Cross-bridges In Smooth Muscle Research Articles

An in silico study examining the role of airway smooth muscle dynamics and airway compliance on the rate of airway re-narrowing after deep inspiration

Deep inspirations are a widely studied topic due to their varied effectiveness as a bronchodilator in asthmatic and non-asthmatic patients. Specifically, they are known to be effective at reversing bronchoconstriction in non-asthmatic patients but may fail to prevent bronchoconstriction in asthmatic patients. Inspired by a recent study on the effect of deep inspirations on the rate of re-narrowing of an isolated airway, we investigate whether the latch-bridge dynamics of smooth muscle cross-bridge theory, coupled with non-linear compliance of the airway wall, can account for the reported results: namely that only the rate of renarrowing after DI is sensitive to the interval between deep inspirations, while other measures are unaffected. We develop and present length- and pressure-controlled protocols which mimic both the experiments performed in the study, as well as simulate in vivo conditions respectively. Both protocols are simulated and show qualitative agreement with the results reported by the experiments, suggesting that latch-bridge dynamics coupled with airway wall non-compliance may be sufficient to explain these results. Moreover pressure- and length-controlled protocols show important differences which should be considered when designing in vitro experiments to mimic in vivo conditions.

Experiments and mechanochemical modeling of smooth muscle contraction: Significance of filament overlap

The main function of smooth muscle is to maintain/regulate the size of different hollow organs through contraction and relaxation. The magnitude of the active force during contraction is dependent on the number of attached cross-bridges, which can be linked to the overlap between the thin and thick filaments. The relevance of filament overlap and the active cross-bridges in smooth muscle is investigated through a mechanical model founded on Hill's three-element model. The mechanical model describes a sarcomere-equivalent contractile unit supported by structural observations with a distinct filament overlap and a realistic framework for the filament sliding behavior based on force–velocity experiments. The mechanical model is coupled to the four-state latch-model by Hai and Murphy to capture the electromechanical activation from intracellular calcium concentration to load-bearing cross-bridges. The model is fitted to isometric experiments performed on the pig carotid media and on isotonic quick-release experiments found in the literature. The proposed coupled mechanochemical model with the description of the filament overlap, which has a significant influence on the results, is able to predict isometric experimental data performed at different muscle lengths. The relevance of the filament overlap and the load-bearing cross-bridges is investigated through the model by simulating additional scenarios that has been documented in the literature.

Regulation of the smooth muscle contractile phenotype by nonmuscle myosin

The contractile phenotype of a smooth muscle can broadly be classified as phasic or tonic. Following activation, phasic smooth muscle exhibits an initial period of rapid force activation, following which force falls to a lower steady state level. In contrast, force generated by tonic smooth muscle rises slowly to a sustained steady state. The differences in contractile patterns cannot be explained by the time course of either the Ca(2+) transient or phosphorylation of the 20-kDa regulatory myosin light chain (MLC(20)). Therefore, a molecular marker that defines tonic and phasic smooth muscle contractile properties remains elusive. Further, smooth muscle can maintain force at low levels of MLC(20) phosphorylation; often referred to as the latch state. The mechanism for the latch state is unknown and has been hypothesized to be due to a number of mechanisms including the formation of slowly cycling dephosphorylated or latch cross-bridges (Hai and Murphy, Am J Physiol 253:H1365-H1371, 1988). This review will focus evidence suggesting that nonmuscle myosin IIB (NMIIB) are the latch cross-bridges in smooth muscle and NMIIB content could define the tonic contractile phenotype.

Electron Tomography of Swollen Rigor Fibers of Insect Flight Muscle Reveals a Short and Variably Angled S2 Domain

Subfragment 2 (S2), the segment that links the two myosin heads to the thick filament backbone, may serve as a swing-out adapter allowing crossbridge access to actin, as the elastic component of crossbridges and as part of a phosphorylation-regulated on-off switch for crossbridges in smooth muscle. Low-salt expansion increases interfilament spacing (from 52 nm to 67 nm) of rigor insect flight muscle fibers and exposes a tethering segment of S2 in many crossbridges. Docking an actoS1 atomic model into EM tomograms of swollen rigor fibers identifies in situ for the first time the location, length and angle assignable to a segment of S2. Correspondence analysis of 1831 38.7 nm crossbridge repeats grouped self-similar forms from which class averages could be computed. The full range of the variability in angles and lengths of exposed S2 was displayed by using class averages for atomic fittings of acto-S1, while S2 was modeled by fitting a length of coiled-coil to unaveraged individual repeats. This hybrid modeling shows that the average length of S2 tethers along the thick filament (except near the tapered ends) is ∼10 nm, or 16% of S2′s total length, with an angular range encompassing 90° axially and 120° azimuthally. The large range of S2 angles indicates that some rigor bridges produce positive force that must be balanced by others producing drag force. The short tethering segment clarifies constraints on the function of S2 in accommodating variable myosin head access to actin. We suggest that the short length of S2 may also favor intermolecular head−head interactions in IFM relaxed thick filaments.

