Immunoelectron microscopic localization of albumin in smooth and striated muscle tissues of rat

Summary Localization of serum albumin in the striated and smooth muscles of rat was studied by an improved immunocytochemical method. Diaphragm, ventricular myocardium, and smooth muscle of stomach were examined. In all of these tissues, albumin was found in the interstitial space and small subsarcolemmal caveolae and vesicles. In addition, the transverse tubular system of the striated muscle stained positive for albumin. The subsarcolemmal vesicles containing albumin did not show any evidence of fusion with lysosomes. Furthermore, in smooth muscle, most of these vesicles were open to the extracellular space. These results demonstrate that albumin in smooth and striated muscle is confined to the extracellular space suggesting that substances such as fatty acids which are carried by albumin are split from it and taken up at the level of the plasma membrane.     

The homeobox gene Msx in development and transdifferentiation of jellyfish striated muscle

Bilaterian Msx homeobox genes are generally expressed in areas of cell proliferation and in association with multipotent progenitor cells. Likewise, jellyfish Msx is expressed in progenitor cells of the developing entocodon, a cell layer giving rise to the striated and smooth muscles of the medusa. However, in contrast to the bilaterian homologs, Msx gene expression is maintained at high levels in the differentiated striated muscle of the medusa in vivo and in vitro. This tissue exhibits reprogramming competence. Upon induction, the Msx gene is immediately switched off in the isolated striated muscle undergoing transdifferentiation, to be upregulated again in the emerging smooth muscle cells which, in a stem cell like manner, undergo quantal cell divisions producing two cell types, a proliferating smooth muscle cell and a differentiating nerve cell. This study indicates that the Msx protein may be a key component of the reprogramming machinery responsible for the extraordinary transdifferentation and regeneration potential of striated muscle in the hydrozoan jellyfish.     

Immunocytochemical electron microscopic study and Western blot analysis of paramyosin in different invertebrate muscle cell types of the fruit flyDrosophila melanogaster, the earthwormEisenia foetida, and the snailHelix aspersa

Summary The presence and distribution pattern of paramyosin have been examined in different invertebrate muscle cell types by means of Western blot analysis and electron microscopy immunogold labelling. the muscles studied were: transversely striated muscle with continuous Z lines (flight muscle fromDrosophila melanogaster), transversely striated muscle with discontinuous Z lines (heart muscle from the snailHelix aspersa), obliquely striated body wall muscle from the earthwormEisenia foetida, and smooth muscles (retractor muscle from the snail and pseudoheart outer muscular layer from the earthworm). Paramyosin-like immunoreactivity was localized in thick filaments of all muscles studied. Immunogold particle density was similar along the whole thick filament length in insect flight muscle but it predominated in filament tips of fusiform thick filaments in both snail heart and earthworm body wall musculature when these filaments were observed in longitudinal sections. In obliquely sectioned thick filaments, immunolabelling was more abundant at the sites where filaments disappeared from the section. These results agree with the notion that paramyosin extended along the whole filament length, but that it can only be immunolabelled when it is not covered by myosin. In all muscles examined, immunolabelling density was lower in cross-sectioned myofilaments than in longitudinally sectioned myofilaments. This suggests that paramyosin does not form a continuous filament. The results of a semiquantitative analysis of paramyosin-like immunoreactivity indicated that it was more abundant in striated than in smooth muscles, and that, within striated muscles, transversely striated muscles contain more paramyosin than obliquely striated muscles.     

Transdifferentiation and regeneration in vitro

Striated muscle tissue and endoderm can be isolated from the anthomedusa Podocoryne carnea. The isolates are uncontaminated by other cell types and can be cultivated in artificial seawater for months without undergoing autonomous regeneration. However, if the endoderm is combined with collagenase-treated striated muscle, a regeneration process is initiated which leads to the formation of the sexual and feeding organ (manubrium) of the medusa. The original endoderm and striated muscle are replaced in the regenerate by at least seven new cell types, including gametes. Labeling experiments with [³H]thymidine and experiments in which mitosis is inhibited in either the striated muscle or the endoderm with mitomycin C demonstrate that the striated muscle is able to transdifferentiate into all the cell types found in the regenerate. With the possible exception of ectodermal smooth muscle this statement is also valid for the endoderm.     

