Actomyosin Cross-bridge Cycling Research Articles

Insights into posttranslational regulation of skeletal muscle contractile function by the acetyltransferases, p300 and CBP.

Mice with skeletal muscle-specific and inducible double knockout of the lysine acetyltransferases, p300 (E1A binding protein p300) and CBP (cAMP-response element-binding protein binding protein), referred to as i-mPCKO, demonstrate a dramatic loss of contractile function in skeletal muscle and ultimately die within 7 days. Given that many proteins involved in ATP generation and cross-bridge cycling are acetylated, we investigated whether these processes are dysregulated in skeletal muscle from i-mPCKO mice and, thus, whether they could underlie the rapid loss of muscle contractile function. Just 4-5 days after inducing knockout of p300 and CBP in skeletal muscle from adult i-mPCKO mice, there was ∼90% reduction in ex vivo contractile function in the extensor digitorum longus (EDL) and a ∼65% reduction in in vivo ankle dorsiflexion torque, as compared with wild type (WT; i.e., Cre negative) littermates. Despite this profound loss of contractile force in i-mPCKO mice, there were no genotype-driven differences in fatigability during repeated contractions, nor were there genotype differences in mitochondrial-specific pathway enrichment of the proteome, intermyofibrillar mitochondrial volume, or mitochondrial respiratory function. As it relates to cross-bridge cycling, remarkably, the overt loss of contractile function in i-mPCKO muscle was reversed in permeabilized fibers supplied with exogenous Ca2+ and ATP, with active tension being similar between i-mPCKO and WT mice, regardless of Ca2+ concentration. Actin-myosin motility was also similar in skeletal muscle from i-mPCKO and WT mice. In conclusion, neither mitochondrial abundance/function, nor actomyosin cross-bridge cycling, are the underlying driver of contractile dysfunction in i-mPCKO mice.NEW & NOTEWORTHY The mechanism underlying dramatic loss of muscle contractile function with inducible deletion of both E1A binding protein p300 (p300) and cAMP-response element-binding protein binding protein (CBP) in skeletal muscle remains unknown. Here, we find that impairments in mitochondrial function or cross-bridge cycling are not the underlying mechanism of action. Future work will investigate other aspects of excitation-contraction coupling, such as Ca2+ handling and membrane excitability, as contractile function could be rescued by permeabilizing skeletal muscle, which provides exogenous Ca2+ and bypasses membrane depolarization.

Focal adhesion kinase activation is involved in contractile stimulation-induced detrusor muscle contraction in mice

Recent studies suggested smooth muscle contraction may involve mechanisms besides the myosin regulatory light chain (MLC) phosphorylation-induced actomyosin crossbridge cycling. This study aims to determine if focal adhesion kinase (FAK) activation is involved in mouse detrusor muscle contraction. The mouse detrusor muscle strips were preincubated with PF-573228 (2 μM), latrunculin B (1 μM), or the same volume of vehicle (DMSO) for 30 min. The contractile responses to KCl (90 mM), electrical field stimulation (EFS, 2–32 Hz), or carbachol (CCh, 10−7.5-10−4.5 M) were measured. In a separate experiment, the phosphorylated FAK (p-FAK) and MLC (p-MLC) levels were measured in the detrusor strips stimulated with CCh (10 μM) after incubation with PF-573228 or vehicle (DMSO) compared to those with vehicle incubation but without CCh stimulation. KCl-induced contractile responses decreased significantly after incubation with PF-573228 or latrunculin B compared to the corresponding vehicle-treated strips (p < 0.0001). The contractile responses induced by EFS were markedly inhibited by preincubation with PF-573228 at 8, 16, and 32 Hz (p < 0.05) or latrunculin B at 16 and 32 Hz (p < 0.01). Following the application of PF-573228 or Latrunculin B, CCh-induced dose-response contractions were lower than the corresponding vehicle group (p = 0.0021 and 0.0003, respectively). Western blot examination showed that CCh stimulation enhanced the expression of p-FAK and p-MLC, while preincubation with PF-573228 prevented the increase of p-FAK but not p-MLC. In conclusion, FAK activation involves tension development induced by contractile stimulation in the mouse detrusor muscle. This effect is likely caused by promoting actin polymerization rather than elevating MLC phosphorylation.

