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.     

Calcium-independent phospholipase A2 modulates cytosolic oxidant activity and contractile function in murine skeletal muscle cells.

Phospholipase A2 (PLA2) activity supports production of reactive oxygen species (ROS) by mammalian cells. In skeletal muscle, endogenous ROS modulate the force of muscle contraction. We tested the hypothesis that skeletal muscle cells constitutively express the calcium-independent PLA2 (iPLA2) isoform and that iPLA2 modulates both cytosolic oxidant activity and contractile function. Experiments utilized differentiated C2C12 myotubes and a panel of striated muscles isolated from adult mice. Muscle preparations were processed for measurement of mRNA by real-time PCR, protein by immunoblot, cytosolic oxidant activity by the dichlorofluorescein oxidation assay, and contractile function by in vitro testing. We found that iPLA2 was constitutively expressed by all muscles tested (myotubes, diaphragm, soleus, extensor digitorum longus, gastrocnemius, heart) and that mRNA and protein levels were generally similar among muscles. Selective iPLA2 blockade by use of bromoenol lactone (10 microM) decreased cytosolic oxidant activity in myotubes and intact soleus muscle fibers. iPLA2 blockade also inhibited contractile function of unfatigued soleus muscles, shifting the force-frequency relationship rightward and depressing force production during acute fatigue. Each of these changes could be reproduced by selective depletion of superoxide anions using superoxide dismutase (1 kU/ml). These findings suggest that constitutively expressed iPLA2 modulates oxidant activity in skeletal muscle fibers by supporting ROS production, thereby influencing contractile properties and fatigue characteristics.     

Some physiological and biochemical features of striated muscles reinnervated by preganglionic sympathetic fibers

Skeletal muscle fibers can be reinnervated by motor cholinergic fibers, that is, functional connection can be achieved. However, functional connection implies not only the capacity of the nerve impulse to elicit a contractile response but also the capability of the reinnervating neurons to evoke particular modifications of the physiological and biological features of the muscles. In order to search for some of the modifications due to reinnervation by preganglionic sympathetic fibers, muscle contraction time was studied in three different preparations of adult cats: a) cricothyroid muscle reinnervated by preganglionic fibers; b) cricothyroideus reinnervated by its own nerve; and c) the corresponding normal neuromuscular preparation. The activities of malic dehydrogenase, of aldolase and pyruvic kinase were studied in these three preparations as well as in the denervated cricothyroid muscles. Reinnervation by preganglionic fibers prolonged the twich contraction time, whereas, self-reinnervation did not alter it. On the other hand, the activities of the three enzymes decreased as a result of denervation. In contrast, the muscle reinnervated with sympathetic preganglionic fibers partially recovered the normal level of malic dehydrogenase and the aldolase activities; but showed no modification in the level of pyruvic kinase activity. Conversely, in the muscle fibers reinnervated by their own nerve, the activity of the three enzymes returned to normal levels. The shortening of contraction time of the preganglionic reinnervated muscle correlates well with the features of the enzymic activities found in these muscles. It can be concluded: a) preganglionic sympathetic axons are able to achieve functional connections with striated muscles and b) considering the trophic effect, preganglionic fibers resemble the motor nerve supplying slow muscles.     

Paramyosin in invertebrate muscles. I. Identification and localization

By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodiffusion, we identified paramyosin in two smooth invertebrate "catch" muscles (Mytilus anterior byssus retractor and Mercenaria opaque adductor) and five invertebrate striated muscles (Limulus telson levator, Homarus claw muscle, Balanus scutal depressor, Lethocerus air tube retractor, and Aequipecten striated adductor). We show that (a) the paramyosins in all of these muscles have the same chain weights and (b) they are immunologically similar. We stained all of these muscles with specific antibody to Limulus paramyosin using the indirect fluorescent antibody technique. Paramyosin was localized to the A bands of the glycerinated striated muscles, and diffus fluorescence was seen throughout the glycerinated fibers of the smooth catch muscles. The presence of paramyosin in Homarus claw muscle, Balanus scutal depressor, and Lethocerus air tube retractor is shown here for the first time. Of the muscles in this study, Limulus telson levator is the only one for which the antiparamyosin staining pattern has been previously reported.     

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.     

