Sarcoplasmic Reticulum Tubules Research Articles

Long-Term Exercise Reduces Formation of Tubular Aggregates and Promotes Maintenance of Ca2+ Entry Units in Aged Muscle.

Tubular aggregates (TAs) in skeletal muscle fibers are unusual accumulation of sarcoplasmic reticulum (SR) tubes that are found in different disorders including TA myopathy (TAM). TAM is a muscular disease characterized by muscle pain, cramping, and weakness that has been recently linked to mutations in STIM1 and ORAI1. STIM1 and ORAI1 are the two main proteins mediating store-operated Ca2+ entry (SOCE), a mechanism activated by depletion of intracellular Ca2+ stores (e.g., SR) that allows recovery of Ca2+ from the extracellular space during repetitive muscle activity. We have recently shown that exercise triggers the formation of unique intracellular junctions between SR and transverse tubules named Ca2+ entry units (CEUs). CEUs promote colocalization of STIM1 with ORAI1 and improve muscle function in presence of external Ca2+. TAs virtually identical to those of TAM patients are also found in fast-twitch fibers of aging male mice. Here, we used a combination of electron and confocal microscopy, Western blotting, and ex vivo stimulation protocols (in presence or absence of external Ca2+) to evaluate the presence of TAs, STIM1-ORAI1 localization and expression and fatigue resistance of intact extensor digitorum longus (EDL) muscles in wild-type male adult (4-month-old) and aged (24-month-old) mice and in mice trained in wheel cages for 15 months (from 9 to 24 months of age). The results collected indicate that (i) aging causes STIM1 and ORAI1 to accumulate in TAs and (ii) long-term exercise significantly reduced formation of TAs. In addition, (iii) EDL muscles from aged mice exhibited a faster decay of contractile force than adult muscles, likely caused by their inability to refill intracellular Ca2+ stores, and (iv) exercise in wheel cages restored the capability of aged EDL muscles to use external Ca2+ by promoting maintenance of CEUs. In conclusion, exercise prevented improper accumulation of STIM1 and ORAI1 in TAs during aging, maintaining the capability of aged muscle to refill intracellular Ca2+ stores via SOCE.

Association with SERCA2a directs phospholamban trafficking to sarcoplasmic reticulum from a nuclear envelope pool

AimsPhospholamban (PLB) stoichiometrically regulates the cardiac Ca2+ pump (SERCA2a) in the sarcoplasmic reticulum (SR); but in the nuclear envelope (NE) of cardiomyocytes (CMs), the PLB to SERCA2a molar ratio is higher, which highlights our poor understanding of how SR proteins distribute to their functional subcompartments. By tracking newly made PLB and SERCA2a in CMs, we will elucidate underlying cellular pathways responsible for their unique intracellular distributions. Methods and resultsHighly specific monoclonal antibodies were used to compare the subcellular distributions of SERCA2a, PLB, and junctin (JCN) in dog heart tissue. The data supported a view that both non-junctional and junctional SR proteins are all prominently enriched in transverse stretches of SR tubules, along the edges of sarcomeres (SR z-tubules). To understand the genesis of these steady state distributions, we analyzed confocal immunofluorescence images of adult rat CMs after acute expression (12-48 h) of the dog ortholog of PLB (dPLB) or dSERCA2a. Newly made dog proteins in rat CMs were detected using dog–specific monoclonal antibodies. By 12-24 h, dSERCA2a had accumulated within the NE in a punctate pattern, presumably reflecting initial sites of biosynthesis. Over the next 24-48 h, higher levels of dSERCA2a immunofluorescence accumulated in transverse/radial SR tubules, aligned along sarcolemmal transverse (T)-tubules, and extending from NE puncta. The patterns of SR tubules carrying dSERCA2a overlapped with those for newly made JCN, suggesting a common Nuclear Envelope to SR along T-tubules or NEST pathway for SR proteins. In contrast to the SERCA2a distribution pattern, dPLB accumulated uniformly in the NE, without visible puncta. With co-expression of dSERCA2a, however, PLB no longer uniformly filled the NE, but instead moved together with SERCA2a to form bright NE puncta, from which the two proteins then trafficked anterogradely. ConclusionExpression of dog SR protein orthologs (dSERCA2a, dPLB, and dJCN) for as little as 48 h reproduces their characteristic steady state distributions. Detailed analyses of the time courses of protein accumulation suggest a possible mechanism by which PLB distributes to both the NE and SR, unlike SERCA2a. SERCA2a moves in SR z-tubules directly from rough ER, along pathways that are in common with those used by junctional SR proteins. A different trafficking route for PLB away the rough ER/NE led to its accumulation in the NE, a process that may account for its enrichment in NE in situ. Association of SERCA2a with PLB from this NE pool enhanced PLB trafficking along the NEST pathway, contributing to steady state stoichiometry and physiologically regulated SERCA2a.

