Role of vimentin in smooth muscle force development

Vimentin intermediate filaments undergo spatial reorganization in cultured smooth muscle cells in response to contractile activation; however, the role of vimentin in the physiological properties of smooth muscle has not been well elucidated. Tracheal smooth muscle strips were loaded with antisense oligonucleotides (ODNs) against vimentin and then cultured for 2 days to allow for protein degradation. Treatment with vimentin antisense, but not sense, ODNs suppressed vimentin protein expression; neither vimentin antisense nor sense ODNs affected protein levels of desmin and actin. Force development in response to ACh stimulation or KCl depolarization was lower in vimentin-deficient tissues than in vimentin sense ODN- or non-ODN-treated muscle strips. Passive tension was also depressed in vimentin-depleted muscle tissues. Vimentin downregulation did not attenuate increases in myosin light chain (MLC) phosphorylation in response to contractile stimulation or basal MLC phosphorylation. In vimentin sense ODN-treated or non-ODN-treated smooth muscle strips, the desmosomal protein plakoglobin was primarily localized in the cell periphery. The membrane-associated localization of plakoglobin was reduced in vimentin-depleted muscle tissues. These studies suggest that vimentin filaments play an important role in mediating active force development and passive tension, which are not regulated by MLC phosphorylation. Vimentin downregulation impairs the structural organization of desmosomes, which may be associated with the decrease in force development. intermediate filaments; cytoskeleton; contraction; desmin     

Activation of the Arp2/3 complex by N-WASp is required for actin polymerization and contraction in smooth muscle

Contractile stimulation has been shown to initiate actin polymerization in smooth muscle tissues, and this actin polymerization is required for active tension development. We evaluated whether neuronal Wiskott-Aldrich syndrome protein (N-WASp)-mediated activation of the actin-related proteins 2 and 3 (Arp2/3) complex regulates actin polymerization and tension development initiated by muscarinic stimulation in canine tracheal smooth muscle tissues. In vitro, the COOH-terminal CA domain of N-WASp acts as an inhibitor of N-WASp-mediated actin polymerization; whereas the COOH-terminal VCA domain of N-WASp is constitutively active and is sufficient by itself to catalyze actin polymerization. Plasmids encoding EGFP-tagged wild-type N-WASp, the N-WASp VCA and CA domains, or enhanced green fluorescent protein (EGFP) were introduced into tracheal smooth muscle strips by reversible permeabilization, and the tissues were incubated for 2 days to allow for expression of the proteins. Expression of the CA domain inhibited actin polymerization and tension development in response to ACh, whereas expression of the wild-type N-WASp, the VCA domain, or EGFP did not. The increase in myosin light-chain (MLC) phosphorylation in response to contractile stimulation was not affected by expression of either the CA or VCA domain of N-WASp. Stimulation of the tissues with ACh increased the association of the Arp2/3 complex with N-WASp, and this association was inhibited by expression of the CA domain. The results demonstrate that 1) N-WASp-mediated activation of the Arp2/3 complex is necessary for actin polymerization and tension development in response to muscarinic stimulation in tracheal smooth muscle and 2) these effects are independent of the regulation of MLC phosphorylation. Wiskott-Aldrich syndrome protein; actin-related protein; tracheal muscle; cytoskeleton     

The small GTPase Cdc42 regulates actin polymerization and tension development during contractile stimulation of smooth muscle

