Contractile System Research Articles

The contractile system as a negative regulator of the connexin 43 hemichannel

The molecular mechanisms underlying the regulation of gap junction (GJ) channels based on the 43-kDa connexin isoform (Cx43) have been studied extensively. GJ channels are formed by the docking of opposed hemichannels in adjacent cells. Mounting data indicate that unopposed Cx43 hemichannels are also functional in the plasma membrane. However, our understanding of how Cx43-hemichannel opening and closing is regulated at the molecular level is only poorly understood. Recent work elucidated that actomyosin contractility inhibits potently Cx43 hemichannels. It is known that intracellular Ca²⁺ exerts a bell-shaped-dependent effect on Cx43-hemichannel opening. While low-intracellular [Ca²⁺] (<500 nM) provokes opening of the channel, high-intracellular [Ca²⁺] (> 500 nM) favours closing of the channel. The mechanism underlying this negative regulation of Cx43-hemichannel activity by high-intracellular [Ca²⁺] seems to be dependent on the activation of the actomyosin contractile system. The activity of Cx43 hemichannels is critically controlled by molecular interactions between the intracellular loop and the C-terminal tail. These interactions are essential for Cx43-hemichannel opening in response to triggers such as cytosolic [Ca²⁺] rise or external [Ca²⁺] lowering. In this review, we present the hypothesis that the actomyosin contractile system can function as an important brake mechanism on Cx43-hemichannel opening. By controlling loop-tail interactions, the contractile system would prevent aberrant or excessive opening of Cx43 hemichannels.

Spatiotemporal expression pattern of KIF21A during normal embryonic development and in congenital fibrosis of the extraocular muscles type 1 (CFEOM1)

Congenital fibrosis of the extraocular muscles type 1 (CFEOM1) is a rare inherited strabismus syndrome characterized by non-progressive ophthalmoplegia. We previously identified that CFEOM1 results from heterozygous missense mutations in KIF21A, which encodes a kinesin motor protein. Here we evaluate the expression pattern of KIF21A in human brain and muscles of control and CFEOM1 patients, and during human and mouse embryonic development. KIF21A is expressed in the cell bodies, axons, and dendrites of many neuronal populations including those in the hippocampus, cerebral cortex, cerebellum, striatum, and motor neurons of the oculomotor, trochlear, and abducens nuclei from early development into maturity, and its spatial distribution is not altered in the CFEOM1 tissues available for study. Multiple splice isoforms of KIF21A are identified in human fetal brain, but none of the reported CFEOM1 mutations are located in or near the alternatively spliced exons. KIF21A immunoreactivity is also observed in extraocular and skeletal muscle biopsies of control and CFEOM1 patients, where it co-localizes with triadin, a marker of the excitation–contractile coupling system. The diffuse and widespread expression of KIF21A in the developing human and mouse central and peripheral nervous system as well as in extraocular muscle does not account for the restricted ocular phenotype observed in CFEOM1, nor does it permit the formal exclusion of a myogenic etiology based on expression patterns alone.

Twitchin can regulate the ATPase cycle of actomyosin in a phosphorylation-dependent manner in skinned mammalian skeletal muscle fibres

The effect of twitchin, a thick filament protein of molluscan muscles, on the actin–myosin interaction at several mimicked sequential steps of the ATPase cycle was investigated using the polarized fluorescence of 1.5-IAEDANS bound to myosin heads, FITC-phalloidin attached to actin and acrylodan bound to twitchin in the glycerol-skinned skeletal muscle fibres of mammalian. The phosphorylation-dependent multi-step changes in mobility and spatial arrangement of myosin SH1 helix, actin subunit and twitchin during the ATPase cycle have been revealed. It was shown that nonphosphorylated twitchin inhibited the movements of SH1 helix of the myosin heads and actin subunits and decreased the affinity of myosin to actin by freezing the position and mobility of twitchin in the muscle fibres. The phosphorylation of twitchin reverses this effect by changing the spatial arrangement and mobility of the actin-binding portions of twitchin. In this case, enhanced movements of SH1 helix of the myosin heads and actin subunits are observed. The data imply a novel property of twitchin incorporated into organized contractile system: its ability to regulate the ATPase cycle in a phosphorylation-dependent fashion by changing the affinity and spatial arrangement of the actin-binding portions of twitchin.