Calcium Homeostasis Is Modified in Skeletal Muscle Fibers of Small Ankyrin1 Knockout Mice.

Small Ankyrins (sAnk1) are muscle-specific isoforms generated by the Ank1 gene that participate in the organization of the sarcoplasmic reticulum (SR) of striated muscles. Accordingly, the volume of SR tubules localized around the myofibrils is strongly reduced in skeletal muscle fibers of 4- and 10-month-old sAnk1 knockout (KO) mice, while additional structural alterations only develop with aging. To verify whether the lack of sAnk1 also alters intracellular Ca2+ handling, cytosolic Ca2+ levels were analyzed in stimulated skeletal muscle fibers from 4- and 10-month-old sAnk1 KO mice. The SR Ca2+ content was reduced in sAnk1 KO mice regardless of age. The amplitude of the Ca2+ transients induced by depolarizing pulses was decreased in myofibers of sAnk1 KO with respect to wild type (WT) fibers, while their voltage dependence was not affected. Furthermore, analysis of spontaneous Ca2+ release events (sparks) on saponin-permeabilized muscle fibers indicated that the frequency of sparks was significantly lower in fibers from 4-month-old KO mice compared to WT. Furthermore, both the amplitude and spatial spread of sparks were significantly smaller in muscle fibers from both 4- and 10-month-old KO mice compared to WT. These data suggest that the absence of sAnk1 results in an impairment of SR Ca2+ release, likely as a consequence of a decreased Ca2+ store due to the reduction of the SR volume in sAnk1 KO muscle fibers.

Endoplasmic reticulum stress induces cardiac dysfunction through architectural modifications and alteration of mitochondrial function in cardiomyocytes.

Endoplasmic reticulum (ER) stress has recently emerged as an important mechanism involved in the pathogenesis of cardiovascular diseases. However, the molecular mechanisms by which ER stress leads to cardiac dysfunction remain poorly understood. In this study, we evaluated the early cardiac effects of ER stress induced by tunicamycin (TN) in mice. Echocardiographic analysis indicated that TN-induced ER stress led to a significant impairment of the cardiac function. Electron microscopic observations revealed that ultrastructural changes of cardiomyocytes in response to ER stress manifested extensively at the level of the reticular membrane system. Smooth tubules of sarcoplasmic reticulum in connection with short sections of rough ER were observed. The presence of rough instead of smooth reticulum was increased at the interfibrillar space, at the level of dyads, and in the vicinity of mitochondria. At the transcriptional level, ER stress resulted in a substantial decrease in the expression of the major regulator of mitochondrial biogenesis PGC-1α and of its targets NRF1, Tfam, CS, and COXIV. At the functional level, ER stress also induced an impairment of mitochondrial Ca2+ uptake, an alteration of mitochondrial oxidative phosphorylation, and a metabolic remodelling characterized by a shift from fatty acid to glycolytic substrate consumption. Our findings show that ER stress induces cytoarchitectural and metabolic alterations in cardiomyocytes and provide evidences that ER stress could represent a primary mechanism that contributes to the impairment of energy metabolism reported in most cardiac diseases.