Contractile stimulation induces actin polymerization in smooth muscle tissues and cells, and the inhibition of actin polymerization depresses smooth muscle force development. In the present study, the role of Cdc42 in the regulation of actin polymerization and tension development in smooth muscle was evaluated. Acetylcholine stimulation of tracheal smooth muscle tissues increased the activation of Cdc42. Plasmids encoding wild type Cdc42 or a dominant negative Cdc42 mutant, Asn-17 Cdc42, were introduced into tracheal smooth muscle strips by reversible permeabilization, and tissues were incubated for 2 days to allow for protein expression. Expression of recombinant proteins was confirmed by immunoblot analysis. The expression of the dominant negative Cdc42 mutant inhibited contractile force and the increase in actin polymerization in response to acetylcholine stimulation but did not inhibit the increase in myosin light chain phosphorylation. The expression of wild type Cdc42 had no significant effect on force, actin polymerization, or myosin light chain phosphorylation. Contractile stimulation increased the association of neuronal Wiskott-Aldrich syndrome protein with Cdc42 and the Arp2/3 (actin-related protein) complex in smooth muscle tissues expressing wild type Cdc42. The agonist-induced increase in these protein interactions was inhibited in tissues expressing the inactive Cdc42 mutant. We conclude that Cdc42 activation regulates active tension development and actin polymerization during contractile stimulation. Cdc42 may regulate the activation of neuronal Wiskott-Aldrich syndrome protein and the actin related protein complex, which in turn regulate actin filament polymerization initiated by the contractile stimulation of smooth muscle.     

Myosin phosphorylation triggers actin polymerization in vascular smooth muscle

A variety of contractile stimuli increases actin polymerization, which is essential for smooth muscle contraction. However, the mechanism(s) of actin polymerization associated with smooth muscle contraction is not fully understood. We tested the hypothesis that phosphorylated myosin triggers actin polymerization. The present study was conducted in isolated intact or beta-escin-permeabilized rat small mesenteric arteries. Reductions in the 20-kDa myosin regulatory light chain (MLC20) phosphorylation were achieved by inhibiting MLC kinase with ML-7. Increases in MLC20 phosphorylation were achieved by inhibiting myosin light chain phosphatase with microcystin. Isometric force, the degree of actin polymerization as indicated by the F-actin-to-G-actin ratio, and MLC20 phosphorylation were determined. Reductions in MLC20 phosphorylation were associated with a decreased force development and actin polymerization. Increased MLC20 phosphorylation was associated with an increased force generation and actin polymerization. We also found that a heptapeptide that mimics the actin-binding motif of myosin II enhanced microcystin-induced force generation and actin polymerization without affecting MLC20 phosphorylation in beta-escin-permeabilized vessels. Collectively, our data demonstrate that MLC20 phosphorylation is capable of triggering actin polymerization. We further suggest that the binding of myosin to actin triggers actin polymerization and enhances the force development in arterial smooth muscle.     

Depletion of focal adhesion kinase by antisense depresses contractile activation of smooth muscle

Focal adhesion kinase (FAK)undergoes tyrosine phosphorylation in response to the contractilestimulation of tracheal smooth muscle. We hypothesized that FAK mayplay an important role in signaling pathways that regulate smoothmuscle contraction. FAK antisense or FAK sense was introduced intomuscle strips by reversible permeabilization, and strips were incubatedwith antisense or sense for 7 days. Antisense decreased FAK expressioncompared with that in untreated and sense-treated tissues, but it didnot affect the expression of vinculin or myosin light chain kinase. Increases in force, intracellular free Ca²⁺, and myosinlight chain phosphorylation in response to stimulation with ACh or KClwere depressed in FAK-depleted tissues, but FAK depletion did notaffect the activation of permeabilized tracheal muscle strips withCa²⁺. The tyrosine phosphorylation of paxillin, a substratefor FAK, was also significantly reduced in FAK-depleted strips. Weconclude that FAK is a necessary component of the signaling pathwaysthat regulate smooth muscle contraction and that FAK plays a role in regulating intracellular free Ca²⁺ and myosin light chain phosphorylation.

