Localization of talin in skeletal and cardiac muscles

Antibodies to talin and vinculin were used for localization of these proteins in skeletal and cardiac muscles by the indirect immunofluorescence method. We have found that talin is localized in intercalated discs of cardiac muscle and in costameres of skeletal and cardiac muscles. It is suggested that in striated muscles talin and vinculin play an important role in interactions between actin filaments and membranes.     

Ultrastructural features of cardiac muscle cells in a tarantula spider

The ultrastructural features of cardiac muscle cells and their innervation were examined in the tarantula spider Eurypelma marxi Simon. The cells are transversely striated and have an A band length of about three microns. H zones are indistinct and M lines are absent. Thick and thin myofilament diameters are approximately 200 and 70 Å respectively. Eight to 12 thin filaments usually surround each thick one. Accumulations of thick and thin myofilaments occur perpendicular to the bulk of the myofilaments in some cells. The Z line is discontinuous and thick filaments may pass through the spaces in the Z line. Extensive systems of sarcoplasmic reticulum and transverse tubules are present; these form numerous dyadic junctions in both A and I band regions. Sarcolemmal invaginations form Z line tubules; lateral extensions of the plasma membrane portion of these invaginations form dyads. Nerve branches of the cardiac ganglion make multiple neuromuscular synapses with at least some of the cardiac muscle cells. Both large granular and small agranular vesicles are present in the presynaptic terminals. Intercalated discs similar to those present in other arthropod hearts occur between the ends of adjacent cardiac muscle cells.     

Immunoelectron microscopic studies of desmin (skeletin) localization and intermediate filament organization in chicken cardiac muscle

We studied the localization of desmin (skeletin), the major protein subunit of muscle-type intermediate filaments, in adult chicken cardiac muscle by high resolution immunoelectron microscopic labeling of ultrathin frozen sections of the intact fixed tissues. We carried out single labeling for desmin and double labeling for both desmin and either vinculin or alpha-actinin. In areas removed from the intercalated disk membranes, we observed desmin labeling between adjacent Z-bands in every interfibrillar space. Where these spaces were wide and contained mitochondria, convoluted strands of desmin labeling bridged between the periphery of neighboring Z-bands and the mitochondria. The intermediate filaments appeared to be organized in a more three-dimensional manner within the interfibrillar spaces of cardiac as compared to skeletal muscle. Near the intercalated disks, desmin labeling was intense within the interfibrillar spaces, but was completely segregated from the microfilament attachment sites (fascia adherens) where vinculin and alpha-actinin were localized. Desmin therefore appears to play no role in the attachment of microfilaments to the intercalated disk membrane. We discuss the role of intermediate filaments in the organization of cardiac and skeletal striated muscle in the light of these and other results.     

Ultrastructural localization of actin in muscle, epithelial and secretory cells by applying the protein A-gold immunocytochemical technique

Actin-immunoreactive sites have been localized at the electron microscope level by the protein A-gold technique in striated and smooth muscle cells as well as in epithelial and secretory cells. The combination of the highly sensitive protein A-gold technique with the good ultrastructural preservation and retention of antigenicity obtained using low-temperature embedding conditions has allowed a very precise identification of the labelled structures with high resolution. In striated muscle cells the labelling was obtained over the myofilaments and the Z-band, mainly at its periphery. Labelling was also observed at the edge of the intercalated discs of the cardiac muscle cells. In smooth muscle cells the labelling was present over the myofilaments; the dense plaques associated with the plasma membrane were labelled at their periphery where actin filaments have been reported to anchor. In epithelial cells of the duodenum and the renal convoluted proximal tubule, the labelling occurred over the filamentous core of the microvilli and over the cell web. Gold particles were often present over, or closely associated with, the cell membrane at the tip of the microvilli or of invaginations and vesicular structures. At the level of the junctional complexes the gold particles were aligned at the edge of the dense zones. In pancreatic endocrine and exocrine secretory cells, actin-immunoreactive sites were revealed over the Golgi apparatus, mainly at the level of the inner cisternae in the maturing face over or closely associated with the membranes of the condensing vacuoles and secretory granules, and also over the plasma membrane. Microvilli and cell web were also labelled. Finally, in fibroblasts, gold particles were associated with the membrane of vesicular structures. The consistent finding of actin-immunoreactive sites closely associated with membranes of secretory granules and vesicular structures brings support to the proposal that contractile proteins might play an important role in transcellular transport and protein secretion.     