Smooth muscle alpha-actinin interaction with smitin

Actin-myosin II filament-based contractile structures in striated muscle, smooth muscle, and nonmuscle cells also contain the actin filament-crosslinking protein alpha-actinin. In striated muscle sarcomeres, interactions between the myosin-binding protein titin and alpha-actinin in the Z-line provide an important structural linkage. We previously discovered a titin-like protein, smitin, associated with the contractile apparatus of smooth muscle cells. Purified native smooth muscle alpha-actinin binds with nanomolar affinity to smitin in smitin-myosin coassemblies in vitro. Smooth muscle alpha-actinin also interacts with striated muscle titin. In contrast to striated muscle alpha-actinin interaction with titin and smitin, which is significantly enhanced by PIP2, smooth muscle alpha-actinin interacts with smitin and titin equally well in the presence and absence of PIP2. Using expressed regions of smooth muscle alpha-actinin, we have demonstrated smitin-binding sites in the smooth muscle alpha-actinin R2-R3 spectrin-like repeat rod domain and a C-terminal domain formed by cryptic EF-hand structures. These smitin-binding sites are highly homologous to the titin-binding sites of striated muscle alpha-actinin. Our results suggest that direct interaction between alpha-actinin and titin or titin-like proteins is a common feature of actin-myosin II contractile structures in striated muscle and smooth muscle cells and that the molecular bases for alpha-actinin interaction with these proteins are similar, although regulation of these interactions may differ according to tissue.     

Role of myosin light chain kinase in muscle contraction

In resting striated muscles of the rabbit muscle in vivo, the phosphorylatable light chain is partially phosphorylated. Tetanic stimulation increased the level of phosphorylation more rapidly in fast twitch than in slow twitch muscle. In both types of muscle the rate of dephosphorylation was relatively slow. In rabbit fast twitch muscles, phosphorylation levels persisted significantly above the resting value for some time after posttetanic potentiation had disappeared. The role of myosin light chain kinase in modulating contractile response in striated muscle is uncertain. In vertebrate smooth muscle the role of myosin phosphorylation appears to be different from that in striated muscle despite the general similarity of the actomyosin system in both tissues. Although phosphorylation in vitro increases the Mg2+ -ATPase of actomyosin, a number of features imply that a somewhat complex relationship exists between the level of phosphorylation and the actin activation of the Mg2+ -ATPase in vertebrate smooth muscle. Contrary to many earlier reports, preparations of smooth muscle actomyosin can be obtained with Mg2+ -ATPase activities comparable to those of actomyosin from skeletal muscle. Preliminary evidence is presented that suggests that phosphorylation changes the Ca2+ sensitivity of the Mg2+ -ATPase of smooth muscle actomyosin.     

Ultrastructural basis of airway smooth muscle contraction

The sliding filament theory of contraction that was developed for striated muscle is generally believed to be also applicable to smooth muscle. However, the well-organized myofilament lattice (i.e., the sarcomeric structure) found in striated muscle has never been clearly delineated in smooth muscle. There is evidence that the myofilament lattice in some smooth muscles, such as airway smooth muscle, is malleable; it can be reshaped to fit a large range of cell dimensions while the maximal overlap between the contractile filaments is maintained. In this review, some early models of the structurally static contractile apparatus of smooth muscle are described. The focus of the review, however, is on the recent findings supporting a model of structurally dynamic contractile apparatus and cytoskeleton for airway smooth muscle. A list of unanswered questions regarding smooth muscle ultrastructure is also proposed in this review, in the hope that it will provide some guidance for future research.     

Ultrastructure of striated muscle fibers in the middle third of the human esophagus

Striated muscle fibers and their spatial relationship to smooth muscle cells have been studied in the middle third of human esophagus. Biopsies were obtained from 3 patients during surgery. In both the circular and longitudinal layers, the muscle coat of this transition zone was composed of fascicles of uniform dimension (100-200 microns of diameter); some of these bundles were made up of striated muscle fibers, others were pure bundles of smooth muscle cells and some were of the mixed type. Striated muscle fibers represented three different types, which were considered as intermediate, with certain structural features characteristic of the fast fiber type. Of these, the most frequently-found fibers were most similar to the fast fiber type. Satellite cells were numerous; in mixed fascicles they were gradually replaced by smooth muscle cells. The gap between striated muscle fiber and smooth muscle cells was more than 200 nm wide. It contained the respective basal laminae and a delicate layer of amorphous connective tissue. No specialized junctions were formed between consecutive striated muscle fibers, or between striated muscle fibers and smooth muscle cells. Interstitial cells of Cajal were never situated as close to striated muscle fibers as to smooth muscle cells.     

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.     