     

The adapter protein CrkII regulates neuronal Wiskott-Aldrich syndrome protein, actin polymerization, and tension development during contractile stimulation of smooth muscle

Actin polymerization has been shown to occur in tracheal smooth muscle tissues and cells in response to contractile stimulation, and there is evidence that the polymerization of actin is required for contraction. In tracheal smooth muscle, agonist-induced actin polymerization is mediated by activation of neuronal Wiskott-Aldrich syndrome protein (N-WASp) and the Arp (actin-related protein) 2/3 complex, and activation of the small GTPase Cdc42 regulates the activation of N-WASp. In the present study, the role of the adapter protein CrkII in the regulation of N-WASp and Cdc42 activation, actin polymerization, and tension development in smooth muscle tissues was evaluated. Stimulation of tracheal smooth muscle tissues with acetylcholine increased the association of CrkII with N-WASp. Plasmids encoding wild type CrkII or a CrkII mutant lacking the SH3 effector-binding ability, CrkII SH3N, were introduced into tracheal smooth muscle tissues, and the tissues were incubated for 2 days to allow for protein expression. Expression of the CrkII SH3N mutant in smooth muscle tissues inhibited the association of CrkII with N-WASp and the activation of Cdc42. The CrkII SH3N mutant also inhibited the increase in the association of N-WASp with Arp2, a major component of the Arp2/3 complex, in response to contractile stimulation, indicating inhibition of N-WASp activation. Expression of the CrkII SH3N mutant also inhibited tension generation and actin polymerization in response to contractile stimulation; however, it did not inhibit myosin light chain phosphorylation. These results suggest that CrkII plays a critical role in the regulation of N-WASp activation, perhaps by regulating the activation of Cdc42, and that it thereby regulates actin polymerization and active tension generation in tracheal smooth muscle. These studies suggest a novel signaling pathway for the regulation of N-WASp activation and active contraction in smooth muscle tissues.     

Upregulation of RGS4 and downregulation of CPI-17 mediate inhibition of colonic muscle contraction by interleukin-1beta

The pro-inflammatory cytokine IL-1beta contributes to the reduced contractile responses of gut smooth muscle observed in both animal colitis models and human inflammatory bowel diseases. However, the mechanisms are not well understood. The effects of IL-1beta on the signaling targets mediating acetylcholine (ACh)-induced initial and sustained contraction were examined using rabbit colonic circular muscle strips and cultured muscle cells. The contraction was assessed through cell length decrease, myosin light chain (MLC(20)) phosphorylation, and activation of PLC-beta and Rho kinase. Expression levels of the signaling targets were determined by Western blot analysis and real-time RT-PCR. Short interfering RNAs (siRNAs) for regulator of G protein signaling 4 (RGS4) were used to silence endogenous RGS4 in muscle strips or cultured muscle cells. IL-1beta treatment of muscle strips inhibited both initial and sustained contraction and MLC(20) phosphorylation in isolated muscle cells. IL-1beta treatment increased RGS4 expression but had no effect on muscarinic receptor binding or Galpha(q) expression. In contrast, IL-1beta decreased the expression and phosphorylation of CPI-17 but had no effect on RhoA expression or ACh-induced Rho kinase activity. Upregulation of RGS4 and downregulation of CPI-17 by IL-1beta in muscle strips were corroborated in cultured muscle cells. Knockdown of RGS4 by siRNA in both muscle strips and cultured muscle cells blocked the inhibitory effect of IL-1beta on initial contraction and PLC-beta activation, whereas overexpression of RGS4 inhibited PLC-beta activation. These data suggest that IL-1beta upregulates RGS4 expression, resulting in the inhibition of initial contraction and downregulation of CPI-17 expression during sustained contraction in colonic smooth muscle.     

Recruitment of β-Catenin to N-Cadherin Is Necessary for Smooth Muscle Contraction

β-Catenin is a key component that connects transmembrane cadherin with the actin cytoskeleton at the cell-cell interface. However, the role of the β-catenin/cadherin interaction in smooth muscle has not been well characterized. Here stimulation with acetylcholine promoted the recruitment of β-catenin to N-cadherin in smooth muscle cells/tissues. Knockdown of β-catenin by lentivirus-mediated shRNA attenuated smooth muscle contraction. Nevertheless, myosin light chain phosphorylation at Ser-19 and actin polymerization in response to contractile activation were not reduced by β-catenin knockdown. In addition, the expression of the β-catenin armadillo domain disrupted the recruitment of β-catenin to N-cadherin. Force development, but not myosin light chain phosphorylation and actin polymerization, was reduced by the expression of the β-catenin armadillo domain. Furthermore, actin polymerization and microtubules have been implicated in intracellular trafficking. In this study, the treatment with the inhibitor latrunculin A diminished the interaction of β-catenin with N-cadherin in smooth muscle. In contrast, the exposure of smooth muscle to the microtubule depolymerizer nocodazole did not affect the protein-protein interaction. Together, these findings suggest that smooth muscle contraction is mediated by the recruitment of β-catenin to N-cadherin, which may facilitate intercellular mechanotransduction. The association of β-catenin with N-cadherin is regulated by actin polymerization during contractile activation.     

