Bistable front dynamics in a contractile medium: travelling wave and cortical advection define stable zones of RhoA signaling at epithelial adherens junctions

NASA Astrophysics Data System (ADS)

Neufeld, Zoltan

Recent studies have demonstrated that mechanical forces can lead to novel mechanisms of pattern formation such as clustering and oscillations in contractile systems. We investigate how contractile forces in mechanically active media can affect bistable front propagation. We found that contraction regulates the front speed or can fully suppress its propagation in space to create a static localized zone. We demonstrate how the interplay between biochemical signaling through positive feedback, combined with diffusion on the cell membrane and mechanical forces generated in the actomyosin cortex, can determine the spatial distribution of RhoA signaling at cell-cell junctions. The dynamical mechanism relies on the balance between a propagating bistable signal that is opposed by an advective flow generated by an actomyosin stress gradient. Experimental observations on the behaviour of the system when contractility is inhibited are in qualitative agreement with the predictions of the model. In collaboration with: Zoltan Neufeld, Guillermo A. Gomez, and Alpha S. Yap, University of Queensland, Brisbane, Australia

Mechanical Coordination of Single-Cell and Collective-Cell Amoeboid Migration

NASA Astrophysics Data System (ADS)

Del Alamo, Juan Carlos

Amoeboid migration consists of the sequential repetition of pseudopod extensions and retractions driven by actin polymerization and actomyosin contraction, and requires cells to apply mechanical forces on their surroundings. We measure the three-dimensional forces exerted by chemotaxing Dictyostelium cells, and examine wild-type cells as well as mutants with defects in contractility, F-actin polymerization, internal F-actin crosslinking, and cortical integrity. We find that cells pull on their substrate adhesions using two distinct, yet interconnected mechanisms: axial actomyosin contractility and cortical tension. The 3D pulling forces generated by both mechanisms are internally balanced by an increase in cytoplasmic pressure that allows cells to push on their substrate, and we show that these pushing forces are relevant for cell invasion and migration in three-dimensional environments. We observe that cells migrate mainly by forming two stationary adhesion sites at the front and back of the cell, over which the cell body moves forward in a step-wise fashion. During this process, the traction forces at each adhesion site are switched off and subsequently their direction is reversed. The cell migration speed is found to be proportional to the rate at which cells are able regulate these forces to produce the cell shape changes needed for locomotion, which is increased when axial contractility overcomes the stabilizing effect of cortical tension. This spatiotemporal coordination is conserved in streams of multiple migratory cells connected head to tail, which also migrate by exerting traction forces on stationary sites. Furthermore, we observe that trailing cells reuse the adhesion sites of the leading cells. Finally, we provide evidence that the above modes of migration may be conserved in a range of other amoeboid-type moving cells such as neutrophils.

An EMMPRIN-γ-catenin-Nm23 complex drives ATP production and actomyosin contractility at endothelial junctions.

PubMed

Moreno, Vanessa; Gonzalo, Pilar; Gómez-Escudero, Jesús; Pollán, Ángela; Acín-Pérez, Rebeca; Breckenridge, Mark; Yáñez-Mó, María; Barreiro, Olga; Orsenigo, Fabrizio; Kadomatsu, Kenji; Chen, Christopher S; Enríquez, José A; Dejana, Elisabetta; Sánchez-Madrid, Francisco; Arroyo, Alicia G

2014-09-01

Cell-cell adhesions are important sites through which cells experience and resist forces. In endothelial cells, these forces regulate junction dynamics and determine endothelial barrier strength. We identify the Ig superfamily member EMMPRIN (also known as basigin) as a coordinator of forces at endothelial junctions. EMMPRIN localization at junctions correlates with endothelial junction strength in different mouse vascular beds. Accordingly, EMMPRIN-deficient mice show altered junctions and increased junction permeability. Lack of EMMPRIN alters the localization and function of VE-cadherin (also known as cadherin-5) by decreasing both actomyosin contractility and tugging forces at endothelial cell junctions. EMMPRIN ensures proper actomyosin-driven maturation of competent endothelial junctions by forming a molecular complex with γ-catenin (also known as junction plakoglobin) and Nm23 (also known as NME1), a nucleoside diphosphate kinase, thereby locally providing ATP to fuel the actomyosin machinery. These results provide a novel mechanism for the regulation of actomyosin contractility at endothelial junctions and might have broader implications in biological contexts such as angiogenesis, collective migration and tissue morphogenesis by coupling compartmentalized energy production to junction assembly. © 2014. Published by The Company of Biologists Ltd.

Contractility of the cell rear drives invasion of breast tumor cells in 3D Matrigel

PubMed Central

Poincloux, Renaud; Collin, Olivier; Lizárraga, Floria; Romao, Maryse; Debray, Marcel; Piel, Matthieu; Chavrier, Philippe

2011-01-01

Cancer cells use different modes of migration, including integrin-dependent mesenchymal migration of elongated cells along elements of the 3D matrix as opposed to low-adhesion-, contraction-based amoeboid motility of rounded cells. We report that MDA-MB-231 human breast adenocarcinoma cells invade 3D Matrigel with a characteristic rounded morphology and with F-actin and myosin-IIa accumulating at the cell rear in a uropod-like structure. MDA-MB-231 cells display neither lamellipodia nor bleb extensions at the leading edge and do not require Arp2/3 complex activity for 3D invasion in Matrigel. Accumulation of phospho-MLC and blebbing activity were restricted to the uropod as reporters of actomyosin contractility, and velocimetric analysis of fluorescent beads embedded within the 3D matrix showed that pulling forces exerted to the matrix are restricted to the side and rear of cells. Inhibition of actomyosin contractility or β1 integrin function interferes with uropod formation, matrix deformation, and invasion through Matrigel. These findings support a model whereby actomyosin-based uropod contractility generates traction forces on the β1 integrin adhesion system to drive cell propulsion within the 3D matrix, with no contribution of lamellipodia extension or blebbing to movement. PMID:21245302

Noncontact minimally invasive technique for the assessment of mechanical properties of single cardiac myocyte via magnetic field loading

NASA Astrophysics Data System (ADS)

Yin, Shizhuo; Zhang, Xueqian; Cheung, Joseph; Wu, Juntao; Zhan, Chun; Xue, Jinchao

2004-07-01

In this paper, a unique non-contact, minimum invasive technique for the assessment of mechanical properties of single cardiac myocyte is presented. The assessment process includes following major steps: (1) attach a micro magnetic bead to the cell to be measured, (2) measure the contractile performance of the cell under the different magnetic field loading, (3) calculate mechanical loading force, and (4) derive the contractile force from the measured contraction data under different magnetic field loading.

An EMMPRIN–γ-catenin–Nm23 complex drives ATP production and actomyosin contractility at endothelial junctions

PubMed Central

Moreno, Vanessa; Gonzalo, Pilar; Gómez-Escudero, Jesús; Pollán, Ángela; Acín-Pérez, Rebeca; Breckenridge, Mark; Yáñez-Mó, María; Barreiro, Olga; Orsenigo, Fabrizio; Kadomatsu, Kenji; Chen, Christopher S.; Enríquez, José A.; Dejana, Elisabetta; Sánchez-Madrid, Francisco; Arroyo, Alicia G.

2014-01-01

ABSTRACT Cell–cell adhesions are important sites through which cells experience and resist forces. In endothelial cells, these forces regulate junction dynamics and determine endothelial barrier strength. We identify the Ig superfamily member EMMPRIN (also known as basigin) as a coordinator of forces at endothelial junctions. EMMPRIN localization at junctions correlates with endothelial junction strength in different mouse vascular beds. Accordingly, EMMPRIN-deficient mice show altered junctions and increased junction permeability. Lack of EMMPRIN alters the localization and function of VE-cadherin (also known as cadherin-5) by decreasing both actomyosin contractility and tugging forces at endothelial cell junctions. EMMPRIN ensures proper actomyosin-driven maturation of competent endothelial junctions by forming a molecular complex with γ-catenin (also known as junction plakoglobin) and Nm23 (also known as NME1), a nucleoside diphosphate kinase, thereby locally providing ATP to fuel the actomyosin machinery. These results provide a novel mechanism for the regulation of actomyosin contractility at endothelial junctions and might have broader implications in biological contexts such as angiogenesis, collective migration and tissue morphogenesis by coupling compartmentalized energy production to junction assembly. PMID:24994937

Global architecture of the F-actin cytoskeleton regulates cell shape-dependent endothelial mechanotransduction.

PubMed

Shao, Yue; Mann, Jennifer M; Chen, Weiqiang; Fu, Jianping

2014-03-01

Uniaxial stretch is an important biophysical regulator of cell morphology (or shape) and functions of vascular endothelial cells (ECs). However, it is unclear whether and how cell shape can independently regulate EC mechanotransductive properties under uniaxial stretch. Herein, utilizing a novel uniaxial cell-stretching device integrated with micropost force sensors, we reported the first experimental evidence showing cell shape-dependent EC mechanotransduction via cytoskeleton (CSK) contractile forces in response to uniaxial stretch. Combining experiments and theoretical modeling from first principles, we showed that it was the global architecture of the F-actin CSK that instructed the cell shape-dependent EC mechanotransductive process. Furthermore, a cell shape-dependent nature was relayed in EC mechanotransduction via dynamic focal adhesion (FA) assembly. Our results suggested a novel mechanotransductive process in ECs wherein the global architecture of the F-actin CSK, governed by cell shape, controls mechanotransduction via CSK contractile forces and force-dependent FA assembly under uniaxial stretch.

Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism.

PubMed

Riveline, D; Zamir, E; Balaban, N Q; Schwarz, U S; Ishizaki, T; Narumiya, S; Kam, Z; Geiger, B; Bershadsky, A D

2001-06-11

The transition of cell-matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II-driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein-tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. Nat. Cell Biol. 1:136-143), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force.

Differences in Contractile Function of Myofibrils within Human Embryonic Stem Cell-Derived Cardiomyocytes vs. Adult Ventricular Myofibrils Are Related to Distinct Sarcomeric Protein Isoforms

PubMed Central

Iorga, Bogdan; Schwanke, Kristin; Weber, Natalie; Wendland, Meike; Greten, Stephan; Piep, Birgit; dos Remedios, Cristobal G.; Martin, Ulrich; Zweigerdt, Robert; Kraft, Theresia; Brenner, Bernhard

2018-01-01

Characterizing the contractile function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is key for advancing their utility for cellular disease models, promoting cell based heart repair, or developing novel pharmacological interventions targeting cardiac diseases. The aim of the present study was to understand whether steady-state and kinetic force parameters of β-myosin heavy chain (βMyHC) isoform-expressing myofibrils within human embryonic stem cell-derived cardiomyocytes (hESC-CMs) differentiated in vitro resemble those of human ventricular myofibrils (hvMFs) isolated from adult donor hearts. Contractile parameters were determined using the same micromechanical method and experimental conditions for both types of myofibrils. We identified isoforms and phosphorylation of main sarcomeric proteins involved in the modulation of force generation of both, chemically demembranated hESC-CMs (d-hESC-CMs) and hvMFs. Our results indicate that at saturating Ca2+ concentration, both human-derived contractile systems developed forces with similar rate constants (0.66 and 0.68 s−1), reaching maximum isometric force that was significantly smaller for d-hESC-CMs (42 kPa) than for hvMFs (94 kPa). At submaximal Ca2+-activation, where intact cardiomyocytes normally operate, contractile parameters of d-hESC-CMs and hvMFs exhibited differences. Ca2+ sensitivity of force was higher for d-hESC-CMs (pCa50 = 6.04) than for hvMFs (pCa50 = 5.80). At half-maximum activation, the rate constant for force redevelopment was significantly faster for d-hESC-CMs (0.51 s−1) than for hvMFs (0.28 s−1). During myofibril relaxation, kinetics of the slow force decay phase were significantly faster for d-hESC-CMs (0.26 s−1) than for hvMFs (0.21 s−1), while kinetics of the fast force decay were similar and ~20x faster. Protein analysis revealed that hESC-CMs had essentially no cardiac troponin-I, and partially non-ventricular isoforms of some other sarcomeric proteins, explaining the functional discrepancies. The sarcomeric protein isoform pattern of hESC-CMs had features of human cardiomyocytes at an early developmental stage. The study indicates that morphological and ultrastructural maturation of βMyHC isoform-expressing hESC-CMs is not necessarily accompanied by ventricular-like expression of all sarcomeric proteins. Our data suggest that hPSC-CMs could provide useful tools for investigating inherited cardiac diseases affecting contractile function during early developmental stages. PMID:29403388

Focal Contacts as Mechanosensors

PubMed Central

Riveline, Daniel; Zamir, Eli; Balaban, Nathalie Q.; Schwarz, Ulrich S.; Ishizaki, Toshimasa; Narumiya, Shuh; Kam, Zvi; Geiger, Benjamin; Bershadsky, Alexander D.

2001-01-01

The transition of cell–matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II–driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein–tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. Nat. Cell Biol. 1:136–143), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force. PMID:11402062

Wrinkling of a spherical lipid interface induced by actomyosin cortex

NASA Astrophysics Data System (ADS)

Ito, Hiroaki; Nishigami, Yukinori; Sonobe, Seiji; Ichikawa, Masatoshi

2015-12-01

Actomyosin actively generates contractile forces that provide the plasma membrane with the deformation stresses essential to carry out biological processes. Although the contractile property of purified actomyosin has been extensively studied, to understand the physical contribution of the actomyosin contractile force on a deformable membrane is still a challenging problem and of great interest in the field of biophysics. Here, we reconstitute a model system with a cell-sized deformable interface that exhibits anomalous curvature-dependent wrinkling caused by the actomyosin cortex underneath the spherical closed interface. Through a shape analysis of the wrinkling deformation, we find that the dominant contributor to the wrinkled shape changes from bending elasticity to stretching elasticity of the reconstituted cortex upon increasing the droplet curvature radius of the order of the cell size, i.e., tens of micrometers. The observed curvature dependence is explained by the theoretical description of the cortex elasticity and contractility. Our present results provide a fundamental insight into the deformation of a curved membrane induced by the actomyosin cortex.

A global, myosin light chain kinase-dependent increase in myosin II contractility accompanies the metaphase-anaphase transition in sea urchin eggs.

PubMed

Lucero, Amy; Stack, Christianna; Bresnick, Anne R; Shuster, Charles B

2006-09-01

Myosin II is the force-generating motor for cytokinesis, and although it is accepted that myosin contractility is greatest at the cell equator, the temporal and spatial cues that direct equatorial contractility are not known. Dividing sea urchin eggs were placed under compression to study myosin II-based contractile dynamics, and cells manipulated in this manner underwent an abrupt, global increase in cortical contractility concomitant with the metaphase-anaphase transition, followed by a brief relaxation and the onset of furrowing. Prefurrow cortical contractility both preceded and was independent of astral microtubule elongation, suggesting that the initial activation of myosin II preceded cleavage plane specification. The initial rise in contractility required myosin light chain kinase but not Rho-kinase, but both signaling pathways were required for successful cytokinesis. Last, mobilization of intracellular calcium during metaphase induced a contractile response, suggesting that calcium transients may be partially responsible for the timing of this initial contractile event. Together, these findings suggest that myosin II-based contractility is initiated at the metaphase-anaphase transition by Ca2+-dependent myosin light chain kinase (MLCK) activity and is maintained through cytokinesis by both MLCK- and Rho-dependent signaling. Moreover, the signals that initiate myosin II contractility respond to specific cell cycle transitions independently of the microtubule-dependent cleavage stimulus.

A Global, Myosin Light Chain Kinase-dependent Increase in Myosin II Contractility Accompanies the Metaphase–Anaphase Transition in Sea Urchin Eggs

PubMed Central

Lucero, Amy; Stack, Christianna; Bresnick, Anne R.

2006-01-01

Myosin II is the force-generating motor for cytokinesis, and although it is accepted that myosin contractility is greatest at the cell equator, the temporal and spatial cues that direct equatorial contractility are not known. Dividing sea urchin eggs were placed under compression to study myosin II-based contractile dynamics, and cells manipulated in this manner underwent an abrupt, global increase in cortical contractility concomitant with the metaphase–anaphase transition, followed by a brief relaxation and the onset of furrowing. Prefurrow cortical contractility both preceded and was independent of astral microtubule elongation, suggesting that the initial activation of myosin II preceded cleavage plane specification. The initial rise in contractility required myosin light chain kinase but not Rho-kinase, but both signaling pathways were required for successful cytokinesis. Last, mobilization of intracellular calcium during metaphase induced a contractile response, suggesting that calcium transients may be partially responsible for the timing of this initial contractile event. Together, these findings suggest that myosin II-based contractility is initiated at the metaphase–anaphase transition by Ca2+-dependent myosin light chain kinase (MLCK) activity and is maintained through cytokinesis by both MLCK- and Rho-dependent signaling. Moreover, the signals that initiate myosin II contractility respond to specific cell cycle transitions independently of the microtubule-dependent cleavage stimulus. PMID:16837551

Multicellular contractility contributes to the emergence of mesothelioma nodules

NASA Astrophysics Data System (ADS)

Czirok, Andras

Malignant pleural mesothelioma (MPM) nodules arise from the mesothelial lining of the pleural cavity by a poorly understood mechanism. We demonstrate that macroscopic multicellular aggregates, reminiscent of the MPM nodules found in patients, develop when MPM cell lines are cultured at high cell densities for several weeks. Surprisingly, the nodule-like aggregates do not arise by excessive local cell proliferation, but by myosin II-driven cell contractility. Contractile nodules contain prominent actin cables that can span several cells. Several features of the in vitro MPM nodule development can be explained by a computational model that assumes uniform and steady intercellular contractile forces within a monolayer of cells, and a mechanical load-dependent lifetime of cell-cell contacts. The model behaves as a self-tensioned Maxwell fluid and exhibits an instability that leads to pattern formation. Altogether, our findings suggest that inhibition of the actomyosin system may provide a hitherto not utilized therapeutic approach to affect MPM growth. NIH R01-GM102801.

Single-Cell Functional Analysis of Stem-Cell Derived Cardiomyocytes on Micropatterned Flexible Substrates.

PubMed

Kijlstra, Jan David; Hu, Dongjian; van der Meer, Peter; Domian, Ibrahim J

2017-11-15

Human pluripotent stem-cell derived cardiomyocytes (hPSC-CMs) hold great promise for applications in human disease modeling, drug discovery, cardiotoxicity screening, and, ultimately, regenerative medicine. The ability to study multiple parameters of hPSC-CM function, such as contractile and electrical activity, calcium cycling, and force generation, is therefore of paramount importance. hPSC-CMs cultured on stiff substrates like glass or polystyrene do not have the ability to shorten during contraction, making them less suitable for the study of hPSC-CM contractile function. Other approaches require highly specialized hardware and are difficult to reproduce. Here we describe a protocol for the preparation of hPSC-CMs on soft substrates that enable shortening, and subsequently the simultaneous quantitative analysis of their contractile and electrical activity, calcium cycling, and force generation at single-cell resolution. This protocol requires only affordable and readily available materials and works with standard imaging hardware. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Synaptopodin couples epithelial contractility to α-actinin-4–dependent junction maturation

PubMed Central

Kannan, Nivetha

2015-01-01

The epithelial junction experiences mechanical force exerted by endogenous actomyosin activities and from interactions with neighboring cells. We hypothesize that tension generated at cell–cell adhesive contacts contributes to the maturation and assembly of the junctional complex. To test our hypothesis, we used a hydraulic apparatus that can apply mechanical force to intercellular junction in a confluent monolayer of cells. We found that mechanical force induces α-actinin-4 and actin accumulation at the cell junction in a time- and tension-dependent manner during junction development. Intercellular tension also induces α-actinin-4–dependent recruitment of vinculin to the cell junction. In addition, we have identified a tension-sensitive upstream regulator of α-actinin-4 as synaptopodin. Synaptopodin forms a complex containing α-actinin-4 and β-catenin and interacts with myosin II, indicating that it can physically link adhesion molecules to the cellular contractile apparatus. Synaptopodin depletion prevents junctional accumulation of α-actinin-4, vinculin, and actin. Knockdown of synaptopodin and α-actinin-4 decreases the strength of cell–cell adhesion, reduces the monolayer permeability barrier, and compromises cellular contractility. Our findings underscore the complexity of junction development and implicate a control process via tension-induced sequential incorporation of junctional components. PMID:26504173

Traction force microscopy in rapidly moving cells reveals separate roles for ROCK and MLCK in the mechanics of retraction.

PubMed

Morin, Timothy R; Ghassem-Zadeh, Sean A; Lee, Juliet

2014-08-15

Retraction is a major rate-limiting step in cell motility, particularly in slow moving cell types that form large stable adhesions. Myosin II dependent contractile forces are thought to facilitate detachment by physically pulling up the rear edge. However, retraction can occur in the absence of myosin II activity in cell types that form small labile adhesions. To investigate the role of contractile force generation in retraction, we performed traction force microscopy during the movement of fish epithelial keratocytes. By correlating changes in local traction stress at the rear with the area retracted, we identified four distinct modes of retraction. "Recoil" retractions are preceded by a rise in local traction stress, while rear edge is temporarily stuck, followed by a sharp drop in traction stress upon detachment. This retraction type was most common in cells generating high average traction stress. In "pull" type retractions local traction stress and area retracted increase concomitantly. This was the predominant type of retraction in keratocytes and was observed mostly in cells generating low average traction stress. "Continuous" type retractions occur without any detectable change in traction stress, and are seen in cells generating low average traction stress. In contrast, to many other cell types, "release" type retractions occur in keratocytes following a decrease in local traction stress. Our identification of distinct modes of retraction suggests that contractile forces may play different roles in detachment that are related to rear adhesion strength. To determine how the regulation of contractility via MLCK or Rho kinase contributes to the mechanics of detachment, inhibitors were used to block or augment these pathways. Modulation of MLCK activity led to the most rapid change in local traction stress suggesting its importance in regulating attachment strength. Surprisingly, Rho kinase was not required for detachment, but was essential for localizing retraction to the rear. We suggest that in keratocytes MLCK and Rho kinase play distinct, complementary roles in the respective temporal and spatial control of rear detachment that is essential for maintaining rapid motility. Copyright © 2014 Elsevier Inc. All rights reserved.

Three-dimensional morphogenesis of MDCK cells induced by cellular contractile forces on a viscous substrate

PubMed Central

Imai, Misako; Furusawa, Kazuya; Mizutani, Takeomi; Kawabata, Kazushige; Haga, Hisashi

2015-01-01

Substrate physical properties are essential for many physiological events such as embryonic development and 3D tissue formation. Physical properties of the extracellular matrix such as viscoelasticity and geometrical constraints are understood as factors that affect cell behaviour. In this study, we focused on the relationship between epithelial cell 3D morphogenesis and the substrate viscosity. We observed that Madin-Darby Canine Kidney (MDCK) cells formed 3D structures on a viscous substrate (Matrigel). The structures appear as a tulip hat. We then changed the substrate viscosity by genipin (GP) treatment. GP is a cross-linker of amino groups. Cells cultured on GP-treated-matrigel changed their 3D morphology in a substrate viscosity-dependent manner. Furthermore, to elucidate the spatial distribution of the cellular contractile force, localization of mono-phosphorylated and di-phosphorylated myosin regulatory light chain (P-MRLCs) was visualized by immunofluorescence. P-MRLCs localized along the periphery of epithelial sheets. Treatment with Y-27632, a Rho-kinase inhibitor, blocked the P-MRLCs localization at the edge of epithelial sheets and halted 3D morphogenesis. Our results indicate that the substrate viscosity, the substrate deformation, and the cellular contractile forces induced by P-MRLCs play crucial roles in 3D morphogenesis. PMID:26374384

Three-dimensional morphogenesis of MDCK cells induced by cellular contractile forces on a viscous substrate.

PubMed

Imai, Misako; Furusawa, Kazuya; Mizutani, Takeomi; Kawabata, Kazushige; Haga, Hisashi

2015-09-16

Substrate physical properties are essential for many physiological events such as embryonic development and 3D tissue formation. Physical properties of the extracellular matrix such as viscoelasticity and geometrical constraints are understood as factors that affect cell behaviour. In this study, we focused on the relationship between epithelial cell 3D morphogenesis and the substrate viscosity. We observed that Madin-Darby Canine Kidney (MDCK) cells formed 3D structures on a viscous substrate (Matrigel). The structures appear as a tulip hat. We then changed the substrate viscosity by genipin (GP) treatment. GP is a cross-linker of amino groups. Cells cultured on GP-treated-matrigel changed their 3D morphology in a substrate viscosity-dependent manner. Furthermore, to elucidate the spatial distribution of the cellular contractile force, localization of mono-phosphorylated and di-phosphorylated myosin regulatory light chain (P-MRLCs) was visualized by immunofluorescence. P-MRLCs localized along the periphery of epithelial sheets. Treatment with Y-27632, a Rho-kinase inhibitor, blocked the P-MRLCs localization at the edge of epithelial sheets and halted 3D morphogenesis. Our results indicate that the substrate viscosity, the substrate deformation, and the cellular contractile forces induced by P-MRLCs play crucial roles in 3D morphogenesis.

Effects of Mechanical Coupling Between Cardiomyocytes and Cardiac Fibroblasts on Myocardium

NASA Astrophysics Data System (ADS)

Zorlutuna, Pinar; Nguyen, Trung Dung; Nagarajan, Neerajha

Cardiomyocytes show excitatory responses to stimulation solely by mechanical forces through their stretch-activated ion channels, and can fire action potentials upon mechanical stimulation through a pathway known as mechano-electric feedback. Furthermore, cardiomyocyte (CM) - cardiac fibroblasts (CF) can couple mechanically through cell-cell junctions. Here we investigated the effects of CM and CF mechanical coupling on myocardial physiology and pathology using a bio-nanoindentered coupled with fast calcium imaging and microelectrode arrays. In order to study mechanical signal transmission, we measured the contractile forces generated by CMs, as well as by CFs that were coupled to the CMs. We observed that CFs were beating with the same frequency but at smaller magnitude compared to CMs, and their contractility was dependent on the substrate stiffness. Our results showed that beating CMs actively stretched neighbouring CFs through the deformation of the substrate the cells were seeded on, which promoted the myocardial contractility through mechanical coupling. The results also revealed that CM contractility was propagated greater on soft substrates than stiff ones. Results of this study could help identify the role of the infarcted tissue stiffness and size on heart failure. This study is supported by NSF Grant No: 1530884.

Novel approaches to determine contractile function of the isolated adult zebrafish ventricular cardiac myocyte.

PubMed

Dvornikov, Alexey V; Dewan, Sukriti; Alekhina, Olga V; Pickett, F Bryan; de Tombe, Pieter P

2014-05-01

The zebrafish (Danio rerio) has been used extensively in cardiovascular biology, but mainly in the study of heart development. The relative ease of its genetic manipulation may indicate the suitability of this species as a cost-effective model system for the study of cardiac contractile biology. However, whether the zebrafish heart is an appropriate model system for investigations pertaining to mammalian cardiac contractile structure-function relationships remains to be resolved. Myocytes were isolated from adult zebrafish hearts by enzymatic digestion, attached to carbon rods, and twitch force and intracellular Ca(2+) were measured. We observed the modulation of twitch force, but not of intracellular Ca(2+), by both extracellular [Ca(2+)] and sarcomere length. In permeabilized cells/myofibrils, we found robust myofilament length-dependent activation. Moreover, modulation of myofilament activation-relaxation and force redevelopment kinetics by varied Ca(2+) activation levels resembled that found previously in mammalian myofilaments. We conclude that the zebrafish is a valid model system for the study of cardiac contractile structure-function relationships.

A device for rapid and quantitative measurement of cardiac myocyte contractility

NASA Astrophysics Data System (ADS)

Gaitas, Angelo; Malhotra, Ricky; Li, Tao; Herron, Todd; Jalife, José

2015-03-01

Cardiac contractility is the hallmark of cardiac function and is a predictor of healthy or diseased cardiac muscle. Despite advancements over the last two decades, the techniques and tools available to cardiovascular scientists are limited in their utility to accurately and reliably measure the amplitude and frequency of cardiomyocyte contractions. Isometric force measurements in the past have entailed cumbersome attachment of isolated and permeabilized cardiomyocytes to a force transducer followed by measurements of sarcomere lengths under conditions of submaximal and maximal Ca2+ activation. These techniques have the inherent disadvantages of being labor intensive and costly. We have engineered a micro-machined cantilever sensor with an embedded deflection-sensing element that, in preliminary experiments, has demonstrated to reliably measure cardiac cell contractions in real-time. Here, we describe this new bioengineering tool with applicability in the cardiovascular research field to effectively and reliably measure cardiac cell contractility in a quantitative manner. We measured contractility in both primary neonatal rat heart cardiomyocyte monolayers that demonstrated a beat frequency of 3 Hz as well as human embryonic stem cell-derived cardiomyocytes with a contractile frequency of about 1 Hz. We also employed the β-adrenergic agonist isoproterenol (100 nmol l-1) and observed that our cantilever demonstrated high sensitivity in detecting subtle changes in both chronotropic and inotropic responses of monolayers. This report describes the utility of our micro-device in both basic cardiovascular research as well as in small molecule drug discovery to monitor cardiac cell contractions.

Applications of Traction Force Microscopy in Measuring Adhesion Molecule Dependent Cell Contractility

ERIC Educational Resources Information Center

Mann, Cynthia Marie

2009-01-01

This work describes the use of polyacrylamide hydrogels as controlled elastic modulus substrates for single cell traction force microscopy studies. The first section describes the use of EDC/NHS chemistry to convalently link microbeads to the hydrogel matrix for the purpose of performing long-term traction force studies (7 days). The final study…

Boundaries steer the contraction of active gels

NASA Astrophysics Data System (ADS)

Schuppler, Matthias; Keber, Felix C.; Kröger, Martin; Bausch, Andreas R.

2016-10-01

Cells set up contractile actin arrays to drive various shape changes and to exert forces to their environment. To understand their assembly process, we present here a reconstituted contractile system, comprising F-actin and myosin II filaments, where we can control the local activation of myosin by light. By stimulating different symmetries, we show that the force balancing at the boundaries determine the shape changes as well as the dynamics of the global contraction. Spatially anisotropic attachment of initially isotropic networks leads to a self-organization of highly aligned contractile fibres, being reminiscent of the order formation in muscles or stress fibres. The observed shape changes and dynamics are fully recovered by a minimal physical model.

PKCδ Regulates Force Signaling during VEGF/CXCL4 Induced Dissociation of Endothelial Tubes

PubMed Central

Jamison, Joshua; Wang, James H-C.; Wells, Alan

2014-01-01

Wound healing requires the vasculature to re-establish itself from the severed ends; endothelial cells within capillaries must detach from neighboring cells before they can migrate into the nascent wound bed to initiate angiogenesis. The dissociation of these endothelial capillaries is driven partially by platelets' release of growth factors and cytokines, particularly the chemokine CXCL4/platelet factor-4 (PF4) that increases cell-cell de-adherence. As this retraction is partly mediated by increased transcellular contractility, the protein kinase c-δ/myosin light chain-2 (PKCδ/MLC-2) signaling axis becomes a candidate mechanism to drive endothelial dissociation. We hypothesize that PKCδ activation induces contractility through MLC-2 to promote dissociation of endothelial cords after exposure to platelet-released CXCL4 and VEGF. To investigate this mechanism of contractility, endothelial cells were allowed to form cords following CXCL4 addition to perpetuate cord dissociation. In this study, CXCL4-induced dissociation was reduced by a VEGFR inhibitor (sunitinib malate) and/or PKCδ inhibition. During combined CXCL4+VEGF treatment, increased contractility mediated by MLC-2 that is dependent on PKCδ regulation. As cellular force is transmitted to focal adhesions, zyxin, a focal adhesion protein that is mechano-responsive, was upregulated after PKCδ inhibition. This study suggests that growth factor regulation of PKCδ may be involved in CXCL4-mediated dissociation of endothelial cords. PMID:24699667

PKCδ regulates force signaling during VEGF/CXCL4 induced dissociation of endothelial tubes.

PubMed

Jamison, Joshua; Wang, James H-C; Wells, Alan

2014-01-01

Wound healing requires the vasculature to re-establish itself from the severed ends; endothelial cells within capillaries must detach from neighboring cells before they can migrate into the nascent wound bed to initiate angiogenesis. The dissociation of these endothelial capillaries is driven partially by platelets' release of growth factors and cytokines, particularly the chemokine CXCL4/platelet factor-4 (PF4) that increases cell-cell de-adherence. As this retraction is partly mediated by increased transcellular contractility, the protein kinase c-δ/myosin light chain-2 (PKCδ/MLC-2) signaling axis becomes a candidate mechanism to drive endothelial dissociation. We hypothesize that PKCδ activation induces contractility through MLC-2 to promote dissociation of endothelial cords after exposure to platelet-released CXCL4 and VEGF. To investigate this mechanism of contractility, endothelial cells were allowed to form cords following CXCL4 addition to perpetuate cord dissociation. In this study, CXCL4-induced dissociation was reduced by a VEGFR inhibitor (sunitinib malate) and/or PKCδ inhibition. During combined CXCL4+VEGF treatment, increased contractility mediated by MLC-2 that is dependent on PKCδ regulation. As cellular force is transmitted to focal adhesions, zyxin, a focal adhesion protein that is mechano-responsive, was upregulated after PKCδ inhibition. This study suggests that growth factor regulation of PKCδ may be involved in CXCL4-mediated dissociation of endothelial cords.

Generation of contractile actomyosin bundles depends on mechanosensitive actin filament assembly and disassembly

PubMed Central

Tojkander, Sari; Gateva, Gergana; Husain, Amjad; Krishnan, Ramaswamy; Lappalainen, Pekka

2015-01-01

Adhesion and morphogenesis of many non-muscle cells are guided by contractile actomyosin bundles called ventral stress fibers. While it is well established that stress fibers are mechanosensitive structures, physical mechanisms by which they assemble, align, and mature have remained elusive. Here we show that arcs, which serve as precursors for ventral stress fibers, undergo lateral fusion during their centripetal flow to form thick actomyosin bundles that apply tension to focal adhesions at their ends. Importantly, this myosin II-derived force inhibits vectorial actin polymerization at focal adhesions through AMPK-mediated phosphorylation of VASP, and thereby halts stress fiber elongation and ensures their proper contractility. Stress fiber maturation additionally requires ADF/cofilin-mediated disassembly of non-contractile stress fibers, whereas contractile fibers are protected from severing. Taken together, these data reveal that myosin-derived tension precisely controls both actin filament assembly and disassembly to ensure generation and proper alignment of contractile stress fibers in migrating cells. DOI: http://dx.doi.org/10.7554/eLife.06126.001 PMID:26652273

Generation of contractile actomyosin bundles depends on mechanosensitive actin filament assembly and disassembly.

PubMed

Tojkander, Sari; Gateva, Gergana; Husain, Amjad; Krishnan, Ramaswamy; Lappalainen, Pekka

2015-12-10

Adhesion and morphogenesis of many non-muscle cells are guided by contractile actomyosin bundles called ventral stress fibers. While it is well established that stress fibers are mechanosensitive structures, physical mechanisms by which they assemble, align, and mature have remained elusive. Here we show that arcs, which serve as precursors for ventral stress fibers, undergo lateral fusion during their centripetal flow to form thick actomyosin bundles that apply tension to focal adhesions at their ends. Importantly, this myosin II-derived force inhibits vectorial actin polymerization at focal adhesions through AMPK-mediated phosphorylation of VASP, and thereby halts stress fiber elongation and ensures their proper contractility. Stress fiber maturation additionally requires ADF/cofilin-mediated disassembly of non-contractile stress fibers, whereas contractile fibers are protected from severing. Taken together, these data reveal that myosin-derived tension precisely controls both actin filament assembly and disassembly to ensure generation and proper alignment of contractile stress fibers in migrating cells.

Contractile forces originating from Cancer Diskiod regulated by geometrical ECM properties

NASA Astrophysics Data System (ADS)

Alobaidi, Amani; Sun, Bo

Cancer cell migration in three-dimensional extracellular matrix is a major cause of death for cancer patients. Although extensive studies have enlightened detailed mechanism of single cell 3D invasion and cell-ECM interaction, 3D collective cancer invasion is still poorly understood. To capture collective cancer invasion with more realistic, we developed a novel 3D invasion assay, Diskiod In Geometrically Micropatterned ECM (DIGME). DIGME allows us to independently controlling the shape the shape of tumor organoids, microstructure and spatial heterogeneity of the extracellular matrix all at the same time. Here we study the affect of contractile forces originating from different geometrical cancer diskiods. We show that cancer invasion could be regulated by geometrical ECM properties.

Stochastic Ratcheting on a Funneled Energy Landscape Is Necessary for Highly Efficient Contractility of Actomyosin Force Dipoles

NASA Astrophysics Data System (ADS)

Komianos, James E.; Papoian, Garegin A.

2018-04-01

Current understanding of how contractility emerges in disordered actomyosin networks of nonmuscle cells is still largely based on the intuition derived from earlier works on muscle contractility. In addition, in disordered networks, passive cross-linkers have been hypothesized to percolate force chains in the network, hence, establishing large-scale connectivity between local contractile clusters. This view, however, largely overlooks the free energy of cross-linker binding at the microscale, which, even in the absence of active fluctuations, provides a thermodynamic drive towards highly overlapping filamentous states. In this work, we use stochastic simulations and mean-field theory to shed light on the dynamics of a single actomyosin force dipole—a pair of antiparallel actin filaments interacting with active myosin II motors and passive cross-linkers. We first show that while passive cross-linking without motor activity can produce significant contraction between a pair of actin filaments, driven by thermodynamic favorability of cross-linker binding, a sharp onset of kinetic arrest exists at large cross-link binding energies, greatly diminishing the effectiveness of this contractility mechanism. Then, when considering an active force dipole containing nonmuscle myosin II, we find that cross-linkers can also serve as a structural ratchet when the motor dissociates stochastically from the actin filaments, resulting in significant force amplification when both molecules are present. Our results provide predictions of how actomyosin force dipoles behave at the molecular level with respect to filament boundary conditions, passive cross-linking, and motor activity, which can explicitly be tested using an optical trapping experiment.

Mechanical influences in bacterial morphogenesis and cell division

NASA Astrophysics Data System (ADS)

Sun, Sean

2010-03-01

Bacterial cells utilize a ring-like organelle (the Z-ring) to accomplish cell division. The Z-ring actively generates a contractile force and influences cell wall growth. We will discuss a general model of bacterial morphogenesis where mechanical forces are coupled to the growth dynamics of the cell wall. The model suggests a physical mechanism that determines the shapes of bacteria cells. The roles of several bacterial cytoskeletal proteins and the Z-ring are discussed. We will also explore molecular mechanisms of force generation by the Z-ring and how cells can generate mechanical forces without molecular motors.

Revealing Early Steps of α2β1 Integrin-mediated Adhesion to Collagen Type I by Using Single-Cell Force Spectroscopy

PubMed Central

Taubenberger, Anna; Cisneros, David A.; Friedrichs, Jens; Puech, Pierre-Henri; Muller, Daniel J.

2007-01-01

We have characterized early steps of α2β1 integrin-mediated cell adhesion to a collagen type I matrix by using single-cell force spectroscopy. In agreement with the role of α2β1 as a collagen type I receptor, α2β1-expressing Chinese hamster ovary (CHO)-A2 cells spread rapidly on the matrix, whereas α2β1-negative CHO wild-type cells adhered poorly. Probing CHO-A2 cell detachment forces over a contact time range of 600 s revealed a nonlinear adhesion response. During the first 60 s, cell adhesion increased slowly, and forces associated with the smallest rupture events were consistent with the breakage of individual integrin–collagen bonds. Above 60 s, a fraction of cells rapidly switched into an activated adhesion state marked by up to 10-fold increased detachment forces. Elevated overall cell adhesion coincided with a rise of the smallest rupture forces above the value required to break a single-integrin–collagen bond, suggesting a change from single to cooperative receptor binding. Transition into the activated adhesion mode and the increase of the smallest rupture forces were both blocked by inhibitors of actomyosin contractility. We therefore propose a two-step mechanism for the establishment of α2β1-mediated adhesion as weak initial, single-integrin–mediated binding events are superseded by strong adhesive interactions involving receptor cooperativity and actomyosin contractility. PMID:17314408

Traction in smooth muscle cells varies with cell spreading

NASA Technical Reports Server (NTRS)

Tolic-Norrelykke, Iva Marija; Wang, Ning

2005-01-01

Changes in cell shape regulate cell growth, differentiation, and apoptosis. It has been suggested that the regulation of cell function by the cell shape is a result of the tension in the cytoskeleton and the distortion of the cell. Here we explore the association between cell-generated mechanical forces and the cell morphology. We hypothesized that the cell contractile force is associated with the degree of cell spreading, in particular with the cell length. We measured traction fields of single human airway smooth muscle cells plated on a polyacrylamide gel, in which fluorescent microbeads were embedded to serve as markers of gel deformation. The traction exerted by the cells at the cell-substrate interface was determined from the measured deformation of the gel. The traction was measured before and after treatment with the contractile agonist histamine, or the relaxing agonist isoproterenol. The relative increase in traction induced by histamine was negatively correlated with the baseline traction. On the contrary, the relative decrease in traction due to isoproterenol was independent of the baseline traction, but it was associated with cell shape: traction decreased more in elongated than in round cells. Maximum cell width, mean cell width, and projected area of the cell were the parameters most tightly coupled to both baseline and histamine-induced traction in this study. Wide and well-spread cells exerted larger traction than slim cells. These results suggest that cell contractility is controlled by cell spreading.

Expansion and concatenation of nonmuscle myosin IIA filaments drive cellular contractile system formation during interphase and mitosis

PubMed Central

Fenix, Aidan M.; Taneja, Nilay; Buttler, Carmen A.; Lewis, John; Van Engelenburg, Schuyler B.; Ohi, Ryoma; Burnette, Dylan T.

2016-01-01

Cell movement and cytokinesis are facilitated by contractile forces generated by the molecular motor, nonmuscle myosin II (NMII). NMII molecules form a filament (NMII-F) through interactions of their C-terminal rod domains, positioning groups of N-terminal motor domains on opposite sides. The NMII motors then bind and pull actin filaments toward the NMII-F, thus driving contraction. Inside of crawling cells, NMIIA-Fs form large macromolecular ensembles (i.e., NMIIA-F stacks), but how this occurs is unknown. Here we show NMIIA-F stacks are formed through two non–mutually exclusive mechanisms: expansion and concatenation. During expansion, NMIIA molecules within the NMIIA-F spread out concurrent with addition of new NMIIA molecules. Concatenation occurs when multiple NMIIA-Fs/NMIIA-F stacks move together and align. We found that NMIIA-F stack formation was regulated by both motor activity and the availability of surrounding actin filaments. Furthermore, our data showed expansion and concatenation also formed the contractile ring in dividing cells. Thus interphase and mitotic cells share similar mechanisms for creating large contractile units, and these are likely to underlie how other myosin II–based contractile systems are assembled. PMID:26960797

Assembly Mechanism of the Contractile Ring for Cytokinesis by Fission Yeast

NASA Astrophysics Data System (ADS)

Vavylonis, Dimitrios; Wu, Jian-Qiu; Huang, Xiaolei; O'Shaughnessy, Ben; Pollard, Thomas

2008-03-01

Animals and fungi assemble a contractile ring of actin filaments and the motor protein myosin to separate into individual daughter cells during cytokinesis. We studied the mechanism of contractile ring assembly in fission yeast with high time resolution confocal microscopy, computational image analysis methods, and numerical simulations. Approximately 63 nodes containing myosin, broadly distributed around the cell equator, assembled into a ring through stochastic motions, making many starts, stops, and changes of direction as they condense into a ring. Estimates of node friction coefficients from the mean square displacement of stationary nodes imply forces for node movement are greater than ˜ 4 pN, similarly to forces by a few molecular motors. Skeletonization and topology analysis of images of cells expressing fluorescent actin filament markers showed transient linear elements extending in all directions from myosin nodes and establishing connections among them. We propose a model with traction between nodes depending on transient connections established by stochastic search and capture (``search, capture, pull and release''). Numerical simulations of the model using parameter values obtained from experiment succesfully condense nodes into a continuous ring.

Substrate stiffness regulates cadherin-dependent collective migration through myosin-II contractility

PubMed Central

Ng, Mei Rosa; Besser, Achim

2012-01-01

The mechanical microenvironment is known to influence single-cell migration; however, the extent to which mechanical cues affect collective migration of adherent cells is not well understood. We measured the effects of varying substrate compliance on individual cell migratory properties in an epithelial wound-healing assay. Increasing substrate stiffness increased collective cell migration speed, persistence, and directionality as well as the coordination of cell movements. Dynamic analysis revealed that wounding initiated a wave of motion coordination from the wound edge into the sheet. This was accompanied by a front-to-back gradient of myosin-II activation and establishment of cell polarity. The propagation was faster and farther reaching on stiff substrates, indicating that substrate stiffness affects the transmission of directional cues. Manipulation of myosin-II activity and cadherin–catenin complexes revealed that this transmission is mediated by coupling of contractile forces between neighboring cells. Thus, our findings suggest that the mechanical environment integrates in a feedback with cell contractility and cell–cell adhesion to regulate collective migration. PMID:23091067

Increased CCT-eta expression is a marker of latent and active disease and a modulator of fibroblast contractility in Dupuytren's contracture.

PubMed

Satish, Latha; O'Gorman, David B; Johnson, Sandra; Raykha, Christina; Gan, Bing Siang; Wang, James H-C; Kathju, Sandeep

2013-07-01

Dupuytren's contracture (DC) is a fibroproliferative disorder of unknown etiology characterized by a scar-like contracture that develops in the palm and/or digits. We have previously reported that the eta subunit of the chaperonin containing T-complex polypeptide (CCT-eta) is increased in fibrotic wound healing, and is essential for the accumulation of α-smooth muscle actin (α-SMA) in fibroblasts. The purpose of this study was to determine if CCT-eta is similarly implicated in the aberrant fibrosis seen in DC and to investigate the role of CCT-eta in the behavior of myo/fibroblasts in DC. Fibroblasts were obtained from DC-affected palmar fascia, from adjacent phenotypically normal palmar fascia in the same DC patients (PF), and from non-DC palmar fascial tissues in patients undergoing carpal tunnel (CT) release. Inherent contractility in these three populations was examined using fibroblast-populated collagen lattices (FPCLs) and by cell traction force microscopy. Expression of CCT-eta and α-SMA protein was determined by Western blot. The effect of CCT-eta inhibition on the contractility of DC cells was determined by deploying an siRNA versus CCT-eta. DC cells were significantly more contractile than both matching palmar fascial (PF) cells and CT cells in both assays, with PF cells demonstrating an intermediate contractility in the FPCL assay. Whereas α-SMA protein was significantly increased only in DC cells compared to PF and CT cells, CCT-eta protein was significantly increased in both PF and DC cells compared to CT cells. siRNA-mediated depletion of CCT-eta inhibited the accumulation of both CCT-eta and α-SMA protein in DC cells, and also significantly decreased the contractility of treated DC cells. These observations suggest that increased expression of CCT-eta appears to be a marker for latent and active disease in these patients and to be essential for the increased contractility exhibited by these fibroblasts.

Emergence of airway smooth muscle mechanical behavior through dynamic reorganization of contractile units and force transmission pathways

PubMed Central

2014-01-01

Airway hyperresponsiveness (AHR) in asthma remains poorly understood despite significant research effort to elucidate relevant underlying mechanisms. In particular, a significant body of experimental work has focused on the effect of tidal fluctuations on airway smooth muscle (ASM) cells, tissues, lung slices, and whole airways to understand the bronchodilating effect of tidal breathing and deep inspirations. These studies have motivated conceptual models that involve dynamic reorganization of both cytoskeletal components as well as contractile machinery. In this article, a biophysical model of the whole ASM cell is presented that combines 1) crossbridge cycling between actin and myosin; 2) actin-myosin disconnectivity, under imposed length changes, to allow dynamic reconfiguration of “force transmission pathways”; and 3) dynamic parallel-to-serial transitions of contractile units within these pathways that occur through a length fluctuation. Results of this theoretical model suggest that behavior characteristic of experimentally observed force-length loops of maximally activated ASM strips can be explained by interactions among the three mechanisms. Crucially, both sustained disconnectivity and parallel-to-serial transitions are necessary to explain the nature of hysteresis and strain stiffening observed experimentally. The results provide strong evidence that dynamic rearrangement of contractile machinery is a likely mechanism underlying many of the phenomena observed at timescales associated with tidal breathing. This theoretical cell-level model captures many of the salient features of mechanical behavior observed experimentally and should provide a useful starting block for a bottom-up approach to understanding tissue-level mechanical behavior. PMID:24481961

Local 3D matrix microenvironment regulates cell migration through spatiotemporal dynamics of contractility-dependent adhesions

NASA Astrophysics Data System (ADS)

Doyle, Andrew D.; Carvajal, Nicole; Jin, Albert; Matsumoto, Kazue; Yamada, Kenneth M.

2015-11-01

The physical properties of two-dimensional (2D) extracellular matrices (ECMs) modulate cell adhesion dynamics and motility, but little is known about the roles of local microenvironmental differences in three-dimensional (3D) ECMs. Here we generate 3D collagen gels of varying matrix microarchitectures to characterize their regulation of 3D adhesion dynamics and cell migration. ECMs containing bundled fibrils demonstrate enhanced local adhesion-scale stiffness and increased adhesion stability through balanced ECM/adhesion coupling, whereas highly pliable reticular matrices promote adhesion retraction. 3D adhesion dynamics are locally regulated by ECM rigidity together with integrin/ECM association and myosin II contractility. Unlike 2D migration, abrogating contractility stalls 3D migration regardless of ECM pore size. We find force is not required for clustering of activated integrins on 3D native collagen fibrils. We propose that efficient 3D migration requires local balancing of contractility with ECM stiffness to stabilize adhesions, which facilitates the detachment of activated integrins from ECM fibrils.

BK Channel-Mediated Relaxation of Urinary Bladder Smooth Muscle: A Novel Paradigm for Phosphodiesterase Type 4 Regulation of Bladder Function

PubMed Central

Xin, Wenkuan; Li, Ning; Cheng, Qiuping

2014-01-01

Elevation of intracellular cAMP and activation of protein kinase A (PKA) lead to activation of large conductance voltage- and Ca2+-activated K+ (BK) channels, thus attenuation of detrusor smooth muscle (DSM) contractility. In this study, we investigated the mechanism by which pharmacological inhibition of cAMP-specific phosphodiesterase 4 (PDE4) with rolipram or Ro-20-1724 (C15H22N2O3) suppresses guinea pig DSM excitability and contractility. We used high-speed line-scanning confocal microscopy, ratiometric fluorescence Ca2+ imaging, and perforated whole-cell patch-clamp techniques on freshly isolated DSM cells, along with isometric tension recordings of DSM isolated strips. Rolipram caused an increase in the frequency of Ca2+ sparks and the spontaneous transient BK currents (TBKCs), hyperpolarized the cell membrane potential (MP), and decreased the intracellular Ca2+ levels. Blocking BK channels with paxilline reversed the hyperpolarizing effect of rolipram and depolarized the MP back to the control levels. In the presence of H-89 [N-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide dihydrochloride], a PKA inhibitor, rolipram did not cause MP hyperpolarization. Rolipram or Ro-20-1724 reduced DSM spontaneous and carbachol-induced phasic contraction amplitude, muscle force, duration, and frequency, and electrical field stimulation-induced contraction amplitude, muscle force, and tone. Paxilline recovered DSM contractility, which was suppressed by pretreatment with PDE4 inhibitors. Rolipram had reduced inhibitory effects on DSM contractility in DSM strips pretreated with paxilline. This study revealed a novel cellular mechanism whereby pharmacological inhibition of PDE4 leads to suppression of guinea pig DSM contractility by increasing the frequency of Ca2+ sparks and the functionally coupled TBKCs, consequently hyperpolarizing DSM cell MP. Collectively, this decreases the global intracellular Ca2+ levels and DSM contractility in a BK channel-dependent manner. PMID:24459245

Roles of Formin Nodes and Myosin Motor Activity in Mid1p-dependent Contractile-Ring Assembly during Fission Yeast Cytokinesis

PubMed Central

Coffman, Valerie C.; Nile, Aaron H.; Lee, I-Ju; Liu, Huayang

2009-01-01

Two prevailing models have emerged to explain the mechanism of contractile-ring assembly during cytokinesis in the fission yeast Schizosaccharomyces pombe: the spot/leading cable model and the search, capture, pull, and release (SCPR) model. We tested some of the basic assumptions of the two models. Monte Carlo simulations of the SCPR model require that the formin Cdc12p is present in >30 nodes from which actin filaments are nucleated and captured by myosin-II in neighboring nodes. The force produced by myosin motors pulls the nodes together to form a compact contractile ring. Live microscopy of cells expressing Cdc12p fluorescent fusion proteins shows for the first time that Cdc12p localizes to a broad band of 30–50 dynamic nodes, where actin filaments are nucleated in random directions. The proposed progenitor spot, essential for the spot/leading cable model, usually disappears without nucleating actin filaments. α-Actinin ain1 deletion cells form a normal contractile ring through nodes in the absence of the spot. Myosin motor activity is required to condense the nodes into a contractile ring, based on slower or absent node condensation in myo2-E1 and UCS rng3-65 mutants. Taken together, these data provide strong support for the SCPR model of contractile-ring formation in cytokinesis. PMID:19864459

Comparing contractile apparatus-driven cytokinesis mechanisms across kingdoms.

PubMed

Balasubramanian, Mohan K; Srinivasan, Ramanujam; Huang, Yinyi; Ng, Kian-Hong

2012-11-01

Cytokinesis is the final stage of the cell cycle during which a cell physically divides into two daughters through the assembly of new membranes (and cell wall in some cases) between the forming daughters. New membrane assembly can either proceed centripetally behind a contractile apparatus, as in the case of prokaryotes, archaea, fungi, and animals or expand centrifugally, as in the case of higher plants. In this article, we compare the mechanisms of cytokinesis in diverse organisms dividing through the use of a contractile apparatus. While an actomyosin ring participates in cytokinesis in almost all centripetally dividing eukaryotes, the majority of bacteria and archaea (except Crenarchaea) divide using a ring composed of the tubulin-related protein FtsZ. Curiously, despite molecular conservation of the division machinery components, division site placement and its cell cycle regulation occur by a variety of unrelated mechanisms even among organisms from the same kingdom. While molecular motors and cytoskeletal polymer dynamics contribute to force generation during eukaryotic cytokinesis, cytoskeletal polymer dynamics alone appears to be sufficient for force generation during prokaryotic cytokinesis. Intriguingly, there are life forms on this planet that appear to lack molecules currently known to participate in cytokinesis and how these cells perform cytokinesis remains a mystery waiting to be unravelled. Copyright © 2012 Wiley Periodicals, Inc.

The contractile ring coordinates curvature-dependent septum assembly during fission yeast cytokinesis

PubMed Central

Zhou, Zhou; Munteanu, Emilia Laura; He, Jun; Ursell, Tristan; Bathe, Mark; Huang, Kerwyn Casey; Chang, Fred

2015-01-01

The functions of the actin-myosin–based contractile ring in cytokinesis remain to be elucidated. Recent findings show that in the fission yeast Schizosaccharomyces pombe, cleavage furrow ingression is driven by polymerization of cell wall fibers outside the plasma membrane, not by the contractile ring. Here we show that one function of the ring is to spatially coordinate septum cell wall assembly. We develop an improved method for live-cell imaging of the division apparatus by orienting the rod-shaped cells vertically using microfabricated wells. We observe that the septum hole and ring are circular and centered in wild-type cells and that in the absence of a functional ring, the septum continues to ingress but in a disorganized and asymmetric manner. By manipulating the cleavage furrow into different shapes, we show that the ring promotes local septum growth in a curvature-dependent manner, allowing even a misshapen septum to grow into a more regular shape. This curvature-dependent growth suggests a model in which contractile forces of the ring shape the septum cell wall by stimulating the cell wall machinery in a mechanosensitive manner. Mechanical regulation of the cell wall assembly may have general relevance to the morphogenesis of walled cells. PMID:25355954

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

PubMed Central

Park, Ki Ho; Brotto, Leticia; Lehoang, Oanh; Brotto, Marco; Ma, Jianjie; Zhao, Xiaoli

2012-01-01

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle. PMID:23149471

TGF-β-Induced Transcription Sustains Amoeboid Melanoma Migration and Dissemination

PubMed Central

Cantelli, Gaia; Orgaz, Jose L.; Rodriguez-Hernandez, Irene; Karagiannis, Panagiotis; Maiques, Oscar; Matias-Guiu, Xavier; Nestle, Frank O.; Marti, Rosa M.; Karagiannis, Sophia N.; Sanz-Moreno, Victoria

2015-01-01

Summary Cell migration underlies metastatic dissemination of cancer cells, and fast “amoeboid” migration in the invasive fronts of tumors is controlled by high levels of actomyosin contractility. How amoeboid migration is regulated by extracellular signals and sustained over time by transcriptional changes is not fully understood. Transforming growth factor β (TGF-β) is well known to promote epithelial-to-mesenchymal transition (EMT) and contribute to metastasis, but melanocytes are neural crest derivatives that have undergone EMT during embryonic development. Surprisingly, we find that in melanoma, TGF-β promotes amoeboid features such as cell rounding, membrane blebbing, high levels of contractility, and increased invasion. Using genome-wide transcriptomics, we find that amoeboid melanoma cells are enriched in a TGF-β-driven signature. We observe that downstream of TGF-β, SMAD2 and its adaptor CITED1 control amoeboid behavior by regulating the expression of key genes that activate contractile forces. Moreover, CITED1 is highly upregulated during melanoma progression, and its high expression is associated with poor prognosis. CITED1 is coupled to a contractile-rounded, amoeboid phenotype in a panel of 16 melanoma cell lines, in mouse melanoma xenografts, and in 47 human melanoma patients. Its expression is also enriched in the invasive fronts of lesions. Functionally, we show how the TGF-β-SMAD2-CITED1 axis promotes different steps associated with progression: melanoma detachment from keratinocytes, 2D and 3D migration, attachment to endothelial cells, and in vivo lung metastatic initial colonization and outgrowth. We propose a novel mechanism by which TGF-β-induced transcription sustains actomyosin force in melanoma cells and thereby promotes melanoma progression independently of EMT. PMID:26526369

Modeling the two-way feedback between contractility and matrix realignment reveals a nonlinear mode of cancer cell invasion

PubMed Central

Ahmadzadeh, Hossein; Webster, Marie R.; Behera, Reeti; Jimenez Valencia, Angela M.; Wirtz, Denis; Weeraratna, Ashani T.; Shenoy, Vivek B.

2017-01-01

Cancer cell invasion from primary tumors is mediated by a complex interplay between cellular adhesions, actomyosin-driven contractility, and the physical characteristics of the extracellular matrix (ECM). Here, we incorporate a mechanochemical free-energy–based approach to elucidate how the two-way feedback loop between cell contractility (induced by the activity of chemomechanical interactions such as Ca2+ and Rho signaling pathways) and matrix fiber realignment and strain stiffening enables the cells to polarize and develop contractile forces to break free from the tumor spheroids and invade into the ECM. Interestingly, through this computational model, we are able to identify a critical stiffness that is required by the matrix to break intercellular adhesions and initiate cell invasion. Also, by considering the kinetics of the cell movement, our model predicts a biphasic invasiveness with respect to the stiffness of the matrix. These predictions are validated by analyzing the invasion of melanoma cells in collagen matrices of varying concentration. Our model also predicts a positive correlation between the elongated morphology of the invading cells and the alignment of fibers in the matrix, suggesting that cell polarization is directly proportional to the stiffness and alignment of the matrix. In contrast, cells in nonfibrous matrices are found to be rounded and not polarized, underscoring the key role played by the nonlinear mechanics of fibrous matrices. Importantly, our model shows that mechanical principles mediated by the contractility of the cells and the nonlinearity of the ECM behavior play a crucial role in determining the phenotype of the cell invasion. PMID:28196892

Spatial differences of cellular origins and in vivo hypoxia modify contractile properties of pulmonary artery smooth muscle cells: lessons for arterial tissue engineering.

PubMed

Hall, S M; Soueid, A; Smith, T; Brown, R A; Haworth, S G; Mudera, V

2007-01-01

Tissue engineering of functional arteries is challenging. Within the pulmonary artery wall, smooth muscle cells (PASMCs) have site-specific developmental and functional phenotypes, reflecting differing contractile roles. The force generated by PASMCs isolated from the inner 25% and outer 50% of the media of intrapulmonary elastic arteries from five normal and eight chronically hypoxic (hypertensive) 14 day-old piglets was quantified in a three-dimensional (3D) collagen construct, using a culture force monitor. Outer medial PASMCs from normal piglets exerted more force (528 +/- 50 dynes) than those of hypoxic piglets (177 +/- 42 dynes; p < 0.01). Force generation by inner medial PASMCs from normal and hypoxic piglets was similar (349 +/- 35 and 239 +/- 60 dynes). In response to agonist (thromboxane) stimulation, all PASMCs from normal and hypoxic piglets contracted, but the increase in force generated by outer and inner hypoxic PASMCs (ranges 13-72 and 14-56 dynes) was less than by normal PASMCs (ranges 27-154 and 34-159 dynes, respectively; p < 0.05 for both). All hypoxic PASMCs were unresponsive to antagonist (sodium nitroprusside) stimulation, all normal PASMCs relaxed (range - 87 to - 494 dynes). Myosin heavy chain expression by both hypoxic PASMC phenotypes was less than normal (p < 0.05 for both), as was the activity of focal adhesion kinase, regulating contraction, in hypoxic inner PASMCs (p < 0.01). Chronic hypoxia resulted in the development of abnormal PASMC phenotypes, which in collagen constructs exhibited a reduction in contractile force and reactivity to agonists. Characterization of the mechanical response of spatially distinct cells and modification of their behaviour by hypoxia is critical for successful tissue engineering of major blood vessels.

Contractile dysfunctions in ATP-dependent K+ channel-deficient mouse muscle during fatigue involve excessive depolarization and Ca2+ influx through L-type Ca2+ channels.

PubMed

Cifelli, Carlo; Boudreault, Louise; Gong, Bing; Bercier, Jean-Philippe; Renaud, Jean-Marc

2008-10-01

Muscles deficient in ATP-dependent potassium (KATP) channels develop contractile dysfunctions during fatigue that may explain their apparently faster rate of fatigue compared with wild-type muscles. The objectives of this study were to determine: (1) whether the contractile dysfunctions, namely unstimulated force and depressed force recovery, result from excessive membrane depolarization and Ca2+ influx through L-type Ca2+ channels; and (2) whether reducing the magnitude of these two contractile dysfunctions reduces the rate of fatigue in KATP channel-deficient muscles. To reduce Ca2+ influx, we lowered the extracellular Ca2+ concentration ([Ca2+]o) from 2.4 to 0.6 mM or added 1 microM verapamil, an L-type Ca2+ channel blocker. Flexor digitorum brevis (FDB) muscles deficient in KATP channels were obtained by exposing wild-type muscles to 10 microM glibenclamide or by using FDB from Kir6.2-/- mice. Fatigue was elicited with one contraction per second for 3 min at 37 degrees C. In wild-type FDB, lowered [Ca2+]o or verapamil did not affect the decrease in peak tetanic force and unstimulated force during fatigue and force recovery following fatigue. In KATP channel-deficient FDB, lowered [Ca2+]o or verapamil slowed down the decrease in peak tetanic force recovery, reduced unstimulated force and improved force recovery. In Kir6.2-/- FDB, the rate of fatigue became slower than in wild-type FDB in the presence of verapamil. The cell membrane depolarized from -83 to -57 mV in normal wild-type FDB. The depolarizations in some glibenclamide-exposed fibres were similar to those of normal FDB, while in other fibres the cell membrane depolarized to -31 mV in 80 s, which was also the time when these fibres supercontracted. It is concluded that: (1) KATP channels are crucial in preventing excessive membrane depolarization and Ca2+ influx through L-type Ca2+ channels; and (2) they contribute to the decrease in force during fatigue.

A free-boundary model of a motile cell explains turning behavior.

PubMed

Nickaeen, Masoud; Novak, Igor L; Pulford, Stephanie; Rumack, Aaron; Brandon, Jamie; Slepchenko, Boris M; Mogilner, Alex

2017-11-01

To understand shapes and movements of cells undergoing lamellipodial motility, we systematically explore minimal free-boundary models of actin-myosin contractility consisting of the force-balance and myosin transport equations. The models account for isotropic contraction proportional to myosin density, viscous stresses in the actin network, and constant-strength viscous-like adhesion. The contraction generates a spatially graded centripetal actin flow, which in turn reinforces the contraction via myosin redistribution and causes retraction of the lamellipodial boundary. Actin protrusion at the boundary counters the retraction, and the balance of the protrusion and retraction shapes the lamellipodium. The model analysis shows that initiation of motility critically depends on three dimensionless parameter combinations, which represent myosin-dependent contractility, a characteristic viscosity-adhesion length, and a rate of actin protrusion. When the contractility is sufficiently strong, cells break symmetry and move steadily along either straight or circular trajectories, and the motile behavior is sensitive to conditions at the cell boundary. Scanning of a model parameter space shows that the contractile mechanism of motility supports robust cell turning in conditions where short viscosity-adhesion lengths and fast protrusion cause an accumulation of myosin in a small region at the cell rear, destabilizing the axial symmetry of a moving cell.

Effects of Matrix Alignment and Mechanical Constraints on Cellular Behavior in 3D Engineered Microtissues

NASA Astrophysics Data System (ADS)

Bose, Prasenjit; Eyckmans, Jeroen; Chen, Christopher; Reich, Daniel

The adhesion of cells to the extracellular matrix (ECM) plays a crucial role in a variety of cellular functions. The main building blocks of the ECM are 3D networks of fibrous proteins whose structure and alignments varies with tissue type. However, the impact of ECM alignment on cellular behaviors such as cell adhesion, spreading, extension and mechanics remains poorly understood. We present results on the development of a microtissue-based system that enables control of the structure, orientation, and degree of fibrillar alignment in 3D fibroblast-populated collagen gels. The tissues self-assemble from cell-laden collagen gels placed in micro-fabricated wells containing sets of elastic pillars. The contractile action of the cells leads to controlled alignment of the fibrous collagen, depending on the number and location of the pillars in each well. The pillars are elastic, and are utilized to measure the contractile forces of the microtissues, and by incorporating magnetic material in selected pillars, time-varying forces can be applied to the tissues for dynamic stimulation and measurement of mechanical properties. Results on the effects of varying pillar shape, spacing, location, and stiffness on microtissue organization and contractility will be presented. This work is supported by NSF CMMI-1463011.

Cell density and actomyosin contractility control the organization of migrating collectives within an epithelium

PubMed Central

Loza, Andrew J.; Koride, Sarita; Schimizzi, Gregory V.; Li, Bo; Sun, Sean X.; Longmore, Gregory D.