The Effects of Isoflurane on Airway Smooth Muscle Crossbridge Kinetics in Fisher and Lewis Rats

Our aim was to determine how isoflurane modified crossbridge (CB) number and kinetics in airway smooth muscle (ASM) and to compare its effects in Fisher and Lewis rats, two strains with differences in airway responsiveness. The effects of isoflurane (2 MAC) on isotonic and isometric contractility in tracheal ASM strips were investigated after methacholine (10(-6) M)-induced contraction. CB mechanics and kinetics were analyzed using the formalism of Huxley's equations adapted to ASM. After isoflurane, maximum velocity did not differ from baseline in Lewis rats, whereas it was significantly less than baseline in Fisher rats ( approximately 25%), the most reactive strain. Isoflurane totally reversed methacholine-induced increase in active CB number in Lewis rats (2.4 +/- 0.5 versus 1.8 +/- 0.4 10(9)/mm(2) after methacholine and isoflurane, respectively) whereas reversal was only partial in Fisher rats (2.7 +/- 0.4 versus 2.1 +/- 0.3 10(9)/mm(2) after methacholine and isoflurane, respectively). Isoflurane induced a 40% increase in attachment step duration in both strains and an almost twofold increase in the CB cycle duration compared with baseline in Lewis rats. The isoflurane-induced increase in detachment step duration was less in Lewis than in Fisher rats (P < 0.05). We concluded that isoflurane modulated CB number and CB cycling rates of isolated rat ASM differently depending on the level of airway responsiveness.

The Smooth Muscle Cross-bridge Cycle Studied Using Sinusoidal Length Perturbations

The mechanical characteristics of smooth muscle can be broadly defined as either phasic, or fast contracting, and tonic, or slow contracting (Somlyo and Somlyo, 1968, Pharmacol. Rev. 20:197–272). To determine if differences in the cross-bridge cycle and/or distribution of the cross-bridge states could contribute to differences in the mechanical properties of smooth muscle, we determined force and stiffness as a function of frequency in Triton-permeabilized strips of rabbit portal vein (phasic) and aorta (tonic). Permeabilized muscle strips were mounted between a piezoelectric length driver and a piezoresistive force transducer. Muscle length was oscillated from 1 to 100 Hz, and the stiffness was determined as a function of frequency from the resulting force response. During calcium activation (pCa 4, 5 mM MgATP), force and stiffness increased to steady-state levels consistent with the attachment of actively cycling cross-bridges. In smooth muscle, because the cross-bridge states involved in force production have yet to be elucidated, the effects of elevation of inorganic phosphate (P i) and MgADP on steady-state force and stiffness were examined. When portal vein strips were transferred from activating solution (pCa 4, 5 mM MgATP) to activating solution with 12 mM P i, force and stiffness decreased proportionally, suggesting that cross-bridge attachment is associated with P i release. For the aorta, elevating P i decreased force more than stiffness, suggesting the existence of an attached, low-force actin-myosin-ADP- P i state. When portal vein strips were transferred from activating solution (pCa 4, 5 mM MgATP) to activating solution with 5 mM MgADP, force remained relatively constant, while stiffness decreased ∼50%. For the aorta, elevating MgADP decreased force and stiffness proportionally, suggesting for tonic smooth muscle that a significant portion of force production is associated with ADP release. These data suggest that in the portal vein, force is produced either concurrently with or after P i release but before MgADP release, whereas in aorta, MgADP release is associated with a portion of the cross-bridge powerstroke. These differences in cross-bridge properties could contribute to the mechanical differences in properties of phasic and tonic smooth muscle.