Allele-Selective Knockdown of MYH7 Using Antisense Oligonucleotides

Hundreds of dominant-negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy, and the biomechanical link between mutation and disease is heterogeneous across this patient population. To increase the therapeutic feasibility of treating this diverse genetic population, we investigated the ability of locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to selectively knock down mutant myosin transcripts by targeting single-nucleotide polymorphisms (SNPs) that were found to be common in the myosin heavy chain 7 (MYH7) gene. We identified three SNPs in MYH7 and designed ASO libraries to selectively target either the reference or alternate MYH7 sequence. We identified ASOs that selectively knocked down either the reference or alternate allele at all three SNP regions. We also show allele-selective knockdown in a mouse model that was humanized on one allele. These results suggest that SNP-targeting ASOs are a promising therapeutic modality for treating cardiac pathology.

Association of cardiac myosin-binding protein-C with the ryanodine receptor channel - putative retrograde regulation?

ABSTRACTThe cardiac muscle ryanodine receptor-Ca2+ release channel (RyR2) constitutes the sarcoplasmic reticulum (SR) Ca2+ efflux mechanism that initiates myocyte contraction, while cardiac myosin-binding protein-C (cMyBP-C; also known as MYBPC3) mediates regulation of acto-myosin cross-bridge cycling. In this paper, we provide the first evidence for the presence of direct interaction between these two proteins, forming a RyR2–cMyBP-C complex. The C-terminus of cMyBP-C binds with the RyR2 N-terminus in mammalian cells and the interaction is not mediated by a fibronectin-like domain. Notably, we detected complex formation between both recombinant cMyBP-C and RyR2, as well as between the native proteins in cardiac tissue. Cellular Ca2+ dynamics in HEK293 cells is altered upon co-expression of cMyBP-C and RyR2, with lowered frequency of RyR2-mediated spontaneous Ca2+ oscillations, suggesting that cMyBP-C exerts a potential inhibitory effect on RyR2-dependent Ca2+ release. Discovery of a functional RyR2 association with cMyBP-C provides direct evidence for a putative mechanistic link between cytosolic soluble cMyBP-C and SR-mediated Ca2+ release, via RyR2. Importantly, this interaction may have clinical relevance to the observed cMyBP-C and RyR2 dysfunction in cardiac pathologies, such as hypertrophic cardiomyopathy.

Association of Cardiac Myosin Binding Protein-C with the Ryanodine Receptor-Ca2+ Release Channel: Putative Retrograde Regulation?

Cardiac ryanodine receptor (RyR2) mediates the sarcoplasmic reticulum (SR) Ca2+ efflux that induces cardiomyocyte contraction, whereas sarcomeric myosin binding protein-C (cMyBP-C) regulates actomyosin cross-bridge cycling. We report the first evidence for direct interaction between these two essential proteins using several complementary methods, suggesting existence of a RyR2:cMyBP-C complex. Our studies indicate the C-terminus of cMyBP-C binds to the RyR2 N-terminus in mammalian cells with minimal requirement of a fibronectin-like type 3 domain. Importantly, we observe that specific binding between both recombinant cMyBP-C and RyR2 also occurs with the native proteins extracted from cardiac tissue. Co-expression of cMyBP-C and RyR2 in HEK293 cells alters cellular Ca2+ dynamics, with reduced frequency of RyR2-mediated spontaneous Ca2+ oscillations, suggesting a potential inhibitory effect of cMyBP-C on RyR2 Ca2+ release. Our discovery of direct, functional cMyBP-C association with RyR2 provides novel evidence for putative linkage between sarcomeric cMyBP-C regulation of myofilament contraction and SR-mediated Ca2+ release via RyR2. A distinct mechanism for retrograde sarcomere regulation of SR RyR2 channels may exist, with significant relevance to cMyBP-C and RyR2 dysfunction observed in inherited cardiac disorders.

Non-muscle (NM) myosin heavy chain phosphorylation regulates the formation of NM myosin filaments, adhesome assembly and smooth muscle contraction.