2016-01-01

The mechanisms underlying collective migration are important for understanding development, wound healing, and tumor invasion. Here we focus on cell density to determine its role in collective migration. Our findings show that increasing cell density, as might be seen in cancer, transforms groups from broad collectives to small, narrow streams. Conversely, diminishing cell density, as might occur at a wound front, leads to large, broad collectives with a distinct leader–follower structure. Simulations identify force-sensitive contractility as a mediator of how density affects collectives, and guided by this prediction, we find that the baseline state of contractility can enhance or reduce organization. Finally, we test predictions from these data in an in vivo epithelium by using genetic manipulations to drive collective motion between predicted migratory phases. This work demonstrates how commonly altered cellular properties can prime groups of cells to adopt migration patterns that may be harnessed in health or exploited in disease. PMID:27605707

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers.

PubMed

Roche, Stuart M; Gumucio, Jonathan P; Brooks, Susan V; Mendias, Christopher L; Claflin, Dennis R

2015-06-16

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

MEMS piezoresistive cantilever for the direct measurement of cardiomyocyte contractile force

NASA Astrophysics Data System (ADS)

Matsudaira, Kenei; Nguyen, Thanh-Vinh; Hirayama Shoji, Kayoko; Tsukagoshi, Takuya; Takahata, Tomoyuki; Shimoyama, Isao

2017-10-01

This paper reports on a method to directly measure the contractile forces of cardiomyocytes using MEMS (micro electro mechanical systems)-based force sensors. The fabricated sensor chip consists of piezoresistive cantilevers that can measure contractile forces with high frequency (several tens of kHz) and high sensing resolution (less than 0.1 nN). Moreover, the proposed method does not require a complex observation system or image processing, which are necessary in conventional optical-based methods. This paper describes the design, fabrication, and evaluation of the proposed device and demonstrates the direct measurements of contractile forces of cardiomyocytes using the fabricated device.

A WAVE2–Arp2/3 actin nucleator apparatus supports junctional tension at the epithelial zonula adherens

PubMed Central

Verma, Suzie; Han, Siew Ping; Michael, Magdalene; Gomez, Guillermo A.; Yang, Zhe; Teasdale, Rohan D.; Ratheesh, Aparna; Kovacs, Eva M.; Ali, Radiya G.; Yap, Alpha S.

2012-01-01

The epithelial zonula adherens (ZA) is a specialized adhesive junction where actin dynamics and myosin-driven contractility coincide. The junctional cytoskeleton is enriched in myosin II, which generates contractile force to support junctional tension. It is also enriched in dynamic actin filaments, which are replenished by ongoing actin assembly. In this study we sought to pursue the relationship between actin assembly and junctional contractility. We demonstrate that WAVE2–Arp2/3 is a major nucleator of actin assembly at the ZA and likely acts in response to junctional Rac signaling. Furthermore, WAVE2–Arp2/3 is necessary for junctional integrity and contractile tension at the ZA. Maneuvers that disrupt the function of either WAVE2 or Arp2/3 reduced junctional tension and compromised the ability of cells to buffer side-to-side forces acting on the ZA. WAVE2–Arp2/3 disruption depleted junctions of both myosin IIA and IIB, suggesting that dynamic actin assembly may support junctional tension by facilitating the local recruitment of myosin. PMID:23051739

A WAVE2-Arp2/3 actin nucleator apparatus supports junctional tension at the epithelial zonula adherens.

PubMed

Verma, Suzie; Han, Siew Ping; Michael, Magdalene; Gomez, Guillermo A; Yang, Zhe; Teasdale, Rohan D; Ratheesh, Aparna; Kovacs, Eva M; Ali, Radiya G; Yap, Alpha S

2012-12-01

The epithelial zonula adherens (ZA) is a specialized adhesive junction where actin dynamics and myosin-driven contractility coincide. The junctional cytoskeleton is enriched in myosin II, which generates contractile force to support junctional tension. It is also enriched in dynamic actin filaments, which are replenished by ongoing actin assembly. In this study we sought to pursue the relationship between actin assembly and junctional contractility. We demonstrate that WAVE2-Arp2/3 is a major nucleator of actin assembly at the ZA and likely acts in response to junctional Rac signaling. Furthermore, WAVE2-Arp2/3 is necessary for junctional integrity and contractile tension at the ZA. Maneuvers that disrupt the function of either WAVE2 or Arp2/3 reduced junctional tension and compromised the ability of cells to buffer side-to-side forces acting on the ZA. WAVE2-Arp2/3 disruption depleted junctions of both myosin IIA and IIB, suggesting that dynamic actin assembly may support junctional tension by facilitating the local recruitment of myosin.

Nine unanswered questions about cytokinesis

PubMed Central

2017-01-01

Experiments on model systems have revealed that cytokinesis in cells with contractile rings (amoebas, fungi, and animals) depends on shared molecular mechanisms in spite of some differences that emerged during a billion years of divergent evolution. Understanding these fundamental mechanisms depends on identifying the participating proteins and characterizing the mechanisms that position the furrow, assemble the contractile ring, anchor the ring to the plasma membrane, trigger ring constriction, produce force to form a furrow, disassemble the ring, expand the plasma membrane in the furrow, and separate the daughter cell membranes. This review reveals that fascinating questions remain about each step. PMID:28807993

Nine unanswered questions about cytokinesis.

PubMed

Pollard, Thomas D

2017-10-02

Experiments on model systems have revealed that cytokinesis in cells with contractile rings (amoebas, fungi, and animals) depends on shared molecular mechanisms in spite of some differences that emerged during a billion years of divergent evolution. Understanding these fundamental mechanisms depends on identifying the participating proteins and characterizing the mechanisms that position the furrow, assemble the contractile ring, anchor the ring to the plasma membrane, trigger ring constriction, produce force to form a furrow, disassemble the ring, expand the plasma membrane in the furrow, and separate the daughter cell membranes. This review reveals that fascinating questions remain about each step. © 2017 Pollard.

Unsteady motion, finite Reynolds numbers, and wall effect on Vorticella convallaria contribute contraction force greater than the stokes drag.

PubMed

Ryu, Sangjin; Matsudaira, Paul

2010-06-02

Contraction of Vorticella convallaria, a sessile ciliated protozoan, is completed within a few milliseconds and results in a retraction of its cell body toward the substratum by coiling its stalk. Previous studies have modeled the cell body as a sphere and assumed a drag force that satisfies Stokes' law. However, the contraction-induced flow of the medium is transient and bounded by the substrate, and the maximum Reynolds number is larger than unity. Thus, calculations of contractile force from the drag force are incomplete. In this study, we analyzed fluid flow during contraction by the particle tracking velocimetry and computational fluid dynamics simulations to estimate the contractile force. Particle paths show that the induced flow is limited by the substrate. Simulation-based force estimates suggest that the combined effect of the flow unsteadiness, the finite Reynolds number, and the substrate comprises 35% of the total force. The work done in the early stage of contraction and the maximum power output are similar regardless of the medium viscosity. These results suggest that, during the initial development of force, V. convallaria uses a common mechanism for performing mechanical work irrespective of viscous loading conditions. Copyright (c) 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Store-Operated Ca2+ Entry (SOCE) Contributes to Normal Skeletal Muscle Contractility in young but not in aged skeletal muscle

PubMed Central

Brotto, Leticia S.; Bougoin, Sylvain; Nosek, Thomas M.; Reid, Michael; Hardin, Brian; Pan, Zui; Ma, Jianjie; Parness, Jerome

2011-01-01

Muscle atrophy alone is insufficient to explain the significant decline in contractile force of skeletal muscle during normal aging. One contributing factor to decreased contractile force in aging skeletal muscle could be compromised excitation-contraction (E-C) coupling, without sufficient available Ca2+ to allow for repetitive muscle contractility, skeletal muscles naturally become weaker. Using biophysical approaches, we previously showed that store-operated Ca2+ entry (SOCE) is compromised in aged skeletal muscle but not in young ones. While important, a missing component from previous studies is whether or not SOCE function correlates with contractile function during aging. Here we test the contribution of extracellular Ca2+ to contractile function of skeletal muscle during aging. First, we demonstrate graded coupling between SR Ca2+ release channel-mediated Ca2+ release and activation of SOCE. Inhibition of SOCE produced significant reduction of contractile force in young skeletal muscle, particularly at high frequency stimulation, and such effects were completely absent in aged skeletal muscle. Our data indicate that SOCE contributes to the normal physiological contractile response of young healthy skeletal muscle and that defective extracellular Ca2+ entry through SOCE contributes to the reduced contractile force characteristic of aged skeletal muscle. PMID:21666285

Store-operated Ca(2+) entry (SOCE) contributes to normal skeletal muscle contractility in young but not in aged skeletal muscle.

PubMed

Thornton, Angela M; Zhao, Xiaoli; Weisleder, Noah; Brotto, Leticia S; Bougoin, Sylvain; Nosek, Thomas M; Reid, Michael; Hardin, Brian; Pan, Zui; Ma, Jianjie; Parness, Jerome; Brotto, Marco

2011-06-01

Muscle atrophy alone is insufficient to explain the significant decline in contractile force of skeletal muscle during normal aging. One contributing factor to decreased contractile force in aging skeletal muscle could be compromised excitation-contraction (E-C) coupling, without sufficient available Ca(2+) to allow for repetitive muscle contractility, skeletal muscles naturally become weaker. Using biophysical approaches, we previously showed that store-operated Ca(2+) entry (SOCE) is compromised in aged skeletal muscle but not in young ones. While important, a missing component from previous studies is whether or not SOCE function correlates with contractile function during aging. Here we test the contribution of extracellular Ca(2+) to contractile function of skeletal muscle during aging. First, we demonstrate graded coupling between SR Ca(2+) release channel-mediated Ca(2+) release and activation of SOCE. Inhibition of SOCE produced significant reduction of contractile force in young skeletal muscle, particularly at high frequency stimulation, and such effects were completely absent in aged skeletal muscle. Our data indicate that SOCE contributes to the normal physiological contractile response of young healthy skeletal muscle and that defective extracellular Ca(2+) entry through SOCE contributes to the reduced contractile force characteristic of aged skeletal muscle.

Stalk-length-dependence of the contractility of Vorticella convallaria

NASA Astrophysics Data System (ADS)

Gul Chung, Eun; Ryu, Sangjin

2017-12-01

Vorticella convallaria is a sessile protozoan of which the spasmoneme contracts on a millisecond timescale. Because this contraction is induced and powered by the binding of calcium ions (Ca2+), the spasmoneme showcases Ca2+-powered cellular motility. Because the isometric tension of V. convallaria increases linearly with its stalk length, it is hypothesized that the contractility of V. convallaria during unhindered contraction depends on the stalk length. In this study, the contractile force and energetics of V. convallaria cells of different stalk lengths were evaluated using a fluid dynamic drag model which accounts for the unsteadiness and finite Reynolds number of the water flow caused by contracting V. convallaria and the wall effect of the no-slip substrate. It was found that the contraction displacement, peak contraction speed, peak contractile force, total mechanical work, and peak power depended on the stalk length. The observed stalk-length-dependencies were simulated using a damped spring model, and the model estimated that the average spring constant of the contracting stalk was 1.34 nN µm-1. These observed length-dependencies of Vorticella’s key contractility parameters reflect the biophysical mechanism of the spasmonemal contraction, and thus they should be considered in developing a theoretical model of the Vorticella spasmoneme.

Actomyosin tension as a determinant of metastatic cancer mechanical tropism

NASA Astrophysics Data System (ADS)

McGrail, Daniel J.; Kieu, Quang Minh N.; Iandoli, Jason A.; Dawson, Michelle R.

2015-04-01

Despite major advances in the characterization of molecular regulators of cancer growth and metastasis, patient survival rates have largely stagnated. Recent studies have shown that mechanical cues from the extracellular matrix can drive the transition to a malignant phenotype. Moreover, it is also known that the metastatic process, which results in over 90% of cancer-related deaths, is governed by intracellular mechanical forces. To better understand these processes, we identified metastatic tumor cells originating from different locations which undergo inverse responses to altered matrix elasticity: MDA-MB-231 breast cancer cells that prefer rigid matrices and SKOV-3 ovarian cancer cells that prefer compliant matrices as characterized by parameters such as tumor cell proliferation, chemoresistance, and migration. Transcriptomic analysis revealed higher expression of genes associated with cytoskeletal tension and contractility in cells that prefer stiff environments, both when comparing MDA-MB-231 to SKOV-3 cells as well as when comparing bone-metastatic to lung-metastatic MDA-MB-231 subclones. Using small molecule inhibitors, we found that blocking the activity of these pathways mitigated rigidity-dependent behavior in both cell lines. Probing the physical forces exerted by cells on the underlying substrates revealed that though force magnitude may not directly correlate with functional outcomes, other parameters such as force polarization do correlate directly with cell motility. Finally, this biophysical analysis demonstrates that intrinsic levels of cell contractility determine the matrix rigidity for maximal cell function, possibly influencing tissue sites for metastatic cancer cell engraftment during dissemination. By increasing our understanding of the physical interactions of cancer cells with their microenvironment, these studies may help develop novel therapeutic strategies.

Blebbistatin, a myosin II inhibitor, suppresses Ca(2+)-induced and "sensitized"-contraction of skinned tracheal muscles from guinea pig.

PubMed

Yumoto, Masatoshi; Watanabe, Masaru

2013-01-01

Blebbistatin, a potent inhibitor of myosin II, has inhibiting effects on Ca(2+)-induced contraction and contractile filament organization without affecting the Ca(2+)-sensitivity to the force and phosphorylation level of myosin regulatory light chain (MLC20) in skinned (cell membrane permeabilized) taenia cecum from the guinea pig (Watanabe et al., Am J Physiol Cell Physiol. 2010; 298: C1118-26). In the present study, we investigated blebbistatin effects on the contractile force of skinned tracheal muscle, in which myosin filaments organization is more labile than that in the taenia cecum. Blebbistatin at 10 μM or higher suppressed Ca(2+)-induced tension development at any given Ca(2+) concentration, but had little effects on the Ca(2+)- induced myosin light chain phosphorylation. Also blebbistatin at 10 μM and higher significantly suppressed GTP-γS-induced "sensitized" force development. Since the force inhibiting effects of blebbistatin on the skinned trachea were much stronger than those in skinned taenia cecum, blebbistatin might directly affect myosin filaments organization.

Thin-film dielectric elastomer sensors to measure the contraction force of smooth muscle cells

NASA Astrophysics Data System (ADS)

Araromi, O.; Poulin, A.; Rosset, S.; Favre, M.; Giazzon, M.; Martin-Olmos, C.; Liley, M.; Shea, H.

2015-04-01

The development of thin-film dielectric elastomer strain sensors for the characterization of smooth muscle cell (SMC) contraction is presented here. Smooth muscle disorders are an integral part of diseases such as asthma and emphysema. Analytical tools enabling the characterization of SMC function i.e. contractile force and strain, in a low-cost and highly parallelized manner are necessary for toxicology screening and for the development of new and more effective drugs. The main challenge with the design of such tools is the accurate measurement of the extremely low contractile cell forces expected as a result of SMC monolayer contraction (as low as ~ 100 μN). Our approach utilizes ultrathin (~5 μm) and soft elastomer membranes patterned with elastomer-carbon composite electrodes, onto which the SMCs are cultured. The cell contraction induces an in-plane strain in the elastomer membrane, predicted to be in the order 1 %, which can be measured via the change in the membrane capacitance. The cell force can subsequently be deduced knowing the mechanical properties of the elastomer membrane. We discuss the materials and fabrication methods selected for our system and present preliminary results indicating their biocompatibility. We fabricate functional capacitive senor prototypes with good signal stability over the several hours (~ 0.5% variation). We succeed in measuring in-plane strains of 1 % with our fabricated devices with good repeatability and signal to noise ratio.

Contractility in type III cochlear fibrocytes is dependent on non-muscle myosin II and intercellular gap junctional coupling.

PubMed

Kelly, John J; Forge, Andrew; Jagger, Daniel J

2012-08-01

The cochlear spiral ligament is a connective tissue that plays diverse roles in normal hearing. Spiral ligament fibrocytes are classified into functional sub-types that are proposed to carry out specialized roles in fluid homeostasis, the mediation of inflammatory responses to trauma, and the fine tuning of cochlear mechanics. We derived a secondary sub-culture from guinea pig spiral ligament, in which the cells expressed protein markers of type III or "tension" fibrocytes, including non-muscle myosin II (nmII), α-smooth muscle actin (αsma), vimentin, connexin43 (cx43), and aquaporin-1. The cells formed extensive stress fibers containing αsma, which were also associated intimately with nmII expression, and the cells displayed the mechanically contractile phenotype predicted by earlier modeling studies. cx43 immunofluorescence was evident within intercellular plaques, and the cells were coupled via dye-permeable gap junctions. Coupling was blocked by meclofenamic acid (MFA), an inhibitor of cx43-containing channels. The contraction of collagen lattice gels mediated by the cells could be prevented reversibly by blebbistatin, an inhibitor of nmII function. MFA also reduced the gel contraction, suggesting that intercellular coupling modulates contractility. The results demonstrate that these cells can impart nmII-dependent contractile force on a collagenous substrate, and support the hypothesis that type III fibrocytes regulate tension in the spiral ligament-basilar membrane complex, thereby determining auditory sensitivity.

Mapping the dynamics of force transduction at cell–cell junctions of epithelial clusters

PubMed Central

Ng, Mei Rosa; Besser, Achim; Brugge, Joan S; Danuser, Gaudenz

2014-01-01

Force transduction at cell-cell adhesions regulates tissue development, maintenance and adaptation. We developed computational and experimental approaches to quantify, with both sub-cellular and multi-cellular resolution, the dynamics of force transmission in cell clusters. Applying this technology to spontaneously-forming adherent epithelial cell clusters, we found that basal force fluctuations were coupled to E-cadherin localization at the level of individual cell-cell junctions. At the multi-cellular scale, cell-cell force exchange depended on the cell position within a cluster, and was adaptive to reconfigurations due to cell divisions or positional rearrangements. Importantly, force transmission through a cell required coordinated modulation of cell-matrix adhesion and actomyosin contractility in the cell and its neighbors. These data provide insights into mechanisms that could control mechanical stress homeostasis in dynamic epithelial tissues, and highlight our methods as a resource for the study of mechanotransduction in cell-cell adhesions. DOI: http://dx.doi.org/10.7554/eLife.03282.001 PMID:25479385

Fluid flows and forces in development: functions, features and biophysical principles

PubMed Central

Freund, Jonathan B.; Goetz, Jacky G.; Hill, Kent L.; Vermot, Julien

2012-01-01

Throughout morphogenesis, cells experience intracellular tensile and contractile forces on microscopic scales. Cells also experience extracellular forces, such as static forces mediated by the extracellular matrix and forces resulting from microscopic fluid flow. Although the biological ramifications of static forces have received much attention, little is known about the roles of fluid flows and forces during embryogenesis. Here, we focus on the microfluidic forces generated by cilia-driven fluid flow and heart-driven hemodynamics, as well as on the signaling pathways involved in flow sensing. We discuss recent studies that describe the functions and the biomechanical features of these fluid flows. These insights suggest that biological flow determines many aspects of cell behavior and identity through a specific set of physical stimuli and signaling pathways. PMID:22395739

Analysis of microtubule growth dynamics arising from altered actin network structure and contractility in breast tumor cells

NASA Astrophysics Data System (ADS)

Ory, Eleanor C.; Bhandary, Lekhana; E Boggs, Amanda; Chakrabarti, Kristi R.; Parker, Joshua; Losert, Wolfgang; Martin, Stuart S.

2017-04-01

The periphery of epithelial cells is shaped by opposing cytoskeletal physical forces generated predominately by two dynamic force generating systems—growing microtubule ends push against the boundary from the cell center, and the actin cortex contracts the attached plasma membrane. Here we investigate how changes to the structure and dynamics of the actin cortex alter the dynamics of microtubules. Current drugs target actin polymerization and contraction to reduce cell division and invasiveness; however, the impacts on microtubule dynamics remain incompletely understood. Using human MCF-7 breast tumor cells expressing GFP-tagged microtubule end-binding-protein-1 (EB1) and coexpression of cytoplasmic fluorescent protein mCherry, we map the trajectories of growing microtubule ends and cytoplasmic boundary respectively. Based on EB1 tracks and cytoplasmic boundary outlines, we calculate the speed, distance from cytoplasmic boundary, and straightness of microtubule growth. Actin depolymerization with Latrunculin-A reduces EB1 growth speed as well as allows the trajectories to extend beyond the cytoplasmic boundary. Blebbistatin, a direct myosin-II inhibitor, reduced EB1 speed and yielded less straight EB1 trajectories. Inhibiting signaling upstream of myosin-II contractility via the Rho-kinase inhibitor, Y-27632, altered EB1 dynamics differently from Blebbistatin. These results indicate that reduced actin cortex integrity can induce distinct alterations in microtubule dynamics. Given recent findings that tumor stem cell characteristics are increased by drugs which reduce actin contractility or stabilize microtubules, it remains important to clearly define how cytoskeletal drugs alter the interactions between these two filament systems in tumor cells.

In vitro cardiomyocyte-driven biogenerator based on aligned piezoelectric nanofibers

NASA Astrophysics Data System (ADS)

Liu, Xia; Zhao, Hui; Lu, Yingxian; Li, Song; Lin, Liwei; Du, Yanan; Wang, Xiaohong

2016-03-01

Capturing the body's mechanical energy from the heart, lungs, and diaphragm can probably meet the requirements for in vivo applications of implantable biomedical devices. In this work, we present a novel contractile cardiomyocyte (CM)-driven biogenerator based on piezoelectric nanofibers (NFs) uniaxially aligned on a PDMS thin film. Flexible nanostructures interact with the CMs, as a physical cue to guide the CMs to align in a specific way, and create mechanical interfaces of contractile CMs and piezoelectric NFs. As such, the cellular construct features specific alignment and synchronous contraction, which realizes the maximal resultant force to drive the NFs to bend periodically. Studies on contraction mapping show that neonatal rat CMs self-assemble into a functional bio-bot film with well-defined axes of force generation. Consequently, the biogenerator produces an average voltage of 200 mV and current of 45 nA at the cell concentration of 1.0 million per ml, offering a biocompatible and scalable platform for biological energy conversion.Capturing the body's mechanical energy from the heart, lungs, and diaphragm can probably meet the requirements for in vivo applications of implantable biomedical devices. In this work, we present a novel contractile cardiomyocyte (CM)-driven biogenerator based on piezoelectric nanofibers (NFs) uniaxially aligned on a PDMS thin film. Flexible nanostructures interact with the CMs, as a physical cue to guide the CMs to align in a specific way, and create mechanical interfaces of contractile CMs and piezoelectric NFs. As such, the cellular construct features specific alignment and synchronous contraction, which realizes the maximal resultant force to drive the NFs to bend periodically. Studies on contraction mapping show that neonatal rat CMs self-assemble into a functional bio-bot film with well-defined axes of force generation. Consequently, the biogenerator produces an average voltage of 200 mV and current of 45 nA at the cell concentration of 1.0 million per ml, offering a biocompatible and scalable platform for biological energy conversion. Electronic supplementary information (ESI) available: Includes the ESI methods and figures, and videos of cell contraction and biogenerator bending. See DOI: 10.1039/c5nr08430j

Flow caused by the stalk contraction of Vorticella

NASA Astrophysics Data System (ADS)

Ryu, Sangjin; Chung, Eun-Gul; Admiraal, David

2016-11-01

Vorticella is a stalked protozoan, and its ultrafast stalk contraction moves the spherically-shrunken cell body (zooid) and thus causes surrounding water to flow. Because the fluid dynamics of this water flow is important for understanding the motility of Vorticella, we investigated the flow based on various fluid dynamics approaches. To find why Vorticella contracts its stalk, we propose a hypothesis that the protist utilizes the contraction-induced water flow to augment transport of food particles. This hypothesis was investigated using a computational fluid dynamics (CFD) model, which was validated with an experimental scale model of Vorticella. The CFD model enabled calculating the motion of particles around Vorticella and thus quantifying the transport effect of the stalk contraction. Also, we have developed a hydrodynamic drag model for easier estimation of Vorticella's contractility without using the CFD model. Because the contractile force of the stalk equals the drag on the moving zooid, the model enabled evaluating the contractile force and energetics of Vorticella based on its contraction speed. Analyses using the drag model show that the stalk contractility of Vorticella depends on the stalk length. This study was supported by UNL Layman Seed Grant and Nebraska EPSCoR First Award Grant.

Contact inhibition of locomotion determines cell-cell and cell-substrate forces in tissues.

PubMed

Zimmermann, Juliane; Camley, Brian A; Rappel, Wouter-Jan; Levine, Herbert

2016-03-08

Cells organized in tissues exert forces on their neighbors and their environment. Those cellular forces determine tissue homeostasis as well as reorganization during embryonic development and wound healing. To understand how cellular forces are generated and how they can influence the tissue state, we develop a particle-based simulation model for adhesive cell clusters and monolayers. Cells are contractile, exert forces on their substrate and on each other, and interact through contact inhibition of locomotion (CIL), meaning that cell-cell contacts suppress force transduction to the substrate and propulsion forces align away from neighbors. Our model captures the traction force patterns of small clusters of nonmotile cells and larger sheets of motile Madin-Darby canine kidney (MDCK) cells. In agreement with observations in a spreading MDCK colony, the cell density in the center increases as cells divide and the tissue grows. A feedback between cell density, CIL, and cell-cell adhesion gives rise to a linear relationship between cell density and intercellular tensile stress and forces the tissue into a nonmotile state characterized by a broad distribution of traction forces. Our model also captures the experimentally observed tissue flow around circular obstacles, and CIL accounts for traction forces at the edge.

The contractile ring coordinates curvature-dependent septum assembly during fission yeast cytokinesis.

PubMed

Zhou, Zhou; Munteanu, Emilia Laura; He, Jun; Ursell, Tristan; Bathe, Mark; Huang, Kerwyn Casey; Chang, Fred

2015-01-01

The functions of the actin-myosin-based contractile ring in cytokinesis remain to be elucidated. Recent findings show that in the fission yeast Schizosaccharomyces pombe, cleavage furrow ingression is driven by polymerization of cell wall fibers outside the plasma membrane, not by the contractile ring. Here we show that one function of the ring is to spatially coordinate septum cell wall assembly. We develop an improved method for live-cell imaging of the division apparatus by orienting the rod-shaped cells vertically using microfabricated wells. We observe that the septum hole and ring are circular and centered in wild-type cells and that in the absence of a functional ring, the septum continues to ingress but in a disorganized and asymmetric manner. By manipulating the cleavage furrow into different shapes, we show that the ring promotes local septum growth in a curvature-dependent manner, allowing even a misshapen septum to grow into a more regular shape. This curvature-dependent growth suggests a model in which contractile forces of the ring shape the septum cell wall by stimulating the cell wall machinery in a mechanosensitive manner. Mechanical regulation of the cell wall assembly may have general relevance to the morphogenesis of walled cells. © 2015 Zhou et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Effect of Substrate Mechanics on Cardiomyocyte Maturation and Growth

PubMed Central

Tallawi, Marwa; Rai, Ranjana; Boccaccini, Aldo. R.

2015-01-01

Cardiac tissue engineering constructs are a promising therapeutic treatment for myocardial infarction, which is one of the leading causes of death. In order to further advance the development and regeneration of engineered cardiac tissues using biomaterial platforms, it is important to have a complete overview of the effects that substrates have on cardiomyocyte (CM) morphology and function. This article summarizes recent studies that investigate the effect of mechanical cues on the CM differentiation, maturation, and growth. In these studies, CMs derived from embryos, neonates, and mesenchymal stem cells were seeded on different substrates of various elastic modulus. Measuring the contractile function by force production, work output, and calcium handling, it was seen that cell behavior on substrates was optimized when the substrate stiffness mimicked that of the native tissue. The contractile function reflected changes in the sarcomeric protein confirmation and organization that promoted the contractile ability. The analysis of the literature also revealed that, in addition to matrix stiffness, mechanical stimulation, such as stretching the substrate during cell seeding, also played an important role during cell maturation and tissue development. PMID:25148904

Bladder smooth muscle organ culture preparation maintains the contractile phenotype

PubMed Central

Wang, Tanchun; Kendig, Derek M.; Chang, Shaohua; Trappanese, Danielle M.; Chacko, Samuel

2012-01-01

Smooth muscle cells, when subjected to culture, modulate from a contractile to a secretory phenotype. This has hampered the use of cell culture for molecular techniques to study the regulation of smooth muscle biology. The goal of this study was to develop a new organ culture model of bladder smooth muscle (BSM) that would maintain the contractile phenotype and aid in the study of BSM biology. Our results showed that strips of BSM subjected to up to 9 days of organ culture maintained their contractile phenotype, including the ability to achieve near-control levels of force with a temporal profile similar to that of noncultured tissues. The technical aspects of our organ culture preparation that were responsible, in part, for the maintenance of the contractile phenotype were a slight longitudinal stretch during culture and subjection of the strips to daily contraction-relaxation. The tissues contained viable cells throughout the cross section of the strips. There was an increase in extracellular collagenous matrix, resulting in a leftward shift in the passive length-tension relationship. There were no significant changes in the content of smooth muscle-specific α-actin, calponin, h-caldesmon, total myosin heavy chain, protein kinase G, Rho kinase-I, or the ratio of SM1 to SM2 myosin isoforms. Moreover the organ cultured tissues maintained functional voltage-gated calcium channels and large-conductance calcium-activated potassium channels. Therefore, we propose that this novel BSM organ culture model maintains the contractile phenotype and will be a valuable tool for the use in cellular/molecular biology studies of bladder myocytes. PMID:22896042

Initial contact guidance during cell spreading is contractility-independent.

PubMed

Sales, Adrià; Holle, Andrew W; Kemkemer, Ralf

2017-08-02

A wide variety of cell types exhibit substrate topography-based behavior, also known as contact guidance. However, the precise cellular mechanisms underlying this process are still unknown. In this study, we investigated contact guidance by studying the reaction of human endothelial cells (ECs) to well-defined microgroove topographies, both during and after initial cell spreading. As the cytoskeleton plays a major role in cellular adaptation to topographical features, two methods were used to perturb cytoskeletal structures. Inhibition of actomyosin contractility with the chemical inhibitor blebbistatatin demonstrated that initial contact guidance events are independent of traction force generation. However, cell alignment to the grooved substrate was altered at later time points, suggesting an initial 'passive' phase of contact guidance, followed by a contractility-dependent 'active' phase that relies on mechanosensitive feedback. The actin cytoskeleton was also perturbed in an indirect manner by culturing cells upside down, resulting in decreased levels of contact guidance and suggesting that a possible loss of contact between the actin cytoskeleton and the substrate could lead to cytoskeleton impairment. The process of contact guidance at the microscale was found to be primarily lamellipodia driven, as no bias in filopodia extension was observed on micron-scale grooves.

Regulation of cortical contractility and spindle positioning by the protein phosphatase 6 PPH-6 in one-cell stage C. elegans embryos

PubMed Central

Afshar, Katayoun; Werner, Michael E.; Tse, Yu Chung; Glotzer, Michael; Gönczy, Pierre

2010-01-01

Modulation of the microtubule and the actin cytoskeleton is crucial for proper cell division. Protein phosphorylation is known to be an important regulatory mechanism modulating these cytoskeletal networks. By contrast, there is a relative paucity of information regarding how protein phosphatases contribute to such modulation. Here, we characterize the requirements for protein phosphatase PPH-6 and its associated subunit SAPS-1 in one-cell stage C. elegans embryos. We establish that the complex of PPH-6 and SAPS-1 (PPH-6/SAPS-1) is required for contractility of the actomyosin network and proper spindle positioning. Our analysis demonstrates that PPH-6/SAPS-1 regulates the organization of cortical non-muscle myosin II (NMY-2). Accordingly, we uncover that PPH-6/SAPS-1 contributes to cytokinesis by stimulating actomyosin contractility. Furthermore, we demonstrate that PPH-6/SAPS-1 is required for the proper generation of pulling forces on spindle poles during anaphase. Our results indicate that this requirement is distinct from the role in organizing the cortical actomyosin network. Instead, we uncover that PPH-6/SAPS-1 contributes to the cortical localization of two positive regulators of pulling forces, GPR-1/2 and LIN-5. Our findings provide the first insights into the role of a member of the PP6 family of phosphatases in metazoan development. PMID:20040490

Thrombopoietin modulates cardiac contractility in vitro and contributes to myocardial depressing activity of septic shock serum.

PubMed

Lupia, Enrico; Spatola, Tiziana; Cuccurullo, Alessandra; Bosco, Ornella; Mariano, Filippo; Pucci, Angela; Ramella, Roberta; Alloatti, Giuseppe; Montrucchio, Giuseppe

2010-09-01

Thrombopoietin (TPO) is a humoral growth factor that has been shown to increase platelet activation in response to several agonists. Patients with sepsis have increased circulating TPO levels, which may enhance platelet activation, potentially participating to the pathogenesis of multi-organ failure. Aim of this study was to investigate whether TPO affects myocardial contractility and participates to depress cardiac function during sepsis. We showed the expression of the TPO receptor c-Mpl on myocardial cells and tissue by RT-PCR, immunofluorescence and western blotting. We then evaluated the effect of TPO on the contractile function of rat papillary muscle and isolated heart. TPO did not change myocardial contractility in basal conditions, but, when followed by epinephrine (EPI) stimulation, it blunted the enhancement of contractile force induced by EPI both in papillary muscle and isolated heart. An inhibitor of TPO prevented TPO effect on cardiac inotropy. Treatment of papillary muscle with pharmacological inhibitors of phosphatidylinositol 3-kinase, NO synthase, and guanilyl cyclase abolished TPO effect, indicating NO as the final mediator. We finally studied the role of TPO in the negative inotropic effect exerted by human septic shock (HSS) serum and TPO cooperation with TNF-alpha and IL-1beta. Pre-treatment with the TPO inhibitor prevented the decrease in contractile force induced by HSS serum. Moreover, TPO significantly amplified the negative inotropic effect induced by TNF-alpha and IL-1beta in papillary muscle. In conclusion, TPO negatively modulates cardiac inotropy in vitro and contributes to the myocardial depressing activity of septic shock serum.

ROCK inhibition promotes microtentacles that enhance reattachment of breast cancer cells

PubMed Central

Bhandary, Lekhana; Whipple, Rebecca A.; Vitolo, Michele I.; Charpentier, Monica S.; Boggs, Amanda E.; Chakrabarti, Kristi R.; Thompson, Keyata N.; Martin, Stuart S.

2015-01-01

The presence of circulating tumor cells (CTCs) in blood predicts poor patient outcome and CTC frequency is correlated with higher risk of metastasis. Recently discovered, novel microtubule-based structures, microtentacles, can enhance reattachment of CTCs to the vasculature. Microtentacles are highly dynamic membrane protrusions formed in detached cells and occur when physical forces generated by the outwardly expanding microtubules overcome the contractile force of the actin cortex. Rho-associated kinase (ROCK) is a major regulator of actomyosin contractility and Rho/ROCK over-activation is implicated in tumor metastasis. ROCK inhibitors are gaining popularity as potential cancer therapeutics based on their success in reducing adherent tumor cell migration and invasion. However, the effect of ROCK inhibition on detached cells in circulation is largely unknown. In this study, we use breast tumor cells in suspension to mimic detached CTCs and show that destabilizing the actin cortex through ROCK inhibition in suspended cells promotes the formation of microtentacles and enhances reattachment of cells from suspension. Conversely, increasing actomyosin contraction by Rho over-activation reduces microtentacle frequency and reattachment. Although ROCK inhibitors may be effective in reducing adherent tumor cell behavior, our results indicate that they could inadvertently increase metastatic potential of non-adherent CTCs by increasing their reattachment efficacy. PMID:25749040

Locomotive forces produced by single leukocytes in vivo and in vitro.

PubMed

Guilford, W H; Lantz, R C; Gore, R W

1995-05-01

We report here the first time-resolved measurements of the forces produced during the migration of single leukocytes in vivo and in vitro. Pulmonary macrophages from hamsters and mice, in vitro, and Nembutal (pentobarbital sodium)-anesthetized hamster neutrophils, in vivo, generated maximum locomotive forces ranging from 1.9 to 10.7 nN or tenths of microdynes. Force production was periodic and correlated with the length of the leading lamellipod but not with generalized cell ruffling. Although the extension of the leading lamella is critical to locomotive force generation, these direct measurements suggest that lamellar extension may not arise from the same contractile processes driving forward motion of the cell mass. Indeed, cell ruffling, lamellar extension, and locomotive force generation may be differentially controlled and have different origins. This technique may be extended to test numerous hypotheses of how these and other nonmuscle cells crawl.

High Resolution Quantification of Cellular Forces for Rigidity Sensing

NASA Astrophysics Data System (ADS)

Liu, Shuaimin

This thesis describes a comprehensive study of understanding the mechanism of rigidity sensing by quantitative analysis using submicron pillar array substrates. From mechanobiology perspective, we explore and study molecular pathways involved in rigidity and force sensing at cell-matrix adhesions with regard to cancer, regeneration, and development by quantification methods. In Chapter 2 and 3, we developed fabrication and imaging techniques to enhance the performance of a submicron pillar device in terms of spatial and temporal measurement ability, and we discovered a correlation of rigidity sensing forces and corresponding proteins involved in the early rigidity sensing events. In Chapter 2, we introduced optical effect arising from submicron structure imaging, and we described a technique to identify the correct focal plane of pillar tip by fabricating a substrate with designed-offset pillars. From calibration result, we identified the correct focal plane that was previously overlooked, and verified our findings by other imaging techniques. In Chapter 3, we described several techniques to selectively functionalize elastomeric pillars top and compared these techniques in terms of purposes and fabrication complexity. Techniques introduced in this chapter included direct labeling, such as stamping of fluorescent substances (organic dye, nano-diamond, q-dot) to pillars top, as well as indirect labeling that selectively modify the surface of molds with either metal or fluorescent substances. In Chapter 4, we examined the characteristics of local contractility forces and identified the components formed a sarcomere like contractile unit (CU) that cells use to sense rigidity. CUs were found to be assembled at cell edge, contain myosin II, alpha-actinin, tropomodulin and tropomyosin (Tm), and resemble sarcomeres in size (˜2 mum) and function. Then we performed quantitative analysis of CUs to evaluate rigidity sensing activity over ˜8 hours time course and found that density of CUs decrease with time after spreading on stiff substrate. However addition of EGF dramatically increased local contraction activity such that about 30% of the total contractility was in the contraction units. This stimulatory effect was only observed on stiff substrate not on soft. Moreover, we find that in the early interactions of cells with rigid substrates that EGFR activity is needed for normal spreading and the assembly of local contraction units in media lacking serum and any soluble EGF. In Chapter 5, we performed high temporal- and spatial-resolution tracking of contractile forces exerted by cells on sub-micron elastomeric pillars. We found that actomyosin-based sarcomere-like CUs simultaneously moved opposing pillars in net steps of ˜2.5 nm, independent of rigidity. What correlated with rigidity was the number of steps taken to reach a force level that activated recruitment of alpha-actinin to the CUs. When we removed actomyosin restriction by depleting tropomyosin 2.1, we observed larger steps and higher forces that resulted in aberrant rigidity sensing and growth of non-transformed cells on soft matrices. Thus, we conclude that tropomyosin 2.1 acts as a suppressor of growth on soft matrices by supporting proper rigidity sensing.

E-cadherin-mediated force transduction signals regulate global cell mechanics

PubMed Central

Muhamed, Ismaeel; Wu, Jun; Sehgal, Poonam; Kong, Xinyu; Tajik, Arash; Wang, Ning

2016-01-01

ABSTRACT This report elucidates an E-cadherin-based force-transduction pathway that triggers changes in cell mechanics through a mechanism requiring epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase (PI3K), and the downstream formation of new integrin adhesions. This mechanism operates in addition to local cytoskeletal remodeling triggered by conformational changes in the E-cadherin-associated protein α-catenin, at sites of mechanical perturbation. Studies using magnetic twisting cytometry (MTC), together with traction force microscopy (TFM) and confocal imaging identified force-activated E-cadherin-specific signals that integrate cadherin force transduction, integrin activation and cell contractility. EGFR is required for the downstream activation of PI3K and myosin-II-dependent cell stiffening. Our findings also demonstrated that α-catenin-dependent cytoskeletal remodeling at perturbed E-cadherin adhesions does not require cell stiffening. These results broaden the repertoire of E-cadherin-based force transduction mechanisms, and define the force-sensitive signaling network underlying the mechano-chemical integration of spatially segregated adhesion receptors. PMID:26966187

Femtosecond laser patterning of biological materials

NASA Astrophysics Data System (ADS)

Grigoropoulos, Costas P.; Jeon, Hojeong; Hidai, Hirofumi; Hwang, David J.

2011-03-01

This paper aims at presenting a review of work at the Laser Thermal Laboratory on the microscopic laser modification of biological materials using ultrafast laser pulses. We have devised a new method for fabricating high aspect ratio patterns of varying height by using two-photon polymerization process in order to study contact guidance and directed growth of biological cells. Studies using NIH-3T3 and MDCK cells indicate that cell morphology on fiber scaffolds is influenced by the pattern of actin microfilament bundles. Cells experienced different strength of contact guidance depending on the ridge height. Cell morphology and motility was investigated on micronscale anisotropic cross patterns and parallel line patterns having different aspect ratios. A significant effect on cell alignment and directionality of migration was observed. Cell morphology and motility were influenced by the aspect ratio of the cross pattern, the grid size, and the ridge height. Cell contractility was examined microscopically in order to measure contractile forces generated by individual cells on self-standing fiber scaffolds.

Contact inhibition of locomotion determines cell–cell and cell–substrate forces in tissues

PubMed Central

Zimmermann, Juliane; Camley, Brian A.; Rappel, Wouter-Jan; Levine, Herbert

2016-01-01

Cells organized in tissues exert forces on their neighbors and their environment. Those cellular forces determine tissue homeostasis as well as reorganization during embryonic development and wound healing. To understand how cellular forces are generated and how they can influence the tissue state, we develop a particle-based simulation model for adhesive cell clusters and monolayers. Cells are contractile, exert forces on their substrate and on each other, and interact through contact inhibition of locomotion (CIL), meaning that cell–cell contacts suppress force transduction to the substrate and propulsion forces align away from neighbors. Our model captures the traction force patterns of small clusters of nonmotile cells and larger sheets of motile Madin–Darby canine kidney (MDCK) cells. In agreement with observations in a spreading MDCK colony, the cell density in the center increases as cells divide and the tissue grows. A feedback between cell density, CIL, and cell–cell adhesion gives rise to a linear relationship between cell density and intercellular tensile stress and forces the tissue into a nonmotile state characterized by a broad distribution of traction forces. Our model also captures the experimentally observed tissue flow around circular obstacles, and CIL accounts for traction forces at the edge. PMID:26903658

Disordered Actomyosin Is Sufficient to Promote Cooperative and Telescopic Contractility

NASA Astrophysics Data System (ADS)

Murrell, Michael; Linsmeier, Ian; Banerjee, Shiladitya; Kim, Tae Yoon; Jung, Wonyeong; Oakes, Patrick

While the molecular interactions between myosin motors and F-actin are well known, the relationship between F-actin organization and myosin-mediated force generation remains poorly understood. Here, we explore the accumulation of myosin-induced stresses within a 2D biomimetic model of the actomyosin cortex, where myosin activity is controlled spatially and temporally using light. By controlling the geometry and the duration of myosin activation, we show that contraction of disordered actomyosin is highly cooperative, telescopic with the activation area and generates a pattern of mechanical stresses consistent with those observed in contractile cells. We quantitatively reproduce these properties using an in vitro isotropic model of the actomyosin cytoskeleton, and explore the physical origins of telescopic contractility in disordered networks using agent-based simulations. NSF CMMI-1525316.

Actin Filament Elasticity and Retrograde Flow Shape the Force-Velocity Relation of Motile Cells

PubMed Central

Zimmermann, Juliane; Brunner, Claudia; Enculescu, Mihaela; Goegler, Michael; Ehrlicher, Allen; Käs, Josef; Falcke, Martin

2012-01-01

Cells migrate through a crowded environment during processes such as metastasis or wound healing, and must generate and withstand substantial forces. The cellular motility responses to environmental forces are represented by their force-velocity relation, which has been measured for fish keratocytes but remains unexplained. Even pN opposing forces slow down lamellipodium motion by three orders of magnitude. At larger opposing forces, the retrograde flow of the actin network accelerates until it compensates for polymerization, and cell motion stalls. Subsequently, the lamellipodium adapts to the stalled state. We present a mechanism quantitatively explaining the cell's force-velocity relation and its changes upon application of drugs that hinder actin polymerization or actomyosin-based contractility. Elastic properties of filaments, close to the lamellipodium leading edge, and retrograde flow shape the force-velocity relation. To our knowledge, our results shed new light on how these migratory responses are regulated, and on the mechanics and structure of the lamellipodium. PMID:22339865

Three-Dimensional Culture Model of Skeletal Muscle Tissue with Atrophy Induced by Dexamethasone.

PubMed

Shimizu, Kazunori; Genma, Riho; Gotou, Yuuki; Nagasaka, Sumire; Honda, Hiroyuki

2017-06-15

Drug screening systems for muscle atrophy based on the contractile force of cultured skeletal muscle tissues are required for the development of preventive or therapeutic drugs for atrophy. This study aims to develop a muscle atrophy model by inducing atrophy in normal muscle tissues constructed on microdevices capable of measuring the contractile force and to verify if this model is suitable for drug screening using the contractile force as an index. Tissue engineered skeletal muscles containing striated myotubes were prepared on the microdevices for the study. The addition of 100 µM dexamethasone (Dex), which is used as a muscle atrophy inducer, for 24 h reduced the contractile force significantly. An increase in the expression of Atrogin-1 and MuRF-1 in the tissues treated with Dex was established. A decrease in the number of striated myotubes was also observed in the tissues treated with Dex. Treatment with 8 ng/mL Insulin-like Growth Factor (IGF-I) for 24 h significantly increased the contractile force of the Dex-induced atrophic tissues. The same treatment, though, had no impact on the force of the normal tissues. Thus, it is envisaged that the atrophic skeletal muscle tissues induced by Dex can be used for drug screening against atrophy.

Three-Dimensional Culture Model of Skeletal Muscle Tissue with Atrophy Induced by Dexamethasone

PubMed Central

Shimizu, Kazunori; Genma, Riho; Gotou, Yuuki; Nagasaka, Sumire; Honda, Hiroyuki

2017-01-01

Drug screening systems for muscle atrophy based on the contractile force of cultured skeletal muscle tissues are required for the development of preventive or therapeutic drugs for atrophy. This study aims to develop a muscle atrophy model by inducing atrophy in normal muscle tissues constructed on microdevices capable of measuring the contractile force and to verify if this model is suitable for drug screening using the contractile force as an index. Tissue engineered skeletal muscles containing striated myotubes were prepared on the microdevices for the study. The addition of 100 µM dexamethasone (Dex), which is used as a muscle atrophy inducer, for 24 h reduced the contractile force significantly. An increase in the expression of Atrogin-1 and MuRF-1 in the tissues treated with Dex was established. A decrease in the number of striated myotubes was also observed in the tissues treated with Dex. Treatment with 8 ng/mL Insulin-like Growth Factor (IGF-I) for 24 h significantly increased the contractile force of the Dex-induced atrophic tissues. The same treatment, though, had no impact on the force of the normal tissues. Thus, it is envisaged that the atrophic skeletal muscle tissues induced by Dex can be used for drug screening against atrophy. PMID:28952535

A new orthodontic force system for moment control utilizing the flexibility of common wires: Evaluation of the effect of contractile force and hook length.

PubMed

Lai, Wei-Jen; Midorikawa, Yoshiyuki; Kanno, Zuisei; Takemura, Hiroshi; Suga, Kazuhiro; Soga, Kohei; Ono, Takashi; Uo, Motohiro

2018-01-01

The application of an appropriate force system is indispensable for successful orthodontic treatments. Second-order moment control is especially important in many clinical situations, so we developed a new force system composed of a straight orthodontic wire and two crimpable hooks of different lengths to produce the second-order moment. The objective of this study was to evaluate this new force system and determine an optimum condition that could be used in clinics. We built a premolar extraction model with two teeth according to the concept of a modified orthodontic simulator. This system was activated by applying contractile force from two hooks that generated second-order moment and force. The experimental device incorporated two sensors, and forces and moments were measured along six axes. We changed the contractile force and hook length to elucidate their effects. Three types of commercial wires were tested. The second-order moment was greater on the longer hook side of the model. Vertical force balanced the difference in moments between the two teeth. Greater contractile force generated a greater second-order moment, which reached a limit of 150 g. Excessive contractile force induced more undesired reactions in the other direction. Longer hooks induced greater moment generation, reaching their limit at 10 mm in length. The system acted similar to an off-center V-bend and can be applied in clinical practice as an unconventional loop design. We suggest that this force system has the potential for second-order moment control in clinical applications. Copyright © 2017. Published by Elsevier B.V.

Effects of geometry and cell-matrix interactions on the mechanics of 3D engineered microtissues

NASA Astrophysics Data System (ADS)

Bose, Prasenjit; Eyckmans, Jeroen; Chen, Christopher; Reich, Daniel

Approaches to measure and control cell-extracellular matrix (ECM) interactions in a dynamic mechanical environment are important both for studies of mechanobiology and for tissue design for bioengineering applications. We have developed a microtissue-based platform capable of controlling the ECM alignment of 3D engineered microtissues while simultaneously permitting measurement of cellular contractile forces and the tissues' mechanical properties. The tissues self-assemble from cell-laden collagen gels placed in micro-fabricated wells containing sets of flexible elastic pillars. Tissue geometry and ECM alignment are controlled by the pillars' number, shape and location. Optical tracking of the pillars provides readout of the tissues' contractile forces. Magnetic materials bound to selected pillars allow quasi-static or dynamic stretching of the tissue, and together with simultaneous measurements of the tissues' local dynamic strain field, enable characterization of the mechanical properties of the system, including their degree of anisotropy. Results on the effects of symmetry and degree of ECM alignment and organization on the role of cell-ECM interactions in determining tissue mechanical properties will be discussed. This work is supported by NSF CMMI-1463011 and CMMI-1462710.

Assessment of the Contractile Properties of Permeabilized Skeletal Muscle Fibers.

PubMed

Claflin, Dennis R; Roche, Stuart M; Gumucio, Jonathan P; Mendias, Christopher L; Brooks, Susan V

2016-01-01

Permeabilized individual skeletal muscle fibers offer the opportunity to evaluate contractile behavior in a system that is greatly simplified, yet physiologically relevant. Here we describe the steps required to prepare, permeabilize and preserve small samples of skeletal muscle. We then detail the procedures used to isolate individual fiber segments and attach them to an experimental apparatus for the purpose of controlling activation and measuring force generation. We also describe our technique for estimating the cross-sectional area of fiber segments. The area measurement is necessary for normalizing the absolute force to obtain specific force, a measure of the intrinsic force-generating capability of the contractile system.

Skeletal muscle contractile properties in a novel murine model for limb girdle muscular dystrophy 2i.

PubMed

Rehwaldt, Jordan D; Rodgers, Buel D; Lin, David C

2017-12-01

Limb-girdle muscular dystrophy (LGMD) 2i results from mutations in fukutin-related protein and aberrant α-dystroglycan glycosylation. Although this significantly compromises muscle function and ambulation, the comprehensive characteristics of contractile dysfunction are unknown. Therefore, we quantified the in situ contractile properties of the medial gastrocnemius in young adult P448L mice, an affected muscle of a novel model of LGMD2i. Normalized maximal twitch force, tetanic force, and power were significantly smaller in P448L mice, compared with sex-matched, wild-type mice. These differences were consistent with the replacement of contractile fibers by passive tissue. The shape of the active force-length relationships were similar in both groups, regardless of sex, consistent with an intact sarcomeric structure in P448L mice. Passive force-length curves normalized to maximal isometric force were steeper in P448L mice, and passive elements contribute disproportionately more to total contractile force in P448L mice. Sex differences were mostly noted in the force-velocity curves, as normalized values for maximal and optimal velocities were significantly slower in P448L males, compared with wild-type, but not in P448L females. This suggests that the dystrophic phenotype, which may include possible changes in cross-bridge kinetics and fiber-type proportions, progresses more quickly in P448L males. These results together indicate that active force and power generation are compromised in both sexes of P448L mice, while passive forces increase. More importantly, the results identified several functional markers of disease pathophysiology that could aid in developing and assessment of novel therapeutics for LGMD2i and possibly other dystroglycanopathies as well. NEW & NOTEWORTHY Comprehensive assessments of muscle contractile function have, until now, never been performed in an animal model for any dystroglycanopathy. This study suggests that skeletal muscle contractile properties are significantly compromised in a recently developed model for limb-girdle muscular dystrophy 2i, the P448L mouse. It further identifies novel pathological markers of muscle function that are suitable for developing therapeutics and for better understanding of disease pathogenesis.

[The relationship between contractile characteristics and fiber type conversion in hind-limb unloading mice soleus].

PubMed

Li, Li; Liu, Hong-Ju; Yang, Ming-Hao; Li, Jing-Long; Wang, Lu; Chen, Xiao-Ping; Fan, Ming

2012-03-01

To explore the relationship between contractile characteristics and fiber type conversion in hind-limb unloading mice soleus. After 28-day hind-limb unloading and muscle atrophy, we used the method of isolated muscle perfusion with different stimulated protocols to determine the changes in contractile characteristics including the isometric twitch force and tetanus force and fatigue index of slow twitch muscle in mice. The muscle myofibrillar composition and fiber type conversion were detected by immunofluorescence staining and real-time PCR. The isometric twitch force and the tetanus force and fatigue index were decreased progressively in 28-day unloaded mice soleus, with the increase in fast twitch fiber subtype and the decrease in slow twitch fiber subtype. The alteration of contractile characteristics is relevant to the slow-to-fast fiber conversion in mice soleus after 28-day hind-limb unloading.

LET-99 functions in the astral furrowing pathway, where it is required for myosin enrichment in the contractile ring

PubMed Central

Price, Kari L.; Rose, Lesilee S.

2017-01-01

The anaphase spindle determines the position of the cytokinesis furrow, such that the contractile ring assembles in an equatorial zone between the two spindle poles. Contractile ring formation is mediated by RhoA activation at the equator by the centralspindlin complex and midzone microtubules. Astral microtubules also inhibit RhoA accumulation at the poles. In the Caenorhabditis elegans one-cell embryo, the astral microtubule–dependent pathway requires anillin, NOP-1, and LET-99. LET-99 is well characterized for generating the asymmetric cortical localization of the Gα-dependent force-generating complex that positions the spindle during asymmetric division. However, whether the role of LET-99 in cytokinesis is specific to asymmetric division and whether it acts through Gα to promote furrowing are unclear. Here we show that LET-99 contributes to furrowing in both asymmetrically and symmetrically dividing cells, independent of its function in spindle positioning and Gα regulation. LET-99 acts in a pathway parallel to anillin and is required for myosin enrichment into the contractile ring. These and other results suggest a positive feedback model in which LET-99 localizes to the presumptive cleavage furrow in response to the spindle and myosin. Once positioned there, LET-99 enhances myosin accumulation to promote furrowing in both symmetrically and asymmetrically dividing cells. PMID:28701343

Changes in fibroblast mechanostat set point and mechanosensitivity: an adaptive response to mechanical stress in floppy eyelid syndrome.

PubMed

Ezra, Daniel G; Ellis, James S; Beaconsfield, Michèle; Collin, Richard; Bailly, Maryse

2010-08-01

Floppy eyelid syndrome (FES) is an acquired hyperelasticity disorder affecting the upper eyelid. The tarsal plate becomes hyperelastic with a loss of intrinsic rigidity. As a result, the eyelid is subjected to cyclic mechanical stress. This condition was used as a model to investigate changes in dynamic fibroblast contractility in the context of chronic cyclic mechanical stress. Contractile efficiency was investigated in a free-floating, three-dimensional collagen matrix model. Intrinsic cellular force measurements and responses to changes in gel tension were explored using a tensioning culture force monitor (t-CFM). Gene expression differences between cell lines exhibiting differences in contractile phenotype were explored with a genome level microarray platform and RT-PCR. FES tarsal plate fibroblasts (TFs) showed an increased contractile efficiency compared with the control, and t-CFM measurements confirmed a higher intrinsic cellular force at plateau levels. Cyclic stretch/relaxation experiments determined that TFs in FES maintained a functional tensional homeostasis response but with an altered sensitivity, operating around a higher mechanostat set point. Gene expression array and RT-PCR analysis identified V-CAM1 and PPP1R3C as being upregulated in FES TFs. These changes may represent an adaptive response that allows tensional homeostasis to be maintained at the high levels of tissue stress experienced in FES. Gene expression studies point to a role for V-CAM1 and PPP1R3C in mediating changes in the dynamic range of mechanosensitivity of TFs. This work identifies FES as a useful model for the study of adaptive physiological responses to mechanical stress.

Shortening actin filaments cause force generation in actomyosin network to change from contractile to extensile

NASA Astrophysics Data System (ADS)

Kumar, Nitin; Gardel, Margaret

Motor proteins in conjunction with filamentous proteins convert biochemical energy into mechanical energy which serves a number of cellular processes including cell motility, force generation and intracellular cargo transport. In-vitro experiments suggest that the forces generated by kinesin motors on microtubule bundles are extensile in nature whereas myosin motors on actin filaments are contractile. It is not clear how qualitatively similar systems can show completely different behaviors in terms of the nature of force generation. In order to answer this question, we carry out in vitro experiments where we form quasi 2D filamentous actomyosin networks and vary the length of actin filaments by adding capping protein. We show that when filaments are much shorter than their typical persistence length (approximately 10 microns), the forces generated are extensile and we see active nematic defect propagation, as seen in the microtubule-kinesin system. Based on this observation, we claim that the rigidity of rods plays an important role in dictating the nature of force generation in such systems. In order to understand this transition, we selectively label individual filaments and find that longer filaments show considerable bending and buckling, making them difficult to slide and extend along their length.

Contractile and mechanical properties of epithelia with perturbed actomyosin dynamics.

PubMed

Fischer, Sabine C; Blanchard, Guy B; Duque, Julia; Adams, Richard J; Arias, Alfonso Martinez; Guest, Simon D; Gorfinkiel, Nicole

2014-01-01

Mechanics has an important role during morphogenesis, both in the generation of forces driving cell shape changes and in determining the effective material properties of cells and tissues. Drosophila dorsal closure has emerged as a reference model system for investigating the interplay between tissue mechanics and cellular activity. During dorsal closure, the amnioserosa generates one of the major forces that drive closure through the apical contraction of its constituent cells. We combined quantitation of live data, genetic and mechanical perturbation and cell biology, to investigate how mechanical properties and contraction rate emerge from cytoskeletal activity. We found that a decrease in Myosin phosphorylation induces a fluidization of amnioserosa cells which become more compliant. Conversely, an increase in Myosin phosphorylation and an increase in actin linear polymerization induce a solidification of cells. Contrary to expectation, these two perturbations have an opposite effect on the strain rate of cells during DC. While an increase in actin polymerization increases the contraction rate of amnioserosa cells, an increase in Myosin phosphorylation gives rise to cells that contract very slowly. The quantification of how the perturbation induced by laser ablation decays throughout the tissue revealed that the tissue in these two mutant backgrounds reacts very differently. We suggest that the differences in the strain rate of cells in situations where Myosin activity or actin polymerization is increased arise from changes in how the contractile forces are transmitted and coordinated across the tissue through ECadherin-mediated adhesion. Altogether, our results show that there is an optimal level of Myosin activity to generate efficient contraction and suggest that the architecture of the actin cytoskeleton and the dynamics of adhesion complexes are important parameters for the emergence of coordinated activity throughout the tissue.

Contractile and Mechanical Properties of Epithelia with Perturbed Actomyosin Dynamics

PubMed Central

Fischer, Sabine C.; Blanchard, Guy B.; Duque, Julia; Adams, Richard J.; Arias, Alfonso Martinez; Guest, Simon D.; Gorfinkiel, Nicole

2014-01-01

Mechanics has an important role during morphogenesis, both in the generation of forces driving cell shape changes and in determining the effective material properties of cells and tissues. Drosophila dorsal closure has emerged as a reference model system for investigating the interplay between tissue mechanics and cellular activity. During dorsal closure, the amnioserosa generates one of the major forces that drive closure through the apical contraction of its constituent cells. We combined quantitation of live data, genetic and mechanical perturbation and cell biology, to investigate how mechanical properties and contraction rate emerge from cytoskeletal activity. We found that a decrease in Myosin phosphorylation induces a fluidization of amnioserosa cells which become more compliant. Conversely, an increase in Myosin phosphorylation and an increase in actin linear polymerization induce a solidification of cells. Contrary to expectation, these two perturbations have an opposite effect on the strain rate of cells during DC. While an increase in actin polymerization increases the contraction rate of amnioserosa cells, an increase in Myosin phosphorylation gives rise to cells that contract very slowly. The quantification of how the perturbation induced by laser ablation decays throughout the tissue revealed that the tissue in these two mutant backgrounds reacts very differently. We suggest that the differences in the strain rate of cells in situations where Myosin activity or actin polymerization is increased arise from changes in how the contractile forces are transmitted and coordinated across the tissue through ECadherin-mediated adhesion. Altogether, our results show that there is an optimal level of Myosin activity to generate efficient contraction and suggest that the architecture of the actin cytoskeleton and the dynamics of adhesion complexes are important parameters for the emergence of coordinated activity throughout the tissue. PMID:24759936

Development and characterization of a 3D multicell microtissue culture model of airway smooth muscle.

PubMed

West, Adrian R; Zaman, Nishat; Cole, Darren J; Walker, Matthew J; Legant, Wesley R; Boudou, Thomas; Chen, Christopher S; Favreau, John T; Gaudette, Glenn R; Cowley, Elizabeth A; Maksym, Geoffrey N

2013-01-01

Airway smooth muscle (ASM) cellular and molecular biology is typically studied with single-cell cultures grown on flat 2D substrates. However, cells in vivo exist as part of complex 3D structures, and it is well established in other cell types that altering substrate geometry exerts potent effects on phenotype and function. These factors may be especially relevant to asthma, a disease characterized by structural remodeling of the airway wall, and highlights a need for more physiologically relevant models of ASM function. We utilized a tissue engineering platform known as microfabricated tissue gauges to develop a 3D culture model of ASM featuring arrays of ∼0.4 mm long, ∼350 cell "microtissues" capable of simultaneous contractile force measurement and cell-level microscopy. ASM-only microtissues generated baseline tension, exhibited strong cellular organization, and developed actin stress fibers, but lost structural integrity and dissociated from the cantilevers within 3 days. Addition of 3T3-fibroblasts dramatically improved survival times without affecting tension development or morphology. ASM-3T3 microtissues contracted similarly to ex vivo ASM, exhibiting reproducible responses to a range of contractile and relaxant agents. Compared with 2D cultures, microtissues demonstrated identical responses to acetylcholine and KCl, but not histamine, forskolin, or cytochalasin D, suggesting that contractility is regulated by substrate geometry. Microtissues represent a novel model for studying ASM, incorporating a physiological 3D structure, realistic mechanical environment, coculture of multiple cells types, and comparable contractile properties to existing models. This new model allows for rapid screening of biochemical and mechanical factors to provide insight into ASM dysfunction in asthma.

Omecamtiv mercabil and blebbistatin modulate cardiac contractility by perturbing the regulatory state of the myosin filament.

PubMed

Kampourakis, Thomas; Zhang, Xuemeng; Sun, Yin-Biao; Irving, Malcolm

2018-01-01

Omecamtiv mecarbil and blebbistatin perturb the regulatory state of the thick filament in heart muscle. Omecamtiv mecarbil increases contractility at low levels of activation by stabilizing the ON state of the thick filament. Omecamtiv mecarbil decreases contractility at high levels of activation by disrupting the acto-myosin ATPase cycle. Blebbistatin reduces contractility by stabilizing the thick filament OFF state and inhibiting acto-myosin ATPase. Thick filament regulation is a promising target for novel therapeutics in heart disease. Contraction of heart muscle is triggered by a transient rise in intracellular free calcium concentration linked to a change in the structure of the actin-containing thin filaments that allows the head or motor domains of myosin from the thick filaments to bind to them and induce filament sliding. It is becoming increasingly clear that cardiac contractility is also regulated through structural changes in the thick filaments, although the molecular mechanisms underlying thick filament regulation are still relatively poorly understood. Here we investigated those mechanisms using small molecules - omecamtiv mecarbil (OM) and blebbistatin (BS) - that bind specifically to myosin and respectively activate or inhibit contractility in demembranated cardiac muscle cells. We measured isometric force and ATP utilization at different calcium and small-molecule concentrations in parallel with in situ structural changes determined using fluorescent probes on the myosin regulatory light chain in the thick filaments and on troponin C in the thin filaments. The results show that BS inhibits contractility and actin-myosin ATPase by stabilizing the OFF state of the thick filament in which myosin head domains are more parallel to the filament axis. In contrast, OM stabilizes the ON state of the thick filament, but inhibits contractility at high intracellular calcium concentration by disrupting the actin-myosin ATPase pathway. The effects of BS and OM on the calcium sensitivity of isometric force and filament structural changes suggest that the co-operativity of calcium activation in physiological conditions is due to positive coupling between the regulatory states of the thin and thick filaments. © 2017 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Omecamtiv mercabil and blebbistatin modulate cardiac contractility by perturbing the regulatory state of the myosin filament

PubMed Central

Kampourakis, Thomas; Zhang, Xuemeng; Sun, Yin‐Biao

2017-01-01

Key points Omecamtiv mecarbil and blebbistatin perturb the regulatory state of the thick filament in heart muscle.Omecamtiv mecarbil increases contractility at low levels of activation by stabilizing the ON state of the thick filament.Omecamtiv mecarbil decreases contractility at high levels of activation by disrupting the acto‐myosin ATPase cycle.Blebbistatin reduces contractility by stabilizing the thick filament OFF state and inhibiting acto‐myosin ATPase.Thick filament regulation is a promising target for novel therapeutics in heart disease. Abstract Contraction of heart muscle is triggered by a transient rise in intracellular free calcium concentration linked to a change in the structure of the actin‐containing thin filaments that allows the head or motor domains of myosin from the thick filaments to bind to them and induce filament sliding. It is becoming increasingly clear that cardiac contractility is also regulated through structural changes in the thick filaments, although the molecular mechanisms underlying thick filament regulation are still relatively poorly understood. Here we investigated those mechanisms using small molecules – omecamtiv mecarbil (OM) and blebbistatin (BS) – that bind specifically to myosin and respectively activate or inhibit contractility in demembranated cardiac muscle cells. We measured isometric force and ATP utilization at different calcium and small‐molecule concentrations in parallel with in situ structural changes determined using fluorescent probes on the myosin regulatory light chain in the thick filaments and on troponin C in the thin filaments. The results show that BS inhibits contractility and actin‐myosin ATPase by stabilizing the OFF state of the thick filament in which myosin head domains are more parallel to the filament axis. In contrast, OM stabilizes the ON state of the thick filament, but inhibits contractility at high intracellular calcium concentration by disrupting the actin‐myosin ATPase pathway. The effects of BS and OM on the calcium sensitivity of isometric force and filament structural changes suggest that the co‐operativity of calcium activation in physiological conditions is due to positive coupling between the regulatory states of the thin and thick filaments. PMID:29052230

Effects of Using Tricaine Methanesulfonate and Metomidate before Euthanasia on the Contractile Properties of Rainbow Trout (Oncorhynchus mykiss) Myocardium

PubMed Central

Roberts, Jordan C; Syme, Douglas A

2016-01-01

Because many anesthetics work through depressing cell excitability, unanesthetized euthanasia has become common for research involving excitable tissues (for example muscle and nerve) to avoid these depressive effects. However, anesthetic use during euthanasia may be indicated for studies involving isolated tissues if the potential depressive effects of brief anesthetic exposure dissipate after subsequent tissue isolation, washout, and saline perfusion. We explore this here by measuring whether, when applied prior to euthanasia, standard immersion doses of 2 fish anesthetics, tricaine methanesulfonate (TMS; 100 mg/L, n = 6) and methyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate (metomidate, 10 mg/L, n = 6), have residual effects on the contractile properties (force and work output) of isolated and saline-perfused ventricular compact myocardium from rainbow trout (Oncorhynchus mykiss). Results suggest that direct exposure of muscle to immersion doses of TMS—but not metomidate—impairs muscle contractile performance. However, brief exposure (2 to 3 min) to either anesthetic during euthanasia only—providing that the agent is washed out prior to tissue experimentation—does not have an effect on the contractile properties of the myocardium. Therefore, the use of TMS, metomidate, and perhaps other anesthetics that depress cell excitability during euthanasia may be indicated when conducting research on isolated and rinsed tissues. PMID:27657711

Effects of Using Tricaine Methanesulfonate and Metomidate before Euthanasia on the Contractile Properties of Rainbow Trout (Oncorhynchus mykiss) Myocardium.

