Response training shortens visuo-motor related time in athletes.

In the present study, we aimed to determine whether response training shortens visuo-motor related time in athletes performing a simple reaction task. 14 healthy male athletes were included in the study. Subjects were randomly divided into 2 groups: a training group, which underwent response training consisting of a mastication task in response to a visual signal, and a non-training (control) group, which did not undergo response training. Pre-motor time and transcranial magnetic stimulation over the primary motor cortex for recording motor evoked potentials were measured in the control group, and before and after the response training session in the training group. Both pre-motor time and visuo-motor related time, but not motor evoked potential latency, were significantly reduced after response training in the training group. Subjects who had a longer visuo-motor related time before training showed a greater reduction in visuo-motor related time after training. These results suggest that visuo-motor related time before training could be useful as a predictor of the reduction in reaction time following response training.

Effects of aerobic exercise training on brain structure and psychological well-being in young adults.

AIM: There is convergent evidence that exercise increases psychological well-being; however, the mechanism of this psychological effect of exercise is not yet completely understood. The purpose of this study was to examine the effects of aerobic exercise training on brain structure and psychological well-being in young adults. METHODS: University students who had not regularly exercised were divided into training group (N.=15) and control group (N.=15). The training group performed a total 30 periods of aerobic exercise training, while the control group never performed. Whole-brain magnetic resonance imaging scans and mental health questionnaire examinations were performed before and after the exercise training period for all of the participants. A voxel-based morphometry (VBM) analysis was used to compare the changes in gray-matter volumes in the two groups. VBM is an objective whole-brain technique for characterization of regional cerebral volume and tissue concentration differences in structural magnetic resonance images. RESULTS: The results of VBM analysis revealed no change in gray-matter volume in the training group, although the gray-matter volume of the left insula was significantly decreased in the control group after the exercise training period. The training group exhibited significant improvement in some scores on the mental health questionnaire after the exercise training period, compared with the control group. CONCLUSIONS: These findings suggest that aerobic exercise training may inhibit gray-matter volume loss in the insula, and that a relationship may exist between preservation of insula gray-matter and improvement of psychological well-being by aerobic exercise training.

Eccentric exercise-induced morphological changes in the membrane systems involved in excitation-contraction coupling in rat skeletal muscle.

1. Physiological evidence suggests that excitation-contraction (E-C) coupling failure results from eccentric contraction-induced muscle injury because of structural and morphological damage to membrane systems directly associated with the E-C coupling processes within skeletal muscle fibres. In this study using rats, we observed the ultrastructural features of the membrane systems of fast-twitch (FT) and slow-twitch (ST) muscle fibres involved in E-C coupling following level and downhill running exercise. Our aim was to find out whether mechanically mediated events following eccentric exercise caused disorder in the membrane systems involved in E-C coupling, and how soon after exercise such disorder occurred. We also compared the morphological changes of the membrane systems between ST and FT muscle fibres within the same muscles. 2. Single muscle fibres were dissected from triceps brachii muscles of male Fischer 344 rats after level or downhill (16 deg decline) motor-driven treadmill running (18 m min(-1), 5 min running with 2 min rest interval, 18 bouts). All single muscle fibres were histochemically classified into ST or FT fibres. The membrane systems were visualized using Ca(2+)-K(3)Fe(CN)(6)-OsO(4) techniques, and observed by high voltage electron microscopy (120-200 kV). 3. There were four obvious ultrastructural changes in the arrangement of the transverse (t)-tubules and the disposition of triads after the downhill running exercise: (1) an increase in the number of longitudinal segments of the t-tubule network, (2) changes in the direction and disposition of triads, (3) the appearance of caveolar clusters, and (4) the appearance of pentads and heptads (close apposition of two or three t-tubule elements with three or four elements of terminal cisternae of the sarcoplasmic reticulum). The caveolar clusters appeared almost exclusively in the ST fibres immediately after downhill running exercise and again 16 h later. The pentads and heptads appeared almost exclusively in the FT fibres, and their numbers increased dramatically 2-3 days after the downhill running exercise. 4. The eccentric exercise led to the formation of abnormal membrane systems involved in E-C coupling processes. These systems have unique morphological features, which differ between ST and FT fibres, even within the same skeletal muscle, and the damage appears to be concentrated in the FT fibres. These observations also support the idea that eccentric exercise- induced E-C coupling failure is due to physical and chemical disruption of the membrane systems involved in the E-C coupling process in skeletal muscle.