Non-muscle (NM) and smooth muscle (SM) myosin II are both expressed in smooth muscle tissues, however the role of NM myosin in SM contraction is unknown. Contractile stimulation of tracheal smooth muscle tissues stimulates phosphorylation of the NM myosin heavy chain on Ser1943 and causes NM myosin filament assembly at the SM cell cortex. Expression of a non-phosphorylatable NM myosin mutant, NM myosin S1943A, in SM tissues inhibits ACh-induced NM myosin filament assembly and SM contraction, and also inhibits the assembly of membrane adhesome complexes during contractile stimulation. NM myosin regulatory light chain (RLC) phosphorylation but not SM myosin RLC phosphorylation is regulated by RhoA GTPase during ACh stimulation, and NM RLC phosphorylation is required for NM myosin filament assembly and SM contraction. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin. The molecular function of non-muscle (NM) isoforms of myosin II in smooth muscle (SM) tissues and their possible role in contraction are largely unknown. We evaluated the function of NM myosin during contractile stimulation of canine tracheal SM tissues. Stimulation with ACh caused NM myosin filament assembly, as assessed by a Triton solubility assay and a proximity ligation assay aiming to measure interactions between NM myosin monomers. ACh stimulated the phosphorylation of NM myosin heavy chain on Ser1943 in tracheal SM tissues, which can regulate NM myosin IIA filament assembly in vitro. Expression of the non-phosphorylatable mutant NM myosin S1943A in SM tissues inhibited ACh-induced endogenous NM myosin Ser1943 phosphorylation, NM myosin filament formation, the assembly of membrane adhesome complexes and tension development. The NM myosin cross-bridge cycling inhibitor blebbistatin suppressed adhesome complex assembly and SM contraction without inhibiting NM myosin Ser1943 phosphorylation or NM myosin filament assembly. RhoA inactivation selectively inhibited phosphorylation of the NM myosin regulatory light chain (RLC), NM myosin filament assembly and contraction, although it did not inhibit SM RLC phosphorylation. We conclude that the assembly and activation of NM myosin II is regulated during contractile stimulation of airway SM tissues by RhoA-mediated NM myosin RLC phosphorylation and by NM myosin heavy chain Ser1943 phosphorylation. NM myosin II actomyosin cross-bridge cycling regulates the assembly of membrane adhesome complexes that mediate the cytoskeletal processes required for tension generation. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin.

MP94-10 THE VISCOELASTIC, REVERSIBLY PLASTIC, BEHAVIOR OF DETRUSOR SMOOTH MUSCLE EXPLAINS BLADDER HIGH COMPLIANCE

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology II1 Apr 2017MP94-10 THE VISCOELASTIC, REVERSIBLY PLASTIC, BEHAVIOR OF DETRUSOR SMOOTH MUSCLE EXPLAINS BLADDER HIGH COMPLIANCE Randy Vince, John Speich, Adam Klausner, Christopher Neal, Amy Miner, and Paul Ratz Randy VinceRandy Vince More articles by this author , John SpeichJohn Speich More articles by this author , Adam KlausnerAdam Klausner More articles by this author , Christopher NealChristopher Neal More articles by this author , Amy MinerAmy Miner More articles by this author , and Paul RatzPaul Ratz More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2913AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES : Biological soft tissues are viscoelastic materials because they display time-independent pseudo-elasticity and time-dependent viscosity. Upon an imposed ramp increase then decrease in strain, the resultant increase and decrease in stress, termed loading and unloading respectively, produce nonlinear stress-strain curves, and a reversible reduction in the stress-strain work area that identifies the viscous component. However, there is evidence that bladder tissue may also display plastic behavior, defined as an increase in strain that is unrecoverable unless work is done by the material. In the present study, an electronic lever was used to induce controlled changes in stress and strain to determine whether the material properties of rabbit detrusor smooth muscle (rDSM) represent a viscoelastic or viscoelastic-plastic material. METHODS Strips of DSM free from underlying mucosa were removed from rabbit bladders. Each tissue was placed in an organ bath connected to an electronic lever and length-adjuster and subjected to a length-tension protocol to identify the length (Lref) that produced the strongest active force induced by KCl. Each ring was subsequently set to 80% Lref and subjected to sequential ramp loading and unloading cycles, stress-strain and stiffness-stress analyses, and a step-loading, load-clamp, step-unloading (creep) protocol. RESULTS Ramp loading-unloading cycles revealed that rDSM displayed reversible viscoelasticity. The viscous component was responsible for establishing a high stiffness at low stresses that increased only modestly with increasing stress compared to the large increase produced when the viscosity was absent and only pseudo-elasticity governed tissue behavior. The creep protocol revealed that rDSM underwent extensive softening correlating with plastic deformation and creep that was reversible upon activation of muscle contraction. Softening reversal was prevented by inhibitors of actomyosin crossbridge cycling. CONCLUSIONS Together, the data support a model of DSM as exhibiting not only viscoelasticity, but also plasticity, the degree of which is controlled by the degree of motor protein activation. This model explains the mechanism of instrinsic bladder compliance as “slipping” crossbridges, predicts that wall tension is dependent not only on vesicle pressure and radius but also on actomyosin crossbridge activity, and identifies a novel molecular target for compliance regulation both physiologically and therapeutically. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1249 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Randy Vince More articles by this author John Speich More articles by this author Adam Klausner More articles by this author Christopher Neal More articles by this author Amy Miner More articles by this author Paul Ratz More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Muscle weakness inTPM3-myopathy is due to reduced Ca2+-sensitivity and impaired acto-myosin cross-bridge cycling in slow fibres