Cellular distribution of smooth muscle actins during mammalian embryogenesis: expression of the alpha-vascular but not the gamma- enteric isoform in differentiating striated myocytes

The cellular distribution of the alpha-vascular and gamma-enteric smooth muscle actin isoforms was analyzed in rat embryos from gestational day (gd) 8 through the first neonatal week by in situ antigen localization using isoactin specific monoclonal antibodies. The alpha-vascular actin isoform was first detected on gd 10 in discrete cells lining the embryonic vasculature. By gd 14, this isoform was also present in the inner layers of mesenchymal cells condensing around the developing airways and gut. The gamma-enteric actin, however, was not detected until gd 15 when cells surrounding the developing aorta, airways, and gut labeled with the gamma-enteric-specific probe. There was continued expression of these two actin isoforms in regions of developing smooth muscle through the remainder of gestation and first neonatal week at which time their distribution coincided with that found in the adult. In addition to developing smooth muscle, the alpha- vascular actin isoform was expressed in differentiating striated muscle cells. On gd 10, there was intense labeling with the alpha-vascular specific probe in developing myocardiocytes and, within 24 h, in somitic myotomal cells. Although significant levels of this smooth muscle actin were present in striated myocytes through gd 17, by the end of the first postnatal week, alpha-vascular actin was no longer detectable in either cardiac or skeletal muscle. Thus, the normal developmental sequence of striated muscle cells includes the transient expression of the alpha-vascular smooth muscle actin isoform. In contrast, the gamma-enteric smooth muscle actin was not detected at any time in embryonic striated muscle. The differential timing of appearance and distribution of these two smooth muscle isoforms indicates that their expression is independently regulated during development.     

The transformational potential of striated muscle in hydromedusae.

Isolated striated muscle tissue of the Anthomedusa Podocoryne carnea participates in the regeneration of a functional manubrium (the feeding organ of medusae) when it is combined homoclonally with endodermal cells of the medusa umbrella. The morphogenetic potential of striated muscle cells in this regeneration process was evaluated by combining nuclear labeled striated muscle cells with some unlabeled endoderm cells. Histological and autoradiographical results demonstrate that transformation of striated muscle cells into smooth muscle cells of the ectoderm and also into endoderm cells must have occurred in the regenerate. The potential for cell transformation of isolated striated muscle cells of Podocoryne carnea is discussed and it is postulated that under appropriate conditions all cell types necessary for the regeneration of a manubrium can be formed from striated muscle cells.     

Structural proteins in the myofilaments and regulation of contraction in vertebrate smooth muscle.

The contractile systems of vertebrate smooth and striated muscles are compared. Smooth muscles contain relatively large amounts of actin and tropomyosin organized into thin filaments, and smaller amounts of myosin in the form of thick filaments. The protein contents are consistent with observed thin:thick filament ratios of about 15-18:1 in smooth compared to 2:1 in striated muscle. The basic characteristics of both types of contractile proteins are similar; but there are a variety of quantitative differences in protein structures, enzymatic activities and filament stabilities. Biochemical and X-ray diffraction data generally support recent ultrastructural evidence concerning the organization of the myofilaments in smooth muscle, although a basic contractile unit comparable to the sarcomere in striated muscle has not been discerned. Myofilament interactions and contraction in smooth muscle are controlled by changes in the Ca2+ concentration. Recent evidence suggests the Ca2+-binding regulatory site is associated with the myosin in vertebrate smooth muscle (as in a variety of invertebrate muscles), rather than with troponin which is the regulatory protein associated with the thin filament in vertebrate striated muscle.     

Structure and flexibility of the tropomyosin overlap junction

To be effective as a gatekeeper regulating the access of binding proteins to the actin filament, adjacent tropomyosin molecules associate head-to-tail to form a continuous super-helical cable running along the filament surface. Chimeric head-to-tail structures have been solved by NMR and X-ray crystallography for N- and C-terminal segments of smooth and striated muscle tropomyosin spliced onto non-native coiled-coil forming peptides. The resulting 4-helix complexes have a tight coiled-coil N-terminus inserted into a separated pair of C-terminal helices, with some helical unfolding of the terminal chains in the striated muscle peptides. These overlap complexes are distinctly curved, much more so than elsewhere along the superhelical tropomyosin cable. To verify whether the non-native protein adducts (needed to stabilize the coiled-coil chimeras) perturb the overlap, we carried out Molecular Dynamics simulations of head-to-tail structures having only native tropomyosin sequences. We observe that the splayed chains all refold and become helical. Significantly, the curvature of both the smooth and the striated muscle overlap domain is reduced and becomes comparable to that of the rest of the tropomyosin cable. Moreover, the measured flexibility across the junction is small. This and the reduced curvature ensure that the super-helical cable matches the contours of F-actin without manifesting localized kinking and excessive flexibility, thus enabling the high degree of cooperativity in the regulation of myosin accessibility to actin filaments.     