ER stress induces cardiac dysfunction through cardiomyocytes architectural modifications and alteration of mitochondrial function

Endoplasmic reticulum (ER) stress has recently emerged as an important mechanism involved in the pathogenesis of cardiovascular diseases including heart failure. However, the molecular mechanisms by which ER stress leads to cardiac dysfunction remain poorly understood. In the present study, we evaluated the early cardiac effects of ER stress induced by tunicamycin (TN) in mice. Electrocardiographic analysis indicated that TN-induced ER stress led to a significant impairment of the cardiac function. Electron microscopic observations revealed that ultrastructural changes of cardiomyocytes in response to ER stress manifested extensively at the level of the sarco-endoplasmic reticulum membranes. Smooth tubules of sarcoplasmic reticulum in connection with short sections of rough endoplasmic reticulum were observed. The presence of rough instead of smooth reticulum was increased at the interfibrillar space, at the level of dyads and in the vicinity of mitochondria. At a functional level, ER stress resulted in a substantial decrease in mitochondrial biogenesis as demonstrated by the decrease of the expression of PGC-1α and of its targets NRF1, Tfam, CS and COXIV. ER stress also led to an impairment of mitochondrial oxidative phosphorylation and to a metabolic remodeling characterized by a shift from fatty acid to glycolytic substrate consumption. Our findings show that ER stress induces cytoarchitectural and metabolic alterations in cardiomyocytes and provide evidences that ER stress could represent a primary mechanism that contributes to the impairment of energy metabolism reported in most cardiac diseases.

Organization of junctional sarcoplasmic reticulum proteins in skeletal muscle fibers.

The sarcoplasmic reticulum (SR) of striated muscles is specialized for releasing Ca(2+) following sarcolemma depolarization in order to activate muscle contraction. To this end, the SR forms a network of longitudinal tubules and cisternae that surrounds the myofibrils and, at the same time, participates to the assembly of the triadic junctional membrane complexes formed by the close apposition of one t-tubule, originated from the sarcolemma, and two SR terminal cisternae. Advancements in understanding the molecular basis of the SR structural organization have identified an interaction between sAnk1, a transmembrane protein located on the longitudinal SR (l-SR) tubules, and obscurin, a myofibrillar protein. The direct interaction between these two proteins results in molecular contacts that have the overall effect to stabilize the l-SR tubules along myofibrils in skeletal muscle fibers. Less known is the structural organization of the sites in the SR that are specialized for Ca(2+) release and are positioned at the junctional SR (j-SR), i.e. the region of the terminal cisternae that faces the t-tubule at triads. At the j-SR, several trans-membrane proteins like triadin, junctin, or intra-luminal SR proteins like calsequestrin, are assembled together with the ryanodine receptor, the SR Ca(2+) release channel, into a macromolecular complex specialized in releasing Ca(2+). At triads, the 12nm-wide gap between the t-tubule and the j-SR allows the ryanodine receptor on the j-SR to be functionally coupled with the voltage-gated L-type calcium channel on the t-tubule in order to allow the transduction of the voltage-induced signal into Ca(2+) release through the ryanodine receptor channels. The muscle-specific junctophilin isoforms (JPH1 and JPH2) are anchored to the j-SR with a trans-membrane segment present at the C-terminus and are capable to bind the sarcolemma with a series of phospholipid-binding motifs localized at the N-terminus. Accordingly, through this dual interaction, JPH1 and JPH2 are responsible for the assembly of the triadic junctional membrane complexes. Recent data indicate that junctophilins seem also to interact with other proteins of the excitation-contraction machinery, suggesting that they may contribute to hold excitation-contraction coupling proteins to the sites where the j-SR is being organized.

Sequential stages in the age-dependent gradual formation and accumulation of tubular aggregates in fast twitch muscle fibers: SERCA and calsequestrin involvement

Tubular aggregates (TAs), ordered arrays of elongated sarcoplasmic reticulum (SR) tubules, are present in skeletal muscle from patients with myopathies and are also experimentally induced by extreme anoxia. In wild-type mice TAs develop in a clear age-, sex- (male), and fiber type- (fast twitch) dependence. However, the events preceding the appearance of TAs have not been explored. We investigated the sequential stages leading to the initial appearance and maturation of TAs in EDL from male mice. TAs' formation requires two temporally distinct steps that operate via different mechanisms. Initially (before 1year of age), the SR Ca(2+) binding protein calsequestrin (CASQ) accumulates specifically at the I band level causing swelling of free SR cisternae. In the second stage, the enlarged SR sacs at the I band level extend into multiple, longitudinally oriented tubules with a full complement of sarco(endo)plasmic reticulum Ca(2+) ATPases (SERCA) in the membrane and CASQ in the lumen. Tubules gradually acquire a regular cylindrical shape and uniform size apparently in concert with partial crystallization of SERCA. Multiple, small TAs associate to form fewer mature TAs of very large size. Interestingly, in fibers from CASQ1-knockout mice abnormal aggregates of SR tubules have different conformation and never develop into ordered aggregates of straight cylinders, possibly due to lack of CASQ accumulation. We conclude that TAs do not arise abruptly but are the final result of a gradually changing SR architecture and we suggest that the crystalline ATPase within the aggregates may be inactive.

