Localization of ank1.5 in the sarcoplasmic reticulum precedes that of SERCA and RyR: relationship with the organization of obscurin in developing sarcomeres

Ank1.5 is a muscle-specific isoform of ankyrin1 localized on the sarcoplasmic reticulum (SR) membrane that has been shown to interact with obscurin, a sarcomeric protein. We report here studies on the localization of obscurin and ank1.5 in embryonic and postnatal rodent skeletal muscles. Using two antibodies against epitopes in the N- and C-terminus of obscurin, two distinct patterns of localization were observed. Before birth, the antibodies against the N- and the C-terminus of obscurin stained the Z-disk and M-band, respectively. At the same time, ank1.5 was detected at the Z-disk, rising the possibility that obscurin molecules at M-band may not be able to interact with ank1.5. Localization of ank1.5 at Z-disks in E14 muscle fibers revealed that ank1.5 is among the earliest SR proteins to assemble, since its organization preceded that of other SR proteins, like SERCA and RyR. After birth, the antibody against the N-terminus of obscurin stained the M-band while that against the C-terminus stained both M-bands and the Z-disks. Starting from postnatal day 1, ank1.5 was found at the level of both M-bands and Z-disks. Altogether, from these results we infer that exposure of some obscurin epitopes changes during skeletal muscle development, resulting in distinct, antibody-specific, localization pattern. Why this occurs is not clear, yet these data indicate that the organization of obscurin at different locations in the sarcomere changes during muscle development and that this might affect the interaction with ank1.5.     

IgG from patients with liver diseases inhibit mitochondrial respiration in permeabilized oxidative muscle cells: Impaired function of intracellular energetic units?

The effect of IgG purified from the sera of healthy persons and patients with primary biliary cirrhosis (PBC) and chronic hepatitis (CH) on ADP dependent respiration (oxidative phosphorylation) in skinned muscle fibers from rat oxidative muscles (heart and M. soleus) and glycolytic skeletal muscle (M. gastrocnemius) was studied. The results show that IgG from three different sources inhibited the rate of respiration by 13, 44 and 42%, respectively, these effects being equally expressed in both types of oxidative muscles, whereas no inhibition was observed in glycolytic muscle. The following washout of unbound IgG did not abolish the inhibition of respiration suggesting that the specific interaction of IgG with antigens had taken place. Laser confocal analysis revealed binding of IgG predominantly to the sarcomeric structures such as Z-disk and M-lines in the cardiomyocytes. The staining of IgG within Z-disks and intermitochondrial space coincided throughout the muscle cells so that transversally serial spaces, each containing mitochondria and adjacent sarcomere, became clearly visible. When the IgG from a CH patient was incubated with the skinned myocardial fibers of the desmin knockout mice, its binding to Z-disks and the sarcomeric area was found to be similar to that in normal cardiac muscle. However, the transversal staining pattern was disintegrated, because of the slippage of the myofibrils in relation to each other and accumulation of mitochondria between them. These observations support the recent hypothesis that in oxidative muscles the mitochondria and adjacent sarcomeres form complexes, termed as the intracellular energetic units, ICEUs. Moreover, they indicate that human autoantibodies can be useful tools for localizing the proteins responsible for formation of ICEUs and modulation of their function. Thus, it appears that the proteins associated with the Z-disks and M-lines may participate in formation of ICEUs and that binding of IgG to these proteins decreases the access of exogenous adenine nucleotides to mitochondria, which manifests as decreased rate of ADP-dependent respiration.     

The Rho-Guanine Nucleotide Exchange Factor Domain of Obscurin Activates RhoA Signaling in Skeletal Muscle

