. Therefore, the junctional face is different from the remainder of the sarcoplasmic reticulum membrane. Junctional feet as viewed by thin section or negative staining have similar periodicity and extend approximately 100 A from the surface of the membrane. Freeze-fracture of isolated triads reveals blocklike structures associated with the membrane of the terminal cisternae at the junctional face, interjunctional connections between the terminal cisternae and t-tubule, and intragap particles. The intragap particles can be observed to be closely associated with the t-tubule. The structure of isolated triads is susceptible to osmotic and salt perturbation, and examples are given regarding differential effects on transverse tubules and terminal cisternae. Conditions that adversely affect morphology must be considered in experimentation with triads as well as in their preparation and handling.     

Deficiency of triad formation in developing skeletal muscle cells lacking junctophilin type 1

Junctophilins (JP-1, JP-2, and JP-3) are transmembrane proteins expressed in the junctional membrane complexes in excitable cells. Both JP-1 and JP-2 are co-expressed in the triads of skeletal muscle, but only JP-2 is expressed in cardiac muscle. We analyzed the roles played by JP-1 and JP-2 in triad formation in skeletal muscle by comparing developing skeletal muscles in wild-type and JP-1-knockout (KO) mice (both before and after birth). In the skeletal muscles of embryos, most of the couplings between sarcoplasmic reticulum (SR) and transverse tubule (T-tubule) were diads, with triads being very scarce. The number of triads increased markedly after birth in wild-type mice. However, there was no increase in the number of triads in the neonates of JP-1-KO mice, and they died within 1 day after birth. JP-2 expression was constant before and after birth, while expression of JP-1 increased with birth. Quantitative and morphological differences were not seen between wild-type and JP-1-KO mice in the formation of diads in the period just before the JP-1-KO mice died. The SR swelled and developed large vacuoles in skeletal muscle cells just before the JP-1-KO mice died. The present results strongly suggest that JP-1 and JP-2 play important roles in the formation of triads and diads, respectively, during the development of skeletal muscle in mouse.     

Structural evidence for direct interaction between the molecular components of the transverse tubule/sarcoplasmic reticulum junction in skeletal muscle

The architecture of the junctional sarcoplasmic reticulum (SR) and transverse tubule (T tubule) membranes and the morphology of the two major proteins isolated from these membranes, the ryanodine receptor (or foot protein) and the dihydropyridine receptor, have been examined in detail. Evidence for a direct interaction between the foot protein and a protein component of the junctional T tubule membrane is presented. Comparisons between freeze-fracture images of the junctional SR and rotary-shadowed images of isolated triads and of the isolated foot protein, show that the foot protein has two domains. One is the large hydrophilic foot which spans the junctional gap and is composed of four subunits. The other is a hydrophobic domain which presumably forms the SR Ca2+-release channel and which also has a fourfold symmetry. Freeze-fracture images of the junctional T tubule membranes demonstrate the presence of diamond-shaped clusters of particles that correspond exactly in position to the subunits of the feet protein. These results suggest the presence of a large junctional complex spanning the two junctional membranes and intervening gap. This junctional complex is an ideal candidate for a mechanical coupling hypothesis of excitation-contraction coupling at the triadic junction.     

The structure of the membrane systems in a novel muscle cell modified for heat production

A thermogenic organ, modified from an eye muscle, warms the brain and eyes of several oceanic fish. The extraocular muscles associated with thermogenesis are composed of modified muscle cells that are structurally distinct from all other types of muscle previously described. In "heater" cells, contractile filaments are virtually absent and the cell volume is packed with mitochondria and smooth membranes. Freeze-fracture studies and negative staining of microsomal fractions treated with vanadate indicate that most of the membrane system of heater cells has a high Ca2+-ATPase density and is equivalent to skeletal muscle sarcoplasmic reticulum (SR). High voltage electron micrographs of heater cells infiltrated with the Golgi stain demonstrate that the cells also have an extensive transverse tubule system with a complicated three-dimensional structure. Junctional regions between transverse tubules and SR occur in the heater cell and contain feet protein. Activation of thermogenesis in heater cells may occur through the same protein components involved in excitation-contraction coupling and appears to be associated with the ATP-dependent cycling of calcium at the SR.     

Sodium Transport in Triads Isolated from Rabbit Skeletal Muscle

Triads and transverse tubules isolated from mammalian skeletal muscle actively accumulated Na+ in the presence of K+ and Mg-ATP. Active Na+ transport exhibited a fast single-exponential phase, lasting 2 min, followed by slower linear uptake that continued for 10 minutes. Valinomycin stimulated Na+ uptake, suggesting it decreased a pump-generated membrane potential gradient (Vm) that prevented further Na+ accumulation. At the end of the fast uptake phase transverse tubule vesicles incubated in 30 mM external [Na+] attained a ratio [Na+]in/[Na+]out=13.4. From this ratio and the transverse tubule volume of 0.35 microl/mg protein measured in this work, [Na+]in=400 mM was calculated. Determinations of active K+ transport in triads, using 86Rb+ as tracer, showed a 30% decrease in vesicular 86Rb+ content two minutes after initiating the reaction, followed by a slower uptake phase during which vesicles regained their initial 86Rb+ content after 10 minutes. Transverse tubule volume increase during active Na+ transport-as shown by light scattering changes of isolated vesicles--presumably accounted for the secondary Na+ and 86Rb+ uptake phases. These combined results indicate that isolated triads have highly sealed transverse tubules that can be polarized effectively by the Na+ pump through the generation of significant Na+ gradients.     

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.