Gradual Formation and Accumulation of Tubular Aggregates in Fast Twitch Muscle Fibers: SERCA and Calsequestrin Involvement

Tubular aggregates (TAs), ordered arrays of elongated sarcoplasmic reticulum (SR) membranes, are present in skeletal muscle fibers from patients with various myopathies. TAs have been also described in ageing wild type (WT) mice, where they display a dependence on sex (male), and fiber type (fast twitch). The mechanism(s) leading to TAs formation are, though, not yet clear. Here, we investigated the sequential stages leading to maturation of TAs in extensor digitorum longus (EDL) from male WT and calsequestrin knockout (CASQ-null) mice. A crucial step in the formation of TAs, seems to be the swelling of free SR cisternae at the sarcomere I band. This dilated SR, which is abundant at 1 year of age contains electron-dense material, which likely indicates abnormal accumulation of CASQ. Lately, the enlarged SR sacs mature into multiple and longitudinally oriented tubules containing CASQ, which first elongates into the A band, and then gradually acquire cylindrical shape and uniform size. Apparently the latter changes occur in concert with partial crystallization of sarco(endo)plasmic reticulum Ca2+ ATPases (SERCA) on its surface, as suggested by freeze-fracture (FF) evidence indicative of an inactive ATPase. Finally, multiple small TAs associate to form fewer mature aggregates of very large size. Interestingly, in fibers from CASQ1-knockout mice the initial swelling of the SR does not occur, possibly due to lack of CASQ accumulation, and aggregates of SR tubules remain small/wavy and never develop into ordered aggregates of straight cylinders. Based on these results, we propose the hypothesis that TAs do not participate actively in the fibers’ Ca2+ homeostasis, but may simply act as deposit sites for accumulated proteins.

Characterization and temporal development of cores in a mouse model of malignant hyperthermia

Malignant hyperthermia (MH) and central core disease are related skeletal muscle diseases often linked to mutations in the type 1 ryanodine receptor (RYR1) gene, encoding for the Ca(2+) release channel of the sarcoplasmic reticulum (SR). In humans, the Y522S RYR1 mutation is associated with malignant hyperthermia susceptibility (MHS) and the presence in skeletal muscle fibers of core regions that lack mitochondria. In heterozygous Y522S knock-in mice (RYR1(Y522S/WT)), the mutation causes SR Ca(2+) leak and MHS. Here, we identified mitochondrial-deficient core regions in skeletal muscle fibers from RYR1(Y522S/WT) knock-in mice and characterized the structural and temporal aspects involved in their formation. Mitochondrial swelling/disruption, the initial detectable structural change observed in young-adult RYR1(Y522S/WT) mice (2 months), does not occur randomly but rather is confined to discrete areas termed presumptive cores. This localized mitochondrial damage is followed by local disruption/loss of nearby SR and transverse tubules, resulting in early cores (2-4 months) and small contracture cores characterized by extreme sarcomere shortening and lack of mitochondria. At later stages (1 year), contracture cores are extended, frequent, and accompanied by areas in which contractile elements are also severely compromised (unstructured cores). Based on these observations, we propose a possible series of events leading to core formation in skeletal muscle fibers of RYR1(Y522S/WT) mice: Initial mitochondrial/SR disruption in confined areas causes significant loss of local Ca(2+) sequestration that eventually results in the formation of contractures and progressive degradation of the contractile elements.