Sequential docking, molecular differentiation, and positioning of T-Tubule/SR junctions in developing mouse skeletal muscle.

Skeletal muscle Ca(2+) release units (CRUs) are junctions of the surface membrane/T-tubule system and the sarcoplasmic reticulum (SR) that function in excitation-contraction coupling. They contain high concentrations of dihydropyridine receptors (DHPRs) in the T-tubules and of ryanodine receptors (RyR) in the SR and they are positioned at specific locations in the sarcomere. In order to characterize the sequence of developmental steps leading to the specific molecular and structural organization of CRUs, we applied a range of imaging techniques that allowed us to follow the differentiation of the membrane compartments and the expression of junctional proteins in developing mouse diaphragm muscle. We find that docking of the two membrane systems precedes the incorporation of the RyRs into the junctions, and that T-tubule/SR junctions are formed and positioned at the I-A interface at a stage when the orientation of T-tubule is predominantly longitudinal. Thus, the sequence of developmental events is first the docking of T-tubules and SR, secondly the incorporation of RyR in the junctions, thirdly the positioning of the junctions in the sarcomere, and only much later the transverse orientation of the T-tubules. These sequential stages suggests an order of inductive processes for the molecular differentiation and structural organization of the CRUs in skeletal muscle development.

RYR1 and RYR3 have different roles in the assembly of calcium release units of skeletal muscle.

Calcium release units (CRUs) are junctions between the sarcoplasmic reticulum (SR) and exterior membranes that mediates excitation contraction (e-c) coupling in muscle cells. In skeletal muscle CRUs contain two isoforms of the sarcoplasmic reticulum Ca(2+)release channel: ryanodine receptors type 1 and type 3 (RyR1 and RyR3). 1B5s are a mouse skeletal muscle cell line that carries a null mutation for RyR1 and does not express either RyR1 or RyR3. These cells develop dyspedic SR/exterior membrane junctions (i.e., dyspedic calcium release units, dCRUs) that contain dihydropyridine receptors (DHPRs) and triadin, two essential components of CRUs, but no RyRs (or feet). Lack of RyRs in turn affects the disposition of DHPRs, which is normally dictated by a linkage to RyR subunits. In the dCRUs of 1B5 cells, DHPRs are neither grouped into tetrads nor aligned in two orthogonal directions. We have explored the structural role of RyR3 in the assembly of CRUs in 1B5 cells independently expressing either RyR1 or RyR3. Either isoform colocalizes with DHPRs and triadin at the cell periphery. Electron microscopy shows that expression of either isoform results in CRUs containing arrays of feet, indicating the ability of both isoforms to be targeted to dCRUs and to assemble in ordered arrays in the absence of the other. However, a significant difference between RyR1- and RyR3-rescued junctions is revealed by freeze fracture. While cells transfected with RyR1 show restoration of DHPR tetrads and DHPR orthogonal alignment indicative of a link to RyRs, those transfected with RyR3 do not. This indicates that RyR3 fails to link to DHPRs in a specific manner. This morphological evidence supports the hypothesis that activation of RyR3 in skeletal muscle cells must be indirect and provides the basis for failure of e-c coupling in muscle cells containing RyR3 but lacking RyR1 (see the accompanying report, ).

Differential response of the membrane systems involved in excitation-contraction coupling to early and later postnatal denervation in rat skeletal muscle.

We compared the morphological features of the membrane systems involved in excitation-contraction (E-C) coupling during early postnatal development stages in rat skeletal muscles (tibialis anterior) denervated either at birth or 7 days after birth. Four obvious structural changes are observed in the arrangement of the transverse (T) tubule network and the disposition of triads following early postnatal denervation: (1) an increase in the longitudinal segments of the T tubule network, (2) changes in the direction and disposition of triads, (3) the appearance of caveolae clusters, (4) the appearance of pentads and heptads (i.e. a close apposition of two or three T tubule elements with three or four elements of terminal cisternae of sarcoplasmic reticulum). The increased presence of longitudinal T tubules parallels the loss of cross striations, and this in turn is due to misalignment of the myofibrils. The clusters of caveolae appear almost exclusively in muscle fibres denervated at birth, and pentads and heptads are more frequently observed in muscles denervated at 7 days. The differential growth of muscle fibres in response to denervation leads to the formation of abnormal membrane systems involved in the E-C coupling with very unique morphological features, which differ from the case of denervation in adult muscle fibres.

