Models of contractile units and their assembly in smooth muscle

It is believed that the contractile filaments in smooth muscle are organized into arrays of contractile units (similar to the sarcomeric structure in striated muscle), and that such an organization is crucial for transforming the mechanical activities of actomyosin interaction into cell shortening and force generation. Details of the filament organization, however, are still poorly understood. Several models of contractile filament architecture are discussed here. To account for the linear relationship observed between the force generated by a smooth muscle and the muscle length at the plateau of an isotonic contraction, a model of contractile unit is proposed. The model consists of 2 dense bodies with actin (thin) filaments attached, and a myosin (thick) filament lying between the parallel thin filaments. In addition, the thick filament is assumed to span the whole contractile unit length, from dense body to dense body, so that when the contractile unit shortens, the amount of overlap between the thick and thin filaments (i.e., the distance between the dense bodies) decreases in exact proportion to the amount of shortening. Assembly of the contractile units into functional contractile apparatus is assumed to involve a group of cells that form a mechanical syncytium. The contractile apparatus is assumed malleable in that the number of contractile units in series and in parallel can be altered to accommodate strains on the muscle and to maintain the muscle's optimal mechanical function.     

Dense bodies and actin polarity in vertebrate smooth muscle

The arrangement of cytoplasmic dense bodies in vertebrate smooth muscle and their relationship to the thin filaments was studied in cells from rabbit vas deferens and portal vein which were made hyperpermeable (skinned) with saponin and incubated with myosin subfragment 1 (S-1). The dense bodies were obliquely oriented, elongated structures sometimes appearing as chains up to 1.5 microns in length; they were often continuous across the cell for 200 to 300 nm and were interconnected by an oblique network of 10-nm filaments. The arrowheads, formed by S-1 decoration of actins, which inserted into both the sides and ends of dense bodies, always pointed away from the dense body, similar to the polarity of the thin filaments at the Z- bands of skeletal muscle. These results show that the cytoplasmic dense bodies function as anchoring sites for the thin filaments and indicate that the thin filaments, thick filaments, and dense bodies constitute a contractile unit.     

Fine structure of the nephridial muscle layer cells of Phascolosoma granulatum (Sipuncula)

The nephridial muscle layer of Phascolosoma granulatum consists of a network of longitudinal and circular cells separated by connective tissue matrix. The muscle fibers are densely packed with thick and thin myofilaments, among which are scattered cytoplasmic dense bodies. The nucleus and noncontractile cytoplasmic organelles occupy a lateral projection from the contractile portion of the fiber. Cytoplasmic dense bodies are the result of a clustering of an indeterminate number of the thin actin filaments that fill the cytoplasm between thick filaments. Attached to the cytoplasmic face of the cell membrane are membrane-associated electron-dense plaques. These sites are linked to the contractile myofilaments by narrow filamentous bridges. Extracellular narrow filaments extend from these plaques to collagen fibers of the connective tissue matrix. Differences in length of the dense plaques may be related to differences in thick myofilament diameter in three types of muscle fiber, types A, B and C, statistically distinguished by mean fiber size differences. The plaques may serve as connecting links for the transmission of tension from contractile units to the connective tissue of the muscle layer. © 1993 Wiley-Liss, Inc.     

Contribution of muscle series elasticity to maximum performance in drop jumping

The contribution of muscle in-series compliance on maximum performance of the muscle tendon complex was investigated using a forward dynamic computer simulation. The model of the human body contains 8 Hill-type muscles of the lower extremities. Muscle activation is optimized as a function of time, so that maximum drop jump height is achieved by the model. It is shown that the muscle series elastic energy stored in the downward phase provides a considerable contribution (32%) to the total muscle energy in the push-off phase. Furthermore, by the return of stored elastic energy all muscle contractile elements can reduce their shortening velocity up to 63% during push-off to develop a higher force due to their force velocity properties. The additional stretch taken up by the muscle series elastic element allows only m. rectus femoris to work closer to its optimal length, due to its force length properties. Therefore the contribution of the series elastic element to muscle performance in maximum height drop jumping is to store and return energy, and at the same time to increase the force producing ability of the contractile elements during push-off.     