Absence of utrophin in intercalated discs of human cardiac muscle

Utrophin is the autosomal homologue of dystrophin. In normal skeletal muscle it is localised only to neuromuscular and myotendinous junctions, nerves and vascular tissue. In Xp21 muscular dystrophies, utrophin is also detected on the sarcolemma of skeletal and cardiac muscle, while dystrophin is absent or reduced. In normal cardiac muscle, some reports have demonstrated utrophin at intercalated discs and T-tubules. We have re-examined the distribution of utrophin in normal human cardiac muscle using a panel of eight monoclonal antibodies against different epitopes in N- and C-terminal domains. In contrast to previous studies, utrophin was not detected at the intercalated discs or T-tubules, although labelling of blood vessels was strong. We conclude that the primary location of utrophin in normal heart is in the vascular system. In addition, our results show that the utrophin on cardiac blood vessels is full length, similar to that of skeletal muscle blood vessels.     

Cardiac Fine Structure in Selected Arthropods and Molluscs

The ultrastructure of the single-chambered hearts of selectedarthropods is compared with that of the multi-chambered heartsof three molluscs. I used the following four systems to makethe comparison: (1) contractile apparatus, (2) sarcoplasmicreticulum and surface invaginations, (3) cell to cell junctions,and (4) nerves. The contractile apparatus is composed of thinand thick filaments. While the thin filaments have the samediameter, the diameter of the thick filaments differs from oneheart to another. Evidence is presented to indicate that thisis due to varying amounts of paramyosin in the thick filaments.The arthropod cardiac cells have an extensive system of sarcoplasmicreticulum, the terminal vesicles of which are coupled to theplasmalemma and to the invaginations of the plasmalemma, theT-system. The molluscan cardiac cells lack a typical T-system,which is presumably due to their small cell size (about 10 µm).They possess, however, an elaborate system of sarcoplasmic reticulumwhich extends from just under the plasmalemma to the middleof the cell. In addition to elaborate sarcoplasmic reticulum,the heart of the whelk (Busycon canaliculatum) possess manysmall invaginations of the plasmalemma, called sarcolemmic tubules.These invaginations of the cell surface are not found in thehearts of the few bivalves examined. All arthropod and molluscanhearts have intercalated discs which can be seen in the lightmicroscope. Two types of junctions can be distinguished in theelectron microscope. The mechanical junction is at the levelof the terminal sarcomere where the thin filaments are embeddedin the cell wall and dense granular material appears to causethe two adjacent cells to adhere to each other. The electricaljunction is found along the lateral borders of cells of boththe molluscan and arthropod hearts. Finally, while nerves appearto be absent in the myogenic moth heart, they are abundant inthe myogenic cockroach heart and in the neurogenic lobster heart.Furthermore, two types of nerves appear very prominently inthe myogenic molluscan hearts.     

Ultrastructural and cytochemical localization of calcium in a rat cardiac muscle in normal and experimental conditions.

The experiment carried out by us on the, stimulated by adrenaline, cardiac muscle allowed the activation of calcium localized mainly in the mitochondria, MA and FA of the intercalated discs and SR to be translocated in the direction of the sarcomere myofilaments and this especially to the thin actin filaments. The authors' experimental proves, that during the contraction--relaxation function of the cardiac muscle there exists a circulation rythm or a oscillatory functional flow of calcium ions between the mitochondria, intercalated discs and SR and the contractile fibrillae of the sarcomere.     

