Actin cytoskeletal dynamics in smooth muscle: a new paradigm for the regulation of smooth muscle contraction

A growing body of data supports a view of the actin cytoskeleton of smooth muscle cells as a dynamic structure that plays an integral role in regulating the development of mechanical tension and the material properties of smooth muscle tissues. The increase in the proportion of filamentous actin that occurs in response to the stimulation of smooth muscle cells and the essential role of stimulus-induced actin polymerization and cytoskeletal dynamics in the generation of mechanical tension has been convincingly documented in many smooth muscle tissues and cells using a wide variety of experimental approaches. Most of the evidence suggests that the functional role of actin polymerization during contraction is distinct and separately regulated from the actomyosin cross-bridge cycling process. The molecular basis for the regulation of actin polymerization and its physiological roles may vary in diverse types of smooth muscle cells and tissues. However, current evidence supports a model for smooth muscle contraction in which contractile stimulation initiates the assembly of cytoskeletal/extracellular matrix adhesion complex proteins at the membrane, and proteins within this complex orchestrate the polymerization and organization of a submembranous network of actin filaments. This cytoskeletal network may serve to strengthen the membrane for the transmission of force generated by the contractile apparatus to the extracellular matrix, and to enable the adaptation of smooth muscle cells to mechanical stresses. Better understanding of the physiological function of these dynamic cytoskeletal processes in smooth muscle may provide important insights into the physiological regulation of smooth muscle tissues.     

The first three minutes: smooth muscle contraction, cytoskeletal events, and soft glasses.

Smooth muscle exhibits biophysical characteristics and physiological behaviors that are not readily explained by present paradigms of cytoskeletal and cross-bridge mechanics. There is increasing evidence that contractile activation of the smooth muscle cell involves an array of cytoskeletal processes that extend beyond cross-bridge cycling and the sliding of thick and thin filaments. We review here the evidence suggesting that the biophysical and mechanical properties of the smooth muscle cell reflect the integrated interactions of an array of highly dynamic cytoskeletal processes that both react to and transform the dynamics of cross-bridge interactions over the course of the contraction cycle. The activation of the smooth muscle cell is proposed to trigger dynamic remodeling of the actin filament lattice within cellular microdomains in response to local mechanical and pharmacological events, enabling the cell to adapt to its external environment. As the contraction progresses, the cytoskeletal lattice stabilizes, solidifies, and forms a rigid structure well suited for transmission of tension generated by the interaction of myosin and actin. The integrated molecular transitions that occur within the contractile cycle are interpreted in the context of microscale agitation mechanisms and resulting remodeling events within the intracellular microenvironment. Such an interpretation suggests that the cytoskeleton may behave as a glassy substance whose mechanical function is governed by an effective temperature.     

Length adaptation of airway smooth muscle: a stochastic model of cytoskeletal dynamics.

To account for cytoskeleton remodeling as well as smooth muscle length adaptation, here we represent the cytoskeleton as a two-dimensional network of links (contractile filaments or stress fibers) that connect nodes (dense plaques or focal adhesions). The network evolves in continuous turnover with probabilities of link formation and dissolution. The probability of link formation increases with the available fraction of contractile units, increases with the degree of network activation, and decreases with increasing distance between nodes, d, as 1/d(s), where s controls the distribution of link lengths. The probability of link dissolution decays with time to mimic progressive cytoskeleton stabilization. We computed network force (F) as the vector summation of link forces exerted at all nodes, unloaded shortening velocity (V) as being proportional to the average link length, and network compliance (C) as the change in network length per change in elastic force. Imposed deformation caused F to decrease transiently and then recover dynamically; recovery ability decreased with increasing time after activation, mimicking observed biological behavior. Isometric contractions showed small sensitivity of F to network length, thus maintaining high force over a wide range of lengths; V and C increased with increasing length. In these behaviors, link length regulation, as described by the parameter s, was found to be crucial. Concerning length adaptation, all phenomena reported thus far in the literature were captured by this extremely simple network model.     