Dominant mutations in TPM3, encoding α-tropomyosinslow, cause a congenital myopathy characterized by generalized muscle weakness. Here, we used a multidisciplinary approach to investigate the mechanism of muscle dysfunction in 12 TPM3-myopathy patients. We confirm that slow myofibre hypotrophy is a diagnostic hallmark of TPM3-myopathy, and is commonly accompanied by skewing of fibre-type ratios (either slow or fast fibre predominance). Patient muscle contained normal ratios of the three tropomyosin isoforms and normal fibre-type expression of myosins and troponins. Using 2D-PAGE, we demonstrate that mutant α-tropomyosinslow was expressed, suggesting muscle dysfunction is due to a dominant-negative effect of mutant protein on muscle contraction. Molecular modelling suggested mutant α-tropomyosinslow likely impacts actin-tropomyosin interactions and, indeed, co-sedimentation assays showed reduced binding of mutant α-tropomyosinslow (R168C) to filamentous actin. Single fibre contractility studies of patient myofibres revealed marked slow myofibre specific abnormalities. At saturating [Ca(2+)] (pCa 4.5), patient slow fibres produced only 63% of the contractile force produced in control slow fibres and had reduced acto-myosin cross-bridge cycling kinetics. Importantly, due to reduced Ca(2+)-sensitivity, at sub-saturating [Ca(2+)] (pCa 6, levels typically released during in vivo contraction) patient slow fibres produced only 26% of the force generated by control slow fibres. Thus, weakness in TPM3-myopathy patients can be directly attributed to reduced slow fibre force at physiological [Ca(2+)], and impaired acto-myosin cross-bridge cycling kinetics. Fast myofibres are spared; however, they appear to be unable to compensate for slow fibre dysfunction. Abnormal Ca(2+)-sensitivity in TPM3-myopathy patients suggests Ca(2+)-sensitizing drugs may represent a useful treatment for this condition.

Myosin MgADP Release Rate Decreases at Longer Sarcomere Length to Prolong Myosin Attachment in Skinned Rat Myocardial Strips

Cardiac contractility increases as sarcomere length (SL) increases, suggesting that intrinsic molecular mechanisms underlie the Frank-Starling relationship to confer increased cardiac output with greater ventricular filling. Myosin's capacity to generate force is Ca2+-regulated by thin-filament proteins and sarcomere length, which dictates the number of potential actin-myosin cross-bridge interactions. One mechanism underlying greater cardiac contractility at longer SL could involve longer myosin attachment duration (ton). To test this idea, we used stochastic length-perturbation analysis in skinned rat papillary muscle strips to measure ton as [MgATP] varied (0.05-5 mM) at 1.9 and 2.2 µm SL. From this ton-MgATP relationship, we calculated cross-bridge MgADP release rate (k-ADP) and MgATP binding rate (k+ATP). As MgATP increased ton decreased hyperbolically for both SL, but ton was roughly 50% longer for 2.2 vs. 1.9 µm SL at each [MgATP] (25±3 vs. 16±1 ms at 5 mM MgATP, 17° C, p<0.05). These ton differences arose from slower k-ADP at 2.2 µm SL (42±3 vs. 74±8 s−1, p<0.001), as MgATP binding rates did not differ with SL (281±56 vs. 327±93 mM−1 s−1). Absolute tension values were greater at 2.2 vs. 1.9 µm SL for relaxed (4.4±0.7 vs. 0.8±0.2 kPa at pCa 8.0, p<0.001) and maximally activated (20.0±1.4 vs. 14.2±1.6 kPa at pCa 4.8, p<0.001) conditions, and the force-pCa relationship was more sensitive to Ca2+ at 2.2 µm SL (pCa50=5.45±0.01 vs. 5.36±0.01, p<0.05). These increased tension values suggest that cross-bridges may bear greater loads at longer SL, which diminishes MgADP release to prolong ton and amplify cooperative cross-bridge contributions to thin filament activation. Therefore, load-dependent rates of the actomyosin cross-bridge cycle may vary with SL to contribute, in part, to the Frank-Starling relationship in the heart.