The cytoskeletal and contractile apparatus of smooth muscle: contraction bands and segmentation of the contractile elements

Confocal laser scanning microscopy of isolated and antibody-labeled avian gizzard smooth muscle cells has revealed the global organization of the contractile and cytoskeletal elements. The cytoskeleton, marked by antibodies to desmin and filamin is composed of a mainly longitudinal, meandering and branched system of fibrils that contrasts with the plait-like, interdigitating arrangement of linear fibrils of the contractile apparatus, labeled with antibodies to myosin and tropomyosin. Although desmin and filamin were colocalized in the body of the cell, filamin antibodies labeled additionally the vinculin- containing surface plaques. In confocal optical sections the contractile fibrils showed a continuous label for myosin for at least 5 microns along their length: there was no obvious or regular interruption of label as might be expected for registered myosin filaments. The cytoplasmic dense bodies, labeled with antibodies to alpha-actinin exhibited a regular, diagonal arrangement in both extended cells and in cells shortened in solution to one-fifth of their extended length: after the same shortening, the fibrils of the cytoskeleton that showed colocalization with the dense bodies in extended cells became crumpled and disordered. It is concluded that the dense bodies serve as coupling elements between the cytoskeletal and contractile systems. After extraction with Triton X-100, isolated cells bound so firmly to a glass substrate that they were unable to shorten as a whole when exposed to exogenous Mg ATP. Instead, they contracted internally, producing integral of 10 regularly spaced contraction nodes along their length. On the basis of differences of actin distribution two types of nodes could be distinguished: actin-positive nodes, in which actin straddled the node, and actin-negative nodes, characterized by an actin-free center flanked by actin fringes of 4.5 microns minimum length on either side. Myosin was concentrated in the center of the node in both cases. The differences in node morphology could be correlated with different degrees of coupling of the contractile with the cytoskeletal elements, effected by a preparation-dependent variability of proteolysis of the cells. The nodes were shown to be closely related to the supercontracted cell fragments shown in the accompanying paper (Small et al., 1990) and furnished further evidence for long actin filaments in smooth muscle. Further, the segmentation of the contractile elements pointed to a hierarchial organization of the myofilaments governed by as yet undetected elements.     

Assembly of contractile and cytoskeletal elements in developing smooth muscle cells.

Specific developmental changes in smooth muscle were studied in gizzards obtained from 6-, 8-, 10-, 12-, 14-, 16-, 18-, and 20-day chick embryos and from 1- and 7-day posthatch chicks. Myoblasts were actively replicating in tissue from 6-day embryos. Cytoplasmic dense bodies (CDBs) first appeared at Embryonic Day 8 (E8) and were recognized as patches of increased electron density that consisted of actin filaments (AFs), intermediate filaments (IFs), and cross-connecting filaments (CCFs). Although the assembly of CDBs was not synchronized within a cell, the number, size, and electron density of CDBs increased as age increased. Membrane-associated dense bodies (MADBs) also could be recognized at E8. The number and size of MADBs increased as age increased, especially after E16. Filaments with the diameter of thick filaments first appeared at E12. Smooth muscle cells were able to divide as late as E20. The axial intermediate filament bundle (IFB) could first be identified in 1-day posthatch cells and became larger and more prominent in 7-day posthatch cells. Immunogold labeling of 1- and 7-day posthatch cells with anti-desmin showed that the IFB contained desmin IFs. The developmental events during this 23-day period were classified into seven stages, based primarily on the appearance and the growth of contractile and cytoskeletal elements. These stages are myoblast proliferation, dense body appearance, thick filament appearance, dense body growth, muscle cell replication, IFB appearance, and appearance of adult type cells. Smooth muscle cells in each stage express similar developmental characteristics. The mechanism of assembly of myofilaments and cytoskeletal elements in smooth muscle in vivo indicates that myofilaments (AFs and thick filaments) and filament attachment sites (CDBs and MADBs) are assembled before the axial IFB, a major cytoskeletal element.     