Structure of autorhythmical activity of contractile systems

In the autorhythmical activity underlying many visceral and in early ontogenesis also somatomotor systems, three kinds of rhythms are to be distinguished: basic, reflecting activity of individual organs and of systems of organs, the secondary ones representing result of the frequency and amplitude modulation of the basic rhythms, and the rhythmical periodic activity whose distinguishing feature is alternation of the activity and rest phases. Each kind has principally different frequency characteristics, different organization and localization of sources. The frequency of basic rhythms is determined by the generator inserted into the system. It serves an individual characteristics of the current state of function of the organ, the degree of maturity of its motor apparatus. The secondary rhythm and the rhythm of the periodic activity cycles are provided by oscillatory processes common to the whole organism. Universality of these rhythms promotes integration of functions.

The adhesive disc in the mobilid ciliate Trichodina pediculus: Evidence for centrin‐related, calcium‐sensitive filaments

The adhesive disc is a highly complex apparatus that allows mobilid ciliates to attach to the tissues of a variety of aquatic invertebrates and vertebrates. The disc comprises concentric rings of rigid skeletal pieces interconnected by filamentous material. This study explored the biochemical properties of the filamentous disc material in the trichodinid Trichodina pediculus. Calcium sensitivity of this material was suggested in vitro by the appearance of transverse cross-striation along bundles of filaments following calcium shock, and complete solubilization of the filamentous material in the presence of EGTA. A 23-kDa immunoanalog of centrins was immunoprecipitated from the EGTA extract. The protein binds calcium as indicated by (45) Ca(2+) blot overlay and Ca(2+) -induced shifts in electrophoretic mobility. Using Ca(2+) /EGTA buffers, we demonstrated a direct relationship between extraction of the filaments and solubilization of the protein. Immunofluorescence and immunoelectron microscopy confirmed that the protein localized to the filamentous disc material and revealed cross-reactivity with the spasmoneme, which is the prototype of ion-sensitive, centrin-like contractile systems in ciliates. The possibility that the filamentous disc material may be a novel example of Ca(2+) -sensitive, centrin-based systems found in ciliates is discussed.

Ca2+ Signaling in Rat Ventricular Myocytes

We employ a coupled electromechanical mathematical model to better understand the biophysical basis of the force-frequency response (FFR) in rat ventricular myocytes under voltage clamp conditions. The model extends our previous work on calcium signaling in the cardiac dyad, and the regulation of Ca2+-concentration in the myoplasm. The present work is focused on achieving a better understanding of mechanisms involved in the rat FFR. Specifically, we examine the role of calmodulin (CaM) in modulating the key control variables Ca2+/calmodulin-dependent protein kinase-II (CaMKII), calcineurin (CaN), and cyclic adenosine monophosphate (cAMP), as they mediate a rate-dependent effect on various intracellular targets controlling the FFR.Our electrochemical model consists of an electrical-equivalent model for the cell membrane; dyadic, myoplasmic and sarcoplasmic reticulum (SR) fluid-compartments; and a modified model of the contractile system by Rice et al. We incorporate frequency-dependent CaM-mediated and spatially heterogenous interaction of the proteins CaMKII and CaN with their principal targets (dihydropyridine (DHPR) and ryanodine (RyR) receptors, and the SERCA pump). Also included are the rate-dependent effects of phospholamban (PLB) on SERCA pump; cAMP on the DHP-sensitive Ca2+ channel; and the enhancement in SERCA pump activity via phosphorylation of PLB.Investigators using multicellular rat ventricular preparations have recorded both positive and negative peak FFRs. Under specific conditions, our VC model can generate either a positive or a negative FFR, while providing mechanistic understanding of its genesis. In addition, the model provides quantitative insight into rate-dependence of CICR by investigating the frequency-dependence of each contributing factor. Since several aforementioned multicellular studies were conducted at different temperatures, we also investigated the temperature-dependence of FFR.Our modeling study suggests that cAMP-mediated stimulation and rate-dependent CaMKII-mediated up-regulation of the trigger current ICa,L are key mechanisms underlying the inconsistency in FFR observations in multicellular rat ventricular tissue.