Obscurin is a large (∼800-kDa), modular protein of striated muscle that concentrates around the M-bands and Z-disks of each sarcomere, where it is well positioned to sense contractile activity. Obscurin contains several signaling domains, including a rho-guanine nucleotide exchange factor (rhoGEF) domain and tandem pleckstrin homology domain, consistent with a role in rho signaling in muscle. We investigated the ability of obscurin''s rhoGEF domain to interact with and activate small GTPases. Using a combination of in vitro and in vivo approaches, we found that the rhoGEF domain of obscurin binds selectively to rhoA, and that rhoA colocalizes with obscurin at the M-band in skeletal muscle. Other small GTPases, including rac1 and cdc42, neither associate with the rhoGEF domain of obscurin nor concentrate at the level of the M-bands. Furthermore, overexpression of the rhoGEF domain of obscurin in adult skeletal muscle selectively increases rhoA expression and activity in this tissue. Overexpression of obscurin''s rhoGEF domain and its effects on rhoA alter the expression of rho kinase and citron kinase, both of which can be activated by rhoA in other tissues. Injuries to rodent hindlimb muscles caused by large-strain lengthening contractions increases rhoA activity and displaces it from the M-bands to Z-disks, similar to the effects of overexpression of obscurin''s rhoGEF domain. Our results suggest that obscurin''s rhoGEF domain signals at least in part by inducing rhoA expression and activation, and altering the expression of downstream kinases in vitro and in vivo.     

The molecular composition of the sarcomeric M-band correlates with muscle fiber type

The M-band is the transverse structure that cross-links the thick filaments in the center and provides a perfect alignment of the A-band in the activated sarcomere. The molecular composition of the M-bands in adult mouse skeletal muscle is fiber-type dependent. All M-bands in fast fibers contain M-protein while M-bands in slow fibers contain a significant proportion of the EH-myomesin isoform, previously detected only in embryonic heart muscle. This fiber-type specificity develops during the first postnatal weeks. However, the ratio between the amounts of myosin and of myomesin, taken as sum of both isoforms, remains nearly constant in all studied muscles. Ultrastructural analysis demonstrates that some of the soleus fibers show a diffuse appearance of the M-band, resembling the situation in the embryonic heart. A model is proposed to explain the functional consequence of differential M-band composition for the physiological and morphological properties of sarcomeres in different muscle types.     

Simultaneous imaging and functional assessment of cytoskeletal protein connections in passively loaded single muscle cells.

We describe a novel system that permits simultaneous confocal imaging of protein interactions and measurement of cell mechanical properties during passive loading. A mechanical apparatus was designed to replace the stage of a confocal microscope, enabling cell manipulation, force transduction, and imaging. In addition, image processing algorithms were developed to quantify the degree of connectivity between subcellular structures. Using this system, we examined the interactions among three cellular structures thought to be linked by the muscle's intermediate filament system: Z-disks, nuclei, and the costamere protein complexes located at the muscle cell surface. Fast Fourier transforms (FFTs) and autocorrelations (ACs) were implemented to quantify image periodicity and relative phase shifts among structures. We demonstrated in sample wild-type muscle cells that there was significant connectivity among Z-disks in the same fiber at various sarcomere lengths, as well as between Z-disks and the costamere complexes. This approach can be applied to any cell system in which structural periodicity and mechanical connectivity are of interest.     

Spatial distribution of the transverse stiffness of relaxed and activated rat soleus muscle fibers

The transverse stiffness of glycerinated and demembranated fibers from the soleus muscle of Wistar rats in different functional states was measured by atomic force microscopy. It was demonstrated that the transverse stiffness of relaxed fibers near the Z disk is approximately twofold higher as compared with the M-line region. However, the stiffness of glycerinated fibers in the Z-disk and M-line regions is considerably lower than that of demembranated fibers. The values of mechanical parameters of activated fibers are significantly higher as compared with the relaxed fibers. However, the stiffness of activated glycerinated fibers near the Z disk approximately doubled as compared with the relaxed state, whereas the stiffness of the Z-disk region in demembranated fibers increased more than fourfold. The stiffness of both glycerinated and demembranized fibers near the M-line increased approximately threefold.     

