Reorganization of myofilament proteins and decreased cGMP-dependent protein kinase in the human uterus during pregnancy

Excessive or premature contractions of uterine smooth muscle may contribute to preterm labor. Contractile stimuli induce myosin and actin filament interactions through calcium-dependent myosin phosphorylation. The mechanisms that maintain myometrial quiescence until term are not well established, but may include control of calcium levels by nitric oxide and cGMP signaling and thin filament (caldesmon and calponin) regulation. Previously, we reported that myometrial tissues from pregnant rats are not responsive to cGMP due to decreases in cGMP-dependent protein kinase. Considering the well documented differences in the endocrinology of parturition among species, this study was conducted to test the hypothesis that the levels and subcellular distribution of caldesmon, calponin, and cGMP-dependent protein kinase are regulated with the hormonal milieu of human pregnancy. Whereas cGMP-dependent protein kinase was significantly reduced in the human uterus during pregnancy, caldesmon expression was significantly increased, and both caldesmon and calponin were redistributed to a readily extractable subcellular pool. These data suggest that cGMP-dependent protein kinase does not mediate gestational quiescence. Redistribution of thin filament-associated proteins, however, may alter uterine smooth muscle tone or the cytoskeletal framework of myocytes to maintain gestation despite the substantial distention that accompanies all intrauterine pregnancies.     

Enhanced force generation by smooth muscle myosin in vitro.

To determine whether the apparent enhanced force-generating capabilities of smooth muscle relative to skeletal muscle are inherent to the myosin cross-bridge, the isometric steady-state force produced by myosin in the in vitro motility assay was measured. In this assay, myosin adhered to a glass surface pulls on an actin filament that is attached to an ultracompliant (50-200 nm/pN) glass microneedle. The number of myosin cross-bridge heads able to interact with a length of actin filament was estimated by measuring the density of biochemically active myosin adhered to the surface; with this estimate, the average force per cross-bridge head of smooth and skeletal muscle myosins is 0.6 pN and 0.2 pN, respectively. Surprisingly, smooth muscle myosin generates approximately three times greater average force per cross-bridge head than does skeletal muscle myosin.     

Role of Crk-associated substrate in the regulation of vascular smooth muscle contraction

A pool of actin monomers is induced to polymerize into actin filaments during contractile stimulation of smooth muscle. The inhibition of actin dynamics by actin polymerization inhibitors depresses active force generation in smooth muscle. In this study, we hypothesized that Crk-associated substrate plays a role in the regulation of contraction and actin dynamics in vascular smooth muscle. Antisense or sense oligodeoxynucleotides for Crk-associated substrate were introduced into carotid smooth muscle tissues by chemical loading. The treatment of smooth muscle strips with antisense oligodeoxynucleotides inhibited the expression of Crk-associated substrates; it did not influence the expression of actin, myosin heavy chain, and paxillin. Sense oligodeoxynucleotides did not affect the expression of these proteins in smooth muscle tissues. Force generation in response to stimulation with norepinephrine or KCl was significantly lower in antisense-treated muscle strips than in sense-treated strips or in muscle strips not treated with oligodeoxynucleotides. The downregulation of Crk-associated substrate did not attenuate increases in phosphorylation of the 20-kDa regulatory light chain of myosin in response to stimulation with norepinephrine. The increase in F-actin/G-actin ratio during contractile stimulation was significantly inhibited in antisense-treated smooth muscle strips. Contractile activation of smooth muscle increased the association of profilin with actin monomers; the depletion of Crk-associated substrate inhibited the increases in the profilin-actin complex in response to contractile stimulation. These results suggest that Crk-associated substrate is a necessary molecule of signaling cascades that regulate active force generation in smooth muscle. This molecule may regulate actin dynamics in smooth muscle in response to contractile stimulation.     

