Reticulum Of Skeletal Muscle Fibers Research Articles

Cytoplasmic γ-actin and tropomodulin isoforms link to the sarcoplasmic reticulum in skeletal muscle fibers

Cytoplasmic γ-actin and tropomodulin isoforms link to the sarcoplasmic reticulum in skeletal muscle fibers

Cytoplasmic γ-actin and tropomodulin isoforms link to the sarcoplasmic reticulum in skeletal muscle fibers

The sarcoplasmic reticulum (SR) serves as the Ca(2+) reservoir for muscle contraction. Tropomodulins (Tmods) cap filamentous actin (F-actin) pointed ends, bind tropomyosins (Tms), and regulate F-actin organization. In this paper, we use a genetic targeting approach to examine the effect of Tmod1 deletion on the organization of cytoplasmic γ-actin (γ(cyto)-actin) in the SR of skeletal muscle. In wild-type muscle fibers, γ(cyto)-actin and Tmod3 defined an SR microdomain that was distinct from another Z line-flanking SR microdomain containing Tmod1 and Tmod4. The γ(cyto)-actin/Tmod3 microdomain contained an M line complex composed of small ankyrin 1.5 (sAnk1.5), γ(cyto)-actin, Tmod3, Tm4, and Tm5NM1. Tmod1 deletion caused Tmod3 to leave its SR compartment, leading to mislocalization and destabilization of the Tmod3-γ(cyto)-actin-sAnk1.5 complex. This was accompanied by SR morphological defects, impaired Ca(2+) release, and an age-dependent increase in sarcomere misalignment. Thus, Tmod3 regulates SR-associated γ(cyto)-actin architecture, mechanically stabilizes the SR via a novel cytoskeletal linkage to sAnk1.5, and maintains the alignment of adjacent myofibrils.

Phenol increases twitch tension by increasing Ca2+ transients in intact single Xenopus myofibers

Phenol is a neurolytic agent used for management of spasticity in patients with neuron injury. The effects of phenol on skeletal muscle function are unknown. We investigated the effects of phenol on force production and Ca2+ release in intact single Xenopus skeletal muscle fibers. To determine the relationship between force production and Ca2+ release in the absence and presence of phenol (0.005% – 0.05% w/v), intact single muscle fibers were dissected and microinjected with FURA‐2 followed by electrically stimulated single‐twitches and tetanic contractions (1–150Hz, 250 ms train, 2 ms stimulus, 8–10V). During single‐twitches at activation frequencies that elicited far less force than maximal, phenol reversibly increased the force and peak Ca2+ ratios compared to the untreated condition in a concentration‐dependent manner (from 10 ± 4% to 57 ± 10% of maximum tetanic force and from 47 ± 11% to 68 ± 10% of the maximum FURA‐2 ratio, both at 0.05% phenol, P<0.01). At higher concentrations (>0.05%), phenol completely blocked force production. These results suggest that phenol increases twitch force by increasing Ca2+ release from the sarcoplasmic reticulum in skeletal muscle fibers. This mechanism remains to be elucidated; however, phenol may prove to be a new agent used to study skeletal muscle function.Supported by NIH grant 1P01HL091830‐01A18101

Calsequestrin targeting to sarcoplasmic reticulum of skeletal muscle fibers

Calsequestrin (CS) is the low-affinity, high-capacity calcium binding protein segregated to the lumen of terminal cisternae (TC) of the sarcoplasmic reticulum (SR). The physiological role of CS in controlling calcium release from the SR depends on both its intrinsic properties and its localization. The mechanisms of CS targeting were investigated in skeletal muscle fibers and C2C12 myotubes, a model of SR differentiation, with four deletion mutants of epitope (hemagglutinin, HA)-tagged CS: CS-HA24NH2, CS-HA2D, CS-HA3D, and CS-HAHT, a double mutant of the NH2 terminus and domain III. As judged by immunofluorescence of transfected skeletal muscle fibers, only the double CS-HA mutant showed a homogeneous distribution at the sarcomeric I band, i.e., it did not segregate to TC. As shown by subfractionation of microsomes derived from transfected skeletal muscles, CS-HAHT was largely associated to longitudinal SR whereas CS-HA was concentrated in TC. In C2C12 myotubes, as judged by immunofluorescence, not only CS-HAHT but also CS-HA3D and CS-HA2D were not sorted to developing SR. Condensation competence, a property referable to CS oligomerization, was monitored for the several CS-HA mutants in C2C12 myoblasts, and only CS-HA3D was found able to condense. Together, the results indicate that 1) there are at least two targeting sequences at the NH2 terminus and domain III of CS, 2) SR-specific target and structural information is contained in these sequences, 3) heterologous interactions with junctional SR proteins are relevant for segregation, 4) homologous CS-CS interactions are involved in the overall targeting process, and 5) different targeting mechanisms prevail depending on the stage of SR differentiation.