3-D structural analysis of the crucial intermediate of skeletal muscle myosin and its role in revised actomyosin cross-bridge cycle.

Skeletal myosin S1 consists of two functional segments, a catalytic-domain and a lever-arm. Since the crystal structure of ADP/Vi-bound S1 exhibits a strong intramolecular flexure between two segments, inter-conversion between bent and extended forms; i.e. “tilting of the lever-arm” has been accepted as the established molecular mechanism of skeletal muscle contraction. We utilized quick-freeze deep-etch replica electron microscopy to directly visualize the structure of in vitro actin-sliding myosin, and found the existence of a novel oppositely-bent configuration, instead of the expected ADP/Vi-bound form. We also noticed that SH1–SH2 cross-linked myosin gives an aberrant appearance similar to the above structure. Since SH1–SH2-cross-linked myosin is a well-studied analogue of the transient intermediate of the actomyosin cross-bridge cycle, we devised a new image-processing procedure to define the relative view-angles between the catalytic-domain and the lever-arm from those averaged images, and built a 3-D model of the new conformer. The lever-arm in that model was bent oppositely to the ADP/Vi-bound form, in accordance with observed actin-sliding cross-bridge structure. Introducing this conformer as the crucial intermediate that transiently appears during sliding, we propose a revised scheme of the cross-bridge cycle. In the scenario, the novel conformer keeps actin-binding in two different modes until it forms a primed configuration. The final extension of the lever-arm back to the original rigor-state constitutes the “power-stroke”. Various images observed during sliding could be easily interpreted by the new conformer. Even the enigmatic behavior of the cross-bridges reported as “loose chemo-mechanical coupling” might be adequately explained under some assumptions.

Slowed Dynamics of Thin Filament Regulatory Units Reduces Ca2+-Sensitivity of Cardiac Biomechanical Function

Actomyosin kinetics in both skinned skeletal muscle fibers at maximum Ca2+-activation and unregulated in vitro motility assays are modulated by solvent microviscosity in a manner consistent with a diffusion limited process. Viscosity might also influence cardiac thin filament Ca2+-regulatory protein dynamics. In vitro motility assays were conducted using thin filaments reconstituted with recombinant human cardiac troponin and tropomyosin; solvent microviscosity was varied by addition of sucrose or glucose. At saturating Ca2+, filament sliding speed (s) was inversely proportional to viscosity. Ca2+-sensitivity (pCa50 ) of s decreased markedly with elevated viscosity (η/η0 ≥ ~1.3). For comparison with unloaded motility assays, steady-state isometric force (F) and kinetics of isometric tension redevelopment (kTR ) were measured in single, permeabilized porcine cardiomyocytes when viscosity surrounding the myofilaments was altered. Maximum Ca2+-activated F changed little for sucrose ≤ 0.3 M (η/η0 ~1.4) or glucose ≤ 0.875 M (η/η0 ~1.66), but decreased at higher concentrations. Sucrose (0.3 M) or glucose (0.875 M) decreased pCa50 for F. kTR at saturating Ca2+ decreased steeply and monotonically with increased viscosity but there was little effect on kTR at sub-maximum Ca2+. Modeling indicates that increased solutes affect dynamics of cardiac muscle Ca2+-regulatory proteins to a much greater extent than actomyosin cross-bridge cycling.