Ca2+-induced Ca2+ desensitization of myosin light chain phosphorylation and contraction in phasic smooth muscle

The temporal relationship between Ca2+-induced contraction and phosphorylation of 20 kDa myosin light chain (MLC) during a step increase in Ca2+ was investigated using permeabilized phasic smooth muscle from rabbit portal vein and guinea-pig ileum at 25°C. We describe here a Ca2+-induced Ca2+ desensitization phenomenon in which a transient rise in MLC phosphorylation is followed by a transient rise in contractile force. During and after the peak contraction, the force to phosphorylation ratio remained constant. Further treatment with cytochalasin D, an actin fragmenting agent, did not affect the transient increase in phosphorylation, but blocked force development. Together, these results indicate that the transient phosphorylation causes the transient contraction and that neither inhomogeneous contractility nor reduced thin filament integrity effects the transient phosphorylation. Lastly, we show that known inhibitors to MLC kinase kinases and to a Ca2+-dependent protein phosphatase did not eliminate the desensitized contractile force. This study suggests that the Ca2+-induced Ca2+ desensitization phenomenon in phasic smooth muscle does not result from any of the known intrinsic mechanisms involved with other aspects of smooth muscle contractility.     

The pro-apoptotic protein Par-4 facilitates vascular contractility by cytoskeletal targeting of ZIPK

Par-4 (prostate apoptosis response 4) is a pro-apoptotic protein and tumour suppressor that was originally identified as a gene product up-regulated during apoptosis in prostate cancer cells. Here, we show, for the first time, that Par-4 is expressed and co-localizes with the actin filament bundles in vascular smooth muscle. Furthermore, we demonstrate that targeting of ZIPK to the actin filaments, as observed upon PGF-2α stimulation, is inhibited by the presence of a cell permeant Par-4 decoy peptide. The same decoy peptide also significantly inhibits PGF-2α induced contractions of smooth muscle tissue. Moreover, knockdown of Par-4 using antisense morpholino nucleotides results in significantly reduced contractility, and myosin light chain and myosin phosphatase target subunit phosphorylation. These results indicate that Par-4 facilitates contraction by targeting ZIPK to the vicinity of its substrates, myosin light chain and MYPT, which are located on the actin filaments. These results identify Par-4 as a novel regulator of myosin light chain phosphorylation in differentiated, contractile vascular smooth muscle.     

Phosphatidylinositol 3-kinase modulates vascular smooth muscle contraction by calcium and myosin light chain phosphorylation-independent and -dependent pathways

Regulation of smooth muscle contraction involves a number of signaling mechanisms that include both kinase and phosphatase reactions. The goal of the present study was to determine the role of one such kinase, phosphatidylinositol (PI)3-kinase, in vascular smooth muscle excitation-contraction coupling. Using intact medial strips of the swine carotid artery, we found that inhibition of PI3-kinase by LY-294002 resulted in a concentration-dependent decrease in the contractile response to both agonist stimulation and membrane depolarization-dependent contractions and a decrease in Ca(2+)-dependent myosin light chain (MLC) phosphorylation, the primary step in the initiation of smooth muscle contraction. Inhibition of PI3-kinase also depressed phorbol dibutyrate-induced contractions, which are not dependent on either Ca(2+) or MLC phosphorylation but are dependent on protein kinase C. To determine the Ca(2+)-dependent site of action of PI3-kinase, we determined the effect of several inhibitors of calcium metabolism on LY-294002-dependent inhibition of contraction. These inhibitors included nifedipine, SK&F-96365, and caffeine. Only SK&F-96365 blocked the LY-294002-dependent inhibition of contraction. Interestingly, all compounds blocked the LY-294002-dependent inhibition of MLC phosphorylation. Our results suggest that activation of PI3-kinase is involved in a Ca(2+)- and MLC phosphorylation-independent pathway for contraction likely to involve protein kinase C. In addition, our results also suggest that activation of PI3-kinase is involved in Ca(2+)-dependent signaling at the level of receptor-operated calcium channels.     