PubMed

Roberts, Jordan C; Syme, Douglas A

2016-01-01

Because many anesthetics work through depressing cell excitability, unanesthetized euthanasia has become common for research involving excitable tissues (for example muscle and nerve) to avoid these depressive effects. However, anesthetic use during euthanasia may be indicated for studies involving isolated tissues if the potential depressive effects of brief anesthetic exposure dissipate after subsequent tissue isolation, washout, and saline perfusion. We explore this here by measuring whether, when applied prior to euthanasia, standard immersion doses of 2 fish anesthetics, tricaine methanesulfonate (TMS; 100 mg/L, n = 6) and methyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate (metomidate, 10 mg/L, n = 6), have residual effects on the contractile properties (force and work output) of isolated and saline-perfused ventricular compact myocardium from rainbow trout (Oncorhynchus mykiss). Results suggest that direct exposure of muscle to immersion doses of TMS-but not metomidate-impairs muscle contractile performance. However, brief exposure (2 to 3 min) to either anesthetic during euthanasia only-providing that the agent is washed out prior to tissue experimentation-does not have an effect on the contractile properties of the myocardium. Therefore, the use of TMS, metomidate, and perhaps other anesthetics that depress cell excitability during euthanasia may be indicated when conducting research on isolated and rinsed tissues.

Serotonin-induced contractile responses of esophageal smooth muscle in the house musk shrew (Suncus murinus).

PubMed

Shiina, T; Naitou, K; Nakamori, H; Suzuki, Y; Horii, K; Sano, Y; Shimaoka, H; Shimizu, Y

2016-11-01

Serotonin (5-hydroxytryptamine, 5-HT) is a regulatory factor in motility of the gastrointestinal tract including the esophagus. Although we proposed that vagal cholinergic and mast cell-derived non-cholinergic components including serotonin coordinately shorten the esophagus, the precise mechanism of serotonin-induced contractions in the suncus esophagus is still unclear. Therefore, the aims of this study were to determine characteristics of contractile responses induced by serotonin and to identify 5-HT receptor subtypes responsible for regulating motility in the suncus esophagus. An isolated segment of the suncus esophagus was placed in an organ bath, and longitudinal or circular mechanical responses were recorded using a force transducer. Serotonin evoked contractile responses of the suncus esophagus in the longitudinal direction but not in the circular direction. Tetrodotoxin did not affect the serotonin-induced contractions. Pretreatment with a non-selective 5-HT receptor antagonist or double application of 5-HT 1 and 5-HT 2 receptor antagonists blocked the serotonin-induced contractions. 5-HT 1 and 5-HT 2 receptor agonists, but not a 5-HT 3 receptor agonist, evoked contractile responses in the suncus esophagus. The findings suggest that serotonin induces contractile responses of the longitudinal smooth muscle in the muscularis mucosae of the suncus esophagus that are mediated via 5-HT 1 and 5-HT 2 receptors on muscle cells. The serotonin-induced contractions might contribute to esophageal peristalsis and emetic response. © 2016 John Wiley & Sons Ltd.

Cell sheet mechanics: How geometrical constraints induce the detachment of cell sheets from concave surfaces.

PubMed

Yamashita, Tadahiro; Kollmannsberger, Philip; Mawatari, Kazuma; Kitamori, Takehiko; Vogel, Viola

2016-11-01

Despite of the progress made to engineer structured microtissues such as BioMEMS and 3D bioprinting, little control exists how microtissues transform as they mature, as the misbalance between cell-generated forces and the strength of cell-cell and cell-substrate contacts can result in unintended tissue deformations and ruptures. To develop a quantitative perspective on how cellular contractility, scaffold curvature and cell-substrate adhesion control such rupture processes, human aortic smooth muscle cells were grown on glass substrates with submillimeter semichannels. We quantified cell sheet detachment from 3D confocal image stacks as a function of channel curvature and cell sheet tension by adding different amounts of Blebbistatin and TGF-β to inhibit or enhance cell contractility, respectively. We found that both higher curvature and higher contractility increased the detachment probability. Variations of the adhesive strength of the protein coating on the substrate revealed that the rupture plane was localized along the substrate-extracellular matrix interface for non-covalently adsorbed adhesion proteins, while the collagen-integrin interface ruptured when collagen I was covalently crosslinked to the substrate. Finally, a simple mechanical model is introduced that quantitatively explains how the tuning of substrate curvature, cell sheet contractility and adhesive strength can be used as tunable parameters as summarized in a first semi-quantitative phase diagram. These parameters can thus be exploited to either inhibit or purposefully induce a collective detachment of sheet-like microtissues for the use in tissue engineering and regenerative therapies. Despite of the significant progress in 3D tissue fabrication technologies at the microscale, there is still no quantitative model that can predict if cells seeded on a 3D structure maintain the imposed geometry while they form a continuous microtissue. Especially, detachment or loss of shape control of growing tissue is a major concern when designing 3D-structured scaffolds. Utilizing semi-cylindrical channels and vascular smooth muscle cells, we characterized how geometrical and mechanical parameters such as curvature of the substrate, cellular contractility, or protein-substrate adhesion strength tune the catastrophic detachment of microtissue. Observed results were rationalized by a theoretical model. The phase diagram showing how unintended tissue detachment progresses would help in designing of mechanically-balanced 3D scaffolds in future tissue engineering applications. Copyright © 2016. Published by Elsevier Ltd.

Cellular Contraction and Polarization Drive Collective Cellular Motion.

PubMed

Notbohm, Jacob; Banerjee, Shiladitya; Utuje, Kazage J C; Gweon, Bomi; Jang, Hwanseok; Park, Yongdoo; Shin, Jennifer; Butler, James P; Fredberg, Jeffrey J; Marchetti, M Cristina

2016-06-21

Coordinated motions of close-packed multicellular systems typically generate cooperative packs, swirls, and clusters. These cooperative motions are driven by active cellular forces, but the physical nature of these forces and how they generate collective cellular motion remain poorly understood. Here, we study forces and motions in a confined epithelial monolayer and make two experimental observations: 1) the direction of local cellular motion deviates systematically from the direction of the local traction exerted by each cell upon its substrate; and 2) oscillating waves of cellular motion arise spontaneously. Based on these observations, we propose a theory that connects forces and motions using two internal state variables, one of which generates an effective cellular polarization, and the other, through contractile forces, an effective cellular inertia. In agreement with theoretical predictions, drugs that inhibit contractility reduce both the cellular effective elastic modulus and the frequency of oscillations. Together, theory and experiment provide evidence suggesting that collective cellular motion is driven by at least two internal variables that serve to sustain waves and to polarize local cellular traction in a direction that deviates systematically from local cellular velocity. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The inverted pendulum model of bipedal standing cannot be stabilized through direct feedback of force and contractile element length and velocity at realistic series elastic element stiffness.

PubMed

van Soest, A J Knoek; Rozendaal, Leonard A

2008-07-01

Control of bipedal standing is typically analyzed in the context of a single-segment inverted pendulum model. The stiffness K (SE) of the series elastic element that transmits the force generated by the contractile elements of the ankle plantarflexors to the skeletal system has been reported to be smaller in magnitude than the destabilizing gravitational stiffness K ( g ). In this study, we assess, in case K (SE) + K ( g ) < 0, if bipedal standing can be locally stable under direct feedback of contractile element length, contractile element velocity (both sensed by muscle spindles) and muscle force (sensed by Golgi tendon organs) to alpha-motoneuron activity. A theoretical analysis reveals that even though positive feedback of force may increase the stiffness of the muscle-tendon complex to values well over the destabilizing gravitational stiffness, dynamic instability makes it impossible to obtain locally stable standing under the conditions assumed.

Contractile function is unaltered in diaphragm from mice lacking calcium release channel isoform 3

NASA Technical Reports Server (NTRS)

Clancy, J. S.; Takeshima, H.; Hamilton, S. L.; Reid, M. B.

1999-01-01

Skeletal muscle expresses at least two isoforms of the calcium release channel in the sarcoplasmic reticulum (RyR1 and RyR3). Whereas the function of RyR1 is well defined, the physiological significance of RyR3 is unclear. Some authors have suggested that RyR3 participates in excitation-contraction coupling and that RyR3 may specifically confer resistance to fatigue. To test this hypothesis, we measured contractile function of diaphragm strips from adult RyR3-deficient mice (exon 2-targeted mutation) and their heterozygous and wild-type littermates. In unfatigued diaphragm, there were no differences in isometric contractile properties (twitch characteristics, force-frequency relationships, maximal force) among the three groups. Our fatigue protocol (30 Hz, 0.25 duty cycle, 37 degrees C) depressed force to 25% of the initial force; however, lack of RyR3 did not accelerate the decline in force production. The force-frequency relationship was shifted to higher frequencies and was depressed in fatigued diaphragm; lack of RyR3 did not exaggerate these changes. We therefore provide evidence that RyR3 deficiency does not alter contractile function of adult muscle before, during, or after fatigue.

Transcriptional and Hormonal Regulation of Gravitropism of Woody Stems in Populus

Treesearch

Suzanne Gerttula; Matthew S. Zinkgraf; Gloria K. Muday; Daniel R. Lewis; Farid M. Ibatullin; Harry Brumer; Foster Hart; Shawn D. Mansfield; Vladimir Filkov; Andrew Groover

2015-01-01

Angiosperm trees reorient their woody stems by asymmetrically producing a specialized xylem tissue, tension wood, which exerts a strong contractile force resulting in negative gravitropism of the stem. Here, we show, in Populus trees, that initial gravity perception and response occurs in specialized cells through sedimentation of starch-filled...

Rigidity Sensing Explained by Active Matter Theory

PubMed Central

Marcq, Philippe; Yoshinaga, Natsuhiko; Prost, Jacques

2011-01-01

The magnitude of traction forces exerted by living animal cells on their environment is a monotonically increasing and approximately sigmoidal function of the stiffness of the external medium. We rationalize this observation using active matter theory, and propose that adaptation to substrate rigidity results from an interplay between passive elasticity and active contractility. PMID:21943439

Cardiomyocytes from late embryos and neonates do optimal work and striate best on substrates with tissue-level elasticity: metrics and mathematics.

PubMed

Majkut, Stephanie F; Discher, Dennis E

2012-11-01

In this review, we discuss recent studies on the mechanosensitive morphology and function of cardiomyocytes derived from embryos and neonates. For early cardiomyocytes cultured on substrates of various stiffnesses, contractile function as measured by force production, work output and calcium handling is optimized when the culture substrate stiffness mimics that of the tissue from which the cells were obtained. This optimal contractile function corresponds to changes in sarcomeric protein conformation and organization that promote contractile ability. In light of current models for myofibillogenesis, a recent mathematical model of striation and alignment on elastic substrates helps to illuminate how substrate stiffness modulates early myofibril formation and organization. During embryonic heart formation and maturation, cardiac tissue mechanics change dynamically. Experiments and models highlighted here have important implications for understanding cardiomyocyte differentiation and function in development and perhaps in regeneration processes.

Mast cells regulate myofilament calcium sensitization and heart function after myocardial infarction.

PubMed

Ngkelo, Anta; Richart, Adèle; Kirk, Jonathan A; Bonnin, Philippe; Vilar, Jose; Lemitre, Mathilde; Marck, Pauline; Branchereau, Maxime; Le Gall, Sylvain; Renault, Nisa; Guerin, Coralie; Ranek, Mark J; Kervadec, Anaïs; Danelli, Luca; Gautier, Gregory; Blank, Ulrich; Launay, Pierre; Camerer, Eric; Bruneval, Patrick; Menasche, Philippe; Heymes, Christophe; Luche, Elodie; Casteilla, Louis; Cousin, Béatrice; Rodewald, Hans-Reimer; Kass, David A; Silvestre, Jean-Sébastien

2016-06-27

Acute myocardial infarction (MI) is a severe ischemic disease responsible for heart failure and sudden death. Inflammatory cells orchestrate postischemic cardiac remodeling after MI. Studies using mice with defective mast/stem cell growth factor receptor c-Kit have suggested key roles for mast cells (MCs) in postischemic cardiac remodeling. Because c-Kit mutations affect multiple cell types of both immune and nonimmune origin, we addressed the impact of MCs on cardiac function after MI, using the c-Kit-independent MC-deficient (Cpa3(Cre/+)) mice. In response to MI, MC progenitors originated primarily from white adipose tissue, infiltrated the heart, and differentiated into mature MCs. MC deficiency led to reduced postischemic cardiac function and depressed cardiomyocyte contractility caused by myofilament Ca(2+) desensitization. This effect correlated with increased protein kinase A (PKA) activity and hyperphosphorylation of its targets, troponin I and myosin-binding protein C. MC-specific tryptase was identified to regulate PKA activity in cardiomyocytes via protease-activated receptor 2 proteolysis. This work reveals a novel function for cardiac MCs modulating cardiomyocyte contractility via alteration of PKA-regulated force-Ca(2+) interactions in response to MI. Identification of this MC-cardiomyocyte cross-talk provides new insights on the cellular and molecular mechanisms regulating the cardiac contractile machinery and a novel platform for therapeutically addressable regulators. ©2016 Ngkelo et al.

The β3 -adrenoceptor agonist mirabegron increases human atrial force through β1 -adrenoceptors: an indirect mechanism?

PubMed

Mo, Weilan; Michel, Martin C; Lee, Xiang Wen; Kaumann, Alberto J; Molenaar, Peter

2017-08-01

Mirabegron has been classified as a β 3 -adrenoceptor agonist approved for overactive bladder syndrome. We investigated possible cardiac effects of mirabegron in the absence or presence of β-adrenoceptor subtype antagonists. In view of its phenylethanolamine structure, we investigated whether mirabegron has indirect sympathomimetic activity by using neuronal uptake blockers. Right atrial trabeculae, from non-failing hearts, were paced and contractile force measured at 37°C. Single concentrations of mirabegron were added in the absence or presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), β 3 (L-748,337), β 1 (CGP 20712A), β 2 (ICI 118,551) -adrenoceptor antagonists, neuronal uptake inhibitors desipramine or phenoxybenzamine. Mirabegron significantly increased contractile force in human right atrium (1 μM, 7.6 ± 2.6%, n = 7; 10 μM, 10.2 ± 1.5%, n = 22 compared with (-)-isoprenaline P < 0.05). In the presence of IBMX, mirabegron (10 μM) caused a greater contraction. L-748,337 (100 nM) had no effect on the increase in contractile force caused by mirabegron (10 μM). In contrast, mirabegron (10 μM) reduced contractile force in the presence of CGP 20712A, which was not affected by L-748,337 (100 nM) or ICI 118,551 (50 nM). Mirabegron (10 μM) also reduced contractile force in the presence of desipramine or phenoxybenzamine. Mirabegron increases human atrial force through β 1 - but not β 3 -adrenoceptors. Desipramine and phenoxybenzamine block neuronal uptake and conceivably prevent mirabegron from releasing noradrenaline. A non-specific cardiodepressant effect is not mediated through β 3 (or β 2 )-adrenoceptors, consistent with lack of β 3 -adrenoceptor function on human atrial contractility. © 2017 The British Pharmacological Society.

Ca2+ sensitizers: An emerging class of agents for counterbalancing weakness in skeletal muscle diseases?

PubMed

Ochala, Julien

2010-02-01

Ca(2+) ions are key regulators of skeletal muscle contraction. By binding to contractile proteins, they initiate a cascade of molecular events leading to cross-bridge formation and ultimately, muscle shortening and force production. The ability of contractile proteins to respond to Ca(2+) attachment, also known as Ca(2+) sensitivity, is often compromised in acquired and congenital skeletal muscle disorders. It constitutes, undoubtedly, a major physiological cause of weakness for patients. In this review, we discuss recent studies giving strong molecular and cellular evidence that pharmacological modulators of some of the contractile proteins, also termed Ca(2+) sensitizers, are efficient agents to improve Ca(2+) sensitivity and function in diseased skeletal muscle cells. In fact, they compensate for the impaired contractile proteins response to Ca(2+) binding. Currently, such Ca(2+) sensitizing compounds are successfully used for reducing problems in cardiac disorders. Therefore, in the future, under certain conditions, these agents may represent an emerging class of agents to enhance the quality of life of patients suffering from skeletal muscle weakness. Copyright 2009 Elsevier B.V. All rights reserved.

Active properties of living tissues lead to size-dependent dewetting

NASA Astrophysics Data System (ADS)

Perez-Gonzalez, Carlos; Alert, Ricard; Blanch-Mercader, Carles; Gomez-Gonzalez, Manuel; Casademunt, Jaume; Trepat, Xavier

Key biological processes such as cancer and development are characterized by drastic transitions from 2D to a 3D geometry. These rearrangements have been classically studied as a wetting problem. According to this theory, wettability of a substrate by an epithelium is determined by the competition between cell-cell and cell-substrate adhesion energies. In contrast, we found that, far from a passive process, tissue dewetting is an active process driven by tissue internal forces. Experimentally, we reproduced epithelial dewetting by promoting a progressive formation of intercellular junctions in a monolayer of epithelial cells. Interestingly, the formation of intercellular junctions produces an increase in cell contractility, with the subsequent increase in traction and intercellular stress. At a certain time, tissue tension overcomes cell-substrate maximum adhesion and the monolayer spontaneously dewets the substrate. We developed an active polar fluid model, finding both theoretically and experimentally that critical contractility to promote wetting-dewetting transition depends on cell-substrate adhesion and, unexpectedly, on tissue size. As a whole, this work generalizes wetting theory to living tissues, unveiling unprecedented properties due to their unique active nature.

Blood pressure and the contractility of a human leg muscle.

PubMed

Luu, Billy L; Fitzpatrick, Richard C

2013-11-01

These studies investigate the relationships between perfusion pressure, force output and pressor responses for the contracting human tibialis anterior muscle. Eight healthy adults were studied. Changing the height of tibialis anterior relative to the heart was used to control local perfusion pressure. Electrically stimulated tetanic force output was highly sensitive to physiological variations in perfusion pressure showing a proportionate change in force output of 6.5% per 10 mmHg. This perfusion-dependent change in contractility begins within seconds and is reversible with a 53 s time constant, demonstrating a steady-state equilibrium between contractility and perfusion pressure. These stimulated contractions did not produce significant cardiovascular responses, indicating that the muscle pressor response does not play a major role in cardiovascular regulation at these workloads. Voluntary contractions at forces that would require constant motor drive if perfusion pressure had remained constant generated a central pressor response when perfusion pressure was lowered. This is consistent with a larger cortical drive being required to compensate for the lost contractility with lower perfusion pressure. The relationship between contractility and perfusion for this large postural muscle was not different from that of a small hand muscle (adductor pollicis) and it responded similarly to passive peripheral and active central changes in arterial pressure, but extended over a wider operating range of pressures. If we consider that, in a goal-oriented motor task, muscle contractility determines central motor output and the central pressor response, these results indicate that muscle would fatigue twice as fast without a pressor response. From its extent, timing and reversibility we propose a testable hypothesis that this change in contractility arises through contraction- and perfusion-dependent changes in interstitial K(+) concentration.

Blood pressure and the contractility of a human leg muscle

PubMed Central

Luu, Billy L; Fitzpatrick, Richard C

2013-01-01

These studies investigate the relationships between perfusion pressure, force output and pressor responses for the contracting human tibialis anterior muscle. Eight healthy adults were studied. Changing the height of tibialis anterior relative to the heart was used to control local perfusion pressure. Electrically stimulated tetanic force output was highly sensitive to physiological variations in perfusion pressure showing a proportionate change in force output of 6.5% per 10 mmHg. This perfusion-dependent change in contractility begins within seconds and is reversible with a 53 s time constant, demonstrating a steady-state equilibrium between contractility and perfusion pressure. These stimulated contractions did not produce significant cardiovascular responses, indicating that the muscle pressor response does not play a major role in cardiovascular regulation at these workloads. Voluntary contractions at forces that would require constant motor drive if perfusion pressure had remained constant generated a central pressor response when perfusion pressure was lowered. This is consistent with a larger cortical drive being required to compensate for the lost contractility with lower perfusion pressure. The relationship between contractility and perfusion for this large postural muscle was not different from that of a small hand muscle (adductor pollicis) and it responded similarly to passive peripheral and active central changes in arterial pressure, but extended over a wider operating range of pressures. If we consider that, in a goal-oriented motor task, muscle contractility determines central motor output and the central pressor response, these results indicate that muscle would fatigue twice as fast without a pressor response. From its extent, timing and reversibility we propose a testable hypothesis that this change in contractility arises through contraction- and perfusion-dependent changes in interstitial K+ concentration. PMID:24018946

Contractile forces at tricellular contacts modulate epithelial organization and monolayer integrity

PubMed Central

Salomon, Julie; Gaston, Cécile; Magescas, Jérémy; Duvauchelle, Boris; Canioni, Danielle; Sengmanivong, Lucie; Mayeux, Adeline; Michaux, Grégoire; Campeotto, Florence; Lemale, Julie; Viala, Jérôme; Poirier, Françoise; Minc, Nicolas; Schmitz, Jacques; Brousse, Nicole; Ladoux, Benoit; Goulet, Olivier; Delacour, Delphine

2017-01-01

Monolayered epithelia are composed of tight cell assemblies that ensure polarized exchanges. EpCAM, an unconventional epithelial-specific cell adhesion molecule, is assumed to modulate epithelial morphogenesis in animal models, but little is known regarding its cellular functions. Inspired by the characterization of cellular defects in a rare EpCAM-related human intestinal disease, we find that the absence of EpCAM in enterocytes results in an aberrant apical domain. In the course of this pathological state, apical translocation towards tricellular contacts (TCs) occurs with striking tight junction belt displacement. These unusual cell organization and intestinal tissue defects are driven by the loss of actomyosin network homoeostasis and contractile activity clustering at TCs, yet is reversed by myosin-II inhibitor treatment. This study reveals that adequate distribution of cortical tension is crucial for individual cell organization, but also for epithelial monolayer maintenance. Our data suggest that EpCAM modulation protects against epithelial dysplasia and stabilizes human tissue architecture. PMID:28084299

Contractile properties of early human embryonic stem cell-derived cardiomyocytes: beta-adrenergic stimulation induces positive chronotropy and lusitropy but not inotropy.

PubMed

Pillekamp, Frank; Haustein, Moritz; Khalil, Markus; Emmelheinz, Markus; Nazzal, Rewa; Adelmann, Roland; Nguemo, Filomain; Rubenchyk, Olga; Pfannkuche, Kurt; Matzkies, Matthias; Reppel, Michael; Bloch, Wilhelm; Brockmeier, Konrad; Hescheler, Juergen

2012-08-10

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) provide the unique opportunity to study the very early development of the human heart. The aim of this study was to investigate the effect of calcium and beta-adrenergic stimulation on the contractile properties of early hESC-CMs. Beating clusters containing hESC-CMs were co-cultured in vitro with noncontractile slices of neonatal murine ventricles. After 5-7 days, when beating clusters had integrated morphologically into the damaged tissue, isometric force measurements were performed during spontaneous beating as well as during electrical field stimulation. Spontaneous beating stopped when extracellular calcium ([Ca²⁺](ec)) was removed or after administration of the Ca²⁺ channel blocker nifedipine. During field stimulation at a constant rate, the developed force increased with incremental concentrations of [Ca²⁺](ec). During spontaneous beating, rising [Ca²⁺](ec) increased beating rate and developed force up to a [Ca²⁺](ec) of 2.5 mM. When [Ca²⁺](ec) was increased further, spontaneous beating rate decreased, whereas the developed force continued to increase. The beta-adrenergic agonist isoproterenol induced a dose-dependent increase of the frequency of spontaneous beating; however, it did not significantly change the developed force during spontaneous contractions or during electrical stimulation at a constant rate. Force developed by early hESC-CMs depends on [Ca²⁺](ec) and on the L-type Ca²⁺ channel. The lack of an inotropic reaction despite a pronounced chronotropic response after beta-adrenergic stimulation most likely indicates immaturity of the sarcoplasmic reticulum. For cell-replacement strategies, further maturation of cardiac cells has to be achieved either in vitro before or in vivo after transplantation.

Multiparity modifies contractile properties of pelvic muscles affecting the genesis of vaginal pressure in rabbits.

PubMed

López-Juárez, Rhode; Zempoalteca, René; Corona-Quintanilla, Dora Luz; Jiménez-Estrada, Ismael; Castelán, Francisco; Martínez-Gómez, Margarita

2018-01-01

To characterize the contractile properties of the bulbospongiosus (Bsm), isquiocavernosus (Ism), and pubococcygeus muscles (Pcm), and their involvement in the genesis of vaginal pressure in nulliparous and multiparous rabbits. Age-matched nulliparous and multiparous rabbits were used to record the isometric contractile responses of each muscle as well as the intravaginal pressure evoked by single square electrical pulses and stimulation trains of ascending frequency. To establish significant differences between groups, two-tail unpaired Student t tests were carried out. The linear correlation between intravaginal pressure and muscle contractile force was analyzed with Pearson correlation tests. For all cases, a P ≤ 0.05 was set as statistically significant. Multiparity decreased the contractile force of Bsm and Ism generated by high-frequency stimulation trains. The normalized force of the Pcm increased when evoked at 1, 4, and 10 Hz while this decreased at higher frequencies (20, 50, and 100 Hz). The contraction of both Bsm and Ism raised particularly the pressure on the perineal vagina while that of the Pcm increased the pressure in the pelvic vagina. Such a functional segregation is still present in multiparous rabbits albeit it was modified. Multiparity induces changes in the contractile responses of Bsm, Ism, and Pcm, which alterates the vaginal pressure. © 2017 Wiley Periodicals, Inc.

Slack length reduces the contractile phenotype of the Swine carotid artery.

PubMed

Rembold, Christopher M; Garvey, Sean M; Tejani, Ankit D

2013-01-01

Contraction is the primary function of adult arterial smooth muscle. However, in response to vessel injury or inflammation, arterial smooth muscle is able to phenotypically modulate from the contractile state to several 'synthetic' states characterized by proliferation, migration and/or increased cytokine secretion. We examined the effect of tissue length (L) on the phenotype of intact, isometrically held, initially contractile swine carotid artery tissues. Tissues were studied (1) without prolonged incubation at the optimal length for force generation (1.0 Lo, control), (2) with prolonged incubation for 17 h at 1.0 Lo, or (3) with prolonged incubation at slack length (0.6 Lo) for 16 h and then restoration to 1.0 Lo for 1 h. Prolonged incubation at 1.0 Lo minimally reduced the contractile force without substantially altering the mediators of contraction (crossbridge phosphorylation, shortening velocity or stimulated actin polymerization). Prolonged incubation of tissues at slack length (0.6 Lo), despite return of length to 1.0 Lo, substantially reduced contractile force, reduced crossbridge phosphorylation, nearly abolished crossbridge cycling (shortening velocity) and abolished stimulated actin polymerization. These data suggest that (1) slack length treatment significantly alters the contractile phenotype of arterial tissue, and (2) slack length treatment is a model to study acute phenotypic modulation of intact arterial smooth muscle. Copyright © 2013 S. Karger AG, Basel.

Remodeling the zonula adherens in response to tension and the role of afadin in this response

PubMed Central

Acharya, Bipul R.; Peyret, Grégoire; Fardin, Marc-Antoine; Mège, René-Marc; Ladoux, Benoit; Yap, Alpha S.; Fanning, Alan S.

2016-01-01

Morphogenesis requires dynamic coordination between cell–cell adhesion and the cytoskeleton to allow cells to change shape and move without losing tissue integrity. We used genetic tools and superresolution microscopy in a simple model epithelial cell line to define how the molecular architecture of cell–cell zonula adherens (ZA) is modified in response to elevated contractility, and how these cells maintain tissue integrity. We previously found that depleting zonula occludens 1 (ZO-1) family proteins in MDCK cells induces a highly organized contractile actomyosin array at the ZA. We find that ZO knockdown elevates contractility via a Shroom3/Rho-associated, coiled-coil containing protein kinase (ROCK) pathway. Our data suggest that each bicellular border is an independent contractile unit, with actin cables anchored end-on to cadherin complexes at tricellular junctions. Cells respond to elevated contractility by increasing junctional afadin. Although ZO/afadin knockdown did not prevent contractile array assembly, it dramatically altered cell shape and barrier function in response to elevated contractility. We propose that afadin acts as a robust protein scaffold that maintains ZA architecture at tricellular junctions. PMID:27114502

Cell stiffness, contractile stress and the role of extracellular matrix

DOE Office of Scientific and Technical Information (OSTI.GOV)

An, Steven S., E-mail: san@jhsph.edu; Kim, Jina; Ahn, Kwangmi

Here we have assessed the effects of extracellular matrix (ECM) composition and rigidity on mechanical properties of the human airway smooth muscle (ASM) cell. Cell stiffness and contractile stress showed appreciable changes from the most relaxed state to the most contracted state: we refer to the maximal range of these changes as the cell contractile scope. The contractile scope was least when the cell was adherent upon collagen V, followed by collagen IV, laminin, and collagen I, and greatest for fibronectin. Regardless of ECM composition, upon adherence to increasingly rigid substrates, the ASM cell positively regulated expression of antioxidant genesmore » in the glutathione pathway and heme oxygenase, and disruption of a redox-sensitive transcription factor, nuclear erythroid 2 p45-related factor (Nrf2), culminated in greater contractile scope. These findings provide biophysical evidence that ECM differentially modulates muscle contractility and, for the first time, demonstrate a link between muscle contractility and Nrf2-directed responses.« less

Superparamagnetic iron oxide nanoparticles regulate smooth muscle cell phenotype

PubMed Central

Angelopoulos, Ioannis; Southern, Paul; Pankhurst, Quentin A.

2016-01-01

Abstract Superparamagnetic iron oxide nanoparticles (SPION) are used for an increasing range of biomedical applications, from imaging to mechanical actuation of cells and tissue. The aim of this study was to investigate the loading of smooth muscle cells (SMC) with SPION and to explore what effect this has on the phenotype of the cells. Adherent human SMC were loaded with ∼17 pg of unconjugated, negatively charged, 50 nm SPION. Clusters of the internalized SPION particles were held in discrete cytoplasmic vesicles. Internalized SPION did not cause any change in cell morphology, proliferation, metabolic activity, or staining pattern of actin and calponin, two of the muscle contractile proteins involved in force generation. However, internalized SPION inhibited the increased gene expression of actin and calponin normally observed when cells are incubated under differentiation conditions. The observed change in the control of gene expression of muscle contractile apparatus by SPION has not previously been described. This finding could offer novel approaches for regulating the phenotype of SMC and warrants further investigation. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2412–2419, 2016. PMID:27176658

Hair Follicle-Derived Smooth Muscle Cells and Small Intestinal Submucosa for Engineering Mechanically Robust and Vasoreactive Vascular Media

PubMed Central

Peng, Hao-Fan; Liu, Jin Yu

2011-01-01

Our laboratory recently reported a new source of smooth muscle cells (SMCs) derived from hair follicle (HF) mesenchymal stem cells. HF-SMCs demonstrated high proliferation and clonogenic potential as well as contractile function. In this study, we aimed at engineering the vascular media using HF-SMCs and a natural biomaterial, namely small intestinal submucosa (SIS). Engineering functional vascular constructs required application of mechanical force, resulting in actin reorganization and cellular alignment. In turn, cell alignment was necessary for development of receptor- and nonreceptor-mediated contractility as soon as 24 h after cell seeding. Within 2 weeks in culture, the cells migrated into SIS and secreted collagen and elastin, the two major extracellular matrix components of the vessel wall. At 2 weeks, vascular reactivity increased significantly up to three- to fivefold and mechanical properties were similar to those of native ovine arteries. Taken together, our data demonstrate that the combination of HF-SMCs with SIS resulted in mechanically strong, biologically functional vascular media with potential for arterial implantation. PMID:21083418

Coupling between apical tension and basal adhesion allow epithelia to collectively sense and respond to substrate topography over long distances.

PubMed

Broaders, Kyle E; Cerchiari, Alec E; Gartner, Zev J

2015-12-01

Epithelial sheets fold into complex topographies that contribute to their function in vivo. Cells can sense and respond to substrate topography in their immediate vicinity by modulating their interfacial mechanics, but the extent to which these mechanical properties contribute to their ability to sense substrate topography across length scales larger than a single cell has not been explored in detail. To study the relationship between the interfacial mechanics of single cells and their collective behavior as tissues, we grew cell-sheets on substrates engraved with surface features spanning macroscopic length-scales. We found that many epithelial cell-types sense and respond to substrate topography, even when it is locally nearly planar. Cells clear or detach from regions of local negative curvature, but not from regions with positive or no curvature. We investigated this phenomenon using a finite element model where substrate topography is coupled to epithelial response through a balance of tissue contractility and adhesive forces. The model correctly predicts the focal sites of cell-clearing and epithelial detachment. Furthermore, the model predicts that local tissue response to substrate curvature is a function of the surrounding topography of the substrate across long distances. Analysis of cell-cell and cell-substrate contact angles suggests a relationship between these single-cell interfacial properties, epithelial interfacial properties, and collective epithelial response to substrate topography. Finally, we show that contact angles change upon activation of oncogenes or inhibition of cell-contractility, and that these changes correlate with collective epithelial response. Our results demonstrate that in mechanically integrated epithelial sheets, cell contractility can be transmitted through multiple cells and focused by substrate topography to affect a behavioral response at distant sites.

[The theory of postmortem rigidity: the history and an original concept].

PubMed

Kil'diushov, E M; Tumanov, É V; Sokolova, Z Iu

2012-01-01

The original theory of postmortem rigidity has been developed and substantiated based on the concept of postmortem muscular contracture. It is postulated that the unrestricted growth of Ca2+ concentration in myoplasm of contractile cells during the immediate postmortal period brings the actin-myosine complex to the force generation state without subsequent relaxation.

Endogenous Sheet-Averaged Tension Within a Large Epithelial Cell Colony.

PubMed

Dumbali, Sandeep P; Mei, Lanju; Qian, Shizhi; Maruthamuthu, Venkat

2017-10-01

Epithelial cells form quasi-two-dimensional sheets that function as contractile media to effect tissue shape changes during development and homeostasis. Endogenously generated intrasheet tension is a driver of such changes, but has predominantly been measured in the presence of directional migration. The nature of epithelial cell-generated forces transmitted over supracellular distances, in the absence of directional migration, is thus largely unclear. In this report, we consider large epithelial cell colonies which are archetypical multicell collectives with extensive cell-cell contacts but with a symmetric (circular) boundary. Using the traction force imbalance method (TFIM) (traction force microscopy combined with physical force balance), we first show that one can determine the colony-level endogenous sheet forces exerted at the midline by one half of the colony on the other half with no prior assumptions on the uniformity of the mechanical properties of the cell sheet. Importantly, we find that this colony-level sheet force exhibits large variations with orientation-the difference between the maximum and minimum sheet force is comparable to the average sheet force itself. Furthermore, the sheet force at the colony midline is largely tensile but the shear component exhibits significantly more variation with orientation. We thus show that even an unperturbed epithelial colony with a symmetric boundary shows significant directional variation in the endogenous sheet tension and shear forces that subsist at the colony level.

LET-99 functions in the astral furrowing pathway, where it is required for myosin enrichment in the contractile ring.

PubMed

Price, Kari L; Rose, Lesilee S

2017-09-01

The anaphase spindle determines the position of the cytokinesis furrow, such that the contractile ring assembles in an equatorial zone between the two spindle poles. Contractile ring formation is mediated by RhoA activation at the equator by the centralspindlin complex and midzone microtubules. Astral microtubules also inhibit RhoA accumulation at the poles. In the Caenorhabditis elegans one-cell embryo, the astral microtubule-dependent pathway requires anillin, NOP-1, and LET-99. LET-99 is well characterized for generating the asymmetric cortical localization of the Gα-dependent force-generating complex that positions the spindle during asymmetric division. However, whether the role of LET-99 in cytokinesis is specific to asymmetric division and whether it acts through Gα to promote furrowing are unclear. Here we show that LET-99 contributes to furrowing in both asymmetrically and symmetrically dividing cells, independent of its function in spindle positioning and Gα regulation. LET-99 acts in a pathway parallel to anillin and is required for myosin enrichment into the contractile ring. These and other results suggest a positive feedback model in which LET-99 localizes to the presumptive cleavage furrow in response to the spindle and myosin. Once positioned there, LET-99 enhances myosin accumulation to promote furrowing in both symmetrically and asymmetrically dividing cells. © 2017 Price and Rose. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes-derived from induced pluripotent stem cells.

PubMed

Yang, Xiulan; Rodriguez, Marita; Pabon, Lil; Fischer, Karin A; Reinecke, Hans; Regnier, Michael; Sniadecki, Nathan J; Ruohola-Baker, Hannele; Murry, Charles E

2014-07-01

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) have great potential as a cell source for therapeutic applications such as regenerative medicine, disease modeling, drug screening, and toxicity testing. This potential is limited, however, by the immature state of the cardiomyocytes acquired using current protocols. Tri-iodo-l-thyronine (T3) is a growth hormone that is essential for optimal heart growth. In this study, we investigated the effect of T3 on hiPSC-CM maturation. A one-week treatment with T3 increased cardiomyocyte size, anisotropy, and sarcomere length. T3 treatment was associated with reduced cell cycle activity, manifest as reduced DNA synthesis and increased expression of the cyclin-dependent kinase inhibitor p21. Contractile force analyses were performed on individual cardiomyocytes using arrays of microposts, revealing an almost two-fold higher force per-beat after T3 treatment and also an enhancement in contractile kinetics. This improvement in force generation was accompanied by an increase in rates of calcium release and reuptake, along with a significant increase in sarcoendoplasmic reticulum ATPase expression. Finally, although mitochondrial genomes were not numerically increased, extracellular flux analysis showed a significant increase in maximal mitochondrial respiratory capacity and respiratory reserve capability after T3 treatment. Using a broad spectrum of morphological, molecular, and functional parameters, we conclude that T3 is a driver for hiPSC-CM maturation. T3 treatment may enhance the utility of hiPSC-CMs for therapy, disease modeling, or drug/toxicity screens. Copyright © 2014 Elsevier Ltd. All rights reserved.

How deep cells feel: Mean-field Computations and Experiments

NASA Astrophysics Data System (ADS)

Buxboim, Amnon; Sen, Shamik; Discher, Dennis E.

2009-03-01

Most cells in solid tissues exert contractile forces that mechanically couple them to elastic surroundings and that significantly influence cell adhesion, cytoskeletal organization and differentiation. However, strains within the depths of matrices are often unclear and are likely relevant to thin matrices, such as basement membranes, relative to cell size as well as to defining how far cells can ``feel.'' We present experimental results for cell spreading on thin, ligand- coated gels and for prestress in stem cells in relation to gel stiffness. Matrix thickness affects cell spread area, focal adhesions and cytoskeleton organization in stem cells, which we will compare to differentiated cells. We introduce a finite element computation to estimate the elastostatic deformations within the matrix on which a cell is placed. Interfacial strains between cell and matrix show large deviations only when soft matrices are a fraction of cell dimensions, proving consistent with experiments. 3-D cell morphologies that model stem cell-derived neurons, myoblasts, and osteoblasts show that a cylinder-shaped myoblast induces the highest strains, consistent with the prominent contractility of muscle. Groups of such cells show a weak crosstalk via matrix strains only when cells are much closer than a cell-width. Cells thus feel on length scales closer to that of adhesions than on cellular scales.

Dissecting the Impact of Matrix Anchorage and Elasticity in Cell Adhesion

PubMed Central

Pompe, Tilo; Glorius, Stefan; Bischoff, Thomas; Uhlmann, Ina; Kaufmann, Martin; Brenner, Sebastian; Werner, Carsten

2009-01-01

Abstract Extracellular matrices determine cellular fate decisions through the regulation of intracellular force and stress. Previous studies suggest that matrix stiffness and ligand anchorage cause distinct signaling effects. We show herein how defined noncovalent anchorage of adhesion ligands to elastic substrates allows for dissection of intracellular adhesion signaling pathways related to matrix stiffness and receptor forces. Quantitative analysis of the mechanical balance in cell adhesion using traction force microscopy revealed distinct scalings of the strain energy imparted by the cells on the substrates dependent either on matrix stiffness or on receptor force. Those scalings suggested the applicability of a linear elastic theoretical framework for the description of cell adhesion in a certain parameter range, which is cell-type-dependent. Besides the deconvolution of biophysical adhesion signaling, site-specific phosphorylation of focal adhesion kinase, dependent either on matrix stiffness or on receptor force, also demonstrated the dissection of biochemical signaling events in our approach. Moreover, the net contractile moment of the adherent cells and their strain energy exerted on the elastic substrate was found to be a robust measure of cell adhesion with a unifying power-law scaling exponent of 1.5 independent of matrix stiffness. PMID:19843448

Temporal Adaptive Changes in Contractility and Fatigability of Diaphragm Muscles from Streptozotocin-Diabetic Rats

PubMed Central

Brotto, Marco; Brotto, Leticia; Jin, J.-P.; Nosek, Thomas M.; Romani, Andrea

2010-01-01

Diabetes is characterized by ventilatory depression due to decreased diaphragm (DPH) function. This study investigated the changes in contractile properties of rat DPH muscles over a time interval encompassing from 4 days to 14 weeks after the onset of streptozotocin-induced diabetes, with and without insulin treatment for 2 weeks. Maximum tetanic force in intact DPH muscle strips and recovery from fatiguing stimulation were measured. An early (4-day) depression in contractile function in diabetic DPH was followed by gradual improvement in muscle function and fatigue recovery (8 weeks). DPH contractile function deteriorated again at 14 weeks, a process that was completely reversed by insulin treatment. Maximal contractile force and calcium sensitivity assessed in Triton-skinned DPH fibers showed a similar bimodal pattern and the same beneficial effect of insulin treatment. While an extensive analysis of the isoforms of the contractile and regulatory proteins was not conducted, Western blot analysis of tropomyosin suggests that the changes in diabetic DPH response depended, at least in part, on a switch in fiber type. PMID:20467472

Temporal adaptive changes in contractility and fatigability of diaphragm muscles from streptozotocin-diabetic rats.

PubMed

Brotto, Marco; Brotto, Leticia; Jin, J-P; Nosek, Thomas M; Romani, Andrea

2010-01-01

Diabetes is characterized by ventilatory depression due to decreased diaphragm (DPH) function. This study investigated the changes in contractile properties of rat DPH muscles over a time interval encompassing from 4 days to 14 weeks after the onset of streptozotocin-induced diabetes, with and without insulin treatment for 2 weeks. Maximum tetanic force in intact DPH muscle strips and recovery from fatiguing stimulation were measured. An early (4-day) depression in contractile function in diabetic DPH was followed by gradual improvement in muscle function and fatigue recovery (8 weeks). DPH contractile function deteriorated again at 14 weeks, a process that was completely reversed by insulin treatment. Maximal contractile force and calcium sensitivity assessed in Triton-skinned DPH fibers showed a similar bimodal pattern and the same beneficial effect of insulin treatment. While an extensive analysis of the isoforms of the contractile and regulatory proteins was not conducted, Western blot analysis of tropomyosin suggests that the changes in diabetic DPH response depended, at least in part, on a switch in fiber type.

Regional Differences in Rat Vaginal Smooth Muscle Contractility and Morphology

PubMed Central

Skoczylas, Laura C.; Jallah, Zegbeh; Sugino, Yoshio; Stein, Suzan E.; Feola, Andrew; Yoshimura, Naoki

2013-01-01

The objective of this study was to define the regional differences in rat vaginal smooth muscle contractility and morphology. We evaluated circumferential segments from the proximal, middle, and distal rat vagina (n = 21) in vitro. Contractile responses to carbachol, phenylephrine, potassium chloride, and electrical field stimulation (EFS) were measured. Immunohistochemical analyses were also performed. The dose–response curves for carbachol- and phenylephrine-dependent contractions were different in the distal (P = .05, P = .04) compared to the proximal/middle regions. Adjusted for region-dependent changes in contractility, the distal vagina generated lower force in response to carbachol and higher force in response to phenylephrine. There was less force with increasing EFS frequency in the distal (P = .03), compared to the proximal/middle regions. Cholinergic versus adrenergic nerves were more frequent in the proximal region (P = .03). In summary, the results indicate that functional and morphological differences in smooth muscle and nerve fibers of the distal versus proximal/middle regions of the vagina exist. PMID:23298869

Treatment of Tourniquet-Induced Ischemia Reperfusion Injury with Muscle Progenitor Cells

DTIC Science & Technology

2011-09-01

application. Muscle mass, isometric contractile properties, and selected histologic properties were evaluated at 2 wk after ischemia. Results. IRI...results showed that a small number of trans- planted cells differentiated and formed muscle fibers , which could potentially contribute to force genera...the wet weight of the muscle (in g); q is the angle of fiber pinnation (12.8 for TA); Lf is the mean fiber length (57% of TAmuscle length); and r is

The Effects of Combined Cyclic Stretch and Pressure on the Aortic Valve Interstitial Cell Phenotype

PubMed Central

Thayer, Patrick; Balachandran, Kartik; Rathan, Swetha; Yap, Choon Hwai; Arjunon, Sivakkumar; Jo, Hanjoong; Yoganathan, Ajit P.

2017-01-01

Aortic valve interstitial cells (VIC) can exhibit phenotypic characteristics of fibroblasts, myofibroblasts, and smooth muscle cells. Others have proposed that valve cells become activated and exhibit myofibroblast or fibroblast characteristics during disease initiation and progression; however, the cues that modulate this phenotypic change remain unclear. We hypothesize that the mechanical forces experienced by the valve play a role in regulating the native phenotype of the valve and that altered mechanical forces result in an activated phenotype. Using a novel ex vivo cyclic stretch and pressure bioreactor, we subjected porcine aortic valve (AV) leaflets to combinations of normal and pathological stretch and pressure magnitudes. The myofibroblast markers α-SMA and Vimentin, along with the smooth muscle markers Calponin and Caldesmon, were analyzed using immunohistochemistry and immunoblotting. Tissue structure was analyzed using Movat’s pentachrome staining. We report that pathological stretch and pressure inhibited the contractile and possibly myofibroblast phenotypes as indicated by downregulation of the proteins α-SMA, Vimentin, and Calponin. In particular, Calponin downregulation implies depolymerization of actin filaments and possible conversion to a more synthetic (non-contractile) phenotype. This agreed well with the increase in spongiosa and fibrosa thickness observed under elevated pressure and stretch that are typically indicative of increased matrix synthesis. Our study therefore demonstrates how cyclic stretch and pressure may possibly act together to modulate the AVIC phenotype. PMID:21347552

Apical constriction: themes and variations on a cellular mechanism driving morphogenesis

PubMed Central

Martin, Adam C.; Goldstein, Bob

2014-01-01

Apical constriction is a cell shape change that promotes tissue remodeling in a variety of homeostatic and developmental contexts, including gastrulation in many organisms and neural tube formation in vertebrates. In recent years, progress has been made towards understanding how the distinct cell biological processes that together drive apical constriction are coordinated. These processes include the contraction of actin-myosin networks, which generates force, and the attachment of actin networks to cell-cell junctions, which allows forces to be transmitted between cells. Different cell types regulate contractility and adhesion in unique ways, resulting in apical constriction with varying dynamics and subcellular organizations, as well as a variety of resulting tissue shape changes. Understanding both the common themes and the variations in apical constriction mechanisms promises to provide insight into the mechanics that underlie tissue morphogenesis. PMID:24803648

Solo and keratin filaments regulate epithelial tubule morphology.

PubMed

Nishimura, Ryosuke; Kato, Kagayaki; Fujiwara, Sachiko; Ohashi, Kazumasa; Mizuno, Kensaku

2018-04-28

Epithelial tubules, consisting of the epithelial cell sheet with a central lumen, are the basic structure of many organs. Mechanical forces play an important role in epithelial tubulogenesis; however, little is known about the mechanisms controlling the mechanical forces during epithelial tubule morphogenesis. Solo (also known as ARHGEF40) is a RhoA-targeting guanine-nucleotide exchange factor that is involved in mechanical force-induced RhoA activation and stress fiber formation. Solo binds to keratin-8/keratin-18 (K8/K18) filaments, and this interaction plays a crucial role in mechanotransduction. In this study, we examined the roles of Solo and K8/K18 filaments in epithelial tubulogenesis using MDCK cells cultured in 3D collagen gels. Knockdown of either Solo or K18 resulted in rounder tubules with increased lumen size, indicating that Solo and K8/K18 filaments play critical roles in forming the elongated morphology of epithelial tubules. Moreover, knockdown of Solo or K18 decreased the level of diphosphorylated myosin light chain (a marker of contractile force) at the luminal and outer surfaces of tubules, suggesting that Solo and K8/K18 filaments are involved in the generation of the myosin II-mediated contractile force during epithelial tubule morphogenesis. In addition, K18 filaments were normally oriented along the long axis of the tubule, but knockdown of Solo perturbed their orientation. These results suggest that Solo plays crucial roles in forming the elongated morphology of epithelial tubules and in regulating myosin II activity and K18 filament organization during epithelial tubule formation.

E258K HCM-causing mutation in cardiac MyBP-C reduces contractile force and accelerates twitch kinetics by disrupting the cMyBP-C and myosin S2 interaction.

PubMed

De Lange, Willem J; Grimes, Adrian C; Hegge, Laura F; Spring, Alexander M; Brost, Taylor M; Ralphe, J Carter

2013-09-01

Mutations in cardiac myosin binding protein C (cMyBP-C) are prevalent causes of hypertrophic cardiomyopathy (HCM). Although HCM-causing truncation mutations in cMyBP-C are well studied, the growing number of disease-related cMyBP-C missense mutations remain poorly understood. Our objective was to define the primary contractile effect and molecular disease mechanisms of the prevalent cMyBP-C E258K HCM-causing mutation in nonremodeled murine engineered cardiac tissue (mECT). Wild-type and human E258K cMyBP-C were expressed in mECT lacking endogenous mouse cMyBP-C through adenoviral-mediated gene transfer. Expression of E258K cMyBP-C did not affect cardiac cell survival and was appropriately incorporated into the cardiac sarcomere. Functionally, expression of E258K cMyBP-C caused accelerated contractile kinetics and severely compromised twitch force amplitude in mECT. Yeast two-hybrid analysis revealed that E258K cMyBP-C abolished interaction between the N terminal of cMyBP-C and myosin heavy chain sub-fragment 2 (S2). Furthermore, this mutation increased the affinity between the N terminal of cMyBP-C and actin. Assessment of phosphorylation of three serine residues in cMyBP-C showed that aberrant phosphorylation of cMyBP-C is unlikely to be responsible for altering these interactions. We show that the E258K mutation in cMyBP-C abolishes interaction between N-terminal cMyBP-C and myosin S2 by directly disrupting the cMyBP-C-S2 interface, independent of cMyBP-C phosphorylation. Similar to cMyBP-C ablation or phosphorylation, abolition of this inhibitory interaction accelerates contractile kinetics. Additionally, the E258K mutation impaired force production of mECT, which suggests that in addition to the loss of physiological function, this mutation disrupts contractility possibly by tethering the thick and thin filament or acting as an internal load.

Mechanosensing of matrix by stem cells: From matrix heterogeneity, contractility, and the nucleus in pore-migration to cardiogenesis and muscle stem cells in vivo.

PubMed

Smith, Lucas; Cho, Sangkyun; Discher, Dennis E

2017-11-01

Stem cells are particularly 'plastic' cell types that are induced by various cues to become specialized, tissue-functional lineages by switching on the expression of specific gene programs. Matrix stiffness is among the cues that multiple stem cell types can sense and respond to. This seminar-style review focuses on mechanosensing of matrix elasticity in the differentiation or early maturation of a few illustrative stem cell types, with an intended audience of biologists and physical scientists. Contractile forces applied by a cell's acto-myosin cytoskeleton are often resisted by the extracellular matrix and transduced through adhesions and the cytoskeleton ultimately into the nucleus to modulate gene expression. Complexity is added by matrix heterogeneity, and careful scrutiny of the evident stiffness heterogeneity in some model systems resolves some controversies concerning matrix mechanosensing. Importantly, local stiffness tends to dominate, and 'durotaxis' of stem cells toward stiff matrix reveals a dependence of persistent migration on myosin-II force generation and also rigid microtubules that confer directionality. Stem and progenitor cell migration in 3D can be further affected by matrix porosity as well as stiffness, with nuclear size and rigidity influencing niche retention and fate choices. Cell squeezing through rigid pores can even cause DNA damage and genomic changes that contribute to de-differentiation toward stem cell-like states. Contraction of acto-myosin is the essential function of striated muscle, which also exhibit mechanosensitive differentiation and maturation as illustrated in vivo by beating heart cells and by the regenerative mobilization of skeletal muscle stem cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mechanisms underlying hypothermia-induced cardiac contractile dysfunction.

PubMed

Han, Young-Soo; Tveita, Torkjel; Prakash, Y S; Sieck, Gary C

2010-03-01

Rewarming patients after profound hypothermia may result in acute heart failure and high mortality (50-80%). However, the underlying pathophysiological mechanisms are largely unknown. We characterized cardiac contractile function in the temperature range of 15-30 degrees C by measuring the intracellular Ca(2+) concentration ([Ca(2+)](i)) and twitch force in intact left ventricular rat papillary muscles. Muscle preparations were loaded with fura-2 AM and electrically stimulated during cooling at 15 degrees C for 1.5 h before being rewarmed to the baseline temperature of 30 degrees C. After hypothermia/rewarming, peak twitch force decreased by 30-40%, but [Ca(2+)](i) was not significantly altered. In addition, we assessed the maximal Ca(2+)-activated force (F(max)) and Ca(2+) sensitivity of force in skinned papillary muscle fibers. F(max) was decreased by approximately 30%, whereas the pCa required for 50% of F(max) was reduced by approximately 0.14. In rewarmed papillary muscle, both total cardiac troponin I (cTnI) phosphorylation and PKA-mediated cTnI phosphorylation at Ser23/24 were significantly increased compared with controls. We conclude that after hypothermia/rewarming, myocardial contractility is significantly reduced, as evidenced by reduced twitch force and F(max). The reduced myocardial contractility is attributed to decreased Ca(2+) sensitivity of force rather than [Ca(2+)](i) itself, resulting from increased cTnI phosphorylation.

A positional code and anisotropic forces control tissue remodeling in Drosophila

NASA Astrophysics Data System (ADS)

Zallen, Jennifer

A major challenge in developmental biology is to understand how tissue-scale changes in organism structure arise from events that occur on a cellular and molecular level. We are using cell biological, biophysical, and quantitative live-embryo imaging approaches to understand how genes encode the forces that shape tissues, and to identify the mechanisms that modulate cell behavior in response to local forces. In many animals, the elongated head-to-tail body axis is achieved by rapid and coordinated movements of hundreds of cells. We found that in the fruit fly, these cell movements are regulated by subcellular asymmetries in the localization of proteins that generate contractile and adhesive forces between cells. Asymmetries in the force-generating machinery are in turn controlled by a positional code of spatial information provided by an ancient family of Toll-related receptors that are widely used for pathogen recognition by the innate immune system. I will describe how this spatial system systematically orients local cell movements and collective rosette-like clusters in the Drosophila embryo. Rosettes have now also been shown to shape the body axis in chicks, frogs, and mice, demonstrating that rosette behaviors are a general mechanism linking cellular asymmetry to tissue reorganization.

Progesterone Metabolites Produced by Cytochrome P450 3A Modulate Uterine Contractility in a Murine Model

PubMed Central

Patil, Avinash S.; Swamy, Geeta K.; Murtha, Amy P.; Heine, R. Phillips; Zheng, Xiaomei; Grotegut, Chad A.

2015-01-01

Objective: We seek to characterize the effect of progesterone metabolites on spontaneous and oxytocin-induced uterine contractility. Study Design: Spontaneous contractility was studied in mouse uterine horns after treatment with progesterone, 2α-hydroxyprogesterone, 6β-hydroxyprogesterone (6β-OHP), 16α-hydroxyprogesterone (16α-OHP), or 17-hydroxyprogesterone caproate (17-OHPC) at 10−9 to 10−6 mol/L. Uterine horns were exposed to progestins (10−6 mol/L), followed by increasing concentrations of oxytocin (1-100 nmol/L) to study oxytocin-induced contractility. Contraction parameters were compared for each progestin and matched vehicle control using repeated measures 2-way analysis of variance. In vitro metabolism of progesterone by recombinant cytochrome P450 3A (CYP3A) microsomes (3A5, 3A5, and 3A7) identified major metabolites. Results: Oxytocin-induced contractile frequency was decreased by 16α-OHP (P = .03) and increased by 6β-OHP (P = .05). Progesterone and 17-OHPC decreased oxytocin-induced contractile force (P = .02 and P = .04, respectively) and frequency (P = .02 and P = .03, respectively). Only progesterone decreased spontaneous contractile force (P = .02). Production of 16α-OHP and 6β-OHP metabolites were confirmed in all CYP3A isoforms tested. Conclusion: Progesterone metabolites produced by maternal or fetal CYP3A enzymes influence uterine contractility. PMID:26037300

Effects of hypoxia and hypercapnia on geniohyoid contractility and endurance.

PubMed

Salmone, R J; Van Lunteren, E

1991-08-01

Sleep apnea and other respiratory diseases produce hypoxemia and hypercapnia, factors that adversely affect skeletal muscle performance. To examine the effects of these chemical alterations on force production by an upper airway dilator muscle, the contractile and endurance characteristics of the geniohyoid muscle were examined in situ during severe hypoxia (arterial PO2 less than 40 Torr), mild hypoxia (PO2 45-65 Torr), and hypercapnia (PCO2 55-80 Torr) and compared with hyperoxic-normocapnic conditions in anesthetized cats. Muscles were studied at optimal length, and contractile force was assessed in response to supramaximal electrical stimulation of the hypoglossal nerve (n = 7 cats) or geniohyoid muscle (n = 2 cats). There were no significant changes in the twitch kinetics or force-frequency curve of the geniohyoid muscle during hypoxia or hypercapnia. However, the endurance of the geniohyoid, as reflected in the fatigue index (ratio of force at 2 min to initial force in response to 40-Hz stimulation at a duty cycle 0.33), was significantly reduced by severe hypoxia but not by hypercapnia or mild hypoxia. In addition, the downward shift in the force-frequency curve after the repetitive stimulation protocol was greater during hypoxia than hyperoxia, especially at higher frequencies. In conclusion, the ability of the geniohyoid muscle to maintain force output during high levels of activation is adversely affected by severe hypoxia but not mild hypoxia or hypercapnia. However, none of these chemical perturbations affected muscle contractility acutely.

Ultraslow myosin molecular motors of placental contractile stem villi in humans.

PubMed

Lecarpentier, Yves; Claes, Victor; Lecarpentier, Edouard; Guerin, Catherine; Hébert, Jean-Louis; Arsalane, Abdelilah; Moumen, Abdelouahab; Krokidis, Xénophon; Michel, Francine; Timbely, Oumar

2014-01-01

Human placental stem villi (PSV) present contractile properties. In vitro mechanics were investigated in 40 human PSV. Contraction of PSV was induced by both KCl exposure (n = 20) and electrical tetanic stimulation (n = 20). Isotonic contractions were registered at several load levels ranging from zero-load up to isometric load. The tension-velocity relationship was found to be hyperbolic. This made it possible to apply the A. Huxley formalism for determining the rate constants for myosin cross-bridge (CB) attachment and detachment, CB single force, catalytic constant, myosin content, and maximum myosin ATPase activity. These molecular characteristics of myosin CBs did not differ under either KCl exposure or tetanus. A comparative approach was established from studies previously published in the literature and driven by mean of a similar method. As compared to that described in mammalian striated muscles, we showed that in human PSV, myosin CB rate constants for attachment and detachment were about 103 times lower whereas myosin ATPase activity was 105 times lower. Up to now, CB kinetics of contractile cells arranged along the long axis of the placental sheath appeared to be the slowest ever observed in any mammalian contractile tissue.

A node organization in the actomyosin contractile ring generates tension and aids stability

PubMed Central

Thiyagarajan, Sathish; Wang, Shuyuan; O’Shaughnessy, Ben

2017-01-01

During cytokinesis, a contractile actomyosin ring constricts and divides the cell in two. How the ring marshals actomyosin forces to generate tension is not settled. Recently, a superresolution microscopy study of the fission yeast ring revealed that myosins and formins that nucleate actin filaments colocalize in plasma membrane-anchored complexes called nodes in the constricting ring. The nodes move bidirectionally around the ring. Here we construct and analyze a coarse-grained mathematical model of the fission yeast ring to explore essential consequences of the recently discovered ring ultrastructure. The model reproduces experimentally measured values of ring tension, explains why nodes move bidirectionally, and shows that tension is generated by myosin pulling on barbed-end-anchored actin filaments in a stochastic sliding-filament mechanism. This mechanism is not based on an ordered sarcomeric organization. We show that the ring is vulnerable to intrinsic contractile instabilities, and protection from these instabilities and organizational homeostasis require both component turnover and anchoring of components to the plasma membrane. PMID:28954859

A biomechanical model of agonist-initiated contraction in the asthmatic airway.

PubMed

Brook, B S; Peel, S E; Hall, I P; Politi, A Z; Sneyd, J; Bai, Y; Sanderson, M J; Jensen, O E

2010-01-31

This paper presents a modelling framework in which the local stress environment of airway smooth muscle (ASM) cells may be predicted and cellular responses to local stress may be investigated. We consider an elastic axisymmetric model of a layer of connective tissue and circumferential ASM fibres embedded in parenchymal tissue and model the active contractile force generated by ASM via a stress acting along the fibres. A constitutive law is proposed that accounts for active and passive material properties as well as the proportion of muscle to connective tissue. The model predicts significantly different contractile responses depending on the proportion of muscle to connective tissue in the remodelled airway. We find that radial and hoop-stress distributions in remodelled muscle layers are highly heterogenous with distinct regions of compression and tension. Such patterns of stress are likely to have important implications, from a mechano-transduction perspective, on contractility, short-term cytoskeletal adaptation and long-term airway remodelling in asthma. Copyright 2009 Elsevier B.V. All rights reserved.

Effect of hypokinesia on contractile function of cardiac muscle

NASA Technical Reports Server (NTRS)

Meyerson, F. Z.; Kapelko, V. I.; Trikhpoyeva, A. M.; Gorina, M. S.

1980-01-01

Rats were subjected to hypokinesia for two months and the contractile function of isolated papillary muscle was studied. Hypokinesia reduced significantly the isotonic contraction rate which depended on the ATPase activity of the myofibrils; it also reduced the rate and index of relaxation which depended on the functional capacity of the Ca(++) pump of the sarcoplasmic reticulum. The maximum force of isometric contraction determined by the quantity of actomyosin bridges in the myofibrils did not change after hypokinesia. This complex of changes is contrary to that observed in adaptation to exercise when the rate of isotonic contraction and relaxation increases while the force of isometric contraction does not change. The possible mechanism of this stability of the contractile force during adaptation and readaptation of the heart is discussed.

Stress and strain in the contractile and cytoskeletal filaments of airway smooth muscle.

PubMed

Deng, Linhong; Bosse, Ynuk; Brown, Nathan; Chin, Leslie Y M; Connolly, Sarah C; Fairbank, Nigel J; King, Greg G; Maksym, Geoffrey N; Paré, Peter D; Seow, Chun Y; Stephen, Newman L

2009-10-01

Stress and strain are omnipresent in the lung due to constant lung volume fluctuation associated with respiration, and they modulate the phenotype and function of all cells residing in the airways including the airway smooth muscle (ASM) cell. There is ample evidence that the ASM cell is very sensitive to its physical environment, and can alter its structure and/or function accordingly, resulting in either desired or undesired consequences. The forces that are either conferred to the ASM cell due to external stretching or generated inside the cell must be borne and transmitted inside the cytoskeleton (CSK). Thus, maintaining appropriate levels of stress and strain within the CSK is essential for maintaining normal function. Despite the importance, the mechanisms regulating/dysregulating ASM cytoskeletal filaments in response to stress and strain remained poorly understood until only recently. For example, it is now understood that ASM length and force are dynamically regulated, and both can adapt over a wide range of length, rendering ASM one of the most malleable living tissues. The malleability reflects the CSK's dynamic mechanical properties and plasticity, both of which strongly interact with the loading on the CSK, and all together ultimately determines airway narrowing in pathology. Here we review the latest advances in our understanding of stress and strain in ASM cells, including the organization of contractile and cytoskeletal filaments, range and adaptation of functional length, structural and functional changes of the cell in response to mechanical perturbation, ASM tone as a mediator of strain-induced responses, and the novel glassy dynamic behaviors of the CSK in relation to asthma pathophysiology.