Postnatal changes in vagal control of esophageal muscle contractions in rats

AimsReplacement of smooth muscles by striated muscles occurs in the esophagus during the early postnatal period. The aim of this study was to clarify postnatal changes in vagal control of esophageal muscle contractions in rats.Main methodsAn isolated segment of the neonatal rat esophagus was placed in an organ bath and the contractile responses were recorded using a force transducer.Key findingsElectrical stimulation of the vagus trunk evoked a biphasic contractile response in the neonatal esophageal segment. The first and second components of the contractions were inhibited by α-bungarotoxin and atropine, respectively. Ganglion blockers, hexamethonium and mecamylamine, did not affect vagally mediated contractions. The first component gradually enlarged with age in days, whereas the second component declined during the first week after birth. Application of d-tubocurarine or acetylcholine caused an apparent contraction in the esophageal striated muscle at postnatal day 0, but responses to these drugs were not observed at 1 week after birth. The neonatal esophagus expressed the γ-subunit of nicotinic acetylcholine receptors. In contrast, the ε-subunit was dominantly expressed in the adult esophagus.SignificanceThe vagus nerves directly innervate both the esophageal striated muscles and smooth muscles in the early neonatal period. During the process of muscle rearrangement, the property of the striated muscles is altered substantially. The specific features of striated muscles in the neonatal rat esophagus might compensate for immature formation of neuromuscular junctions. Unsuccessful conversion of the striated muscle property during postnatal muscle rearrangement would be related to disorders of esophageal motility.     

Phylogenetic relationship of muscle tissues deduced from superimposition of gene trees.

Muscle tissues can be divided into six classes; smooth, fast skeletal, slow skeletal and cardiac muscle tissues for vertebrates, and striated and smooth muscle tissues for invertebrates. We reconstructed phylogenetic trees of six protein genes that are expressed in muscle tissues and, using a newly developed program, inferred the phylogeny of muscle tissues by superimposition of five of those gene trees. The proteins used are troponin C, myosin essential light chain, myosin regulatory light chain, myosin heavy chain, actin, and muscle regulatory factor (MRF) families. Our results suggest that the emergence of skeletal-cardiac muscle type tissues preceded the vertebrate/arthropod divergence (ca. 700 MYA), while vertebrate smooth muscle seemed to evolve independent of other muscles. In addition, skeletal muscle is not monophyletic, but cardiac and slow skeletal muscles make a cluster. Furthermore, arthropod striated muscle, urochordate smooth muscle, and vertebrate muscles except for smooth muscle share a common ancestor. On the other hand, arthropod nonmuscle and vertebrate smooth muscle and nonmuscle share a common ancestor.     

Notch and bone morphogenetic protein differentially act on dermomyotome cells to generate endothelium, smooth, and striated muscle

We address the mechanisms underlying generation of skeletal muscle, smooth muscle, and endothelium from epithelial progenitors in the dermomyotome. Lineage analysis shows that of all epithelial domains, the lateral region is the most prolific producer of smooth muscle and endothelium. Importantly, individual labeled lateral somitic cells give rise to only endothelial or mural cells (not both), and endothelial and mural cell differentiation is driven by distinct signaling systems. Notch activity is necessary for smooth muscle production while inhibiting striated muscle differentiation, yet it does not affect initial development of endothelial cells. On the other hand, bone morphogenetic protein signaling is required for endothelial cell differentiation and/or migration but inhibits striated muscle differentiation and fails to impact smooth muscle cell production. Hence, although different mechanisms are responsible for smooth muscle and endothelium generation, the choice to become smooth versus striated muscle depends on a single signaling system. Altogether, these findings underscore the spatial and temporal complexity of lineage diversification in an apparently homogeneous epithelium.     

Smitin, a novel smooth muscle titin-like protein, interacts with myosin filaments in vivo and in vitro.

Smooth muscle cells use an actin-myosin II-based contractile apparatus to produce force for a variety of physiological functions, including blood pressure regulation and gut peristalsis. The organization of the smooth muscle contractile apparatus resembles that of striated skeletal and cardiac muscle, but remains much more poorly understood. We have found that avian vascular and visceral smooth muscles contain a novel, megadalton protein, smitin, that is similar to striated muscle titin in molecular morphology, localization in a contractile apparatus, and ability to interact with myosin filaments. Smitin, like titin, is a long fibrous molecule with a globular domain on one end. Specific reactivities of an anti-smitin polyclonal antibody and an anti-titin monoclonal antibody suggest that smitin and titin are distinct proteins rather than differentially spliced isoforms encoded by the same gene. Smitin immunofluorescently colocalizes with myosin in chicken gizzard smooth muscle, and interacts with two configurations of smooth muscle myosin filaments in vitro. In physiological ionic strength conditions, smitin and smooth muscle myosin coassemble into irregular aggregates containing large sidepolar myosin filaments. In low ionic strength conditions, smitin and smooth muscle myosin form highly ordered structures containing linear and polygonal end-to-end and side-by-side arrays of small bipolar myosin filaments. We have used immunogold localization and sucrose density gradient cosedimentation analyses to confirm association of smitin with both the sidepolar and bipolar smooth muscle myosin filaments. These findings suggest that the titin-like protein smitin may play a central role in organizing myosin filaments in the contractile apparatus and perhaps in other structures in smooth muscle cells.