Correct targeting of dihydropyridine receptors and triadin in dyspedic mouse skeletal muscle in vivo.

Excitation-contraction coupling in skeletal muscle involves junctions (triads and dyads) between sarcoplasmic reticulum (SR) and transverse (T) -tubules. Two proteins of the junctional SR, ryanodine receptors (RyRs) and triadin and one protein of T tubules, dihydropyridine receptors (DHPRs) are located at these junctions. We studied the targeting of DHPRs and triadin to T-tubules and SR in skeletal muscles of dyspedic mouse embryos lacking RyR1. In normal differentiating muscle fibers DHPRs, triadin and RyRs are located in intensely immunolabeled foci that are randomly distributed across the fiber. Correlation with electron microscopy and with previous studies indicates that the foci represent the location of triads and dyads. In dyspedic fibers DHPRs and triadin antibodies stain internal foci of the two proteins; RyR antibodies are completely negative. The appearance and location of the foci in dyspedic fibers is similar to that of normal muscle, but their fluorescent intensity is weaker. The SR Ca-ATPase has more diffuse distribution than triadin in both normal and dyspedic fibers. These observations indicate that an interaction with RyRs is not necessary for the appropriate targeting of DHPRs or triadin to junctional domains of T tubules and SR respectively.

Morphological changes in the triads and sarcoplasmic reticulum of rat slow and fast muscle fibres following denervation and immobilization.

We observed the morphological features of the membrane systems (sarcoplasmic reticulum, transverse tubules and triads) involved with the excitation-contraction coupling in rat soleus and extensor digitorum longus muscle following two disuse protocols: denervation and immobilization. The immobilized positions were: maximum dorsal flexor (soleus were stretched and extensor digitorum longus were shortened), maximum plantar flexor (soleus were shortened and extensor digitorum longus were stretched), and midway between the dorsal flexor and plantar flexor. The arrangement of the membrane systems was disordered following both disuse conditions. Increases in transverse tubule network were apparent; there were clearly more triads than in normal fibres, and pentadic and heptadic structures (i.e., a close approximation of two or three transverse tubule elements with three or four elements of terminal cisternae of sarcoplasmic reticulum) were frequently appeared following both denervation and immobilization. The most notable difference between the influence of denervation and immobilization on the membrane systems is the time at which the pentads and heptads appeared. They appeared much earlier (1 week after denervation) in denervated than in immobilized (3 or 4 weeks after immobilization) muscle fibres. On the other hand, the frequency of pentads and heptads is clearly related to the fibre type (significantly higher in extensor digitorum longus) and to extent of atrophy. The different influences of immobilization in each leg position suggest that disuse, but with neurotrophic factor(s), influences on the membrane systems were affected by sarcomere length, and the neurotrophic factor(s) and muscle activity were not always necessary to form new membrane systems in disuse skeletal muscle fibres.

Influences of sarcomere length and selective elimination of myosin filaments on the localization and orientation of triads in rat muscle fibres.

Ultrastructural features of internal membrane systems directly concerned with the excitation-contraction coupling were observed in chemically skinned muscle bundles prepared from Wistar rat extensor digitorum longus muscle to clarify two questions: (1) whether triads localization and orientation are influenced by the sarcomere length and (2) whether triads localisation and orientation are influenced by the selective elimination of myosin filaments. The distance between triads and Z-lines depends on the sarcomere length: it increase with sarcomere length. There is a highly significant (p < 0.01) positive correlation between sarcomere length and the distance between triads and Z-line. The distance between Z-line and triads is dependent on sarcomere length, but the width of junctional gap remains constant when the sarcomere length was changed. Incubation in a concentration of KCI, which dissolves the myosin filaments. The localization and orientation of triads was not altered by the elimination of myosin filaments, however, the distance between the Z-line and triads becomes shorter when the myosin filaments was completely eliminated. There were significant differences (p < 0.01) between control and myosin filament eliminated fibres in the distances between Z-lines and triads (over 2 microns). These results indicate that the distance between triads and Z-lines depend on the sarcomere length and that there may be some connection(s) between triads and the myofibrils. There is that the elastic component responsible for tethering the triads in their normal position is interrupted either because it is normally attached to the myosin filaments, or because it is extracted by the conditions that dissociate myosin filaments.