The integrin alphav beta3 increases cellular stiffness and cytoskeletal remodeling dynamics to facilitate cancer cell invasion

NASA Astrophysics Data System (ADS)

Mierke, Claudia Tanja

2013-01-01

The process of cancer cell invasion through the extracellular matrix (ECM) of connective tissue plays a prominent role in tumor progression and is based fundamentally on biomechanics. Cancer cell invasion usually requires cell adhesion to the ECM through the cell-matrix adhesion receptors integrins. The expression of the αvβ3 integrin is increased in several tumor types and is consistently associated with increased metastasis formation in patients. The hypothesis was that the αvβ3 integrin expression increases the invasiveness of cancer cells through increased cellular stiffness, and increased cytoskeletal remodeling dynamics. Here, the invasion of cancer cells with different αvβ3 integrin expression levels into dense three-dimensional (3D) ECMs has been studied. Using a cell sorter, two subcell lines expressing either high or low amounts of αvβ3 integrins (αvβ3high or αvβ3low cells, respectively) have been isolated from parental MDA-MB-231 breast cancer cells. αvβ3high cells showed a threefold increased cell invasion compared to αvβ3low cells. Similar results were obtained for A375 melanoma, 786-O kidney and T24 bladder carcinoma cells, and cells in which the β3 integrin subunit was knocked down using specific siRNA. To investigate whether contractile forces are essential for αvβ3 integrin-mediated increased cellular stiffness and subsequently enhanced cancer cell invasion, invasion assays were performed in the presence of myosin light chain kinase inhibitor ML-7 and Rho kinase inhibitor Y27632. Indeed, cancer cell invasiveness was reduced after addition of ML-7 and Y27632 in αvβ3high cells but not in αvβ3low cells. Moreover, after addition of the contractility enhancer calyculin A, an increase in pre-stress in αvβ3low cells was observed, which enhanced cellular invasiveness. In addition, inhibition of the Src kinase, STAT3 or Rac1 strongly reduced the invasiveness of αvβ3high cells, whereas the invasiveness of β3 specific knock-down cells and αvβ3low cells was not altered. In summary, these results suggest that the αvβ3 integrin enhances cancer cell invasion through increased cellular stiffness and enhanced cytoskeletal remodeling dynamics, which enables the cells to generate and transmit contractile forces to overcome the steric hindrance of 3D ECMs.

Store-operated Ca2+ entry supports contractile function in hearts of hibernators

PubMed Central

Nakipova, Olga V.; Averin, Alexey S.; Evdokimovskii, Edward V.; Pimenov, Oleg Yu.; Kosarski, Leonid; Ignat’ev, Dmitriy; Anufriev, Andrey; Kokoz, Yuri M.; Reyes, Santiago; Terzic, Andre; Alekseev, Alexey E.

2017-01-01

Hibernators have a distinctive ability to adapt to seasonal changes of body temperature in a range between 37°C and near freezing, exhibiting, among other features, a unique reversibility of cardiac contractility. The adaptation of myocardial contractility in hibernation state relies on alterations of excitation contraction coupling, which becomes less-dependent from extracellular Ca2+ entry and is predominantly controlled by Ca2+ release from sarcoplasmic reticulum, replenished by the Ca2+-ATPase (SERCA). We found that the specific SERCA inhibitor cyclopiazonic acid (CPA), in contrast to its effect in papillary muscles (PM) from rat hearts, did not reduce but rather potentiated contractility of PM from hibernating ground squirrels (GS). In GS ventricles we identified drastically elevated, compared to rats, expression of Orai1, Stim1 and Trpc1/3/4/5/6/7 mRNAs, putative components of store operated Ca2+ channels (SOC). Trpc3 protein levels were found increased in winter compared to summer GS, yet levels of Trpc5, Trpc6 or Trpc7 remained unchanged. Under suppressed voltage-dependent K+, Na+ and Ca2+ currents, the SOC inhibitor 2-aminoethyl diphenylborinate (2-APB) diminished whole-cell membrane currents in isolated cardiomyocytes from hibernating GS, but not from rats. During cooling-reheating cycles (30°C–7°C–30°C) of ground squirrel PM, 2-APB did not affect typical CPA-sensitive elevation of contractile force at low temperatures, but precluded the contractility at 30°C before and after the cooling. Wash-out of 2-APB reversed PM contractility to control values. Thus, we suggest that SOC play a pivotal role in governing the ability of hibernator hearts to maintain their function during the transition in and out of hibernating states. PMID:28531217

Effects of temperature and calcium availability on ventricular myocardium from rainbow trout.

PubMed

Coyne, M D; Kim, C S; Cameron, J S; Gwathmey, J K

2000-06-01

We studied the mechanical and electrophysiological properties of ventricular myocardium from rainbow trout (Oncorhynchus mykiss) in vitro at 4, 10, and 18 degrees C from fish acclimated at 10 degrees C. Temperature alone did not significantly alter the contractile force of the myocardium, but the time to peak tension and time to 80% relaxation were prolonged at 4 degrees C and shortened at 18 degrees C. The duration of the action potential was also prolonged at 4 degrees C and progressively shortened at higher temperatures. An alteration of the stimulation frequency did not affect contraction amplitude at any temperature. Calcium influx via L-type calcium channels was increased by raising extracellular calcium concentration (¿Ca(2+)(o)) or including Bay K 8644 (Bay K) and isoproterenol in the bathing medium. These treatments significantly enhanced the contractile force at all temperatures. Calcium channel blockers had a reverse-negative inotropic effect. Unexpectedly, the duration of the action potential at 10 degrees C was shortened as ¿Ca(2+)(o) increased. However, Bay K prolonged the plateau phase at 4 degrees C. Caffeine, which promotes the release of sarcoplasmic reticulum (SR) calcium, increased contractile force eightfold at all three temperatures, but the SR blocker ryanodine was only inhibitory at 4 degrees C. Our results suggest that contractile force in ventricular myocardium from Oncorhynchus mykiss is primarily regulated by sarcolemmal calcium influx and that ventricular contractility is maintained during exposure to a wide range of temperatures.

The myofibroblast, multiple origins for major roles in normal and pathological tissue repair

PubMed Central

2012-01-01

Myofibroblasts differentiate, invade and repair injured tissues by secreting and organizing the extracellular matrix and by developing contractile forces. When tissues are damaged, tissue homeostasis must be re-established, and repair mechanisms have to rapidly provide harmonious mechanical tissue organization, a process essentially supported by (myo)fibroblasts. Under physiological conditions, the secretory and contractile activities of myofibroblasts are terminated when the repair is complete (scar formation) but the functionality of the tissue is only rarely perfectly restored. At the end of the normal repair process, myofibroblasts disappear by apoptosis but in pathological situations, myofibroblasts likely remain leading to excessive scarring. Myofibroblasts originate from different precursor cells, the major contribution being from local recruitment of connective tissue fibroblasts. However, local mesenchymal stem cells, bone marrow-derived mesenchymal stem cells and cells derived from an epithelial-mesenchymal transition process, may represent alternative sources of myofibroblasts when local fibroblasts are not able to satisfy the requirement for these cells during repair. These diverse cell types probably contribute to the appearance of myofibroblast subpopulations which show specific biological properties and which are important to understand in order to develop new therapeutic strategies for treatment of fibrotic and scarring diseases. PMID:23259712

Mechanics and contraction dynamics of single platelets and implications for clot stiffening

NASA Astrophysics Data System (ADS)

Lam, Wilbur A.; Chaudhuri, Ovijit; Crow, Ailey; Webster, Kevin D.; Li, Tai-De; Kita, Ashley; Huang, James; Fletcher, Daniel A.

2011-01-01

Platelets interact with fibrin polymers to form blood clots at sites of vascular injury. Bulk studies have shown clots to be active materials, with platelet contraction driving the retraction and stiffening of clots. However, neither the dynamics of single-platelet contraction nor the strength and elasticity of individual platelets, both of which are important for understanding clot material properties, have been directly measured. Here we use atomic force microscopy to measure the mechanics and dynamics of single platelets. We find that platelets contract nearly instantaneously when activated by contact with fibrinogen and complete contraction within 15 min. Individual platelets can generate an average maximum contractile force of 29 nN and form adhesions stronger than 70 nN. Our measurements show that when exposed to stiffer microenvironments, platelets generated higher stall forces, which indicates that platelets may be able to contract heterogeneous clots more uniformly. The high elasticity of individual platelets, measured to be 10 kPa after contraction, combined with their high contractile forces, indicates that clots may be stiffened through direct reinforcement by platelets as well as by strain stiffening of fibrin under tension due to platelet contraction. These results show how the mechanosensitivity and mechanics of single cells can be used to dynamically alter the material properties of physiologic systems.

Rigidity sensing explained by active matter theory.

PubMed

Marcq, Philippe; Yoshinaga, Natsuhiko; Prost, Jacques

2011-09-21

The magnitude of traction forces exerted by living animal cells on their environment is a monotonically increasing and approximately sigmoidal function of the stiffness of the external medium. We rationalize this observation using active matter theory, and propose that adaptation to substrate rigidity results from an interplay between passive elasticity and active contractility. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Tissue mechanics regulate brain development, homeostasis and disease

PubMed Central

Barnes, J. Matthew

2017-01-01

ABSTRACT All cells sense and integrate mechanical and biochemical cues from their environment to orchestrate organismal development and maintain tissue homeostasis. Mechanotransduction is the evolutionarily conserved process whereby mechanical force is translated into biochemical signals that can influence cell differentiation, survival, proliferation and migration to change tissue behavior. Not surprisingly, disease develops if these mechanical cues are abnormal or are misinterpreted by the cells – for example, when interstitial pressure or compression force aberrantly increases, or the extracellular matrix (ECM) abnormally stiffens. Disease might also develop if the ability of cells to regulate their contractility becomes corrupted. Consistently, disease states, such as cardiovascular disease, fibrosis and cancer, are characterized by dramatic changes in cell and tissue mechanics, and dysregulation of forces at the cell and tissue level can activate mechanosignaling to compromise tissue integrity and function, and promote disease progression. In this Commentary, we discuss the impact of cell and tissue mechanics on tissue homeostasis and disease, focusing on their role in brain development, homeostasis and neural degeneration, as well as in brain cancer. PMID:28043968

Stuck in gear: age-related loss of variable gearing in skeletal muscle.

PubMed

Holt, Natalie C; Danos, Nicole; Roberts, Thomas J; Azizi, Emanuel

2016-04-01

Skeletal muscles power a broad diversity of animal movements, despite only being able to produce high forces over a limited range of velocities. Pennate muscles use a range of gear ratios, the ratio of muscle shortening velocity to fiber shortening velocity, to partially circumvent these force-velocity constraints. Muscles operate with a high gear ratio at low forces; fibers rotate to greater angles of pennation, enhancing velocity but compromising force. At higher forces, muscles operate with a lower gear ratio; fibers rotate little so limiting muscle shortening velocity, but helping to preserve force. This ability to shift gears is thought to be due to the interplay of contractile force and connective tissue constraints. In order to test this hypothesis, gear ratios were determined in the medial gastrocnemius muscles of both healthy young rats, and old rats where the interaction between contractile and connective tissue properties was assumed to be disrupted. Muscle fiber and aponeurosis stiffness increased with age (P<0.05) from 19.1±5.0 kPa and 188.5±24.2 MPa, respectively, in young rats to 39.1±4.2 kPa and 328.0±48.3 MPa in old rats, indicating a mechanical change in the interaction between contractile and connective tissues. Gear ratio decreased with increasing force in young (P<0.001) but not old (P=0.72) muscles, indicating that variable gearing is lost in old muscle. These findings support the hypothesis that variable gearing results from the interaction between contractile and connective tissues and suggest novel explanations for the decline in muscle performance with age. © 2016. Published by The Company of Biologists Ltd.

Optogenetic control of RhoA reveals zyxin-mediated elasticity of stress fibres

NASA Astrophysics Data System (ADS)

Oakes, Patrick W.; Wagner, Elizabeth; Brand, Christoph A.; Probst, Dimitri; Linke, Marco; Schwarz, Ulrich S.; Glotzer, Michael; Gardel, Margaret L.

2017-06-01

Cytoskeletal mechanics regulates cell morphodynamics and many physiological processes. While contractility is known to be largely RhoA-dependent, the process by which localized biochemical signals are translated into cell-level responses is poorly understood. Here we combine optogenetic control of RhoA, live-cell imaging and traction force microscopy to investigate the dynamics of actomyosin-based force generation. Local activation of RhoA not only stimulates local recruitment of actin and myosin but also increased traction forces that rapidly propagate across the cell via stress fibres and drive increased actin flow. Surprisingly, this flow reverses direction when local RhoA activation stops. We identify zyxin as a regulator of stress fibre mechanics, as stress fibres are fluid-like without flow reversal in its absence. Using a physical model, we demonstrate that stress fibres behave elastic-like, even at timescales exceeding turnover of constituent proteins. Such molecular control of actin mechanics likely plays critical roles in regulating morphodynamic events.

Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions

PubMed Central

Swaminathan, Vinay; Kalappurakkal, Joseph Mathew; Moore, Travis I.; Koga, Nobuyasu; Baker, David A.; Oldenbourg, Rudolf; Tani, Tomomi; Springer, Timothy A.; Waterman, Clare M.

2017-01-01

Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization- or contractility-driven “retrograde flow” of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-αVβ3 integrins in fibroblasts shows that integrins are coaligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modeling, suggest that integrins in FA are coaligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues. PMID:29073038

Mechanisms of Plastic Deformation in Collagen Networks Induced by Cellular Forces.

PubMed

Ban, Ehsan; Franklin, J Matthew; Nam, Sungmin; Smith, Lucas R; Wang, Hailong; Wells, Rebecca G; Chaudhuri, Ovijit; Liphardt, Jan T; Shenoy, Vivek B

2018-01-23

Contractile cells can reorganize fibrous extracellular matrices and form dense tracts of fibers between neighboring cells. These tracts guide the development of tubular tissue structures and provide paths for the invasion of cancer cells. Here, we studied the mechanisms of the mechanical plasticity of collagen tracts formed by contractile premalignant acinar cells and fibroblasts. Using fluorescence microscopy and second harmonic generation, we quantified the collagen densification, fiber alignment, and strains that remain within the tracts after cellular forces are abolished. We explained these observations using a theoretical fiber network model that accounts for the stretch-dependent formation of weak cross-links between nearby fibers. We tested the predictions of our model using shear rheology experiments. Both our model and rheological experiments demonstrated that increasing collagen concentration leads to substantial increases in plasticity. We also considered the effect of permanent elongation of fibers on network plasticity and derived a phase diagram that classifies the dominant mechanisms of plasticity based on the rate and magnitude of deformation and the mechanical properties of individual fibers. Plasticity is caused by the formation of new cross-links if moderate strains are applied at small rates or due to permanent fiber elongation if large strains are applied over short periods. Finally, we developed a coarse-grained model for plastic deformation of collagen networks that can be employed to simulate multicellular interactions in processes such as morphogenesis, cancer invasion, and fibrosis. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

An Equatorial Contractile Mechanism Drives Cell Elongation but not Cell Division

PubMed Central

Denker, Elsa; Bhattachan, Punit; Deng, Wei; Mathiesen, Birthe T.; Jiang, Di

2014-01-01

Cell shape changes and proliferation are two fundamental strategies for morphogenesis in animal development. During embryogenesis of the simple chordate Ciona intestinalis, elongation of individual notochord cells constitutes a crucial stage of notochord growth, which contributes to the establishment of the larval body plan. The mechanism of cell elongation is elusive. Here we show that although notochord cells do not divide, they use a cytokinesis-like actomyosin mechanism to drive cell elongation. The actomyosin network forming at the equator of each notochord cell includes phosphorylated myosin regulatory light chain, α-actinin, cofilin, tropomyosin, and talin. We demonstrate that cofilin and α-actinin are two crucial components for cell elongation. Cortical flow contributes to the assembly of the actomyosin ring. Similar to cytokinetic cells, membrane blebs that cause local contractions form at the basal cortex next to the equator and participate in force generation. We present a model in which the cooperation of equatorial actomyosin ring-based constriction and bleb-associated contractions at the basal cortex promotes cell elongation. Our results demonstrate that a cytokinesis-like contractile mechanism is co-opted in a completely different developmental scenario to achieve cell shape change instead of cell division. We discuss the occurrences of actomyosin rings aside from cell division, suggesting that circumferential contraction is an evolutionally conserved mechanism to drive cell or tissue elongation. PMID:24503569

Smooth muscle architecture within cell-dense vascular tissues influences functional contractility.

PubMed

Win, Zaw; Vrla, Geoffrey D; Steucke, Kerianne E; Sevcik, Emily N; Hald, Eric S; Alford, Patrick W

2014-12-01

The role of vascular smooth muscle architecture in the function of healthy and dysfunctional vessels is poorly understood. We aimed at determining the relationship between vascular smooth muscle architecture and contractile output using engineered vascular tissues. We utilized microcontact printing and a microfluidic cell seeding technique to provide three different initial seeding conditions, with the aim of influencing the cellular architecture within the tissue. Cells seeded in each condition formed confluent and aligned tissues but within the tissues, the cellular architecture varied. Tissues with a more elongated cellular architecture had significantly elevated basal stress and produced more contractile stress in response to endothelin-1 stimulation. We also found a correlation between the contractile phenotype marker expression and the cellular architecture, contrary to our previous findings in non-confluent tissues. Taken with previous results, these data suggest that within cell-dense vascular tissues, smooth muscle contractility is strongly influenced by cell and tissue architectures.

IGF-II and IGFBP-6 regulate cellular contractility and proliferation in Dupuytren's disease.

PubMed

Raykha, Christina; Crawford, Justin; Gan, Bing Siang; Fu, Ping; Bach, Leon A; O'Gorman, David B

2013-10-01

Dupuytren's disease (DD) is a common and heritable fibrosis of the palmar fascia that typically manifests as permanent finger contractures. The molecular interactions that induce the development of hyper-contractile fibroblasts, or myofibroblasts, in DD are poorly understood. We have identified IGF2 and IGFBP6, encoding insulin-like growth factor (IGF)-II and IGF binding protein (IGFBP)-6 respectively, as reciprocally dysregulated genes and proteins in primary cells derived from contracture tissues (DD cells). Recombinant IGFBP-6 inhibited the proliferation of DD cells, patient-matched control (PF) cells and normal palmar fascia (CT) cells. Co-treatments with IGF-II, a high affinity IGFBP-6 ligand, were unable to rescue these effects. A non-IGF-II binding analog of IGFBP-6 also inhibited cellular proliferation, implicating IGF-II-independent roles for IGFBP-6 in this process. IGF-II enhanced the proliferation of CT cells, but not DD or PF cells, and significantly enhanced DD and PF cell contractility in stressed collagen lattices. While IGFBP-6 treatment did not affect cellular contractility, it abrogated the IGF-II-induced contractility of DD and PF cells in stressed collagen lattices. IGF-II also significantly increased the contraction of DD cells in relaxed lattices, however this effect was not evident in relaxed collagen lattices containing PF cells. The disparate effects of IGF-II on DD and PF cells in relaxed and stressed contraction models suggest that IGF-II can enhance lattice contractility through more than one mechanism. This is the first report to implicate IGFBP-6 as a suppressor of cellular proliferation and IGF-II as an inducer of cellular contractility in this connective tissue disease. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Acute radiation impacts contractility of guinea-pig bladder strips affecting mucosal-detrusor interactions.

PubMed

McDonnell, Bronagh M; Buchanan, Paul J; Prise, Kevin M; McCloskey, Karen D

2018-01-01

Radiation-induced bladder toxicity is associated with radiation therapy for pelvic malignancies, arising from unavoidable irradiation of neighbouring normal bladder tissue. This study aimed to investigate the acute impact of ionizing radiation on the contractility of bladder strips and identify the radiation-sensitivity of the mucosa vs the detrusor. Guinea-pig bladder strips (intact or mucosa-free) received ex vivo sham or 20Gy irradiation and were studied with in vitro myography, electrical field stimulation and Ca2+-fluorescence imaging. Frequency-dependent, neurogenic contractions in intact strips were reduced by irradiation across the force-frequency graph. The radiation-difference persisted in atropine (1μM); subsequent addition of PPADs (100μM) blocked the radiation effect at higher stimulation frequencies and decreased the force-frequency plot. Conversely, neurogenic contractions in mucosa-free strips were radiation-insensitive. Radiation did not affect agonist-evoked contractions (1μM carbachol, 5mM ATP) in intact or mucosa-free strips. Interestingly, agonist-evoked contractions were larger in irradiated mucosa-free strips vs irradiated intact strips suggesting that radiation may have unmasked an inhibitory mucosal element. Spontaneous activity was larger in control intact vs mucosa-free preparations; this difference was absent in irradiated strips. Spontaneous Ca2+-transients in smooth muscle cells within tissue preparations were reduced by radiation. Radiation affected neurogenic and agonist-evoked bladder contractions and also reduced Ca2+-signalling events in smooth muscle cells when the mucosal layer was present. Radiation eliminated a positive modulatory effect on spontaneous activity by the mucosa layer. Overall, the findings suggest that radiation impairs contractility via mucosal regulatory mechanisms independent of the development of radiation cystitis.

I-Wire Heart-on-a-Chip II: Biomechanical analysis of contractile, three-dimensional cardiomyocyte tissue constructs.

PubMed

Schroer, Alison K; Shotwell, Matthew S; Sidorov, Veniamin Y; Wikswo, John P; Merryman, W David

2017-01-15

This companion study presents the biomechanical analysis of the "I-Wire" platform using a modified Hill model of muscle mechanics that allows for further characterization of construct function and response to perturbation. The I-Wire engineered cardiac tissue construct (ECTC) is a novel experimental platform to investigate cardiac cell mechanics during auxotonic contraction. Whereas passive biomaterials often exhibit nonlinear and dissipative behavior, active tissue equivalents, such as ECTCs, also expend metabolic energy to perform mechanical work that presents additional challenges in quantifying their properties. The I-Wire model uses the passive mechanical response to increasing applied tension to measure the inherent stress and resistance to stretch of the construct before, during, and after treatments. Both blebbistatin and isoproterenol reduced prestress and construct stiffness; however, blebbistatin treatment abolished subsequent force-generating potential while isoproterenol enhanced this property. We demonstrate that the described model can replicate the response of these constructs to intrinsic changes in force-generating potential in response to both increasing frequency of stimulation and decreasing starting length. This analysis provides a useful mathematical model of the I-Wire platform, increases the number of parameters that can be derived from the device, and serves as a demonstration of quantitative characterization of nonlinear, active biomaterials. We anticipate that this quantitative analysis of I-Wire constructs will prove useful for qualifying patient-specific cardiomyocytes and fibroblasts prior to their utilization for cardiac regenerative medicine. Passive biomaterials may have non-linear elasticity and losses, but engineered muscle tissue also exhibits time- and force-dependent contractions. Historically, mathematical muscle models include series-elastic, parallel-elastic, contractile, and viscous elements. While hearts-on-a-chip can demonstrate in vitro the contractile properties of engineered cardiac constructs and their response to drugs, most of these use cellular monolayers that cannot be readily probed with controlled forces. The I-Wire platform described in the preceding paper by Sidorov et al. addresses these limitations with three-dimensional tissue constructs to which controlled forces can be applied. In this companion paper, we show how to characterize I-Wire constructs using a non-linear, active Hill model, which should be useful for qualifying cells prior to their use in cardiac regenerative medicine. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Myopathic changes in murine skeletal muscle lacking synemin

PubMed Central

García-Pelagio, Karla P.; Muriel, Joaquin; O'Neill, Andrea; Desmond, Patrick F.; Lovering, Richard M.; Lund, Linda; Bond, Meredith

2015-01-01

Diseases of striated muscle linked to intermediate filament (IF) proteins are associated with defects in the organization of the contractile apparatus and its links to costameres, which connect the sarcomeres to the cell membrane. Here we study the role in skeletal muscle of synemin, a type IV IF protein, by examining mice null for synemin (synm-null). Synm-null mice have a mild skeletal muscle phenotype. Tibialis anterior (TA) muscles show a significant decrease in mean fiber diameter, a decrease in twitch and tetanic force, and an increase in susceptibility to injury caused by lengthening contractions. Organization of proteins associated with the contractile apparatus and costameres is not significantly altered in the synm-null. Elastimetry of the sarcolemma and associated contractile apparatus in extensor digitorum longus myofibers reveals a reduction in tension consistent with an increase in sarcolemmal deformability. Although fatigue after repeated isometric contractions is more marked in TA muscles of synm-null mice, the ability of the mice to run uphill on a treadmill is similar to controls. Our results suggest that synemin contributes to linkage between costameres and the contractile apparatus and that the absence of synemin results in decreased fiber size and increased sarcolemmal deformability and susceptibility to injury. Thus synemin plays a moderate but distinct role in fast twitch skeletal muscle. PMID:25567810

Biomaterials patterned with discontinuous microwalls for vascular smooth muscle cell culture: biodegradable small diameter vascular grafts and stable cell culture substrates.

PubMed

Heath, Daniel E; Kang, Gavin C W; Cao, Ye; Poon, Yin Fun; Chan, Vincent; Chan-Park, Mary B

2016-10-01

The medial layer of small diameter blood vessels contains circumferentially aligned vascular smooth muscle cells (vSMC) that possess contractile phenotype. In tissue-engineered constructs, these cellular characteristics are usually achieved by seeding planar scaffolds with vSMC, rolling the cell-laden scaffold into a tubular structure, and maturing the construct in a pulsatile bioreactor, a lengthy process that can take up to two months. During the maturation phase, the cells circumferentially orient, their contractile protein expression increases, and they obtain a contractile phenotype. Generating cell culture platforms that enable the rapid production of directionally oriented vSMC with increased contractile protein expression would be a major step forward for blood vessel tissue engineering and would greatly facilitate the in vitro study of vSMC biology. Previously, we developed a micropatterned cell culture surface that promotes orientation and contractile protein expression of vSMC. Herein, we explore two potential applications of this technology. First, we fabricate tubular and biodegradable scaffolds that possess the micropatterning on their exterior surface. When vSMC are seeded on these scaffolds, they initially proliferate in order to fill the microchannels and as confluence is reached the cells align in the direction of the micropatterning resulting in a biodegradable scaffold that is inhabited by circumferentially aligned vSMC within a week. Second, we illustrate that we can generate biostable cell culture surfaces that allow the in vitro study of the cells in a more contractile state. Specifically, we explore contractile protein expression of cells cultured on the micropatterned surfaces with the addition of soluble transforming growth factor beta one (TGFβ1).

Reduced force of diaphragm muscle fibers in patients with chronic thromboembolic pulmonary hypertension

PubMed Central

Manders, Emmy; Bonta, Peter I.; Kloek, Jaap J.; Symersky, Petr; Bogaard, Harm-Jan; Hooijman, Pleuni E.; Jasper, Jeff R.; Malik, Fady I.; Stienen, Ger J. M.; Vonk-Noordegraaf, Anton; de Man, Frances S.

2016-01-01

Patients with pulmonary hypertension (PH) suffer from inspiratory muscle weakness. However, the pathophysiology of inspiratory muscle dysfunction in PH is unknown. We hypothesized that weakness of the diaphragm, the main inspiratory muscle, is an important contributor to inspiratory muscle dysfunction in PH patients. Our objective was to combine ex vivo diaphragm muscle fiber contractility measurements with measures of in vivo inspiratory muscle function in chronic thromboembolic pulmonary hypertension (CTEPH) patients. To assess diaphragm muscle contractility, function was studied in vivo by maximum inspiratory pressure (MIP) and ex vivo in diaphragm biopsies of the same CTEPH patients (N = 13) obtained during pulmonary endarterectomy. Patients undergoing elective lung surgery served as controls (N = 15). Muscle fiber cross-sectional area (CSA) was determined in cryosections and contractility in permeabilized muscle fibers. Diaphragm muscle fiber CSA was not significantly different between control and CTEPH patients in both slow-twitch and fast-twitch fibers. Maximal force-generating capacity was significantly lower in slow-twitch muscle fibers of CTEPH patients, whereas no difference was observed in fast-twitch muscle fibers. The maximal force of diaphragm muscle fibers correlated significantly with MIP. The calcium sensitivity of force generation was significantly reduced in fast-twitch muscle fibers of CTEPH patients, resulting in a ∼40% reduction of submaximal force generation. The fast skeletal troponin activator CK-2066260 (5 μM) restored submaximal force generation to levels exceeding those observed in control subjects. In conclusion, diaphragm muscle fiber contractility is hampered in CTEPH patients and contributes to the reduced function of the inspiratory muscles in CTEPH patients. PMID:27190061

Collective Cell Behavior in Mechanosensing of Substrate Thickness.

PubMed

Tusan, Camelia G; Man, Yu-Hin; Zarkoob, Hoda; Johnston, David A; Andriotis, Orestis G; Thurner, Philipp J; Yang, Shoufeng; Sander, Edward A; Gentleman, Eileen; Sengers, Bram G; Evans, Nicholas D

2018-06-05

Extracellular matrix stiffness has a profound effect on the behavior of many cell types. Adherent cells apply contractile forces to the material on which they adhere and sense the resistance of the material to deformation-its stiffness. This is dependent on both the elastic modulus and the thickness of the material, with the corollary that single cells are able to sense underlying stiff materials through soft hydrogel materials at low (<10 μm) thicknesses. Here, we hypothesized that cohesive colonies of cells exert more force and create more hydrogel deformation than single cells, therefore enabling them to mechanosense more deeply into underlying materials than single cells. To test this, we modulated the thickness of soft (1 kPa) elastic extracellular-matrix-functionalized polyacrylamide hydrogels adhered to glass substrates and allowed colonies of MG63 cells to form on their surfaces. Cell morphology and deformations of fluorescent fiducial-marker-labeled hydrogels were quantified by time-lapse fluorescence microscopy imaging. Single-cell spreading increased with respect to decreasing hydrogel thickness, with data fitting to an exponential model with half-maximal response at a thickness of 3.2 μm. By quantifying cell area within colonies of defined area, we similarly found that colony-cell spreading increased with decreasing hydrogel thickness but with a greater half-maximal response at 54 μm. Depth-sensing was dependent on Rho-associated protein kinase-mediated cellular contractility. Surface hydrogel deformations were significantly greater on thick hydrogels compared to thin hydrogels. In addition, deformations extended greater distances from the periphery of colonies on thick hydrogels compared to thin hydrogels. Our data suggest that by acting collectively, cells mechanosense rigid materials beneath elastic hydrogels at greater depths than individual cells. This raises the possibility that the collective action of cells in colonies or sheets may allow cells to sense structures of differing material properties at comparatively large distances. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Development and maintenance of force and stiffness in airway smooth muscle.

PubMed

Lan, Bo; Norris, Brandon A; Liu, Jeffrey C-Y; Paré, Peter D; Seow, Chun Y; Deng, Linhong

2015-03-01

Airway smooth muscle (ASM) plays a central role in the excessive narrowing of the airway that characterizes the primary functional impairment in asthma. This phenomenon is known as airway hyper-responsiveness (AHR). Emerging evidence suggests that the development and maintenance of ASM force involves dynamic reorganization of the subcellular filament network in both the cytoskeleton and the contractile apparatus. In this review, evidence is presented to support the view that regulation of ASM contraction extends beyond the classical actomyosin interaction and involves processes within the cytoskeleton and at the interfaces between the cytoskeleton, the contractile apparatus, and the extracellular matrix. These processes are initiated when the muscle is activated, and collectively they cause the cytoskeleton and the contractile apparatus to undergo structural transformation, resulting in a more connected and solid state that allows force generated by the contractile apparatus to be transmitted to the extracellular domain. Solidification of the cytoskeleton also serves to stiffen the muscle and hence the airway. Oscillatory strain from tidal breathing and deep inspiration is believed to be the counter balance that prevents hypercontraction and stiffening of ASM in vivo. Dysregulation of this balance could lead to AHR seen in asthma.

Correlating confocal microscopy and atomic force indentation reveals metastatic cancer cells stiffen during invasion into collagen I matrices

NASA Astrophysics Data System (ADS)

Staunton, Jack R.; Doss, Bryant L.; Lindsay, Stuart; Ros, Robert

2016-01-01

Mechanical interactions between cells and their microenvironment dictate cell phenotype and behavior, calling for cell mechanics measurements in three-dimensional (3D) extracellular matrices (ECM). Here we describe a novel technique for quantitative mechanical characterization of soft, heterogeneous samples in 3D. The technique is based on the integration of atomic force microscopy (AFM) based deep indentation, confocal fluorescence microscopy, finite element (FE) simulations and analytical modeling. With this method, the force response of a cell embedded in 3D ECM can be decoupled from that of its surroundings, enabling quantitative determination of the elastic properties of both the cell and the matrix. We applied the technique to the quantification of the elastic properties of metastatic breast adenocarcinoma cells invading into collagen hydrogels. We found that actively invading and fully embedded cells are significantly stiffer than cells remaining on top of the collagen, a clear example of phenotypical change in response to the 3D environment. Treatment with Rho-associated protein kinase (ROCK) inhibitor significantly reduces this stiffening, indicating that actomyosin contractility plays a major role in the initial steps of metastatic invasion.

Myosin IIA interacts with the spectrin-actin membrane skeleton to control red blood cell membrane curvature and deformability.

PubMed

Smith, Alyson S; Nowak, Roberta B; Zhou, Sitong; Giannetto, Michael; Gokhin, David S; Papoin, Julien; Ghiran, Ionita C; Blanc, Lionel; Wan, Jiandi; Fowler, Velia M

2018-05-08

The biconcave disk shape and deformability of mammalian RBCs rely on the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin-F-actin network of RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by superresolution fluorescence microscopy. MgATP disrupts NMIIA association with the membrane skeleton, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical properties. In addition, the phosphorylation of the RBC NMIIA heavy and light chains in vivo indicates active regulation of NMIIA motor activity and filament assembly, while reduced heavy chain phosphorylation of membrane skeleton-associated NMIIA indicates assembly of stable filaments at the membrane. Treatment of RBCs with blebbistatin, an inhibitor of NMII motor activity, decreases the number of NMIIA filaments associated with the membrane and enhances local, nanoscale membrane oscillations, suggesting decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deformability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disk cell shape. As structures similar to the RBC membrane skeleton exist in many metazoan cell types, these data demonstrate a general function for NMII in controlling specialized membrane morphology and mechanical properties through contractile interactions with short F-actin in spectrin-F-actin networks.

Generation of Compartmentalized Pressure by a Nuclear Piston Governs Cell Motility in 3D Matrix*

PubMed Central

Petrie, Ryan J.; Koo, Hyun; Yamada, Kenneth M.

2017-01-01

Cells use actomyosin contractility to move through three-dimensional (3D) extracellular matrix. Contractility affects the type of protrusions cells use to migrate in 3D, but the mechanisms are unclear. Here we found that contractility generated high-pressure lobopodial protrusions in cells migrating in a 3D matrix. In these cells, the nucleus physically divided the cytoplasm into forward and rear compartments. Actomyosin contractility with the nucleoskeleton-intermediate filament linker protein nesprin 3 pulled the nucleus forward and pressurized the front of the cell. Reducing expression of nesprin 3 reduced and equalized the intracellular pressure. Thus, the nucleus can act as a piston that physically compartmentalizes the cytoplasm and increases the hydrostatic pressure between the nucleus and the leading edge to drive lamellipodia-independent 3D cell migration. PMID:25170155

Model of myosin node aggregation into a contractile ring: the effect of local alignment

NASA Astrophysics Data System (ADS)

Ojkic, Nikola; Wu, Jian-Qiu; Vavylonis, Dimitrios

2011-09-01

Actomyosin bundles frequently form through aggregation of membrane-bound myosin clusters. One such example is the formation of the contractile ring in fission yeast from a broad band of cortical nodes. Nodes are macromolecular complexes containing several dozens of myosin-II molecules and a few formin dimers. The condensation of a broad band of nodes into the contractile ring has been previously described by a search, capture, pull and release (SCPR) model. In SCPR, a random search process mediated by actin filaments nucleated by formins leads to transient actomyosin connections among nodes that pull one another into a ring. The SCPR model reproduces the transport of nodes over long distances and predicts observed clump-formation instabilities in mutants. However, the model does not generate transient linear elements and meshwork structures as observed in some wild-type and mutant cells during ring assembly. As a minimal model of node alignment, we added short-range aligning forces to the SCPR model representing currently unresolved mechanisms that may involve structural components, cross-linking and bundling proteins. We studied the effect of the local node alignment mechanism on ring formation numerically. We varied the new parameters and found viable rings for a realistic range of values. Morphologically, transient structures that form during ring assembly resemble those observed in experiments with wild-type and cdc25-22 cells. Our work supports a hierarchical process of ring self-organization involving components drawn together from distant parts of the cell followed by progressive stabilization.

Matrix stiffness reverses the effect of actomyosin tension on cell proliferation.

PubMed

Mih, Justin D; Marinkovic, Aleksandar; Liu, Fei; Sharif, Asma S; Tschumperlin, Daniel J

2012-12-15

The stiffness of the extracellular matrix exerts powerful effects on cell proliferation and differentiation, but the mechanisms transducing matrix stiffness into cellular fate decisions remain poorly understood. Two widely reported responses to matrix stiffening are increases in actomyosin contractility and cell proliferation. To delineate their relationship, we modulated cytoskeletal tension in cells grown across a physiological range of matrix stiffnesses. On both synthetic and naturally derived soft matrices, and across a panel of cell types, we observed a striking reversal of the effect of inhibiting actomyosin contractility, switching from the attenuation of proliferation on rigid substrates to the robust promotion of proliferation on soft matrices. Inhibiting contractility on soft matrices decoupled proliferation from cytoskeletal tension and focal adhesion organization, but not from cell spread area. Our results demonstrate that matrix stiffness and actomyosin contractility converge on cell spreading in an unexpected fashion to control a key aspect of cell fate.

Matrix stiffness reverses the effect of actomyosin tension on cell proliferation

PubMed Central

Mih, Justin D.; Marinkovic, Aleksandar; Liu, Fei; Sharif, Asma S.; Tschumperlin, Daniel J.

2012-01-01

Summary The stiffness of the extracellular matrix exerts powerful effects on cell proliferation and differentiation, but the mechanisms transducing matrix stiffness into cellular fate decisions remain poorly understood. Two widely reported responses to matrix stiffening are increases in actomyosin contractility and cell proliferation. To delineate their relationship, we modulated cytoskeletal tension in cells grown across a physiological range of matrix stiffnesses. On both synthetic and naturally derived soft matrices, and across a panel of cell types, we observed a striking reversal of the effect of inhibiting actomyosin contractility, switching from the attenuation of proliferation on rigid substrates to the robust promotion of proliferation on soft matrices. Inhibiting contractility on soft matrices decoupled proliferation from cytoskeletal tension and focal adhesion organization, but not from cell spread area. Our results demonstrate that matrix stiffness and actomyosin contractility converge on cell spreading in an unexpected fashion to control a key aspect of cell fate. PMID:23097048

Image-based evaluation of contraction-relaxation kinetics of human-induced pluripotent stem cell-derived cardiomyocytes: Correlation and complementarity with extracellular electrophysiology.

PubMed

Hayakawa, Tomohiro; Kunihiro, Takeshi; Ando, Tomoko; Kobayashi, Seiji; Matsui, Eriko; Yada, Hiroaki; Kanda, Yasunari; Kurokawa, Junko; Furukawa, Tetsushi

2014-12-01

In this study, we used high-speed video microscopy with motion vector analysis to investigate the contractile characteristics of hiPS-CM monolayer, in addition to further characterizing the motion with extracellular field potential (FP), traction force and the Ca(2+) transient. Results of our traction force microscopy demonstrated that the force development of hiPS-CMs correlated well with the cellular deformation detected by the video microscopy with motion vector analysis. In the presence of verapamil and isoproterenol, contractile motion of hiPS-CMs showed alteration in accordance with the changes in fluorescence peak of the Ca(2+) transient, i.e., upstroke, decay, amplitude and full-width at half-maximum. Simultaneously recorded hiPS-CM motion and FP showed that there was a linear correlation between changes in the motion and field potential duration in response to verapamil (30-150nM), isoproterenol (0.1-10μM) and E-4031 (10-50nM). In addition, tetrodotoxin (3-30μM)-induced delay of sodium current was corresponded with the delay of the contraction onset of hiPS-CMs. These results indicate that the electrophysiological and functional behaviors of hiPS-CMs are quantitatively reflected in the contractile motion detected by this image-based technique. In the presence of 100nM E-4031, the occurrence of early after-depolarization-like negative deflection in FP was also detected in the hiPS-CM motion as a characteristic two-step relaxation pattern. These findings offer insights into the interpretation of the motion kinetics of the hiPS-CMs, and are relevant for understanding electrical and mechanical relationship in hiPS-CMs. Copyright © 2014. Published by Elsevier Ltd.

Aim44p regulates phosphorylation of Hof1p to promote contractile ring closure during cytokinesis in budding yeast

PubMed Central

Wolken, Dana M. Alessi; McInnes, Joseph; Pon, Liza A.

2014-01-01

Whereas actomyosin and septin ring organization and function in cytokinesis are thoroughly described, little is known regarding the mechanisms by which the actomyosin ring interacts with septins and associated proteins to coordinate cell division. Here we show that the protein product of YPL158C, Aim44p, undergoes septin-dependent recruitment to the site of cell division. Aim44p colocalizes with Myo1p, the type II myosin of the contractile ring, throughout most of the cell cycle. The Aim44p ring does not contract when the actomyosin ring closes. Instead, it forms a double ring that associates with septin rings on mother and daughter cells after cell separation. Deletion of AIM44 results in defects in contractile ring closure. Aim44p coimmunoprecipitates with Hof1p, a conserved F-BAR protein that binds both septins and type II myosins and promotes contractile ring closure. Deletion of AIM44 results in a delay in Hof1p phosphorylation and altered Hof1p localization. Finally, overexpression of Dbf2p, a kinase that phosphorylates Hof1p and is required for relocalization of Hof1p from septin rings to the contractile ring and for Hof1p-triggered contractile ring closure, rescues the cytokinesis defect observed in aim44∆ cells. Our studies reveal a novel role for Aim44p in regulating contractile ring closure through effects on Hof1p. PMID:24451263

On the origins of the universal dynamics of endogenous granules in mammalian cells.

PubMed

Vanapalli, Siva A; Li, Yixuan; Mugele, Frieder; Duits, Michel H G

2009-12-01

Endogenous granules (EGs) that consist of lipid droplets and mitochondria have been commonly used to assess intracellular mechanical properties via multiple particle tracking microrheology (MPTM). Despite their widespread use, the nature of interaction of EGs with the cytoskeletal network and the type of forces driving their dynamics--both of which are crucial for the interpretation of the results from MPTM technique--are yet to be resolved. In this report, we study the dynamics of endogenous granules in mammalian cells using particle tracking methods. We find that the ensemble dynamics of EGs is diffusive in three types of mammalian cells (endothelial cells, smooth muscle cells and fibroblasts), thereby suggesting an apparent universality in their dynamical behavior. Moreover, in a given cell, the amplitude of the mean-squared displacement for EGs is an order of magnitude larger than that of injected particles. This observation along with results from ATP depletion and temperature intervention studies suggests that cytoskeletal active forces drive the dynamics of EGs. To elucidate the dynamical origin of the diffusive-like nonthermal motion, we consider three active force generation mechanisms--molecular motor transport, actomyosin contractility and microtubule polymerization forces. We test these mechanisms using pharmacological interventions. Experimental evidence and model calculations suggest that EGs are intimately linked to microtubules and that microtubule polymerization forces drive their dynamics. Thus, endogenous granules could serve as non-invasive probes for microtubule network dynamics in mammalian cells.

Mitosis-Specific Mechanosensing and Contractile Protein Redistribution Control Cell Shape

PubMed Central

Effler, Janet C.; Kee, Yee-Seir; Berk, Jason M.; Tran, Minhchau N.; Iglesias, Pablo A.; Robinson, Douglas N.

2008-01-01

Summary Because cell division failure is deleterious, promoting tumorigenesis in mammals [1], cells utilize numerous mechanisms to control their cell-cycle progression [2–4]. Though cell division is considered a well-ordered sequence of biochemical events [5], cytokinesis, an inherently mechanical process, must also be mechanically controlled to ensure that two equivalent daughter cells are produced with high fidelity. Since cells respond to their mechanical environment [6, 7], we hypothesized that cells utilize mechanosensing and mechanical feedback to sense and correct shape asymmetries during cytokinesis. Because the mitotic spindle and myosin-II are vital to cell division [8, 9], we explored their roles in responding to shape perturbations during cell division. We demonstrate that the contractile proteins, myosin-II and cortexillin-I, redistribute in response to intrinsic and externally induced shape asymmetries. In early cytokinesis, mechanical load overrides spindle cues and slows cytokinesis progression while contractile proteins accumulate and correct shape asymmetries. In late cytokinesis, mechanical perturbation also directs contractile proteins but without apparently disrupting cytokinesis. Significantly, this response only occurs during anaphase through cytokinesis, does not require microtubules, is independent of spindle orientation, but is dependent on myosin-II. Our data provide evidence for a mechanosensory system that directs contractile proteins to regulate cell shape during mitosis. PMID:17027494

Chronic clenbuterol treatment compromises force production without directly altering skeletal muscle contractile machinery

PubMed Central

Py, G; Ramonatxo, C; Sirvent, P; Sanchez, A M J; Philippe, A G; Douillard, A; Galbès, O; Lionne, C; Bonnieu, A; Chopard, A; Cazorla, O; Lacampagne, A; Candau, R B

2015-01-01

Clenbuterol is a β2-adrenergic receptor agonist known to induce skeletal muscle hypertrophy and a slow-to-fast phenotypic shift. The aim of the present study was to test the effects of chronic clenbuterol treatment on contractile efficiency and explore the underlying mechanisms, i.e. the muscle contractile machinery and calcium-handling ability. Forty-three 6-week-old male Wistar rats were randomly allocated to one of six groups that were treated with either subcutaneous equimolar doses of clenbuterol (4 mg kg−1 day−1) or saline solution for 9, 14 or 21 days. In addition to the muscle hypertrophy, although an 89% increase in absolute maximal tetanic force (Po) was noted, specific maximal tetanic force (sPo) was unchanged or even depressed in the slow twitch muscle of the clenbuterol-treated rats (P < 0.05). The fit of muscle contraction and relaxation force kinetics indicated that clenbuterol treatment significantly reduced the rate constant of force development and the slow and fast rate constants of relaxation in extensor digitorum longus muscle (P < 0.05), and only the fast rate constant of relaxation in soleus muscle (P < 0.05). Myofibrillar ATPase activity increased in both relaxed and activated conditions in soleus (P < 0.001), suggesting that the depressed specific tension was not due to the myosin head alteration itself. Moreover, action potential-elicited Ca2+ transients in flexor digitorum brevis fibres (fast twitch fibres) from clenbuterol-treated animals demonstrated decreased amplitude after 14 days (−19%, P < 0.01) and 21 days (−25%, P < 0.01). In conclusion, we showed that chronic clenbuterol treatment reduces contractile efficiency, with altered contraction and relaxation kinetics, but without directly altering the contractile machinery. Lower Ca2+ release during contraction could partially explain these deleterious effects. PMID:25656230

Mechanobiological induction of long-range contractility by diffusing biomolecules and size scaling in cell assemblies

NASA Astrophysics Data System (ADS)

Dasbiswas, K.; Alster, E.; Safran, S. A.

2016-06-01

Mechanobiological studies of cell assemblies have generally focused on cells that are, in principle, identical. Here we predict theoretically the effect on cells in culture of locally introduced biochemical signals that diffuse and locally induce cytoskeletal contractility which is initially small. In steady-state, both the concentration profile of the signaling molecule as well as the contractility profile of the cell assembly are inhomogeneous, with a characteristic length that can be of the order of the system size. The long-range nature of this state originates in the elastic interactions of contractile cells (similar to long-range “macroscopic modes” in non-living elastic inclusions) and the non-linear diffusion of the signaling molecules, here termed mechanogens. We suggest model experiments on cell assemblies on substrates that can test the theory as a prelude to its applicability in embryo development where spatial gradients of morphogens initiate cellular development.

Mesenchymal stem cell mechanobiology and emerging experimental platforms

PubMed Central

MacQueen, Luke; Sun, Yu; Simmons, Craig A.

2013-01-01

Experimental control over progenitor cell lineage specification can be achieved by modulating properties of the cell's microenvironment. These include physical properties of the cell adhesion substrate, such as rigidity, topography and deformation owing to dynamic mechanical forces. Multipotent mesenchymal stem cells (MSCs) generate contractile forces to sense and remodel their extracellular microenvironments and thereby obtain information that directs broad aspects of MSC function, including lineage specification. Various physical factors are important regulators of MSC function, but improved understanding of MSC mechanobiology requires novel experimental platforms. Engineers are bridging this gap by developing tools to control mechanical factors with improved precision and throughput, thereby enabling biological investigation of mechanics-driven MSC function. In this review, we introduce MSC mechanobiology and review emerging cell culture platforms that enable new insights into mechanobiological control of MSCs. Our main goals are to provide engineers and microtechnology developers with an up-to-date description of MSC mechanobiology that is relevant to the design of experimental platforms and to introduce biologists to these emerging platforms. PMID:23635493

Parvalbumin Gene Transfer Impairs Skeletal Muscle Contractility in Old Mice

PubMed Central

Murphy, Kate T.; Ham, Daniel J.; Church, Jarrod E.; Naim, Timur; Trieu, Jennifer; Williams, David A.

2012-01-01

Abstract Sarcopenia is the progressive age-related loss of skeletal muscle mass associated with functional impairments that reduce mobility and quality of life. Overt muscle wasting with sarcopenia is usually preceded by a slowing of the rate of relaxation and a reduction in maximum force production. Parvalbumin (PV) is a cytosolic Ca2+ buffer thought to facilitate relaxation in muscle. We tested the hypothesis that restoration of PV levels in muscles of old mice would increase the magnitude and hasten relaxation of submaximal and maximal force responses. The tibialis anterior (TA) muscles of young (6 month), adult (13 month), and old (26 month) C57BL/6 mice received electroporation-assisted gene transfer of plasmid encoding PV or empty plasmid (pcDNA3.1). Contractile properties of TA muscles were assessed in situ 14 days after transfer. In old mice, muscles with increased PV expression had a 40% slower rate of tetanic force development (p<0.01), and maximum twitch and tetanic force were 22% and 16% lower than control values, respectively (p<0.05). Muscles with increased PV expression from old mice had an 18% lower maximum specific (normalized) force than controls, and absolute force was ∼26% lower at higher stimulation frequencies (150–300 Hz, p<0.05). In contrast, there was no effect of increased PV expression on TA muscle contractile properties in young and adult mice. The impairments in skeletal muscle function in old mice argue against PV overexpression as a therapeutic strategy for ameliorating aspects of contractile dysfunction with sarcopenia and help clarify directions for therapeutic interventions for age-related changes in skeletal muscle structure and function. PMID:22455364

Changes in force and calcium sensitivity in the developing avian heart.

PubMed

Godt, R E; Fogaça, R T; Nosek, T M

1991-11-01

The aim of this study was to characterize the development of the contractile properties of intact and chemically skinned muscle from chicken heart and to compare these characteristics with those of developing mammalian heart reported by others. Small trabeculae were dissected from left ventricles of Arbor Acre chickens between embryonic day 7 and young adulthood (7 weeks post-hatching). At all ages, increasing extracellular calcium (0.45-3.6 mM) progressively increased twitch force of electrically stimulated trabeculae. Twitch force at 1.8 mM extracellular calcium, normalized to cross-sectional area, increased to a maximum at 1 day post-hatching, remained constant through 3 weeks post-hatching, but then decreased at 7 weeks post-hatching. The maximal calcium-activated force of trabeculae chemically skinned with Triton X-100 detergent increased to a maximum 2 days before the time of hatching and was not significantly changed up to 7 weeks post-hatching. Over the ages studied, average twitch force in 1.8 mM calcium was between 26 and 66% of maximal calcium-activated force after skinning, suggesting that the contractile apparatus is not fully activated during the twitch in normal Ringer. In skinned trabeculae, the calcium sensitivity of the contractile apparatus was higher in the embryo than in the young adult. These age-dependent changes in calcium sensitivity are correlated with isoform switching in troponin T. A decrease in pH from 7.0 to 6.5 decreased the calcium sensitivity of the contractile apparatus to a greater degree in skinned trabeculae from young adult hearts than in those from embryonic hearts. This change in susceptibility to acidosis is temporally associated with isoform switching in troponin I.(ABSTRACT TRUNCATED AT 250 WORDS)

Combining cell sheet technology and electrospun scaffolding for engineered tubular, aligned, and contractile blood vessels.

PubMed

Rayatpisheh, Shahrzad; Heath, Daniel E; Shakouri, Amir; Rujitanaroj, Pim-On; Chew, Sing Yian; Chan-Park, Mary B

2014-03-01

Herein we combine cell sheet technology and electrospun scaffolding to rapidly generate circumferentially aligned tubular constructs of human aortic smooth muscles cells with contractile gene expression for use as tissue engineered blood vessel media. Smooth muscle cells cultured on micropatterned and N-isopropylacrylamide-grafted (pNIPAm) polydimethylsiloxane (PDMS), a small portion of which was covered by aligned electrospun scaffolding, resulted in a single sheet of unidirectionally aligned cells. Upon cooling to room temperature, the scaffold, its adherent cells, and the remaining cell sheet detached and were collected on a mandrel to generating tubular constructs with circumferentially aligned smooth muscle cells which possess contractile gene expression and a single layer of electrospun scaffold as an analogue to a small diameter blood vessel's internal elastic lamina (IEL). This method improves cell sheet handling, results in rapid circumferential alignment of smooth muscle cells which immediately express contractile genes, and introduction of an analogue to small diameter blood vessel IEL. Copyright © 2013 Elsevier Ltd. All rights reserved.

Repeated high-intensity exercise modulates Ca(2+) sensitivity of human skeletal muscle fibers.

PubMed

Gejl, K D; Hvid, L G; Willis, S J; Andersson, E; Holmberg, H-C; Jensen, R; Frandsen, U; Hansen, J; Plomgaard, P; Ørtenblad, N

2016-05-01

The effects of short-term high-intensity exercise on single fiber contractile function in humans are unknown. Therefore, the purposes of this study were: (a) to access the acute effects of repeated high-intensity exercise on human single muscle fiber contractile function; and (b) to examine whether contractile function was affected by alterations in the redox balance. Eleven elite cross-country skiers performed four maximal bouts of 1300 m treadmill skiing with 45 min recovery. Contractile function of chemically skinned single fibers from triceps brachii was examined before the first and following the fourth sprint with respect to Ca(2+) sensitivity and maximal Ca(2+) -activated force. To investigate the oxidative effects of exercise on single fiber contractile function, a subset of fibers was incubated with dithiothreitol (DTT) before analysis. Ca(2+) sensitivity was enhanced by exercise in both MHC I (17%, P < 0.05) and MHC II (15%, P < 0.05) fibers. This potentiation was not present after incubation of fibers with DTT. Specific force of both MHC I and MHC II fibers was unaffected by exercise. In conclusion, repeated high-intensity exercise increased Ca(2+) sensitivity in both MHC I and MHC II fibers. This effect was not observed in a reducing environment indicative of an exercise-induced oxidation of the human contractile apparatus. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Effects of N-acetylcysteine on isolated skeletal muscle contractile properties after an acute bout of aerobic exercise.

PubMed

Jannig, Paulo R; Alves, Christiano R R; Voltarelli, Vanessa A; Bozi, Luiz H M; Vieira, Janaina S; Brum, Patricia C; Bechara, Luiz R G

2017-12-15

The current study tested the hypotheses that 1) an acute bout of aerobic exercise impairs isolated skeletal muscle contractile properties and 2) N-acetylcysteine (a thiol antioxidant; NAC) administration can restore the impaired muscle contractility after exercise. At rest or immediately after an acute bout of aerobic exercise, extensor digitorum longus (EDL) and soleus muscles from male Wistar rats were harvested for ex vivo skeletal muscle contraction experiments. Muscles from exercised animals were incubated in Krebs Ringer's buffer in absence or presence of 20mM of NAC. Force capacity and fatigue properties were evaluated. Exercised EDL and soleus displayed lower force production across various stimulation frequencies (p<0.001), indicating that skeletal muscle force production was impaired after an acute bout of exercise. However, NAC treatment restored the loss of force production in both EDL and soleus after fatiguing exercise (p<0.05). Additionally, NAC treatment increased relative force production at different time points during a fatigue-induced protocol, suggesting that NAC treatment mitigates fatigue induced by successive contractions. NAC treatment improves force capacity and fatigue properties in ex vivo skeletal muscle from rats submitted to an acute bout of aerobic exercise. Copyright © 2017 Elsevier Inc. All rights reserved.

Improved throughput traction microscopy reveals pivotal role for matrix stiffness in fibroblast contractility and TGF-β responsiveness

PubMed Central

Marinković, Aleksandar; Mih, Justin D.; Park, Jin-Ah; Liu, Fei

2012-01-01

Lung fibroblast functions such as matrix remodeling and activation of latent transforming growth factor-β1 (TGF-β1) are associated with expression of the myofibroblast phenotype and are directly linked to fibroblast capacity to generate force and deform the extracellular matrix. However, the study of fibroblast force-generating capacities through methods such as traction force microscopy is hindered by low throughput and time-consuming procedures. In this study, we improved at the detail level methods for higher-throughput traction measurements on polyacrylamide hydrogels using gel-surface-bound fluorescent beads to permit autofocusing and automated displacement mapping, and transduction of fibroblasts with a fluorescent label to streamline cell boundary identification. Together these advances substantially improve the throughput of traction microscopy and allow us to efficiently compute the forces exerted by lung fibroblasts on substrates spanning the stiffness range present in normal and fibrotic lung tissue. Our results reveal that lung fibroblasts dramatically alter the forces they transmit to the extracellular matrix as its stiffness changes, with very low forces generated on matrices as compliant as normal lung tissue. Moreover, exogenous TGF-β1 selectively accentuates tractions on stiff matrices, mimicking fibrotic lung, but not on physiological stiffness matrices, despite equivalent changes in Smad2/3 activation. Taken together, these results demonstrate a pivotal role for matrix mechanical properties in regulating baseline and TGF-β1-stimulated contraction of lung fibroblasts and suggest that stiff fibrotic lung tissue may promote myofibroblast activation through contractility-driven events, whereas normal lung tissue compliance may protect against such feedback amplification of fibroblast activation. PMID:22659883

Crosstalk between MLO-Y4 osteocytes and C2C12 muscle cells is mediated by the Wnt/β-catenin pathway.

PubMed

Huang, Jian; Romero-Suarez, Sandra; Lara, Nuria; Mo, Chenglin; Kaja, Simon; Brotto, Leticia; Dallas, Sarah L; Johnson, Mark L; Jähn, Katharina; Bonewald, Lynda F; Brotto, Marco

2017-10-01

We examined the effects of osteocyte secreted factors on myogenesis and muscle function. MLO-Y4 osteocyte-like cell conditioned media (CM) (10%) increased ex vivo soleus muscle contractile force by ~25%. MLO-Y4 and primary osteocyte CM (1-10%) stimulated myogenic differentiation of C2C12 myoblasts, but 10% osteoblast CMs did not enhance C2C12 cell differentiation. Since WNT3a and WNT1 are secreted by osteocytes, and the expression level of Wnt3a is increased in MLO-Y4 cells by fluid flow shear stress, both were compared, showing WNT3a more potent than WNT1 in inducing myogenesis. Treatment of C2C12 myoblasts with WNT3a at concentrations as low as 0.5ng/mL mirrored the effects of both primary osteocyte and MLO-Y4 CM by inducing nuclear translocation of β-catenin with myogenic differentiation, suggesting that Wnts might be potential factors secreted by osteocytes that signal to muscle cells. Knocking down Wnt3a in MLO-Y4 osteocytes inhibited the effect of CM on C2C12 myogenic differentiation. Sclerostin (100ng/mL) inhibited both the effects of MLO-Y4 CM and WNT3a on C2C12 cell differentiation. RT-PCR array results supported the activation of the Wnt/β-catenin pathway by MLO-Y4 CM and WNT3a. These results were confirmed by qPCR showing up-regulation of myogenic markers and two Wnt/β-catenin downstream genes, Numb and Flh1 . We postulated that MLO-Y4 CM/WNT3a could modulate intracellular calcium homeostasis as the trigger mechanism for the enhanced myogenesis and contractile force. MLO-Y4 CM and WNT3a increased caffeine-induced Ca 2+ release from the sarcoplasmic reticulum (SR) of C2C12 myotubes and the expression of genes directly associated with intracellular Ca 2+ signaling and homeostasis. Together, these data show that in vitro and ex vivo , osteocytes can stimulate myogenesis and enhance muscle contractile function and suggest that Wnts could be mediators of bone to muscle signaling, likely via modulation of intracellular Ca 2+ signaling and the Wnt/β-Catenin pathway.

Force Exertion and Transmission in Cross-Linked Actin Networks

NASA Astrophysics Data System (ADS)

Stam, Samantha

Cells are responsive to external cues in their environment telling them to proliferate or migrate within their surrounding tissue. Sensing of cues that are mechanical in nature, such stiffness of a tissue or forces transmitted from other cells, is believed to involve the cytoskeleton of a cell. The cytoskeleton is a complex network of proteins consisting of polymers that provide structural support, motor proteins that remodel these structures, and many others. We do not yet have a complete understanding of how cytoskeletal components respond to either internal or external mechanical force and stiffness. Such an understanding should involve mechanisms by which constituent molecules, such as motor proteins, are responsive to mechanics. Additionally, physical models of how forces are transmitted through biopolymer networks are necessary. My research has focused on networks formed by the cytoskeletal filament actin and the molecular motor protein myosin II. Actin filaments form networks and bundles that form a structural framework of the cell, and myosin II slides actin filaments. In this thesis, we show that stiffness of an elastic load that opposes myosin-generated actin sliding has a very sharp effect on the myosin force output in simulations. Secondly, we show that the stiffness and connectivity of cytoskeletal filaments regulates the contractility and anisotropy of network deformations that transmit force on material length scales. Together, these results have implications for predicting and interpreting the deformations and forces in biopolymeric active materials.

A node organization in the actomyosin contractile ring generates tension and aids stability.

PubMed

Thiyagarajan, Sathish; Wang, Shuyuan; O'Shaughnessy, Ben

2017-11-07

During cytokinesis, a contractile actomyosin ring constricts and divides the cell in two. How the ring marshals actomyosin forces to generate tension is not settled. Recently, a superresolution microscopy study of the fission yeast ring revealed that myosins and formins that nucleate actin filaments colocalize in plasma membrane-anchored complexes called nodes in the constricting ring. The nodes move bidirectionally around the ring. Here we construct and analyze a coarse-grained mathematical model of the fission yeast ring to explore essential consequences of the recently discovered ring ultrastructure. The model reproduces experimentally measured values of ring tension, explains why nodes move bidirectionally, and shows that tension is generated by myosin pulling on barbed-end-anchored actin filaments in a stochastic sliding-filament mechanism. This mechanism is not based on an ordered sarcomeric organization. We show that the ring is vulnerable to intrinsic contractile instabilities, and protection from these instabilities and organizational homeostasis require both component turnover and anchoring of components to the plasma membrane. © 2017 Thiyagarajan, Wang, and O’Shaughnessy. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Effect of Aging on Tongue Protrusion Forces in Rats

PubMed Central

Nagai, Hiromi; Russell, John A.; Jackson, Michelle A.

2010-01-01

The purpose of this study was to ascertain the effect of aging on muscle contractile properties associated with tongue protrusion in a rat model. Fischer 344/Brown Norway hybrid rats, ten young (9 months old) and ten old (32 months old), were used to measure protrusive contractile properties. Results showed a significant reduction in tetanic forces in the old animals. The following measures of muscle contraction were not different between age groups: mean twitch contraction force, twitch contraction time, twitch contraction half-decay time, and a calculated measure of fatigability. In conclusion, aging influenced protrusive tongue muscle contractions in a rat model such that tetanic forces were reduced. The reduction of tetanus force may parallel findings in human subjects relative to isometric tongue force generation and may be associated with age-related disorders of swallowing. PMID:17694408

Direct measurement of Vorticella contraction force by micropipette deflection.

PubMed

France, Danielle; Tejada, Jonathan; Matsudaira, Paul

2017-02-01

The ciliated protozoan Vorticella convallaria is noted for its exceptionally fast adenosine triphosphate-independent cellular contraction, but direct measurements of contractile force have proven difficult given the length scale, speed, and forces involved. We used high-speed video microscopy to image live Vorticella stalled in midcontraction by deflection of an attached micropipette. Stall forces correlate with both distance contracted and the resting stalk length. Estimated isometric forces range from 95 to 177 nanonewtons (nN), or 1.12 nN·μm -1 of the stalk. Maximum velocity and work are also proportional to distance contracted. These parameters constrain proposed biochemical/physical models of the contractile mechanism. © Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Reconstitution of Contractile FtsZ Rings in Liposomes

PubMed Central

Osawa, Masaki; Anderson, David E.; Erickson, Harold P.

2009-01-01

FtsZ is a tubulin homolog and the major cytoskeletal protein in bacterial cell division. It assembles into the Z ring, which contains FtsZ and a dozen other division proteins, and constricts to divide the cell. We have constructed a membrane-targeted FtsZ (FtsZ-mts) by splicing an amphipathic helix to its C terminus. When mixed with lipid vesicles, FtsZ-mts was incorporated into the interior of some tubular vesicles. There it formed multiple Z rings that could move laterally in both directions along the length of the liposome and coalesce into brighter Z rings. Brighter Z rings produced visible constrictions in the liposome, suggesting that FtsZ itself can assemble the Z ring and generate a force. No other proteins were needed for assembly and force generation. PMID:18420899

Smitin, a novel smooth muscle titin-like protein, interacts with myosin filaments in vivo and in vitro.

PubMed

Kim, Kyoungtae; Keller, Thomas C S

2002-01-07

Smooth muscle cells use an actin-myosin II-based contractile apparatus to produce force for a variety of physiological functions, including blood pressure regulation and gut peristalsis. The organization of the smooth muscle contractile apparatus resembles that of striated skeletal and cardiac muscle, but remains much more poorly understood. We have found that avian vascular and visceral smooth muscles contain a novel, megadalton protein, smitin, that is similar to striated muscle titin in molecular morphology, localization in a contractile apparatus, and ability to interact with myosin filaments. Smitin, like titin, is a long fibrous molecule with a globular domain on one end. Specific reactivities of an anti-smitin polyclonal antibody and an anti-titin monoclonal antibody suggest that smitin and titin are distinct proteins rather than differentially spliced isoforms encoded by the same gene. Smitin immunofluorescently colocalizes with myosin in chicken gizzard smooth muscle, and interacts with two configurations of smooth muscle myosin filaments in vitro. In physiological ionic strength conditions, smitin and smooth muscle myosin coassemble into irregular aggregates containing large sidepolar myosin filaments. In low ionic strength conditions, smitin and smooth muscle myosin form highly ordered structures containing linear and polygonal end-to-end and side-by-side arrays of small bipolar myosin filaments. We have used immunogold localization and sucrose density gradient cosedimentation analyses to confirm association of smitin with both the sidepolar and bipolar smooth muscle myosin filaments. These findings suggest that the titin-like protein smitin may play a central role in organizing myosin filaments in the contractile apparatus and perhaps in other structures in smooth muscle cells.