Dense bodies of the contractile system of cardiac muscle in Venus mercenaria

Dense bodies in the heart muscle of Venus mercenaria exist in two forms, free and attached. Free dense bodies morphologically consist of fascicles of thin filaments in parallel array and bound together by a dense, amorphous proteinaceous material. The binding of dense bodies to the cell membrane is effected via connecting filaments of the amorphous material of the dense body which join a condensation of morphologically similar material attached to the inner osmiophilic layer of the unit membrane. This composite of dense body, connecting filaments, membrane condensation and unit cell membrane has been termed collectively the attachment plaque. The attachment plaque is part of an extensive network on the cell surface which obligates that surface to a role in the contractile process. Moreover, this set of attachment plaques imposes an organization and an orientation to most thin filaments of the cell and preserves the contractile axis of the cell.     

Electron microscopic study of actin polymerization in airway smooth muscle

Actin polymerization as part of the normal smooth muscle response to various stimuli has been reported. The actin dynamics are believed to be necessary for cytoskeletal remodeling in smooth muscle in its adaptation to external stress and strain and for maintenance of optimal contractility. We have shown in our previous studies in airway smooth muscle that myosins polymerized in response to contractile activation as well as to adaptation at longer cell lengths. We postulated that the same response could be elicited from actins under the same conditions. In the present study, actin filament formation was quantified electron microscopically in cell cross sections. Nanometer resolution allowed us to examine regional distribution of filaments in a cell cross section. Airway smooth muscle bundles were fixed in relaxed and activated states at two lengths; muscle preparations were also fixed after a period of oscillatory strain, a condition known to cause depolymerization of myosin filaments. The results indicate that contractile activation and increased cell length nonsynergistically enhanced actin polymerization; the extent of actin polymerization was substantially less than that of myosin polymerization. Oscillatory strain increased thin filament formation. Although thin filament density was found higher in cytoplasmic areas near dense bodies, contractile activation did not preferentially enhance actin polymerization in these areas. It is concluded that actin thin filaments are dynamic structures whose length and number are regulated by the cell in response to changes in extracellular environment and that polymerization and depolymerization of thin filaments occur uniformly across the whole cell cross section.     

Use of actin isoform-specific antibodies to probe the domain structure in three smooth muscles

We investigated the location of actin isoforms in relation to each other and to filament attachment sites by studying the edge-to-edge distribution of both immunofluorescence and immunogold probes in smooth muscle cells from three sources. Antibodies to alpha- or alpha,gamma-actin labeled uniformly across smooth muscle cells from each source. Antibodies to beta-cytoplasmic actin were concentrated on and near dense bodies, especially in gizzard smooth muscle, but were also located throughout the filament compartment. Double immunofluorescent labeling with antibodies to alpha- or alpha/gamma- and to beta-actin shows overlap of label at dense bodies and attachment plaques. Double immunofluorescent labeling with antibodies to alpha-actinin and to beta-actin identified dense bodies and attachment plaques as sites of colocalization. Immunogold labeling with anti-desmin was most prominent near dense bodies in the gizzard and was widely dispersed in vas deferens and arterial smooth muscle cells. Our results indicate that there is extensive overlap between the locations of contractile and cytoskeletal elements and, thus, do not support the two-domain model of smooth muscle structure. Tissue-specific organizational motif differences were seen when gizzard, vas deferens, and artery were compared and suggest that one model may not apply to these three smooth muscles.     