Half-Sarcomeres with Heterogeneous Crossbridge Populations can be Stabilized by Accompanying Variations in Titin Filaments

Residual force enhancement occurs when the steady-state force produced by a contracting muscle is increased following stretch even though the average actin-myosin overlap within sarcomeres is reduced. We recently used a computational model to examine the mechanical properties of a system of serially-connected half-sarcomeres. The simulations demonstrated that such a model exhibits force enhancement when the active force generating capacity of the individual half-sarcomeres (the number of functioning myosin heads) varies by as little as 2% (Campbell et al., PLoS Comput Biol 2011). Variability at this level would be hard to detect in real experiments but is difficult to discount in biological systems. We have now performed additional simulations in which we vary the passive mechanical properties of the individual half-sarcomeres (the non-linear stiffness of their titin filaments) as well as their active mechanics. The results show that when both active and passive properties are varied, lengths of half-sarcomeres prior to stretch do not always correlate with post-stretch values. This finding may explain experimental observations of ‘stronger’ (shorter) half-sarcomeres prior to stretch that yield to a greater degree than ‘weaker’ (longer) half-sarcomeres. Data published by other groups (Labeit et al., PNAS 2003; Bagni et al., Biophys J 2002) suggest that the stiffness of titin molecules increases with the intracellular Ca2+ concentration. We predict that when we add Ca2+-dependent titin stiffness to our model it will further stabilize half sarcomeres after stretch (reduce half-sarcomere length variability). This might help to prevent stretch-induced damage, thus increasing the resiliency of the contractile system. A computational model that combines active and passive half-sarcomere variability with a Ca2+-activated stiffness may therefore offer a more complete explanation of residual force enhancement and related phenomena.

Muscle Cells from Lancelet: An Emerging Model for Sarcomere Dynamic Research

The sarcomere and its networks are the mechanical driving mechanism of the animal kingdom. To overcome the fundamental limitations involved (network size and structural complexity) for the investigation of sarcomere dynamics in mammalian muscle, a uniquely small contractile system was introduced. The sarcomere network of lancelet is simply structured and exceptionally small. The aim of the study was to introduce cells from lancelet for the investigation of sarcomere function in an intact, self-regulated system. Cells and myofibrils can be isolated and repeatedly activated. They can be adhered for force-length experiments and sarcomere tracking. Mononuclear cells vary in length from 102 to 103 μm and 2-10 μm in diameter. The network is arranged in 1-20 myofibrils. The structure closely resembles the mammalian sarcomere having a rest-length (L0) of 2 μm. Force response of myofibrils to stretch-release resembles the behavior of mammalian myofibrils. Although the stretch response at 0.4 L0∗s−1 exceeds the isometric force by a factor of five. This is remarkably high indicating a distinct stabilization mechanism. In conclusion muscle from lancelet is a potential model for the study of interaction between sarcomere function and the behavior of the cell.

Actin Fusion Proteins Alter the Dynamics of Mechanically Induced Cytoskeleton Rearrangement

Mechanical forces can regulate various functions in living cells. The cytoskeleton is a crucial element for the transduction of forces in cell-internal signals and subsequent biological responses. Accordingly, many studies in cellular biomechanics have been focused on the role of the contractile acto-myosin system in such processes. A widely used method to observe the dynamic actin network in living cells is the transgenic expression of fluorescent proteins fused to actin. However, adverse effects of GFP-actin fusion proteins on cell spreading, migration and cell adhesion strength have been reported. These shortcomings were shown to be partly overcome by fusions of actin binding peptides to fluorescent proteins. Nevertheless, it is not understood whether direct labeling by actin fusion proteins or indirect labeling via these chimaeras alters biomechanical responses of cells and the cytoskeleton to forces. We investigated the dynamic reorganization of actin stress fibers in cells under cyclic mechanical loading by transiently expressing either egfp-Lifeact or eyfp-actin in rat embryonic fibroblasts and observing them by means of live cell microscopy. Our results demonstrate that mechanically-induced actin stress fiber reorganization exhibits very different kinetics in EYFP-actin cells and EGFP-Lifeact cells, the latter showing a remarkable agreement with the reorganization kinetics of non-transfected cells under the same experimental conditions.