Regional differences in actomyosin contraction shape the primary vesicles in the embryonic chicken brain

NASA Astrophysics Data System (ADS)

Filas, Benjamen A.; Oltean, Alina; Majidi, Shabnam; Bayly, Philip V.; Beebe, David C.; Taber, Larry A.

2012-12-01

In the early embryo, the brain initially forms as a relatively straight, cylindrical epithelial tube composed of neural stem cells. The brain tube then divides into three primary vesicles (forebrain, midbrain, hindbrain), as well as a series of bulges (rhombomeres) in the hindbrain. The boundaries between these subdivisions have been well studied as regions of differential gene expression, but the morphogenetic mechanisms that generate these constrictions are not well understood. Here, we show that regional variations in actomyosin-based contractility play a major role in vesicle formation in the embryonic chicken brain. In particular, boundaries did not form in brains exposed to the nonmuscle myosin II inhibitor blebbistatin, whereas increasing contractile force using calyculin or ATP deepened boundaries considerably. Tissue staining showed that contraction likely occurs at the inner part of the wall, as F-actin and phosphorylated myosin are concentrated at the apical side. However, relatively little actin and myosin was found in rhombomere boundaries. To determine the specific physical mechanisms that drive vesicle formation, we developed a finite-element model for the brain tube. Regional apical contraction was simulated in the model, with contractile anisotropy and strength estimated from contractile protein distributions and measurements of cell shapes. The model shows that a combination of circumferential contraction in the boundary regions and relatively isotropic contraction between boundaries can generate realistic morphologies for the primary vesicles. In contrast, rhombomere formation likely involves longitudinal contraction between boundaries. Further simulations suggest that these different mechanisms are dictated by regional differences in initial morphology and the need to withstand cerebrospinal fluid pressure. This study provides a new understanding of early brain morphogenesis.

Computation of forces from deformed visco-elastic biological tissues

NASA Astrophysics Data System (ADS)

Muñoz, José J.; Amat, David; Conte, Vito

2018-04-01

We present a least-squares based inverse analysis of visco-elastic biological tissues. The proposed method computes the set of contractile forces (dipoles) at the cell boundaries that induce the observed and quantified deformations. We show that the computation of these forces requires the regularisation of the problem functional for some load configurations that we study here. The functional measures the error of the dynamic problem being discretised in time with a second-order implicit time-stepping and in space with standard finite elements. We analyse the uniqueness of the inverse problem and estimate the regularisation parameter by means of an L-curved criterion. We apply the methodology to a simple toy problem and to an in vivo set of morphogenetic deformations of the Drosophila embryo.

Actomyosin drives cancer cell nuclear dysmorphia and threatens genome stability.

PubMed

Takaki, Tohru; Montagner, Marco; Serres, Murielle P; Le Berre, Maël; Russell, Matt; Collinson, Lucy; Szuhai, Karoly; Howell, Michael; Boulton, Simon J; Sahai, Erik; Petronczki, Mark

2017-07-24

Altered nuclear shape is a defining feature of cancer cells. The mechanisms underlying nuclear dysmorphia in cancer remain poorly understood. Here we identify PPP1R12A and PPP1CB, two subunits of the myosin phosphatase complex that antagonizes actomyosin contractility, as proteins safeguarding nuclear integrity. Loss of PPP1R12A or PPP1CB causes nuclear fragmentation, nuclear envelope rupture, nuclear compartment breakdown and genome instability. Pharmacological or genetic inhibition of actomyosin contractility restores nuclear architecture and genome integrity in cells lacking PPP1R12A or PPP1CB. We detect actin filaments at nuclear envelope rupture sites and define the Rho-ROCK pathway as the driver of nuclear damage. Lamin A protects nuclei from the impact of actomyosin activity. Blocking contractility increases nuclear circularity in cultured cancer cells and suppresses deformations of xenograft nuclei in vivo. We conclude that actomyosin contractility is a major determinant of nuclear shape and that unrestrained contractility causes nuclear dysmorphia, nuclear envelope rupture and genome instability.

Automated Video-Based Analysis of Contractility and Calcium Flux in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes Cultured over Different Spatial Scales.

PubMed

Huebsch, Nathaniel; Loskill, Peter; Mandegar, Mohammad A; Marks, Natalie C; Sheehan, Alice S; Ma, Zhen; Mathur, Anurag; Nguyen, Trieu N; Yoo, Jennie C; Judge, Luke M; Spencer, C Ian; Chukka, Anand C; Russell, Caitlin R; So, Po-Lin; Conklin, Bruce R; Healy, Kevin E

2015-05-01

Contractile motion is the simplest metric of cardiomyocyte health in vitro, but unbiased quantification is challenging. We describe a rapid automated method, requiring only standard video microscopy, to analyze the contractility of human-induced pluripotent stem cell-derived cardiomyocytes (iPS-CM). New algorithms for generating and filtering motion vectors combined with a newly developed isogenic iPSC line harboring genetically encoded calcium indicator, GCaMP6f, allow simultaneous user-independent measurement and analysis of the coupling between calcium flux and contractility. The relative performance of these algorithms, in terms of improving signal to noise, was tested. Applying these algorithms allowed analysis of contractility in iPS-CM cultured over multiple spatial scales from single cells to three-dimensional constructs. This open source software was validated with analysis of isoproterenol response in these cells, and can be applied in future studies comparing the drug responsiveness of iPS-CM cultured in different microenvironments in the context of tissue engineering.

Actomyosin drives cancer cell nuclear dysmorphia and threatens genome stability

PubMed Central

Takaki, Tohru; Montagner, Marco; Serres, Murielle P.; Le Berre, Maël; Russell, Matt; Collinson, Lucy; Szuhai, Karoly; Howell, Michael; Boulton, Simon J.; Sahai, Erik; Petronczki, Mark

2017-01-01

Altered nuclear shape is a defining feature of cancer cells. The mechanisms underlying nuclear dysmorphia in cancer remain poorly understood. Here we identify PPP1R12A and PPP1CB, two subunits of the myosin phosphatase complex that antagonizes actomyosin contractility, as proteins safeguarding nuclear integrity. Loss of PPP1R12A or PPP1CB causes nuclear fragmentation, nuclear envelope rupture, nuclear compartment breakdown and genome instability. Pharmacological or genetic inhibition of actomyosin contractility restores nuclear architecture and genome integrity in cells lacking PPP1R12A or PPP1CB. We detect actin filaments at nuclear envelope rupture sites and define the Rho-ROCK pathway as the driver of nuclear damage. Lamin A protects nuclei from the impact of actomyosin activity. Blocking contractility increases nuclear circularity in cultured cancer cells and suppresses deformations of xenograft nuclei in vivo. We conclude that actomyosin contractility is a major determinant of nuclear shape and that unrestrained contractility causes nuclear dysmorphia, nuclear envelope rupture and genome instability. PMID:28737169

Age-related peculiarities of contractile activity of rat myocardium during blockade of hyperpolarization-activated currents.

PubMed

Zefirov, T L; Gibina, A E; Sergejeva, A M; Ziyatdinova, N I; Zefirov, A L

2007-09-01

Contractile activity of atrial and ventricular myocardial strips isolated from rats of various age was examined under conditions of blockade of non-selective hyperpolarization-activated cation currents. Addition of ZD7288, a blocker of non-selective hyperpolarization-activated cation currents, to the perfusion solution increased the contraction force of atrial and ventricular strips in 1-, 8-, and 20-week rats, but produced an opposite effect on contractile activity of atrial and ventricular strips in 3-week rats.

Intracellular pressure is a motive force for cell motion in Amoeba proteus.

PubMed

Yanai, M; Kenyon, C M; Butler, J P; Macklem, P T; Kelly, S M

1996-01-01

The cortical filament layer of free-living amoebae contains concentrated actomyosin, suggesting that it can contract and produce an internal hydrostatic pressure. We report here on direct and dynamic intracellular pressure (P(ic)) measurements in Amoeba proteus made using the servo-null technique. In resting apolar A. proteus, P(ic) increased while the cells remained immobile and at apparently constant volume. P(ic) then decreased approximately coincident with pseudopod formation. There was a positive correlation between P(ic) at the onset of movement and the rate of pseudopod formation. These results are the first direct evidence that hydrostatic pressure may be a motive force for cell motion. We postulate that contractile elements in the amoeba's cortical layer contract and increase P(ic) and that this P(ic) is utilized to overcome the viscous flow resistance of the intracellular contents during pseudopod formation.

A Vertex Model of Drosophila Ventral Furrow Formation

PubMed Central

Spahn, Philipp; Reuter, Rolf

2013-01-01

Ventral furrow formation in Drosophila is an outstanding model system to study the mechanisms involved in large-scale tissue rearrangements. Ventral cells accumulate myosin at their apical sides and, while being tightly coupled to each other via apical adherens junctions, execute actomyosin contractions that lead to reduction of their apical cell surface. Thereby, a band of constricted cells along the ventral epithelium emerges which will form a tissue indentation along the ventral midline (the ventral furrow). Here we adopt a 2D vertex model to simulate ventral furrow formation in a surface view allowing easy comparison with confocal live-recordings. We show that in order to reproduce furrow morphology seen in vivo, a gradient of contractility must be assumed in the ventral epithelium which renders cells more contractile the closer they lie to the ventral midline. The model predicts previous experimental findings, such as the gain of eccentric morphology of constricting cells and an incremental fashion of apical cell area reduction. Analysis of the model suggests that this incremental area reduction is caused by the dynamical interplay of cell elasticity and stochastic contractility as well as by the opposing forces from contracting neighbour cells. We underpin results from the model through in vivo analysis of ventral furrow formation in wildtype and twi mutant embryos. Our results show that ventral furrow formation can be accomplished as a “tug-of-war” between stochastically contracting, mechanically coupled cells and may require less rigorous regulation than previously thought. Summary For the developmental biologist it is a fascinating question how cells can coordinate major tissue movements during embryonic development. The so-called ventral furrow of the Drosophila embryo is a well-studied example of such a process when cells from a ventral band, spanning nearly the entire length of the embryo, undergo dramatic shape change by contracting their tips and then fold inwards into the interior of the embryo. Although numerous genes have been identified that are critical for ventral furrow formation, it is an open question how cells work together to elicit this tissue rearrangement. We use a computational model to mimic the physical properties of cells in the ventral epithelium and simulate the formation of the furrow. We find that the ventral furrow can form through stochastic self-organisation and that previous experimental observations can be readily explained in our model by considering forces that arise when cells execute contractions while being coupled to each other in a mechanically coherent epithelium. The model highlights the importance of a physical perspective when studying tissue morphogenesis and shows that only a minimal genetic regulation may be required to drive complex processes in embryonic development. PMID:24066163

Differential effects of peroxynitrite on contractile protein properties in fast- and slow-twitch skeletal muscle fibers of rat.

PubMed

Dutka, T L; Mollica, J P; Lamb, G D

2011-03-01

Oxidative modification of contractile proteins is thought to be a key factor in muscle weakness observed in many pathophysiological conditions. In particular, peroxynitrite (ONOO(-)), a potent short-lived oxidant, is a likely candidate responsible for this contractile dysfunction. In this study ONOO(-) or 3-morpholinosydnonimine (Sin-1, a ONOO(-) donor) was applied to rat skinned muscle fibers to characterize the effects on contractile properties. Both ONOO(-) and Sin-1 exposure markedly reduced maximum force in slow-twitch fibers but had much less effect in fast-twitch fibers. The rate of isometric force development was also reduced without change in the number of active cross bridges. Sin-1 exposure caused a disproportionately large decrease in Ca(2+) sensitivity, evidently due to coproduction of superoxide, as it was prevented by Tempol, a superoxide dismutase mimetic. The decline in maximum force with Sin-1 and ONOO(-) treatments could be partially reversed by DTT, provided it was applied before the fiber was activated. Reversal by DTT indicates that the decrease in maximum force was due at least in part to oxidation of cysteine residues. Ascorbate caused similar reversal, further suggesting that the cysteine residues had undergone S-nitrosylation. The reduction in Ca(2+) sensitivity, however, was not reversed by either DTT or ascorbate. Western blot analysis showed cross-linking of myosin heavy chain (MHC) I, appearing as larger protein complexes after ONOO(-) exposure. The findings suggest that ONOO(-) initially decreases maximum force primarily by oxidation of cysteine residues on the myosin heads, and that the accompanying decrease in Ca(2+) sensitivity is likely due to other, less reversible actions of hydroxyl or related radicals.

Development of a Cyclic Strain Bioreactor for Mechanical Enhancement and Assessment of Bioengineered Myocardial Constructs

PubMed Central

Salazar, Betsy H.; Cashion, Avery T.; Dennis, Robert G.; Birla, Ravi K.

2015-01-01

Purpose The purpose of this study was to develop enabling bioreactor technologies using a novel voice coil actuator system for investigating the effects of periodic strain on cardiac patches fabricated with rat cardiomyocytes. Methods The bioengineered muscle constructs used in this study were formed by culturing rat neonatal primary cardiac cells on a fibrin gel. The physical design of the bioreactor was initially conceived using Solidworks to test clearances and perform structural strain analysis. Once the software design phase was completed the bioreactor was assembled using a combination of commercially available, custom machined, and 3-D printed parts. We utilized the bioreactor to evaluate the effect of a 4-hour stretch protocol on the contractile properties of the tissue after which immunohistological assessment of the tissue was also performed. Results An increase in contractile force was observed after the strain protocol of 10% stretch at 1Hz, with no significant increase observed in the control group. Additionally, an increase in cardiac myofibril alignment, connexin 43 expression, and collagen type I distribution were noted. Conclusion In this study we demonstrated the effectiveness of a new bioreactor design to improve contractility of engineered cardiac muscle tissue. PMID:26577484

Development of a Cyclic Strain Bioreactor for Mechanical Enhancement and Assessment of Bioengineered Myocardial Constructs.

PubMed

Salazar, Betsy H; Cashion, Avery T; Dennis, Robert G; Birla, Ravi K

2015-12-01

The purpose of this study was to develop enabling bioreactor technologies using a novel voice coil actuator system for investigating the effects of periodic strain on cardiac patches fabricated with rat cardiomyocytes. The bioengineered muscle constructs used in this study were formed by culturing rat neonatal primary cardiac cells on a fibrin gel. The physical design of the bioreactor was initially conceived using Solidworks to test clearances and perform structural strain analysis. Once the software design phase was completed the bioreactor was assembled using a combination of commercially available, custom machined, and 3-D printed parts. We utilized the bioreactor to evaluate the effect of a 4-h stretch protocol on the contractile properties of the tissue after which immunohistological assessment of the tissue was also performed. An increase in contractile force was observed after the strain protocol of 10% stretch at 1 Hz, with no significant increase observed in the control group. Additionally, an increase in cardiac myofibril alignment, connexin 43 expression, and collagen type I distribution were noted. In this study we demonstrated the effectiveness of a new bioreactor design to improve contractility of engineered cardiac muscle tissue.

Contractile actin cables induced by Bacillus anthracis lethal toxin depend on the histone acetylation machinery.

PubMed

Rolando, Monica; Stefani, Caroline; Doye, Anne; Acosta, Maria I; Visvikis, Orane; Yevick, Hannah G; Buchrieser, Carmen; Mettouchi, Amel; Bassereau, Patricia; Lemichez, Emmanuel

2015-10-01

It remains a challenge to decode the molecular basis of the long-term actin cytoskeleton rearrangements that are governed by the reprogramming of gene expression. Bacillus anthracis lethal toxin (LT) inhibits mitogen-activated protein kinase (MAPK) signaling, thereby modulating gene expression, with major consequences for actin cytoskeleton organization and the loss of endothelial barrier function. Using a laser ablation approach, we characterized the contractile and tensile mechanical properties of LT-induced stress fibers. These actin cables resist pulling forces that are transmitted at cell-matrix interfaces and at cell-cell discontinuous adherens junctions. We report that treating the cells with trichostatin A (TSA), a broad range inhibitor of histone deacetylases (HDACs), or with MS-275, which targets HDAC1, 2 and 3, induces stress fibers. LT decreased the cellular levels of HDAC1, 2 and 3 and reduced the global HDAC activity in the nucleus. Both the LT and TSA treatments induced Rnd3 expression, which is required for the LT-mediated induction of actin stress fibers. Furthermore, we reveal that treating the LT-intoxicated cells with garcinol, an inhibitor of histone acetyl-transferases (HATs), disrupts the stress fibers and limits the monolayer barrier dysfunctions. These data demonstrate the importance of modulating the flux of protein acetylation in order to control actin cytoskeleton organization and the endothelial cell monolayer barrier. © 2015 Wiley Periodicals, Inc.

[Subcellular basis of disorders of the contractile capacity of the heart in L-thyroxine-induced thyrotoxicosis].

PubMed

Karsanov, N V; Melashvili, N O; Khugashvili, Z G; Mamulashvili, L D; Azrumelashvili, M I; Khaindrava, G K; Kapanadze, R V

1990-02-01

In experiments on dogs, the authors examined the functional activity of three cardiomyocyte systems responsible for contraction-relaxation (the systems of contractile proteins, calcium transport and energy supply) in the dynamics of L-thyroxine-induced toxicosis. A fall in the capacity of the contractile protein system to generate energy and to perform was shown to play the leading role in decrease of myocardial reserve forces and reduction in cardiac contractility. There was a drop in the intensity of calcium transport through the membranes of the sarcoplasmic reticulum and mitochondria and a deficiency of the direct energy source for contraction only in the late period of the disease.

Continuum mechanical model for cross-linked actin networks with contractile bundles

NASA Astrophysics Data System (ADS)

Ferreira, J. P. S.; Parente, M. P. L.; Natal Jorge, R. M.

2018-01-01

In the context of a mechanical approach to cell biology, there is a close relationship between cellular function and mechanical properties. In recent years, an increasing amount of attention has been given to the coupling between biochemical and mechanical signals by means of constitutive models. In particular, on the active contractility of the actin cytoskeleton. Given the importance of the actin contraction on the physiological functions, this study propose a constitutive model to describe how the filamentous network controls its mechanics actively. Embedded in a soft isotropic ground substance, the network behaves as a viscous mechanical continuum, comprised of isotropically distributed cross-linked actin filaments and actomyosin bundles. Trough virtual rheometry experiments, the present model relates the dynamics of the myosin motors with the network stiffness, which is to a large extent governed by the time-scale of the applied deformations/forces.

Cerebrospinal fluid from subarachnoid haemorrhage patients causes excessive oxidative metabolism compared to vascular smooth muscle force generation.

PubMed

Pyne, G J; Cadoux-Hudson, T A; Clark, J F

2001-01-01

Cerebrospinal fluid (CSF) from subarachnoid haemorrhage (SAH) patients can stimulate vascular smooth muscle to generate force in vitro. CSF from SAH patients suffering from delayed ischaemic neurological deficits due to cerebral vasospasm can generate near maximal force in vitro and previous experiments have ascribed this generation of force to be a calcium mediated event. The intracellular calcium concentration has been demonstrated to rise during the vasospastic process. Calcium also stimulates oxidative metabolism as does adenosine diphosphate (ADP), the product of adenosine triphosphate (ATP) hydrolysis. Significant alteration in high energy metabolites such as ATP, ADP and phosphocreatine have also been demonstrated in various models of SAH mediated vasospasm. Vascular smooth muscle predominantly uses oxidative metabolism for force generation and reserves glycolytic metabolism for ion homeostasis. A decrease in oxidative metabolism during force generation would imply failing mitochondria and increased glycolytic high-energy phosphate supply. Increased oxidative metabolism would imply a decreased efficiency of the contractile apparatus or mitochondria. The aim of this study was to see if SAH CSF stimulation of porcine carotid artery oxidative metabolism was altered during force generation when compared with incremental calcium stimulation with potassium chloride depolarisation. CSF from patients (n = 10) who had subarachnoid haemorrhage stimulated force generation but with a significant 'right shift' in oxygen consumption. This 'right shift' is indicative of an increased energy cost for contractile work. These results suggest that vascular smooth muscle contractile apparatus, when stimulated by subarachnoid cerebrospinal fluid, is consuming excess adenosine triphosphate during force generation.

Accuracy of gastrocnemius muscles forces in walking and running goats predicted by one-element and two-element Hill-type models.

PubMed

Lee, Sabrina S M; Arnold, Allison S; Miara, Maria de Boef; Biewener, Andrew A; Wakeling, James M

2013-09-03

Hill-type models are commonly used to estimate muscle forces during human and animal movement-yet the accuracy of the forces estimated during walking, running, and other tasks remains largely unknown. Further, most Hill-type models assume a single contractile element, despite evidence that faster and slower motor units, which have different activation-deactivation dynamics, may be independently or collectively excited. This study evaluated a novel, two-element Hill-type model with "differential" activation of fast and slow contractile elements. Model performance was assessed using a comprehensive data set (including measures of EMG intensity, fascicle length, and tendon force) collected from the gastrocnemius muscles of goats during locomotor experiments. Muscle forces predicted by the new two-element model were compared to the forces estimated using traditional one-element models and to the forces measured in vivo using tendon buckle transducers. Overall, the two-element model resulted in the best predictions of in vivo gastrocnemius force. The coefficient of determination, r(2), was up to 26.9% higher and the root mean square error, RMSE, was up to 37.4% lower for the two-element model than for the one-element models tested. All models captured salient features of the measured muscle force during walking, trotting, and galloping (r(2)=0.26-0.51), and all exhibited some errors (RMSE=9.63-32.2% of the maximum in vivo force). These comparisons provide important insight into the accuracy of Hill-type models. The results also show that incorporation of fast and slow contractile elements within muscle models can improve estimates of time-varying, whole muscle force during locomotor tasks. Copyright © 2013 Elsevier Ltd. All rights reserved.

Accuracy of gastrocnemius muscles forces in walking and running goats predicted by one-element and two-element Hill-type models

PubMed Central

Lee, Sabrina S.M.; Arnold, Allison S.; Miara, Maria de Boef; Biewener, Andrew A.; Wakeling, James M.

2013-01-01

Hill-type models are commonly used to estimate muscle forces during human and animal movement —yet the accuracy of the forces estimated during walking, running, and other tasks remains largely unknown. Further, most Hill-type models assume a single contractile element, despite evidence that faster and slower motor units, which have different activation-deactivation dynamics, may be independently or collectively excited. This study evaluated a novel, two-element Hill-type model with “differential” activation of fast and slow contractile elements. Model performance was assessed using a comprehensive data set (including measures of EMG intensity, fascicle length, and tendon force) collected from the gastrocnemius muscles of goats during locomotor experiments. Muscle forces predicted by the new two-element model were compared to the forces estimated using traditional one-element models and to the forces measured in vivo using tendon buckle transducers. Overall, the two-element model resulted in the best predictions of in vivo gastrocnemius force. The coefficient of determination, r2, was up to 26.9% higher and the root mean square error, RMSE, was up to 37.4% lower for the two-element model than for the one-element models tested. All models captured salient features of the measured muscle force during walking, trotting, and galloping (r2 = 0.26 to 0.51), and all exhibited some errors (RMSE = 9.63 to 32.2% of the maximum in vivo force). These comparisons provide important insight into the accuracy of Hill-type models. The results also show that incorporation of fast and slow contractile elements within muscle models can improve estimates of time-varying, whole muscle force during locomotor tasks. PMID:23871235

Coordination of contractility, adhesion and flow in migrating Physarum amoebae.

PubMed

Lewis, Owen L; Zhang, Shun; Guy, Robert D; del Álamo, Juan C

2015-05-06

This work examines the relationship between spatio-temporal coordination of intracellular flow and traction stress and the speed of amoeboid locomotion of microplasmodia of Physarum polycephalum. We simultaneously perform particle image velocimetry and traction stress microscopy to measure the velocity of cytoplasmic flow and the stresses applied to the substrate by migrating Physarum microamoebae. In parallel, we develop a mathematical model of a motile cell which includes forces from the viscous cytosol, a poro-elastic, contractile cytoskeleton and adhesive interactions with the substrate. Our experiments show that flow and traction stress exhibit back-to-front-directed waves with a distinct phase difference. The model demonstrates that the direction and speed of locomotion are determined by this coordination between contraction, flow and adhesion. Using the model, we identify forms of coordination that generate model predictions consistent with experiments. We demonstrate that this coordination produces near optimal migration speed and is insensitive to heterogeneity in substrate adhesiveness. While it is generally thought that amoeboid motility is robust to changes in extracellular geometry and the nature of extracellular adhesion, our results demonstrate that coordination of adhesive forces is essential to producing robust migration. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

Automated Video-Based Analysis of Contractility and Calcium Flux in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes Cultured over Different Spatial Scales

PubMed Central

Huebsch, Nathaniel; Loskill, Peter; Mandegar, Mohammad A.; Marks, Natalie C.; Sheehan, Alice S.; Ma, Zhen; Mathur, Anurag; Nguyen, Trieu N.; Yoo, Jennie C.; Judge, Luke M.; Spencer, C. Ian; Chukka, Anand C.; Russell, Caitlin R.; So, Po-Lin

2015-01-01

Contractile motion is the simplest metric of cardiomyocyte health in vitro, but unbiased quantification is challenging. We describe a rapid automated method, requiring only standard video microscopy, to analyze the contractility of human-induced pluripotent stem cell-derived cardiomyocytes (iPS-CM). New algorithms for generating and filtering motion vectors combined with a newly developed isogenic iPSC line harboring genetically encoded calcium indicator, GCaMP6f, allow simultaneous user-independent measurement and analysis of the coupling between calcium flux and contractility. The relative performance of these algorithms, in terms of improving signal to noise, was tested. Applying these algorithms allowed analysis of contractility in iPS-CM cultured over multiple spatial scales from single cells to three-dimensional constructs. This open source software was validated with analysis of isoproterenol response in these cells, and can be applied in future studies comparing the drug responsiveness of iPS-CM cultured in different microenvironments in the context of tissue engineering. PMID:25333967

Effect of spaceflight on skeletal muscle: Mechanical properties and myosin isoform content of a slow muscle

NASA Technical Reports Server (NTRS)

Caiozzo, Vincent J.; Baker, Michael J.; Herrick, Robert E.; Tao, Ming; Baldwin, Kenneth M.

1994-01-01

This study examined changes in contractile, biochemical, and histochemical properties of slow antigravity skeletal muscle after a 6-day spaceflight mission. Twelve male Sprague-Dawley rats were randomly divided into two groups: flight and ground-based control. Approximately 3 h after the landing, in situ contractile measurements were made on the soleus muscles of the flight animals. The control animals were studied 24 h later. The contractile measurements included force-velocity relationship, force-frequency relationship, and fatigability. Biochemical measurements focused on the myosin heavy chain (MHC) and myosin light chain profiles. Adenosinetriphosphatase histochemistry was performed to identify cross-sectional area of slow and fast muscle fibers and to determine the percent fiber type distribution. The force-velocity relationships of the flight muscles were altered such that maximal isometric tension P(sub o) was decreased by 24% and maximal shortening velocity was increased by 14% (P less than 0.05). The force-frequency relationship of the flight muscles was shifted to the right of the control muscles. At the end of the 2-min fatigue test, the flight muscles generated only 34% of P(sub o), whereas the control muscles generated 64% of P(sub o). The flight muscles exhibited de novo expression of the type IIx MHC isoform as well as a slight decrease in the slow type I and fast type IIa MHC isoforms. Histochemical analyses of flight muscles demonstrated a small increase in the percentage of fast type II fibers and a greater atrophy of the slow type I fibers. The results demonstrate that contractile properties of slow antigravity skeletal muscle are sensitive to the microgravity environment and that changes begin to occur within the 1st wk. These changes were at least, in part, associated with changes in the amount and type of contractile protein expressed.

Fasudil inhibits the proliferation and contractility and induces cell cycle arrest and apoptosis of human endometriotic stromal cells: a promising agent for the treatment of endometriosis.

PubMed

Tsuno, Akitoshi; Nasu, Kaei; Kawano, Yukie; Yuge, Akitoshi; Li, Haili; Abe, Wakana; Narahara, Hisashi

2011-12-01

During the development of endometriotic lesions, excess fibrosis may lead to scarring and to the alterations of tissue function that are the characteristic features of this disease. Enhanced extracellular matrix contractility of endometriotic stromal cells (ECSC) mediated by the mevalonate-Ras homology (Rho)/Rho-associated coiled-coil-forming protein kinase (ROCK) pathway has been shown to contribute to the pathogenesis of endometriosis. To assess the use of fasudil, a selective ROCK inhibitor, for the medical treatment of endometriosis-associated fibrosis, the effects of this agent on the cell proliferation, apoptosis, cell cycle, morphology, cell density, and contractility of ECSC were investigated. The effects of fasudil on the expression of contractility-related, apoptosis-related, and cell cycle-related molecules in ECSC were also evaluated. Fasudil significantly inhibited the proliferation and contractility of ECSC and induced the cell cycle arrest in the G2/M phase and apoptosis of these cells. Morphological observation revealed the suppression of ECSC attachment to collagen fibers and decrease of cell density by fasudil. The expression of α-smooth muscle actin, RhoA, ROCK-I, and ROCK-II proteins was inhibited by fasudil administration. The expression of the antiapoptotic factors, Bcl-2 and Bcl-X(L), in two-dimensional cultured ECSC were down-regulated by the addition of fasudil, whereas, the expression of p16(INK4a) and p21(Waf1/Cip1) was up-regulated by the addition of fasudil. The present findings suggest that fasudil is a promising agent for the treatment of endometriosis. The inhibition of cell proliferation, contractility, and myofibroblastic differentiation, the attenuation of attachment to collagen fibers, the decrease of cell density, and the induction of cell cycle arrest and apoptosis of ECSC are involved in the active mechanisms of fasudil.

Border Forces and Friction Control Epithelial Closure Dynamics

PubMed Central

Cochet-Escartin, Olivier; Ranft, Jonas; Silberzan, Pascal; Marcq, Philippe

2014-01-01

We study the closure dynamics of a large number of well-controlled circular apertures within an epithelial monolayer, where the collective cell migration responsible for epithelization is triggered by the removal of a spatial constraint rather than by scratching. Based on experimental observations, we propose a physical model that takes into account border forces, friction with the substrate, and tissue rheology. Border protrusive activity drives epithelization despite the presence of a contractile actomyosin cable at the periphery of the wound. The closure dynamics is quantified by an epithelization coefficient, defined as the ratio of protrusive stress to tissue-substrate friction, that allows classification of different phenotypes. The same analysis demonstrates a distinct signature for human cells bearing the oncogenic RasV12 mutation, demonstrating the potential of the approach to quantitatively characterize metastatic transformations. PMID:24411238

Altered calcium handling and increased contraction force in human embryonic stem cell derived cardiomyocytes following short term dexamethasone exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kosmidis, Georgios; Bellin, Milena; Ribeiro, Marcelo C.

One limitation in using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) for disease modeling and cardiac safety pharmacology is their immature functional phenotype compared with adult cardiomyocytes. Here, we report that treatment of human embryonic stem cell derived cardiomyocytes (hESC-CMs) with dexamethasone, a synthetic glucocorticoid, activated glucocorticoid signaling which in turn improved their calcium handling properties and contractility. L-type calcium current and action potential properties were not affected by dexamethasone but significantly faster calcium decay, increased forces of contraction and sarcomeric lengths, were observed in hESC-CMs after dexamethasone exposure. Activating the glucocorticoid pathway can thus contribute to mediating hPSC-CMs maturation.more » - Highlights: • Dexamethasone accelerates Ca{sup 2+} transient decay in hESC-CMs. • Dexamethasone enhances SERCA and NCX function in hESC-CMs. • Dexamethasone increases force of contraction and sarcomere length in hESC-CMs. • Dexamethasone does not alter I{sub Ca,L} and action potential characteristics in hESC-CMs.« less

Quantification of surface tension and internal pressure generated by single mitotic cells

NASA Astrophysics Data System (ADS)

Fischer-Friedrich, Elisabeth; Hyman, Anthony A.; Jülicher, Frank; Müller, Daniel J.; Helenius, Jonne

2014-08-01

During mitosis, adherent cells round up, by increasing the tension of the contractile actomyosin cortex while increasing the internal hydrostatic pressure. In the simple scenario of a liquid cell interior, the surface tension is related to the local curvature and the hydrostatic pressure difference by Laplace's law. However, verification of this scenario for cells requires accurate measurements of cell shape. Here, we use wedged micro-cantilevers to uniaxially confine single cells and determine confinement forces while concurrently determining cell shape using confocal microscopy. We fit experimentally measured confined cell shapes to shapes obeying Laplace's law with uniform surface tension and find quantitative agreement. Geometrical parameters derived from fitting the cell shape, and the measured force were used to calculate hydrostatic pressure excess and surface tension of cells. We find that HeLa cells increase their internal hydrostatic pressure excess and surface tension from ~ 40 Pa and 0.2 mNm-1 during interphase to ~ 400 Pa and 1.6 mNm-1 during metaphase. The method introduced provides a means to determine internal pressure excess and surface tension of rounded cells accurately and with minimal cellular perturbation, and should be applicable to characterize the mechanical properties of various cellular systems.

Molecular determinants of force production in human skeletal muscle fibers: effects of myosin isoform expression and cross-sectional area.

PubMed

Miller, Mark S; Bedrin, Nicholas G; Ades, Philip A; Palmer, Bradley M; Toth, Michael J

2015-03-15

Skeletal muscle contractile performance is governed by the properties of its constituent fibers, which are, in turn, determined by the molecular interactions of the myofilament proteins. To define the molecular determinants of contractile function in humans, we measured myofilament mechanics during maximal Ca(2+)-activated and passive isometric conditions in single muscle fibers with homogenous (I and IIA) and mixed (I/IIA and IIA/X) myosin heavy chain (MHC) isoforms from healthy, young adult male (n = 5) and female (n = 7) volunteers. Fibers containing only MHC II isoforms (IIA and IIA/X) produced higher maximal Ca(2+)-activated forces over the range of cross-sectional areas (CSAs) examined than MHC I fibers, resulting in higher (24-42%) specific forces. The number and/or stiffness of the strongly bound myosin-actin cross bridges increased in the higher force-producing MHC II isoforms and, in all isoforms, better predicted force than CSA. In men and women, cross-bridge kinetics, in terms of myosin attachment time and rate of myosin force production, were independent of CSA, although women had faster (7-15%) kinetics. The relative proportion of cross bridges and/or their stiffness was reduced as fiber size increased, causing a decline in specific force. Results from our examination of molecular mechanisms across the range of physiological CSAs explain the variation in specific force among the different fiber types in human skeletal muscle, which may have relevance to understanding how various physiological and pathophysiological conditions modulate single-fiber and whole muscle contractility. Copyright © 2015 the American Physiological Society.

Molecular determinants of force production in human skeletal muscle fibers: effects of myosin isoform expression and cross-sectional area

PubMed Central

Bedrin, Nicholas G.; Ades, Philip A.; Palmer, Bradley M.; Toth, Michael J.

2015-01-01

Skeletal muscle contractile performance is governed by the properties of its constituent fibers, which are, in turn, determined by the molecular interactions of the myofilament proteins. To define the molecular determinants of contractile function in humans, we measured myofilament mechanics during maximal Ca2+-activated and passive isometric conditions in single muscle fibers with homogenous (I and IIA) and mixed (I/IIA and IIA/X) myosin heavy chain (MHC) isoforms from healthy, young adult male (n = 5) and female (n = 7) volunteers. Fibers containing only MHC II isoforms (IIA and IIA/X) produced higher maximal Ca2+-activated forces over the range of cross-sectional areas (CSAs) examined than MHC I fibers, resulting in higher (24–42%) specific forces. The number and/or stiffness of the strongly bound myosin-actin cross bridges increased in the higher force-producing MHC II isoforms and, in all isoforms, better predicted force than CSA. In men and women, cross-bridge kinetics, in terms of myosin attachment time and rate of myosin force production, were independent of CSA, although women had faster (7–15%) kinetics. The relative proportion of cross bridges and/or their stiffness was reduced as fiber size increased, causing a decline in specific force. Results from our examination of molecular mechanisms across the range of physiological CSAs explain the variation in specific force among the different fiber types in human skeletal muscle, which may have relevance to understanding how various physiological and pathophysiological conditions modulate single-fiber and whole muscle contractility. PMID:25567808

Extracellular UDP enhances P2X-mediated bladder smooth muscle contractility via P2Y6 activation of the phospholipase C/inositol trisphosphate pathway

PubMed Central

Yu, Weiqun; Sun, Xiaofeng; Robson, Simon C.; Hill, Warren G.

2013-01-01

Bladder dysfunction characterized by abnormal bladder smooth muscle (BSM) contractions is pivotal to the disease process in overactive bladder, urge incontinence, and spinal cord injury. Purinergic signaling comprises one key pathway in modulating BSM contractility, but molecular mechanisms remain unclear. Here we demonstrate, using myography, that activation of P2Y6 by either UDP or a specific agonist (MRS 2693) induced a sustained increase in BSM tone (up to 2 mN) in a concentration-dependent manner. Notably, activation of P2Y6 enhanced ATP-mediated BSM contractile force by up to 45%, indicating synergistic interactions between P2X and P2Y signaling. P2Y6-activated responses were abolished by phospholipase C (PLC) and inositol trisphosphate (IP3) receptor antagonists U73122 and xestospongin C, demonstrating involvement of the PLC/IP3 signal pathway. Mice null for Entpd1, an ectonucleotidase on BSM, demonstrated increased force generation on P2Y6 activation (150%). Thus, in vivo perturbations to purinergic signaling resulted in altered P2Y6 activity and bladder contractility. We conclude that UDP, acting on P2Y6, regulates BSM tone and in doing so selectively maximizes P2X1-mediated contraction forces. This novel neurotransmitter pathway may play an important role in urinary voiding disorders characterized by abnormal bladder motility.—Yu, W., Sun, X., Robson, S. C., Hill, W. G. Extracellular UDP enhances P2X-mediated bladder smooth muscle contractility via P2Y6 activation of the phospholipase C/inositol trisphosphate pathway. PMID:23362118

MRCK-1 drives apical constriction in C. elegans by linking developmental patterning to force generation

PubMed Central

Marston, Daniel J.; Higgins, Christopher D.; Peters, Kimberly A.; Cupp, Timothy D.; Dickinson, Daniel J.; Pani, Ariel M.; Moore, Regan P.; Cox, Amanda H.; Kiehart, Daniel P.; Goldstein, Bob

2016-01-01

Summary Apical constriction is a change in cell shape that drives key morphogenetic events including gastrulation and neural tube formation. Apical force-producing actomyosin networks drive apical constriction by contracting while connected to cell-cell junctions. The mechanisms by which developmental patterning regulates these actomyosin networks and associated junctions with spatial precision are not fully understood. Here, we identify a myosin light chain kinase MRCK-1 as a key regulator of C. elegans gastrulation that integrates spatial and developmental patterning information. We show that MRCK-1 is required for activation of contractile actomyosin dynamics and elevated cortical tension in the apical cell cortex of endodermal precursor cells. MRCK-1 is apically localized by active Cdc42 at the external, cell-cell contact-free surfaces of apically constricting cells, downstream of cell fate determination mechanisms. We establish that the junctional components α-catenin, β-catenin, and cadherin become highly enriched at the apical junctions of apically-constricting cells, and that MRCK-1 and myosin activity are required in vivo for this enrichment. Taken together, our results define mechanisms that position a myosin activator to a specific cell surface where it both locally increases cortical tension and locally enriches junctional components to facilitate apical constriction. These results reveal crucial links that can tie spatial information to local force generation to drive morphogenesis. PMID:27451898

Increased Ca2+ sensitivity of contractile elements via protein kinase C in alpha-toxin permeabilized SMA from young spontaneously hypertensive rats.

PubMed

Sasajima, H; Shima, H; Toyoda, Y; Kimura, K; Yoshikawa, A; Hano, T; Nishio, I

1997-10-01

The purpose of the present investigation was to examine the Ca2+ sensitivity of the contractile elements via protein kinase C (PKC) in superior mesenteric artery (SMA) from young (5-6 weeks old) spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Staphylococcal aureus alpha-toxin, which produces pores in the plasma membrane too small to allow passage of proteins such as PKC, was used to investigate the signal transduction system in vascular smooth muscle cells. We investigated the Ca2+ sensitivity of the contractile apparatus via PKC in intact and alpha-toxin skinned SMA from young SHR and WKY. In intact SMA, high K+ responses were not different between SHR and WKY. However, phorbol 12,13-dibutyrate (PDBu, a PKC activator) augmented high K(+)-evoked contractions and PKC inhibitors, such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and calphostin C, suppressed them more in SHR as compared with WKY. In alpha-toxin skinned SMA, the [Ca2+]i-force relationship curve was not significantly different between SHR and WKY. However, PDBu augmented [Ca2+]i-evoked contractions and PKC inhibitors suppressed them more in SHR than in WKY. These results suggest that the Ca2+ sensitivity of the contractile elements via PKC is significantly greater in prehypertensive SHR than in age-matched WKY. This abnormality in small muscular arteries may be involved in the pathogenesis of hypertension in SHR.

Born to run: creating the muscle fiber.

PubMed

Schejter, Eyal D; Baylies, Mary K

2010-10-01

From the muscles that control the blink of your eye to those that allow you to walk, the basic architecture of muscle is the same: muscles consist of bundles of the unit muscle cell, the muscle fiber. The unique morphology of the individual muscle fiber is dictated by the functional demands necessary to generate and withstand the forces of contraction, which in turn leads to movement. Contractile muscle fibers are elongated, syncytial cells, which interact with both the nervous and skeletal systems to govern body motion. In this review, we focus on three key cell-cell and cell-matrix contact processes, that are necessary to create this exquisitely specialized cell: cell fusion, cell elongation, and establishment of a myotendinous junction. We address these processes by highlighting recent findings from the Drosophila model system. Copyright © 2010 Elsevier Ltd. All rights reserved.

Use of arginine-glycine-aspartic acid adhesion peptides coupled with a new collagen scaffold to engineer a myocardium-like tissue graft.

PubMed

Schussler, O; Coirault, C; Louis-Tisserand, M; Al-Chare, W; Oliviero, P; Menard, C; Michelot, R; Bochet, P; Salomon, D R; Chachques, J C; Carpentier, A; Lecarpentier, Y

2009-03-01

Cardiac tissue engineering might be useful in treatment of diseased myocardium or cardiac malformations. The creation of functional, biocompatible contractile tissues, however, remains challenging. We hypothesized that coupling of arginine-glycine-aspartic acid-serine (RGD+) adhesion peptides would improve cardiomyocyte viability and differentiation and contractile performance of collagen-cell scaffolds. Clinically approved collagen scaffolds were functionalized with RGD+ cells and seeded with cardiomyocytes. Contractile performance, cardiomyocyte viability and differentiation were analyzed at days 1 and 8 and/or after culture for 1 month. The method used for the RGD+ cell-collagen scaffold coupling enabled the following features: high coupling yields and complete washout of excess reagent and by-products with no need for chromatography; spectroscopic quantification of RGD+ coupling; a spacer arm of 36 A, a length reported as optimal for RGD+-peptide presentation and favorable for integrin-receptor clustering and subsequent activation. Isotonic and isometric mechanical parameters, either spontaneous or electrostimulated, exhibited good performance in RGD+ constructs. Cell number and viability was increased in RGD+ scaffolds, and we saw good organization of cell contractile apparatus with occurrence of cross-striation. We report a novel method of engineering a highly effective collagen-cell scaffold based on RGD+ peptides cross-linked to a clinically approved collagen matrix. The main advantages were cell contractile performance, cardiomyocyte viability and differentiation.

Eigenstrain as a mechanical set-point of cells.

PubMed

Lin, Shengmao; Lampi, Marsha C; Reinhart-King, Cynthia A; Tsui, Gary; Wang, Jian; Nelson, Carl A; Gu, Linxia

2018-02-05

Cell contraction regulates how cells sense their mechanical environment. We sought to identify the set-point of cell contraction, also referred to as tensional homeostasis. In this work, bovine aortic endothelial cells (BAECs), cultured on substrates with different stiffness, were characterized using traction force microscopy (TFM). Numerical models were developed to provide insights into the mechanics of cell-substrate interactions. Cell contraction was modeled as eigenstrain which could induce isometric cell contraction without external forces. The predicted traction stresses matched well with TFM measurements. Furthermore, our numerical model provided cell stress and displacement maps for inspecting the fundamental regulating mechanism of cell mechanosensing. We showed that cell spread area, traction force on a substrate, as well as the average stress of a cell were increased in response to a stiffer substrate. However, the cell average strain, which is cell type-specific, was kept at the same level regardless of the substrate stiffness. This indicated that the cell average strain is the tensional homeostasis that each type of cell tries to maintain. Furthermore, cell contraction in terms of eigenstrain was found to be the same for both BAECs and fibroblast cells in different mechanical environments. This implied a potential mechanical set-point across different cell types. Our results suggest that additional measurements of contractility might be useful for monitoring cell mechanosensing as well as dynamic remodeling of the extracellular matrix (ECM). This work could help to advance the understanding of the cell-ECM relationship, leading to better regenerative strategies.

Cytoskeletal Role in the Contractile Dysfunction of Hypertrophied Myocardium

NASA Astrophysics Data System (ADS)

Tsutsui, Hiroyuki; Ishihara, Kazuaki; Cooper, George

1993-04-01

Cardiac hypertrophy in response to systolic pressure loading frequently results in contractile dysfunction of unknown cause. In the present study, pressure loading increased the microtubule component of the cardiac muscle cell cytoskeleton, which was responsible for the cellular contractile dysfunction observed. The linked microtubule and contractile abnormalities were persistent and thus may have significance for the deterioration of initially compensatory cardiac hypertrophy into congestive heart failure.

PTP1B triggers integrin-mediated repression of myosin activity and modulates cell contractility

PubMed Central

González Wusener, Ana E.; González, Ángela; Nakamura, Fumihiko; Arregui, Carlos O.

2016-01-01

ABSTRACT Cell contractility and migration by integrins depends on precise regulation of protein tyrosine kinase and Rho-family GTPase activities in specific spatiotemporal patterns. Here we show that protein tyrosine phosphatase PTP1B cooperates with β3 integrin to activate the Src/FAK signalling pathway which represses RhoA-myosin-dependent contractility. Using PTP1B null (KO) cells and PTP1B reconstituted (WT) cells, we determined that some early steps following cell adhesion to fibronectin and vitronectin occurred robustly in WT cells, including aggregation of β3 integrins and adaptor proteins, and activation of Src/FAK-dependent signalling at small puncta in a lamellipodium. However, these events were significantly impaired in KO cells. We established that cytoskeletal strain and cell contractility was highly enhanced at the periphery of KO cells compared to WT cells. Inhibition of the Src/FAK signalling pathway or expression of constitutive active RhoA in WT cells induced a KO cell phenotype. Conversely, expression of constitutive active Src or myosin inhibition in KO cells restored the WT phenotype. We propose that this novel function of PTP1B stimulates permissive conditions for adhesion and lamellipodium assembly at the protruding edge during cell spreading and migration. PMID:26700725

Loss of Gα12/13 exacerbates apical area dependence of actomyosin contractility

PubMed Central

Xie, Shicong; Mason, Frank M.; Martin, Adam C.

2016-01-01

During development, coordinated cell shape changes alter tissue shape. In the Drosophila ventral furrow and other epithelia, apical constriction of hundreds of epithelial cells folds the tissue. Genes in the Gα12/13 pathway coordinate collective apical constriction, but the mechanism of coordination is poorly understood. Coupling live-cell imaging with a computational approach to identify contractile events, we discovered that differences in constriction behavior are biased by initial cell shape. Disrupting Gα12/13 exacerbates this relationship. Larger apical area is associated with delayed initiation of contractile pulses, lower apical E-cadherin and F-actin levels, and aberrantly mobile Rho-kinase structures. Our results suggest that loss of Gα12/13 disrupts apical actin cortex organization and pulse initiation in a size-dependent manner. We propose that Gα12/13 robustly organizes the apical cortex despite variation in apical area to ensure the timely initiation of contractile pulses in a tissue with heterogeneity in starting cell shape. PMID:27489340

Protective effects of anisodamine on cigarette smoke extract-induced airway smooth muscle cell proliferation and tracheal contractility

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Guang-Ni; Yang, Kai; Xu, Zu-Peng

2012-07-01

Anisodamine, an antagonist of muscarinic acetylcholine receptors (mAChRs), has been used therapeutically to improve smooth muscle function, including microvascular, intestinal and airway spasms. Our previous studies have revealed that airway hyper-reactivity could be prevented by anisodamine. However, whether anisodamine prevents smoking-induced airway smooth muscle (ASM) cell proliferation remained unclear. In this study, a primary culture of rat ASM cells was used to evaluate an ASM phenotype through the ability of the cells to proliferate and express contractile proteins in response to cigarette smoke extract (CSE) and intervention of anisodamine. Our results showed that CSE resulted in an increase in cyclinmore » D1 expression concomitant with the G0/G1-to-S phase transition, and high expression of M2 and M3. Functional studies showed that tracheal hyper-contractility accompanied contractile marker α-SMA high-expression. These changes, which occur only after CSE stimulation, were prevented and reversed by anisodamine, and CSE-induced cyclin D1 expression was significantly inhibited by anisodamine and the specific inhibitor U0126, BAY11-7082 and LY294002. Thus, we concluded that the protective and reversal effects and mechanism of anisodamine on CSE-induced events might involve, at least partially, the ERK, Akt and NF-κB signaling pathways associated with cyclin D1 via mAChRs. Our study validated that anisodamine intervention on ASM cells may contribute to anti-remodeling properties other than bronchodilation. -- Highlights: ► CSE induces tracheal cell proliferation, hyper-contractility and α-SMA expression. ► Anisodamine reverses CSE-induced tracheal hyper-contractility and cell proliferation. ► ERK, PI3K, and NF-κB pathways and cyclin D1 contribute to the reversal effect.« less

Directional Bleb Formation in Spherical Cells under Temperature Gradient

PubMed Central

Oyama, Kotaro; Arai, Tomomi; Isaka, Akira; Sekiguchi, Taku; Itoh, Hideki; Seto, Yusuke; Miyazaki, Makito; Itabashi, Takeshi; Ohki, Takashi; Suzuki, Madoka; Ishiwata, Shin'ichi

2015-01-01

Living cells sense absolute temperature and temporal changes in temperature using biological thermosensors such as ion channels. Here, we reveal, to our knowledge, a novel mechanism of sensing spatial temperature gradients within single cells. Spherical mitotic cells form directional membrane extensions (polar blebs) under sharp temperature gradients (≥∼0.065°C μm−1; 1.3°C temperature difference within a cell), which are created by local heating with a focused 1455-nm laser beam under an optical microscope. On the other hand, multiple nondirectional blebs are formed under gradual temperature gradients or uniform heating. During heating, the distribution of actomyosin complexes becomes inhomogeneous due to a break in the symmetry of its contractile force, highlighting the role of the actomyosin complex as a sensor of local temperature gradients. PMID:26200871

Dietary nitrate increases tetanic [Ca2+]i and contractile force in mouse fast-twitch muscle

PubMed Central

Hernández, Andrés; Schiffer, Tomas A; Ivarsson, Niklas; Cheng, Arthur J; Bruton, Joseph D; Lundberg, Jon O; Weitzberg, Eddie; Westerblad, Håkan

2012-01-01

Dietary inorganic nitrate has profound effects on health and physiological responses to exercise. Here, we examined if nitrate, in doses readily achievable via a normal diet, could improve Ca2+ handling and contractile function using fast- and slow-twitch skeletal muscles from C57bl/6 male mice given 1 mm sodium nitrate in water for 7 days. Age matched controls were provided water without added nitrate. In fast-twitch muscle fibres dissected from nitrate treated mice, myoplasmic free [Ca2+] was significantly greater than in Control fibres at stimulation frequencies from 20 to 150 Hz, which resulted in a major increase in contractile force at ≤50 Hz. At 100 Hz stimulation, the rate of force development was ∼35% faster in the nitrate group. These changes in nitrate treated mice were accompanied by increased expression of the Ca2+ handling proteins calsequestrin 1 and the dihydropyridine receptor. No changes in force or calsequestrin 1 and dihydropyridine receptor expression were measured in slow-twitch muscles. In conclusion, these results show a striking effect of nitrate supplementation on intracellular Ca2+ handling in fast-twitch muscle resulting in increased force production. A new mechanism is revealed by which nitrate can exert effects on muscle function with applications to performance and a potential therapeutic role in conditions with muscle weakness. PMID:22687611

Dietary nitrate increases tetanic [Ca2+]i and contractile force in mouse fast-twitch muscle.

PubMed

Hernández, Andrés; Schiffer, Tomas A; Ivarsson, Niklas; Cheng, Arthur J; Bruton, Joseph D; Lundberg, Jon O; Weitzberg, Eddie; Westerblad, Håkan

2012-08-01

Dietary inorganic nitrate has profound effects on health and physiological responses to exercise. Here, we examined if nitrate, in doses readily achievable via a normal diet, could improve Ca(2+) handling and contractile function using fast- and slow-twitch skeletal muscles from C57bl/6 male mice given 1 mm sodium nitrate in water for 7 days. Age matched controls were provided water without added nitrate. In fast-twitch muscle fibres dissected from nitrate treated mice, myoplasmic free [Ca(2+)] was significantly greater than in Control fibres at stimulation frequencies from 20 to 150 Hz, which resulted in a major increase in contractile force at ≤ 50 Hz. At 100 Hz stimulation, the rate of force development was ∼35% faster in the nitrate group. These changes in nitrate treated mice were accompanied by increased expression of the Ca(2+) handling proteins calsequestrin 1 and the dihydropyridine receptor. No changes in force or calsequestrin 1 and dihydropyridine receptor expression were measured in slow-twitch muscles. In conclusion, these results show a striking effect of nitrate supplementation on intracellular Ca(2+) handling in fast-twitch muscle resulting in increased force production. A new mechanism is revealed by which nitrate can exert effects on muscle function with applications to performance and a potential therapeutic role in conditions with muscle weakness.

Fropofol decreases force development in cardiac muscle.

PubMed

Ren, Xianfeng; Schmidt, William; Huang, Yiyuan; Lu, Haisong; Liu, Wenjie; Bu, Weiming; Eckenhoff, Roderic; Cammarato, Anthony; Gao, Wei Dong

2018-03-09

Supranormal contractile properties are frequently associated with cardiac diseases. Anesthetic agents, including propofol, can depress myocardial contraction. We tested the hypothesis that fropofol, a propofol derivative, reduces force development in cardiac muscles via inhibition of cross-bridge cycling and may therefore have therapeutic potential. Force and intracellular Ca 2+ ([Ca 2+ ] i ) transients of rat trabecular muscles were determined. Myofilament ATPase, actin-activated myosin ATPase, and velocity of actin filaments propelled by myosin were also measured. Fropofol dose dependently decreased force without altering [Ca 2+ ] i in normal and pressure-induced hypertrophied-hypercontractile muscles. Similarly, fropofol depressed maximum Ca 2+ -activated force ( F max ) and increased the [Ca 2+ ] i required for 50% activation at steady-state (Ca 50 ) without affecting the Hill coefficient in both intact and skinned cardiac fibers. The drug also depressed cardiac myofibrillar and actin-activated myosin ATPase activity. In vitro actin sliding velocity was significantly reduced when fropofol was introduced during rigor binding of cross-bridges. The data suggest that the depressing effects of fropofol on cardiac contractility are likely to be related to direct targeting of actomyosin interactions. From a clinical standpoint, these findings are particularly significant, given that fropofol is a nonanesthetic small molecule that decreases myocardial contractility specifically and thus may be useful in the treatment of hypercontractile cardiac disorders.-Ren, X., Schmidt, W., Huang, Y., Lu, H., Liu, W., Bu, W., Eckenhoff, R., Cammarato, A., Gao, W. D. Fropofol decreases force development in cardiac muscle.

Internal viscoelastic loading in cat papillary muscle.

PubMed Central

Chiu, Y L; Ballou, E W; Ford, L E

1982-01-01

The passive mechanical properties of myocardium were defined by measuring force responses to rapid length ramps applied to unstimulated cat papillary muscles. The immediate force changes following these ramps recovered partially to their initial value, suggesting a series combination of viscous element and spring. Because the stretched muscle can bear force at rest, the viscous element must be in parallel with an additional spring. The instantaneous extension-force curves measured at different lengths were nonlinear, and could be made to superimpose by a simple horizontal shift. This finding suggests that the same spring was being measured at each length, and that this spring was in series with both the viscous element and its parallel spring (Voigt configuration), so that the parallel spring is held nearly rigid by the viscous element during rapid steps. The series spring in the passive muscle could account for most of the series elastic recoil in the active muscle, suggesting that the same spring is in series with both the contractile elements and the viscous element. It is postulated that the viscous element might be coupled to the contractile elements by a compliance, so that the load imposed on the contractile elements by the passive structures is viscoelastic rather than purely viscous. Such a viscoelastic load would give the muscle a length-independent, early diastolic restoring force. The possibility is discussed that the length-independent restoring force would allow some of the energy liberated during active shortening to be stored and released during relaxation. Images FIGURE 7 FIGURE 8 PMID:7171707

Theory of Epithelial Cell Shape Transitions Induced by Mechanoactive Chemical Gradients.

PubMed

Dasbiswas, Kinjal; Hannezo, Edouard; Gov, Nir S

2018-02-27

Cell shape is determined by a balance of intrinsic properties of the cell as well as its mechanochemical environment. Inhomogeneous shape changes underlie many morphogenetic events and involve spatial gradients in active cellular forces induced by complex chemical signaling. Here, we introduce a mechanochemical model based on the notion that cell shape changes may be induced by external diffusible biomolecules that influence cellular contractility (or equivalently, adhesions) in a concentration-dependent manner-and whose spatial profile in turn is affected by cell shape. We map out theoretically the possible interplay between chemical concentration and cellular structure. Besides providing a direct route to spatial gradients in cell shape profiles in tissues, we show that the dependence on cell shape helps create robust mechanochemical gradients. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Myosin IIA-related Actomyosin Contractility Mediates Oxidative Stress-induced Neuronal Apoptosis

PubMed Central

Wang, Yan; Xu, Yingqiong; Liu, Qian; Zhang, Yuanyuan; Gao, Zhen; Yin, Mingzhu; Jiang, Nan; Cao, Guosheng; Yu, Boyang; Cao, Zhengyu; Kou, Junping

2017-01-01

Oxidative stress-induced neuronal apoptosis plays an important role in the progression of central nervous system (CNS) diseases. In our study, when neuronal cells were exposed to hydrogen peroxide (H2O2), an exogenous oxidant, cell apoptosis was observed with typical morphological changes including membrane blebbing, neurite retraction and cell contraction. The actomyosin system is considered to be responsible for the morphological changes, but how exactly it regulates oxidative stress-induced neuronal apoptosis and the distinctive functions of different myosin II isoforms remain unclear. We demonstrate that myosin IIA was required for neuronal contraction, while myosin IIB was required for neuronal outgrowth in normal conditions. During H2O2-induced neuronal apoptosis, myosin IIA, rather than IIB, interacted with actin filaments to generate contractile forces that lead to morphological changes. Moreover, myosin IIA knockout using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) reduced H2O2-induced neuronal apoptosis and the associated morphological changes. We further demonstrate that caspase-3/Rho-associated kinase 1 (ROCK1) dependent phosphorylation of myosin light chain (MLC) was required for the formation of the myosin IIA-actin complex. Meanwhile, either inhibition of myosin II ATPase with blebbistatin or knockdown of myosin IIA with siRNA reversely attenuated caspase-3 activation, suggesting a positive feedback loop during oxidative stress-induced apoptosis. Based on our observation, myosin IIA-actin complex contributes to actomyosin contractility and is associated with the positive feedback loop of caspase-3/ROCK1/MLC pathway. This study unravels the biochemical and mechanistic mechanisms during oxidative stress-induced neuronal apoptosis and may be applicable for the development of therapies for CNS diseases. PMID:28352215

Tissue Cells Feel and Respond to the Stiffness of Their Substrate

NASA Astrophysics Data System (ADS)

Discher, Dennis E.; Janmey, Paul; Wang, Yu-li

2005-11-01

Normal tissue cells are generally not viable when suspended in a fluid and are therefore said to be anchorage dependent. Such cells must adhere to a solid, but a solid can be as rigid as glass or softer than a baby's skin. The behavior of some cells on soft materials is characteristic of important phenotypes; for example, cell growth on soft agar gels is used to identify cancer cells. However, an understanding of how tissue cells-including fibroblasts, myocytes, neurons, and other cell types-sense matrix stiffness is just emerging with quantitative studies of cells adhering to gels (or to other cells) with which elasticity can be tuned to approximate that of tissues. Key roles in molecular pathways are played by adhesion complexes and the actin-myosin cytoskeleton, whose contractile forces are transmitted through transcellular structures. The feedback of local matrix stiffness on cell state likely has important implications for development, differentiation, disease, and regeneration.

Length adaptation of airway smooth muscle.

PubMed

Bossé, Ynuk; Sobieszek, Apolinary; Paré, Peter D; Seow, Chun Y

2008-01-01

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

A cdk1 gradient guides surface contraction waves in oocytes.

PubMed

Bischof, Johanna; Brand, Christoph A; Somogyi, Kálmán; Májer, Imre; Thome, Sarah; Mori, Masashi; Schwarz, Ulrich S; Lénárt, Péter

2017-10-11

Surface contraction waves (SCWs) in oocytes and embryos lead to large-scale shape changes coupled to cell cycle transitions and are spatially coordinated with the cell axis. Here, we show that SCWs in the starfish oocyte are generated by a traveling band of myosin II-driven cortical contractility. At the front of the band, contractility is activated by removal of cdk1 inhibition of the RhoA/RhoA kinase/myosin II signaling module, while at the rear, contractility is switched off by negative feedback originating downstream of RhoA kinase. The SCW's directionality and speed are controlled by a spatiotemporal gradient of cdk1-cyclinB. This gradient is formed by the release of cdk1-cyclinB from the asymmetrically located nucleus, and progressive degradation of cyclinB. By combining quantitative imaging, biochemical and mechanical perturbations with mathematical modeling, we demonstrate that the SCWs result from the spatiotemporal integration of two conserved regulatory modules, cdk1-cyclinB for cell cycle regulation and RhoA/Rok/NMYII for actomyosin contractility.Surface contraction waves (SCWs) are prominent shape changes coupled to cell cycle transitions in oocytes. Here the authors show that SCWs are patterned by the spatiotemporal integration of two conserved modules, cdk1-cyclinB for cell cycle regulation and RhoA/Rok/NMYII for actomyosin contractility.

Cell division requires a direct link between microtubule-bound RacGAP and Anillin in the contractile ring.

PubMed

Gregory, Stephen L; Ebrahimi, Saman; Milverton, Joanne; Jones, Whitney M; Bejsovec, Amy; Saint, Robert

2008-01-08

The mitotic microtubule array plays two primary roles in cell division. It acts as a scaffold for the congression and separation of chromosomes, and it specifies and maintains the contractile-ring position. The current model for initiation of Drosophila and mammalian cytokinesis [1-5] postulates that equatorial localization of a RhoGEF (Pbl/Ect2) by a microtubule-associated motor protein complex creates a band of activated RhoA [6], which subsequently recruits contractile-ring components such as actin, myosin, and Anillin [1-3]. Equatorial microtubules are essential for continued constriction, but how they interact with the contractile apparatus is unknown. Here, we report the first direct molecular link between the microtubule spindle and the actomyosin contractile ring. We find that the spindle-associated component, RacGAP50C, which specifies the site of cleavage [1-5], interacts directly with Anillin, an actin and myosin binding protein found in the contractile ring [7-10]. Both proteins depend on this interaction for their localization. In the absence of Anillin, the spindle-associated RacGAP loses its association with the equatorial cortex, and cytokinesis fails. These results account for the long-observed dependence of cytokinesis on the continual presence of microtubules at the cortex.

Aging alters contractile properties and fiber morphology in pigeon skeletal muscle.

PubMed

Pistilli, Emidio E; Alway, Stephen E; Hollander, John M; Wimsatt, Jeffrey H

2014-12-01

In this study, we tested the hypothesis that skeletal muscle from pigeons would display age-related alterations in isometric force and contractile parameters as well as a shift of the single muscle fiber cross-sectional area (CSA) distribution toward smaller fiber sizes. Maximal force output, twitch contraction durations and the force-frequency relationship were determined in tensor propatagialis pars biceps muscle from young 3-year-old pigeons, middle-aged 18-year-old pigeons, and aged 30-year-old pigeons. The fiber CSA distribution was determined by planimetry from muscle sections stained with hematoxylin and eosin. Maximal force output of twitch and tetanic contractions was greatest in muscles from young pigeons, while the time to peak force of twitch contractions was longest in muscles from aged pigeons. There were no changes in the force-frequency relationship between the age groups. Interestingly, the fiber CSA distribution in aged muscles revealed a greater number of larger sized muscle fibers, which was verified visually in histological images. Middle-aged and aged muscles also displayed a greater amount of slow myosin containing muscle fibers. These data demonstrate that muscles from middle-aged and aged pigeons are susceptible to alterations in contractile properties that are consistent with aging, including lower force production and longer contraction durations. These functional changes were supported by the appearance of slow myosin containing muscle fibers in muscles from middle-aged and aged pigeons. Therefore, the pigeon may represent an appropriate animal model for the study of aging-related alterations in skeletal muscle function and structure.