Evidence that unphosphorylated smooth muscle myosin supports smooth muscle contraction.

Unphosphorylated smooth muscle myosin filaments do not disassemble in MgATP, provided that the solution is supplemented either by 25% serum albumin or by 6% polyethylene glycol 6000. These filaments are able to support actomyosin retraction but their ATPase activity is not activated by tropomyosin-decorated F-actin.     

Intracellular calcium and smooth muscle contraction

Excitation-contraction coupling in smooth muscle involves many processes, some of which are outlined in this article. The total amount of Ca2+ released on excitation is considerably in excess of the free Ca2+ concentration and this implies a high capacity, high affinity Ca2+ buffer system. The two major Ca2+-binding proteins are calmodulin and myosin. Only calmodulin has the appropriate binding affinity to act as a component of the Ca2+-buffer system. The Ca2+-calmodulin complex activates myosin light chain kinase and thus is involved in the regulation of contractile activity. Phosphorylation of myosin stabilizes an active conformation and promotes cross bridge cycling and is essential for the initiation of contraction. During the initial contractile response phosphorylation correlates to tension development and velocity of shortening. However, as contraction continues the extent of myosin phosphorylation and velocity often decreases but tension is maintained. In general, the Ca2+ transient is reflected by the extent of phosphorylation that in turn correlates with shortening velocity. Maintenance of tension at low phosphorylation levels is not accounted for within our understanding of the phosphorylation theory and thus alternative regulatory mechanisms have been implicated. Some of the possibilities are discussed.     

Nox regulation of smooth muscle contraction

The catalytic subunit gp91phox (Nox2) of the NADPH oxidase of mammalian phagocytes is activated by microbes and immune mediators to produce large amounts of reactive oxygen species (ROS) which participate in microbial killing. Homologs of gp91phox, the Nox and Duox enzymes, were recently described in a range of organisms, including plants, vertebrates, and invertebrates such as Drosophila melanogaster. While their enzymology and cell biology are being extensively studied in many laboratories, little is known about in vivo functions of Noxes. Here, we establish and use an inducible system for RNAi to discover functions of dNox, an ortholog of human Nox5 in Drosophila. We report here that depletion of dNox in musculature causes retention of mature eggs within ovaries, leading to female sterility. In dNox-depleted ovaries and ovaries treated with a Nox inhibitor, muscular contractions induced by the neuropeptide proctolin are markedly inhibited. This functional defect results from a requirement for dNox-for the proctolin-induced calcium flux in Drosophila ovaries. Thus, these studies demonstrate a novel biological role for Nox-generated ROS in mediating agonist-induced calcium flux and smooth muscle contraction.     

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.     

The latch-bridge hypothesis of smooth muscle contraction

In contrast to striated muscle, both normalized force and shortening velocities are regulated functions of cross-bridge phosphorylation in smooth muscle. Physiologically this is manifested as relatively fast rates of contraction associated with transiently high levels of cross-bridge phosphorylation. In sustained contractions, Ca2+, cross-bridge phosphorylation, and ATP consumption rates fall, a phenomenon termed "latch". This review focuses on the Hai and Murphy (1988a) model that predicted the highly non-linear dependence of force on phosphorylation and a directly proportional dependence of shortening velocity on phosphorylation. This model hypothesized that (i) cross-bridge phosphorylation was obligatory for cross-bridge attachment, but also that (ii) dephosphorylation of an attached cross-bridge reduced its detachment rate. The resulting variety of cross-bridge cycles as predicted by the model could explain the observed dependencies of force and velocity on cross-bridge phosphorylation. New evidence supports modifications for more general applicability. First, myosin light chain phosphatase activity is regulated. Activation of myosin phosphatase is best demonstrated with inhibitory regulatory mechanisms acting via nitric oxide. The second modification of the model incorporates cooperativity in cross-bridge attachment to predict improved data on the dependence of force on phosphorylation. The molecular basis for cooperativity is unknown, but may involve thin filament proteins absent in striated muscle.     