Pharmacological reactivity of granulation tissue.

The contractile system of the myofibroblast has well defined characteristics. The findings suggest the presence of 5-hydroxytryptaminergic as well as alpha and beta-adreno-receptors, although both appear to be immature. It does not have cholinergic or histaminergic receptors. The contractile system is inhibited by cytochalasin B and is not sensitive to colchicine. The contractile machinery is suggested to be the microfilament system, as it occurs in the contractile apparatus of primitive cells. This is in accordance with the role of myofibroblasts in granulation tissue neoformation.

Mechanism of inhibition of contraction by cadmium in guinea-pig taenia coli.

Further evidence about the mechanism of the inhibition of contractions caused by cadmium ions (Cd2+) in guinea-pig taenia coli has been sought. Cd2+ at a concentration of 0.5 mM completely inhibited the high-K (40 mM)-induced contraction within 3-5 min. Cd2+ did not shift the Ca2+-induced concentration-response curve to the right in Ca2+-free K+ depolarized muscle, although it reduced the Ca2+ response size. The K+-induced increase in tissue calcium content and 45Ca uptake determined by the lanthanum method was significantly reduced in the presence of Cd2+ (0.5 mM) and the contractions of the glycerolated taenia coli were inhibited with increasing Cd2+ (0.001-0.5 mM). Muscle strips, incubated in a medium containing 0.5 mM Cd2+, accumulated greater amounts of cadmium than within the extracellular space. It is suggested that the inhibitory action on tension produced by Cd2+ in taenia coli may result from the interference of calcium permeability at the cell membrane. There is the possibility that Cd2+ acts on the contractile system of the muscle.

Relaxation of intestinal smooth muscle and calcium movements.

Abstract The effects of papaverine, isoprenaline and N6,2′-O-dibutyryl 3′,5′-cyclic adenosine monophosphate (DiBu.C-AMP) on calcium movements in the taenia from the guinea-pig caecum have been investigated and compared with the effects of phenylephrine. Papaverine and DiBu.C-AMP antagonized the contraction of KCl-depolarized muscle induced by CaCl2 and increased 45Ca-efflux from the taenia. Papaverine also significantly depressed 45Ca-uptake by this preparation. These findings suggest that papaverine impairs the availability of calcium to the contractile system and that there may be a correlation between relaxation and increase of 45Ca-efflux. The action of DiBu.C-AMP resembles that of papaverine but its potency is less.

Detection of a troponin I-like protein in non-striated muscle of the tardigrades (water bears)

Tardigrades, also known as water bears, have somatic muscle fibers that are responsible for movement of their body and legs. These muscle fibers contain thin and thick filaments in a non-striated pattern. However, the regulatory mechanism of muscle contraction in tardigrades is unknown. In the absence of extensive molecular and genomic information, we detected a protein of 31 kDa in whole lysates of tardigrades that cross-reacted with the antibody raised against nematode troponin I (TnI). TnI is a component of the troponin complex that regulates actin-myosin interaction in a Ca(2+)-dependent and actin-linked manner. This TnI-like protein was co-extracted with actin in a buffer containing ATP and EGTA, which is known to induce relaxation of a troponin-regulated contractile system. The TnI-like protein was specifically expressed in the somatic muscle fibers in adult animals and partially co-localized with actin filaments in a non-striated manner. Interestingly, the pharyngeal muscle did not express this protein. These observations suggest that the non-striated somatic muscle of tardigrades has an actin-linked and troponin-regulated system for muscle contraction.