Anisotropic shear stress patterns predict the orientation of convergent tissue movements in the embryonic heart

PubMed Central

2017-01-01

Myocardial contractility and blood flow provide essential mechanical cues for the morphogenesis of the heart. In general, endothelial cells change their migratory behavior in response to shear stress patterns, according to flow directionality. Here, we assessed the impact of shear stress patterns and flow directionality on the behavior of endocardial cells, the specialized endothelial cells of the heart. At the early stages of zebrafish heart valve formation, we show that endocardial cells are converging to the valve-forming area and that this behavior depends upon mechanical forces. Quantitative live imaging and mathematical modeling allow us to correlate this tissue convergence with the underlying flow forces. We predict that tissue convergence is associated with the direction of the mean wall shear stress and of the gradient of harmonic phase-averaged shear stresses, which surprisingly do not match the overall direction of the flow. This contrasts with the usual role of flow directionality in vascular development and suggests that the full spatial and temporal complexity of the wall shear stress should be taken into account when studying endothelial cell responses to flow in vivo. PMID:29183943

Methods for the Organogenesis of Skeletal Muscle in Tissue Culture

NASA Technical Reports Server (NTRS)

Vandenburgh, Herman; Shansky, Janet; DelTatto, Michael; Chromiak, Joseph

1997-01-01

Skeletal muscle structure is regulated by many factors, including nutrition, hormones, electrical activity, and tension. The muscle cells are subjected to both passive and active mechanical forces at all stages of development and these forces play important but poorly understood roles in regulating muscle organogenesis and growth. For example, during embryogenesis, the rapidly growing skeleton places large passive mechanical forces on the attached muscle tissue. These forces not only help to organize the proliferating mononucleated myoblasts into the oriented, multinucleated myofibers of a functional muscle but also tightly couple the growth rate of muscle to that of bone. Postnatally, the actively contracting, innervated muscle fibers are subjected to different patterns of active and passive tensions which regulate longitudinal and cross sectional myofiber growth. These mechanically-induced organogenic processes have been difficult to study under normal tissue culture conditions, resulting in the development of numerous methods and specialized equipment to simulate the in vivo mechanical environment.These techniques have led to the "engineering" of bioartificial muscles (organoids) which display many of the characteristics of in vivo muscle including parallel arrays of postmitotic fibers organized into fascicle-like structures with tendon-like ends. They are contractile, express adult isoforms of contractile proteins, perform directed work, and can be maintained in culture for long periods. The in vivo-like characteristics and durability of these muscle organoids make them useful for long term in vitro studies on mechanotransduction mechanisms and on muscle atrophy induced by decreased tension. In this report, we described a simple method for generating muscle organoids from either primary embrionic avain or neonatal rodent myoblasts.

Micropatterning tractional forces in living cells

NASA Technical Reports Server (NTRS)

Wang, Ning; Ostuni, Emanuele; Whitesides, George M.; Ingber, Donald E.

2002-01-01

Here we describe a method for quantifying traction in cells that are physically constrained within micron-sized adhesive islands of defined shape and size on the surface of flexible polyacrylamide gels that contain fluorescent microbeads (0.2-microm diameter). Smooth muscle cells were plated onto square (50 x 50 microm) or circular (25- or 50-microm diameter) adhesive islands that were created on the surface of the gels by applying a collagen coating through microengineered holes in an elastomeric membrane that was later removed. Adherent cells spread to take on the size and shape of the islands and cell tractions were quantitated by mapping displacement fields of the fluorescent microbeads within the gel. Cells on round islands did not exhibit any preferential direction of force application, but they exerted their strongest traction at sites where they formed protrusions. When cells were confined to squares, traction was highest in the corners both in the absence and presence of the contractile agonist, histamine, and cell protrusions were also observed in these regions. Quantitation of the mean traction exerted by cells cultured on the different islands revealed that cell tension increased as cell spreading was promoted. These results provide a mechanical basis for past studies that demonstrated a similar correlation between spreading and growth within various anchorage-dependent cells. This new approach for analyzing the spatial distribution of mechanical forces beneath individual cells that are experimentally constrained to defined sizes and shapes may provide additional insight into the biophysical basis of cell regulation. Copyright 2002 Wiley-Liss, Inc.

Functional vascular smooth muscle cells derived from human induced pluripotent stem cells via mesenchymal stem cell intermediates

PubMed Central

Bajpai, Vivek K.; Mistriotis, Panagiotis; Loh, Yuin-Han; Daley, George Q.; Andreadis, Stelios T.

2012-01-01

Aims Smooth muscle cells (SMC) play an important role in vascular homeostasis and disease. Although adult mesenchymal stem cells (MSC) have been used as a source of contractile SMC, they suffer from limited proliferation potential and culture senescence, particularly when originating from older donors. By comparison, human induced pluripotent stem cells (hiPSC) can provide an unlimited source of functional SMC for autologous cell-based therapies and for creating models of vascular disease. Our goal was to develop an efficient strategy to derive functional, contractile SMC from hiPSC. Methods and results We developed a robust, stage-wise, feeder-free strategy for hiPSC differentiation into functional SMC through an intermediate stage of multipotent MSC, which could be coaxed to differentiate into fat, bone, cartilage, and muscle. At this stage, the cells were highly proliferative and displayed higher clonogenic potential and reduced senescence when compared with parental hair follicle mesenchymal stem cells. In addition, when exposed to differentiation medium, the myogenic proteins such as α-smooth muscle actin, calponin, and myosin heavy chain were significantly upregulated and displayed robust fibrillar organization, suggesting the development of a contractile phenotype. Indeed, tissue constructs prepared from these cells exhibited high levels of contractility in response to receptor- and non-receptor-mediated agonists. Conclusion We developed an efficient stage-wise strategy that enabled hiPSC differentiation into contractile SMC through an intermediate population of clonogenic and multipotent MSC. The high yield of MSC and SMC derivation suggests that our strategy may facilitate an acquisition of the large numbers of cells required for regenerative medicine or for studying vascular disease pathophysiology. PMID:22941255

Microfabricated Nanotopological Surfaces for Study of Adhesion-dependent Cell mechanosensitivity**

PubMed Central

Chen, Weiqiang; Sun, Yubing

2014-01-01

Cells display high sensitivity and exhibit diverse responses to the intrinsic nanotopography of the extracellular matrix through their nanoscale cellular sensing machinery. Here, we reported a simple microfabrication method for precise control and spatial patterning of the local nanoroughness on glass surfaces using photolithography and reactive ion etching (RIE). Using RIE-generated nanorough glass surfaces, we demonstrated that local nanoroughness could provide a potent biophysical signal to regulate a diverse array of NIH/3T3 fibroblast behaviors, including cell morphology, adhesion, proliferation and migration. We further showed that cellular responses to nanotopography might be regulated by cell adhesion signaling and actin cytoskeleton remodeling. To further investigate the role of cytoskeleton contractility in nanoroughness sensing, we applied the RIE method to generate nanoroughness on the tops of an array of elastomeric poly-dimethylsiloxane (PDMS) microposts. We utilized the PDMS microposts as force sensors and demonstrated that nanoroughness could indeed regulate the cytoskeleton contractility of NIH/3T3 fibroblasts. Our results suggested that a feedback regulation and mechano-chemical integration mechanism involving adhesion signaling, actin cytoskeleton, and intracellular mechanosensory components might play an important role in regulating mechanosensitive behaviors of NIH/3T3 fibroblasts. The capability to control and further predict cellular responses to nanoroughness might suggest novel methods for developing biomaterials mimicking nanotopographic structures in vivo and suitable local cellular microenvironments for functional tissue engineering. PMID:22887768

Histone deacetylase 8 regulates cortactin deacetylation and contraction in smooth muscle tissues

PubMed Central

Li, Jia; Chen, Shu; Cleary, Rachel A.; Wang, Ruping; Gannon, Olivia J.; Seto, Edward

2014-01-01

Histone deacetylases (HDACs) are a family of enzymes that mediate nucleosomal histone deacetylation and gene expression. Some members of the HDAC family have also been implicated in nonhistone protein deacetylation, which modulates cell-cycle control, differentiation, and cell migration. However, the role of HDACs in smooth muscle contraction is largely unknown. Here, HDAC8 was localized both in the cytoplasm and the nucleus of mouse and human smooth muscle cells. Knockdown of HDAC8 by lentivirus-encoding HDAC8 shRNA inhibited force development in response to acetylcholine. Treatment of smooth muscle tissues with HDAC8 inhibitor XXIV (OSU-HDAC-44) induced relaxation of precontracted smooth muscle tissues. In addition, cortactin is an actin-regulatory protein that undergoes deacetylation during migration of NIH 3T3 cells. In this study, acetylcholine stimulation induced cortactin deacetylation in mouse and human smooth muscle tissues, as evidenced by immunoblot analysis using antibody against acetylated lysine. Knockdown of HDAC8 by RNAi or treatment with the inhibitor attenuated cortactin deacetylation and actin polymerization without affecting myosin activation. Furthermore, expression of a charge-neutralizing cortactin mutant inhibited contraction and actin dynamics during contractile activation. These results suggest a novel mechanism for the regulation of smooth muscle contraction. In response to contractile stimulation, HDAC8 may mediate cortactin deacetylation, which subsequently promotes actin filament polymerization and smooth muscle contraction. PMID:24920679

Mechanically Induced Chromatin Condensation Requires Cellular Contractility in Mesenchymal Stem Cells.

PubMed

Heo, Su-Jin; Han, Woojin M; Szczesny, Spencer E; Cosgrove, Brian D; Elliott, Dawn M; Lee, David A; Duncan, Randall L; Mauck, Robert L

2016-08-23

Mechanical cues play important roles in directing the lineage commitment of mesenchymal stem cells (MSCs). In this study, we explored the molecular mechanisms by which dynamic tensile loading (DL) regulates chromatin organization in this cell type. Our previous findings indicated that the application of DL elicited a rapid increase in chromatin condensation through purinergic signaling mediated by ATP. Here, we show that the rate and degree of condensation depends on the frequency and duration of mechanical loading, and that ATP release requires actomyosin-based cellular contractility. Increases in baseline cellular contractility via the addition of an activator of G-protein coupled receptors (lysophosphatidic acid) induced rapid ATP release, resulting in chromatin condensation independent of loading. Conversely, inhibition of contractility through pretreatment with either a RhoA/Rock inhibitor (Y27632) or MLCK inhibitor (ML7) abrogated ATP release in response to DL, blocking load-induced chromatin condensation. With loading, ATP release occurred very rapidly (within the first 10-20 s), whereas changes in chromatin occurred at a later time point (∼10 min), suggesting a downstream biochemical pathway mediating this process. When cells were pretreated with blockers of the transforming growth factor (TGF) superfamily, purinergic signaling in response to DL was also eliminated. Further analysis showed that this pretreatment decreased contractility, implicating activity in the TGF pathway in the establishment of the baseline contractile state of MSCs (in the absence of exogenous ligands). These data indicate that chromatin condensation in response to DL is regulated through the interplay between purinergic and RhoA/Rock signaling, and that ligandless activity in the TGF/bone morphogenetic proteins signaling pathway contributes to the establishment of baseline contractility in MSCs. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Active unjamming of confluent cell layers

NASA Astrophysics Data System (ADS)

Marchetti, M. Cristina

Cell motion inside dense tissues governs many biological processes, including embryonic development and cancer metastasis, and recent experiments suggest that these tissues exhibit collective glassy behavior. Motivated by these observations, we have studied a model of dense tissues that combines self-propelled particle models and vertex models of confluent cell layers. In this model, referred to as self-propelled Voronoi (SPV), cells are described as polygons in a Voronoi tessellation with directed noisy cell motility and interactions governed by a shape energy that incorporates the effects of cell volume incompressibility, contractility and cell-cell adhesion. Using this model, we have demonstrated a new density-independent solid-liquid transition in confluent tissues controlled by cell motility and a cell-shape parameter measuring the interplay of cortical tension and cell-cell adhesion. An important insight of this work is that the rigidity and dynamics of cell layers depends sensitively on cell shape. We have also used the SPV model to test a new method developed by our group to determine cellular forces and tissue stresses from experimentally accessible cell shapes and traction forces, hence providing the spatio-temporal distribution of stresses in motile dense tissues. This work was done with Dapeng Bi, Lisa Manning and Xingbo Yang. MCM was supported by NSF-DMR-1305184 and by the Simons Foundation.

Changes in contractile activation characteristics of rat fast and slow skeletal muscle fibres during regeneration.

PubMed

Gregorevic, Paul; Plant, David R; Stupka, Nicole; Lynch, Gordon S

2004-07-15

Damaged skeletal muscle fibres are replaced with new contractile units via muscle regeneration. Regenerating muscle fibres synthesize functionally distinct isoforms of contractile and regulatory proteins but little is known of their functional properties during the regeneration process. An advantage of utilizing single muscle fibre preparations is that assessment of their function is based on the overall characteristics of the contractile apparatus and regulatory system and as such, these preparations are sensitive in revealing not only coarse, but also subtle functional differences between muscle fibres. We examined the Ca(2+)- and Sr(2+)-activated contractile characteristics of permeabilized fibres from rat fast-twitch (extensor digitorum longus) and slow-twitch (soleus) muscles at 7, 14 and 21 days following myotoxic injury, to test the hypothesis that fibres from regenerating fast and slow muscles have different functional characteristics to fibres from uninjured muscles. Regenerating muscle fibres had approximately 10% of the maximal force producing capacity (P(o)) of control (uninjured) fibres, and an altered sensitivity to Ca(2+) and Sr(2+) at 7 days post-injury. Increased force production and a shift in Ca(2+) sensitivity consistent with fibre maturation were observed during regeneration such that P(o) was restored to 36-45% of that in control fibres by 21 days, and sensitivity to Ca(2+) and Sr(2+) was similar to that of control (uninjured) fibres. The findings support the hypothesis that regenerating muscle fibres have different contractile activation characteristics compared with mature fibres, and that they adopt properties of mature fast- or slow-twitch muscle fibres in a progressive manner as the regeneration process is completed.

Fine Tuning of Tissues' Viscosity and Surface Tension through Contractility Suggests a New Role for α-Catenin

PubMed Central

Stirbat, Tomita Vasilica; Mgharbel, Abbas; Bodennec, Selena; Ferri, Karine; Mertani, Hichem C.; Rieu, Jean-Paul; Delanoë-Ayari, Hélène

2013-01-01

What governs tissue organization and movement? If molecular and genetic approaches are able to give some answers on these issues, more and more works are now giving a real importance to mechanics as a key component eventually triggering further signaling events. We chose embryonic cell aggregates as model systems for tissue organization and movement in order to investigate the origin of some mechanical constraints arising from cells organization. Steinberg et al. proposed a long time ago an analogy between liquids and tissues and showed that indeed tissues possess a measurable tissue surface tension and viscosity. We question here the molecular origin of these parameters and give a quantitative measurement of adhesion versus contractility in the framework of the differential interfacial tension hypothesis. Accompanying surface tension measurements by angle measurements (at vertexes of cell-cell contacts) at the cell/medium interface, we are able to extract the full parameters of this model: cortical tensions and adhesion energy. We show that a tunable surface tension and viscosity can be achieved easily through the control of cell-cell contractility compared to cell-medium one. Moreover we show that -catenin is crucial for this regulation to occur: these molecules appear as a catalyser for the remodeling of the actin cytoskeleton underneath cell-cell contact, enabling a differential contractility between the cell-medium and cell-cell interface to take place. PMID:23390488

Calcium-responsive contractility during fertilization in sea urchin eggs.

PubMed

Stack, Christianna; Lucero, Amy J; Shuster, Charles B

2006-04-01

Fertilization triggers a reorganization of oocyte cytoskeleton, and in sea urchins, there is a dramatic increase in cortical F-actin. However, the role that myosin II plays during fertilization remains largely unexplored. Myosin II is localized to the cortical cytoskeleton both before and after fertilization and to examine myosin II contractility in living cells, Lytechinus pictus eggs were observed by time-lapse microscopy. Upon sperm binding, a cell surface deflection traversed the egg that was followed by and dependent on the calcium wave. The calcium-dependence of surface contractility could be reproduced in unfertilized eggs, where mobilization of intracellular calcium in unfertilized eggs under compression resulted in a marked contractile response. Lastly, inhibition of myosin II delayed absorption of the fertilization cone, suggesting that myosin II not only responds to the same signals that activate eggs but also participates in the remodeling of the cortical actomyosin cytoskeleton during the first zygotic cell cycle. (c) 2006 Wiley-Liss, Inc.

Calcium-Responsive Contractility During Fertilization in Sea Urchin Eggs

PubMed Central

Stack, Christianna; Lucero, Amy J.; Shuster, Charles B.

2008-01-01

Fertilization triggers a reorganization of oocyte cytoskeleton, and in sea urchins there is a dramatic increase in cortical F-actin. However, the role that myosin II plays during fertilization remains largely unexplored. Myosin II is localized to the cortical cytoskeleton both prior to- and following fertilization, and to examine myosin II contractility in living cells, Lytechinus pictus eggs were observed by time-lapse microscopy. Upon sperm binding, a cell surface deflection traversed the egg that was followed- and dependent on the calcium wave. The calcium-dependence of surface contractility could be reproduced in unfertilized eggs, where mobilization of intracellular calcium in unfertilized eggs under compression resulted in a marked contractile response. Lastly, inhibition of myosin II delayed absorption of the fertilization cone, suggesting that myosin II not only responds to the same signals that activate eggs, but also participates in the remodeling of the cortical actomyosin cytoskeleton during the first zygotic cell cycle. PMID:16470603

Actomyosin-based tissue folding requires a multicellular myosin gradient

PubMed Central

Miller, Pearson W.; Chanet, Soline; Stoop, Norbert; Dunkel, Jörn

2017-01-01

Tissue folding promotes three-dimensional (3D) form during development. In many cases, folding is associated with myosin accumulation at the apical surface of epithelial cells, as seen in the vertebrate neural tube and the Drosophila ventral furrow. This type of folding is characterized by constriction of apical cell surfaces, and the resulting cell shape change is thought to cause tissue folding. Here, we use quantitative microscopy to measure the pattern of transcription, signaling, myosin activation and cell shape in the Drosophila mesoderm. We found that cells within the ventral domain accumulate different amounts of active apical non-muscle myosin 2 depending on the distance from the ventral midline. This gradient in active myosin depends on a newly quantified gradient in upstream signaling proteins. A 3D continuum model of the embryo with induced contractility demonstrates that contractility gradients, but not contractility per se, promote changes to surface curvature and folding. As predicted by the model, experimental broadening of the myosin domain in vivo disrupts tissue curvature where myosin is uniform. Our data argue that apical contractility gradients are important for tissue folding. PMID:28432215

Orosomucoid-like 3 (ORMDL3) upregulates airway smooth muscle proliferation, contraction, and Ca2+ oscillations in asthma.

PubMed

Chen, Jun; Miller, Marina; Unno, Hirotoshi; Rosenthal, Peter; Sanderson, Michael J; Broide, David H

2017-09-07

Airway hyperresponsiveness is a major feature of asthma attributed predominantly to an extrinsic immune/inflammatory response increasing airway smooth muscle (ASM) contractility. We investigated whether increased ASM expression of orosomucoid-like 3 (ORMDL3), a gene on chromosome 17q21 highly linked to asthma, induced increased ASM proliferation and contractility in vitro and influenced airway contractility and calcium flux in ASM in precision-cut lung slices (PCLSs) from wild-type and hORMDL3 Zp3-Cre mice (which express increased levels of human ORMDL3 [hORMDL3]). Levels of ASM proliferation and contraction were assessed in ASM cells transfected with ORMDL3 in vitro. In addition, airway contractility and calcium oscillations were quantitated in ASM cells in PCLSs derived from naive wild-type and naive hORMDL3 Zp3-Cre mice, which do not have a blood supply. Increased ASM expression of ORMDL3 in vitro resulted in increased ASM proliferation and contractility. PCLSs derived from naive hORMDL3 Zp3-Cre mice, which do not have airway inflammation, exhibit increased airway contractility with increased calcium oscillations in ASM cells. Increased ASM ORMDL3 expression increases levels of ASM sarcoplasmic reticulum Ca 2+ ATPase 2b (SERCA2b), which increases ASM proliferation and contractility. Overall, these studies provide evidence that an intrinsic increase in ORMDL3 expression in ASM can induce increased ASM proliferation and contractility, which might contribute to increased airway hyperresponsiveness in the absence of airway inflammation in asthmatic patients. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Chronic treatment with fluoxetine and sertraline prevents forced swimming test-induced hypercontractility of rat detrusor muscle.

PubMed

Bilge, Sirri; Bozkurt, Ayhan; Bas, Duygu B; Aksoz, Elif; Savli, Evren; Ilkaya, Fatih; Kesim, Yuksel

2008-01-01

Serotonin (5-hydroxytryptamine, 5-HT) reuptake inhibitors represent important targets for the development of new treatments for detrusor overactivity and urinary incontinence. The present study was undertaken to investigate the effects of the forced swimming test (FST) on the contractile response of isolated rat detrusor muscle and to examine the effects of in vivo treatments of fluoxetine and sertraline on altered detrusor muscle contractility. Fluoxetine (20 mg/kg ip) and sertraline (10 mg/kg ip) were administered once a day for 14 days. Rats were exposed to the FST on the 15th day. After the test, detrusor muscles were removed and placed in organ baths, and the contraction responses induced by carbachol, potassium chloride (KCl) and electrical field stimulation (EFS) were recorded. The contractile responses of detrusor muscle strips to carbachol and electrical field stimulation were found to be increased at all carbachol doses and frequencies, respectively. FST also increased the contractile responses to KCl, which is used to test the differences in postreceptor-mediated contractions. The hypercontractile responses of detrusor strips to carbachol, EFS and KCl were abolished by treatment with both fluoxetine and sertraline. These treatments also decreased the immobility duration in the FST consistent with an antidepressant-like effect in this test. The results of this study provide the first evidence that FST increases contractility of the rat detrusor muscle, and this hypercontractility was abolished by chronic treatments of fluoxetine and sertraline at antidepressant doses by decreasing the postreceptor-mediated events.

Alpha-actinin binding kinetics modulate cellular dynamics and force generation

PubMed Central

Ehrlicher, Allen J.; Krishnan, Ramaswamy; Guo, Ming; Bidan, Cécile M.; Weitz, David A.; Pollak, Martin R.

2015-01-01

The actin cytoskeleton is a key element of cell structure and movement whose properties are determined by a host of accessory proteins. Actin cross-linking proteins create a connected network from individual actin filaments, and though the mechanical effects of cross-linker binding affinity on actin networks have been investigated in reconstituted systems, their impact on cellular forces is unknown. Here we show that the binding affinity of the actin cross-linker α-actinin 4 (ACTN4) in cells modulates cytoplasmic mobility, cellular movement, and traction forces. Using fluorescence recovery after photobleaching, we show that an ACTN4 mutation that causes human kidney disease roughly triples the wild-type binding affinity of ACTN4 to F-actin in cells, increasing the dissociation time from 29 ± 13 to 86 ± 29 s. This increased affinity creates a less dynamic cytoplasm, as demonstrated by reduced intracellular microsphere movement, and an approximate halving of cell speed. Surprisingly, these less motile cells generate larger forces. Using traction force microscopy, we show that increased binding affinity of ACTN4 increases the average contractile stress (from 1.8 ± 0.7 to 4.7 ± 0.5 kPa), and the average strain energy (0.4 ± 0.2 to 2.1 ± 0.4 pJ). We speculate that these changes may be explained by an increased solid-like nature of the cytoskeleton, where myosin activity is more partitioned into tension and less is dissipated through filament sliding. These findings demonstrate the impact of cross-linker point mutations on cell dynamics and forces, and suggest mechanisms by which such physical defects lead to human disease. PMID:25918384

Deletion of Mbtps1 (Pcsk8, S1p, Ski-1) Gene in Osteocytes Stimulates Soleus Muscle Regeneration and Increased Size and Contractile Force with Age*

PubMed Central

Gorski, Jeff P.; Huffman, Nichole T.; Vallejo, Julian; Brotto, Leticia; Chittur, Sridar V.; Breggia, Anne; Stern, Amber; Huang, Jian; Mo, Chenglin; Seidah, Nabil G.; Bonewald, Lynda; Brotto, Marco

2016-01-01

Conditional deletion of Mbtps1 (cKO) protease in bone osteocytes leads to an age-related increase in mass (12%) and in contractile force (30%) in adult slow twitch soleus muscles (SOL) with no effect on fast twitch extensor digitorum longus muscles. Surprisingly, bone from 10–12-month-old cKO animals was indistinguishable from controls in size, density, and morphology except for a 25% increase in stiffness. cKO SOL exhibited increased expression of Pax7, Myog, Myod1, Notch, and Myh3 and 6-fold more centralized nuclei, characteristics of postnatal regenerating muscle, but only in type I myosin heavy chain-expressing cells. Increased expression of gene pathways mediating EGF receptor signaling, circadian exercise, striated muscle contraction, and lipid and carbohydrate oxidative metabolism were also observed in cKO SOL. This muscle phenotype was not observed in 3-month-old mice. Although Mbtps1 mRNA and protein expression was reduced in cKO bone osteocytes, no differences in Mbtps1 or cre recombinase expression were observed in cKO SOL, explaining this age-related phenotype. Understanding bone-muscle cross-talk may provide a fresh and novel approach to prevention and treatment of age-related muscle loss. PMID:26719336

Activity-induced regulation of myosin isoform distribution - Comparison of two contractile activity programs

NASA Technical Reports Server (NTRS)

Diffee, Gary M.; Caiozzo, Vince J.; Mccue, Samuel A.; Herrick, Robert E.; Baldwin, Kenneth M.

1993-01-01

This study examined the role of specific types of contractile activity in regulating myosin heavy chain (MHC) isoform expression in rodent soleus. A combination of hindlimb suspension (SN) and two programmed contractile training activity paradigms, either isometric contractile activity (ST-IM) or high-load slowly shortening isovelocity activity, were utilized. Both training paradigms increased muscle mass compared with SN alone. However, only ST-IM resulted in a partial prevention of the suspension-induced decrease in type I MHC. With the use of a fluorescently labeled antibody to type IIa MHC, the distribution of MHCs among fibers was examined immunohistochemically. In SN, the percentage of cells staining positive for type IIa MHC was increased but the staining intensity of the positively staining cells was unchanged compared with control cells. In the ST-IM soleus, the percentage of positively staining fibers was unchanged but the intensity of the positively staining cells was decreased compared with SN values. These results suggest that 1) isometric contractile activity is more effective than isovelocity activity in preventing suspension-induced shifts in soleus MHC distribution and 2) changes associated with both suspension and training occur in only a small number of fibers, with the majority of fibers apparently unresponsive to these interventions.

Transcellular tunnel dynamics: Control of cellular dewetting by actomyosin contractility and I-BAR proteins.

PubMed

Lemichez, Emmanuel; Gonzalez-Rodriguez, David; Bassereau, Patricia; Brochard-Wyart, Françoise

2013-03-01

Dewetting is the spontaneous withdrawal of a liquid film from a non-wettable surface by nucleation and growth of dry patches. Two recent reports now propose that the principles of dewetting explain the physical phenomena underpinning the opening of transendothelial cell macroaperture (TEM) tunnels, referred to as cellular dewetting. This was discovered by studying a group of bacterial toxins endowed with the property of corrupting actomyosin cytoskeleton contractility. For both liquid and cellular dewetting, the growth of holes is governed by a competition between surface forces and line tension. We also discuss how the dynamics of TEM opening and closure represent remarkable systems to investigate actin cytoskeleton regulation by sensors of plasma membrane curvature and investigate the impact on membrane tension and the role of TEM in vascular dysfunctions. Copyright © 2013 Soçiété Française des Microscopies and Soçiété de Biologie Cellulaire de France.

Excitation-contraction coupling in zebrafish ventricular myocardium is regulated by trans-sarcolemmal Ca2+ influx and sarcoplasmic reticulum Ca2+ release.

PubMed

Haustein, Moritz; Hannes, Tobias; Trieschmann, Jan; Verhaegh, Rabea; Köster, Annette; Hescheler, Jürgen; Brockmeier, Konrad; Adelmann, Roland; Khalil, Markus

2015-01-01

Zebrafish (Danio rerio) have become a popular model in cardiovascular research mainly due to identification of a large number of mutants with structural defects. In recent years, cardiomyopathies and other diseases influencing contractility of the heart have been studied in zebrafish mutants. However, little is known about the regulation of contractility of the zebrafish heart on a tissue level. The aim of the present study was to elucidate the role of trans-sarcolemmal Ca(2+)-flux and sarcoplasmic reticulum Ca(2+)-release in zebrafish myocardium. Using isometric force measurements of fresh heart slices, we characterised the effects of changes of the extracellular Ca(2+)-concentration, trans-sarcolemmal Ca(2+)-flux via L-type Ca(2+)-channels and Na(+)-Ca(2+)-exchanger, and Ca(2+)-release from the sarcoplasmic reticulum as well as beating frequency and β-adrenergic stimulation on contractility of adult zebrafish myocardium. We found an overall negative force-frequency relationship (FFR). Inhibition of L-type Ca(2+)-channels by verapamil (1 μM) decreased force of contraction to 22 ± 7% compared to baseline (n=4, p<0.05). Ni(2+) was the only substance to prolong relaxation (5 mM, time after peak to 50% relaxation: 73 ± 3 ms vs. 101 ± 8 ms, n=5, p<0.05). Surprisingly though, inhibition of the sarcoplasmic Ca(2+)-release decreased force development to 54 ± 3% in ventricular (n=13, p<0.05) and to 52 ± 8% in atrial myocardium (n=5, p<0.05) suggesting a substantial role of SR Ca(2+)-release in force generation. In line with this finding, we observed significant post pause potentiation after pauses of 5 s (169 ± 7% force compared to baseline, n=8, p<0.05) and 10 s (198 ± 9% force compared to baseline, n=5, p<0.05) and mildly positive lusitropy after β-adrenergic stimulation. In conclusion, force development in adult zebrafish ventricular myocardium requires not only trans-sarcolemmal Ca2+-flux, but also intact sarcoplasmic reticulum Ca(2+)-cycling. In contrast to mammals, FFR is strongly negative in the zebrafish heart. These aspects need to be considered when using zebrafish to model human diseases of myocardial contractility.

Excitation-Contraction Coupling in Zebrafish Ventricular Myocardium Is Regulated by Trans-Sarcolemmal Ca2+ Influx and Sarcoplasmic Reticulum Ca2+ Release

PubMed Central

Trieschmann, Jan; Verhaegh, Rabea; Köster, Annette; Hescheler, Jürgen; Brockmeier, Konrad; Adelmann, Roland; Khalil, Markus

2015-01-01

Zebrafish (Danio rerio) have become a popular model in cardiovascular research mainly due to identification of a large number of mutants with structural defects. In recent years, cardiomyopathies and other diseases influencing contractility of the heart have been studied in zebrafish mutants. However, little is known about the regulation of contractility of the zebrafish heart on a tissue level. The aim of the present study was to elucidate the role of trans-sarcolemmal Ca2+-flux and sarcoplasmic reticulum Ca2+-release in zebrafish myocardium. Using isometric force measurements of fresh heart slices, we characterised the effects of changes of the extracellular Ca2+-concentration, trans-sarcolemmal Ca2+-flux via L-type Ca2+-channels and Na+-Ca2+-exchanger, and Ca2+-release from the sarcoplasmic reticulum as well as beating frequency and β-adrenergic stimulation on contractility of adult zebrafish myocardium. We found an overall negative force-frequency relationship (FFR). Inhibition of L-type Ca2+-channels by verapamil (1 μM) decreased force of contraction to 22±7% compared to baseline (n=4, p<0.05). Ni2+ was the only substance to prolong relaxation (5 mM, time after peak to 50% relaxation: 73±3 ms vs. 101±8 ms, n=5, p<0.05). Surprisingly though, inhibition of the sarcoplasmic Ca2+-release decreased force development to 54±3% in ventricular (n=13, p<0.05) and to 52±8% in atrial myocardium (n=5, p<0.05) suggesting a substantial role of SR Ca2+-release in force generation. In line with this finding, we observed significant post pause potentiation after pauses of 5 s (169±7% force compared to baseline, n=8, p<0.05) and 10 s (198±9% force compared to baseline, n=5, p<0.05) and mildly positive lusitropy after β-adrenergic stimulation. In conclusion, force development in adult zebrafish ventricular myocardium requires not only trans-sarcolemmal Ca2+-flux, but also intact sarcoplasmic reticulum Ca2+-cycling. In contrast to mammals, FFR is strongly negative in the zebrafish heart. These aspects need to be considered when using zebrafish to model human diseases of myocardial contractility. PMID:25938412

In-depth evaluation of commercially available human vascular smooth muscle cells phenotype: Implications for vascular tissue engineering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Timraz, Sara B.H., E-mail: sara.timraz@kustar.ac.ae; Farhat, Ilyas A.H., E-mail: ilyas.farhat@outlook.com; Alhussein, Ghada, E-mail: ghada.alhussein@kustar.ac.ae

In vitro research on vascular tissue engineering has extensively used isolated primary human or animal smooth muscle cells (SMC). Research programs that lack such facilities tend towards commercially available primary cells sources. Here, we aim to evaluate the capacity of commercially available human SMC to maintain their contractile phenotype, and determine if dedifferentiation towards the synthetic phenotype occurs in response to conventional cell culture and passaging without any external biochemical or mechanical stimuli. Lower passage SMC adopted a contractile phenotype marked by a relatively slower proliferation rate, higher expression of proteins of the contractile apparatus and smoothelin, elongated morphology, andmore » reduced deposition of collagen types I and III. As the passage number increased, migratory capacity was enhanced, average cell speed, total distance and net distance travelled increased up to passage 8. Through the various assays, corroborative evidence pinpoints SMC at passage 7 as the transition point between the contractile and synthetic phenotypes, while passage 8 distinctly and consistently exhibited characteristics of synthetic phenotype. This knowledge is particularly useful in selecting SMC of appropriate passage number for the target vascular tissue engineering application, for example, a homeostatic vascular graft for blood vessel replacement versus recreating atherosclerotic blood vessel model in vitro. - Highlights: • Ability of human smooth muscle cells to alter phenotype in culture is evaluated. • Examined the effect of passaging human smooth muscle cells on phenotype. • Phenotype is assessed based on morphology, proliferation, markers, and migration. • Multi-resolution assessment methodology, single-cell and cell-population. • Lower and higher passages than P7 adopted a contractile and synthetic phenotype respectively.« less

Hydrogen peroxide modulates Ca2+-activation of single permeabilized fibres from fast- and slow-twitch skeletal muscles of rats.

PubMed

Plant, D R; Lynch, G S; Williams, D A

2000-01-01

We examined the effects of redox modulation on single membrane-permeabilized fibre segments from the fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus muscles of adult rats to determine whether the contractile apparatus was the redox target responsible for the increased contractility of muscles exposed to low concentrations of H2O2. The effects of H2O2 on maximum Ca2+-activated force were dose-dependent with 30 min exposure to 5 mM H2O2 causing a progressive decrease by 22+/-3 and 13+/-2% in soleus and EDL permeabilized muscle fibres, respectively. Lower concentrations of exogenous H2O2 (100 microM and 1 mM) had no effect on maximum Ca2+-activated force. Subsequent exposure to the reductant dithiothreitol (DTT, 10 mM, 10 min) fully reversed the H2O2-induced depression of force in EDL, but not in soleus muscle fibres. Incubation with DTT alone for 10 min did not alter Ca2+-activated force in either soleus or EDL muscle fibres. The sensitivity of the contractile filaments to Ca2+ (pCa50) was not altered by exposure to any concentration of exogenous H2O2. However, all concentrations of H2O2 diminished the Hill coefficient in permeabilized fibres from the EDL muscle, indicating that the cooperativity of Ca2+ binding to troponin is altered. H2O2 (5 mM) did not affect rigor force, which indicates that the number of crossbridges participating in contraction was not reduced. In conclusion, H2O2 may reduce the maximum Ca2+ activated force production in skinned muscle fibres by decreasing the force per crossbridge.

Matrigel Mattress: A Method for the Generation of Single Contracting Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

PubMed

Feaster, Tromondae K; Cadar, Adrian G; Wang, Lili; Williams, Charles H; Chun, Young Wook; Hempel, Jonathan E; Bloodworth, Nathaniel; Merryman, W David; Lim, Chee Chew; Wu, Joseph C; Knollmann, Björn C; Hong, Charles C

2015-12-04

The lack of measurable single-cell contractility of human-induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery. To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult CMs. Single hiPSC-CMs were cultured for 5 to 7 days on a 0.4- to 0.8-mm thick mattress of undiluted Matrigel (mattress hiPSC-CMs) and compared with hiPSC-CMs maintained on a control substrate (<0.1-mm thick 1:60 diluted Matrigel, control hiPSC-CMs). Compared with control hiPSC-CMs, mattress hiPSC-CMs had more rod-shape morphology and significantly increased sarcomere length. Contractile parameters of mattress hiPSC-CMs measured with video-based edge detection were comparable with those of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CMs were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CMs, mattress hiPSC-CMs display robust contractile responses to positive inotropic agents, such as myofilament calcium sensitizers. Mattress hiPSC-CMs exhibit molecular changes that include increased expression of the maturation marker cardiac troponin I and significantly increased action potential upstroke velocity because of a 2-fold increase in sodium current (INa). The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single-cell level, which should be valuable to disease modeling, drug discovery, and preclinical cardiotoxicity testing. © 2015 American Heart Association, Inc.

A theoretical and computational framework for mechanics of the cortex

NASA Astrophysics Data System (ADS)

Torres-SáNchez, Alejandro; Arroyo, Marino

The cell cortex is a thin network of actin filaments lying beneath the cell surface of animal cells. Myosin motors exert contractile forces in this network leading to active stresses, which play a key role in processes such as cytokinesis or cell migration. Thus, understanding the mechanics of the cortex is fundamental to understand the mechanics of animal cells. Due to the dynamic remodeling of the actin network, the cortex behaves as a viscoelastic fluid. Furthermore, due to the difference between its thickness (tens of nanometers) and its dimensions (tens of microns), the cortex can be regarded a surface. Thus, we can model the cortex as a viscoelastic fluid, confined to a surface, that generates active stresses. Interestingly, geometric confinement results in the coupling between shape generation and material flows. In this work we present a theoretical framework to model the mechanics of the cortex that couples elasticity, hydrodynamics and force generation. We complement our theoretical description with a computational setting to simulate the resulting non-linear equations. We use this methodology to understand different processes such as asymmetric cell division or experimental probing of the rheology of the cortex We acknowledge the support of the Europen Research Council through Grant ERC CoG-681434.

Cardiac myofibrillar contractile properties during the progression from hypertension to decompensated heart failure.

PubMed

Hanft, Laurin M; Emter, Craig A; McDonald, Kerry S

2017-07-01

Heart failure arises, in part, from a constellation of changes in cardiac myocytes including remodeling, energetics, Ca 2+ handling, and myofibrillar function. However, little is known about the changes in myofibrillar contractile properties during the progression from hypertension to decompensated heart failure. The aim of the present study was to provide a comprehensive assessment of myofibrillar functional properties from health to heart disease. A rodent model of uncontrolled hypertension was used to test the hypothesis that myocytes in compensated hearts exhibit increased force, higher rates of force development, faster loaded shortening, and greater power output; however, with progression to overt heart failure, we predicted marked depression in these contractile properties. We assessed contractile properties in skinned cardiac myocyte preparations from left ventricles of Wistar-Kyoto control rats and spontaneous hypertensive heart failure (SHHF) rats at ~3, ~12, and >20 mo of age to evaluate the time course of myofilament properties associated with normal aging processes compared with myofilaments from rats with a predisposition to heart failure. In control rats, the myofilament contractile properties were virtually unchanged throughout the aging process. Conversely, in SHHF rats, the rate of force development, loaded shortening velocity, and power all increased at ~12 mo and then significantly fell at the >20-mo time point, which coincided with a decrease in left ventricular fractional shortening. Furthermore, these changes occurred independent of changes in β-myosin heavy chain but were associated with depressed phosphorylation of myofibrillar proteins, and the fall in loaded shortening and peak power output corresponded with the onset of clinical signs of heart failure. NEW & NOTEWORTHY This novel study systematically examined the power-generating capacity of cardiac myofilaments during the progression from hypertension to heart disease. Previously undiscovered changes in myofibrillar power output were found and were associated with alterations in myofilament proteins, providing potential new targets to exploit for improved ventricular pump function in heart failure. Copyright © 2017 the American Physiological Society.

Phosphoinositide 3-Kinase p110β Regulates Integrin αIIbβ3 Avidity and the Cellular Transmission of Contractile Forces*

PubMed Central

Schoenwaelder, Simone M.; Ono, Akiko; Nesbitt, Warwick S.; Lim, Joanna; Jarman, Kate; Jackson, Shaun P.

2010-01-01

Phosphoinositide (PI) 3-kinase (PI3K) signaling processes play an important role in regulating the adhesive function of integrin αIIbβ3, necessary for platelet spreading and sustained platelet aggregation. PI3K inhibitors are effective at reducing platelet aggregation and thrombus formation in vivo and as a consequence are currently being evaluated as novel antithrombotic agents. PI3K regulation of integrin αIIbβ3 activation (affinity modulation) primarily occurs downstream of Gi-coupled and tyrosine kinase-linked receptors linked to the activation of Rap1b, AKT, and phospholipase C. In the present study, we demonstrate an important role for PI3Ks in regulating the avidity (strength of adhesion) of high affinity integrin αIIbβ3 bonds, necessary for the cellular transmission of contractile forces. Using knock-out mouse models and isoform-selective PI3K inhibitors, we demonstrate that the Type Ia p110β isoform plays a major role in regulating thrombin-stimulated fibrin clot retraction in vitro. Reduced clot retraction induced by PI3K inhibitors was not associated with defects in integrin αIIbβ3 activation, actin polymerization, or actomyosin contractility but was associated with a defect in integrin αIIbβ3 association with the contractile cytoskeleton. Analysis of integrin αIIbβ3 adhesion contacts using total internal reflection fluorescence microscopy revealed an important role for PI3Ks in regulating the stability of high affinity integrin αIIbβ3 bonds. These studies demonstrate an important role for PI3K p110β in regulating the avidity of high affinity integrin αIIbβ3 receptors, necessary for the cellular transmission of contractile forces. These findings may provide new insight into the potential antithrombotic properties of PI3K p110β inhibitors. PMID:19940148

Role of YAP/TAZ in cell-matrix adhesion-mediated signalling and mechanotransduction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dupont, Sirio, E-mail: sirio.dupont@unipd.it

2016-04-10

Signalling from the extracellular matrix (ECM) is a fundamental cellular input that sustains proliferation, opposes cell death and regulates differentiation. Through integrins, cells perceive both the chemical composition and physical properties of the ECM. In particular, cell behaviour is profoundly influenced by the mechanical elasticity or stiffness of the ECM, which regulates the ability of cells to develop forces through their contractile actomyosin cytoskeleton and to mature focal adhesions. This mechanosensing ability affects fundamental cellular functions, such that alterations of ECM stiffness is nowadays considered not a simple consequence of pathology, but a causative input driving aberrant cell behaviours. Wemore » here discuss recent advances on how mechanical signals intersect nuclear transcription and in particular the activity of YAP/TAZ transcriptional coactivators, known downstream transducers of the Hippo pathway and important effectors of ECM mechanical cues.« less

Role of myometrial activity in sperm transport through the genital tract and in fertilization in sows.

PubMed

Langendijk, P; Bouwman, E G; Kidson, A; Kirkwood, R N; Soede, N M; Kemp, B

2002-05-01

The effects of stimulation and suppression of uterine contractility at about the time of insemination on sperm distribution and fertilization in multiparous sows are described. For assessment of fertilization, sows were inseminated about 28 h before (synchronized) ovulation and killed at day 5 after ovulation (n = 53). For assessment of sperm distribution, sows were inseminated about 20 h before expected ovulation and were killed 12 h later (n = 26). At 10 min before insemination, sows received an intrauterine infusion of one of three solutions: (i) saline (control); (ii) 0.60 mg clenbuterol hydrochloride to suppress contractility; or (iii) 1 mg cloprostenol to stimulate contractility. Both clenbuterol and cloprostenol reduced median fertilization rate (P < 0.05) and median number of accessory sperm cells (P < 0.05). Distribution of sperm cells was also affected by treatments. Clenbuterol increased, and cloprostenol decreased, the number of sperm cells (P < 0.05) in the proximal 20 cm of the uterine horn and in the uterotubal junction. In addition, clenbuterol tended to increase and cloprostenol tended to decrease the number of sperm cells in the isthmus, although these effects were not significant. However, relative to the number of sperm cells in the uterus, clenbuterol treatment reduced the number of sperm cells in the uterotubal junction and oviduct, in contrast to cloprostenol. Cloprostenol increased the reflux of semen during insemination. It is hypothesized that suppression of uterine contractility increases transuterine transport time, reducing the ability of sperm cells to enter the uterotubal junction and the oviduct. Stimulation of uterine contractility above a certain level probably increases reflux and impedes transuterine transport of sufficient numbers of sperm cells.

Wounded cells drive rapid epidermal repair in the early Drosophila embryo

PubMed Central

Fernandez-Gonzalez, Rodrigo; Zallen, Jennifer A.

2013-01-01

Epithelial tissues are protective barriers that display a remarkable ability to repair wounds. Wound repair is often associated with an accumulation of actin and nonmuscle myosin II around the wound, forming a purse string. The role of actomyosin networks in generating mechanical force during wound repair is not well understood. Here we investigate the mechanisms of force generation during wound repair in the epidermis of early and late Drosophila embryos. We find that wound closure is faster in early embryos, where, in addition to a purse string around the wound, actomyosin networks at the medial cortex of the wounded cells contribute to rapid wound repair. Laser ablation demonstrates that both medial and purse-string actomyosin networks generate contractile force. Quantitative analysis of protein localization dynamics during wound closure indicates that the rapid contraction of medial actomyosin structures during wound repair in early embryos involves disassembly of the actomyosin network. By contrast, actomyosin purse strings in late embryos contract more slowly in a mechanism that involves network condensation. We propose that the combined action of two force-generating structures—a medial actomyosin network and an actomyosin purse string—contributes to the increased efficiency of wound repair in the early embryo. PMID:23985320

Dynamics of elastic interactions in soft and biological matter.

PubMed

Yuval, Janni; Safran, Samuel A

2013-04-01

Cells probe their mechanical environment and can change the organization of their cytoskeletons when the elastic and viscous properties of their environment are modified. We use a model in which the forces exerted by small, contractile acto-myosin filaments (e.g., nascent stress fibers in stem cells) on the extracellular matrix are modeled as local force dipoles. In some cases, the strain field caused by these force dipoles propagates quickly enough so that only static elastic interactions need be considered. On the other hand, in the case of significant energy dissipation, strain propagation is slower and may be eliminated completely by the relaxation of the cellular cytoskeleton (e.g., by cross-link dissociation). Here, we consider several dissipative mechanisms that affect the propagation of the strain field in adhered cells and consider these effects on the interaction between force dipoles and their resulting mutual orientations. This is a first step in understanding the development of orientational (nematic) or layering (smectic) order in the cytoskeleton. We use the theory to estimate the propagation time of the strain fields over a cellular distance for different mechanisms and find that in some cases it can be of the order of seconds, thus competing with the cytoskeletal relaxation time. Furthermore, for a simple system of two force dipoles, we predict that in some cases the orientation of force dipoles might change significantly with time, e.g., for short times the dipoles exhibit parallel alignment while for later times they align perpendicularly.

Contractile properties of skinned muscle fibres from young and adult normal and dystrophic (mdx) mice.

PubMed Central

Williams, D A; Head, S I; Lynch, G S; Stephenson, D G

1993-01-01

1. Single muscle fibres were enzymatically isolated from the soleus and extensor digitorum longus (EDL) muscles of genetically dystrophic mdx and normal (C57BL/10) mice aged 3-6 or 17-23 weeks. 2. Fibres of both muscles were chemically skinned with the non-ionic detergent Triton X-100 (2% v/v). Ca(2+)- and Sr(2+)-activated contractile responses were recorded and comparisons were made between several contractile parameters of various fibre types of normal and dystrophic mice of similar age. 3. There were no significant differences in the following contractile parameters of skinned fibres of normal and mdx mice of the same age: sensitivity to activating Ca2+ (pCa50) or Sr2+ (pSr50) and differential sensitivity to the activating ions (pCa50-pSr50). However the maximum isometric tension (Po) and the frequency of myofibrillar force oscillations in EDL fast-twitch fibres of young mdx mice were significantly lower than those of soleus fast-twitch fibres of the same animals, or fast-twitch fibres (EDL or soleus) of normal mice. 4. Age-related differences were apparent in some contractile parameters of both normal and mdx mice. In particular the steepness of force-pCa and force-pSr curves increased with age in normal mice, yet decreased with age in fibres of mdx mice. 5. A fluorescent probe, ethidium bromide, which interchelates with DNA, was used with laser-scanning confocal microscopy to determine the distribution of myonuclei in fibres. Fibres isolated from either muscle type of normal animals displayed a characteristic peripheral spiral of myonuclei. Fibres from muscles of mdx mice displayed three major patterns of nuclear distribution; the normal peripheral spiral, long central strands of nuclei, and a mixture of these two patterns. 6. The contractile characteristics of mdx fibres were not markedly influenced by the nuclear distribution pattern in that there were no discernible differences in the major contractile parameters (the Hill coefficients nCa and nSr, which are associated with the steepness of the Ca2+ and Sr2+ activation curves, pCa50, pSr50, pCa50-pSr50) of skinned fibres possessing peripheral or central nuclei. However, except for nSr, these values were all lower in individual fibres which displayed similar proportions of central and peripheral nuclei. The presence of mixed nucleation and absence of fibres with embryonic contractile characteristics in mdx mice suggest that the dystrophin-negative fibres can repair locally occurring muscle damage. Images Fig. 1 Fig. 1(Contd.) Fig. 4 Fig. 5 PMID:8487206

Using Force to Punch Holes: Mechanics of Contractile Nanomachines.

PubMed

Brackmann, Maximilian; Nazarov, Sergey; Wang, Jing; Basler, Marek

2017-09-01

Using physical force to translocate macromolecules across a membrane has the advantage of being a universal solution independent of the properties of the target membrane. However, physically punching a stiff membrane is not a trivial task and three things are necessary for success: a sharp tip, a source of energy, and the ability to strongly bind to the target. In this review we describe the basic mechanism of membrane puncturing by contractile nanomachines with a focus on the T4 phage, R-type pyocin, and the bacterial Type VI secretion system (T6SS) based on recent studies of the structures and dynamics of their assembly. Copyright © 2017 Elsevier Ltd. All rights reserved.

Micro pumping with cardiomyocyte-polymer hybrid.

PubMed

Park, Jungyul; Kim, Il Chaek; Baek, Jeongeun; Cha, Misun; Kim, Jinseok; Park, Sukho; Lee, Junghoon; Kim, Byungkyu

2007-10-01

This paper presents a hybrid micropump actuated by the up-down motion of a dome shaped cell-polymer membrane composite. The contractile force induced from self-beating cardiomyocytes cultured on the membrane causes shrinkage and relaxation of a microchamber, leading to a flow in a microchannel. Flow direction is controlled by the geometry of diffuser/nozzle in the microchannel. The fabrication process is noninvasive to cells, thus, cardiomyocytes can robustly maintain their activity for a long time. The fluid motion in the microchannel was monitored by tracking 2 microm polystyrene beads. A net flow rate of 0.226 nl min(-1) was obtained in our microscale device. Our device demonstrates a unique performance of a cell-microdevice hybrid lab-on-a-chip that does not require any external power source, preventing electrical or heat shock to analytes.

Introduction of non-linear elasticity models for characterization of shape and deformation statistics: application to contractility assessment of isolated adult cardiocytes.

PubMed

Bazan, Carlos; Hawkins, Trevor; Torres-Barba, David; Blomgren, Peter; Paolini, Paul

2011-08-22

We are exploring the viability of a novel approach to cardiocyte contractility assessment based on biomechanical properties of the cardiac cells, energy conservation principles, and information content measures. We define our measure of cell contraction as being the distance between the shapes of the contracting cell, assessed by the minimum total energy of the domain deformation (warping) of one cell shape into another. To guarantee a meaningful vis-à-vis correspondence between the two shapes, we employ both a data fidelity term and a regularization term. The data fidelity term is based on nonlinear features of the shapes while the regularization term enforces the compatibility between the shape deformations and that of a hyper-elastic material. We tested the proposed approach by assessing the contractile responses in isolated adult rat cardiocytes and contrasted these measurements against two different methods for contractility assessment in the literature. Our results show good qualitative and quantitative agreements with these methods as far as frequency, pacing, and overall behavior of the contractions are concerned. We hypothesize that the proposed methodology, once appropriately developed and customized, can provide a framework for computational cardiac cell biomechanics that can be used to integrate both theory and experiment. For example, besides giving a good assessment of contractile response of the cardiocyte, since the excitation process of the cell is a closed system, this methodology can be employed in an attempt to infer statistically significant model parameters for the constitutive equations of the cardiocytes.

Cell prestress. II. Contribution of microtubules

NASA Technical Reports Server (NTRS)

Stamenovic, Dimitrije; Mijailovich, Srboljub M.; Tolic-Norrelykke, Iva Marija; Chen, Jianxin; Wang, Ning; Ingber, D. E. (Principal Investigator)

2002-01-01

The tensegrity model hypothesizes that cytoskeleton-based microtubules (MTs) carry compression as they balance a portion of cell contractile stress. To test this hypothesis, we used traction force microscopy to measure traction at the interface of adhering human airway smooth muscle cells and a flexible polyacrylamide gel substrate. The prediction is that if MTs balance a portion of contractile stress, then, upon their disruption, the portion of stress balanced by MTs would shift to the substrate, thereby causing an increase in traction. Measurements were done first in maximally activated cells (10 microM histamine) and then again after MTs had been disrupted (1 microM colchicine). We found that after disruption of MTs, traction increased on average by approximately 13%. Because in activated cells colchicine induced neither an increase in intracellular Ca(2+) nor an increase in myosin light chain phosphorylation as shown previously, we concluded that the observed increase in traction was a result of load shift from MTs to the substrate. In addition, energy stored in the flexible substrate was calculated as work done by traction on the deformation of the substrate. This result was then utilized in an energetic analysis. We assumed that cytoskeleton-based MTs are slender elastic rods supported laterally by intermediate filaments and that MTs buckle as the cell contracts. Using the post-buckling equilibrium theory of Euler struts, we found that energy stored during buckling of MTs was quantitatively consistent with the measured increase in substrate energy after disruption of MTs. This is further evidence supporting the idea that MTs are intracellular compression-bearing elements.

Cortical PAR polarity proteins promote robust cytokinesis during asymmetric cell division

PubMed Central

Jordan, Shawn N.; Davies, Tim; Zhuravlev, Yelena; Dumont, Julien; Shirasu-Hiza, Mimi

2016-01-01

Cytokinesis, the physical division of one cell into two, is thought to be fundamentally similar in most animal cell divisions and driven by the constriction of a contractile ring positioned and controlled solely by the mitotic spindle. During asymmetric cell divisions, the core polarity machinery (partitioning defective [PAR] proteins) controls the unequal inheritance of key cell fate determinants. Here, we show that in asymmetrically dividing Caenorhabditis elegans embryos, the cortical PAR proteins (including the small guanosine triphosphatase CDC-42) have an active role in regulating recruitment of a critical component of the contractile ring, filamentous actin (F-actin). We found that the cortical PAR proteins are required for the retention of anillin and septin in the anterior pole, which are cytokinesis proteins that our genetic data suggest act as inhibitors of F-actin at the contractile ring. Collectively, our results suggest that the cortical PAR proteins coordinate the establishment of cell polarity with the physical process of cytokinesis during asymmetric cell division to ensure the fidelity of daughter cell formation. PMID:26728855

Myosin phosphorylation improves contractile economy of mouse fast skeletal muscle during staircase potentiation.

PubMed

Bunda, Jordan; Gittings, William; Vandenboom, Rene

2018-01-30

Phosphorylation of the myosin regulatory light chain (RLC) by skeletal myosin light chain kinase (skMLCK) potentiates rodent fast twitch muscle but is an ATP-requiring process. Our objective was to investigate the effect of skMLCK-catalyzed RLC phosphorylation on the energetic cost of contraction and the contractile economy (ratio of mechanical output to metabolic input) of mouse fast twitch muscle in vitro (25°C). To this end, extensor digitorum longus (EDL) muscles from wild-type (WT) and from skMLCK-devoid (skMLCK -/- ) mice were subjected to repetitive low-frequency stimulation (10 Hz for 15 s) to produce staircase potentiation of isometric twitch force, after which muscles were quick frozen for determination of high-energy phosphate consumption (HEPC). During stimulation, WT muscles displayed significant potentiation of isometric twitch force while skMLCK -/- muscles did not (i.e. 23% versus 5% change, respectively). Consistent with this, RLC phosphorylation was increased ∼3.5-fold from the unstimulated control value in WT but not in skMLCK -/- muscles. Despite these differences, the HEPC of WT muscles was not greater than that of skMLCK -/- muscles. As a result of the increased contractile output relative to HEPC, the calculated contractile economy of WT muscles was greater than that of skMLCK -/- muscles. Thus, our results suggest that skMLCK-catalyzed phosphorylation of the myosin RLC increases the contractile economy of WT mouse EDL muscle compared with skMLCK -/- muscles without RLC phosphorylation. © 2018. Published by The Company of Biologists Ltd.

A human in vitro model of Duchenne muscular dystrophy muscle formation and contractility.

PubMed

Nesmith, Alexander P; Wagner, Matthew A; Pasqualini, Francesco S; O'Connor, Blakely B; Pincus, Mark J; August, Paul R; Parker, Kevin Kit

2016-10-10

Tongue weakness, like all weakness in Duchenne muscular dystrophy (DMD), occurs as a result of contraction-induced muscle damage and deficient muscular repair. Although membrane fragility is known to potentiate injury in DMD, whether muscle stem cells are implicated in deficient muscular repair remains unclear. We hypothesized that DMD myoblasts are less sensitive to cues in the extracellular matrix designed to potentiate structure-function relationships of healthy muscle. To test this hypothesis, we drew inspiration from the tongue and engineered contractile human muscle tissues on thin films. On this platform, DMD myoblasts formed fewer and smaller myotubes and exhibited impaired polarization of the cell nucleus and contractile cytoskeleton when compared with healthy cells. These structural aberrations were reflected in their functional behavior, as engineered tongues from DMD myoblasts failed to achieve the same contractile strength as healthy tongue structures. These data suggest that dystrophic muscle may fail to organize with respect to extracellular cues necessary to potentiate adaptive growth and remodeling. © 2016 Nesmith et al.

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

PubMed Central

Wang, Tao; Wang, Ruping; Cleary, Rachel A.; Gannon, Olivia J.; Tang, Dale D.

2015-01-01

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. PMID:25713069

Spontaneous actin dynamics in contractile rings

NASA Astrophysics Data System (ADS)

Kruse, Karsten; Wollrab, Viktoria; Thiagarajan, Raghavan; Wald, Anne; Riveline, Daniel

Networks of polymerizing actin filaments are known to be capable to self-organize into a variety of structures. For example, spontaneous actin polymerization waves have been observed in living cells in a number of circumstances, notably, in crawling neutrophils and slime molds. During later stages of cell division, they can also spontaneously form a contractile ring that will eventually cleave the cell into two daughter cells. We present a framework for describing networks of polymerizing actin filaments, where assembly is regulated by various proteins. It can also include the effects of molecular motors. We show that the molecular processes driven by these proteins can generate various structures that have been observed in contractile rings of fission yeast and mammalian cells. We discuss a possible functional role of each of these patterns. The work was supported by Agence Nationale de la Recherche, France, (ANR-10-LABX-0030-INRT) and by Deutsche Forschungsgemeinschaft through SFB1027.

Smooth muscle length adaptation and actin filament length: a network model of the cytoskeletal dysregulation.

PubMed

Silveira, Paulo S P; Fredberg, Jeffrey J

2005-10-01

Length adaptation of the airway smooth muscle cell is attributable to cytoskeletal remodeling. It has been proposed that dysregulated actin filaments may become longer in asthma, and that such elongation would prevent a parallel-to-series transition of contractile units, thus precluding the well-known beneficial effects of deep inspirations and tidal breathing. To test the potential effect that actin filament elongation could have in overall muscle mechanics, we present an extremely simple model. The cytoskeleton is represented as a 2-D network of links (contractile filaments) connecting nodes (adhesion plaques). Such a network evolves in discrete time steps by forming and dissolving links in a stochastic fashion. Links are formed by idealized contractile units whose properties are either those from normal or elongated actin filaments. Oscillations were then imposed on the network to evaluate both the effects of breathing and length adaptation. In response to length oscillation, a network with longer actin filaments showed smaller decreases of force, smaller increases in compliance, and higher shortening velocities. Taken together, these changes correspond to a network that is refractory to the effects of breathing and therefore approximates an asthmatic scenario. Thus, an extremely simple model seems to capture some relatively complex mechanics of airway smooth muscle, supporting the idea that dysregulation of actin filament length may contribute to excessive airway narrowing.

Estrogen and testosterone in concert with EFNB3 regulate vascular smooth muscle cell contractility and blood pressure.

PubMed

Wang, Yujia; Wu, Zenghui; Thorin, Eric; Tremblay, Johanne; Lavoie, Julie L; Luo, Hongyu; Peng, Junzheng; Qi, Shijie; Wu, Tao; Chen, Fei; Shen, Jianzhong; Hu, Shenjiang; Wu, Jiangping

2016-04-01

EPH kinases and their ligands, ephrins (EFNs), have vital and diverse biological functions, although their function in blood pressure (BP) control has not been studied in detail. In the present study, we report that Efnb3 gene knockout (KO) led to increased BP in female but not male mice. Vascular smooth muscle cells (VSMCs) were target cells for EFNB3 function in BP regulation. The deletion of EFNB3 augmented contractility of VSMCs from female but not male KO mice, compared with their wild-type (WT) counterparts. Estrogen augmented VSMC contractility while testosterone reduced it in the absence of EFNB3, although these sex hormones had no effect on the contractility of VSMCs from WT mice. The effect of estrogen on KO VSMC contractility was via a nongenomic pathway involving GPER, while that of testosterone was likely via a genomic pathway, according to VSMC contractility assays and GPER knockdown assays. The sex hormone-dependent contraction phenotypes in KO VSMCs were reflected in BP in vivo. Ovariectomy rendered female KO mice normotensive. At the molecular level, EFNB3 KO in VSMCs resulted in reduced myosin light chain kinase phosphorylation, an event enhancing sensitivity to Ca(2+)flux in VSMCs. Our investigation has revealed previously unknown EFNB3 functions in BP regulation and show that EFNB3 might be a hypertension risk gene in certain individuals. Copyright © 2016 the American Physiological Society.

Rho A and the Rho kinase pathway regulate fibroblast contraction: Enhanced contraction in constitutively active Rho A fibroblast cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nobe, Koji, E-mail: kojinobe@pharm.showa-u.ac.jp; Nobe, Hiromi; Department of Physical Therapy, Bunkyo-Gakuin University

Research highlights: {yields} Mechanisms of fibroblast cell contraction in collagen matrix. {yields} Assessed an isometric force development using 3D-reconstituted-fibroblast fiber. {yields} Constitutively active Rho A induced the over-contraction of fibroblast cells. {yields} Rho A and Rho kinase pathway has a central role in fibroblast cell contraction. -- Abstract: Fibroblast cells play a central role in the proliferation phase of wound healing processes, contributing to force development. The intracellular signaling pathways regulating this non-muscle contraction are only partially understood. To study the relations between Rho A and contractile responses, constitutively active Rho A (CA-Rho A) fibroblast cells were reconstituted into fibersmore » and the effects of calf serum (CS) on isometric force were studied. CS-induced force in CA-Rho A fibroblast fibers was twice as large as that in wild type (NIH 3T3) fibroblast fibers. During this response, the translocation of Rho A from the cytosol to the membrane was detected by Rho A activity assays and Western blot analysis. Pre-treatment with a Rho specific inhibitor (C3-exoenzyme) suppressed translocation as well as contraction. These results indicate that Rho A activation is essential for fibroblast contraction. The Rho kinase inhibitor ( (Y27632)) inhibited both NIH 3T3 and CA-Rho A fibroblast fiber contractions. Activation of Rho A is thus directly coupled with Rho kinase activity. We conclude that the translocation of Rho A from the cytosol to the membrane and the Rho kinase pathway can regulate wound healing processes mediated by fibroblast contraction.« less

New phenotypic aspects of the decidual spiral artery wall during early post-implantation mouse pregnancy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Elia, Artemis; Charalambous, Fotini; Georgiades, Pantelis, E-mail: pgeor@ucy.ac.cy

Highlights: Black-Right-Pointing-Pointer Spiral artery (SA) wall remodeling (SAR) is ill-defined and clinically important. Black-Right-Pointing-Pointer SA muscular phenotype prior to and during SAR in mice is underexplored. Black-Right-Pointing-Pointer SA muscular wall consists of contractile and non-contractile components. Black-Right-Pointing-Pointer SA wall non-contractile component may be synthetic smooth muscle. Black-Right-Pointing-Pointer Timing and extent of SA wall contractile component loss is revealed. -- Abstract: During pregnancy the walls of decidual spiral arteries (SAs) undergo clinically important structural modifications crucial for embryo survival/growth and maternal health. However, the mechanisms of SA remodeling (SAR) are poorly understood. Although an important prerequisite to this understanding is knowledgemore » about the phenotype of SA muscular wall prior to and during the beginning of mouse SAR, this remains largely unexplored and was the main aim of this work. Using histological and immunohistochemical techniques, this study shows for the first time that during early mouse gestation, from embryonic day 7.5 (E7.5) to E10.5, the decidual SA muscular coat is not a homogeneous structure, but consists of two concentric layers. The first is a largely one cell-thick sub-endothelial layer of contractile mural cells (positive for {alpha}-smooth muscle actin, calponin and SM22{alpha}) with pericyte characteristics (NG2 positive). The second layer is thicker, and evidence is presented that it may be of the synthetic/proliferative smooth muscle phenotype, based on absence ({alpha}-smooth muscle actin and calponin) or weak (SM22{alpha}) expression of contractile mural cell markers, and presence of synthetic smooth muscle characteristics (expression of non-muscle Myosin heavy chain-IIA and of the cell proliferation marker PCNA). Importantly, immunohistochemistry and morphometrics showed that the contractile mural cell layer although prominent at E7.5-E8.5, becomes drastically reduced by E10.5 and is undetectable by E12.5. In conclusion, this study reveals novel aspects of the decidual SA muscular coat phenotype prior to and during early SAR that may have important implications for understanding the mechanisms of SAR.« less

Low concentrations of niflumic acid enhance basal spontaneous and carbachol-induced contractions of the detrusor.

PubMed

Lam, Wai Ping; Tang, Hong Chai; Zhang, Xin; Leung, Ping Chung; Yew, David Tai Wai; Liang, Willmann

2014-02-01

The urinary bladder expresses Ca(2+)-activated Cl(-) channels (CACC), but its physiological role in governing contractility remains to be defined. The CACC modulator niflumic acid (NFA) is widely used despite the variable results arisen from different drug concentrations used. This study was designed to examine the effects of NFA at low concentrations on detrusor strip contractility. Rat detrusor strips with mucosa-intact (+MU) and mucosa-denuded (-MU) were prepared in transverse (Tr) and longitudinal (Lg) with respect to the bladder orientation. Isometric force measurements were made at baseline (for spontaneous phasic contractile activity) and during drug stimulation (by carbachol, CCh) with and without NFA. NFA (1 and 10 μmol/L) pretreatment enhanced CCh-induced contractions more in +MU than -MU strips with no selectivity on contractile direction. For spontaneous phasic contractions, NFA-treated strips in the Tr direction showed increased phasic amplitude, while phasic frequency was unchanged. The findings suggest low concentrations of NFA having a potentiating effect on detrusor contractions that was sensitive to the MU and contractile direction.