The Mechanism of Spontaneous Oscillatory Contractions in Skeletal Muscle

Most striated muscles generate steady contractile tension when activated, but some preparations, notably cardiac myocytes and slow-twitch fibers, may show spontaneous oscillatory contractions (SPOC) at low levels of activation. We have provided what we believe is new evidence that SPOC is a property of the contractile system at low actin-myosin affinity, whether caused by a thin-filament regulatory system or by other means. We present a quantitative single-sarcomere model for isotonic SPOC in skeletal muscle with three basic ingredients: i), actin and myosin filaments initially in partial overlap, ii), stretch activation by length-dependent changes in the lattice spacing, and iii), viscoelastic passive tension. Modeling examples are given for slow-twitch and fast-twitch fibers, with periods of 10 s and 4 s respectively. Isotonic SPOC occurs in a narrow domain of parameter values, with small minimum and maximum values for actin-myosin affinity, a minimum amount of passive tension, and a maximum transient response rate that explains why SPOC is favored in slow-twitch fibers. The model also predicts the contractile, relaxed and SPOC phases as a function of phosphate and ADP levels. The single-sarcomere model can also be applied to a whole fiber under auxotonic and fixed-end conditions if the remaining sarcomeres are treated as a viscoelastic load. Here the model predicts an upper limit for the load stiffness that leads to SPOC; this limit lies above the equivalent loads expected from the rest of the fiber.     

Fluorescence histochemical study on the noradrenergic control to the anterior column of the spinal lumbosacral segments of the rat and dog,with special reference to motoneurons innervating the perineal striated muscles (Onuf's nucleus)

Summary The organization of noradrenergic fibers in the lumbosacral anterior column of rats and dogs was examined in detail using a modification of a highly sensitive glyoxylic acid fluorescence histochemical method. In both rat and dog, there were greater concentrations of fluorescing noradrenergic fibers around the motoneurons innervating the perineal striated muscles (Onuf's nucleus) than around other motoneuronal groups. The preferential accumulation of noradrenergic fibers in Onuf's nucleus may indicate that the noradrenergic neuron system in the spinal cord of rodents and carnivores is closely related to the functional peculiarities of the perineal striated muscles, including the external anal and urethral sphincter muscles.     

Fluorescence histochemical study on the noradrenergic control to the anterior column of the spinal lumbosacral segments of the rat and dog, with special reference to motoneurons innervating the perineal striated muscles (Onuf's nucleus)

The organization of noradrenergic fibers in the lumbosacral anterior column of rats and dogs was examined in detail using a modification of a highly sensitive glyoxylic acid fluorescence histochemical method. In both rat and dog, there were greater concentrations of fluorescing noradrenergic fibers around the motoneurons innervating the perineal striated muscles (Onuf's nucleus) than around other motoneuronal groups. The preferential accumulation of noradrenergic fibers in Onuf's nucleus may indicate that the noradrenergic neuron system in the spinal cord of rodents and carnivores is closely related to the functional peculiarities of the perineal striated muscles, including the external anal and urethral sphincter muscles.     

Fine structure and innervation of an annelid muscle with the longest recorded sarcomere

The fine structure of myoepithelial cells of the proventriculus of a marine annelid (Syllis spongiphila) is described. The contractile system of these muscle fibers includes a single medial Z band. The thick filaments possess a ~140 Å paramyosin-like periodicity, but the filament disposition in these cells corresponds to that of other striated muscles, and actin orbitals in the A band number up to 20. The distance between the centers of the two H bands in the largest cells is ca. 40 µ Dyads involving T-system invaginations and isolated vesicles of the sarcoplasmic reticulum are situated at all sarcomere levels. Insertions of the contractile material onto invaginations of the inner and outer cell surfaces are described. Presumed polyaxonal neuromuscular junctions are established across wide synaptic clefts and include terminals with spherical and non-spherical synaptic vesicles. Inclusions occupying the core of each muscle cell appear to be rich in magnesium.     

Fiber type-specific distribution of M-band proteins in chicken muscle

The functions of two myofibrillar proteins, myomesin (Mr 185,000) and M-protein (Mr 165,000), associated with the M-band are as yet unknown. To extend our knowledge of these proteins, we have examined chicken striated muscles with fast and slow contractile properties, e.g., pectoralis major, PLD, ALD, medial adductor, and lateral adductor, to determine the expression and isoform composition of myomesin and M-protein in various muscles and fiber types. The high molecular weight M-band proteins were characterized and quantitated using monoclonal antibodies in immunoblotting and double-antibody sandwich ELISA. Fiber specificity was determined by immuno- and enzyme histochemistry. In addition to the previously reported Mr 195,000 and 190,000 isoforms of myomesin in heart [Grove et al. (1985): J Cell Biol 101:1431], the Mr 185,000 myomesin in skeletal muscles may represent different isoforms in fast and slow muscles on the basis of distinctive degradation patterns. M-protein has the same molecular weight in striated chicken muscles and degradation patterns indicate only one isoform. The low quantities of M-protein in slow muscles were shown to be due to the absence of M-protein in two of the generally recognized slow fiber types, types I and III. Thus, M-protein was present only in fast type II fibers, whereas myomesin was ubiquitous in all fiber types. Whatever the causal relationship, M-protein appears to function in fast motor units composed of type II fibers.     