Myosin Phosphatase Target Subunit 1 (MYPT1) Regulates the Contraction and Relaxation of Vascular Smooth Muscle and Maintains Blood Pressure

Myosin light chain phosphatase with its regulatory subunit, myosin phosphatase target subunit 1 (MYPT1) modulates Ca²⁺-dependent phosphorylation of myosin light chain by myosin light chain kinase, which is essential for smooth muscle contraction. The role of MYPT1 in vascular smooth muscle was investigated in adult MYPT1 smooth muscle specific knock-out mice. MYPT1 deletion enhanced phosphorylation of myosin regulatory light chain and contractile force in isolated mesenteric arteries treated with KCl and various vascular agonists. The contractile responses of arteries from knock-out mice to norepinephrine were inhibited by Rho-associated kinase (ROCK) and protein kinase C inhibitors and were associated with inhibition of phosphorylation of the myosin light chain phosphatase inhibitor CPI-17. Additionally, stimulation of the NO/cGMP/protein kinase G (PKG) signaling pathway still resulted in relaxation of MYPT1-deficient mesenteric arteries, indicating phosphorylation of MYPT1 by PKG is not a major contributor to the relaxation response. Thus, MYPT1 enhances myosin light chain phosphatase activity sufficient for blood pressure maintenance. Rho-associated kinase phosphorylation of CPI-17 plays a significant role in enhancing vascular contractile responses, whereas phosphorylation of MYPT1 in the NO/cGMP/PKG signaling module is not necessary for relaxation.     

The Association of Cortactin with Profilin-1 Is Critical for Smooth Muscle Contraction

Profilin-1 (Pfn-1) is an actin-regulatory protein that has a role in modulating smooth muscle contraction. However, the mechanisms that regulate Pfn-1 in smooth muscle are not fully understood. Here, stimulation with acetylcholine induced an increase in the association of the adapter protein cortactin with Pfn-1 in smooth muscle cells/tissues. Furthermore, disruption of the protein/protein interaction by a cell-permeable peptide (CTTN-I peptide) attenuated actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser-19. Knockdown of cortactin by lentivirus-mediated RNAi also diminished actin polymerization and smooth muscle force development. However, cortactin knockdown did not affect myosin activation. In addition, cortactin phosphorylation has been implicated in nonmuscle cell migration. In this study, acetylcholine stimulation induced cortactin phosphorylation at Tyr-421 in smooth muscle cells. Phenylalanine substitution at this position impaired cortactin/Pfn-1 interaction in response to contractile activation. c-Abl is a tyrosine kinase that is necessary for actin dynamics and contraction in smooth muscle. Here, c-Abl silencing inhibited the agonist-induced cortactin phosphorylation and the association of cortactin with Pfn-1. Finally, treatment with CTTN-I peptide reduced airway resistance and smooth muscle hyperreactivity in a murine model of asthma. These results suggest that the interaction of cortactin with Pfn-1 plays a pivotal role in regulating actin dynamics, smooth muscle contraction, and airway hyperresponsiveness in asthma. The association of cortactin with Pfn-1 is regulated by c-Abl-mediated cortactin phosphorylation.     

Myosin light-chain kinase regulates endothelial calcium entry and endothelium-dependent vasodilation.