Differential Effects of Bupivacaine and Ropivacaine Enantiomers on Intracellular Ca2+Regulation in Murine Skeletal Muscle Fibers

Increased intracellular Ca concentrations are considered to be a major pathomechanism in local anesthetic myotoxicity. Racemic bupivacaine and S-ropivacaine cause Ca release from the sarcoplasmic reticulum of skeletal muscle fibers and simultaneously inhibit Ca reuptake. Examining the optical isomers of both agents, the authors investigated stereoselective effects on muscular Ca regulation to get a closer insight in subcellular mechanisms of local anesthetic myotoxicity. R- and S-enantiomers as well as racemic mixtures of both agents were tested in concentrations of 1, 5, 10, and 15 mm. Saponin-skinned muscle fibers from the extensor digitorum longus muscle of BALB/c mice were examined according to a standardized procedure. For the assessment of effects on Ca uptake and release from the sarcoplasmic reticulum, agents were added to the loading solution and the release solution, respectively, and force and Ca transients were monitored. The effects of S-enantiomers on both Ca release and reuptake were significantly more pronounced than those of racemic mixtures and R-enantiomers, respectively. In addition, the effects of racemates were markedly stronger than those of R-enantiomers. With regard to Ca release, the effects of bupivacaine isomers were more pronounced than the isomers of ropivacaine. These data show that stereoselectivity is involved in alterations of intracellular Ca regulation by bupivacaine and ropivacaine. S-enantiomers seem to be more potent than R-enantiomers, with intermediate effects of racemic mixtures. In addition, lipophilicity also seems to determine the extent of Ca release by local anesthetics.

Homer proteins and InsP 3 receptors co-localise in the longitudinal sarcoplasmic reticulum of skeletal muscle fibres

Striated muscle represents one of the best models for studies on Ca 2+ signalling. However, although much is known on the localisation and molecular interactions of the ryanodine receptors (RyRs), far less is known on the localisation and on the molecular interactions of the inositol trisphosphate receptors (InsP 3Rs) in striated muscle cells. Recently, members of the Homer protein family have been shown to cluster type 1 metabotropic glutamate receptors (mGluR1) in the plasma membrane and to interact with InsP 3R in the endoplasmic reticulum of neurons. Thus, these scaffolding proteins are good candidates for organising plasma membrane receptors and intracellular effector proteins in signalosomes involved in intracellular Ca 2+ signalling. Homer proteins are also expressed in skeletal muscle, and the type 1 ryanodine receptor (RyR1) contains a specific Homer-binding motif. We report here on the relative sub-cellular localisation of InsP 3Rs and Homer proteins in skeletal muscle cells with respect to the localisation of RyRs. Immunofluorescence analysis showed that both Homer and InsP 3R proteins present a staining pattern indicative of a localisation at the Z-line, clearly distinct from that of RyR1. Consistent herewith, in sub-cellular fractionation experiments, Homer proteins and InsP 3R were both found in the fractions enriched in longitudinal sarcoplasmic reticulum (LSR) but not in fractions of terminal cisternae that are enriched in RyRs. Thus, in skeletal muscle, Homer proteins may play a role in the organisation of a second Ca 2+ signalling compartment containing the InsP 3R, but are apparently not involved in the organisation of RyRs at triads.