Co-expression in CHO cells of two muscle proteins involved in excitation-contraction coupling.

Ryanodine receptors and dihydropyridine receptors are located opposite each other at the junctions between sarcoplasmic reticulum and either the surface membrane or the transverse tubules in skeletal muscle. Ryanodine receptors are the calcium release channels of the sarcoplasmic reticulum and their cytoplasmic domains form the feet, connecting sarcoplasmic reticulum to transverse tubules. Dihydropyridine receptors are L-type calcium channels that act as the voltage sensors of excitation-contraction coupling: they sense surface membrane and transverse tubule depolarization and induce opening of the sarcoplasmic reticulum release channels. In skeletal muscle, ryanodine receptors are arranged in extensive arrays and dihydropyridine receptors are grouped into tetrads, which in turn are associated with the four subunits of ryanodine receptors. The disposition allows for a direct interaction between the two sets of molecules. CHO cells were stably transformed with plasmids for skeletal muscle ryanodine receptors and either the skeletal dihydropyridine receptor, or a skeletal-cardiac dihydropyridine receptor chimera (CSk3) which can functionally substitute for the skeletal dihydropyridine receptor, in addition to plasmids for the alpha 2, beta and gamma subunits. RNA blot hybridization gave positive results for all components. Immunoblots, ryanodine binding, electron microscopy and exposure to caffeine show that the expressed ryanodine receptors forms functional tetrameric channels, which are correctly inserted into the endoplasmic reticulum membrane, and form extensive arrays with the same spacings as in skeletal muscle. Since formation of arrays does not require coexpression of dihydropyridine receptors, we conclude that self-aggregation is an independent property of ryanodine receptors. All dihydropyridine receptor-expressing clones show high affinity binding for dihydropyridine and immunolabelling with antibodies against dihydropyridine receptor. The presence of calcium currents with fast kinetics and immunolabelling for dihydropyridine receptors in the surface membrane of CSk3 clones indicate that CSk3-dihydropyridine receptors are appropriately targeted to the cell's plasmalemma. The expressed skeletal-type dihydropyridine receptors, however, remain mostly located within perinuclear membranes. In cells coexpressing functional dihydropyridine receptors and ryanodine receptors, no junctions between feet-bearing endoplasmic reticulum elements and surface membrane are formed, and dihydropyridine receptors do not assemble into tetrads. A separation between dihydropyridine receptors and ryanodine receptors is not unique to CHO cells, but is found also in cardiac muscle, in muscles of invertebrates and, under certain conditions, in skeletal muscle. We suggest that failure to form junctions in co-transfected CHO cell may be due to lack of an essential protein necessary either for the initial docking of the endoplasmic reticulum to the surface membrane or for maintaining the interaction between dihydropyridine receptors and ryanodine receptors. We also conclude that formation of tetrads requires a close interaction between dihydropyridine receptors and ryanodine receptors.

Ca(2+)-induced Ca2+ release in myocytes from dyspedic mice lacking the type-1 ryanodine receptor.

While subtypes 1 and 2 of the ryanodine receptor (RyR) function as intracellular Ca2+ release channels, little is known about the function of the third subtype (RyR-3), first identified in brain. Myocytes from mice homozygous for a targeted mutation in the RyR-1 gene (dyspedic mice) can now be used for a study on the function of RyR-3, which is predominantly expressed in these cells according to our reverse transcription-polymerase chain reaction analysis. We here demonstrate in these myocytes caffeine-, ryanodine- and adenine nucleotide-sensitive Ca(2+)-induced Ca2+ release with approximately 10 times lower sensitivity to Ca2+ than that of RyR-1. Although RyR-3 does not mediate excitation-contraction coupling of the skeletal muscle type, we propose that RyR-3 may induce intracellular Ca2+ release in response to a Ca2+ rise with a high threshold.

Abnormal junctions between surface membrane and sarcoplasmic reticulum in skeletal muscle with a mutation targeted to the ryanodine receptor.