The ADP Release Step of the Smooth Muscle Cross-Bridge Cycle Is Not Directly Associated with Force Generation

When smooth muscle myosin subfragment 1 (S1) is bound to actin filaments in vitro, the light chain domain tilts upon release of MgADP, producing a ∼3.5-nm axial motion of the head-rod junction (Whittaker et al., 1995. Nature. 378:748–751). If this motion contributes significantly to the power stroke, rigor tension of smooth muscle should decrease substantially in response to cross-bridge binding of MgADP. To test this prediction, we monitored mechanical properties of permeabilized strips of chicken gizzard muscle in rigor and in the presence of MgADP. For comparison, we also tested psoas and soleus muscle fibers. Any residual bound ADP was minimized by incubation in Mg2+-free rigor solution containing 15mM EDTA. The addition of 2mM MgADP, while keeping ionic strength and free Mg2+ concentration constant, resulted in a slight increase in rigor tension in both gizzard and soleus muscles, but a decrease in psoas muscle. In-phase stiffness monitored during small (<0.1%) 500-Hz sinusoidal length oscillations decreased in all three muscle types when MgADP was added. The changes in force and stiffness with the addition of MgADP were similar at ionic strengths from 50 to 200mM and were reversible. The results with gizzard muscle were similar after thiophosphorylation of the regulatory light chain of myosin. These results suggest that the axial motion of smooth muscle S1 bound to actin, upon dissociation of MgADP, is not associated with force generation. The difference between the present mechanical data and previous structural studies of smooth S1 may be explained if geometrical constraints of the intact contractile filament array alter the motions of the myosin heads.

Regulation of the cross-bridge cycle: the effects of MgADP, LC17 isoforms and telokin.

This review summarizes the role of MgADP in force maintenance by dephosphorylated cross-bridges in smooth muscle and a potential physiological role for telokin. In tonic, compared with phasic, smooth muscles the affinity of cross-bridges in approximately 5 times higher for MgADP and the apparent second-order rate constant for MgATP is approximately 3 times lower. This gives rise to a large population of dephosphorylated cross-bridges in tonic smooth muscle. Such cross-bridges are thought to be major determinants of the different relaxation kinetics of the two types of smooth muscle and contribute to force maintenance at low levels of MLC20 phosphorylation, termed 'catch-like state' (Somlyo & Somlyo 1967) or 'latch' (Dillon et al. 1981). The molecular basis of the different affinities for MgADP and MgATP between tonic and phasic smooth muscle myosin was explored by exchange of essential myosin light chain (LC17) isoforms. In phasic bladder smooth muscle the exchange of LC17b for LC17a caused a significant decrease in the unloaded shortening velocity of non-phosphorylated, slowly cycling cross-bridges, suggesting that the LC17 isoforms contribute to the nucleotide affinity of latch bridges. The role of telokin in Ca(2+)-desensitization in phasic smooth muscle is reviewed. Telokin, the independently expressed C-terminus of myosin light chain kinase, is extensively phosphorylated during forskolin- and 8-br-cGMP-induced relaxation in situ. Telokin accelerated dephosphorylation of the regulatory myosin light chain and relaxed rabbit ileum smooth muscle. The results suggest that telokin contributes to cAMP and/or cGMP kinase-mediated Ca(2+)-desensitization of phasic smooth muscles.

The role of MgADP in force maintenance by dephosphorylated cross-bridges in smooth muscle: a flash photolysis study

The effect of [MgADP] on relaxation from isometric tension, initiated by reducing free [Ca2+] through photolysis of the caged photolabile Ca2+ chelator diazo-2, was determined at 20 degrees C in alpha-toxin permeabilized tonic (rabbit femoral artery, Rf) and phasic (rabbit bladder, Rb) smooth muscle. In Rf, the shape of the relaxation curve was clearly biphasic, consisting of a slow "plateau" phase followed by a monotonic exponential decline with rate constant k. The duration of the plateau (d = 44 +/- 4 s, mean +/- SEM, n = 28) was well correlated (R = 0.92) with the total t1/2 of relaxation that was 66 +/- 3 s (n = 28) in the presence of 20 mM creatine phosphate (CP), and was prolonged in the absence of CP (t1/2 = 83 +/- 3 s, n = 7); addition of 100 microM MgADP further slowed relaxation (t1/2 = 132 +/- 7 s, n = 14). In Rb, a plateau was not detectable and t1/2 (= 15 +/- 2 s, n = 6) was not affected by 100 microM MgADP. In Rf the Q10 between 20 degrees C and 30 degrees C was 4.3 +/- 0.4 for d-1 and 2.8 +/- 0.3 for k (n = 8; p = 0.006). The regulatory myosin light chain (MLC20) in Rf was dephosphorylated at 0.07 +/- 0.02 s-1, from 42 +/- 3% before to 20 +/- 2% after photolysis of diazo-2, reaching basal values at a time when force had fallen by only 40%. We conclude that, in the presence of ATP, as during rigor, the affinity of dephosphorylated cross-bridges for MgADP is significantly higher in tonic than in phasic smooth muscle and contributes to the maintenance of force at low levels of phosphorylation. The MgADP dependence of the post-dephosphorylation phase of relaxation is consistent with its being rate-limited by the slow off-rate of ADP from cross-bridges that were dephosphorylated while in force-generating ADP-bound (AM*D) cross-bridge states. The fourfold faster off-rate of ADP from AM*D in the phasic, Rb, compared to tonic, Rf, smooth muscle is a major determinant of the different kinetics of relaxation in the two types of smooth muscle.