Effects of calcium on vascular smooth muscle contraction

Calcium initiates smooth muscle contraction by binding to calmodulin and activating the enzyme myosin light chain kinase. The activated form of myosin light chain kinase phosphorylates myosin on the 20,000-dalton light chain and contractile activity ensues. Calcium may also enhance smooth muscle contractile activity by binding directly to myosin, the main component of the thick filament. Recent studies raise the possibility that the calcium-calmodulin complex may also modulate smooth muscle contractile activity by removing the inhibition imposed by caldesmon, a protein that is bound to the thin (i.e., actin-containing) filaments of smooth muscle. In vitro studies have demonstrated that the calcium-activated, phospholipid-dependent kinase, protein kinase C, can phosphorylate smooth muscle myosin at a different site than does myosin light chain kinase and down-regulate its actin-activated magnesium adenosine triphosphatase activity. This raises the possibility that protein kinase C phosphorylation of myosin may play a role in modulating vascular contractile activity in vivo.     

The contractile apparatus and mechanical properties of airway smooth muscle.

The functional properties of airway smooth muscle are fundamental to the properties of the airways in vivo. However, many of the distinctive characteristics of smooth muscle are not easily accounted for on the basis of molecular models developed to account for the properties of striated muscles. The specialized ultrastructural features and regulatory mechanisms present in smooth muscle are likely to form the basis for many of its characteristic properties. The molecular organization and structure of the contractile apparatus in smooth muscle is consistent with a model of force generation based on the relative sliding of adjacent actin and myosin filaments. In airway smooth muscle, actomyosin activation is initiated by the phosphorylation of the 20 kDa light chain of myosin; but there is conflicting evidence regarding the role of myosin light chain phosphorylation in tension maintenance. Tension generated by the contractile filaments is transmitted throughout the cell via a network of actin filaments anchored at dense plaques at the cell membrane, where force is transmitted to the extracellular matrix via transmembrane integrins. Proteins bound to actin and/or localized to actin filament anchorage sites may participate in regulating the shape of the smooth muscle cell and the organization of its contractile filament system. These proteins may also participate in signalling pathways that regulate the crossbridge activation and other functions of the actin cytoskeleton. The length-dependence of active force and the mechanical plasticity of airway smooth muscle may play an important role in determining airway responsiveness during lung volume changes in vivo. The molecular basis for the length-dependence of tension in smooth muscle differs from that in skeletal muscle, and may involve mechano-transduction mechanisms that modulate contractile filament activation and cytoskeletal organization in response to changes in muscle length. The reorganization of contractile filaments may also underlie the plasticity of the mechanical response of airway smooth muscle. Changes in the structural organization and signalling pathways of airway smooth muscle cells resulting form alterations in mechanical forces in the lung may be important factors in the development of pathophysiological conditions of chronic airway hyperresponsiveness.     

Both N-terminal myosin-binding and C-terminal actin-binding sites on smooth muscle caldesmon are required for caldesmon-mediated inhibition of actin filament velocity

It has been suggested that the tethering caused by binding of the N-terminal region of smooth muscle caldesmon (CaD) to myosin and its C-terminal region to actin contributes to the inhibition of actin-filament movement over myosin heads in an in vitro motility assay. However, direct evidence for this assumption has been lacking. In this study, analysis of baculovirus-generated N-terminal and C-terminal deletion mutants of chicken-gizzard CaD revealed that the major myosin-binding site on the CaD molecule resides in a 30-amino acid stretch between residues 24 and 53, based on the very low level of binding of CaDΔ24–53 lacking the residues 24–53 to myosin compared with the level of binding of CaDΔ54–85 missing the adjacent residues 54–85 or of the full-length CaD. As expected, deletion of the region between residues 24 and 53 or between residues 54 and 85 had no effect on either actin-binding or inhibition of actomyosin ATPase activity. Deletion of residues 24–53 nearly abolished the ability of CaD to inhibit actin filament velocity in the in vitro motility experiments, whereas CaDΔ54–85 strongly inhibited actin filament velocity in a manner similar to that of full-length CaD. Moreover, CaD1–597, which lacks the major actin-binding site(s), did not inhibit actin-filament velocity despite the presence of the major myosin-binding site. These data provide direct evidence for the inhibition of actin filament velocity in the in vitro motility assay caused by the tethering of myosin to actin through binding of both the CaD N-terminal region to myosin and the C-terminal region to actin.     