Junctions that mediate excitation-contraction (e-c) coupling are formed between the sarcoplasmic reticulum (SR) and either the surface membrane or the transverse (T) tubules in normal skeletal muscle. Two structural components of the junctions, the feet of the SR and the tetrads of T tubules, have been identified respectively as ryanodine receptors (RyRs, or SR calcium-release channels), and as groups of four dihydropyridine receptors (DHPRs, or voltage sensors of e-c coupling). A targeted mutation (skrrm1) of the gene for skeletal muscle RyRs in mice results in the absence of e-c coupling in homozygous offspring of transgenic parents. The mutant gene is expected to produce no functional RyRs, and we have named the mutant mice "dyspedic" because they lack feet--the cytoplasmic domain of RyRs anchored in the SR membrane. We have examined the development of junctions in skeletal muscle fibers from normal and dyspedic embryos. Surprisingly, despite the absence of RyRs, junctions are formed in dyspedic myotubes, but the junctional gap between the SR and T tubule is narrow, presumably because the feet are missing. Tetrads are also absent from these junctions. The results confirm the identity of RyRs and feet and a major role for RyRs and tetrads in e-c coupling. Since junctions form in the absence of feet and tetrads, coupling of SR to surface membrane and T tubules appears to be mediated by additional proteins, distinct from either RyRs or DHPRs.

Restoration of junctional tetrads in dysgenic myotubes by dihydropyridine receptor cDNA.

Excitation-contraction coupling was restored in primary cultures of dysgenic myotubes by transfecting the cells with an expression plasmid encoding the rabbit skeletal muscle dihydropyridine receptor. Dishes containing normal, dysgenic, and transfected myotubes were fixed, freeze-fractured, and replicated for electron microscopy. Numerous small domains in the surface membrane of normal myotubes contain ordered arrays of intramembrane particles in groups of four (tetrads). The disposition of tetrads in the arrays is consistent with alternate positioning of tetrads relative to the underlying feet of the sarcoplasmic reticulum. Dysgenic myotubes have no arrays of tetrads. Some myotubes from successfully transfected cultures have arrays of tetrads with spacings equal to those found in normal myotubes. Thus the dihydropyridine receptor appears to be needed for the formation of tetrads and their association with the sarcoplasmic reticulum feet. This result is consistent with the hypothesis that each tetrad is composed of four dihydropyridine receptors.

Excitation-contraction uncoupling and muscular degeneration in mice lacking functional skeletal muscle ryanodine-receptor gene.

Contraction of skeletal muscle is triggered by the release of Ca2+ from the sarcoplasmic reticulum (SR) after depolarization of transverse tubules. The ryanodine receptor exists as a 'foot' protein in the junctional gap between the sarcoplasmic reticulum and the transverse tubule in skeletal muscle, and is proposed to function as a calcium-release channel during excitation-contraction (E-C) coupling. Previous complementary DNA-cloning studies have defined three distinct subtypes of the ryanodine receptor in mammalian tissues, namely skeletal muscle, cardiac and brain types. We report here mice with a targeted mutation in the skeletal muscle ryanodine receptor gene. Mice homozygous for the mutation die perinatally with gross abnormalities of the skeletal muscle. The contractile response to electrical stimulation under physiological conditions is totally abolished in the mutant muscle, although ryanodine receptors other than the skeletal-muscle type seem to exist because the response to caffeine is retained. Our results show that the skeletal muscle ryanodine receptor is essential for both muscular maturation and E-C coupling, and also imply that the function of the skeletal muscle ryanodine receptor during E-C coupling cannot be substituted by other subtypes of the receptor.

Development of the excitation-contraction coupling apparatus in skeletal muscle: peripheral and internal calcium release units are formed sequentially.