Signal transduction and protein phosphorylation in smooth muscle contraction

Smooth muscles are important constituents of vertebrate organisms that provide for contractile activity of internal organs and blood vessels. Basic molecular mechanism of both smooth and striated muscle contractility is the force-producing ATP-dependent interaction of the major contractile proteins, actin and myosin II molecular motor, activated upon elevation of the free intracellular Ca²⁺ concentration ([Ca²⁺]_i). However, whereas striated muscles display a proportionality of generated force to the [Ca²⁺]_i level, smooth muscles feature molecular mechanisms that modulate sensitivity of contractile machinery to [Ca²⁺]_i. Phosphorylation of proteins that regulate functional activity of actomyosin plays an essential role in these modulatory mechanisms. This provides an ability for smooth muscle to contract and maintain tension within a broad range of [Ca²⁺]_i and with a low energy cost, unavailable to a striated muscle. Detailed exploration of these mechanisms is required to understand the molecular organization and functioning of vertebrate contractile systems and for development of novel advances for treating cardiovascular and many other disorders. This review summarizes the currently known and hypothetical mechanisms involved in regulation of smooth muscle Ca²⁺-sensitivity with a special reference to phosphorylation of regulatory proteins of the contractile machinery as a means to modulate their activity.     

Species-independent expression of nitric oxide synthase in the sarcolemma region of visceral and somatic striated muscle fibers

The expression and distribution of nitric oxide synthase (NOS) was studied by use of the newly designed specific histochemical NADPH diaphorase staining method and the indirect immunofluorescence technique employing an antiserum to brain NOS in visceral and somatic striated muscles of several mammalian species. Histochemical activity and immunoreactivity were located in the sarcolemma region of type I and II fibers of all muscles investigated. Visceral muscles were more strongly stained than somatic muscles. Furthermore, type II fibers, identified by staining of myosin adenosine triphosphatase activity after pre-incubation at alkaline pH, were more intensely labeled than type I fibers. In addition, NOS activity was detected in the area of the sarcolemma of intrafusal fibers. No obvious differences between species were observed. It was concluded that NOS of striated muscles probably makes up the richest and most important nitric oxide source in mammals.     

Appearance of Muscle Proteins in Ontogenesis of the Mussel <Emphasis Type="Italic">Mytilus trossulus</Emphasis> (Bivalvia)

The appearance of muscle proteins in the contractile apparatus of the mussel Mytilus trossulus was subjected to comparative analysis during ontogenesis. It was established, with the use of Western blot analysis and electrophoresis in polyacrylamid gel in the presence of sodium dodecylsulfate, that proteins of the contractile apparatus of mussel muscles express long before the formation of the first functionally active muscle system of the veliger larvae. Paramyosin is present in egg cells; twitchin, myorod, and actin appear at the stage of blastula (12 h after fertilization), and myosin appears at the trochophore stage (17 h after fertilization). The quantitative relation of muscle proteins was studied in actomyosin extracts of larvae obtained from different developmental stages. It was shown that the ratios actin/myosin and paramyosin/myosin at the veliger stage (96 h after fertilization) were found to be similar to those in the striated muscles of invertebrates.     

Limitations on locomotor performance in squid

An empirical equation relating O2 consumption (power input) to pressure production during jet-propelled swimming in the squid (Illex illecebrosus) is compared with hydrodynamic estimates of the pressure-flow power output also calculated from pressure data. Resulting estimates of efficiency and stress indicate that the circularly arranged obliquely striated muscles in squid mantle produce maximum tensions about half those of vertebrate cross-striated muscle, that "anaerobic" fibers contribute to aerobic swimming, and that peak pressure production requires an instantaneous power output higher than is thought possible for muscle. Radial muscles probably contribute additional energy via elastic storage in circular collagen fibers. Although higher rates of aerobic power consumption are only found in terrestrial animals at much higher temperatures, the constraint on squid performance is circulation, not ventilation. Anaerobic power consumption is also among the highest ever measured, but the division of labor between "aerobic" and "anaerobic" fibers suggests a system designed to optimize the limited capacity of the circulation.     