Characterization of muscle filamin isoforms suggests a possible role of gamma-filamin/ABP-L in sarcomeric Z-disc formation

Filamin, also called actin binding protein-280, is a dimeric protein that cross-links actin filaments in the cortical cytoplasm. In addition to this ubiquitously expressed isoform (FLN1), a second isoform (ABP-L/gamma-filamin) was recently identified that is highly expressed in mammalian striated muscles. A monoclonal antibody was developed, that enabled us to identify filamin as a Z-disc protein in mammalian striated muscles by immunocytochemistry and immunoelectron microscopy. In addition, filamin was identified as a component of intercalated discs in mammalian cardiac muscle and of myotendinous junctions in skeletal muscle. Northern and Western blots showed that both, ABP-L/gamma-filamin mRNA and protein, are absent from proliferating cultured human skeletal muscle cells. This muscle specific filamin isoform is, however, up-regulated immediately after the induction of differentiation. In cultured myotubes, ABP-L/gamma-filamin localises in Z-discs already at the first stages of Z-disc formation, suggesting that ABP-L/gamma-filamin might play a role in Z-disc assembly.     

Vinculin is a component of an extensive network of myofibril-sarcolemma attachment regions in cardiac muscle fibers

Immunofluorescent staining of bovine and avian cardiac tissue with affinity-purified antibody to chicken gizzard vinculin reveals two new sites of vinculin reactivity. First, vinculin is organized at the sarcolemma in a striking array of rib-like bands, or costameres. The costameres encircle the cardiac muscle cell perpendicular to the long axis of the fiber and overlie the I bands of the immediately subjacent sarcomeres. The second new site of vinculin reactivity is found in bovine cardiocytes at tubular invaginations of the plasma membrane. The frequency and location of these invaginations correspond to the known frequency and distribution of the transverse tubular system in bovine atrial, ventricular, and Purkinje fibers. We do not detect tubular invaginations that stain with antivinculin in avian cardiocytes and, in fact, a transverse tubular system has not been found in avian cardiac fibers. Apparent lateral Z-line attachments to the sarcolemma and its invaginations have been observed in cardiac muscle by electron microscopy in the same regions where we find vinculin. On the basis of these previous ultrastructural findings and our published evidence for a physical connection between costameres and the underlying myofibrils in skeletal muscle, we interpret the immunofluorescence data of this study to mean that, in cardiac muscle, vinculin is a component of an extensive system of lateral attachment of myofibrils to the plasma membrane and its invaginations.     

Observations on the Fine Structure of the Turtle Atrium

The general fine structure of the atrial musculature of the turtle heart is described, including; the nature of the sarcolemma; the cross-banded structure of the myofibrils; the character of the sarcoplasm, and the form and disposition of its organelles. An abundant granular component of the sarcoplasm in this species is tentatively identified as a particulate form of glycogen. The myocardium is composed of individual cells joined end to end at primitive intercalated discs, and side to side at sites of cohesion that resemble the desmosomes of epithelia. Transitional forms are found between desmosomes and intercalated discs. Both consist of a thickened area of the cell membrane with an accumulation of dense material in the subjacent cytoplasm. This dense amorphous component is often continuous with the Z substance of the myofibrils and may be of the same composition. The observations reported reemphasize the basic similarity between desmosomes and terminal bars of epithelia and intercalated discs of cardiac muscle. Numerous unmyelinated nerves are found beneath the endocardium. Some of these occupy recesses in the surface of Schwann cells; others are naked axons. No specialized nerve endings are found. Axons passing near the sarcolemma contain synaptic vesicles, and it is believed that this degree of proximity is sufficient to constitute a functioning myoneural junction.     

The subcellular distribution of dystrophin in mouse skeletal, cardiac, and smooth muscle