Three Dimensional Reaction‐diffusion Model Of Interaction Between Mitochondria And Sarcoplasmic Reticulum In Heart Muscle Cells

Functional interaction between mitochondria and surrounding ATPases has been found from the experiments on permeabilized heart muscle fibers. According to our earlier analysis of the acquired kinetic data, such interaction can be induced by relatively local diffusion restrictions in cardiac muscle cells. The specific causes of these restrictions are not known but intracellular structures are speculated to act as diffusion barriers. Based on the proximity of sarcoplasmic reticulum (SR) to mitochondria, we hypothesize that SR not only utilizes ATP but may also act as a diffusion barrier for adenosine phosphates leading to functional coupling between ATPases and mitochondria. The structural proximity is evident from electron microscope images where thin SR tubules form loose network around and between consecutive mitochondria. With a 3D finite-element model, we attempted to explore SR as the first candidate for diffusion barrier. The geometry for the model was constructed using representative mitochondrial and SR structural organization from confocal microscope images. The reaction-diffusion model was tracking changes for ADP, ATP and Pi in myoplasm, myofibrils, mitochondrial intermembrane space and in the vicinity of mitochondria and SR. To analyze if SR alone could account for diffusion restriction, we compared the model simulation results with the following set of experimental data: mitochondrial respiration rate dependence on exogenous ADP and ATP; influence of pyruvate kinase and phosphoenolpyruvate additions on respiration; stabilization of respiration rate after addition of 2 mM ATP. The research was supported in part by EU Marie Curie Reintegration Grant.

A Comparative Study on the Structural Features of Muscle Fibers in Intrinsic Lingual Muscles of 21 Day Old and 9 Month Old Mice Using Light and Electron Microscopy

Temelli, A. and Geyikoğlu, F. 2006. A comparative study on the structural features of muscle fibers in intrinsic lingual muscles of 21 day old and 9 month old mice using light and electron microscopy. J. Appl. Anim. Res., 30: 47–51. To compare structural features of muscle fibers in intrinsic lingual muscles of 21 day old and 9 month old mice, the semi-thin sections stained with toluidine blue were examined under light microscope and the thin sections stained with uranyl acetate and lead citrate were examined under JEOL 100SX electron microscope. Two types of fibers in both groups of mice were distinguished (Type 2A, Type 2B). When intrinsic lingual muscles of 21 day old and 9 month old mice were compared, differentiations were observed in distribution of muscle fibers, their compositions and the size and shape of sarcosomes. In addition, the sarcoplasmic reticulum tubules were different between two groups. These differences may be the results of the adaptations of intrinsic lingual muscles of mice in different age groups to feeding behaviours.

Muscle cells in the tiny marine Antarctic mite Halacarellus thomasi ?: an ultrastructural and immunocytochemical study

All musculature examined in the tiny, 0.3-mm, marine Antarctic mite Halacarellus thomasi (i.e. body and appendages) appeared ultrastructurally to be of the transversely striated type with continuous Z-lines. Tubules of sarcoplasmic reticulum lay among the myofibrils. The complexity of the sarcotubular system, sarcomere lengths of over 6 μm, and the abundance of mitochondria are interpreted as signs that the mite is slow moving, but capable of considerable and sustained contraction forces, features deemed necessary in the strong currents of the frigid water prevailing in the mite's habitat. Presence and distribution of regulatory (troponin, tropomyosin, caldesmon and calponin), contractile (actin, myosin, paramyosin and miniparamyosin) and structural (alpha-actinin, titin, minititin and nebulin) proteins were determined immunocytochemically. The results are consistent with the notion of a well-functioning contractile machinery but, furthermore, provide evidence for the great importance of the structural proteins alpha-actinin, minititin and nebulin in maintaining muscle-cell stability under the environmental conditions in which the mite has to function.

Immunocytochemical Observations on Muscle Cell Proteins in the Antarctic Mussel Shrimp Acetabulastoma sp. (Crustacea, Ostracoda)

In the antarctic ostracode Acetabulastoma sp., a singular type of muscle cell has tentatively been classified as transversely striated with continuous Z-line, interrupted by tubules of sarcoplasmic reticulum. We investigated the presence and distribution of different regulatory, contractile, and structural proteins in this muscle by electron microscopical immunocytochem- istry. Troponin, caldesmon, and calponin are three proteins suitable to identify muscle cell types that do not show the classical ultrastructural patterns expected of striated or smooth muscles. Reaction to troponin T was positive (the protein was located along the thin filaments), but no immunoreaction was observed to caldesmon and calponin. Thus, the muscle clearly belongs to the striated class. The contractile proteins myosin, paramyosin, and miniparamyosin were lo- cated along the thick filaments. Paramyosin and miniparamyosin are known only from inver- tebrate thick filaments and have no known vertebrate homolog. A nebulin-like protein was found and, as in vertebrates, may be involved in regulating sarcomere length. Instead of the giant protein titin, known from vertebrate striated muscle, minititin, a protein of the same family but lower molecular weight, was developed. The presence and distribution of proteins such as myosin, troponin, and nebulin as well as the absence of caldesmon and calponin suggest that despite its small size and parasitic life style, Acetabulastoma sp. is an active invertebrate quite unlike those in which the contractile proteins were found to be discontinuously distributed or concentrated at the tips of the thick filaments.