Activation of smooth muscle myosin light-chain kinase (MLCK) causes contraction. Here we have proven that MLCK controls Ca2+ entry (CE) in endothelial cells (ECs): MLCK antisense oligonucleotides strongly prevented bradykinin (BK)- and thapsigargin (TG)-induced endothelial Ca2+ response, while MLCK sense did not. We also show that the relevant mechanism is not phosphorylation of myosin light-chain (MLC): MLC phosphorylation by BK required CE, but MLC phosphorylation caused by the phosphatase inhibitor calyculin A did not trigger Ca2+ response. Most important, we provide for the first time strong evidence that, in contrast to its role in smooth muscle cells, activation of MLCK in ECs stimulates the production of important endothelium-derived vascular relaxing factors: MLCK antisense and MLCK inhibitors abolished BK- and TG-induced nitric oxide production, and MLCK inhibitors substantially inhibited acetylcholine-stimulated hyperpolarization of smooth muscle cell membrane in rat mesenteric artery. These results indicate that MLCK controls endothelial CE, but not through MLC phosphorylation, and unveils a hitherto unknown physiological function of the enzyme: vasodilation through its action in endothelial cells. The study discovers a counter-balancing role of MLCK in the regulation of vascular tone.     

Mechanisms of the Ba2+-induced contraction in smooth muscle cells of the rabbit mesenteric artery

The mechanism of the Ba2+-induced contraction was investigated using intact and saponin-treated skinned smooth muscle (skinned muscle) strips of the rabbit mesenteric artery. After depletion of Ca2+ stored in the caffeine-sensitive site, greater than 0.65 mM Ba2+ evoked contraction in muscle strips depolarized with 128 mM K+ in Ca2+-free solution in a dose-dependent fashion, and the ED50 values for Ca2+ and Ba2+ were 0.5 mM and 1.2 mM in intact muscle strips, respectively. Nisoldipine (10 nM) blocked the contraction evoked by high K+ or 10 microM norepinephrine (NE) in the presence of 2.6 mM Ba2+, but did not block the contraction evoked in the presence of 2.6 mM Ca2+. These results may indicate that Ba2+ permeates the voltage-dependent Ca2+ channel. In skinned muscle strips, the ED50 values for Ca2+ and Ba2+ were 0.34 and 90 microM, respectively, as estimated from the pCa- and pBa-tension relationships. Calmodulin enhanced and trifluoperazine inhibited the Ba2+- and Ca2+-induced contractions. After the application of Ba2+ or Ca2+ with ATP gamma S in rigor solution, myosin light chain (MLC) was irreversibly thiophosphorylated, as estimated from the Ba2+- or Ca2+-independent contraction. Furthermore, both divalent cations phosphorylated MLC, as measured using two-dimensional gel electrophoresis, to the extent expected from the amplitudes of the contraction evoked by these cations. Thus, Ba2+ is capable of activating the contractile proteins as Ca2+ does. The amount of Ca2+ or Ba2+ stored in cells was estimated from the caffeine response evoked in Ca2+-free solution in intact and skinned muscle strips. After the application of 0.3 microM Ca2+ or 0.1 mM Ba2+ for 60 s to skinned muscle strips after the depletion of Ca2+ stored in cells, caffeine produced a contraction only upon pretreatment with Ca2+ but not with Ba2+. When Ba2+ was applied successively just after the application of Ca2+, the subsequently evoked caffeine-induced contraction was much smaller than that evoked by pretreatment with Ca2+ alone. The above results indicate that Ba2+ permeates the voltage-dependent Ca2+ channel but may not permeate the receptor-operated Ca2+ channel, it releases Ca2+ from store sites but is not accumulated into the store site, and it directly activates the contractile proteins via formation of a Ba2+-calmodulin complex.     

siRNA knock down of casein kinase 2 increases force and cross-bridge cycling rates in vascular smooth muscle