Muscle fatigue and muscle weakness: what we know and what we wish we did

Muscle fatigue and muscle weakness: what we know and what we wish we did

Tuning the Calcium Sensitivity of Cardiac Muscle

Tuning the Calcium Sensitivity of Cardiac Muscle

Bioenergetic Supply and Demand in the Cardiomyocyte

The mechanisms that regulate the control of energy demand and supply in the myocardium are crucial for maintaining normal cardiac function. Although a number of mechanisms have been proffered by which mitochondrial supply of ATP can change to match varying workload in the myocardium, identifying the underlying regulatory pathways remains controversial. We describe an approach to studying this problem in which thermodynamically consistent mathematical models of the key energy-consuming processes in the cardiomyocyte (sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA) [1], sodium pump [2] and the acto-myosin cross-bridge cycle [3]) are coupled to a model of mitochondrial ATP production within a whole-cell modelling framework for cardiac excitation-contraction coupling [4]. We use the model to investigate the metabolic stability hypothesis, wherein energy demand-supply homeostasis is maintained despite negligible variation in metabolite concentrations at varying cardiac workloads. We find that under physiological workloads cellular metabolite concentrations do not change significantly with increasing workload if a proposed feedback of inorganic phosphate onto mitochondrial oxidative phosphorylation is present, consistent with the proposition that Pi-regulation alone is sufficient to maintain metabolic homeostasis in the absence of other regulatory mechanisms. Finally, we use our model to address the empirically observed linearity of the cardiac ATP vs. Force-Length-Area curve (the cellular equivalent of the VO2 vs. Pressure-Volume-Area relationship). We show that the apparent linearity arises from the near irreversibility of the cross-bridge cycle, but that the linear relationship may disappear at cardiac workloads high enough that cellular metabolite concentrations start to vary.[1] Biophysical Journal 96 (5), 2029-2042, 2009.[2] American Journal of Physiology, Heart and Circulatory Physiology 293, H3036-H3045, 2007.[3] Biophysical Journal 98, 267-276, 2010.[4] Biophysical Journal 90, 3074-3090, 2006.

Activation of Vinculin Induced by Cholinergic Stimulation Regulates Contraction of Tracheal Smooth Muscle Tissue

Vinculin localizes to membrane adhesion junctions where it links actin filaments to the extracellular matrix by binding to the integrin-binding protein talin at its head domain (Vh) and to actin filaments at its tail domain (Vt). Vinculin can assume an inactive (closed) conformation in which Vh and Vt bind to each other, masking the binding sites for actin and talin, and an active (open) conformation in which the binding sites for talin and actin are exposed. We hypothesized that the contractile activation of smooth muscle tissues might regulate the activation of vinculin and thereby contribute to the regulation of contractile tension. Stimulation of tracheal smooth muscle tissues with acetylcholine (ACh) induced the recruitment of vinculin to cell membrane and its interaction with talin and increased the phosphorylation of membrane-localized vinculin at the C-terminal Tyr-1065. Expression of recombinant vinculin head domain peptide (Vh) in smooth muscle tissues, but not the talin-binding deficient mutant head domain, VhA50I, inhibited the ACh-induced recruitment of endogenous vinculin to the membrane and the interaction of vinculin with talin and also inhibited vinculin phosphorylation. Expression of Vh peptide also inhibited ACh-induced smooth muscle contraction and inhibited ACh-induced actin polymerization; however, it did not affect myosin light chain phosphorylation, which is necessary for cross-bridge cycling. Inactivation of RhoA inhibited vinculin activation in response to ACh. We conclude that ACh stimulation regulates vinculin activation in tracheal smooth muscle via RhoA and that vinculin activation contributes to the regulation of active tension by facilitating connections between actin filaments and talin-integrin adhesion complexes and by mediating the initiation of actin polymerization.

Orthovanadate and Orthophosphate Inhibit Muscle Force via Two Different Pathways of the Myosin ATPase Cycle

Measurements of the half-sarcomere stiffness during activation of skinned fibers from rabbit psoas (sarcomere length 2.5 μm, temperature 12°C) indicate that addition of 0.1 mM orthovanadate (Vi) to the solution produces a drop to ∼1/2 in number of force-generating myosin motors, proportional to the drop in steady isometric force (T0), an effect similar to that produced by the addition of 10 mM phosphate (Pi). However, in contrast to Pi, Vi does not change the rate of isometric force development. The depression of T0 in a series of activations in presence of Vi is consistent with an apparent second-order rate constant of ∼1 × 103 M−1 s−1. The rate constant of T0 recovery in a series of activations after removal of Vi is 3.5 × 10−2 s−1. These results, together with the finding in the literature that the ATPase rate is reduced by Vi in proportion to isometric force, are reproduced with a kinetic model of the acto-myosin cross-bridge cycle where binding of Vi to the force-generating actomyosin-ADP state induces detachment from actin to form a stable myosin-ADP-Vi complex that is not able to complete the hydrolysis cycle and reenters the cycle only via reattachment to actin upon activation in Vi-free solution.