Myosin light chain and caldesmon phosphorylation in arterial muscle stimulated with endothelin-1

Endothelin-1 contracts porcine carotid arterial smooth muscle with an ED50 of 10 nM. Contraction is associated with phosphorylation of the 20,000 dalton-regulatory light chain subunits of vascular myosin. Phosphopeptide mapping of light chains isolated from 32PO4-loaded muscle strips stimulated by endothelin-1 (5 x 10(-8) M) and comparison with maps generated from light chains phosphorylated in vitro or muscles stimulated with KCl (110 mM) or angiotensin-II (5 x 10(-8) M) indicates that Ca2(+)-calmodulin activation of myosin light chain kinase is a biochemical pathway stimulated by all three agonists. However, a small amount of phosphate (17%) was detected in a light chain peptide phosphorylated by protein kinase C. Endothelin-1 also stimulated phosphorylation of the thin filament protein, caldesmon, (from 0.35 mol PO4/mol caldesmon to 0.52 mol PO4/mol). Collectively, these results provide evidence that the effects of endothelin-1 on force generation and maintenance in vascular muscle may be dependent upon myosin light chain phosphorylation by Ca2+ calmodulin--requiring myosin light chain kinase and upon a thin filament mechanism that is modulated by phosphorylation of caldesmon.     

Contractile protein interactions in smooth muscle.

Smooth muscle tone and 'holding economy' depend on the rate constants governing the cross-bridge cycle. Thus, calcium activation via calmodulin-dependent myosin light chain phosphorylation may determine the apparent rate constant ('f') at which cross-bridges enter the force-generating state, forming actin-attached, strongly bound cross-bridges. This phosphorylation of the light chain may be inhibited in skinned fibers by a peptide mimic of the calmodulin recognition site of the myosin light chain kinase (RS 20) that relaxes smooth muscle. In smooth muscle, the apparent cross-bridge detachment rate constant ('g') also seems to be variable, a low constant allowing for a high holding economy and low shortening velocity in the 'latch state'. It may also account for force maintenance at low levels of myosin phosphorylation. Additionally, cross-bridge attachment may, however, be also controlled by other regulatory proteins such as calponin and caldesmon.     

Characterization of antibodies to smooth muscle myosin kinase and their use in localizing myosin kinase in nonmuscle cells.

Antibodies to myosin light chain kinase, purified from turkey gizzard smooth muscle, were developed in rabbits and purified by affinity chromatography on a myosin light chain kinase-Sepharose 4B column. The purified antibodies crossreact with purified smooth muscle myosin light chain kinase but not with a variety of contractile or cytoskeletal proteins. The antibodies inhibit the catalytic activity of smooth muscle myosin light chain kinase and there is an inverse relationship between the kinase activity and the amount of antibody present in an assay. Half-maximal inhibition of myosin kinase activity occurs at an antibody/myosin kinase molar ratio of 10:1. The affinity-purified antibodies to smooth muscle myosin kinase were used to study the location of myosin kinase in a variety of nonmuscle cells. Immunofluorescence studies indicate that myosin light chain kinase is localized on microfilament bundles (stress fibers) in cultured fibroblasts. The stress fiber staining pattern is abolished when the antibodies are incubated with purified smooth muscle myosin light chain kinase prior to staining cells, while the staining pattern is unaffected when the antibodies are incubated with actin, myosin, alpha-actinin, or tropomyosin prior to staining. Moreover, the stress fiber staining pattern is periodic in well-spread gerbil fibroma cells and experiments have demonstrated that myosin light chain kinase appears to have the same periodic distribution as myosin but an antiperiodic distribution relative to alpha-actinin. These data indicate that myosin light chain kinase and its substrate, myosin, are in close proximity and are consistent with the hypothesis that myosin light chain kinase regulates actin-myosin interactions in nonmuscle cells.     

Comparison of the caldesmon content of cardiac and smooth muscle.

The high abundance of caldesmon in smooth muscle and its ability to inhibit actomyosin ATPase activity have led to the hypothesis that caldesmon modulates contractile activity. It has also been proposed, however, that caldesmon acts as a structural protein in muscle and non-muscle cells. We have determined the caldesmon content of mammalian cardiac muscle and have found that caldesmon is 200-fold less abundant in cardiac muscle than it is in gizzard smooth muscle. This finding argues against a role for caldesmon in the modulation of cardiac contractility.     