Development of the muscle system and contractile activity in the mussel Mytilus trossulus (Mollusca, Bivalvia)

The development of contractile apparatus was subjected to comparative analysis during ontogenesis of the mussel Mytilus trossulus. Indirect immunofluorescence with the polyclonal antibody against mussel twitchin, a protein of thick filaments, and florescent phalloidin as a marker of filamentous cell actin were used to monitor changes in the developing muscle system at different larval stages. The first definitive muscle structures were found at the late trochophore stage (36 h after fertilization) and starting from the midveliger stage (96 h), striated muscles, which are never present in adult mussels, were distinctly seen. The striated muscle periodicity was 1.25 microm in both mussle larvae and adult scallop. The contractile activities of veliger and adult muscles were measured using an electronic signal-processing videosystem. This work is the first complex study of morphological, biochemical, and physiological characteristics of the muscle system in the larvae and adult mollusks.     

New titin (connectin) isoforms and their functional role in striated muscles of mammals: Facts and suppositions

This review summarizes results of our studies on titin isoform composition in vertebrate striated muscles under normal conditions, during hibernation, real and simulated microgravity, and under pathological conditions (stiff-person syndrome, post-apoplectic spasticity, dilated cardiomyopathy, cardiac hypertrophy). Experimental evidence for the existence in mammalian striated muscles of higher molecular weight isoforms of titin (NT-isoforms) in addition to the known N2A-, N2BA-, and N2B-titin isoforms was obtained. Comparative studies of changes in titin isoform composition and structure-functional properties of human and animal striated muscles during adaptive and pathological processes led to a conclusion about the key role of NT-isoforms of titin in maintenance of sarcomere structure and contractile function of these muscles.     

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.     

甲壳动物横纹肌的收缩蛋白质与调节机制

甲壳动物横纹肌肌原纤维的肌丝陈列,收缩蛋白质和收缩的Ca²⁺依赖性调节机制与脊椎动物横纹肌有不少差异.脊椎动物横纹肌、甲壳动物快肌与慢肌的粗丝与细丝的数量比依次为1:2,1:3和1:6,肌丝阵列各异.甲壳动物粗肌丝由肌球蛋白和副肌球蛋白组成,其分子装配与脊椎动物不同.细肌丝含有肌动蛋白、原肌球蛋白和肌钙蛋白,肌钙蛋白-T分子量较高,肌钙蛋白-C仅1个Ca²⁺结合位点.甲壳动物横纹肌兼有细肌丝调节与粗肌丝调节.     

Smoothelin-like 1 Protein Regulates Myosin Phosphatase-targeting Subunit 1 Expression during Sexual Development and Pregnancy

Pregnancy coordinately alters the contractile properties of both vascular and uterine smooth muscles reducing systemic blood pressure and maintaining uterine relaxation. The precise molecular mechanisms underlying these pregnancy-induced adaptations have yet to be fully defined but are likely to involve changes in the expression of proteins regulating myosin phosphorylation. Here we show that smoothelin like protein 1 (SMTNL1) is a key factor governing sexual development and pregnancy induced adaptations in smooth and striated muscle. A primary target gene of SMTNL1 in these muscles is myosin phosphatase-targeting subunit 1 (MYPT1). Deletion of SMTNL1 increases expression of MYPT1 30–40-fold in neonates and during development expression of both SMTNL1 and MYPT1 increases over 20-fold. Pregnancy also regulates SMTNL1 and MYPT1 expression, and deletion SMTNL1 greatly exaggerates expression of MYPT1 in vascular smooth muscle, producing a profound reduction in force development in response to phenylephrine as well as sensitizing the muscle to acetylcholine. We also show that MYPT1 is expressed in Type2a muscle fibers in mice and humans and its expression is regulated during pregnancy, suggesting unrecognized roles in mediating skeletal muscle plasticity in both species. Our findings define a new conserved pathway in which sexual development and pregnancy mediate smooth and striated muscle adaptations through SMTNL1 and MYPT1.     

Arrangement and structure of sinus hair muscles in the big-clawed shrew,Sorex unguiculatus

The arrangement and structure of sinus hair muscles in the snout of the shrew, Sorex unguiculatus, were studied by electron microscopy and serial section light microscopy. Both striated and smooth muscles are directly associated with sinus hair follicles. The striated muscle fibers originate from the base of a follicle and insert onto the superficial portion of adjoining caudally positioned follicles. Some fibers insert into the corium instead of inserting into a follicle. The fibers show a fine structure typical of red fibers. Smooth muscle cells form a network with elastic fibers beneath the corium. Some cells are directly attached to the capsule of the sinus, thus forming a type of M. arrector pili. Striated muscle fibers that appear to end in the corium are connected with the smooth muscle network through the elastic fibers which appear to function as the tendon of these two types of muscle cell.