The development of calcium release units and of transverse tubules has been studied in skeletal muscle fibres from embryonal and newborn chicken. Three constituents of calcium release units: the tetrads, the feet and an internal protein directly associated with junctional surface of the sarcoplasmic reticulum are visualized by various electron microscope techniques. Evidence in the literature indicates that the three components correspond to the voltage sensors, the sarcoplasmic reticulum calcium release channels and the calcium binding protein calsequestrin respectively. We recognize two stages at which important events in membrane morphogenesis occur. The first stage coincides with early myofibrillogenesis (starting at approximately embryonal day E5.5), and it involves the assembly of calcium release units at the periphery of the muscle fibre in which feet and the internal protein are identified. Groups of tetrads also are present at very early stages and their disposition indicates a relation to the feet of peripheral couplings. Thus three major components of the excitation-contraction coupling pathway are in place as soon as myofibrils develop. The density of groups of tetrads in the surface membrane of primary and secondary fibres is similar, despite differences in developmental stages. The second stage involves the formation of a complex transverse tubule network and of internal sarcoplasmic reticulum-transverse tubule junctions, while peripheral couplings disappear. This stage starts abruptly (between E15 and E16) and simultaneously in primary and secondary fibres. It coincides with the myotube-to-myofibre transition. The two stages are separated by a relatively long intervening period (between E9 and E16). During the latter part of this period some primitive transverse tubules appear, and form junctions with the sarcoplasmic reticulum, but they remain strictly located at the periphery of the fibre and are not numerous. Finally, after the second stage there is a prolonged (up to 4 weeks) period of maturation, during which density of free sarcoplasmic reticulum increases, triads acquire a location at the A-I junction and fibre type differences appear. We conclude that a system for calcium uptake, storage and release exists at the periphery of the myotube during early myogenesis. The complexity of the system and its ability to deliver calcium through the entire fibre develop in parallel to the formation of myofibrils.

Differences in ultrastructural and metabolic profiles within the same type of fibres in various muscles of young and adult rats.

Single fibres from tibialis anterior, extensor digitorum longus, gastrocnemius and soleus muscles in young (4-week-old) and adult (35-week-old) Wistar male rats were classified into three types on the basis of their enzyme-histochemical features: slow-twitch oxidative (SO), fast-twitch oxidative and glycolytic (FOG) and fast-twitch glycolytic (FG) fibres. Ultrastructural (volume density of mitochondria: Vmt and Z line width) and metabolic (phosphofructokinase: PFK and succinate dehydrogenase: SDH activities) profiles were measured. PFK activity in all types of fibres was higher in adult rats, and the difference between the two age-groups (adult/young) was largest between FG, FOG and SO fibres respectively. SDH activity and Vmt were lower in adult rats in a similar way in all fibres. A significant positive correlation was observed between the Vmt and SDH activity in both age-groups. This positive correlation was very specific in fast-twitch and slow-twitch fibres. Changes in the Vmt did not relate directly to the changes in fibre cross-sectional area. The overall pattern indicates that glycolytic capacity of fast-twitch fibres in flexor muscles (TA and EDL) is higher than in extensor muscles (GC and SOL), and that oxidative capacity of all types of fibre in extensor muscles is higher than in flexor muscles. These profiles were changed by growth, and may be related to the specific differences in pattern of activity of each skeletal muscle, and may reflect differences in the recruitment order of different muscles.

Differentiation of membrane systems during development of slow and fast skeletal muscle fibres in chicken.

The disposition of transverse (T) tubules, sarcoplasmic reticulum (SR) and T-SR junctions (triads) and the width of Z lines are matched to contractile properties in adult muscle fibres. We have studied the development of the membrane systems in the slow anterior (ALD) and the fast posterior (PLD) latissimus dorsi of the chicken in ovo (E14-E21) and after hatching (D1-D30). T tubules, SR, triads and Z lines were visualized using DiIC16[3] labelling for confocal microscopy and either Ca-osmium-ferrocyanide or standard procedures for electron microscopy. Anterior latissimus dorsi and PLD have similar, slow twitches in early development (E14-E16), but PLD suddenly becomes faster starting at E17-E18. We find that in coincidence with the differentiation of faster contraction properties (starting at E18-E19) density of triads is significantly higher and width of Z lines is narrower in PLD. The SR also begins to acquire fibre-type specific characteristics at this time. Early development of T tubules, on the other hand, is quite similar in the two muscles. Peripherally-located, longitudinally-oriented T tubules, and the first T networks crossing the fibre center appear earlier in ALD (E14-E15 and E16) than in PLD (E14-E16 and E17), but have similar dispositions. The final fibre-type specific distribution of T tubules is achieved after hatching. Some T tubules-rich fibres in the ALD, presumably future fast fibres, develop extensive T tubules networks at early stages. Location of triads at the Z line in pectoralis occurs in three steps: an initial location of longitudinally oriented triads at the A-I junction; a subsequent move to the Z lines and finally a rotation to a transverse orientation.