Sarcomeric model of stretch-induced stress fiber reorganization

Sarcomeric model of stretch-induced stress fiber reorganization Roland Kaunas1, Hui-Ju Hsu1, Shinji Deguchi21Department of Biomedical Engineering, Texas A&amp;M University, College Station, TX, USA; 2Department of Biomedical Engineering, Tohoku University, Sendai, JapanAbstract: Actin stress fibers (SFs) are mechanosensitive structural elements that respond to applied stress and strain to regulate cell morphology, signal transduction, and cell function. Results from various studies indicate that SFs tend to maintain stress or strain at a constant level. We developed a simple quantitative sarcomeric model of SFs to predict the role of actomyosin crossbridge cycling in SF tension regulation and reorientation in response to cyclic stretching. Under static conditions, the steady-state levels of SF tension were determined by the fiber passive stiffness and the stall force of the constituent myosin II filaments. When subject to cyclic changes in length at low frequencies, SFs change their unloaded reference length levels through myosin sliding to maintain tension at the original level. At high stretch frequencies, myosin cannot respond quickly enough and the SF behaves elastically. Myosin sliding also contributes to SF turnover, resulting in SF reorientation away from the direction of stretching at high, but not low, stretch frequencies. Using model parameters extracted from the literature, our model describes the dependence of cyclic stretch-induced SF alignment on stretch frequency and pattern consistent with experimental findings. This analysis predicts that myosin II plays multiple roles in regulating the ability of SFs to adapt to a dynamic mechanical environment.Keywords: mechanical stretch, cytoskeletal dynamics, myosin, mechanotransduction

Remodeling of Integrated Contractile Tissues and Its Dependence on Strain-Rate Amplitude

Here we investigate the origin of relaxation times governing the mechanical response of an integrated contractile tissue to imposed cyclic changes of length. When strain-rate amplitude is held constant as frequency is varied, fast events are accounted for by actomyosin cross-bridge cycling, but slow events reveal relaxation processes associated with ongoing cytoskeletal length adaptation. Although both relaxation regimes are innately nonlinear, these regimes are unified and their positions along the frequency axis are set by the imposed strain-rate amplitude.

The Plastic Nature of the Vascular Wall: Reply to Folkow

This Letter to the Editor is in response to the comments made by Professor Folkow on our recently published review in Physiology ([7][1]). First, we would like to acknowledge Professor Folkow's numerous and seminal contributions to the field of cardiovascular physiology (e.g., Ref. [3][2]), and in

The Influence of Myosin Converter and Relay Domains on Cross-Bridge Kinetics of Drosophila Indirect Flight Muscle

We are investigating the influence of the converter and relay domains on elementary rate constants of the actomyosin cross-bridge cycle. The converter and relay domains vary between Drosophila myosin heavy chain isoforms due to alternative mRNA splicing. Previously, we found that separate insertions of embryonic myosin isoform (EMB) versions of these domains into the indirect flight muscle (IFM) myosin isoform (IFI) both decreased Drosophila IFM power and slowed muscle kinetics. To determine cross-bridge mechanisms behind the changes, we employed sinusoidal analysis while varying phosphate and MgATP concentrations in skinned Drosophila IFM fibers. Based on a six-state cross-bridge model, the EMB converter decreased myosin rate constants associated with actin attachment and work production, k4, but increased rates related to cross-bridge detachment and work absorption, k2. In contrast, the EMB relay domain had little influence on kinetics, because only k4 decreased. The main alteration was mechanical, in that work production amplitude decreased. That both domains decreased k4 supports the hypothesis that these domains are critical to lever-arm-mediated force generation. Neither domain significantly influenced MgATP affinity. Our modeling suggests the converter domain is responsible for the difference in rate-limiting cross-bridge steps between EMB and IFI myosin—i.e., a myosin isomerization associated with MgADP release for EMB and Pi release for IFI.