Periodic organization of the contractile apparatus in smooth muscle revealed by the motion of dense bodies in single cells

To study the organization of the contractile apparatus in smooth muscle and its behavior during shortening, the movement of dense bodies in contracting saponin skinned, isolated cells was analyzed from digital images collected at fixed time intervals. These cells were optically lucent so that punctate structures, identified immunocytochemically as dense bodies, were visible in them with the phase contrast microscope. Methods were adapted and developed to track the bodies and to study their relative motion. Analysis of their tracks or trajectories indicated that the bodies did not move passively as cells shortened and that nearby bodies often had similar patterns of motion. Analysis of the relative motion of the bodies indicated that some bodies were structurally linked to one another or constrained so that the distance between them remained relatively constant during contraction. Such bodies tended to fall into laterally oriented, semirigid groups found at approximately 6-microns intervals along the cell axis. Other dense bodies moved rapidly toward one another axially during contraction. Such bodies were often members of separate semirigid groups. This suggests that the semirigid groups of dense bodies in smooth muscle cells may provide a framework for the attachment of the contractile structures to the cytoskeleton and the cell surface and indicates that smooth muscle may be more well-ordered than previously thought. The methods described here for the analysis of the motion of intracellular structures should be directly applicable to the study of motion in other cell types.     

The pattern of organization of intermediate filaments and their asymmetrical association with dense bodies in smooth muscle of an ascidian Halocynthia roretzi

Summary An extensive network of intermediate filaments that interconnected cytoplasmic dense bodies and connected the dense bodies to the cell surface was revealed in double-fixed, tannic acid-stained preparations of ascidian smooth muscle. The filament network ran through spaces in the continuous network of myofibrils, connecting them longitudinally, obliquely and transversely to form an intimately associated, dual network. In their transverse passage, the intermediate filaments ran across myofibrils along I-zones exclusively, interconnecting successive dense bodies.The pattern of attachment of intermediate filaments to dense bodies was predominantly one-sided. The filaments, which themselves were not incorporated into the contractile apparatus, remained folded or unfolded between myofibrils and between sarcomere-like structures in synchrony with the contraction-relaxation cycles.These results suggest that the intermediate filaments mechanically maintain the organization and arrangement of myofibrils via an intimate association with the myofibrils in the regions of the dense bodies, in such a way that the filaments do not impede muscle function.Based on these observations, a new model for the network of intermediate filaments in smooth muscle cells is proposed.     

Mathematical description of geometric and kinematic aspects of smooth muscle plasticity and some related morphometrics.

Despite considerable investigation, the mechanisms underlying the functional properties of smooth muscle are poorly understood. This can be attributed, at least in part, to a lack of knowledge about the structure and organization of the contractile apparatus inside the muscle cell. Recent observations of the plasticity of smooth muscle and of morphometry of the cell have provided enough information for us to propose a quantitative, although highly simplified, model for the geometric arrangement of contractile units and their collective kinematic functions in smooth muscle, particularly airway smooth muscle. We propose that, to a considerable extent, contractile machinery restructures upon activation of the muscle and adapts to cell geometry at the time of activation. We assume that, under steady-state conditions, the geometric arrangement of contractile units and the filaments within these units determines the kinematic characteristics of the muscle. The model successfully predicts the results of experiments on airway smooth muscle plasticity relating to maximal force generation, maximal velocity of shortening, and the variation of compliance with adapted length. The model is also concordant with morphometric observations that show an increase in myosin filament density when muscle is adapted to a longer length. The model provides a framework for design of experiments to quantitatively test various aspects of smooth muscle plasticity in terms of geometric arrangement of contractile units and the muscle's mechanical properties.     

Plasticity in canine airway smooth muscle

The large volume changes of some hollow viscera require a greater length range for the smooth muscle of their walls than can be accommodated by a fixed array of sliding filaments. A possible explanation is that smooth muscles adapt to length changes by forming variable numbers of contractile units in series. To test for such plasticity we examined the muscle length dependence of shortening velocity and compliance, both of which will vary directly with the number of thick filaments in series. Dog tracheal smooth muscle was studied because its cells are arrayed in long, straight, parallel bundles that span the length of the preparation. In experiments where muscle length was changed, both compliance and velocity showed a strong dependence on muscle length, varying by 1.7-fold and 2.2-fold, respectively, over a threefold range of length. The variation in isometric force was substantially less, ranging from a 1.2- to 1.3-fold in two series of experiments where length was varied by twofold to an insignificant 4% variation in a third series where a threefold length range was studied. Tetanic force was below its steady level after both stretches and releases, and increased to a steady level with 5-6 tetani at 5 min intervals. These results suggest strongly that the number of contractile units in series varies directly with the adapted muscle length. Temporary force depression after a length change would occur if the change transiently moved the filaments from their optimum overlap. The relative length independence of the adapted force is explained by the reforming of the filament lattice to produce optimum force development, with commensurate changes of velocity and compliance.     