We use a highly specific and sensitive antibody to further characterize the distribution of dystrophin in skeletal, cardiac, and smooth muscle. No evidence for localization other than at the cell surface is apparent in skeletal muscle and no 427-kD dystrophin labeling was detected in sciatic nerve. An elevated concentration of dystrophin appears at the myotendinous junction and the neuromuscular junction, labeling in the latter being more intense specifically in the troughs of the synaptic folds. In cardiac muscle the distribution of dystrophin is limited to the surface plasma membrane but is notably absent from the membrane that overlays adherens junctions of the intercalated disks. In smooth muscle, the plasma membrane labeling is considerably less abundant than in cardiac or skeletal muscle and is found in areas of membrane underlain by membranous vesicles. As in cardiac muscle, smooth muscle dystrophin seems to be excluded from membrane above densities that mark adherens junctions. Dystrophin appears as a doublet on Western blots of skeletal and cardiac muscle, and as a single band of lower abundance in smooth muscle that corresponds most closely in molecular weight to the upper band of the striated muscle doublet. The lower band of the doublet in striated muscle appears to lack a portion of the carboxyl terminus and may represent a dystrophin isoform. Isoform differences and the presence of dystrophin on different specialized membrane surfaces imply multiple functional roles for the dystrophin protein.     

Immunoelectron microscopic localization of alpha-actinin and actin in embryonic hamster heart cells

Myofibrillogenesis in developing cardiac cells of the Syrian hamster from early embryonic stages through newborn was studied by electron microscopy, immunofluorescence microscopy and immunoelectron microscopy. alpha-Actinin and actin were localized at light and electron microscopic levels in embryonic heart cells which had been fixed in a periodate-lysine-paraformaldehyde or a glutaraldehyde-formaldehyde mixture, and embedded in Lowicryl K4M. Indirect staining methods were used for immunofluorescence staining of thick sections and immunoferritin staining of thin sections. The earliest evidence of myofibrillogenesis in embryonic myocardial cells was the presence of many randomly arranged thin (6 nm) filaments and a few scattered thick filaments (15 nm) near the plasma membrane. alpha-Actinin was detected in a semi-continuous, diffuse layer in some portions of the cell just beneath the plasma membrane in association with the filamentous collections. Later in development, alpha-actinin coalesced into Z-plaques at the membrane as the filaments arranged into parallel arrays. Actin was localized in the thin filaments as expected. In later stages of development, alpha-actinin was observed at the Z-lines and intercalated discs of the mature myofibrils while actin was localized at both the I-band and Z-line. Our results suggest that myofibrillogenesis is initiated at the plasma membrane and that Z-plaques are precursors of myofibrillar Z-bands and may serve as organizing centers for myofibrillogenesis in developing cardiomyocytes.     

The localization of VAMP5 in skeletal and cardiac muscle

Vesicle-associated membrane protein 5 (VAMP5) is a member of the SNARE protein family, which is generally thought to regulate the docking and fusion of vesicles with their target membranes. This study investigated the expression and localization of the VAMP5 protein. Immunoblotting analyses detected the VAMP5 protein in skeletal muscle, heart, spleen, lung, liver, and kidney tissue, but not in brain or small intestine tissue. Through the immunofluorescence microscopy of skeletal muscle, we found that the expression level of VAMP5 varies among fibers. Most of the fibers with high expression levels of VAMP5 were categorized as type IIa fibers on the basis of their myosin heavy chain subtypes. In addition, the expression patterns of VAMP5 and glucose transporter 4 (GLUT4) were similar. In cardiac muscle, we determined that VAMP5 was localized to the vicinity of intercalated discs. These results suggest that VAMP5 plays local roles in membrane trafficking in skeletal and cardiac muscle.     

Ultrastructure of the vocal muscle and its morphological differences from the myocardium]

A comparative ultrastructural investigation of the M. vocalis in mammals has been carried out. Morphological differences between the vocal muscle and cardiac tissue are reported; a distinct classification of the M. vocalis according to a typisation of skeletal muscle fibers is presented. In all species investigated (man, dog, cat, guinea-pig and rat) the general ultrastructure of the sarcomeres as well as their mitochondrial content and the innervation pattern allow to classify the M. vocalis as to belong to the "fast twitch (white) skeletal muscle fibers. A single innervation was found with large motor endplates containing numerous synaptic infoldings. Structural specializations known to be characteristic for cardiac tissue, e.g. intercalated discs, T-tubules at the level of the Z-band and nuclei in a midst postion of the muscle cell could not be observed. The m. vocalis, therefore, cannot be considered to have histologically any relationship with cardiac tissue. The vocal muscle is described as a special type of skeletal muscle very similar to the extraocular muscles. The electron microscopic findings are discussed with respect to current theories of phonation. The myoleastic theory of phonation can be favoured according to our ultrastructural results.     