Changes in muscle fiber contractility and extracellular matrix production during skeletal muscle hypertrophy.

PubMed

Mendias, Christopher L; Schwartz, Andrew J; Grekin, Jeremy A; Gumucio, Jonathan P; Sugg, Kristoffer B

2017-03-01

Skeletal muscle can adapt to increased mechanical loads by undergoing hypertrophy. Transient reductions in whole muscle force production have been reported during the onset of hypertrophy, but contractile changes in individual muscle fibers have not been previously studied. Additionally, the extracellular matrix (ECM) stores and transmits forces from muscle fibers to tendons and bones, and determining how the ECM changes during hypertrophy is important in understanding the adaptation of muscle tissue to mechanical loading. Using the synergist ablation model, we sought to measure changes in muscle fiber contractility, collagen content, and cross-linking, and in the expression of several genes and activation of signaling proteins that regulate critical components of myogenesis and ECM synthesis and remodeling during muscle hypertrophy. Tissues were harvested 3, 7, and 28 days after induction of hypertrophy, and nonoverloaded rats served as controls. Muscle fiber specific force (sF o ), which is the maximum isometric force normalized to cross-sectional area, was reduced 3 and 7 days after the onset of mechanical overload, but returned to control levels by 28 days. Collagen abundance displayed a similar pattern of change. Nearly a quarter of the transcriptome changed over the course of overload, as well as the activation of signaling pathways related to hypertrophy and atrophy. Overall, this study provides insight into fundamental mechanisms of muscle and ECM growth, and indicates that although muscle fibers appear to have completed remodeling and regeneration 1 mo after synergist ablation, the ECM continues to be actively remodeling at this time point. NEW & NOTEWORTHY This study utilized a rat synergist ablation model to integrate changes in single muscle fiber contractility, extracellular matrix composition, activation of important signaling pathways in muscle adaption, and corresponding changes in the muscle transcriptome to provide novel insight into the basic biological mechanisms of muscle fiber hypertrophy. Copyright © 2017 the American Physiological Society.

Changes in muscle fiber contractility and extracellular matrix production during skeletal muscle hypertrophy

PubMed Central

Schwartz, Andrew J.; Grekin, Jeremy A.; Gumucio, Jonathan P.; Sugg, Kristoffer B.

2017-01-01

Skeletal muscle can adapt to increased mechanical loads by undergoing hypertrophy. Transient reductions in whole muscle force production have been reported during the onset of hypertrophy, but contractile changes in individual muscle fibers have not been previously studied. Additionally, the extracellular matrix (ECM) stores and transmits forces from muscle fibers to tendons and bones, and determining how the ECM changes during hypertrophy is important in understanding the adaptation of muscle tissue to mechanical loading. Using the synergist ablation model, we sought to measure changes in muscle fiber contractility, collagen content, and cross-linking, and in the expression of several genes and activation of signaling proteins that regulate critical components of myogenesis and ECM synthesis and remodeling during muscle hypertrophy. Tissues were harvested 3, 7, and 28 days after induction of hypertrophy, and nonoverloaded rats served as controls. Muscle fiber specific force (sFo), which is the maximum isometric force normalized to cross-sectional area, was reduced 3 and 7 days after the onset of mechanical overload, but returned to control levels by 28 days. Collagen abundance displayed a similar pattern of change. Nearly a quarter of the transcriptome changed over the course of overload, as well as the activation of signaling pathways related to hypertrophy and atrophy. Overall, this study provides insight into fundamental mechanisms of muscle and ECM growth, and indicates that although muscle fibers appear to have completed remodeling and regeneration 1 mo after synergist ablation, the ECM continues to be actively remodeling at this time point. NEW & NOTEWORTHY This study utilized a rat synergist ablation model to integrate changes in single muscle fiber contractility, extracellular matrix composition, activation of important signaling pathways in muscle adaption, and corresponding changes in the muscle transcriptome to provide novel insight into the basic biological mechanisms of muscle fiber hypertrophy. PMID:27979985

Overexpression of antioxidant enzymes in diaphragm muscle does not alter contraction-induced fatigue or recovery

PubMed Central

McClung, Joseph M.; DeRuisseau, Keith C.; Whidden, Melissa A.; Van Remmen, Holly; Richardson, Arlan; Song, Wook; Vrabas, Ioannis S.; Powers, Scott K.

2010-01-01

Low levels of reactive oxygen species (ROS) production are necessary to optimize muscle force production in unfatigued muscle. In contrast, sustained high levels of ROS production have been linked to impaired muscle force production and contraction-induced skeletal muscle fatigue. Using genetically engineered mice, we tested the hypothesis that the independent transgenic overexpression of catalase (CAT), copper/zinc superoxide dismutase (CuZnSOD; SOD1) or manganese superoxide dismutase (MnSOD; SOD2) antioxidant enzymes would negatively affect force production in unfatigued diaphragm muscle but would delay the development of muscle fatigue and enhance force recovery after fatiguing contractions. Diaphragm muscle from wild-type littermates (WT) and from CAT, SOD1 and SOD2 overexpressing mice were subjected to an in vitro contractile protocol to investigate the force–frequency characteristics, the fatigue properties and the time course of recovery from fatigue. The CAT, SOD1 and SOD2 overexpressors produced less specific force (in N cm−2) at stimulation frequencies of 20–300 Hz and produced lower maximal tetanic force than WT littermates. The relative development of muscle fatigue and recovery from fatigue were not influenced by transgenic overexpression of any antioxidant enzyme. Morphologically, the mean cross-sectional area (in μm2) of diaphragm myofibres expressing myosin heavy chain type IIA was decreased in both CAT and SOD2 transgenic animals, and the percentage of non-contractile tissue increased in diaphragms from all transgenic mice. In conclusion, our results do not support the hypothesis that overexpression of independent antioxidant enzymes protects diaphragm muscle from contraction-induced fatigue or improves recovery from fatigue. Moreover, our data are consistent with the concept that a basal level of ROS is important to optimize muscle force production, since transgenic overexpression of major cellular antioxidants is associated with contractile dysfunction. Finally, the transgenic overexpression of independent endogenous antioxidants alters diaphragm skeletal muscle morphology, and these changes may also contribute to the diminished specific force production observed in these animals. PMID:19783618

Unobtrusive Estimation of Cardiac Contractility and Stroke Volume Changes Using Ballistocardiogram Measurements on a High Bandwidth Force Plate

PubMed Central

Ashouri, Hazar; Orlandic, Lara; Inan, Omer T.

2016-01-01

Unobtrusive and inexpensive technologies for monitoring the cardiovascular health of heart failure (HF) patients outside the clinic can potentially improve their continuity of care by enabling therapies to be adjusted dynamically based on the changing needs of the patients. Specifically, cardiac contractility and stroke volume (SV) are two key aspects of cardiovascular health that change significantly for HF patients as their condition worsens, yet these parameters are typically measured only in hospital/clinical settings, or with implantable sensors. In this work, we demonstrate accurate measurement of cardiac contractility (based on pre-ejection period, PEP, timings) and SV changes in subjects using ballistocardiogram (BCG) signals detected via a high bandwidth force plate. The measurement is unobtrusive, as it simply requires the subject to stand still on the force plate while holding electrodes in the hands for simultaneous electrocardiogram (ECG) detection. Specifically, we aimed to assess whether the high bandwidth force plate can provide accuracy beyond what is achieved using modified weighing scales we have developed in prior studies, based on timing intervals, as well as signal-to-noise ratio (SNR) estimates. Our results indicate that the force plate BCG measurement provides more accurate timing information and allows for better estimation of PEP than the scale BCG (r2 = 0.85 vs. r2 = 0.81) during resting conditions. This correlation is stronger during recovery after exercise due to more significant changes in PEP (r2 = 0.92). The improvement in accuracy can be attributed to the wider bandwidth of the force plate. ∆SV (i.e., changes in stroke volume) estimations from the force plate BCG resulted in an average error percentage of 5.3% with a standard deviation of ±4.2% across all subjects. Finally, SNR calculations showed slightly better SNR in the force plate measurements among all subjects but the small difference confirmed that SNR is limited by motion artifacts rather than instrumentation. PMID:27240380

Actomyosin contractility rotates the cell nucleus

PubMed Central

Kumar, Abhishek; Maitra, Ananyo; Sumit, Madhuresh; Ramaswamy, Sriram; Shivashankar, G. V.

2014-01-01

The cell nucleus functions amidst active cytoskeletal filaments, but its response to their contractile stresses is largely unexplored. We study the dynamics of the nuclei of single fibroblasts, with cell migration suppressed by plating onto micro-fabricated patterns. We find the nucleus undergoes noisy but coherent rotational motion. We account for this observation through a hydrodynamic approach, treating the nucleus as a highly viscous inclusion residing in a less viscous fluid of orientable filaments endowed with active stresses. Lowering actin contractility selectively by introducing blebbistatin at low concentrations drastically reduced the speed and coherence of the angular motion of the nucleus. Time-lapse imaging of actin revealed a correlated hydrodynamic flow around the nucleus, with profile and magnitude consistent with the results of our theoretical approach. Coherent intracellular flows and consequent nuclear rotation thus appear to be an intrinsic property of cells. PMID:24445418

Actomyosin contractility rotates the cell nucleus.

PubMed

Kumar, Abhishek; Maitra, Ananyo; Sumit, Madhuresh; Ramaswamy, Sriram; Shivashankar, G V

2014-01-21

The cell nucleus functions amidst active cytoskeletal filaments, but its response to their contractile stresses is largely unexplored. We study the dynamics of the nuclei of single fibroblasts, with cell migration suppressed by plating onto micro-fabricated patterns. We find the nucleus undergoes noisy but coherent rotational motion. We account for this observation through a hydrodynamic approach, treating the nucleus as a highly viscous inclusion residing in a less viscous fluid of orientable filaments endowed with active stresses. Lowering actin contractility selectively by introducing blebbistatin at low concentrations drastically reduced the speed and coherence of the angular motion of the nucleus. Time-lapse imaging of actin revealed a correlated hydrodynamic flow around the nucleus, with profile and magnitude consistent with the results of our theoretical approach. Coherent intracellular flows and consequent nuclear rotation thus appear to be an intrinsic property of cells.

Preservation of cardiac function by prolonged action potentials in mice deficient of KChIP2.

PubMed

Grubb, Søren; Aistrup, Gary L; Koivumäki, Jussi T; Speerschneider, Tobias; Gottlieb, Lisa A; Mutsaers, Nancy A M; Olesen, Søren-Peter; Calloe, Kirstine; Thomsen, Morten B

2015-08-01

Inherited ion channelopathies and electrical remodeling in heart disease alter the cardiac action potential with important consequences for excitation-contraction coupling. Potassium channel-interacting protein 2 (KChIP2) is reduced in heart failure and interacts under physiological conditions with both Kv4 to conduct the fast-recovering transient outward K(+) current (Ito,f) and with CaV1.2 to mediate the inward L-type Ca(2+) current (ICa,L). Anesthetized KChIP2(-/-) mice have normal cardiac contraction despite the lower ICa,L, and we hypothesized that the delayed repolarization could contribute to the preservation of contractile function. Detailed analysis of current kinetics shows that only ICa,L density is reduced, and immunoblots demonstrate unaltered CaV1.2 and CaVβ₂ protein levels. Computer modeling suggests that delayed repolarization would prolong the period of Ca(2+) entry into the cell, thereby augmenting Ca(2+)-induced Ca(2+) release. Ca(2+) transients in disaggregated KChIP2(-/-) cardiomyocytes are indeed comparable to wild-type transients, corroborating the preserved contractile function and suggesting that the compensatory mechanism lies in the Ca(2+)-induced Ca(2+) release event. We next functionally probed dyad structure, ryanodine receptor Ca(2+) sensitivity, and sarcoplasmic reticulum Ca(2+) load and found that increased temporal synchronicity of the Ca(2+) release in KChIP2(-/-) cardiomyocytes may reflect improved dyad structure aiding the compensatory mechanisms in preserving cardiac contractile force. Thus the bimodal effect of KChIP2 on Ito,f and ICa,L constitutes an important regulatory effect of KChIP2 on cardiac contractility, and we conclude that delayed repolarization and improved dyad structure function together to preserve cardiac contraction in KChIP2(-/-) mice. Copyright © 2015 the American Physiological Society.

Adenoviral gene transfer of Akt enhances myocardial contractility and intracellular calcium handling

PubMed Central

Cittadini, A; Monti, MG; Iaccarino, G; Di Rella, F; Tsichlis, PN; Di Gianni, A; Strömer, H; Sorriento, D; Peschle, C; Trimarco, B; Saccà, L; Condorelli, G

2010-01-01

The serine-threonine kinase Akt/PKB mediates stimuli from different classes of cardiomyocyte receptors, including the growth hormone/insulin like growth factor and the β-adrenergic receptors. Whereas the growth-promoting and antiapoptotic properties of Akt activation are well established, little is known about the effects of Akt on myocardial contractility, intracellular calcium (Ca2+) handling, oxygen consumption, and β-adrenergic pathway. To this aim, Sprague–Dawley rats were subjected to a wild-type Akt in vivo adenoviral gene transfer using a catheter-based technique combined with aortopulmonary crossclamping. Left ventricular (LV) contractility and intracellular Ca2+ handling were evaluated in an isolated isovolumic buffer-perfused, aequorin-loaded whole heart preparations 10 days after the surgery. The Ca2+–force relationship was obtained under steady-state conditions in tetanized muscles. No significant hypertrophy was detected in adenovirus with wild-type Akt (Ad.Akt) versus controls rats (LV-to-body weight ratio 2.6±0.2 versus 2.7±0.1 mg/g, controls versus Ad.Akt, P, NS). LV contractility, measured as developed pressure, increased by 41% in Ad.Akt. This was accounted for by both more systolic Ca2+ available to the contractile machinery (+19% versus controls) and by enhanced myofilament Ca2+ responsiveness, documented by an increased maximal Ca2+-activated pressure (+19% versus controls) and a shift to the left of the Ca2+–force relationship. Such increased contractility was paralleled by a slight increase of myocardial oxygen consumption (14%), while titrated dose of dobutamine providing similar inotropic effect augmented oxygen consumption by 39% (P<0.01). Phospholamban, calsequestrin, and ryanodine receptor LV mRNA and protein content were not different among the study groups, while sarcoplasmic reticulum Ca2+ ATPase protein levels were significantly increased in Ad.Akt rats. β-Adrenergic receptor density, affinity, kinase-1 levels, and adenylyl cyclase activity were similar in the three animal groups. In conclusion, our results support an important role for Akt/PKB in the regulation of myocardial contractility and mechanoenergetics. PMID:16094411

Supersoft lithography: Candy-based fabrication of soft silicone microstructures

PubMed Central

Moraes, Christopher; Labuz, Joseph M.; Shao, Yue; Fu, Jianping; Takayama, Shuichi

2015-01-01

We designed a fabrication technique able to replicate microstructures in soft silicone materials (E < 1 kPa). Sugar-based ‘hard candy’ recipes from the confectionery industry were modified to be compatible with silicone processing conditions, and used as templates for replica molding. Microstructures fabricated in soft silicones can then be easily released by dissolving the template in water. We anticipate that this technique will be of particular importance in replicating physiologically soft, microstructured environments for cell culture, and demonstrate a first application in which intrinsically soft microstructures are used to measure forces generated by fibroblast-laden contractile tissues. PMID:26245893

Supersoft lithography: candy-based fabrication of soft silicone microstructures.

PubMed

Moraes, Christopher; Labuz, Joseph M; Shao, Yue; Fu, Jianping; Takayama, Shuichi

2015-01-01

We designed a fabrication technique able to replicate microstructures in soft silicone materials (E < 1 kPa). Sugar-based 'hard candy' recipes from the confectionery industry were modified to be compatible with silicone processing conditions, and used as templates for replica molding. Microstructures fabricated in soft silicones can then be easily released by dissolving the template in water. We anticipate that this technique will be of particular importance in replicating physiologically soft, microstructured environments for cell culture, and demonstrate a first application in which intrinsically soft microstructures are used to measure forces generated by fibroblast-laden contractile tissues.

Na+,K+-pump stimulation improves contractility in isolated muscles of mice with hyperkalemic periodic paralysis

PubMed Central

Nielsen, Ole Bækgaard; Clausen, Johannes D.; Pedersen, Thomas Holm; Hayward, Lawrence J.

2011-01-01

In patients with hyperkalemic periodic paralysis (HyperKPP), attacks of muscle weakness or paralysis are triggered by K+ ingestion or rest after exercise. Force can be restored by muscle work or treatment with β2-adrenoceptor agonists. A missense substitution corresponding to a mutation in the skeletal muscle voltage-gated Na+ channel (Nav1.4, Met1592Val) causing human HyperKPP was targeted into the mouse SCN4A gene (mutants). In soleus muscles prepared from these mutant mice, twitch, tetanic force, and endurance were markedly reduced compared with soleus from wild type (WT), reflecting impaired excitability. In mutant soleus, contractility was considerably more sensitive than WT soleus to inhibition by elevated [K+]o. In resting mutant soleus, tetrodotoxin (TTX)-suppressible 22Na uptake and [Na+]i were increased by 470 and 58%, respectively, and membrane potential was depolarized (by 16 mV, P < 0.0001) and repolarized by TTX. Na+,K+ pump–mediated 86Rb uptake was 83% larger than in WT. Salbutamol stimulated 86Rb uptake and reduced [Na+]i both in mutant and WT soleus. Stimulating Na+,K+ pumps with salbutamol restored force in mutant soleus and extensor digitorum longus (EDL). Increasing [Na+]i with monensin also restored force in soleus. In soleus, EDL, and tibialis anterior muscles of mutant mice, the content of Na+,K+ pumps was 28, 62, and 33% higher than in WT, respectively, possibly reflecting the stimulating effect of elevated [Na+]i on the synthesis of Na+,K+ pumps. The results confirm that the functional disorders of skeletal muscles in HyperKPP are secondary to increased Na+ influx and show that contractility can be restored by acute stimulation of the Na+,K+ pumps. Calcitonin gene-related peptide (CGRP) restored force in mutant soleus but caused no detectable increase in 86Rb uptake. Repeated excitation and capsaicin also restored contractility, possibly because of the release of endogenous CGRP from nerve endings in the isolated muscles. These observations may explain how mild exercise helps locally to prevent severe weakness during an attack of HyperKPP. PMID:21708955

Effects of platelet activating factor on contractile force and 45Ca fluxes in guinea-pig isolated atria.

PubMed Central

Diez, J.; Delpón, E.; Tamargo, J.

1990-01-01

1. The effects of platelet activating factor (PAF) were studied on the electromechanical properties and 45Ca2+ fluxes of guinea-pig isolated atria. 2 Both in spontaneously beating and electrically driven atria, PAF (10(-12)-10(-7) M) increased atrial rate but produced a biphasic effect on contractile force. At low concentrations (up to 10(-10) M) it produced a positive inotropic effect, while at higher concentrations PAF exerted a negative inotropic effect. A similar biphasic effect was observed in the slow contractions elicited by isoprenaline in K(+)-depolarized atrial fibres. 3. The positive inotropic effect of PAF was prevented by verapamil, whereas pretreatment of atria with propranolol, phentolamine, indomethacin or atropine did not modify its positive and negative inotropic actions. BN 52021, a specific PAF antagonist, abolished both the positive and negative inotropic effects. 4. PAF had no effect on the characteristics of the action potentials recorded in either normally polarized or K(+)-depolarized (slow action potential) atrial fibres. 5. At concentrations at which it increased contractile force, PAF potentiated the contractile responses to Ca2+ (0.9-9 mM), whereas at negative inotropic concentrations it inhibited them. The negative inotropic effect of PAF was partially reversed in 70% Na+ medium. 6. At 10(-11) M, PAF increased 45Ca2+ uptake and reduced the rate coefficient (kcm) for the 45Ca2+ efflux. This increase in 45Ca2+ uptake was abolished in atria pretreated with verapamil or BN 52021. However, 10(-7) M PAF modified neither 45Ca2+ uptake nor efflux in atrial muscle.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2379035

Why do receptor–ligand bonds in cell adhesion cluster into discrete focal-adhesion sites?

DOE PAGES

Gao, Zhiwen; Gao, Yanfei

2016-05-14

We report that cell adhesion often exhibits the clustering of the receptor–ligand bonds into discrete focal-adhesion sites near the contact edge, thus resembling a rosette shape or a contracting membrane anchored by a small number of peripheral forces. The ligands on the extracellular matrix are immobile, and the receptors in the cell plasma membrane consist of two types: high-affinity integrins (that bond to the substrate ligands and are immobile) and low-affinity integrins (that are mobile and not bonded to the ligands). Thus the adhesion energy density is proportional to the high-affinity integrin density. This paper provides a mechanistic explanation formore » the clustering/assembling of the receptor–ligand bonds from two main points: (1) the cellular contractile force leads to the density evolution of these two types of integrins, and results into a large high-affinity integrin density near the contact edge and (2) the front of a propagating crack into a decreasing toughness field will be unstable and wavy. From this fracture mechanics perspective, the chemomechanical equilibrium is reached when a small number of patches with large receptor–ligand bond density are anticipated to form at the cell periphery, as opposed to a uniform distribution of bonds on the entire interface. Finally, cohesive fracture simulations show that the de-adhesion force can be significantly enhanced by this nonuniform bond density field, but the de-adhesion force anisotropy due to the substrate elastic anisotropy is significantly reduced.« less

Why do receptor–ligand bonds in cell adhesion cluster into discrete focal-adhesion sites?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Zhiwen; Gao, Yanfei

We report that cell adhesion often exhibits the clustering of the receptor–ligand bonds into discrete focal-adhesion sites near the contact edge, thus resembling a rosette shape or a contracting membrane anchored by a small number of peripheral forces. The ligands on the extracellular matrix are immobile, and the receptors in the cell plasma membrane consist of two types: high-affinity integrins (that bond to the substrate ligands and are immobile) and low-affinity integrins (that are mobile and not bonded to the ligands). Thus the adhesion energy density is proportional to the high-affinity integrin density. This paper provides a mechanistic explanation formore » the clustering/assembling of the receptor–ligand bonds from two main points: (1) the cellular contractile force leads to the density evolution of these two types of integrins, and results into a large high-affinity integrin density near the contact edge and (2) the front of a propagating crack into a decreasing toughness field will be unstable and wavy. From this fracture mechanics perspective, the chemomechanical equilibrium is reached when a small number of patches with large receptor–ligand bond density are anticipated to form at the cell periphery, as opposed to a uniform distribution of bonds on the entire interface. Finally, cohesive fracture simulations show that the de-adhesion force can be significantly enhanced by this nonuniform bond density field, but the de-adhesion force anisotropy due to the substrate elastic anisotropy is significantly reduced.« less

Optogenetic control of contractile function in skeletal muscle

PubMed Central

Bruegmann, Tobias; van Bremen, Tobias; Vogt, Christoph C.; Send, Thorsten; Fleischmann, Bernd K.; Sasse, Philipp

2015-01-01

Optogenetic stimulation allows activation of cells with high spatial and temporal precision. Here we show direct optogenetic stimulation of skeletal muscle from transgenic mice expressing the light-sensitive channel Channelrhodopsin-2 (ChR2). Largest tetanic contractions are observed with 5-ms light pulses at 30 Hz, resulting in 84% of the maximal force induced by electrical stimulation. We demonstrate the utility of this approach by selectively stimulating with a light guide individual intralaryngeal muscles in explanted larynges from ChR2-transgenic mice, which enables selective opening and closing of the vocal cords. Furthermore, systemic injection of adeno-associated virus into wild-type mice provides sufficient ChR2 expression for optogenetic opening of the vocal cords. Thus, direct optogenetic stimulation of skeletal muscle generates large force and provides the distinct advantage of localized and cell-type-specific activation. This technology could be useful for therapeutic purposes, such as restoring the mobility of the vocal cords in patients suffering from laryngeal paralysis. PMID:26035411

3D bioprinted functional and contractile cardiac tissue constructs

PubMed Central

Wang, Zhan; Lee, Sang Jin; Cheng, Heng-Jie; Yoo, James J.; Atala, Anthony

2018-01-01

Bioengineering of a functional cardiac tissue composed of primary cardiomyocytes has great potential for myocardial regeneration and in vitro tissue modeling. However, its applications remain limited because the cardiac tissue is a highly organized structure with unique physiologic, biomechanical, and electrical properties. In this study, we undertook a proof-of-concept study to develop a contractile cardiac tissue with cellular organization, uniformity, and scalability by using three-dimensional (3D) bioprinting strategy. Primary cardiomyocytes were isolated from infant rat hearts and suspended in a fibrin-based bioink to determine the priting capability for cardiac tissue engineering. This cell-laden hydrogel was sequentially printed with a sacrificial hydrogel and a supporting polymeric frame through a 300-μm nozzle by pressured air. Bioprinted cardiac tissue constructs had a spontaneous synchronous contraction in culture, implying in vitro cardiac tissue development and maturation. Progressive cardiac tissue development was confirmed by immunostaining for α-actinin and connexin 43, indicating that cardiac tissues were formed with uniformly aligned, dense, and electromechanically coupled cardiac cells. These constructs exhibited physiologic responses to known cardiac drugs regarding beating frequency and contraction forces. In addition, Notch signaling blockade significantly accelerated development and maturation of bioprinted cardiac tissues. Our results demonstrated the feasibility of bioprinting functional cardiac tissues that could be used for tissue engineering applications and pharmaceutical purposes. PMID:29452273

Lung Parenchymal Mechanics

PubMed Central

Suki, Béla; Stamenovic, Dimitrije; Hubmayr, Rolf

2014-01-01

The lung parenchyma comprises a large number of thin-walled alveoli, forming an enormous surface area, which serves to maintain proper gas exchange. The alveoli are held open by the transpulmonary pressure, or prestress, which is balanced by tissues forces and alveolar surface film forces. Gas exchange efficiency is thus inextricably linked to three fundamental features of the lung: parenchymal architecture, prestress, and the mechanical properties of the parenchyma. The prestress is a key determinant of lung deformability that influences many phenomena including local ventilation, regional blood flow, tissue stiffness, smooth muscle contractility, and alveolar stability. The main pathway for stress transmission is through the extracellular matrix. Thus, the mechanical properties of the matrix play a key role both in lung function and biology. These mechanical properties in turn are determined by the constituents of the tissue, including elastin, collagen, and proteoglycans. In addition, the macroscopic mechanical properties are also influenced by the surface tension and, to some extent, the contractile state of the adherent cells. This article focuses on the biomechanical properties of the main constituents of the parenchyma in the presence of prestress and how these properties define normal function or change in disease. An integrated view of lung mechanics is presented and the utility of parenchymal mechanics at the bedside as well as its possible future role in lung physiology and medicine are discussed. PMID:23733644

Deletion of Mbtps1 (Pcsk8, S1p, Ski-1) Gene in Osteocytes Stimulates Soleus Muscle Regeneration and Increased Size and Contractile Force with Age.

PubMed

Gorski, Jeff P; Huffman, Nichole T; Vallejo, Julian; Brotto, Leticia; Chittur, Sridar V; Breggia, Anne; Stern, Amber; Huang, Jian; Mo, Chenglin; Seidah, Nabil G; Bonewald, Lynda; Brotto, Marco

2016-02-26

Conditional deletion of Mbtps1 (cKO) protease in bone osteocytes leads to an age-related increase in mass (12%) and in contractile force (30%) in adult slow twitch soleus muscles (SOL) with no effect on fast twitch extensor digitorum longus muscles. Surprisingly, bone from 10-12-month-old cKO animals was indistinguishable from controls in size, density, and morphology except for a 25% increase in stiffness. cKO SOL exhibited increased expression of Pax7, Myog, Myod1, Notch, and Myh3 and 6-fold more centralized nuclei, characteristics of postnatal regenerating muscle, but only in type I myosin heavy chain-expressing cells. Increased expression of gene pathways mediating EGF receptor signaling, circadian exercise, striated muscle contraction, and lipid and carbohydrate oxidative metabolism were also observed in cKO SOL. This muscle phenotype was not observed in 3-month-old mice. Although Mbtps1 mRNA and protein expression was reduced in cKO bone osteocytes, no differences in Mbtps1 or cre recombinase expression were observed in cKO SOL, explaining this age-related phenotype. Understanding bone-muscle cross-talk may provide a fresh and novel approach to prevention and treatment of age-related muscle loss. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Angiotensin II and angiotensin II receptor blocker modulate the arrhythmogenic activity of pulmonary veins.

PubMed

Chen, Yi-Jen; Chen, Yao-Chang; Tai, Ching-Tai; Yeh, Hung-I; Lin, Cheng-I; Chen, Shih-Ann

2006-01-01

Angiotensin II receptor blockers (AIIRBs) have been shown to prevent atrial fibrillation. The pulmonary veins (PVs) are the most important focus for the generation of atrial fibrillation. The aim of this study was to evaluate whether angiotensin II or AIIRB may change the arrhythmogenic activity of the PVs. Conventional microelectrodes and whole-cell patch clamps were used to investigate the action potentials (APs) and ionic currents in isolated rabbit PV tissue and single cardiomyocytes before and after administering angiotensin II or losartan (AIIRB). In the tissue preparations, angiotensin II induced delayed after-depolarizations (1, 10, and 100 nM) and accelerated the automatic rhythm (10 and 100 nM). Angiotensin II (100 nM) prolonged the AP duration and increased the contractile force (10 and 100 nM). Losartan (1 and 10 microM) inhibited the automatic rhythm. Losartan (10 microM) prolonged the AP duration and reduced the contractile force (1 and 10 microM). Angiotensin II reduced the transient outward potassium current (I(to)) but increased the L-type calcium, delayed rectifier potassium (I(K)), transient inward (I(ti)), pacemaker, and Na(+)-Ca(2+) exchanger (NCX) currents in the PV cardiomyocytes. Losartan decreased the I(to), I(K), I(ti), and NCX currents. In conclusion, angiotensin II and AIIRB modulate the PV electrical activity, which may play a role in the pathophysiology of atrial fibrillation.

Up-regulation of MHC class I in transgenic mice results in reduced force-generating capacity in slow-twitch muscle

PubMed Central

Salomonsson, Stina; Grundtman, Cecilia; Zhang, Shi-Jin; Lanner, Johanna T.; Li, Charles; Katz, Abram; Wedderburn, Lucy R.; Nagaraju, Kanneboyina; Lundberg, Ingrid E.; Westerblad, Håkan

2008-01-01

Expression of major histocompatibility complex (MHC) class I in skeletal muscle fibers is an early and consistent finding in inflammatory myopathies. To test if MHC class I has a primary role in muscle impairment; we used transgenic mice with inducible over-expression of MHC class I in their skeletal muscle cells. Contractile function was studied in isolated extensor digitorum longus (EDL, fast-twitch) and soleus (slow-twitch) muscles. We found that EDL was smaller, whereas soleus muscle was slightly larger. Both muscles generated less absolute force in myopathic compared to control mice, however when force was expressed per cross-sectional area, only soleus muscle generated less force. Inflammation was markedly increased, but no changes were found in the activities of key mitochondrial and glycogenolytic enzymes in myopathic mice. The induction of MHC class I results in muscle atrophy and an intrinsic decrease in force-generation capacity. These observations may have important implications for our understanding of the pathophysiological processes of muscle weakness seen in inflammatory myopathies. PMID:19229963

RhoA GTPase inhibition organizes contraction during epithelial morphogenesis

PubMed Central

Mason, Frank M.; Xie, Shicong; Vasquez, Claudia G.; Tworoger, Michael

2016-01-01

During morphogenesis, contraction of the actomyosin cytoskeleton within individual cells drives cell shape changes that fold tissues. Coordination of cytoskeletal contractility is mediated by regulating RhoA GTPase activity. Guanine nucleotide exchange factors (GEFs) activate and GTPase-activating proteins (GAPs) inhibit RhoA activity. Most studies of tissue folding, including apical constriction, have focused on how RhoA is activated by GEFs to promote cell contractility, with little investigation as to how GAPs may be important. Here, we identify a critical role for a RhoA GAP, Cumberland GAP (C-GAP), which coordinates with a RhoA GEF, RhoGEF2, to organize spatiotemporal contractility during Drosophila melanogaster apical constriction. C-GAP spatially restricts RhoA pathway activity to a central position in the apical cortex. RhoGEF2 pulses precede myosin, and C-GAP is required for pulsation, suggesting that contractile pulses result from RhoA activity cycling. Finally, C-GAP expression level influences the transition from reversible to irreversible cell shape change, which defines the onset of tissue shape change. Our data demonstrate that RhoA activity cycling and modulating the ratio of RhoGEF2 to C-GAP are required for tissue folding. PMID:27551058

Actin dynamics mediates the changes of calcium level during the pulvinus movement of Mimosa pudica

PubMed Central

Yao, Heng; Xu, Qiangyi

2008-01-01

The bending movement of the pulvinus of Mimosa pudica is caused by a rapid change in volume of the abaxial motor cells, in response to various environmental stimuli. We investigated the relationship between the actin cytoskeleton and changes in the level of calcium during rapid contractile movement of the motor cells that was induced by electrical stimulation. The bending of the pulvinus was retarded by treatments with actin-affecting reagents and calcium channel inhibitors. The actin filaments in the motor cells were fragmented in response to electrical stimulation. Further investigations were performed using protoplasts from the motor cells of M. pudica pulvini. Calcium-channel inhibitors and EGTA had an inhibitory effect on contractile movement of the protoplasts. The level of calcium increased and became concentrated in the tannin vacuole after electrical stimulation. Ruthenium Red inhibited the increase in the level of calcium in the tannin vacuole and the contractile movement of the protoplasts. However, treatment with latrunculin A abolished the inhibitory effect of Ruthenium Red. Phalloidin inhibited the contractile movement and the increase in the level of calcium in the protoplasts. Our study demonstrates that depolymerization of the actin cytoskeleton in pulvinus motor cells in response to electrical signals results in increased levels of calcium. PMID:19513198

Macrophage migration inhibitory factor plays a permissive role in the maintenance of cardiac contractile function under starvation through regulation of autophagy.

PubMed

Xu, Xihui; Pacheco, Benjamin D; Leng, Lin; Bucala, Richard; Ren, Jun

2013-08-01

The cytokine macrophage migration inhibitory factor (MIF) protects the heart through AMPK activation. Autophagy, a conserved pathway for bulk degradation of intracellular proteins and organelles, helps preserve and recycle energy and nutrients for cells to survive under starvation. This study was designed to examine the role of MIF in cardiac homeostasis and autophagy regulation following an acute starvation challenge. Wild-type (WT) and MIF knockout mice were starved for 48 h. Echocardiographic data revealed little effect of starvation on cardiac geometry, contractile and intracellular Ca²⁺ properties. MIF deficiency unmasked an increase in left ventricular end-systolic diameter, a drop in fractional shortening associated with cardiomyocyte contractile and intracellular Ca²⁺ anomalies following starvation. Interestingly, the unfavourable effect of MIF deficiency was associated with interruption of starvation-induced autophagy. Furthermore, restoration of autophagy using rapamycin partially protected against starvation-induced cardiomyocyte contractile defects. In our in vitro model of starvation, neonatal mouse cardiomyocytes from WT and MIF-/- mice and H9C2 cells were treated with serum free-glucose free DMEM for 2 h. MIF depletion dramatically attenuated starvation-induced autophagic vacuole formation in neonatal mouse cardiomyocytes and exacerbated starvation-induced cell death in H9C2 cells. In summary, these results indicate that MIF plays a permissive role in the maintenance of cardiac contractile function under starvation by regulation of autophagy.

Dynamic culture yields engineered myocardium with near-adult functional output

PubMed Central

Jackman, Christopher P.; Carlson, Aaron L.; Bursac, Nenad

2016-01-01

Engineered cardiac tissues hold promise for cell therapy and drug development, but exhibit inadequate function and maturity. In this study, we sought to significantly improve the function and maturation of rat and human engineered cardiac tissues. We developed dynamic, free-floating culture conditions for engineering “cardiobundles”, 3-dimensional cylindrical tissues made from neonatal rat cardiomyocytes or human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) embedded in fibrin-based hydrogel. Compared to static culture, 2-week dynamic culture of neonatal rat cardiobundles significantly increased expression of sarcomeric proteins, cardiomyocyte size (~2.1-fold), contractile force (~3.5-fold), and conduction velocity of action potentials (~1.4-fold). The average contractile force per cross-sectional area (59.7 mN/mm2) and conduction velocity (52.5 cm/sec) matched or approached those of adult rat myocardium, respectively. The inferior function of statically cultured cardiobundles was rescued by transfer to dynamic conditions, which was accompanied by an increase in mTORC1 activity and decline in AMPK phosphorylation and was blocked by rapamycin. Furthermore, dynamic culture effects did not stimulate ERK1/2 pathway and were insensitive to blockers of mechanosensitive channels, suggesting increased nutrient availability rather than mechanical stimulation as the upstream activator of mTORC1. Direct comparison with phenylephrine treatment confirmed that dynamic culture promoted physiological cardiomyocyte growth rather than pathological hypertrophy. Optimized dynamic culture conditions also augmented function of human cardiobundles made reproducibly from cardiomyocytes derived from multiple hPSC lines, resulting in significantly increased contraction force (~2.5-fold) and conduction velocity (~1.4-fold). The average specific force of 23.2 mN/mm2 and conduction velocity of 25.8 cm/sec approached the functional metrics of adult human myocardium. In conclusion, we have developed a versatile methodology for engineering cardiac tissues with a near-adult functional output without the need for exogenous electrical or mechanical stimulation, and have identified mTOR signaling as an important mechanism for advancing tissue maturation and function in vitro. PMID:27723557

Dynamic culture yields engineered myocardium with near-adult functional output.

PubMed

Jackman, Christopher P; Carlson, Aaron L; Bursac, Nenad

2016-12-01

Engineered cardiac tissues hold promise for cell therapy and drug development, but exhibit inadequate function and maturity. In this study, we sought to significantly improve the function and maturation of rat and human engineered cardiac tissues. We developed dynamic, free-floating culture conditions for engineering "cardiobundles", 3-dimensional cylindrical tissues made from neonatal rat cardiomyocytes or human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) embedded in fibrin-based hydrogel. Compared to static culture, 2-week dynamic culture of neonatal rat cardiobundles significantly increased expression of sarcomeric proteins, cardiomyocyte size (∼2.1-fold), contractile force (∼3.5-fold), and conduction velocity of action potentials (∼1.4-fold). The average contractile force per cross-sectional area (59.7 mN/mm 2 ) and conduction velocity (52.5 cm/s) matched or approached those of adult rat myocardium, respectively. The inferior function of statically cultured cardiobundles was rescued by transfer to dynamic conditions, which was accompanied by an increase in mTORC1 activity and decline in AMPK phosphorylation and was blocked by rapamycin. Furthermore, dynamic culture effects did not stimulate ERK1/2 pathway and were insensitive to blockers of mechanosensitive channels, suggesting increased nutrient availability rather than mechanical stimulation as the upstream activator of mTORC1. Direct comparison with phenylephrine treatment confirmed that dynamic culture promoted physiological cardiomyocyte growth rather than pathological hypertrophy. Optimized dynamic culture conditions also augmented function of human cardiobundles made reproducibly from cardiomyocytes derived from multiple hPSC lines, resulting in significantly increased contraction force (∼2.5-fold) and conduction velocity (∼1.4-fold). The average specific force of 23.2 mN/mm 2 and conduction velocity of 25.8 cm/s approached the functional metrics of adult human myocardium. In conclusion, we have developed a versatile methodology for engineering cardiac tissues with a near-adult functional output without the need for exogenous electrical or mechanical stimulation, and have identified mTOR signaling as an important mechanism for advancing tissue maturation and function in vitro. Copyright © 2016 Elsevier Ltd. All rights reserved.

Impact of stirred suspension bioreactor culture on the differentiation of murine embryonic stem cells into cardiomyocytes.

PubMed

Shafa, Mehdi; Krawetz, Roman; Zhang, Yuan; Rattner, Jerome B; Godollei, Anna; Duff, Henry J; Rancourt, Derrick E

2011-12-14

Embryonic stem cells (ESCs) can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. Under particular in vitro culture conditions, ESCs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors (SSBs). However, in using these systems we must be cognizant of the mechanical forces acting upon the cells. The effect of mechanical forces and shear stress on ESC pluripotency and differentiation has yet to be clarified. The purpose of this study was to investigate the impact of the suspension culture environment on ESC pluripotency during cardiomyocyte differentiation. Murine D3-MHC-neo(r) ESCs formed embyroid bodies (EBs) and differentiated into cardiomyocytes over 25 days in static culture and suspension bioreactors. G418 (Geneticin) was used in both systems from day 10 to enrich for cardiomyocytes by eliminating non-resistant, undifferentiated cells. Treatment of EBs with 1 mM ascorbic acid and 0.5% dimethyl sulfoxide from day 3 markedly increased the number of beating EBs, which displayed spontaneous and cadenced contractile beating on day 11 in the bioreactor. Our results showed that the bioreactor differentiated cells displayed the characteristics of fully functional cardiomyocytes. Remarkably, however, our results demonstrated that the bioreactor differentiated ESCs retained their ability to express pluripotency markers, to form ESC-like colonies, and to generate teratomas upon transplantation, whereas the cells differentiated in adherent culture lost these characteristics. This study demonstrates that although cardiomyocyte differentiation can be achieved in stirred suspension bioreactors, the addition of medium enhancers is not adequate to force complete differentiation as fluid shear forces appear to maintain a subpopulation of cells in a transient pluripotent state. The development of successful ESC differentiation protocols within suspension bioreactors demands a more complete understanding of the impacts of shear forces on the regulation of pluripotency and differentiation in pluripotent stem cells.

Impact of stirred suspension bioreactor culture on the differentiation of murine embryonic stem cells into cardiomyocytes

PubMed Central

2011-01-01

Background Embryonic stem cells (ESCs) can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. Under particular in vitro culture conditions, ESCs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors (SSBs). However, in using these systems we must be cognizant of the mechanical forces acting upon the cells. The effect of mechanical forces and shear stress on ESC pluripotency and differentiation has yet to be clarified. The purpose of this study was to investigate the impact of the suspension culture environment on ESC pluripotency during cardiomyocyte differentiation. Results Murine D3-MHC-neor ESCs formed embyroid bodies (EBs) and differentiated into cardiomyocytes over 25 days in static culture and suspension bioreactors. G418 (Geneticin) was used in both systems from day 10 to enrich for cardiomyocytes by eliminating non-resistant, undifferentiated cells. Treatment of EBs with 1 mM ascorbic acid and 0.5% dimethyl sulfoxide from day 3 markedly increased the number of beating EBs, which displayed spontaneous and cadenced contractile beating on day 11 in the bioreactor. Our results showed that the bioreactor differentiated cells displayed the characteristics of fully functional cardiomyocytes. Remarkably, however, our results demonstrated that the bioreactor differentiated ESCs retained their ability to express pluripotency markers, to form ESC-like colonies, and to generate teratomas upon transplantation, whereas the cells differentiated in adherent culture lost these characteristics. Conclusions This study demonstrates that although cardiomyocyte differentiation can be achieved in stirred suspension bioreactors, the addition of medium enhancers is not adequate to force complete differentiation as fluid shear forces appear to maintain a subpopulation of cells in a transient pluripotent state. The development of successful ESC differentiation protocols within suspension bioreactors demands a more complete understanding of the impacts of shear forces on the regulation of pluripotency and differentiation in pluripotent stem cells. PMID:22168552

Coupled expression of troponin T and troponin I isoforms in single skeletal muscle fibers correlates with contractility.

PubMed

Brotto, Marco A; Biesiadecki, Brandon J; Brotto, Leticia S; Nosek, Thomas M; Jin, Jian-Ping

2006-02-01

Striated muscle contraction is powered by actin-activated myosin ATPase. This process is regulated by Ca(2+) via the troponin complex. Slow- and fast-twitch fibers of vertebrate skeletal muscle express type I and type II myosin, respectively, and these myosin isoenzymes confer different ATPase activities, contractile velocities, and force. Skeletal muscle troponin has also diverged into fast and slow isoforms, but their functional significance is not fully understood. To investigate the expression of troponin isoforms in mammalian skeletal muscle and their functional relationship to that of the myosin isoforms, we concomitantly studied myosin, troponin T (TnT), and troponin I (TnI) isoform contents and isometric contractile properties in single fibers of rat skeletal muscle. We characterized a large number of Triton X-100-skinned single fibers from soleus, diaphragm, gastrocnemius, and extensor digitorum longus muscles and selected fibers with combinations of a single myosin isoform and a single class (slow or fast) of the TnT and TnI isoforms to investigate their role in determining contractility. Types IIa, IIx, and IIb myosin fibers produced higher isometric force than that of type I fibers. Despite the polyploidy of adult skeletal muscle fibers, the expression of fast or slow isoforms of TnT and TnI is tightly coupled. Fibers containing slow troponin had higher Ca(2+) sensitivity than that of the fast troponin fibers, whereas fibers containing fast troponin showed a higher cooperativity of Ca(2+) activation than that of the slow troponin fibers. These results demonstrate distinct but coordinated regulation of troponin and myosin isoform expression in skeletal muscle and their contribution to the contractile properties of muscle.

Coupled expression of troponin T and troponin I isoforms in single skeletal muscle fibers correlates with contractility

PubMed Central

BROTTO, MARCO A.; BIESIADECKI, BRANDON J.; BROTTO, LETICIA S.; NOSEK, THOMAS M; JIN, J.-P.

2005-01-01

(Summary) Brotto, Marco A., Brandon J. Biesiadecki, Leticia S. Brotto, Thomas M. Nosek, and J.-P. Jin. Striated muscle contraction is powered by actin-activated myosin ATPase. This process is regulated by Ca2+ via the troponin complex. Slow and fast twitch fibers of vertebrate skeletal muscle express type I and type II myosin, respectively, and these myosin isoenzymes confer different ATPase activities, contractile velocities and force. Skeletal muscle troponin has also diverged into fast and slow isoforms, but their functional significance is not fully understood. To investigate the expression of troponin isoforms in mammalian skeletal muscle and their functional relationship to that of the myosin isoforms, we concomitantly studied myosin and troponin T (TnT) and troponin I (TnI) isoform contents and isometric contractile properties in single fibers of rat skeletal muscle. We characterized a large number of Triton skinned single fibers from soleus, diaphragm, gastrocnemius and extensor digitorum longus muscles and selected fibers with combinations of a single myosin isoform and a single class (slow or fast) of TnT and TnI isoform to investigate their role in determining contractility. Type IIa, IIx and IIb myosin fibers produced higher isometric force than that of type I fibers. Despite the polyploidy of adult skeletal muscle fibers, the expression of fast or slow isoforms of TnT and TnI is tightly coupled. Fibers containing slow troponin had higher Ca2+ sensitivity than that of the fast troponin fibers, while fibers containing fast troponin showed a higher cooperativity of Ca2+ activation than that of the slow troponin fibers. The results demonstrate distinctive, but coordinated, regulation of troponin and myosin isoform expression in skeletal muscle and their contribution to the contractile properties. PMID:16192301

Spontaneous Contractility-Mediated Cortical Flow Generates Cell Migration in Three-Dimensional Environments

PubMed Central

Hawkins, Rhoda J.; Poincloux, Renaud; Bénichou, Olivier; Piel, Matthieu; Chavrier, Philippe; Voituriez, Raphaël

2011-01-01

We present a model of cell motility generated by actomyosin contraction of the cell cortex. We identify, analytically, dynamical instabilities of the cortex and show that they yield steady-state cortical flows, which, in turn, can induce cell migration in three-dimensional environments. This mechanism relies on the regulation of contractility by myosin, whose transport is explicitly taken into account in the model. Theoretical predictions are compared to experimental data of tumor cells migrating in three-dimensional matrigel and suggest that this mechanism could be a general mode of cell migration in three-dimensional environments. PMID:21889440

Myosin II dynamics are regulated by tension in intercalating cells.

PubMed

Fernandez-Gonzalez, Rodrigo; Simoes, Sérgio de Matos; Röper, Jens-Christian; Eaton, Suzanne; Zallen, Jennifer A

2009-11-01

Axis elongation in Drosophila occurs through polarized cell rearrangements driven by actomyosin contractility. Myosin II promotes neighbor exchange through the contraction of single cell boundaries, while the contraction of myosin II structures spanning multiple pairs of cells leads to rosette formation. Here we show that multicellular actomyosin cables form at a higher frequency than expected by chance, indicating that cable assembly is an active process. Multicellular cables are sites of increased mechanical tension as measured by laser ablation. Fluorescence recovery after photobleaching experiments show that myosin II is stabilized at the cortex in regions of increased tension. Myosin II is recruited in response to an ectopic force and relieving tension leads to a rapid loss of myosin, indicating that tension is necessary and sufficient for cortical myosin localization. These results demonstrate that myosin II dynamics are regulated by tension in a positive feedback loop that leads to multicellular actomyosin cable formation and efficient tissue elongation.

Force generation by titin folding.

PubMed

Mártonfalvi, Zsolt; Bianco, Pasquale; Naftz, Katalin; Ferenczy, György G; Kellermayer, Miklós

2017-07-01

Titin is a giant protein that provides elasticity to muscle. As the sarcomere is stretched, titin extends hierarchically according to the mechanics of its segments. Whether titin's globular domains unfold during this process and how such unfolded domains might contribute to muscle contractility are strongly debated. To explore the force-dependent folding mechanisms, here we manipulated skeletal-muscle titin molecules with high-resolution optical tweezers. In force-clamp mode, after quenching the force (<10 pN), extension fluctuated without resolvable discrete events. In position-clamp experiments, the time-dependent force trace contained rapid fluctuations and a gradual increase of average force, indicating that titin can develop force via dynamic transitions between its structural states en route to the native conformation. In 4 M urea, which destabilizes H-bonds hence the consolidated native domain structure, the net force increase disappeared but the fluctuations persisted. Thus, whereas net force generation is caused by the ensemble folding of the elastically-coupled domains, force fluctuations arise due to a dynamic equilibrium between unfolded and molten-globule states. Monte-Carlo simulations incorporating a compact molten-globule intermediate in the folding landscape recovered all features of our nanomechanics results. The ensemble molten-globule dynamics delivers significant added contractility that may assist sarcomere mechanics, and it may reduce the dissipative energy loss associated with titin unfolding/refolding during muscle contraction/relaxation cycles. © 2017 The Protein Society.

Gastroesophageal reflux disease-associated esophagitis induces endogenous cytokine production leading to motor abnormalities.

PubMed

Rieder, Florian; Cheng, Ling; Harnett, Karen M; Chak, Amitabh; Cooper, Gregory S; Isenberg, Gerard; Ray, Monica; Katz, Jeffry A; Catanzaro, Andrew; O'Shea, Robert; Post, Anthony B; Wong, Richard; Sivak, Michael V; McCormick, Thomas; Phillips, Manijeh; West, Gail A; Willis, Joseph E; Biancani, Piero; Fiocchi, Claudio

2007-01-01

Gastroesophageal reflux disease is a condition frequently associated with esophagitis and motor abnormalities. Recent evidence suggests that proinflammatory cytokines, such as interleukin (IL)-1beta and IL-6, may be implicated because they reduce esophageal muscle contractility, but these results derive from in vitro or animal models of esophagitis. This study used human esophageal cells and tissues to identify the cellular source of cytokines in human esophagitis investigate whether cytokines can be induced by gastric refluxate, and examine whether esophageal tissue- or cell-derived mediators affect muscle contractility. Endoscopic mucosal biopsy specimens were obtained from patients with and without esophagitis, organ-cultured, and undernatants were assessed for cytokine content. The cytokine profile of esophageal epithelial, fibroblast, and muscle cells was analyzed, and esophageal mucosa and cell products were tested in an esophageal circular muscle contraction assay. The mucosa of esophagitis patients produced significantly greater amounts of IL-1beta and IL-6 compared with those of control patients. Cultured esophageal epithelial cells produced IL-6, as did fibroblasts and muscle cells. Epithelial cells exposed to buffered, but not denatured, gastric juice produced IL-6. Undernatants of mucosal biopsy cultures from esophagitis patients reduced esophageal muscle contraction, as did supernatants from esophageal epithelial cell cultures. The human esophagus produces cytokines capable of reducing contractility of esophageal muscle cells. Exposure to gastric juice is sufficient to stimulate esophageal epithelial cells to produce IL-6, a cytokine able to alter esophageal contractility. These results indicate that classic cytokines are important mediators of the motor disturbances associated with human esophageal inflammation.

PKA catalytic subunit compartmentation regulates contractile and hypertrophic responses to β-adrenergic signaling

PubMed Central

Yang, Jason H.; Polanowska-Grabowska, Renata K.; Smith, Jeffrey S.; Shields, Charles W.; Saucerman, Jeffrey J.

2014-01-01

β-adrenergic signaling is spatiotemporally heterogeneous in the cardiac myocyte, conferring exquisite control to sympathetic stimulation. Such heterogeneity drives the formation of protein kinase A (PKA) signaling microdomains, which regulate Ca2+ handling and contractility. Here, we test the hypothesis that the nucleus independently comprises a PKA signaling microdomain regulating myocyte hypertrophy. Spatially-targeted FRET reporters for PKA activity identified slower PKA activation and lower isoproterenol sensitivity in the nucleus (t50 = 10.60±0.68 min; EC50 = 89.00 nmol/L) than in the cytosol (t50 = 3.71±0.25 min; EC50 = 1.22 nmol/L). These differences were not explained by cAMP or AKAP-based compartmentation. A computational model of cytosolic and nuclear PKA activity was developed and predicted that differences in nuclear PKA dynamics and magnitude are regulated by slow PKA catalytic subunit diffusion, while differences in isoproterenol sensitivity are regulated by nuclear expression of protein kinase inhibitor (PKI). These were validated by FRET and immunofluorescence. The model also predicted differential phosphorylation of PKA substrates regulating cell contractility and hypertrophy. Ca2+ and cell hypertrophy measurements validated these predictions and identified higher isoproterenol sensitivity for contractile enhancements (EC50 = 1.84 nmol/L) over cell hypertrophy (EC50 = 85.88 nmol/L). Over-expression of spatially targeted PKA catalytic subunit to the cytosol or nucleus enhanced contractile and hypertrophic responses, respectively. We conclude that restricted PKA catalytic subunit diffusion is an important PKA compartmentation mechanism and the nucleus comprises a novel PKA signaling microdomain, insulating hypertrophic from contractile β-adrenergic signaling responses. PMID:24225179

Cell contact as an independent factor modulating cardiac myocyte hypertrophy and survival in long-term primary culture

NASA Technical Reports Server (NTRS)

Clark, W. A.; Decker, M. L.; Behnke-Barclay, M.; Janes, D. M.; Decker, R. S.

1998-01-01

Cardiac myocytes maintained in cell culture develop hypertrophy both in response to mechanical loading as well as to receptor-mediated signaling mechanisms. However, it has been shown that the hypertrophic response to these stimuli may be modulated through effects of intercellular contact achieved by maintaining cells at different plating densities. In this study, we show that the myocyte plating density affects not only the hypertrophic response and features of the differentiated phenotype of isolated adult myocytes, but also plays a significant role influencing myocyte survival in vitro. The native rod-shaped phenotype of freshly isolated adult myocytes persists in an environment which minimizes myocyte attachment and spreading on the substratum. However, these conditions are not optimal for long-term maintenance of cultured adult cardiac myocytes. Conditions which promote myocyte attachment and spreading on the substratum, on the other hand, also promote the re-establishment of new intercellular contacts between myocytes. These contacts appear to play a significant role in the development of spontaneous activity, which enhances the redevelopment of highly differentiated contractile, junctional, and sarcoplasmic reticulum structures in the cultured adult cardiomyocyte. Although it has previously been shown that adult cardiac myocytes are typically quiescent in culture, the addition of beta-adrenergic agonists stimulates beating and myocyte hypertrophy, and thereby serves to increase the level of intercellular contact as well. However, in densely-plated cultures with intrinsically high levels of intercellular contact, spontaneous contractile activity develops without the addition of beta-adrenergic agonists. In this study, we compare the function, morphology, and natural history of adult feline cardiomyocytes which have been maintained in cultures with different levels of intercellular contact, with and without the addition of beta-adrenergic agonists. Intercellular contact, communication, and transmission of contractile forces between myocytes appears to play a primary role in remodeling the 2-dimensional cell layer into a parallel alignment of elongated myocytes with highly developed intercalated disk-like junctions. This highly differentiated state is very stable, and cultures which achieve this state exhibit significantly greater longevity than more sparsely plated myocytes. These myocytes typically continue beating, and survive from 6 to more than 12 weeks in culture. When this level of contact and differentiation are not achieved, even among beta-adrenergic stimulated myocytes, contractile activity is not sustained, myofibrils atrophy, there is little or no development of junctional complexes, and the period of myocyte viability is typically no more than 5 weeks in vitro.

Electrophysiological effects of FK664, a new cardiotonic agent, on preparations from guinea pig ventricle and from rabbit sino-atrial node.

PubMed

Kodama, I; Anno, T; Sudo, Y; Satake, N; Shibata, S

1989-05-01

Effects of the cardiotonic agent FK664, 6-(3, 4-dimethoxy-phenyl)-1-ethyl-4-mesitylimino-3-methyl-3,4-dihydro-2 (1H)-pyrimidone, on isolated guinea pig ventricular muscles and rabbit sinus node pacemaker cells were studied using micro-electrode techniques. In ventricular muscles driven at 0.5-1.0 Hz, FK664 above 3 mumol.litre-1 caused an increase in contractile force and a shortening of time to peak tension. This positive inotropic effect of FK664 was accompanied by a slight elevation of the early plateau phase of the action potential, while other action potential variables were unaffected. The change in contractile force induced by FK664 was abolished in a low Ca2+ medium (0.12 mmol.litre-1) or by treatment with ryanodine (2 mumol.litre-1), whereas it was relatively well preserved in the preparations pretreated with nefedipine (1 mumol.litre-1). The slow action potentials induced by isoprenaline (0.3 mumol.litre-1) in high K+ medium (30 mmol.litre-1) and the slow inward current measured by single sucrose gap voltage clamp at a holding potential of -40 mV were unaffected by FK664. In sinus node pacemaker cells, FK664 (1-10 mumol.litre-1) caused a dose dependent acceleration of phase 4 depolarisation and a shortening of spontaneous firing cycle length. This positive chronotropic effect of FK664 was markedly inhibited in a low Ca2+ medium (0.3 mmol.litre-1). These findings suggest that FK664 has positive inotropic and chronotropic effects on the heart, due to an enhancement of transsarcolemmal calcium influx through the low threshold, dihydropyridine insensitive Ca2+ channel population.

Leucine elicits myotube hypertrophy and enhances maximal contractile force in tissue engineered skeletal muscle in vitro.

PubMed

Martin, Neil R W; Turner, Mark C; Farrington, Robert; Player, Darren J; Lewis, Mark P

2017-10-01

The amino acid leucine is thought to be important for skeletal muscle growth by virtue of its ability to acutely activate mTORC1 and enhance muscle protein synthesis, yet little data exist regarding its impact on skeletal muscle size and its ability to produce force. We utilized a tissue engineering approach in order to test whether supplementing culture medium with leucine could enhance mTORC1 signaling, myotube growth, and muscle function. Phosphorylation of the mTORC1 target proteins 4EBP-1 and rpS6 and myotube hypertrophy appeared to occur in a dose dependent manner, with 5 and 20 mM of leucine inducing similar effects, which were greater than those seen with 1 mM. Maximal contractile force was also elevated with leucine supplementation; however, although this did not appear to be enhanced with increasing leucine doses, this effect was completely ablated by co-incubation with the mTOR inhibitor rapamycin, showing that the augmented force production in the presence of leucine was mTOR sensitive. Finally, by using electrical stimulation to induce chronic (24 hr) contraction of engineered skeletal muscle constructs, we were able to show that the effects of leucine and muscle contraction are additive, since the two stimuli had cumulative effects on maximal contractile force production. These results extend our current knowledge of the efficacy of leucine as an anabolic nutritional aid showing for the first time that leucine supplementation may augment skeletal muscle functional capacity, and furthermore validates the use of engineered skeletal muscle for highly-controlled investigations into nutritional regulation of muscle physiology. © 2017 The Authors. Journal of Cellular Physiology Published by wiley periodicals, Inc.

Leucine elicits myotube hypertrophy and enhances maximal contractile force in tissue engineered skeletal muscle in vitro

PubMed Central

Martin, Neil R.W.; Turner, Mark C.; Farrington, Robert; Player, Darren J.

2017-01-01

The amino acid leucine is thought to be important for skeletal muscle growth by virtue of its ability to acutely activate mTORC1 and enhance muscle protein synthesis, yet little data exist regarding its impact on skeletal muscle size and its ability to produce force. We utilized a tissue engineering approach in order to test whether supplementing culture medium with leucine could enhance mTORC1 signaling, myotube growth, and muscle function. Phosphorylation of the mTORC1 target proteins 4EBP‐1 and rpS6 and myotube hypertrophy appeared to occur in a dose dependent manner, with 5 and 20 mM of leucine inducing similar effects, which were greater than those seen with 1 mM. Maximal contractile force was also elevated with leucine supplementation; however, although this did not appear to be enhanced with increasing leucine doses, this effect was completely ablated by co‐incubation with the mTOR inhibitor rapamycin, showing that the augmented force production in the presence of leucine was mTOR sensitive. Finally, by using electrical stimulation to induce chronic (24 hr) contraction of engineered skeletal muscle constructs, we were able to show that the effects of leucine and muscle contraction are additive, since the two stimuli had cumulative effects on maximal contractile force production. These results extend our current knowledge of the efficacy of leucine as an anabolic nutritional aid showing for the first time that leucine supplementation may augment skeletal muscle functional capacity, and furthermore validates the use of engineered skeletal muscle for highly‐controlled investigations into nutritional regulation of muscle physiology. PMID:28409828

Excitation model of pacemaker cardiomyocytes of cardiac conduction system

NASA Astrophysics Data System (ADS)

Grigoriev, M.; Babich, L.

2015-11-01

Myocardium includes typical and atypical cardiomyocytes - pacemakers, which form the cardiac conduction system. Excitation from the atrioventricular node in normal conditions is possible only in one direction. Retrograde direction of pulses is impossible. The most important prerequisite for the work of cardiomyocytes is the anatomical integrity of the conduction system. Changes in contractile force of the cardiomyocytes, which appear periodically, are due to two mechanisms of self-regulation - heterometric and homeometric. Graphic course of the excitation pulse propagation along the heart muscle more accurately reveals the understanding of the arrhythmia mechanism. These models have the ability to visualize the essence of excitation dynamics. However, they do not have the proper forecasting function for result estimation. Integrative mathematical model enables further investigation of general laws of the myocardium active behavior, allows for determination of the violation mechanism of electrical and contractile function of cardiomyocytes. Currently, there is no full understanding of the topography of pacemakers and ionic mechanisms. There is a need for the development of direction of mathematical modeling and comparative studies of the electrophysiological arrangement of cells of atrioventricular connection and ventricular conduction system.

Overview of the Muscle Cytoskeleton

PubMed Central

Henderson, Christine A.; Gomez, Christopher G.; Novak, Stefanie M.; Mi-Mi, Lei; Gregorio, Carol C.

2018-01-01

Cardiac and skeletal striated muscles are intricately designed machines responsible for muscle contraction. Coordination of the basic contractile unit, the sarcomere, and the complex cytoskeletal networks are critical for contractile activity. The sarcomere is comprised of precisely organized individual filament systems that include thin (actin), thick (myosin), titin, and nebulin. Connecting the sarcomere to other organelles (e.g., mitochondria and nucleus) and serving as the scaffold to maintain cellular integrity are the intermediate filaments. The costamere, on the other hand, tethers the sarcomere to the cell membrane. Unique structures like the intercalated disc in cardiac muscle and the myotendinous junction in skeletal muscle help synchronize and transmit force. Intense investigation has been done on many of the proteins that make up these cytoskeletal assemblies. Yet the details of their function and how they interconnect have just started to be elucidated. A vast number of human myopathies are contributed to mutations in muscle proteins; thus understanding their basic function provides a mechanistic understanding of muscle disorders. In this review, we highlight the components of striated muscle with respect to their interactions, signaling pathways, functions, and connections to disease. PMID:28640448

Arp2/3 complex–dependent actin networks constrain myosin II function in driving retrograde actin flow

PubMed Central

Yang, Qing; Zhang, Xiao-Feng; Pollard, Thomas D.

2012-01-01

The Arp2/3 complex nucleates actin filaments to generate networks at the leading edge of motile cells. Nonmuscle myosin II produces contractile forces involved in driving actin network translocation. We inhibited the Arp2/3 complex and/or myosin II with small molecules to investigate their respective functions in neuronal growth cone actin dynamics. Inhibition of the Arp2/3 complex with CK666 reduced barbed end actin assembly site density at the leading edge, disrupted actin veils, and resulted in veil retraction. Strikingly, retrograde actin flow rates increased with Arp2/3 complex inhibition; however, when myosin II activity was blocked, Arp2/3 complex inhibition now resulted in slowing of retrograde actin flow and veils no longer retracted. Retrograde flow rate increases induced by Arp2/3 complex inhibition were independent of Rho kinase activity. These results provide evidence that, although the Arp2/3 complex and myosin II are spatially segregated, actin networks assembled by the Arp2/3 complex can restrict myosin II–dependent contractility with consequent effects on growth cone motility. PMID:22711700

The effect of obesity on the contractile performance of isolated mouse soleus, EDL, and diaphragm muscles.

PubMed

Tallis, Jason; Hill, Cameron; James, Rob S; Cox, Val M; Seebacher, Frank

2017-01-01

Obesity affects the major metabolic and cellular processes involved in skeletal muscle contractility. Surprisingly, the effect of obesity on isolated skeletal muscle performance remains unresolved. The present study is the first to examine the muscle-specific changes in contractility following dietary-induced obesity using an isolated muscle work-loop (WL) model that more closely represents in vivo muscle performance. Following 16-wk high-calorific feeding, soleus (SOL), extensor digitorum longus (EDL), and diaphragm (DIA) were isolated from female (CD-1) mice, and contractile performance was compared against a lean control group. Obese SOL produced greater isometric force; however, isometric stress (force per unit muscle area), absolute WL power, and normalized WL power (watts per kilogram muscle mass) were unaffected. Maximal isometric force and absolute WL power of the EDL were similar between groups. For both EDL and DIA, isometric stress and normalized WL power were reduced in the obese groups. Obesity caused a significant reduction in fatigue resistance in all cases. Our findings demonstrate a muscle-specific reduction in contractile performance and muscle quality that is likely related to in vivo mechanical role, fiber type, and metabolic profile, which may in part be related to changes in myosin heavy chain expression and AMP-activated protein kinase activity. These results infer that, beyond the additional requirement of moving a larger body mass, functional performance and quality of life may be further limited by poor muscle function in obese individuals. As such, a reduction in muscle performance may be a substantial contributor to the negative cycle of obesity. The effect of obesity on isolated muscle function is surprisingly underresearched. The present study is the first to examine the effects of obesity on isolated muscle performance using a method that more closely represents real-world muscle function. This work uniquely establishes a muscle-specific profile of mechanical changes in relation to underpinning mechanisms. These findings may be important to understanding the negative cycle of obesity and in designing interventions for improving weight status. Copyright © 2017 the American Physiological Society.