Structure-function correlation in airway smooth muscle adapted to different lengths

Airway smooth muscle is able to adapt and maintain a nearly constant maximal force generation over a large length range. This implies that a fixed filament lattice such as that found in striated muscle may not exist in this tissue and that plastic remodeling of its contractile and cytoskeletal filaments may be involved in the process of length adaptation that optimizes contractile filament overlap. Here, we show that isometric force produced by airway smooth muscle is independent of muscle length over a twofold length change; cell cross-sectional area was inversely proportional to cell length, implying that the cell volume was conserved at different lengths; shortening velocity and myosin filament density varied similarly to length change: increased by 69.4% ± 5.7 (SE) and 76.0% ± 9.8, respectively, for a 100% increase in cell length. Muscle power output, ATPase rate, and myosin filament density also have the same dependence on muscle cell length: increased by 35.4% ± 6.7, 34.6% ± 3.4, and 35.6% ± 10.6, respectively, for a 50% increase in cell length. The data can be explained by a model in which additional contractile units containing myosin filaments are formed and placed in series with existing contractile units when the muscle is adapted at a longer length. muscle contraction; myosin filaments; ATPase activity; electron microscopy     

Interaction between series compliance and sarcomere kinetics determines internal sarcomere shortening during fixed-end contraction

The interaction between contractile force and in-series compliance was investigated for the intact skeletal muscle-tendon unit (MTU) of Rana pipiens semitendinosus muscles during fixed-end contraction. It was hypothesized that internal sarcomere shortening is a function of the length-force characteristics of contractile and series elastic components. The MTUs (n=18) were dissected, and, while submerged in Ringer's solution, muscles were activated at nine muscle lengths (-2 to +6 mm relative to optimal length in 1 mm intervals), while measuring muscle force and sarcomere length (SL) by laser diffraction. The MTU was clamped either at the bone (n=6), or at the proximal and distal ends of the aponeuroses (n=6). Muscle fibers were also trimmed along with aponeuroses down to 5-20 fibers and identical measurements were performed (n=6). The magnitude of shortening decreased as MTU length increased. The magnitude of shortening ranged from -0.08 to 0.3 microm, and there was no significant difference between delta SL as a function of clamp location. When aponeuroses were trimmed, sarcomere shortening was not observed at L(0) and longer. These results suggest that the aponeurosis is the major contributor to in-series compliance. Results also support our hypothesis but there also appear to be other factors affecting internal sarcomere shortening. The functional consequence of internal sarcomere shortening as a function of sarcomere length was to skew the muscle length-tension relationship to longer sarcomere lengths.     

Oocyte-follicle cell relationships in a starfish

Summary The follicle cells which surround the oocytes of starfish are known to both release the hormone 1-methyladenine and to respond to it by an active movement which forms a component of the spawning response to the hormone. In Patiria miniata these flagellated cells are located peripheral to the oocyte and have long cytoplasmic processes which penetrate the vitelline layer to the egg surface to form an adhering zonule-like junction. Within the follicle cell cytoplasm are located elongate filamentous bands which appear to represent a component of the contractile mechanism that mediates follicle cell response to 1-methyladenine. These bands do not resemble the filaments of vertebrate smooth muscle cells (quantity, distribution and size of filaments; lack of dense bodies in the filament mass), nor the contractile units of the superficial epithelium of lower vertebrate follicles.This investigation was supported by grants HD-07194 and HD-12499 from the National Institutes of Health. We are indebted to Mr. James D. Huber for able technical assistance     