Intermediate-sized filaments of the prekeratin type in myoepithelial cells

Myoepithelial cells from mammary glands, the modified sweat glands of bovine muzzle, and salivary glands have been studied by electron microscopy and by immunofluorescence microscopy in frozen sections in an attempt to further characterize the type of intermediate-sized filaments present in these cells. Electron microscopy has shown that all myoepithelial cells contain extensive meshworks of intermediate-sized (7--11-nm) filaments, many of which are anchored at typical desmosomes or hemidesmosomes. The intermediate-sized filaments are also intimately associated with masses of contractile elements, identified as bundles of typical 5--6-nm microfilaments and with characteristically spaced dense bodies. This organization resembles that described for various smooth muscle cells. In immunofluorescence microscopy, using antibodies specific for the various classes of intermediate-sized filaments, the myoepithelial cells are strongly decorated by antibodies to prekeratin. They are not specifically stained by antibodies to vimentin, which stain mesenchymal cells, nor by antibodies to chick gizzard desmin, which decorate fibrils in smooth muscle Z bands and intercalated disks in skeletal and cardiac muscle of mammals. Myoepithelial cells are also strongly stained by antibodies to actin. The observations show (a) that the epithelial character, as indicated by the presence of intermediate-sized filaments of the prekeratin type, is maintained in the differentiated contractile myoepithelial cell, and (b) that desmin and desmin-containing filaments are not generally associated with musclelike cell specialization for contraction but are specific to myogenic differentiation. The data also suggest that in myoepithelial cells prekeratin filaments are arranged--and might function--in a manner similar to the desmin filaments in smooth muscle cells.     

Desmin filaments are stably associated with the outer nuclear surface in chick myoblasts

Eukaryotic cells have highly organized, interconnected intracellular compartments. The nuclear surface and cytoplasmic cytoskeletal filaments represent compartments involved in such an association. Intermediate filaments are the major cytoskeletal elements in this association. Desmin is a muscle-specific structural protein and one of the earliest known muscle-specific genes to be expressed during cardiac and skeletal muscle development. Desmin filaments have been shown to be associated with the nuclear surface in the myogenic cell line C2C12. Previous studies have revealed that mice lacking desmin develop imperfect muscle, exhibiting the loss of nuclear shape and positioning. In the present work, we have analyzed the association between desmin filaments and the outer nuclear surface in nuclei isolated from pectoral skeletal muscle of chick embryos and in primary chick myogenic cell cultures by using immunofluorescence microscopy, negative staining, immunogold, and transmission electron microscopy. We show that desmin filaments remain firmly attached to the outer nuclear surface after the isolation of nuclei. Furthermore, positive localization of desmin persists after gentle washing of the nuclei with high ionic strength solutions. These data suggest that desmin intermediate filaments are stably and firmly connected to the outer nuclear surface in skeletal muscles cells in vivo and in vitro.     

Truncation of vertebrate striated muscle myosin light chains disturbs calcium-induced structural transitions in synthetic myosin filaments

Electron microscopy and negative staining techniques have been used to show that the proteolytic removal of 13 amino acids from the N-terminus of essential light chain 1 and 19 amino acids from the N-terminus of the regulatory light chain of rabbit skeletal and cardiac muscle myosins destroys Ca(2+)-induced reversible movement of subfragment-2 (S2) with heads (S1) away from the backbone of synthetic myosin filaments observed for control assemblies of the myosin under near physiological conditions. This is the direct demonstration of the contribution of the S2 movement to the Ca(2+)-sensitive structural behavior of rabbit cardiac and skeletal myosin filaments and of the necessity of intact light chains for this movement. In muscle, such a mobility might play an important role in proper functioning of the myosin filaments. The impairment of the Ca(2+)-dependent structural behavior of S2 with S1 on the surface of the synthetic myosin filaments observed by us may be of direct relevance to some cardiomyopathies, which are accompanied by proteolytic breakdown or dissociation of myosin light chains.     