Ultrastructure of muscle cells in Acetabulostoma (Crustacea, Ostracoda) - mussel shrimp from the Ross Sea (Antarctica)

This paper gives the first ultrastructural description of muscle cells in Acetabulostoma sp., a small (less than 1 mm in length) Antarctic ostracod. All muscle cells showed the same ultrastructural pattern: this consisted of mononucleated cells containing a single thick myofibril which was surrounded by numerous mitochondria. The nucleus and most cytoplasmic organelles were also located in the cell periphery. The myofibril showed the ultrastructural pattern of transversely striated muscle. Myofilaments formed well-defined sarcomeres, delimited by Z lines. The average sarcomere lengths were: total sarcomere 4.6 ± 0.9 μm, A band 2.86 ± 0.45 μm (0.178 ± 0.06 μm corresponded to the H band), and I band 1.72 ± 0.58 μm. The myofilament diameters were 5.8 ± 0.3 nm (thin myofilaments), and 14.8 ± 0.68 nm (thick myofilaments). In the same myofibril and in cross sections, some thick filaments appeared homogeneously dense whereas others showed a tubular appearance. Each thick filament was surrounded by 12 thin filaments, and the thin/thick filament ratio was 6/1. The sarcotubular system was well developed and consisted of both transverse and longitudinal components, both running within the myofibril among the myofilaments. The transverse components consisted of 20- to 22-nm-diameter tubules that originated from invaginations of the plasma membrane (T tubules). They penetrated radially throughout each A-I interband and branched inside the myofibril. The longitudinal components consisted of bundles of parallel-arranged sarcoplasmic reticulum saccules running parallel to the myofilaments. Each bundle comprised from three to six 30 to 35-nm-diameter saccules. At the levels of the A-I interbands, the saccules of some bundles fused together to form triads with the T tubules. In the triad, the distance between the sarcoplasmic reticulum tubules and the T tubules was 10–14 nm. The mitochondria were large, presented numerous transverse cristae, and were joined one to another by electron-dense plaques. The muscle cell tips ending beneath the cuticle were connected to a modified epidermal cell by a specialized intercellular junction of the fascia adherens type. This junction occurred at the level of a Z line, and displayed an irregular outline with numerous interdigitations that formed conical projections. The ultrastructural characteristics of the myofibrillar apparatus, the plentiful sarcotubular system and the abundance of mitochondria in Acetabulostoma muscles suggest that this muscle type is concurrently a developed model of both the fast-twitch type and the fatigue-resistant type.

Two distinct distribution patterns of sarcoplasmic reticulum in two functionally different giant smooth muscle cells of Beroe ovata.

The sarcoplasmic reticulum has been studied in radial and longitudinal giant smooth muscle fibres of the marine planktonic invertebrate Beroe. Impregnation with heavy metals has revealed that the smooth component is organised in a longitudinally oriented three-dimensional network of tubules running along the myofilaments. An ultrastructural morphometric analysis has shown that the relative volume of the sarcoplasmic reticulum is the same (1% of the myofilament volume) in both fibres but that the size, number and distribution of the sarcoplasmic reticulum tubules differ significantly. The longitudinal fibres are characterised physiologically by an action potential with a short calcium-dependent plateau that can trigger a short contraction; radial fibres produce action potentials without a plateau and their contraction requires a train of spikes. The sarcoplasmic reticulum tubules in longitudinal fibres are thinner (132 nm in diameter) and more numerous than those in radial fibres (160 nm in diameter). Moreover, the tubules are homogeneously distributed among the myofilaments in radial fibres, whereas they are more numerous in the centre of longitudinal muscles.