Self-aggregation of triadin in the sarcoplasmic reticulum of rabbit skeletal muscle

The 95 kDa transmembrane glycoprotein triadin is believed to be an essential component of excitation–contraction coupling in the junctional sarcoplasmic reticulum of skeletal muscle fibers. It is debatable whether triadin mediates intraluminal interactions between calsequestrin and the ryanodine receptor exclusively or whether this junctional protein provides also a cytoplasmic linkage between the Ca 2+-release channel and the dihydropyridine receptor. Here, we could show that native triadin exists as disulfide-linked homo-polymers of above 3000 kDa. Under non-reducing conditions, protein bands representing the α 1-dihydropyridine receptor and calsequestrin did not show an immunodecorative overlap with the extremely high-molecular-mass triadin clusters. Following chemical crosslinking, the ryanodine receptor and triadin exhibited a similarly decreased electrophoretic mobility. However, immunoblotting of diagonal non-reducing/reducing two-dimensional gels clearly demonstrated a lack of overlap between the immunodecorated bands representing triadin, the α 1-dihydropyridine receptor, the ryanodine receptor and calsequestrin. Thus, in native membranes triadin appears to form large self-aggregates primarily. Although triadin exists in a close neighborhood relationship to the Ca 2+-release channel tetramers, it does not seem to be directly linked to the other main triad components implicated in the regulation of the excitation–contraction–relaxation cycle and Ca 2+-homeostasis. This agrees with a proposed role of triadin in the maintenance of overall triad architecture.

The endoplasmic reticulum-sarcoplasmic reticulum connection: distribution of endoplasmic reticulum markers in the sarcoplasmic reticulum of skeletal muscle fibers.

The skeletal muscle sarcoplasmic reticulum (SR) was investigated for the presence of well-known endoplasmic reticulum (ER) markers: the lumenal protein BiP and a group of membrane proteins recognized by an antibody raised against ER membrane vesicles. Western blots of SR fractions revealed the presence of BiP in fast- and slow-twitch muscles of the rabbit as well as in rat and chicken muscles. Analyses of purified SR subfractions, together with cryosection immunofluorescence and immunogold labeling, revealed BiP evenly distributed within the longitudinal SR and the terminal cisternae. Within the terminal cisternae BiP appeared not to be mixed with calsequestrin but to be distributed around the aggregates of the latter Ca2+ binding protein. Of the various membrane markers only calnexin (91 kDa) was found to be distributed within both SR subfractions, whereas the other markers (apparent molecular masses of 64 kDa and 58 kDa and a doublet around 28 kDa) were concentrated in the terminal cisternae. These results suggest that the SR is a specialized ER subcompartment in which general markers, such as the ones we have investigated, coexist with the major SR proteins specifically responsible for Ca2+ uptake, storage, and release. The differential distribution of the ER markers reveals new aspects of the SR molecular structure that might be of importance for the functioning of the endomembrane system.

Calcium dependence of inactivation of calcium release from the sarcoplasmic reticulum in skeletal muscle fibers.

The steady-state calcium dependence of inactivation of calcium release from the sarcoplasmic reticulum was studied in voltage-clamped, cut segments of frog skeletal muscle fibers containing two calcium indicators, fura-2 and anti-pyrylazo III (AP III). Fura-2 fluorescence was used to monitor resting calcium and relatively small calcium transients during small depolarizations. AP III absorbance signals were used to monitor larger calcium transients during larger depolarizations. The rate of release (Rrel) of calcium from the sarcoplasmic reticulum was calculated from the calcium transients. The equilibrium calcium dependence of inactivation of calcium release was determined using 200-ms prepulses of various amplitudes to elevate [Ca2+] to various steady levels. Each prepulse was followed by a constant test pulse. The suppression of peak Rrel during the test pulse provided a measure of the extent of inactivation of release at the end of the prepulse. The [Ca2+] dependence of inactivation indicated that binding of more than one calcium ion was required to inactivate each release channel. Half-maximal inactivation was produced at a [Ca2+] of approximately 0.3 microM. Variation of the prepulse duration and amplitude showed that the suppression of peak release was consistent with calcium-dependent inactivation of calcium release but not with calcium depletion. The same calcium dependence of inactivation was obtained using different amplitude test pulses to determine the degree of inactivation. Prepulses that produced near maximal inactivation of release during the following test pulse produced no suppression of intramembrane charge movement during the test pulse, indicating that inactivation occurred at a step beyond the voltage sensor for calcium release. Three alternative set of properties that were assumed for the rapidly equilibrating calcium-binding sites intrinsic to the fibers gave somewhat different Rrel records, but gave very similar calcium dependence of inactivation. Thus, equilibrium inactivation of calcium release appears to be produced by rather modest increases in [Ca2+] above the resting level and in a steeply calcium-dependent manner. However, the inactivation develops relatively slowly even during marked elevation of [Ca2+], indicating that a calcium-independent transition appears to occur after the initial calcium-binding step.