Nonmuscle myosin II localizes to the Z-lines and intercalated discs of cardiac muscle and to the Z-lines of skeletal muscle

To understand the role of nonmuscle myosin II in cardiac and skeletal muscle, we used a number of polyclonal antibodies, three detecting nonmuscle myosin heavy chain II-B (NMHC II-B) and two detecting NMHC II-A, to examine the localization of these two proteins in fresh-frozen, acetone-fixed sections of normal human and mouse hearts and human skeletal muscles. Results were similar in both species and were confirmed by examination of fresh-frozen sections of human hearts subjected to no fixation or to treatment with either 4% p-formaldehyde or 50% glycerol. NMHC II-B was diffusely distributed in the cytoplasm of cardiac myocytes during development, but after birth it was localized to the Z-lines and intercalated discs. Dual labeling showed almost complete colocalization of NMHC II-B with alpha-actinin. Whereas endothelial cells, smooth muscle cells and fibroblasts showed strong immunoreactivity for NMHC II-A and NMHC II-B, cardiac myocytes only showed reactivity for the latter. The Z-lines of human skeletal muscle cells, in contrast to those of cardiac myocytes, gave positive reactions for both NMHC II-A and NMHC II-B. The presence of a motor protein in the Z-lines and intercalated discs raises the possibility that these structures may play a more dynamic role in the contraction/relaxation mechanism of cardiac and skeletal muscle than has been previously suspected.     

Vinculin localization in cardiac muscle

Vinculin isolated from chicken cardiac muscle crossreacts with antibodies against smooth muscle vinculin. Antibodies to vinculin were used for localization of vinculin in cardiac muscle by indirect immunofluorescence method. In cardiac muscle vinculin was localized in intercalated discs and near plasma membrane at the cell periphery between external myofibrils and sarcolemma. It was suggested that vinculin plays an important role in myofibril-sarcolemma interaction in cardiac muscle.     

Order-disorder structural transitions in synthetic filaments of fast and slow skeletal muscle myosins under relaxing and activating conditions

In the previous study (Podlubnaya et al., 1999, J. Struc. Biol. 127, 1-15) Ca2+-induced reversible structural transitions in synthetic filaments of pure fast skeletal and cardiac muscle myosins were observed under rigor conditions (-Ca2+/+Ca2+). In the present work these studies have been extended to new more order-producing conditions (presence of ATP in the absence of Ca2+) aimed at arresting the relaxed structure in synthetic filaments of both fast and slow skeletal muscle myosin. Filaments were formed from column-purified myosins (rabbit fast skeletal muscle and rabbit slow skeletal semimebranosusproprius muscle). In the presence of 0.1 mM free Ca2+, 3 mM Mg2+ and 2 mM ATP (activating conditions) these filaments had a spread structure with a random arrangement of myosin heads and subfragments 2 protruding from the filament backbone. Such a structure is indistinguishable from the filament structures observed previously for fast skeletal, cardiac (see reference cited above) and smooth (Podlubnaya et al., 1999, J. Muscle Res. Cell Motil. 20, 547-554) muscle myosins in the presence of 0.1 mM free Ca2+. In the absence of Ca2+ and in the presence of ATP (relaxing conditions) the filaments of both studied myosins revealed a compact ordered structure. The fast skeletal muscle myosin filaments exhibited an axial periodicity of about 14.5 nm and which was much more pronounced than under rigor conditions in the absence of Ca2+ (see the first reference cited). The slow skeletal muscle myosin filaments differ slightly in their appearance from those of fast muscle as they exhibit mainly an axial repeat of about 43 nm while the 14.5 nm repeat is visible only in some regions. This may be a result of a slightly different structural properties of slow skeletal muscle myosin. We conclude that, like other filaments of vertebrate myosins, slow skeletal muscle myosin filaments also undergo the Ca2+-induced structural order-disorder transitions. It is very likely that all vertebrate muscle myosins possess such a property.