Effects of nifedipine on the mechanical properties of sarcolemma and modulation of calcium accumulation dynamics in fibers of the rat soleus muscle under short-term hypogravity conditions

It has been shown that the modulation of the mechanical properties of sarcolemma by nifedipine may be related to the dynamics of accumulation of calcium ions under short-term rat hindlimb suspension. The basal calcium level was measured by the fluorescence probe Fluo-4AM, the transversal stiffness of different parts of the contractile apparatus and sarcolemma was estimated by atomic force microscopy, and the content of desmin was determined by gel electrophoresis with immunoblotting. It has been found that nifedipine has a protecting effect on muscle fibers under hypogravity by decreasing the degradation of desmin and proteins that determine the transversal stiffness of sarcolemma and the contractile apparatus, and the intensity of the increase in the basal calcium level. It was shown that the selective blocking of L-channels leads to an increase in the basal calcium level in intact soleus fibers. At the same time, the transversal stiffness of sarcolemma and the contractile apparatus increased. The mechanism of this increase is still unclear; however, it is assumed that it mediates the protecting action of nifedipine.     

Structural and functional roles of desmin in mouse skeletal muscle during passive deformation

Mechanical interactions between desmin and Z-disks, costameres, and nuclei were measured during passive deformation of single muscle cells. Image processing and continuum kinematics were used to quantify the structural connectivity among these structures. Analysis of both wild-type and desmin-null fibers revealed that the costamere protein talin colocalized with the Z-disk protein alpha-actinin, even at very high strains and stresses. These data indicate that desmin is not essential for mechanical coupling of the costamere complex and the sarcomere lattice. Within the sarcomere lattice, significant differences in myofibrillar connectivity were revealed between passively deformed wild-type and desmin-null fibers. Connectivity in wild-type fibers was significantly greater compared to desmin-null fibers, demonstrating a significant functional connection between myofibrils that requires desmin. Passive mechanical analysis revealed that desmin may be partially responsible for regulating fiber volume, and consequently, fiber mechanical properties. Kinematic analysis of alpha-actinin strain fields revealed that knockout fibers transmitted less shear strain compared to wild-type fibers and experienced a slight increase in fiber volume. Finally, linkage of desmin intermediate filaments to muscle nuclei was strongly suggested based on extensive loss of nuclei positioning in the absence of desmin during passive fiber loading.     

Tetanic contractions impair sarcomeric Z-disk of atrophic soleus muscle via calpain pathway

The aim of this study was to determine whether or not over-activation of calpains during running exercise or tetanic contractions was a major factor to induce sarcomere lesions in atrophic soleus muscle. Relationship between the degrees of desmin degradation and sarcomere lesions was also elucidated. We observed ultrastructural changes in soleus muscle fibers after 4-week unloading with or without running exercise. Calpain activity and desmin degradation were measured in atrophic soleus muscles before or after repeated tetani in vitro. Calpain-1 activity was progressively increased and desmin degradation was correspondingly elevated in 1-, 2-, and 4-week of unloaded soleus muscles. Calpain-1 activity and desmin degradation had an additional increase in unloaded soleus muscles after repeated tetani in vitro. PD150606, an inhibitor of calpains, reduced calpain activity and desmin degradation during tetanic contractions in unloaded soleus muscles. The 4-week unloading decreased the width of myofibrils and Z-disk in soleus fibers. After running exercise in unloaded group, Z-disks of adjacent myofibrils were not well in register but instead were longitudinally displaced. Calpain inhibition compromised exercise-induced misalignment of the Z-disks in atrophic soleus muscle. These results suggest that tetanic contractions induce an over-activation of calpains which lead to higher degrees of desmin degradation in unloaded soleus muscle. Desmin degradation may loose connections between adjacent myofibrils, whereas running exercise results in sarcomere injury in unloaded soleus muscle.     

Morphological and histochemical organization of the flexor carpi radialis muscle in the cat.

Most studies concerning the structure and function of skeletal muscle have utilized the hind limb of the experimental animal. However, it has been shown that the number of behavioral tasks performed by the cat's forelimb is greater than that of the hind limb. In addition, the forelimb muscles exhibit a functional complexity not observed in hind-limb musculature. The purpose of this study was to investigate the distribution of fast-twitch and slow-twitch muscle fibers and muscle spindles in the flexor carpi radialis muscle (FCR) and to correlate the distributional patterns in these structures with muscle tendon architecture and muscle function. It was found that the FCR, a wrist flexor, contains 37% slow-twitch fibers and 63% fast-twitch fibers. However, the slow-twitch fibers were concentrated in the deep region located between the tendons of origin and insertion, while the fast-twitch-glycolytic fibers were concentrated more peripherally. Muscle spindles were associated with the slow-twitch region and were never found in the region containing high concentrations of fast-twitch-glycolytic fibers. Fast-twitch-oxidative-glycolytic fibers were uniformly distributed throughout the muscle. It is proposed that the association of muscle spindles with slow-twitch fibers and the differential distribution of muscle fibers into slow-twitch and fast-twitch regions might allow these regions to function independently of one another when called upon to perform complex behavioral tasks.     