The calcium release channel of sarcoplasmic reticulum is modulated by FK-506-binding protein. Dissociation and reconstitution of FKBP-12 to the calcium release channel of skeletal muscle sarcoplasmic reticulum.

The ryanodine receptor/calcium release channel (CRC) of rabbit skeletal muscle terminal cisternae (TC) of sarcoplasmic reticulum (SR) has been found to be tightly associated with FK-506 binding protein (FKBP-12), the cytosolic receptor (immunophilin) for the immunosuppressant drug FK-506 (Jayaraman, T., Brillantes, A. M., Timerman, A. P., Fleischer, S., Erdjument-Bromage, H., Tempst, P., and Marks, A. (1992) J. Biol. Chem. 267, 9474-9477). In this study, a procedure is described to dissociate FKBP from TC and reconstitute human recombinant FKBP-12 back to the ryanodine receptor so that the role of the immunophilin on CRC activity can be assessed. Titration of TC vesicles with FK-506 dissociates FKBP from the ryanodine receptor. Sedimentation of FK-506-treated vesicles effectively separates the TC from the soluble FKBP-FK506 complex which remains in the supernatant. The FKBP-deficient TC vesicles have altered functional characteristics: 1) the ATP-stimulated calcium uptake rate of TC vesicles is reduced 2-fold; and 2) the threshold concentration of caffeine required to induce calcium release from TC vesicles is decreased. These changes appear to reflect modification of the calcium release channel since: 1) severalfold higher concentrations of FK-506 do not alter the calcium uptake rate of either longitudinal tubules of SR, or TC vesicles in the presence of ruthenium red; 2) human recombinant FKBP reassociates with FKBP-deficient TC but not with control TC or longitudinal tubules of SR; and 3) the reduced Ca2+ uptake rate in FKBP-deficient TC is restored to control values in the FKBP-reconstituted TC. These studies demonstrate that FKBP-12 modulates the CRC of rabbit skeletal muscle sarcoplasmic reticulum.

FK506 binding protein associated with the calcium release channel (ryanodine receptor).

The calcium release channel (CRC)/ryanodine receptor (RyRec) has been identified as the foot structure of the sarcoplasmic reticulum (SR) and provides the pathway for calcium efflux required for excitation-contraction coupling in skeletal muscle. The CRC has previously been reported to consist of four identical 565-kDa protomers. We now report the identification of a 12-kDa protein which is tightly associated with highly purified RyRec from rabbit skeletal muscle SR. N-terminal amino acid sequencing and cDNA cloning demonstrates that the 12-kDa protein from fast twitch skeletal muscle is the binding protein for the immunosuppressant drug FK506. In humans, FK506 binds to the 12-kDa FK506-binding protein (FKBP12) and blocks calcium-dependent T cell activation. We find that FKBP12 and the RyRec are tightly associated in skeletal muscle SR on the basis of: 1) co-purification through sequential heparin-agarose, hydroxylapatite, and size exclusion chromatography columns; 2) coimmunoprecipitation of the RyRec and FKBP12 with anti-FKBP12 antibodies; and 3) subcellular localization of both proteins to the terminal cisternae of the SR, and not in the longitudinal tubules of SR, in fast twitch skeletal muscle. The molar ratio of FKBP12 to RyRec in highly purified RyRec preparations is approximately 1:4, indicating that one FKBP12 molecule is associated with each calcium release channel/foot structure.

Ultrastructure of the myocardium of the least shrew, Cryptotis parva say

The heart of the least shrew, Cryptotis parva Say, is an extremely active organ, capable of achieving rates of 800-1,200 beats/minute. The general features of myocardial cell ultrastructure in this insectivore are much like those of other small mammals; no single striking feature of fine structure is present to which the physiological properties of this heart might necessarily be attributed. Still there exist in these myocardial cells a number of atypical properties. These include 1) mitochondria having a wide variety of sizes and internal configurations 2) a pleiomorphic, highly ramified, small-diameter transverse-axial tubular system (TATS) 3) numerous "labyrinths," which are proliferated components of the TATS, and 4) myofibril-free regions, located both in juxtanuclear and other myoplasmic levels and populated by a concentration of TATS elements and fibrillar structures. Features (2) and (3) are also characteristic of another fast-beating heart, that of the mouse. The sinoatrial and atrioventricular nodal regions, as well as a Purkinje system, have been identified in the least shrew heart, along with sparsely distributed atrial cells whose myofibrils contain proliferated Z-band material. A feature frequently encountered in atrial working muscle cells is the occurrence of close appositions between gap junctions and tubules of sarcoplasmic reticulum; such appositions are also present in other regions of the shrew heart, as are complexes composed of gap junctions and mitochondria.