Contraction of smooth muscle involves myosin light chain (MLC) kinase catalyzed phosphorylation of the regulatory MLC, activation of myosin, and the development of force. However, this cannot account for all aspects of a smooth muscle contraction, suggesting that other regulatory mechanisms exist. One potentially important technique to study alternative sites of contractile regulation is the use of small interfering RNA (siRNA). The goal of this study was to determine whether siRNA technology can decrease the levels of a specific protein and allow for the determination of how that protein affects contractile regulation. To achieve this goal, we tested the hypothesis that casein kinase 2 (CK2) is part of the complex regulatory scheme present in vascular smooth muscle. Using intact strips of swine carotid artery, we determined that siRNA against CK2 produced a tissue that resulted in a 60% knockdown after 4 days in organ culture. Intact strips of vascular tissue depleted of CK2 produced greater levels of force and exhibited an increased sensitivity to all stimuli tested. This was accompanied by an increase in cross-bridge cycling rates but not by a change in MLC phosphorylation levels. -Toxin-permeabilized vascular tissue depleted of CK2 also showed an increased sensitivity to calcium compared with control tissues. Our results demonstrate that siRNA is a viable technique with which to study regulatory pathways in intact smooth muscle tissue. Our results also demonstrate that CK2 plays an important role in the mechanism(s) responsible for the development of force and cross-bridge cycling by a MLC phosphorylation-independent pathway. myosin light chain phosphorylation; shortening velocity; -toxin permeabilization; swine carotid artery; caldesmon     

Influence of dispersion in myosin filament orientation and anisotropic filament contractions in smooth muscle

A new constitutive model for the biomechanical behaviour of smooth muscle tissue is proposed. The active muscle contraction is accomplished by the relative sliding between actin and myosin filaments, comprising contractile units in the smooth muscle cells. The orientation of the myosin filaments, and thereby the contractile units, are taken to exhibit a statistical dispersion around a preferred direction. The number of activated cross-bridges between the actin and myosin filaments governs the contractile force generated by the muscle and also the contraction speed. A strain-energy function is used to describe the mechanical behaviour of the smooth muscle tissue. Besides the active contractile apparatus, the mechanical model also incorporates a passive elastic part. The constitutive model was compared to histological and isometric tensile test results for smooth muscle tissue from swine carotid artery. In order to be able to predict the active stress at different muscle lengths, a filament dispersion significantly larger than the one observed experimentally was required. Furthermore, a comparison of the predicted active stress for a case of uniaxially oriented myosin filaments and a case of filaments with a dispersion based on the experimental histological data shows that the difference in generated stress is noticeable but limited. Thus, the results suggest that myosin filament dispersion alone cannot explain the increase in active muscle stress with increasing muscle stretch.     

Role of the Adapter Protein Abi1 in Actin-associated Signaling and Smooth Muscle Contraction

Actin filament polymerization plays a critical role in the regulation of smooth muscle contraction. However, our knowledge regarding modulation of the actin cytoskeleton in smooth muscle just begins to accumulate. In this study, stimulation with acetylcholine (ACh) induced an increase in the association of the adapter protein c-Abl interactor 1 (Abi1) with neuronal Wiskott-Aldrich syndrome protein (N-WASP) (an actin-regulatory protein) in smooth muscle cells/tissues. Furthermore, contractile stimulation activated N-WASP in live smooth muscle cells as evidenced by changes in fluorescence resonance energy transfer efficiency of an N-WASP sensor. Abi1 knockdown by lentivirus-mediated RNAi inhibited N-WASP activation, actin polymerization, and contraction in smooth muscle. However, Abi1 silencing did not affect myosin regulatory light chain phosphorylation at Ser-19 in smooth muscle. In addition, c-Abl tyrosine kinase and Crk-associated substrate (CAS) have been shown to regulate smooth muscle contraction. The interaction of Abi1 with c-Abl and CAS has not been investigated. Here, contractile activation induced formation of a multiprotein complex including c-Abl, CAS, and Abi1. Knockdown of c-Abl and CAS attenuated the activation of Abi1 during contractile activation. More importantly, Abi1 knockdown inhibited c-Abl phosphorylation at Tyr-412 and the interaction of c-Abl with CAS. These results suggest that Abi1 is an important component of the cellular process that regulates N-WASP activation, actin dynamics, and contraction in smooth muscle. Abi1 is activated by the c-Abl-CAS pathway, and Abi1 reciprocally controls the activation of its upstream regulator c-Abl.