Contractile properties, transversal stiffness and cytoskeletal protein content in Mongolian gerbils soleus fibers under long-term hindlimb suspension

Structural and functional changes in Mongolian gerbil soleus fibers were analyzed after a 31-day hindlimb suspension. Contractile properties of muscle fibers were studied by means of tensometry; the transversal stiffness of different parts of the contractile apparatus was measured by atomic force microscopy, resting calcium level was estimated by fluorescence microscopy by using Fluo-4-AM; cytoskeletal protein content was determined by western blotting. It was shown that after gravitational unloading the maximal force of contraction and specific tension of fiber were significantly reduced, as well as calcium sensitivity actually lowered. At the same time, the transversal stiffness of Z-disk in the relaxed and activated state was decreased significantly compared to the control group. Desmin content was at the control level, but alpha-actinin-2, main structural protein of Z-disk, became considerably less after a 31-day hindlimb suspension. Besides, resting calcium level remained at control values during the simulated gravitational unloading. The data suggest that Z-disk destruction, as a result of alpha-actinin-2 content reduction, leads to changes in the lattice spacing and decreases contractile properties.     

Deformation and three-dimensional displacement of fibers in isometrically contracting rat plantaris muscles

In this study, the deformation of different fibers of the rat m. plantaris during "isometric" contractions at different muscle lengths was considered. Because the m. plantaris has an obviously inhomogeneous architecture, its fibers on the medial side of the muscle belly are judged to be shorter than those on the lateral side of it. It was expected that longitudinal deformation of different fibers would vary accordingly. A 3D video analysis of contracting muscle showed that longitudinal strain of fibers as a function of muscle length does not differ between fibers on different sides of the muscle. Apart from longitudinal shortening, the fibers were also displaced laterally during a contraction. The fibers displaced during a contraction in a direction perpendicular to their longitudinal axis. The displacement of the fibers occurred asymmetrically, resulting in a helical deformation of the whole muscle. It is concluded that the asymmetric displacement and the helical deformation must result from transversal forces between the fibers. It is hypothesized that these transversal forces cancel out differences in longitudinal strains that might exist between fibers.     

Possible Role of Non-Muscle Alpha-Actinins in Muscle Cell Mechanosensitivity

The main hypothesis suggested that changes in the external mechanical load would lead to different deformations of the submembranous cytoskeleton and, as a result, dissociation of different proteins from its structure (induced by increased/decreased mechanical stress). The study subjects were fibers of the soleus muscle and cardiomyocytes of Wistar rats. Changes in external mechanical conditions were reconstructed by means of antiorthostatic suspension of the animals by their tails for 6, 12, 18, 24 and 72 hours. Transversal stiffness was measured by atomic force microscopy imaging; beta-, gamma-actin, alpha-actinin 1 and alpha-actinin 4 levels in membranous and cytoplasmic fractions were quantified by Western blot analysis; expression rates of the corresponding genes were studied using RT-PCR. Results: In 6 hours, alpha-actinin 1 and alpha-actinin 4 levels decreased in the membranous fraction of proteins of cardiomyocytes and soleus muscle fibers, respectively, but increased in the cytoplasmic fraction of the abovementioned cells. After 6–12 hours of suspension, the expression rates of beta-, gamma-actin, alpha-actinin 1 and alpha-actinin 4 were elevated in the soleus muscle fibers, but the alpha-actinin 1 expression rate returned to the reference level in 72 hours. After 18–24 hours, the expression rates of beta-actin and alpha-actinin 4 increased in cardiomyocytes, while the alpha-actinin 1 expression rate decreased in soleus muscle fibers. After 12 hours, the beta- and gamma-actin content dropped in the membranous fraction and increased in the cytoplasmic protein fractions from both cardiomyocytes and soleus muscle fibers. The stiffness of both cell types decreased after the same period of time. Further, during the unloading period the concentration of nonmuscle actin and different isoforms of alpha-actinins increased in the membranous fraction from cardiomyocytes. At the same time, the concentration of the abovementioned proteins decreased in the soleus muscle fibers.     