Role of Hyaluronan-Mediated CD44 Signaling in Head and Neck Squamous Cell Carcinoma Progression and Chemoresistance

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy that may involve the oral cavity, pharynx, larynx, and paranasal sinuses. The mechanisms of tumor progression underlying the clinical behavior of HNSCC remain unclear. CD44 comprises a family of transmembrane receptors that can give rise to multiple CD44 variant isoforms. Hyaluronan (HA), a major extracellular matrix component is the primary ligand for CD44 receptors. HA and CD44 signaling play an important role in HNSCC progression. Several CD44 variant isoforms (including v3-, v6-, and v10-containing isoforms) are associated with advanced disease, possibly through unique growth factor interactions with binding domains in the inserted variant regions of the cytoplasmic domain of CD44. In HNSCC, HA mediates the formation of a complex including CD44 and the epidermal growth factor receptor (EGFR) which is overexpressed in a large proportion of HNSCCs. Downstream effectors under EGFR regulation are activated, promoting promote cell growth and tumor survival. The leukemia-associated Rho-guanine nucleotide exchange factor (LARG) also associates with CD44 and EGFR to promote several Ras and RhoA pathway effectors, leading to cell migration, growth, and tumor survival. The secretion of matrix metalloproteinases, necessary for tumor cell invasion, is also regulated by these HA/CD44-mediated pathways. Finally, EGFR-mediated pathways play major roles in the HA/CD44 promotion of chemoresistance in HNSCC. Understanding HA/CD44-mediated signaling pathways may lead to improved treatment of HNSCC.

Sodium channel mechanosensitivity: pay a-tension to voltage sensor movement

Sodium channel mechanosensitivity: pay a-tension to voltage sensor movement

Multiple mitral leaflet contractile systems in the beating heart

Mitral valve closure may be aided by contraction of anterior leaflet (AL) cardiac myocytes located in the annular third of the leaflet. This contraction, observed as a stiffening of the annular region of the AL during isovolumic contraction (IVC), is abolished by beta-blockade (βB). Sub-threshold rapid pacing in the region of aorto-mitral continuity (STIM) also causes AL stiffening, although this increases the stiffness of the entire leaflet during both IVC and isovolumic relaxation (IVR). We investigated whether these contractile events share a common pathway or whether multiple AL contractile mechanisms may be present. Ten sheep had radiopaque-markers implanted: 13 silhouetting the LV, 16 on the mitral annulus, an array of 16 on the AL, and one on each papillary muscle tip. 4-D marker coordinates were obtained from biplane videofluoroscopy during control (C), βB (esmolol) and during βB+STIM. Circumferential and radial stiffness values for three AL regions (Annular, Belly, and free-Edge), were obtained from inverse finite element analysis of AL displacements in response to trans-leaflet pressure changes during IVC and IVR. βB+STIM increased stiffness values in all regions at both IVC and IVR by 35±7% relative to βB ( p<0.001). Thus, even when AL myocyte contraction was blocked by βB, STIM stiffened all regions of the AL during both IVC and IVR. This demonstrates the presence of at least two contractile systems in the AL; one being the AL annular cardiac muscle, involving a β-dependent pathway, others via a β-independent pathway, likely involving valvular interstitial cells and/or AL smooth muscle cells.

Transgenic Overexpression of Heart-specific Adenine Nucleotide Translocase 1 Positively Affects Contractile Function in Cardiomyocytes

Background/Aims: The adenine nucleotide translocase (ANT) exchanges ATP and ADP over the inner mitochondrial membrane, supplying the cells with energy. Interestingly, myocardial ANT1 overexpression preserves cardiac structure and function under pathophysiological conditions. To ascertain whether the contractile system is directly affected by increased ANT1 expression, we analyzed cell morphology, contraction and relaxation parameters of ANT1 transgenic (ANT1-TG) cardiomyocytes, myofibrillar protein expression, and Ca²⁺ handling in ANT1-TG rat hearts. Results: ANT1-TG cardiomyoycytes displayed an elevation in cell volume (52.6±12.0%; p<0.0001) in comparison to wildtype (WT) cells. Concurrently, contractile function in ANT1-TG cells was significantly increased, measured by a decline in time to peak contraction (TTP) and RT50, the time from peak contraction to 50% relaxation, during stimulation with 0.5, 1, and 2 Hz. Quantification of myofibrillar proteins exhibited a marked increase in total cardiac myosin heavy chain (51.8±12.8%) (p<0.03), beta myosin heavy chain (22.9±5.0%; p<0.03), actin (23.8±8.8%; p<0.05), and troponin I (51.5±13.7%; p<0.01). Regarding intracellular Ca²⁺ handling, ANT1-TGs revealed a significant elevation in sarcoplasmic reticulum (SR) Ca²⁺ ATPase (SERCA2a) protein level (22.2±4.7%; p<0.01) associated with increased Ca²⁺ uptake into the SR (34%; p<0.01). Moreover, the plasmalemmal Ca²⁺ ATPase (PMCA) indicated advanced protein expression (23.8±4.8%; p<0.01), whereas the protein amount of the Na⁺/Ca²⁺ exchanger was not altered in ANT1 overexpressing hearts. Conclusion: These data reveal a close association of elevated mitochondrial ATP/ADP transportation via ANT1 with increased contractile function. Furthermore, the ANT1-TGs exhibit an elevation in SR Ca²⁺ transport that contributes to increased cardiac work, which may protect the heart under pathophysiological conditions.