Polymyositis in mice experimentally inoculated with Getah virus.

Mice inoculated intracerebrally with parent, large-plaque (LP) and small-plaque (SP) strains of Kanagawa strain of Getah virus showed clinically recumbency and paralysis. The LP strain caused recumbency more rapidly and killed mice more early after inoculation than the parent and SP strains. Microscopically, skeletal muscles of the whole body were involved showing degenerative or inflammatory changes. In mice inoculated with the parent or SP strains, there were degeneration and necrosis of the muscle fibers with inflammatory cell infiltration and regenerative reaction. The lesions were particularly conspicuous in muscles of the hind legs. In mice inoculated with the LP strain, most of the muscle fibers revealed degeneration and necrosis, but reactive changes were poor. In addition, the periosteum and muscular connective tissue were thickened with karyorrhexis. Electron microscopically, virus particles were recognized mainly in cisternae of sarcoplasmic reticulum in skeletal muscle fibers of mice inoculated with the LP strain, while they were rare in those of animals injected with the parent and SP strains. From these finding, it was suggested that Kanagawa strain of Getah virus has the virulence to skeletal muscles of mice.

Chapter 34 Do sensory cells in the ear have a motile function?

This chapter discusses the motile function of sensory cells in the ear. It has been demonstrated at the light microscopic level that the apical region of cochlear hair cells contains contractile proteins. Actin is now identified and localized by antibody labelling techniques at the electron microscopical level. To investigate if outer hair cells show some form of contractile response, isolated cells were subjected to media that would induce relaxation or contraction in muscle cells. In intact cells, electron microscopy showed that the plasma membrane is intimately associated with subsurface cisternae by membrane-associated rodlets that remind of the connection between the transverse tubule system and the sarcoplasmic reticulum in skeletal muscle fibers. It is suggested that outer hair cells can exhibit motor activity, which is integrated in the electro-mechanical function of the organ of Corti. Inner hair cells, lacking submembraneous actin, are suggested to have a mainly sensory function.

A general procedure for determining the rate of calcium release from the sarcoplasmic reticulum in skeletal muscle fibers

A general procedure for using myoplasmic calcium transients measured with a metallochromic indicator dye to calculate the time course of calcium release from the sarcoplasmic reticulum in voltage-clamped skeletal muscle fibers is described and analyzed. Explicit properties are first assigned to all relatively rapidly equilibrating calcium binding sites in the myoplasm so that the calcium content (CaF) in this pool of "fast" calcium can be calculated from the calcium transient. The overall properties of the transport systems and relatively slowly equilibrating binding sites that remove calcium from CaF are then characterized experimentally from the decay of CaF following fiber repolarization. The rate of calcium release can then be calculated as dCaF/dt plus the rate of removal of calcium from CaF. Two alternatives are assumed for the component of CaF that is due to fast binding sites intrinsic to the fiber: a linear instantaneous buffer or a set of binding sites having properties similar to thin filament troponin. Both assumptions yielded similar calcium release wave forms. Three alternative methods for characterizing the removal system are presented. The choice among these or other methods for characterizing removal can be based entirely on convenience since any method that reproduces the decay of CaF following fiber repolarization will give the same release wave form. The calculated release wave form will be accurate provided that the properties assumed for CaF are correct, that release turns off within a relatively short time after fiber repolarization, that the properties of the slow removal system are the same during and after fiber depolarization, and that possible spatial nonuniformities of free or bound calcium do not introduce major errors.

Two ATPases in the sarcoplasmic reticulum of skeletal muscle fibres.

Two distinct ATPase activities have been localized on the sarcoplasmic reticulum of muscle cells. They may play a part in contraction and relaxation.