What is special about smooth muscle? The significance of covalent crossbridge regulation.

Knowledge of the molecular mechanisms involved in chemomechanical transduction and the regulatory elements determining contraction and relaxation are largely derived from cross-striated muscle. However, it has become clear that vertebrate smooth muscles have features lacking in striated muscle. These derive from regulated crossbridge cycling rates observed as variable shortening velocities and ATP consumption rates. The special properties of smooth muscle are associated with differences in the physiological roles of muscle in the walls of hollow organs and reflect a common molecular motor governed by different regulatory mechanisms. A remarkably simple scheme involving Ca(2+)-dependent phosphorylation of crossbridges in smooth muscle can predict much of the mechanical and energetics behavior characterizing the muscle of hollow organs. Nevertheless, many unresolved issues are identified that are the focus of current research efforts.

ADP release from myosin in permeabilized smooth muscle

The purpose of this study was to determine the nucleotide bound to myosin and its rate of release under relaxed and activated conditions in permeabilized rabbit portal veins. Incubation of the muscles in a relaxing solution containing [3H]-ATP resulted in the formation of 60-70 microM radiolabeled ADP in the muscle whether or not the myosin light chains had been thiophosphorylated. This value was similar to the estimate of the concentration of myosin subfragment 1. Upon transfer of the muscles to a chase solution containing no labeled ATP, there was a very slow loss of labeled ADP when the light chains were unphosphorylated, but a much faster release occurred when the light chains were thiophosphorylated. The results suggest that smooth muscle myosin exists primarily in a complex with ADP under both relaxed and phosphorylated conditions and that phosphorylation of all of the light chains results in a large increase in the rate of release of the products of ATP splitting from all of the myosin. Interestingly, the exponential release of ADP in relaxed muscle shows two components, one of which contains about two-thirds of the total ADP and is 5- to 10-fold faster than the other. If the difference in rates of ADP release observed in relaxed muscle persists when the myosin is phosphorylated, then it is possible that there is a 5- to 10-fold difference in rates of cycling for different phosphorylated cross bridges in smooth muscle.

Cross-bridge elasticity in single smooth muscle cells.

In smooth muscle, a cross-bridge mechanism is believed to be responsible for active force generation and fiber shortening. In the present studies, the viscoelastic and kinetic properties of the cross-bridge were probed by eliciting tension transients in response to small, rapid, step length changes (delta L = 0.3-1.0% Lcell in 2 ms). Tension transients were obtained in a single smooth muscle cell isolated from the toad (Bufo marinus) stomach muscularis, which was tied between a force transducer and a displacement device. To record the transients, which were of extremely small magnitude (0.1 microN), a high-frequency (400 Hz), ultrasensitive force transducer (18 mV/microN) was designed and built. The transients obtained during maximal force generation (Fmax = 2.26 microN) were characterized by a linear elastic response (Emax = 1.26 X 10(4) mN/mm2) coincident with the length step, which was followed by a biphasic tension recovery made up of two exponentials (tau fast = 5-20 ms, tau slow = 50-300 ms). During the development of force upon activation, transients were elicited. The relationship between stiffness and force was linear, which suggests that the transients originate within the cross-bridge and reflect the cross-bridge's viscoelastic and kinetic properties. The observed fiber elasticity suggests that the smooth muscle cross-bridge is considerably more compliant than in fast striated muscle. A thermodynamic model is presented that allows for an analysis of the factors contributing to the increased compliance of the smooth muscle cross-bridge.