A theory on auto-oscillation and contraction in striated muscle

It is widely accepted that muscle cells take either force-generating or relaxing state in an all-or-none fashion through the so-called excitation–contraction coupling. On the other hand, the membrane-less contractile apparatus takes the third state, i.e., the auto-oscillation (SPOC) state, at the activation level that is intermediate between full activation and relaxation. Here, to explain the dynamics of all three states of muscle, we construct a novel theoretical model based on the balance of forces not only parallel but also perpendicular to the long axis of myofibrils, taking into account the experimental fact that the spacing of myofilament lattice changes with sarcomere length and upon contraction. This theory presents a phase diagram composed of several states of the contractile apparatus and explains the dynamic behavior of SPOC, e.g., periodical changes in sarcomere length with the saw-tooth waveform. The appropriate selection of the constant of the molecular friction due to the cross-bridge formation can explain the difference in the SPOC periods observed under various activating conditions and in different muscle types, i.e., skeletal and cardiac. The theory also predicts the existence of a weak oscillation state at the boundary between SPOC and relaxation regions in the phase diagram. Thus, the present theory comprehensively explains the characteristics of auto-oscillation and contraction in the contractile system of striated muscle.

Cytomechanics of oscillatory contractions. Modeling the longitudinal dynamics of Physarum polycephalum protoplasmic strands

A mathematical model of the longitudinal dynamics of an isolated strand of the Physarum polycephalum plasmodium has been constructed. Its contractile system is considered as a continual viscoelastic medium with passive and active components. The mathematical description of the longitudinal dynamics of the plasmodial strand is reduced to a system of three first-order differential equations, whose variables are its active stress, deformation, and the intracellular concentration of calcium ions. The model is based on the hypothesis that there exists a feedback loop, which appears because of the influence of strand stretching on the rate of the release of calcium ions, which in turn controls the active contraction and deformation of the strand. Nonlinear interactions between the variables evoke a loss of the stationary state stability and a self-excitation of mechanochemical autooscillations when the external load exceeds some critical value. The results of numerical solutions of the model with the empirically determined viscoelastic parameters are in good agreement with the available experimental data and testify to the adequacy of the description of strand dynamics by the mathematical model in which the contractile apparatus is a part of the cellular control system. In particular, this model well simulates the form and duration of transient mechanochemical processes observed under isotonic and isometric conditions immediately after strand isolation, as well as the subsequent excitation of autooscillations of the contractile activity and their activation by strand stretching.

Dictyostelium dynamin B modulates cytoskeletal structures and membranous organelles

Dictyostelium discoideum cells produce five dynamin family proteins. Here, we show that dynamin B is the only member of this group of proteins that is initially produced as a preprotein and requires processing by mitochondrial proteases for formation of the mature protein. Our results show that dynamin B-depletion affects many aspects of cell motility, cell-cell and cell-surface adhesion, resistance to osmotic shock, and fatty acid metabolism. The mature form of dynamin B mediates a wide range and unique combination of functions. Dynamin B affects events at the plasma membrane, peroxisomes, the contractile vacuole system, components of the actin-based cytoskeleton, and cell adhesion sites. The modulating effect of dynamin B on the activity of the contractile vacuole system is unique for the Dictyostelium system. Other functions displayed by dynamin B are commonly associated with either classical dynamins or dynamin-related proteins.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-010-0590-5) contains supplementary material, which is available to authorized users.