Caenorhabditis elegans UNC-98, a C2H2 Zn finger protein,is a novel partner of UNC-97/PINCH in muscle adhesion complexes

To further understand the assembly and maintenance of the muscle contractile apparatus, we have identified a new protein, UNC-98, in the muscle of Caenorhabditis elegans. unc-98 mutants display reduced motility and a characteristic defect in muscle structure. We show that the major defect in the mutant muscle is in the M-lines and dense bodies (Z-line analogs). Both functionally and compositionally, nematode M-lines and dense bodies are analogous to focal adhesions of nonmuscle cells. UNC-98 is a novel 310-residue polypeptide consisting of four C2H2 Zn fingers and several possible nuclear localization signal and nuclear export signal sequences. By use of UNC-98 antibodies and green fluorescent protein fusions (to full-length UNC-98 and UNC-98 fragments), we have shown that UNC-98 resides at M-lines, muscle cell nuclei, and possibly at dense bodies. Furthermore, we demonstrated that 1) the N-terminal 106 amino acids are both necessary and sufficient for nuclear localization, and 2) the C-terminal (fourth) Zn finger is required for localization to M-lines and dense bodies. UNC-98 interacts with UNC-97, a C. elegans homolog of PINCH. We propose that UNC-98 is both a structural component of muscle focal adhesions and a nuclear protein that influences gene expression.     

Heterogeneous distribution of isoactins in cultured vascular smooth muscle cells does not reflect segregation of contractile and cytoskeletal domains.

We have previously demonstrated that alpha-smooth muscle (alpha-SM) actin is predominantly distributed in the central region and beta-non-muscle (beta-NM) actin in the periphery of cultured rabbit aortic smooth muscle cells (SMCs). To determine whether this reflects a special form of segregation of contractile and cytoskeletal components in SMCs, this study systematically investigated the distribution relationship of structural proteins using high-resolution confocal laser scanning fluorescent microscopy. Not only isoactins but also smooth muscle myosin heavy chain, alpha-actinin, vinculin, and vimentin were heterogeneously distributed in the cultured SMCs. The predominant distribution of beta-NM actin in the cell periphery was associated with densely distributed vinculin plaques and disrupted or striated myosin and alpha-actinin aggregates, which may reflect a process of stress fiber assembly during cell spreading and focal adhesion formation. The high-level labeling of alpha-SM actin in the central portion of stress fibers was related to continuous myosin and punctate alpha-actinin distribution, which may represent the maturation of the fibrillar structures. The findings also suggest that the stress fibers, in which actin and myosin filaments organize into sarcomere-like units with alpha-actinin-rich dense bodies analogous to Z-lines, are the contractile structures of cultured SMCs that link to the network of vimentin-containing intermediate filaments through the dense bodies and dense plaques.     

The LIM domain protein UNC-95 is required for the assembly of muscle attachment structures and is regulated by the RING finger protein RNF-5 in C. elegans

Here, we describe a new muscle LIM domain protein, UNC-95, and identify it as a novel target for the RING finger protein RNF-5 in the Caenorhabditis elegans body wall muscle. unc-95(su33) animals have disorganized muscle actin and myosin-containing filaments as a result of a failure to assemble normal muscle adhesion structures. UNC-95 is active downstream of PAT-3/beta-integrin in the assembly pathways of the muscle dense body and M-line attachments, and upstream of DEB-1/vinculin in the dense body assembly pathway. The translational UNC-95::GFP fusion construct is expressed in dense bodies, M-lines, and muscle-muscle cell boundaries as well as in muscle cell bodies. UNC-95 is partially colocalized with RNF-5 in muscle dense bodies and its expression and localization are regulated by RNF-5. rnf-5(RNAi) or a RING domain deleted mutant, rnf-5(tm794), exhibit structural defects of the muscle attachment sites. Together, our data demonstrate that UNC-95 constitutes an essential component of muscle adhesion sites that is regulated by RNF-5.