PDGF-receptor concentration is elevated in regenerative muscle fibers in dystrophin-deficient muscle.

Dystrophin-deficient muscle undergoes sudden, postnatal onset of muscle necrosis that is either progressive, as in Duchenne muscular dystrophy, or successfully arrested and followed by regeneration, as in most muscles of mdx mice. The mechanisms regulating regeneration in mdx muscle are unknown, although the possibility that there is renewed expression of genes regulating embryonic muscle cell proliferation and differentiation may provide testable hypotheses. Here, we examine the possibility that necrotic and regenerating mdx muscles exhibit renewed or increased expression of PDGF-receptors. PDGF-binding to receptors on muscle has been shown previously to be associated with myogenic cell proliferation and delay of muscle differentiation. We find that PDGF-receptors are present in 4-week-old mdx mice in muscles that undergo brief, reversible necrosis (hindlimb muscles) or progressive necrosis (diaphragm), as well as in 4-week-old control mouse muscles. Immunoblots indicate that the concentrations of PDGF-receptors in 4-week-old dystrophic (necrotic) and control muscles are similar. Prenecrotic, dystrophic fibers and control fibers possess some cell surface labeling of fibers treated with anti-PDGF-receptor and viewed by indirect immunofluorescence. Necrotic fibers in dystrophic muscle show cytoplasmic labeling for PDGF-receptors and labeling of perinuclear regions at the muscle cell surface. Adult dystrophic muscle displays higher concentrations of PDGF-receptor in both regenerated muscle (hindlimb) and progressively necrotic muscle (diaphragm) than found in controls. Anti-PDGF-receptor labeling of regenerated, dystrophic muscle is observed primarily in granules surrounding central nuclei or surrounding nuclei located at the surface of regenerated fibers. No labeling of perinuclear regions of control muscle or prenecrotic fibers was observed. Myonuclei fractionated from adult mdx hindlimb muscles contained no PDGF-receptor, indicating that PDGF-receptor-positive structures are not tightly associated with nuclei or within nuclei. L6 myoblasts show PDGF-receptor distributed diffusely on the cell surface. Stimulation of L6 myoblasts with 10 ng/ml of PDGF-BB causes receptor internalization and concentration in granules at perinuclear regions. Thus, PDGF stimulation of myoblasts causes a redistribution of PDGF-receptors to resemble receptor localization observed during muscle regeneration. These findings implicate PDGF-mediated mechanisms in regeneration of dystrophic muscle.     

Carbonic anhydrase III in skeletal muscle fibers: an immunocytochemical and biochemical study

The objectives of the present study were to determine if carbonic anhydrase III (CA III) demonstrated a specific association for any particular organelle or structure of the skeletal muscle cell and to quantify the activity and content of this enzyme in different types of skeletal muscle fibers. Ultrastructural localization of CA III in the soleus (SOL), deep vastus lateralis (DVL), and superficial vastus lateralis (SVL), composed of predominantly type I, IIa, and IIb fibers, respectively, was performed using a high-resolution immunocytochemical technique and antibody specific for CA III on ultra-thin sections of skeletal muscle embedded in the water-soluble medium polyvinyl alcohol (PVA). The results indicated a uniform distribution of CA III within the sarcomere. Mitochondria, nuclei, triads, Z-, and M-bands were not specifically labeled. Immunoblotting of washed myofibril preparations did not show any detectable CA III associated with this structure. In addition to quantification of the immunogold labeling, CA III activity and content were assayed in the post-mitochondrial supernatant of the three muscles. In the SOL, these values were found to be 3.6-7.6 times higher than in the DVL. The SVL showed a labeling intensity slightly higher than background level, while the enzyme activity and content were indistinguishable from background levels. We therefore conclude that CA III is randomly distributed in the cytoplasm of the three muscle fiber types and that the relative CA III content and activity in the three muscles studied is SOL greater than DVL greater than SVL approximately equal to 0.     