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.     

A dynamic model of smooth muscle contraction

A dynamic model of smooth muscle contraction is presented and is compared with the mechanical properties of vascular smooth muscle in the rat portal vein. The model is based on the sliding filament theory and the assumption that force is produced by cross-bridges extending from the myosin to the actin filaments. Thus, the fundamental aspects of the model are also potentially applicable to skeletal muscle. The main concept of the model is that the transfer of energy via the cross-bridges can be described as a 'friction clutch' mechanism. It is shown that a mathematical formulation of this concept gives rise to a model that agrees well with experimental observations on smooth muscle mechanics under isotonic as well as isometric conditions. It is noted that the model, without any ad hoc assumptions, displays a nonhyperbolic force-velocity relationship in its high-force portion and that it is able to maintain isometric force in conditions of reduced maximum contraction velocity. Both these findings are consistent with new experimental observations on smooth muscle mechanics cannot be accounted for by the classical Hill model.     

Airway smooth muscle: contraction and beyond

Airway smooth muscle (ASM), an important tissue involved in the regulation of bronchomotor tone, exists in the trachea and in the bronchial tree up to the terminal bronchioles. The physiological relevance of ASM in healthy airways remains unclear. Evidence, however, suggests that ASM undergoes marked phenotypic modulation in lung development and in disease states such as asthma, chronic bronchitis and emphysema. The shortening of ASM regulates airway luminal diameter and modulates airway resistance, which can be augmented by cytokines as well as extracellular matrix alterations. ASM may also serve immunomodulatory functions, which are mediated by the secretion of pro-inflammatory mediators such as cytokines and chemokines. In addition, ASM mass increases in chronic airway diseases and may represent either a pathologic or an injury-repair response due to chronic inflammation. This review will present evidence that ASM, a "passive" contractile tissue, may become an "active participant" in modulating inflammation in chronic lung diseases. Cell facts 1. Found in the trachea and along the bronchial tree. 2. Critically important in regulating bronchomotor tone of the airways. 3. Differentiation state is associated with the expression of various "contractile proteins." 4. Displays phenotypic modulation of mechanical, synthetic and proliferative responses. 5. Secretes cytokines, chemokines and extracellular matrix proteins. 6. May serve as a potential new target for the treatment of chronic lung diseases.     

Reserpine-induced post-receptor reduction in muscarinic-mediated airway smooth muscle contraction

Radioligand binding was conducted on airways of the rat and human, surgically subdivided into trachea, lung airways, and parenchyma. 3H-QNB bound uniformly to receptors in separate sections of the rat and human airway. Receptor densities generally were ranked: lung airways greater than trachea greater than parenchyma. Receptor subtypes were identified mostly by pirenzepine displacement of bound 3H-QNB. The rat trachea, and rat and human lung airways had a uniformly low affinity for pirenzepine while rat and human parenchyma demonstrated both high and low affinity pirenzepine binding. Inhibition of methacholine-stimulated smooth muscle contraction by the M1 receptor antagonist, pirenzepine, and M2 receptor antagonist, gallamine, was studied in rat trachea and bronchus in vitro. Schild plot pA2 values were compatible with low potency antagonism, thereby favoring the presence of M3 receptors at these smooth muscle sites. Reserpine treatment of rats (0.5 mg kg-1 day-1 for 7 days) produced a decrease in peak tension in response to methacholine without changing the muscarinic receptor character (Kd 3H-QNB), population density (Bmax in fmol mg-1 protein), or function (methacholine EC50). These results indicate that muscarinic receptor heterogeneity exists in the airway of both laboratory rat and man. While the muscarinic receptor subserving airway smooth muscle contraction appears to be the M3 subtype, decreased contractile responses to methacholine by trachea and bronchus from reserpine-treated rats were receptor independent.     

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.