Blebbistatin and blebbistatin-inactivated myosin II inhibit myosin II-independent processes in Dictyostelium

Blebbistatin, a cell-permeable inhibitor of class-II myosins, was developed to provide a tool for studying the biologic roles of myosin II. Consistent with this use, we find that blebbistatin inhibits three myosin II-dependent processes in Dictyostelium (growth in suspension culture, capping of Con A receptors, and development to fruiting bodies) and does not inhibit growth on plates, which does not require myosin II. As expected, macropinocytosis (myosin I-dependent), contractile vacuole activity (myosin V-dependent), and phagocytosis (myosin VII-dependent), none of which requires myosin II, are not inhibited by blebbistatin in myosin II-null cells, but, unexpectedly, blebbistatin does inhibit macropinocytosis and phagocytosis by cells expressing myosin II. Expression of catalytically inactive myosin II in myosin II-null cells also inhibits macropinocytosis and phagocytosis. Both blebbistatin-inhibited myosin II and catalytically inactive myosin II form cytoplasmic aggregates, which may be why they inhibit myosin II-independent processes, but neither affects the distribution of actin filaments in vegetative cells or actin and myosin distribution in dividing or polarized cells. Blebbistatin also inhibits cell streaming and plaque expansion in myosin II-null cells. Our results are consistent with myosin II being the only Dictyostelium myosin that is inhibited by blebbistatin but also show that blebbistatin-inactivated myosin II inhibits some myosin II-independent processes and that blebbistatin inhibits other activities in the absence of myosin II.     

Expression of smooth muscle-specific proteins in myoepithelium and stromal myofibroblasts of normal and malignant human breast tissue.

The expression of several differentiation markers in normal human mammary gland myoepithelium and in certain stromal fibroblasts ("myofibroblasts") associated with breast carcinomas was studied by immunofluorescence microscopy of frozen sections. Several antibodies to smooth muscle-specific proteins (smooth muscle alpha-actin, smooth muscle myosin heavy chains, calponin, alpha 1-integrin, and high molecular weight caldesmon) and to epithelial-specific proteins (cytokeratins, E-cadherin, and desmoplakin) were used to show that myoepithelial cells concomitantly express epithelial and smooth muscle markers whereas adjacent luminal cells express only epithelial markers. The same antibodies were used to establish that stromal myofibroblasts exhibit smooth muscle phenotypic properties characterized by the expression of all the smooth muscle markers examined except for high molecular weight caldesmon. In addition, both myoepithelium and myofibroblasts show a significant degree of heterogeneity in smooth muscle protein expression. Thus, myoepithelial cells and stromal myofibroblasts are epithelial and mesenchymal cells, respectively, which coordinately express a set of smooth muscle markers while maintaining their specific original features. The dual nature of myoepithelial cells and the phenotypic transition of fibroblasts to myofibroblasts are examples of the plasticity of the differentiated cell phenotype.     

Isolation and Characterization of Myosin and Two Myosin Fragments from Human Blood Platelets

Platelet myosin (thrombosthenin M) and two additional proteins corresponding to the head and rod portion of the myosin molecule have been prepared from human blood platelets. Characterization of these proteins by SDS-polyacrylamide gel electrophoresis, actin binding studies, assay of enzymic ATPase activity, and electron microscopy has shown that the platelet contractile proteins closely resemble the corresponding muscle proteins. Platelet myosin and platelet myosin-head bind to both muscle and platelet actin and have an EDTA + K-stimulated ATPase activity, which is suppressed by Mg(2+) in high salt concentration, whereas platelet rod does not possess either of these properties; platelet myosin and platelet myosin rod aggregate to form thick filaments at low ionic strength. Both intact platelet myosin and myosin head form typical arrowhead-shaped complexes with either platelet or muscle F-actin.     

Parallel inhibition of active force and relaxed fiber stiffness in skeletal muscle by caldesmon: implications for the pathway to force generation.