Scanning electron microscopy of nerve-muscle contacts in embryonic cell culture.

Scanning electron microscopy (SEM) of cell cultures of dissociated nerve and muscle from chick embryos has shown that developing muscle fibers can be contacted at many sites by one or more than one neuron, and that a single nerve can send branches to several myofibers. At these contact regions of nerve with muscle, the neurons send out terminal or lateral sprouts with fine tips which initially lack terminal swellings, but later acquire small “bouton”-like structures in contact with the sarcolemma, which resemble embryonic synapses. At these points, the sarcolemma does not appear to differ in ultrastructure from other surface regions of the myofiber. Transmission electron microscopy (TEM) has revealed the presence of both electron lucent and dense-cored vesicles at some nerve terminals. However, fluorescence histochemistry (Falck-Hillarp technique) failed to detect the presence of catecholamines in these cultures. The SEM pictures at substantially higher resolutions than the light microscope, and the enhanced three dimensional perspective of this technique, provide additional information about the developmental morphology of the nerve-muscle cell culture system. The results are correlated with previous findings by light microscopy, TEM and electrophysiology, and discussed in relationship to proposed innervation processes of skeletal muscle fibers in vivo.     

Vinculin in subsarcolemmal densities in chicken skeletal muscle: localization and relationship to intracellular and extracellular structures

Using immunocytochemical methods we have studied the distribution of vinculin in the anterior and posterior latissimus dorsi skeletal (ALD and PLD, respectively) muscles of the adult chicken. The ALD muscle is made up of both tonic (85%) and twitch (15%) myofibers, and the PLD muscle is made up entirely of twitch myofibers. In indirect immunofluorescence, antivinculin antibodies stained specific regions adjacent to the sarcolemma of the ALD and PLD muscles. In the central and myotendinous regions of the ALD, staining of the tonic fibers was intense all around the fiber periphery. Staining of the twitch fibers of both ALD and PLD muscles was intense only at neuromuscular junctions and myotendinous regions. Electron microscopy revealed subsarcolemmal, electron-dense plaques associated with the membrane only in those regions where vinculin was localized by immunofluorescence. Using antivinculin antibody and protein A conjugated to colloidal gold, we found that the electron-dense subsarcolemmal densities in the tonic fibers of the ALD contain vinculin; no other structures were labeled. The basal lamina overlying the densities appeared to be connected to the sarcolemma by fine, filamentous structures, more enriched at these sites than elsewhere along the muscle fiber. Increased amounts of endomysial connective tissue were often found just outside the basal lamina near the densities. In tonic ALD muscle fibers, the subsarcolemmal densities were present preferentially over the I-bands. In partially contracted ALD muscle, subsarcolemmal densities adjacent to the Z-disk appeared to be connected to that structure by short filaments. We propose that in the ALD muscle, through their association with the extracellular matrix, the densities stabilize the muscle membrane and perhaps assist in force transmission.     

Theoretical Predictions of the Effects of Force Transmission by Desmin on Intersarcomere Dynamics

Desmin is an intermediate filament protein in skeletal muscle that forms a meshlike network around Z-disks. A model of a muscle fiber was developed to investigate the mechanical role of desmin. A two-dimensional mesh of viscoelastic sarcomere elements was connected laterally by elastic elements representing desmin. The equations of motion for each sarcomere boundary were evaluated at quasiequilibrium to determine sarcomere stresses and strains. Simulations of passive stretch and fixed-end contractions yielded values for sarcomere misalignment and stress in wild-type and desmin null fibers. Passive sarcomere misalignment increased nonlinearly with fiber strain in both wild-type and desmin null simulations and was significantly larger without desmin. During fixed-end contraction, desmin null simulations also demonstrated greater sarcomere misalignment and reduced stress production compared with wild-type. In simulations with only a fraction of wild-type desmin present, fixed-end stress increased as a function of desmin concentration and this relationship was influenced by the cellular location of the desmin filaments. This model suggests that desmin stabilizes Z-disks and enables greater stress production by providing a mechanical tether between adjacent myofibrils and to the extracellular matrix and that the significance of the tether is a function of its location within the cell.