30] Modification of phospholipid environment in sarcoplasmic reticulum using nonspecific phospholipid transfer protein

Publisher Summary This chapter focuses on the modification of phospholipid environment in sarcoplasmic reticulum using nonspecific phospholipid transfer protein. The calcium-pump membrane of sarcoplasmic reticulum (SR) from fast twitch skeletal muscle is highly specialized for calcium transport. The calcium-pump protein accounts for 90% of the protein of the calcium-pump membrane of highly purified light SR, which derives from the longitudinal tubules of SR. Phospholipid comprises 35% of the mass of the membrane of which more than 90% is phospholipid. The Ca 2+ -pump protein is oriented transmembrane with a major portion extending out from the cytoplasmic face. The membrane lipids appear to be required for both hydrolysis of ATP and Ca 2+ -pumping activity. The phospholipid exchange procedure consists of incubating the SR membrane vesicles with the transfer protein in the presence of phospholipid vesicles of defined phospholipid composition. The exchange of phospholipid takes place with retention of the unidirectional orientation of the calcium-pump protein in the membrane. The use of a nonspecific phospholipid transfer protein is an effective and gentle way to modify the phospholipid composition in the SR membrane. With this methodology, the presence of two different classes of phospholipids in the SR membranes is observed and interpreted in terms of membrane sidedness.

Preparation and characterization of longitudinal tubules of sarcoplasmic reticulum from fast skeletal muscle

Sarcoplasmic reticulum (SR) serves a central role in calcium uptake and release, thereby regulating muscle relaxation and contraction, respectively. Recently, we have isolated fractions referable to longitudinal tubules (R2) and terminal cisternae (R4), the two major types of Sarcoplasmic reticulum (A. Saito et al. (1984) J. Cell Biol. 99, 875–885). The terminal cisternae contain two types of membranes, the calcium pump membrane and the junctional face membrane. The terminal cisternae are filled with electronopaque contents which serve as a Ca 2+ reservoir. The longitudinal tubules consist mainly of the calcium pump membrane. In this study, we describe a new longitudinal tubule fraction (F2) and characterize it together with the R2 and R4 SR fractions. The calcium pump membrane of the longitudinal tubules is a highly specialized membrane consisting of about 90% calcium pump protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extensive changes in morphology can be observed in the SR fractions referable to osmotic differences during the fixation conditions using either glutaraldehyde-tannic acid or osmium tetroxide fixatives. The changes include swelling or shrinkage and aggregation of the compartmental contents when the fixative contains calcium ions. The two types of SR have different osmotic permeability to the same medium, as indicated by differential swelling or shrinkage. Both longitudinal tubule and terminal cisternae vesicles of SR appear larger and are spherical vesicles when the glutaraldehyde-tannic acid fixative is isotonic as compared with the “standard” fixation method. We have previously reported that the ruthenium red-sensitive calcium release channels are localized to the terminal cisternae. The terminal cisternae as isolated are leaky to Ca 2+ since these channels are in the “open state” (S. Fleischer et al. (1985) Proc. Natl. Acad. Sci USA 82, 7256–7259). Thus, the Ca 2+,Mg 2+-dependent ATPase (Ca 2+ ATPase) rate is only slightly enhanced in the presence of a Ca 2+ ionophore, which dissipates the Ca 2+ gradient across the SR membrane. We now find that preincubation with ruthenium red restores the tight coupling of the Ca 2+ ATPase activity to Ca 2+ transport. That is to say, ATPase activity is reduced and the addition of ionophore stimulates the Ca 2+ ATPase activity 4– to 7-fold. The Ca 2+ ATPase activity in longitudinal tubules is already tightly coupled. It is minimal after a Ca 2+ gradient has been generated, but can be stimulated 9– to 20-fold when the Ca 2+ gradient is dissipated with ionophore. This finding suggests that the Ca 2+ ATPase activity in SR is tightly coupled to Ca 2+ transport in situ.