Development of the excitation-contraction coupling apparatus in skeletal muscle: association of sarcoplasmic reticulum and transverse tubules with myofibrils.

The formation and maintenance of the highly regular organization of membrane systems and proteins in striated muscle require specific membrane-membrane and membrane-cytoskeleton interactions. The development of T-tubules and sarcoplasmic reticulum (SR) was followed in gastrocnemius muscle fibers from chicken embryos between 12 days (E12) and 21 days (E21) of incubation, with particular attention to their relationship with one another and with the myofibrils. The fluorescent lipid analog DiIC16[3] was used to label either the external membranes (plasmalemma and transverse (T)-tubules) or the internal SR in living and fixed muscle. Short membrane invaginations can first be seen in fibers at E14, and at E15 longitudinal T-tubules appear in the periphery of the fibers. A complex network of T-tubules filling the whole fiber diameter develops suddenly at E16. In contrast, SR is abundant at the earliest observed stage (E12) and forms regularly spaced cross striations located at the I-Z-I bands. These correspond to a specific accumulation of smooth membranes around the Z-discs seen in electron micrographs. While SR is specifically associated with the newly formed myofibrils in the periphery of the fibers, the disposition of early T-tubules shows little specific relationship to either SR or the myofibrils. However, electron microscopy shows that junctions between T-tubules and SR are formed during this period (Takekura and Franzini-Armstrong, submitted for publication). Junctions do not acquire a specific relation to the myofibrils until around hatching when triads begin to reorganize into their mature location, the A-I junction. These findings indicate three key events in the organization of T-tubules and SR in the sarcomeres: (1) early SR/Z-line interactions independent of T-tubules; (2) SR/T-tubule interactions to form the triad junctions, independent from the myofibrils; and (3) the late association of the junctional complexes with the myofibrils at the A-I border.

[Differentiational profiles of skeletal muscle internal membrane systems directly related to excitation-contraction coupling].

The arrangements of internal membrane systems (transverse (T) tubules, sarcoplasmic reticulum (SR) and T-SR junction (triad)) are matched to contractile profiles in adult muscle fibers. The developmental profiles of the these internal membrane systems directly related to the excitation-contraction (EC) coupling were investigated in the chicken anterior (ALD, slow) and posterior (PLD, fast) latissimus dorsi muscles in ovo (14 to 21 days incubation: E 14-E 21) and after hatching (1 to 30 days after hatching: D 1-D 30). These internal membrane systems were visualized using confocal and electron microscopes. ALD and PLD have similar contraction time in early developmental stage (E 14-E 16), however, PLD suddenly becomes faster at E 17-E 18. Following the differentiation of faster contraction properties (E 18-E 19), density of triads increased significantly in PLD. Early development of T-tubules, on the other hand, is quite similar in both ALD and PLD. Peripherally located, longitudinally oriented T tubules, and the first T networks crossing the fiber center appear earlier in ALD (E 14-E 16) than PLD, but have similar dispositions. The final fiber type specific disposition of T-tubules is established after hatching, and three major components of the E-C coupling pathway are in place as soon as myofibrils develop.

The brain ryanodine receptor: a caffeine-sensitive calcium release channel.

The release of stored Ca2+ from intracellular pools triggers a variety of important neuronal processes. Physiological and pharmacological evidence has indicated the presence of caffeine-sensitive intracellular pools that are distinct from the well-characterized inositol 1,4,5,-trisphosphate (IP3)-gated pools. Here we report that the brain ryanodine receptor functions as a caffeine- and ryanodine-sensitive Ca2+ release channel that is distinct from the brain IP3 receptor. The brain ryanodine receptor has been purified 6700-fold with no change in [3H]ryanodine binding affinity and shown to be a homotetramer composed of an approximately 500 kd protein subunit, which is identified by anti-peptide antibodies against the skeletal and cardiac muscle ryanodine receptors. Our results demonstrate that the brain ryanodine receptor functions as a caffeine-sensitive Ca2+ release channel and thus is the likely gating mechanism for intracellular caffeine-sensitive Ca2+ pools in neurons.