In recent hypotheses on muscle contraction, myosin cross-bridges cycle between two types of actin-bound configuration. These two configurations differ greatly in the stability of their actin-myosin complexes ("weak-binding" vs. "strong-binding"), and force generation or movement is the result of structural changes associated with the transition from the weak-binding (preforce generating) configuration to strong-binding (force producing) configuration [cf. Eisenberg, E. & Hill, T. L. (1985) Science 227, 999-1006]. Specifically, in this concept, the main force-generating states are only accessible after initial cross-bridge attachment in a weak-binding configuration. It has been shown that strong and weak cross-bridge attachment can occur in muscle fibers [Brenner, B., Schoenberg, M., Chalovich, J. M., Greene, L. E. & Eisenberg, E. (1982) Proc. Natl. Acad. Sci. USA 79, 7288-7291]. However, there has been no evidence that attachment in the weak-binding states represents an essential step leading to force generation. It is shown here that caldesmon can be used to selectively inhibit attachment of weak-binding cross-bridges in skeletal muscle. Such inhibition causes a parallel decrease in active force, while the kinetics of cross-bridge turnover are unchanged by this procedure. This suggests that (i) cross-bridge attachment in the weak-binding states is specific and (ii) force production can only occur after cross-bridges have first attached to actin in a weakly bound, nonforce-generating configuration.     

Clonogenic multipotent stem cells in human adipose tissue differentiate into functional smooth muscle cells

Smooth muscle is a major component of human tissues and is essential for the normal function of a multitude of organs including the intestine, urinary tract and the vascular system. The use of stem cells for cell-based tissue engineering and regeneration strategies represents a promising alternative for smooth muscle repair. For such strategies to succeed, a reliable source of smooth muscle precursor cells must be identified. Adipose tissue provides an abundant source of multipotent cells. In this study, the capacity of processed lipoaspirate (PLA) and adipose-derived stem cells to differentiate into phenotypic and functional smooth muscle cells was evaluated. To induce differentiation, PLA cells were cultured in smooth muscle differentiation medium. Smooth muscle differentiation of PLA cells induced genetic expression of all smooth muscle markers and further confirmed by increased protein expression of smooth muscle cell-specific alpha actin (ASMA), calponin, caldesmon, SM22, myosin heavy chain (MHC), and smoothelin. Clonal studies of adipose derived multipotent cells demonstrated differentiation of these cells into smooth muscle cells in addition to trilineage differentiation capacity. Importantly, smooth muscle-differentiated cells, but not their precursors, exhibit the functional ability to contract and relax in direct response to pharmacologic agents. In conclusion, adipose-derived cells have the potential to differentiate into functional smooth muscle cells and, thus, adipose tissue can be a useful source of cells for treatment of injured tissues where smooth muscle plays an important role.     

Cardiac myosin binding protein-C modulates actomyosin binding and kinetics in the in vitro motility assay

The modulatory role of whole cardiac myosin binding protein-C (cMyBP-C) on myosin force and motion generation was assessed in an in vitro motility assay. The presence of cMyBP-C at an approximate molar ratio of cMyBP-C to whole myosin of 1:2, resulted in a 25% reduction in thin filament velocity (P < 0.002) with no effect on relative isometric force under maximally activated conditions (pCa 5). Cardiac MyBP-C was capable of inhibiting actin filament velocity in a concentration-dependent manner using either whole myosin, HMM or S1, indicating that the cMyBP-C does not have to bind to myosin LMM or S2 subdomains to exert its effect. The reduction in velocity by cMyBP-C was independent of changes in ionic strength or excess inorganic phosphate. Co-sedimentation experiments demonstrated S1 binding to actin is reduced as a function of cMyBP-C concentration in the presence of ATP. In contrast, S1 avidly bound to actin in the absence of ATP and limited cMyBP-C binding, indicating that cMyBP-C and S1 compete for actin binding in an ATP-dependent fashion. However, based on the relationship between thin filament velocity and filament length, the cMyBP-C induced reduction in velocity was independent of the number of cross-bridges interacting with the thin filament. In conclusion, the effects of cMyBP-C on velocity and force at both maximal and submaximal activation demonstrate that cMyBP-C does not solely act as a tether between the myosin S2 and LMM subdomains but likely affects both the kinetics and recruitment of myosin cross-bridges through its direct interaction with actin and/or myosin head.     

Role of calcium and cyclic adenosine 3':5' monophosphate in regulating smooth muscle contraction. Mechanisms of excitation-contraction coupling in smooth muscle.

Caclium initiates smooth muscle contraction by activating an enzyme, myosin light chain kinase. This enzyme catalyzes the transfer of phosphate from adenosine triphosphate to the 20,000 dalton light chain of myosin. In its phosphorylated form myosin interacts with actin to produce muscle contraction. The mechanism by which calcium activates myosin kinase requires (1) the binding of calcium to a 16,500 dalton calcium-binding protein (calmodulin), and (2) the binding of calmodulin-calcium to a 125,000 dalton catalytic subunit. This two protein complex is the active form of myosin light chain kinase. Smooth muscle relaxation is mediated by cyclic adenosine 3':5' monophosphate (cyclic AMP). One nechanism by which the latter may exert a direct effect on actin-myosin interaction is through the activation of a cyclic AMP-dependent protein kinase that can phosphorylate the 125,000 dalton component of myosin light chain kinase. Phosphorylation of myosin light chain kinase decreases the activity of the enzyme, thus favoring the unphosphorylated form of myosin, which cannot interact with actin to produce smooth muscle contraction.     

Length adaptation of airway smooth muscle

Many types of smooth muscle, including airway smooth muscle (ASM), are capable of generating maximal force over a large length range due to length adaptation, which is a relatively rapid process in which smooth muscle regains contractility after experiencing a force decrease induced by length fluctuation. Although the underlying mechanism is unclear, it is believed that structural malleability of smooth muscle cells is essential for the adaptation to occur. The process is triggered by strain on the cell cytoskeleton that results in a series of yet undefined biochemical and biophysical events leading to restructuring of the cytoskeleton and contractile apparatus and consequently optimization of the overlap between the myosin and actin filaments. Although length adaptability is an intrinsic property of smooth muscle, maladaptation of ASM could result in excessive constriction of the airways and the inability of deep inspirations to dilate them. In this article, we describe the phenomenon of length adaptation in ASM and some possible underlying mechanisms that involve the myosin filament assembly and disassembly. We discuss a possible role of maladaptation of ASM in the pathogenesis of asthma. We believe that length adaptation in ASM is mediated by specific proteins and their posttranslational regulations involving covalent modifications, such as phosphorylation. The discovery of these molecules and the processes that regulate their activity will greatly enhance our understanding of the basic mechanisms of ASM contraction and will suggest molecular targets to alleviate asthma exacerbation related to excessive constriction of the airways.     

Immunohistological and ultrastructural analysis of the intimal thickening in coarctation of human aorta.

OBJECTIVES: The histological nature and characteristics of aortic coarctation are not clearly defined, the aim of this study is to analyse intimal thickening in aortic coarctation. METHODS: In order to characterize the components of intimal thickening in coarctation, narrowed segments of aorta obtained after surgery from ten children were examined immunocytochemically and by electron microscopy. RESULTS: Histological analysis of aortic coarctation demonstrated a widened subendothelial region with separation of endothelial cells from the internal elastic lamina. Masson's trichrome staining showed a marked increase in extracellular matrix and cell numbers in the intimal thickening compared with normal aorta. Cellular component analysis demonstrated invagination of the intima by smooth muscle actin-positive cells, with a fragmentation of the internal elastic lamina. No proliferating smooth muscle and inflammatory cells were identified in the intima. In order to characterize the smooth muscle cell phenotypes, various smooth muscle cell markers were sought using specific monoclonal antibodies: alpha-smooth muscle actin, smooth muscle-myosin heavy chain, heavy caldesmon, desmin. In moderate coarcted aorta, at least two distinct smooth muscle phenotypes were identified. In the juxtamedial part of the intima smooth muscle, cells were differentiated and expressed all smooth muscle markers; in the subendothelial part of the intimal thickening, the majority of smooth muscle cells expressed only alpha-smooth muscle actin and appeared dedifferentiated. In regions of marked stenosis, a strong expression of smooth muscle-myosin heavy chain, and heavy caldesmon in the intimal thickening pointed to the presence of redifferentiated smooth muscle cells, not still expressing desmin. Electron microscopic examination also revealed a variety of smooth muscle cell phenotypes in the intimal thickening. In the superficial layer, smooth muscle cells appeared to be in the synthetic state, while in the deeper part, both synthetic and contractile components were identified. CONCLUSIONS: These observations indicated that human coarctation was characterized by intimal recruitment of non-proliferating smooth muscle cells with dedifferentiated phenotype. However, the presence of smooth muscle cells with an intermediate phenotype in the narrowest part of the coarctation suggest that the redifferentiation process could participate in the pathogenesis of aortic coarctation.