Cylindrical cellular geometry ensures fidelity of division site placement in fission yeast.

PubMed

Mishra, Mithilesh; Huang, Yinyi; Srivastava, Pragya; Srinivasan, Ramanujam; Sevugan, Mayalagu; Shlomovitz, Roie; Gov, Nir; Rao, Madan; Balasubramanian, Mohan

2012-08-15

Successful cytokinesis requires proper assembly of the contractile actomyosin ring, its stable positioning on the cell surface and proper constriction. Over the years, many of the key molecular components and regulators of the assembly and positioning of the actomyosin ring have been elucidated. Here we show that cell geometry and mechanics play a crucial role in the stable positioning and uniform constriction of the contractile ring. Contractile rings that assemble in locally spherical regions of cells are unstable and slip towards the poles. By contrast, actomyosin rings that assemble on locally cylindrical portions of the cell under the same conditions do not slip, but uniformly constrict the cell surface. The stability of the rings and the dynamics of ring slippage can be described by a simple mechanical model. Using fluorescence imaging, we verify some of the quantitative predictions of the model. Our study reveals an intimate interplay between geometry and actomyosin dynamics, which are likely to apply in a variety of cellular contexts.

The emergence of extracellular matrix mechanics and cell traction forces as important regulators of cellular self-organization.

PubMed

Checa, Sara; Rausch, Manuel K; Petersen, Ansgar; Kuhl, Ellen; Duda, Georg N

2015-01-01

Physical cues play a fundamental role in a wide range of biological processes, such as embryogenesis, wound healing, tumour invasion and connective tissue morphogenesis. Although it is well known that during these processes, cells continuously interact with the local extracellular matrix (ECM) through cell traction forces, the role of these mechanical interactions on large scale cellular and matrix organization remains largely unknown. In this study, we use a simple theoretical model to investigate cellular and matrix organization as a result of mechanical feedback signals between cells and the surrounding ECM. The model includes bi-directional coupling through cellular traction forces to deform the ECM and through matrix deformation to trigger cellular migration. In addition, we incorporate the mechanical contribution of matrix fibres and their reorganization by the cells. We show that a group of contractile cells will self-polarize at a large scale, even in homogeneous environments. In addition, our simulations mimic the experimentally observed alignment of cells in the direction of maximum stiffness and the building up of tension as a consequence of cell and fibre reorganization. Moreover, we demonstrate that cellular organization is tightly linked to the mechanical feedback loop between cells and matrix. Cells with a preference for stiff environments have a tendency to form chains, while cells with a tendency for soft environments tend to form clusters. The model presented here illustrates the potential of simple physical cues and their impact on cellular self-organization. It can be used in applications where cell-matrix interactions play a key role, such as in the design of tissue engineering scaffolds and to gain a basic understanding of pattern formation in organogenesis or tissue regeneration.

Nitrergic signalling via interstitial cells of Cajal regulates motor activity in murine colon.

PubMed

Lies, Barbara; Beck, Katharina; Keppler, Jonas; Saur, Dieter; Groneberg, Dieter; Friebe, Andreas

2015-10-15

In the enteric nervous systems, NO is released from nitrergic neurons as a major inhibitory neurotransmitter. NO acts via NO-sensitive guanylyl cyclase (NO-GC), which is found in different gastrointestinal (GI) cell types including smooth muscle cells (SMCs) and interstitial cells of Cajal (ICC). The precise mechanism of nitrergic signalling through these two cell types to regulate colonic spontaneous contractions is not fully understood yet. In the present study we investigated the impact of endogenous and exogenous NO on colonic contractile motor activity using mice lacking nitric oxide-sensitive guanylyl cyclase (NO-GC) globally and specifically in SMCs and ICC. Longitudinal smooth muscle of proximal colon from wild-type (WT) and knockout (KO) mouse strains exhibited spontaneous contractile activity ex vivo. WT and smooth muscle-specific guanylyl cyclase knockout (SMC-GCKO) colon showed an arrhythmic contractile activity with varying amplitudes and frequencies. In contrast, colon from global and ICC-specific guanylyl cyclase knockout (ICC-GCKO) animals showed a regular contractile rhythm with constant duration and amplitude of the rhythmic contractions. Nerve blockade (tetrodotoxin) or specific blockade of NO signalling (L-NAME, ODQ) did not significantly affect contractions of GCKO and ICC-GCKO colon whereas the arrhythmic contractile patterns of WT and SMC-GCKO colon were transformed into uniform motor patterns. In contrast, the response to electric field-stimulated neuronal NO release was similar in SMC-GCKO and global GCKO. In conclusion, our results indicate that basal enteric NO release acts via myenteric ICC to influence the generation of spontaneous contractions whereas the effects of elevated endogenous NO are mediated by SMCs in the murine proximal colon. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Validation of an in vitro contractility assay using canine ventricular myocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harmer, A.R., E-mail: alex.harmer@astrazeneca.com; Abi-Gerges, N.; Morton, M.J.

Measurement of cardiac contractility is a logical part of pre-clinical safety assessment in a drug discovery project, particularly if a risk has been identified or is suspected based on the primary- or non-target pharmacology. However, there are limited validated assays available that can be used to screen several compounds in order to identify and eliminate inotropic liability from a chemical series. We have therefore sought to develop an in vitro model with sufficient throughput for this purpose. Dog ventricular myocytes were isolated using a collagenase perfusion technique and placed in a perfused recording chamber on the stage of a microscopemore » at ∼ 36 °C. Myocytes were stimulated to contract at a pacing frequency of 1 Hz and a digital, cell geometry measurement system (IonOptix™) was used to measure sarcomere shortening in single myocytes. After perfusion with vehicle (0.1% DMSO), concentration–effect curves were constructed for each compound in 4–30 myocytes taken from 1 or 2 dog hearts. The validation test-set was 22 negative and 8 positive inotropes, and 21 inactive compounds, as defined by their effect in dog, cynolomolgous monkey or humans. By comparing the outcome of the assay to the known in vivo contractility effects, the assay sensitivity was 81%, specificity was 75%, and accuracy was 78%. With a throughput of 6–8 compounds/week from 1 cell isolation, this assay may be of value to drug discovery projects to screen for direct contractility effects and, if a hazard is identified, help identify inactive compounds. -- Highlights: ► Cardiac contractility is an important physiological function of the heart. ► Assessment of contractility is a logical part of pre-clinical drug safety testing. ► There are limited validated assays that predict effects of compounds on contractility. ► Using dog myocytes, we have developed an in vitro cardiac contractility assay. ► The assay predicted the in vivo contractility with a good level of accuracy.« less

Regulation of muscle contraction by Drebrin-like protein 1 probed by atomic force microscopy

NASA Astrophysics Data System (ADS)

Garces, Renata; Butkevich, Eugenia; Platen, Mitja; Schmidt, Christoph F.; Biophysics Team

Sarcomeres are the fundamental contractile units of striated muscle cells. They are composed of a variety of structural and regulatory proteins functioning in a precisely orchestrated fashion to enable coordinated force generation in striated muscles. Recently, we have identified a C. elegans drebrin-like protein 1 (DBN-1) as a novel sarcomere component, which stabilizes actin filaments during muscle contraction. To further characterize the function of DBN-1 in muscle cells, we generated a new dbn-1 loss-of-function allele. Absence of DBN-1 resulted in a unique worm movement phenotype, characterized by hyper-bending. It is not clear yet if DBN-1 acts to enhance or reduce the capacity for contraction. We present here an experimental mechanical study on C. elegans muscle mechanics. We measured the stiffness of the worm by indenting living C. eleganswith a micron-sized sphere adhered to the cantilever of an atomic force microscope (AFM). Modeling the worm as a pressurized elastic shell allows us to monitor the axial tension in the muscle through the measured stiffness. We compared responses of wild-type and mutant C. elegans in which DBN-1 is not expressed..

Eukaryotic and Prokaryotic Cytoskeletons: Structure and Mechanics

NASA Astrophysics Data System (ADS)

Gopinathan, Ajay

2013-03-01

The eukaryotic cytoskeleton is an assembly of filamentous proteins and a host of associated proteins that collectively serve functional needs ranging from spatial organization and transport to the production and transmission of forces. These systems can exhibit a wide variety of non-equilibrium, self-assembled phases depending on context and function. While much recent progress has been made in understanding the self-organization, rheology and nonlinear mechanical properties of such active systems, in this talk, we will concentrate on some emerging aspects of cytoskeletal physics that are promising. One such aspect is the influence of cytoskeletal network topology and its dynamics on both active and passive intracellular transport. Another aspect we will highlight is the interplay between chirality of filaments, their elasticity and their interactions with the membrane that can lead to novel conformational states with functional implications. Finally we will consider homologs of cytoskeletal proteins in bacteria, which are involved in templating cell growth, segregating genetic material and force production, which we will discuss with particular reference to contractile forces during cell division. These prokaryotic structures function in remarkably similar yet fascinatingly different ways from their eukaryotic counterparts and can enrich our understanding of cytoskeletal functioning as a whole.

Classification and development of myofiber types in the superior oblique extraocular muscle of chicken.

PubMed

Baryshnikova, Larisa M; Croes, Scott A; von Bartheld, Christopher S

2007-12-01

Precise control of contractile force of extraocular muscles is required for appropriate movements and alignment of the eyes. It is unclear how such precise regulation of contractile force is achieved during development and maturation. By using the posthatch chicken as a model, we describe and quantify critical parameters of the developing superior oblique extraocular muscle from hatching to 16 weeks of age, including contractile force, muscle mass, myofiber diameters, classification of fiber types, and distribution and quantification of mitochondria. Analysis at the light- and electron microscopic levels shows that chicken myofiber types largely correspond to their mammalian counterparts, with four fiber types in the orbital and four types in the global layer. Twitch tension muscle force and muscle mass gradually increase and stabilize at approximately 11 weeks. Tetanic tension continues to increase between 11 and 16 weeks. Myofiber diameters in both the orbital and global layer increase from hatching to six weeks, and then stabilize, whereas the myofiber number is constant after hatching. This finding suggests that muscle mass increases during late maturation due to increasing fiber length rather than fiber diameter. Quantitative ultrastructural analysis reveals continuing changes in the composition of the four muscle fiber types, suggesting ongoing fiber type conversion or differential replacement of myofiber types. Muscle fiber composition continues to change into late juvenile and adult age. Our study provides evidence for gradual, incremental, and continuing changes in avian myofiber composition and function that is similar to postnatal oculomotor maturation in visually oriented mammals such as kitten.

Revisiting the slow force response: the role of the PKG signaling pathway in the normal and the ischemic heart.

PubMed

Castro-Ferreira, Ricardo; Neves, João Sérgio; Ladeiras-Lopes, Ricardo; Leite-Moreira, André M; Neiva-Sousa, Manuel; Almeida-Coelho, João; Ferreira-Martins, João; F Leite-Moreira, Adelino

2014-09-01

The myocardial response to acute stretch consists of a two-phase increase in contractility: an acute increase by the Frank-Starling mechanism and a gradual and time-dependent increase in force generated known as the slow force response (SFR). The SFR is actively modulated by different signaling pathways, but the role of protein kinase G (PKG) signaling is unknown. In this study we aim to characterize the role of the PKG signaling pathway in the SFR under normal and ischemic conditions. Rabbit papillary muscles were stretched from 92 to 100% of maximum length (Lmax) under basal conditions, in the absence (1) or presence of: a PKG agonist (2) and a PKG inhibitor (3); under ischemic conditions in the absence (4) or presence of: a PKG agonist (5); a nitric oxide (NO) donor (6) and a phosphodiesterase 5 (PDE5) inhibitor (7). Under normoxia, the SFR was significantly attenuated by inhibition of PKG and remained unaltered with PKG activation. Ischemia induced a progressive decrease in myocardial contractility after stretch. Neither the PKG agonist nor the NO donor altered the myocardial response to stretch under ischemic conditions. However, the use of a PDE5 inhibitor in ischemia partially reversed the progressive deterioration in contractility. PKG activity is essential for the SFR. During ischemia, a progressive decline in the force is observed in response to acute myocardial stretch. This dysfunctional response can be partially reversed by the use of PDE5 inhibitors. Copyright © 2013 Sociedade Portuguesa de Cardiologia. Published by Elsevier España. All rights reserved.

Cancer-associated fibroblasts promote directional cancer cell migration by aligning fibronectin.

PubMed

Erdogan, Begum; Ao, Mingfang; White, Lauren M; Means, Anna L; Brewer, Bryson M; Yang, Lijie; Washington, M Kay; Shi, Chanjuan; Franco, Omar E; Weaver, Alissa M; Hayward, Simon W; Li, Deyu; Webb, Donna J

2017-11-06

Cancer-associated fibroblasts (CAFs) are major components of the carcinoma microenvironment that promote tumor progression. However, the mechanisms by which CAFs regulate cancer cell migration are poorly understood. In this study, we show that fibronectin (Fn) assembled by CAFs mediates CAF-cancer cell association and directional migration. Compared with normal fibroblasts, CAFs produce an Fn-rich extracellular matrix with anisotropic fiber orientation, which guides the cancer cells to migrate directionally. CAFs align the Fn matrix by increasing nonmuscle myosin II- and platelet-derived growth factor receptor α-mediated contractility and traction forces, which are transduced to Fn through α5β1 integrin. We further show that prostate cancer cells use αv integrin to migrate efficiently and directionally on CAF-derived matrices. We demonstrate that aligned Fn is a prominent feature of invasion sites in human prostatic and pancreatic carcinoma samples. Collectively, we present a new mechanism by which CAFs organize the Fn matrix and promote directional cancer cell migration. © 2017 Erdogan et al.

Cancer-associated fibroblasts promote directional cancer cell migration by aligning fibronectin

PubMed Central

Ao, Mingfang; White, Lauren M.; Means, Anna L.; Yang, Lijie; Washington, M. Kay; Franco, Omar E.; Li, Deyu; Webb, Donna J.

2017-01-01

Cancer-associated fibroblasts (CAFs) are major components of the carcinoma microenvironment that promote tumor progression. However, the mechanisms by which CAFs regulate cancer cell migration are poorly understood. In this study, we show that fibronectin (Fn) assembled by CAFs mediates CAF–cancer cell association and directional migration. Compared with normal fibroblasts, CAFs produce an Fn-rich extracellular matrix with anisotropic fiber orientation, which guides the cancer cells to migrate directionally. CAFs align the Fn matrix by increasing nonmuscle myosin II- and platelet-derived growth factor receptor α–mediated contractility and traction forces, which are transduced to Fn through α5β1 integrin. We further show that prostate cancer cells use αv integrin to migrate efficiently and directionally on CAF-derived matrices. We demonstrate that aligned Fn is a prominent feature of invasion sites in human prostatic and pancreatic carcinoma samples. Collectively, we present a new mechanism by which CAFs organize the Fn matrix and promote directional cancer cell migration. PMID:29021221

Regulation of adult cardiocyte growth: effects of active and passive mechanical loading

NASA Technical Reports Server (NTRS)

Decker, M. L.; Janes, D. M.; Barclay, M. M.; Harger, L.; Decker, R. S.

1997-01-01

Fluctuations in hemodynamic load have been documented to modulate contractile protein turnover and myofibrillar structure in the heart; however, the relative importance of active and passive loading in regulating adult cardiocyte growth remains unresolved. To address this issue at the cellular level, adult feline cardiocytes were cultured either on Silastic membranes or plastic surfaces. Cardiocyte-laden membranes were stretched 10% of their rest length to enhance passive loading, whereas heart cells cultured on plastic or Silastic were field stimulated at 1 Hz to mimic active loading. Turnover of contractile proteins and structural integrity of the contractile-cytoskeletal apparatus were monitored for periods ranging from 4 to 72 h. Active and passive loading elevated contractile protein synthesis nearly equally (approximately 50%) and promoted the attachment of remodeled myofibrils to vinculin-positive focal contacts and/or costameres during the first 24 h of loading. Thereafter, rates of contractile protein synthesis returned to control values in passively stretched heart cells but remained elevated in field-stimulated cultures. The fractional rate of growth was increased significantly (approximately 8%/day) in electrically paced cells, whereas in passively stretched cardiocytes the growth rate rose only modestly (approximately 2%/day). Changes in the rate of myocyte growth appeared more closely correlated with the development of focal contacts and myofibril remodeling than with changes in myofibrillar protein turnover per se. 2,3-Butanedione monoxime, nifedipine, and, to a lesser extent, ryanodine blocked field-stimulated contractile protein synthesis and myofibrillar remodeling but had no impact on protein turnover or myofibril reassembly in passively loaded cardiocytes. The results of these experiments imply that both active and passive loading stimulate contractile protein turnover and myofibril remodeling, but the generation of active tension accelerates cardiocyte growth to a greater extent than passive loading. Furthermore, pharmacological interventions suggest that unique pathways may mediate these cellular events in actively and passively loaded adult cardiocytes.

Regulation of adult cardiocyte growth: effects of active and passive mechanical loading.

PubMed

Decker, M L; Janes, D M; Barclay, M M; Harger, L; Decker, R S

1997-06-01

Fluctuations in hemodynamic load have been documented to modulate contractile protein turnover and myofibrillar structure in the heart; however, the relative importance of active and passive loading in regulating adult cardiocyte growth remains unresolved. To address this issue at the cellular level, adult feline cardiocytes were cultured either on Silastic membranes or plastic surfaces. Cardiocyte-laden membranes were stretched 10% of their rest length to enhance passive loading, whereas heart cells cultured on plastic or Silastic were field stimulated at 1 Hz to mimic active loading. Turnover of contractile proteins and structural integrity of the contractile-cytoskeletal apparatus were monitored for periods ranging from 4 to 72 h. Active and passive loading elevated contractile protein synthesis nearly equally (approximately 50%) and promoted the attachment of remodeled myofibrils to vinculin-positive focal contacts and/or costameres during the first 24 h of loading. Thereafter, rates of contractile protein synthesis returned to control values in passively stretched heart cells but remained elevated in field-stimulated cultures. The fractional rate of growth was increased significantly (approximately 8%/day) in electrically paced cells, whereas in passively stretched cardiocytes the growth rate rose only modestly (approximately 2%/day). Changes in the rate of myocyte growth appeared more closely correlated with the development of focal contacts and myofibril remodeling than with changes in myofibrillar protein turnover per se. 2,3-Butanedione monoxime, nifedipine, and, to a lesser extent, ryanodine blocked field-stimulated contractile protein synthesis and myofibrillar remodeling but had no impact on protein turnover or myofibril reassembly in passively loaded cardiocytes. The results of these experiments imply that both active and passive loading stimulate contractile protein turnover and myofibril remodeling, but the generation of active tension accelerates cardiocyte growth to a greater extent than passive loading. Furthermore, pharmacological interventions suggest that unique pathways may mediate these cellular events in actively and passively loaded adult cardiocytes.

Changes in the interstitial cells of Cajal and neuronal nitric oxide synthase positive neuronal cells with aging in the esophagus of F344 rats.

PubMed

Kim, Hee Jin; Kim, Nayoung; Kim, Yong Sung; Nam, Ryoung Hee; Lee, Sun Min; Park, Ji Hyun; Choi, Daeun; Hwang, Young-Jae; Lee, Jongchan; Lee, Hye Seung; Kim, Min-Seob; Lee, Moon Young; Lee, Dong Ho

2017-01-01

The aging-associated cellular and molecular changes in esophagus have not been established, yet. Thus we evaluated histological structure, interstitial cells of Cajal (ICCs), neuronal nitric oxide synthase (nNOS)-positive cells, and contractility in the esophagus of Fischer 344 rat at different ages (6-, 31-, 74-weeks, and 2-years). The lamina propria thickness and endomysial area were calculated. The immunoreactivity of c-Kit, nNOS and protein gene product (PGP) 9.5 was counted after immunohistochemistry. Expression of c-Kit, stem cell factor (SCF), nNOS and PGP 9.5 mRNA was measured by real-time PCR, and expression of c-Kit and nNOS protein was detected by Western blot. Isovolumetric contractile force measurement and electrical field stimulation (EFS) were conducted. The lamina propria thickness increased (6 week vs 2 year, P = 0.005) and the endomysial area of longitudinal muscle decreased with aging (6 week vs 2 year, P<0.001), while endomysial area of circular muscle did not significantly decrease. The proportions of NOS-immunoreactive cells and c-Kit-immunoreactive areas declined with aging (6 week vs 2 year; P<0.001 and P = 0.004, respectively), but there was no significant change of PGP 9.5-immunopositiviy. The expressions of nNOS, c-Kit and SCF mRNA also reduced with aging (6 week vs 2 year; P = 0.006, P = 0.001 and P = 0.006, respectively), while the change of PGP 9.5 mRNA expression was not significant. Western blot showed the significant decreases of nNOS and c-Kit protein expression with aging (6 week vs 2 year; P = 0.008 and P = 0.012, respectively). The EFS-induced esophageal contractions significantly decreased in 2-yr-old rat compared with 6-wk-old rats, however, L-NG-Nitroarginine methylester did not significantly increase the spontaneous and EFS-induced contractions in the 6-wk- and 2-yr-old rat esophagus. In conclusion, an increase of lamina propria thickness, a decrease of endomysial area, c-Kit, SCF and NOS expression with preserved total enteric neurons, and contractility in aged rat esophagus may explain the aging-associated esophageal dysmotility.

Changes in the interstitial cells of Cajal and neuronal nitric oxide synthase positive neuronal cells with aging in the esophagus of F344 rats

PubMed Central

Kim, Hee Jin; Kim, Yong Sung; Nam, Ryoung Hee; Lee, Sun Min; Park, Ji Hyun; Choi, Daeun; Hwang, Young-Jae; Lee, Jongchan; Lee, Hye Seung; Kim, Min-Seob; Lee, Moon Young; Lee, Dong Ho

2017-01-01

The aging-associated cellular and molecular changes in esophagus have not been established, yet. Thus we evaluated histological structure, interstitial cells of Cajal (ICCs), neuronal nitric oxide synthase (nNOS)-positive cells, and contractility in the esophagus of Fischer 344 rat at different ages (6-, 31-, 74-weeks, and 2-years). The lamina propria thickness and endomysial area were calculated. The immunoreactivity of c-Kit, nNOS and protein gene product (PGP) 9.5 was counted after immunohistochemistry. Expression of c-Kit, stem cell factor (SCF), nNOS and PGP 9.5 mRNA was measured by real-time PCR, and expression of c-Kit and nNOS protein was detected by Western blot. Isovolumetric contractile force measurement and electrical field stimulation (EFS) were conducted. The lamina propria thickness increased (6 week vs 2 year, P = 0.005) and the endomysial area of longitudinal muscle decreased with aging (6 week vs 2 year, P<0.001), while endomysial area of circular muscle did not significantly decrease. The proportions of NOS-immunoreactive cells and c-Kit-immunoreactive areas declined with aging (6 week vs 2 year; P<0.001 and P = 0.004, respectively), but there was no significant change of PGP 9.5-immunopositiviy. The expressions of nNOS, c-Kit and SCF mRNA also reduced with aging (6 week vs 2 year; P = 0.006, P = 0.001 and P = 0.006, respectively), while the change of PGP 9.5 mRNA expression was not significant. Western blot showed the significant decreases of nNOS and c-Kit protein expression with aging (6 week vs 2 year; P = 0.008 and P = 0.012, respectively). The EFS-induced esophageal contractions significantly decreased in 2-yr-old rat compared with 6-wk-old rats, however, L-NG-Nitroarginine methylester did not significantly increase the spontaneous and EFS-induced contractions in the 6-wk- and 2-yr-old rat esophagus. In conclusion, an increase of lamina propria thickness, a decrease of endomysial area, c-Kit, SCF and NOS expression with preserved total enteric neurons, and contractility in aged rat esophagus may explain the aging-associated esophageal dysmotility. PMID:29182640

Mechanical stress and network structure drive protein dynamics during cytokinesis.

PubMed

Srivastava, Vasudha; Robinson, Douglas N

2015-03-02

Cell-shape changes associated with processes like cytokinesis and motility proceed on several-second timescales but are derived from molecular events, including protein-protein interactions, filament assembly, and force generation by molecular motors, all of which occur much faster [1-4]. Therefore, defining the dynamics of such molecular machinery is critical for understanding cell-shape regulation. In addition to signaling pathways, mechanical stresses also direct cytoskeletal protein accumulation [5-7]. A myosin-II-based mechanosensory system controls cellular contractility and shape during cytokinesis and under applied stress [6, 8]. In Dictyostelium, this system tunes myosin II accumulation by feedback through the actin network, particularly through the crosslinker cortexillin I. Cortexillin-binding IQGAPs are major regulators of this system. Here, we defined the short timescale dynamics of key cytoskeletal proteins during cytokinesis and under mechanical stress, using fluorescence recovery after photobleaching and fluorescence correlation spectroscopy, to examine the dynamic interplay between these proteins. Equatorially enriched proteins including cortexillin I, IQGAP2, and myosin II recovered much more slowly than actin and polar crosslinkers. The mobility of equatorial proteins was greatly reduced at the furrow compared to the interphase cortex, suggesting their stabilization during cytokinesis. This mobility shift did not arise from a single biochemical event, but rather from a global inhibition of protein dynamics by mechanical-stress-associated changes in the cytoskeletal structure. Mechanical tuning of contractile protein dynamics provides robustness to the cytoskeletal framework responsible for regulating cell shape and contributes to cytokinesis fidelity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Proteostasis and REDOX state in the heart

PubMed Central

Christians, Elisabeth S.

2012-01-01

Force-generating contractile cells of the myocardium must achieve and maintain their primary function as an efficient mechanical pump over the life span of the organism. Because only half of the cardiomyocytes can be replaced during the entire human life span, the maintenance strategy elicited by cardiac cells relies on uninterrupted renewal of their components, including proteins whose specialized functions constitute this complex and sophisticated contractile apparatus. Thus cardiac proteins are continuously synthesized and degraded to ensure proteome homeostasis, also termed “proteostasis.” Once synthesized, proteins undergo additional folding, posttranslational modifications, and trafficking and/or become involved in protein-protein or protein-DNA interactions to exert their functions. This includes key transient interactions of cardiac proteins with molecular chaperones, which assist with quality control at multiple levels to prevent misfolding or to facilitate degradation. Importantly, cardiac proteome maintenance depends on the cellular environment and, in particular, the reduction-oxidation (REDOX) state, which is significantly different among cardiac organelles (e.g., mitochondria and endoplasmic reticulum). Taking into account the high metabolic activity for oxygen consumption and ATP production by mitochondria, it is a challenge for cardiac cells to maintain the REDOX state while preventing either excessive oxidative or reductive stress. A perturbed REDOX environment can affect protein handling and conformation (e.g., disulfide bonds), disrupt key structure-function relationships, and trigger a pathogenic cascade of protein aggregation, decreased cell survival, and increased organ dysfunction. This review covers current knowledge regarding the general domain of REDOX state and protein folding, specifically in cardiomyocytes under normal-healthy conditions and during disease states associated with morbidity and mortality in humans. PMID:22003057

Proteostasis and REDOX state in the heart.

PubMed

Christians, Elisabeth S; Benjamin, Ivor J

2012-01-01

Force-generating contractile cells of the myocardium must achieve and maintain their primary function as an efficient mechanical pump over the life span of the organism. Because only half of the cardiomyocytes can be replaced during the entire human life span, the maintenance strategy elicited by cardiac cells relies on uninterrupted renewal of their components, including proteins whose specialized functions constitute this complex and sophisticated contractile apparatus. Thus cardiac proteins are continuously synthesized and degraded to ensure proteome homeostasis, also termed "proteostasis." Once synthesized, proteins undergo additional folding, posttranslational modifications, and trafficking and/or become involved in protein-protein or protein-DNA interactions to exert their functions. This includes key transient interactions of cardiac proteins with molecular chaperones, which assist with quality control at multiple levels to prevent misfolding or to facilitate degradation. Importantly, cardiac proteome maintenance depends on the cellular environment and, in particular, the reduction-oxidation (REDOX) state, which is significantly different among cardiac organelles (e.g., mitochondria and endoplasmic reticulum). Taking into account the high metabolic activity for oxygen consumption and ATP production by mitochondria, it is a challenge for cardiac cells to maintain the REDOX state while preventing either excessive oxidative or reductive stress. A perturbed REDOX environment can affect protein handling and conformation (e.g., disulfide bonds), disrupt key structure-function relationships, and trigger a pathogenic cascade of protein aggregation, decreased cell survival, and increased organ dysfunction. This review covers current knowledge regarding the general domain of REDOX state and protein folding, specifically in cardiomyocytes under normal-healthy conditions and during disease states associated with morbidity and mortality in humans.

Intrinsic Cell Stress is Independent of Organization in Engineered Cell Sheets.

PubMed

van Loosdregt, Inge A E W; Dekker, Sylvia; Alford, Patrick W; Oomens, Cees W J; Loerakker, Sandra; Bouten, Carlijn V C

2018-06-01

Understanding cell contractility is of fundamental importance for cardiovascular tissue engineering, due to its major impact on the tissue's mechanical properties as well as the development of permanent dimensional changes, e.g., by contraction or dilatation of the tissue. Previous attempts to quantify contractile cellular stresses mostly used strongly aligned monolayers of cells, which might not represent the actual organization in engineered cardiovascular tissues such as heart valves. In the present study, therefore, we investigated whether differences in organization affect the magnitude of intrinsic stress generated by individual myofibroblasts, a frequently used cell source for in vitro engineered heart valves. Four different monolayer organizations were created via micro-contact printing of fibronectin lines on thin PDMS films, ranging from strongly anisotropic to isotropic. Thin film curvature, cell density, and actin stress fiber distribution were quantified, and subsequently, intrinsic stress and contractility of the monolayers were determined by incorporating these data into sample-specific finite element models. Our data indicate that the intrinsic stress exerted by the monolayers in each group correlates with cell density. Additionally, after normalizing for cell density and accounting for differences in alignment, no consistent differences in intrinsic contractility were found between the different monolayer organizations, suggesting that the intrinsic stress exerted by individual myofibroblasts is independent of the organization. Consequently, this study emphasizes the importance of choosing proper architectural properties for scaffolds in cardiovascular tissue engineering, as these directly affect the stresses in the tissue, which play a crucial role in both the functionality and remodeling of (engineered) cardiovascular tissues.

Influence of stretch-shortening cycle on mechanical behaviour of triceps surae during hopping.

PubMed

Belli, A; Bosco, C

1992-04-01

Six subjects performed a first series of vertical plantar flexions and a second series of vertical rebounds, both involving muscle triceps surae exclusively. Vertical displacements, vertical forces and ankle angles were recorded during the entire work period of 60 seconds per series. In addition, expired gases were collected during the test and recovery for determination of the energy expenditure. Triceps surae was mechanically modelled with a contractile component and with an elastic component. Mechanical behaviour and work of the different muscle components were determined in both series. The net muscular efficiency calculated from the work performed by the centre of gravity was 17.5 +/- 3.0% (mean +/- SD) in plantar flexions and 29.9 +/- 4.8% in vertical rebounds. The net muscle efficiency calculated from the work performed by the contractile component was 17.4 +/- 2.9% in plantar flexions and 16.1 +/- 1.4% in vertical rebounds. These results suggest that the muscular efficiency differences do not reflect muscle contractile component efficiency but essentially the storage and recoil of elastic energy. This is supported by the relationship (P less than 0.01) found in vertical rebounds between the extra work and the elastic component work. A detailed observation of the mechanical behaviour of muscle mechanical components showed that the strategy to maximize the elastic work depends also on the force-velocity characteristics of the movement and that the eccentric-concentric work of the contractile component does not always correspond respectively to the ankle extension-flexion.

Aqueous two-phase printing of cell-containing contractile collagen microgels.

PubMed

Moraes, Christopher; Simon, Arlyne B; Putnam, Andrew J; Takayama, Shuichi

2013-12-01

This work describes the use of aqueous two-phase systems to print cell-containing contractile collagen microdroplets. The fully aqueous conditions enable convenient formation of sub-microliter 'microgels' that are much smaller than otherwise possible to fabricate while maintaining high cell viability. The produced microgels contract over several days, mimicking the behavior of macroscale contraction assays, which have been valued as an important biological readout for over three decades. Use of microgels not only reduces reagent consumption and increases throughput of the assay, but also improves transport of molecules into and out of the collagen matrix, thereby enabling efficient and more precise studies of timed stimulation profiles. Utility of the technology is demonstrated by analyzing the effects of TGF-β1 on gel contraction, and we demonstrate that brief 'burst' stimulation profiles in microgels prompt contraction of the matrix, a feature not observed in the conventional macroscale assay. The fully aqueous process also enables the integration of contractile collagen microgels within existing cell culture systems, and we demonstrate proof-of-principle experiments in which a contractile collagen droplet is fabricated in situ on an existing epithelial monolayer. The simplicity, versatility and ability to robustly produce collagen microgels should allow effective translation of this microengineering technology into a variety of research environments. Copyright © 2013 Elsevier Ltd. All rights reserved.

Hydrogel tissue construct-based high-content compound screening.

PubMed

Lam, Vy; Wakatsuki, Tetsuro

2011-01-01

Current pharmaceutical compound screening systems rely on cell-based assays to identify therapeutic candidates and potential toxicities. However, cells grown on 2D substrata or in suspension do not exhibit the mechanical or physiological properties of cells in vivo. To address this limitation, the authors developed an in vitro, high-throughput, 3D hydrogel tissue construct (HTC)-based assay system to quantify cell and tissue mechanical properties and multiple parameters of physiology. HTC mechanics was quantified using an automated device, and physiological status was assessed using spectroscopy-based indicators that were read on microplate readers. To demonstrate the application of this system, the authors screened 4 test compounds--rotenone (ROT), cytochalasin D (CD), 2,4-dinitrophenol (DNP), and Rho kinase inhibitor (H-1152)--for their ability to modulate HTC contractility without affecting actin integrity, mitochondrial membrane potential (MMP), or viability. All 4 compounds dose-dependently reduced HTC contractility. However, ROT was toxic, DNP dissipated MMP, and CD reduced both intracellular F-actin and viability. H-1152 was found to be the best candidate compound since it reduced HTC contractility with minimal side effects. The authors propose that their HTC-based assay system can be used to screen for compounds that modulate HTC contractility and assess the underlying physiological mechanism(s) of compound activity and toxicity.

Effect of acute gastric dilatation on gastric myoelectic and motor activity in dogs.

PubMed

Hall, J A; Solie, T N; Seim, H B; Twedt, D C

1999-05-01

To investigate the effects of experimentally induced acute gastric dilatation on electrical and mechanical activities of the stomach in dogs. 7 healthy dogs. Electrodes and strain-gauge force transducers were implanted on the serosal surface of the antrum and pylorus. Eight days later, baseline gastric electrical and contractile activities were recorded. The dogs were anesthetized and mechanically ventilated to maintain normocapnia while the stomach was distended (intragastric pressure, 30 mm Hg) for 180 minutes, using a thin compliant bag. Gastric electrical and contractile activities were recorded again on days 1 and 10 after dilatation. Recordings were analyzed to determine gastric slow-wave frequency, slow-wave dysrhythmia, propagation velocity of slow-waves, coupling of contractions to slow waves, motility index on the basis of relative contractile amplitudes, and onset of contractions after a standardized meal. Electrical or contractile activities were not significantly different 18 hours after acute gastric dilatation (day 1). Arrhythmias were evident before and after gastric dilatation in dogs from which food was withheld and in dogs after consumption of a meal. Variables for assessing gastric electrical and contractile activities were unaffected 18 hours after acute gastric dilatation. Analysis of results of this study indicated that altered electrical and contractile activities in dogs with short-term gastric dilatation are not likely to be secondary to the process of acute gastric dilatation.

Myosin II Dynamics during Embryo Morphogenesis

NASA Astrophysics Data System (ADS)

Kasza, Karen

2013-03-01

During embryonic morphogenesis, the myosin II motor protein generates forces that help to shape tissues, organs, and the overall body form. In one dramatic example in the Drosophila melanogaster embryo, the epithelial tissue that will give rise to the body of the adult animal elongates more than two-fold along the head-to-tail axis in less than an hour. This elongation is accomplished primarily through directional rearrangements of cells within the plane of the tissue. Just prior to elongation, polarized assemblies of myosin II accumulate perpendicular to the elongation axis. The contractile forces generated by myosin activity orient cell movements along a common axis, promoting local cell rearrangements that contribute to global tissue elongation. The molecular and mechanical mechanisms by which myosin drives this massive change in embryo shape are poorly understood. To investigate these mechanisms, we generated a collection of transgenic flies expressing variants of myosin II with altered motor function and regulation. We found that variants that are predicted to have increased myosin activity cause defects in tissue elongation. Using biophysical approaches, we found that these myosin variants also have decreased turnover dynamics within cells. To explore the mechanisms by which molecular-level myosin dynamics are translated into tissue-level elongation, we are using time-lapse confocal imaging to observe cell movements in embryos with altered myosin activity. We are utilizing computational approaches to quantify the dynamics and directionality of myosin localization and cell rearrangements. These studies will help elucidate how myosin-generated forces control cell movements within tissues. This work is in collaboration with J. Zallen at the Sloan-Kettering Institute.

Structure of the Elastin-Contractile Units in the Thoracic Aorta and How Genes That Cause Thoracic Aortic Aneurysms and Dissections Disrupt This Structure.

PubMed

Karimi, Ashkan; Milewicz, Dianna M

2016-01-01

The medial layer of the aorta confers elasticity and strength to the aortic wall and is composed of alternating layers of smooth muscle cells (SMCs) and elastic fibres. The SMC elastin-contractile unit is a structural unit that links the elastin fibres to the SMCs and is characterized by the following: (1) layers of elastin fibres that are surrounded by microfibrils; (2) microfibrils that bind to the integrin receptors in focal adhesions on the cell surface of the SMCs; and (3) SMC contractile filaments that are linked to the focal adhesions on the inner side of the membrane. The genes that are altered to cause thoracic aortic aneurysms and aortic dissections encode proteins involved in the structure or function of the SMC elastin-contractile unit. Included in this gene list are the genes encoding protein that are structural components of elastin fibres and microfibrils, FBN1, MFAP5, ELN, and FBLN4. Also included are genes that encode structural proteins in the SMC contractile unit, including ACTA2, which encodes SMC-specific α-actin and MYH11, which encodes SMC-specific myosin heavy chain, along with MYLK and PRKG1, which encode kinases that control SMC contraction. Finally, mutations in the gene encoding the protein linking integrin receptors to the contractile filaments, FLNA, also predispose to thoracic aortic disease. Thus, these data suggest that functional SMC elastin-contractile units are important for maintaining the structural integrity of the aorta. Copyright © 2016 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.

Cytoskeletal role in the transition from compensated to decompensated hypertrophy during adult canine left ventricular pressure overloading

NASA Technical Reports Server (NTRS)

Tagawa, H.; Koide, M.; Sato, H.; Zile, M. R.; Carabello, B. A.; Cooper, G. 4th

1998-01-01

Increased microtubule density causes cardiocyte contractile dysfunction in right ventricular (RV) pressure-overload hypertrophy, and these linked phenotypic and contractile abnormalities persist and progress during the transition to failure. Although more severe in cells from failing than hypertrophied RVs, the mechanical defects are normalized in each case by microtubule depolymerization. To define the role of increased microtubule density in left ventricular (LV) pressure-overload hypertrophy and failure, in a given LV we examined ventricular mechanics, sarcomere mechanics, and free tubulin and microtubule levels in control dogs and in dogs with aortic stenosis both with LV hypertrophy alone and with initially compensated hypertrophy that had progressed to LV muscle failure. In comparing initial values with those at study 8 weeks later, dogs with hypertrophy alone had a very substantial increase in LV mass but preservation of a normal ejection fraction and mean systolic wall stress. Dogs with hypertrophy and associated failure had a substantial but lesser increase in LV mass and a reduction in ejection fraction, as well as a marked increase in mean systolic wall stress. Cardiocyte contractile function was equivalent, and unaffected by microtubule depolymerization, in cells from control LVs and those with compensated hypertrophy. In contrast, cardiocyte contractile function in cells from failing LVs was quite depressed but was normalized by microtubule depolymerization. Microtubules were increased only in failing LVs. These contractile and cytoskeletal changes, when assayed longitudinally in a given dog by biopsy, appeared in failing ventricles only when wall stress began to increase and function began to decrease. Thus, the microtubule-based cardiocyte contractile dysfunction characteristic of pressure-hypertrophied myocardium, originally described in the RV, obtains equally in the LV but is shown here to have a specific association with increased wall stress.

3D engineered cardiac tissue models of human heart disease: learning more from our mice.

PubMed

Ralphe, J Carter; de Lange, Willem J

2013-02-01

Mouse engineered cardiac tissue constructs (mECTs) are a new tool available to study human forms of genetic heart disease within the laboratory. The cultured strips of cardiac cells generate physiologic calcium transients and twitch force, and respond to electrical pacing and adrenergic stimulation. The mECT can be made using cells from existing mouse models of cardiac disease, providing a robust readout of contractile performance and allowing a rapid assessment of genotype-phenotype correlations and responses to therapies. mECT represents an efficient and economical extension to the existing tools for studying cardiac physiology. Human ECTs generated from iPSCMs represent the next logical step for this technology and offer significant promise of an integrated, fully human, cardiac tissue model. Copyright © 2013. Published by Elsevier Inc.

Formation of contractile networks and fibers in the medial cell cortex through myosin-II turnover, contraction, and stress-stabilization

PubMed Central

Nie, Wei; Wei, Ming-Tzo; Ou-Yang, Daniel H.; Jedlicka, Sabrina S.; Vavylonis, Dimitrios

2015-01-01

The morphology of adhered cells depends crucially on the formation of a contractile meshwork of parallel and cross-linked fibers along the contacting surface. The motor activity and minifilament assembly of non-muscle myosin-II is an important component of cortical cytoskeletal remodeling during mechanosensing. We used experiments and computational modeling to study cortical myosin-II dynamics in adhered cells. Confocal microscopy was used to image the medial cell cortex of HeLa cells stably expressing myosin regulatory light chain tagged with GFP (MRLC-GFP). The distribution of MRLC-GFP fibers and focal adhesions was classified into three types of network morphologies. Time-lapse movies show: myosin foci appearance and disappearance; aligning and contraction; stabilization upon alignment. Addition of blebbistatin, which perturbs myosin motor activity, leads to a reorganization of the cortical networks and to a reduction of contractile motions. We quantified the kinetics of contraction, disassembly and reassembly of myosin networks using spatio-temporal image correlation spectroscopy (STICS). Coarse-grained numerical simulations include bipolar minifilaments that contract and align through specified interactions as basic elements. After assuming that minifilament turnover decreases with increasing contractile stress, the simulations reproduce stress-dependent fiber formation in between focal adhesions above a threshold myosin concentration. The STICS correlation function in simulations matches the function measured in experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness. PMID:25641802

β-adrenergic effects on cardiac myofilaments and contraction in an integrated rabbit ventricular myocyte model

PubMed Central

Negroni, Jorge A.; Morotti, Stefano; Lascano, Elena C.; Gomes, Aldrin V.; Grandi, Eleonora; Puglisi, José L; Bers, Donald M.

2015-01-01

A five-state model of myofilament contraction was integrated into a well-established rabbit ventricular myocyte model of ion channels, Ca2+ transporters and kinase signaling to analyze the relative contribution of different phosphorylation targets to the overall mechanical response driven by β-adrenergic stimulation (β-AS). β-AS effect on sarcoplasmic reticulum Ca2+ handling, Ca2+, K+ and Cl− currents, and Na+/K+-ATPase properties were included based on experimental data. The inotropic effect on the myofilaments was represented as reduced myofilament Ca2+ sensitivity (XBCa) and titin stiffness, and increased cross-bridge (XB) cycling rate (XBcy). Assuming independent roles of XBCa and XBcy, the model reproduced experimental β-AS responses on action potentials and Ca2+ transient amplitude and kinetics. It also replicated the behavior of force-Ca2+, release-restretch, length-step, stiffness-frequency and force-velocity relationships, and increased force and shortening in isometric and isotonic twitch contractions. The β-AS effect was then switched off from individual targets to analyze their relative impact on contractility. Preventing β-AS effects on L-type Ca2+ channels or phospholamban limited Ca2+ transients and contractile responses in parallel, while blocking phospholemman and K+ channel (IKs) effects enhanced Ca2+ and inotropy. Removal of β-AS effects from XBCa enhanced contractile force while decreasing peak Ca2+ (due to greater Ca2+ buffering), but had less effect on shortening. Conversely, preventing β-AS effects on XBcy preserved Ca2+ transient effects, but blunted inotropy (both isometric force and especially shortening). Removal of titin effects had little impact on contraction. Finally, exclusion of β-AS from XBCa and XBcy while preserving effects on other targets resulted in preserved peak isometric force response (with slower kinetics) but nearly abolished enhanced shortening. β-AS effects on XBCa vs. XBcy have greater impact on isometric vs. isotonic contraction, respectively. PMID:25724724

Traction force microscopy of engineered cardiac tissues.

PubMed

Pasqualini, Francesco Silvio; Agarwal, Ashutosh; O'Connor, Blakely Bussie; Liu, Qihan; Sheehy, Sean P; Parker, Kevin Kit

2018-01-01

Cardiac tissue development and pathology have been shown to depend sensitively on microenvironmental mechanical factors, such as extracellular matrix stiffness, in both in vivo and in vitro systems. We present a novel quantitative approach to assess cardiac structure and function by extending the classical traction force microscopy technique to tissue-level preparations. Using this system, we investigated the relationship between contractile proficiency and metabolism in neonate rat ventricular myocytes (NRVM) cultured on gels with stiffness mimicking soft immature (1 kPa), normal healthy (13 kPa), and stiff diseased (90 kPa) cardiac microenvironments. We found that tissues engineered on the softest gels generated the least amount of stress and had the smallest work output. Conversely, cardiomyocytes in tissues engineered on healthy- and disease-mimicking gels generated significantly higher stresses, with the maximal contractile work measured in NRVM engineered on gels of normal stiffness. Interestingly, although tissues on soft gels exhibited poor stress generation and work production, their basal metabolic respiration rate was significantly more elevated than in other groups, suggesting a highly ineffective coupling between energy production and contractile work output. Our novel platform can thus be utilized to quantitatively assess the mechanotransduction pathways that initiate tissue-level structural and functional remodeling in response to substrate stiffness.

Spatial and temporal traction response in human airway smooth muscle cells

NASA Technical Reports Server (NTRS)

Tolic-Norrelykke, Iva Marija; Butler, James P.; Chen, Jianxin; Wang, Ning

2002-01-01

Tractions that cells exert on their substrates are essential in cell spreading, migration, and contraction. These tractions can be determined by plating the cells on a flexible gel and measuring the deformation of the gel by using fluorescent beads embedded just below the surface of the gel. In this article we describe the image correlation method (ICM) optimized for determining the displacement field of the gel under a contracting cell. For the calculation of the traction field from the displacement field we use the recently developed method of Fourier transform traction cytometry (FTTC). The ICM and FTTC methods are applied to human airway smooth muscle cells during stimulation with the contractile agonist histamine or the relaxing agonist isoproterenol. The overall intensity of the cell contraction (the median traction magnitude, the energy transferred from the cell to the gel, and the net contractile moment) increased after activation with histamine, and decreased after treatment with isoproterenol. Cells exhibited regional differences in the time course of traction during the treatment. Both temporal evolution and magnitude of traction increase induced by histamine varied markedly among different cell protrusions, whereas the nuclear region showed the smallest response. These results suggest that intracellular mediators of cell adhesion and contraction respond to contractile stimuli with different rates and intensities in different regions of the cell.

Myosin binding protein-C activates thin filaments and inhibits thick filaments in heart muscle cells

PubMed Central

Kampourakis, Thomas; Yan, Ziqian; Gautel, Mathias; Sun, Yin-Biao; Irving, Malcolm

2014-01-01

Myosin binding protein-C (MyBP-C) is a key regulatory protein in heart muscle, and mutations in the MYBPC3 gene are frequently associated with cardiomyopathy. However, the mechanism of action of MyBP-C remains poorly understood, and both activating and inhibitory effects of MyBP-C on contractility have been reported. To clarify the function of the regulatory N-terminal domains of MyBP-C, we determined their effects on the structure of thick (myosin-containing) and thin (actin-containing) filaments in intact sarcomeres of heart muscle. We used fluorescent probes on troponin C in the thin filaments and on myosin regulatory light chain in the thick filaments to monitor structural changes associated with activation of demembranated trabeculae from rat ventricle by the C1mC2 region of rat MyBP-C. C1mC2 induced larger structural changes in thin filaments than calcium activation, and these were still present when active force was blocked with blebbistatin, showing that C1mC2 directly activates the thin filaments. In contrast, structural changes in thick filaments induced by C1mC2 were smaller than those associated with calcium activation and were abolished or reversed by blebbistatin. Low concentrations of C1mC2 did not affect resting force but increased calcium sensitivity and reduced cooperativity of force and structural changes in both thin and thick filaments. These results show that the N-terminal region of MyBP-C stabilizes the ON state of thin filaments and the OFF state of thick filaments and lead to a novel hypothesis for the physiological role of MyBP-C in the regulation of cardiac contractility. PMID:25512492

Myosin binding protein-C activates thin filaments and inhibits thick filaments in heart muscle cells.

PubMed

Kampourakis, Thomas; Yan, Ziqian; Gautel, Mathias; Sun, Yin-Biao; Irving, Malcolm

2014-12-30

Myosin binding protein-C (MyBP-C) is a key regulatory protein in heart muscle, and mutations in the MYBPC3 gene are frequently associated with cardiomyopathy. However, the mechanism of action of MyBP-C remains poorly understood, and both activating and inhibitory effects of MyBP-C on contractility have been reported. To clarify the function of the regulatory N-terminal domains of MyBP-C, we determined their effects on the structure of thick (myosin-containing) and thin (actin-containing) filaments in intact sarcomeres of heart muscle. We used fluorescent probes on troponin C in the thin filaments and on myosin regulatory light chain in the thick filaments to monitor structural changes associated with activation of demembranated trabeculae from rat ventricle by the C1mC2 region of rat MyBP-C. C1mC2 induced larger structural changes in thin filaments than calcium activation, and these were still present when active force was blocked with blebbistatin, showing that C1mC2 directly activates the thin filaments. In contrast, structural changes in thick filaments induced by C1mC2 were smaller than those associated with calcium activation and were abolished or reversed by blebbistatin. Low concentrations of C1mC2 did not affect resting force but increased calcium sensitivity and reduced cooperativity of force and structural changes in both thin and thick filaments. These results show that the N-terminal region of MyBP-C stabilizes the ON state of thin filaments and the OFF state of thick filaments and lead to a novel hypothesis for the physiological role of MyBP-C in the regulation of cardiac contractility.

An Apical MRCK-driven Morphogenetic Pathway Controls Epithelial Polarity

PubMed Central

Zihni, Ceniz; Vlassaks, Evi; Terry, Stephen; Carlton, Jeremy; Leung, Thomas King Chor; Olson, Michael; Pichaud, Franck; Balda, Maria Susana; Matter, Karl

2017-01-01

Polarized epithelia develop distinct cell surface domains, with the apical membrane acquiring characteristic morphological features such as microvilli. Cell polarization is driven by polarity determinants including the evolutionarily conserved partitioning defective (PAR) proteins that are separated into distinct cortical domains. PAR protein segregation is thought to be a consequence of asymmetric actomyosin contractions. The mechanism of activation of apically polarized actomyosin contractility is unknown. Here we show that the Cdc42 effector MRCK activates Myosin-II at the apical pole to segregate aPKC-Par6 from junctional Par3, defining the apical domain. Apically polarized MRCK-activated actomyosin contractility is reinforced by cooperation with aPKC-Par6 downregulating antagonistic RhoA-driven junctional actomyosin contractility, and drives polarization of cytosolic brush border determinants and apical morphogenesis. MRCK-activated polarized actomyosin contractility is required for apical differentiation and morphogenesis in vertebrate epithelia and Drosophila photoreceptors. Our results identify an apical origin of actomyosin-driven morphogenesis that couples cytoskeletal reorganization to PAR polarity signalling. PMID:28825699

In vitro models of tail contraction and cytoplasmic streaming in amoeboid cells.

PubMed

Janson, L W; Taylor, D L

1993-10-01

We have developed a reconstituted gel-sol and contractile model system that mimics the structure and dynamics found at the ectoplasm/endoplasm interface in the tails of many amoeboid cells. We tested the role of gel-sol transformations of the actin-based cytoskeleton in the regulation of contraction and in the generation of endoplasm from ectoplasm. In a model system with fully phosphorylated myosin II, we demonstrated that either decreasing the actin filament length distribution or decreasing the extent of actin filament cross-linking initiated both a weakening of the gel strength and contraction. However, streaming of the solated gel components occurred only under conditions where the length distribution of actin was decreased, causing a self-destruct process of continued solation and contraction of the gel. These results offer significant support that gel strength plays an important role in the regulation of actin/myosin II-based contractions of the tail cortex in many amoeboid cells as defined by the solation-contraction coupling hypothesis (Taylor, D. L., and M. Fechheimer. 1982. Phil. Trans. Soc. Lond. B. 299:185-197). The competing processes of solation and contraction of the gel would appear to be mutually exclusive. However, it is the temporal-spatial balance of the rate and extent of two stages of solation, coupled to contraction, that can explain the conversion of gelled ectoplasm in the tail to a solated endoplasm within the same small volume, generation of a force for the retraction of tails, maintenance of cell polarity, and creation of a positive hydrostatic pressure to push against the newly formed endoplasm. The mechanism of solation-contraction of cortical cytoplasm may be a general component of the normal movement of a variety of amoeboid cells and may also be a component of other contractile events such as cytokinesis.

Cells Lacking β-Actin are Genetically Reprogrammed and Maintain Conditional Migratory Capacity*

PubMed Central

Tondeleir, Davina; Lambrechts, Anja; Müller, Matthias; Jonckheere, Veronique; Doll, Thierry; Vandamme, Drieke; Bakkali, Karima; Waterschoot, Davy; Lemaistre, Marianne; Debeir, Olivier; Decaestecker, Christine; Hinz, Boris; Staes, An; Timmerman, Evy; Colaert, Niklaas; Gevaert, Kris; Vandekerckhove, Joël; Ampe, Christophe

2012-01-01

Vertebrate nonmuscle cells express two actin isoforms: cytoplasmic β- and γ-actin. Because of the presence and localized translation of β-actin at the leading edge, this isoform is generally accepted to specifically generate protrusive forces for cell migration. Recent evidence also implicates β-actin in gene regulation. Cell migration without β-actin has remained unstudied until recently and it is unclear whether other actin isoforms can compensate for this cytoplasmic function and/or for its nuclear role. Primary mouse embryonic fibroblasts lacking β-actin display compensatory expression of other actin isoforms. Consistent with this preservation of polymerization capacity, β-actin knockout cells have unchanged lamellipodial protrusion rates despite a severe migration defect. To solve this paradox we applied quantitative proteomics revealing a broad genetic reprogramming of β-actin knockout cells. This also explains why reintroducing β-actin in knockout cells does not restore the affected cell migration. Pathway analysis suggested increased Rho-ROCK signaling, consistent with observed phenotypic changes. We therefore developed and tested a model explaining the phenotypes in β-actin knockout cells based on increased Rho-ROCK signaling and increased TGFβ production resulting in increased adhesion and contractility in the knockout cells. Inhibiting ROCK or myosin restores migration of β-actin knockout cells indicating that other actins compensate for β-actin in this process. Consequently, isoactins act redundantly in providing propulsive forces for cell migration, but β-actin has a unique nuclear function, regulating expression on transcriptional and post-translational levels, thereby preventing myogenic differentiation. PMID:22448045

An oscillating dynamic model of collective cells in a monolayer

NASA Astrophysics Data System (ADS)

Lin, Shao-Zhen; Xue, Shi-Lei; Li, Bo; Feng, Xi-Qiao

2018-03-01

Periodic oscillations of collective cells occur in the morphogenesis and organogenesis of various tissues and organs. In this paper, an oscillating cytodynamic model is presented by integrating the chemomechanical interplay between the RhoA effector signaling pathway and cell deformation. We show that both an isolated cell and a cell aggregate can undergo spontaneous oscillations as a result of Hopf bifurcation, upon which the system evolves into a limit cycle of chemomechanical oscillations. The dynamic characteristics are tailored by the mechanical properties of cells (e.g., elasticity, contractility, and intercellular tension) and the chemical reactions involved in the RhoA effector signaling pathway. External forces are found to modulate the oscillation intensity of collective cells in the monolayer and to polarize their oscillations along the direction of external tension. The proposed cytodynamic model can recapitulate the prominent features of cell oscillations observed in a variety of experiments, including both isolated cells (e.g., spreading mouse embryonic fibroblasts, migrating amoeboid cells, and suspending 3T3 fibroblasts) and multicellular systems (e.g., Drosophila embryogenesis and oogenesis).

Balance between cell-substrate adhesion and myosin contraction determines the frequency of motility initiation in fish keratocytes.

PubMed

Barnhart, Erin; Lee, Kun-Chun; Allen, Greg M; Theriot, Julie A; Mogilner, Alex

2015-04-21

Cells are dynamic systems capable of spontaneously switching among stable states. One striking example of this is spontaneous symmetry breaking and motility initiation in fish epithelial keratocytes. Although the biochemical and mechanical mechanisms that control steady-state migration in these cells have been well characterized, the mechanisms underlying symmetry breaking are less well understood. In this work, we have combined experimental manipulations of cell-substrate adhesion strength and myosin activity, traction force measurements, and mathematical modeling to develop a comprehensive mechanical model for symmetry breaking and motility initiation in fish epithelial keratocytes. Our results suggest that stochastic fluctuations in adhesion strength and myosin localization drive actin network flow rates in the prospective cell rear above a critical threshold. Above this threshold, high actin flow rates induce a nonlinear switch in adhesion strength, locally switching adhesions from gripping to slipping and further accelerating actin flow in the prospective cell rear, resulting in rear retraction and motility initiation. We further show, both experimentally and with model simulations, that the global levels of adhesion strength and myosin activity control the stability of the stationary state: The frequency of symmetry breaking decreases with increasing adhesion strength and increases with increasing myosin contraction. Thus, the relative strengths of two opposing mechanical forces--contractility and cell-substrate adhesion--determine the likelihood of spontaneous symmetry breaking and motility initiation.

Heart repair by reprogramming non-myocytes with cardiac transcription factors

PubMed Central

Song, Kunhua; Nam, Young-Jae; Luo, Xiang; Qi, Xiaoxia; Tan, Wei; Huang, Guo N.; Acharya, Asha; Smith, Christopher L.; Tallquist, Michelle D.; Neilson, Eric G.; Hill, Joseph A.; Bassel-Duby, Rhonda; Olson, Eric N.

2012-01-01

The adult mammalian heart possesses little regenerative potential following injury. Fibrosis due to activation of cardiac fibroblasts impedes cardiac regeneration and contributes to loss of contractile function, pathological remodeling and susceptibility to arrhythmias. Cardiac fibroblasts account for a majority of cells in the heart and represent a potential cellular source for restoration of cardiac function following injury through phenotypic reprogramming to a myocardial cell fate. Here we show that four transcription factors, GATA4, Hand2, MEF2C and Tbx5 can cooperatively reprogram adult mouse tail-tip and cardiac fibroblasts into beating cardiac-like myocytes in vitro. Forced expression of these factors in dividing non-cardiomyocytes in mice reprograms these cells into functional cardiac-like myocytes, improves cardiac function and reduces adverse ventricular remodeling following myocardial infarction. Our results suggest a strategy for cardiac repair through reprogramming fibroblasts resident in the heart with cardiogenic transcription factors or other molecules. PMID:22660318

Examining platelet-fibrin interactions during traumatic shock in a swine model using platelet contractile force and clot elastic modulus.

PubMed

White, Nathan J; Martin, Erika J; Brophy, Donald F; Ward, Kevin R

2011-07-01

A significant proportion of severely injured patients develop early coagulopathy, characterized by abnormal clot formation, which impairs resuscitation and increases mortality. We have previously demonstrated an isolated decrease in clot strength by thrombelastography in a swine model of nonresuscitated traumatic shock. In order to more closely examine platelet-fibrin interactions in this setting, we define the observed decrease in clot strength in terms of platelet-induced clot contraction and clot elastic modulus using the Hemostasis Analysis System (HAS) (Hemodyne Inc., Richmond, Virginia, USA). Whole blood was sampled for HAS measurements, metabolic measurements, cell counts, and fibrinogen concentration at baseline prior to injury and again at a predetermined level of traumatic shock defined by oxygen debt. Male swine (N=17) received femur fracture and controlled arterial hemorrhage to achieve an oxygen debt of 80 ml/kg. Platelet counts were unchanged, but fibrinogen concentration was reduced significantly during shock (167.6 vs. 66.7 mg/dl, P=0.0007). Platelet contractile force generated during clot formation did not change during shock (11.7 vs. 10.4 kdynes, P=0.41), but clot elastic modulus was dynamically altered, resulting in a lower final value (22.9 vs. 17.3 kdynes/cm, P<0.0001). In this model of traumatic shock, platelet function was preserved, whereas terminal clot elastic modulus was reduced during shock in a manner most consistent with early changes in the mechanical properties of the developing fibrin fiber network.

Slow recovery of the impaired fatigue resistance in postunloading mouse soleus muscle corresponding to decreased mitochondrial function and a compensatory increase in type I slow fibers

PubMed Central

Feng, Han-Zhong; Chen, Xuequn; Malek, Moh H.

2015-01-01

Unloading or disuse rapidly results in skeletal muscle atrophy, switching to fast-type fibers, and decreased resistance to fatigue. The recovery process is of major importance in rehabilitation for various clinical conditions. Here we studied mouse soleus muscle during 60 days of reloading after 4 wk of hindlimb suspension. Unloading produced significant atrophy of soleus muscle with decreased contractile force and fatigue resistance, accompanied by switches of myosin isoforms from IIa to IIx and IIb and fast troponin T to more low-molecular-weight splice forms. The total mass, fiber size, and contractile force of soleus muscle recovered to control levels after 15 days of reloading. However, the fatigue resistance showed a trend of worsening during this period with significant infiltration of inflammatory cells at days 3 and 7, indicating reloading injuries that were accompanied by active regeneration with upregulations of filamin-C, αB-crystallin, and desmin. The fatigue resistance partially recovered after 30–60 days of reloading. The expression of peroxisome proliferator-activated receptor γ coactivator 1α and mitofusin-2 showed changes parallel to that of fatigue resistance after unloading and during reloading, suggesting a causal role of decreased mitochondrial function. Slow fiber contents in the soleus muscle were increased after 30–60 days of reloading to become significantly higher than the normal level, indicating a secondary adaption to compensate for the slow recovery of fatigue resistance. PMID:26447205

Paramagnetic Beads and Magnetically Mediated Strain Enhance Cardiomyogenesis in Mouse Embryoid Bodies

PubMed Central

Geuss, Laura R.; Wu, Douglas C.; Ramamoorthy, Divya; Alford, Corinne D.; Suggs, Laura J.

2014-01-01

Mechanical forces play an important role in proper embryologic development, and similarly such forces can directly impact pluripotency and differentiation of mouse embryonic stem cells (mESC) in vitro. In addition, manipulation of the embryoid body (EB) microenvironment, such as by incorporation of microspheres or microparticles, can similarly influence fate determination. In this study, we developed a mechanical stimulation regimen using permanent neodymium magnets to magnetically attract cells within an EB. Arginine-Glycine-Aspartic Acid (RGD)-conjugated paramagnetic beads were incorporated into the interior of the EBs during aggregation, allowing us to exert force on individual cells using short-term magnetization. EBs were stimulated for one hour at different magnetic field strengths, subsequently exerting a range of force intensity on the cells at different stages of early EB development. Our results demonstrated that following exposure to a 0.2 Tesla magnetic field, ESCs respond to magnetically mediated strain by activating Protein Kinase A (PKA) and increasing phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) expression. The timing of stimulation can also be tailored to guide ESC differentiation: the combination of bone morphogenetic protein 4 (BMP4) supplementation with one hour of magnetic attraction on Day 3 enhances cardiomyogenesis by increasing contractile activity and the percentage of sarcomeric α-actin-expressing cells compared to control samples with BMP4 alone. Interestingly, we also observed that the beads alone had some impact on differentiation by increasingly slightly, albeit not significantly, the percentage of cardiomyocytes. Together these results suggest that magnetically mediated strain can be used to enhance the percentage of mouse ESC-derived cardiomyocytes over current differentiation protocols. PMID:25501004

Transcriptional and Hormonal Regulation of Gravitropism of Woody Stems in Populus[OPEN

PubMed Central

Gerttula, Suzanne; Zinkgraf, Matthew; Lewis, Daniel R.; Brumer, Harry; Hart, Foster; Filkov, Vladimir

2015-01-01

Angiosperm trees reorient their woody stems by asymmetrically producing a specialized xylem tissue, tension wood, which exerts a strong contractile force resulting in negative gravitropism of the stem. Here, we show, in Populus trees, that initial gravity perception and response occurs in specialized cells through sedimentation of starch-filled amyloplasts and relocalization of the auxin transport protein, PIN3. Gibberellic acid treatment stimulates the rate of tension wood formation and gravibending and enhances tissue-specific expression of an auxin-responsive reporter. Gravibending, maturation of contractile fibers, and gibberellic acid (GA) stimulation of tension wood formation are all sensitive to transcript levels of the Class I KNOX homeodomain transcription factor-encoding gene ARBORKNOX2 (ARK2). We generated genome-wide transcriptomes for trees in which gene expression was perturbed by gravistimulation, GA treatment, and modulation of ARK2 expression. These data were employed in computational analyses to model the transcriptional networks underlying wood formation, including identification and dissection of gene coexpression modules associated with wood phenotypes, GA response, and ARK2 binding to genes within modules. We propose a model for gravitropism in the woody stem in which the peripheral location of PIN3-expressing cells relative to the cambium results in auxin transport toward the cambium in the top of the stem, triggering tension wood formation, while transport away from the cambium in the bottom of the stem triggers opposite wood formation. PMID:26410302

Out-of-equilibrium dynamics in the cytoskeleton of the living cell

NASA Astrophysics Data System (ADS)

Lenormand, Guillaume; Bursac, Predrag; Butler, James P.; Fredberg, Jeffrey J.

2007-10-01

We report here measurements of rheological properties of the human airway smooth muscle cell using forced nanoscale motions of Arg-Gly-Asp RGD-coated microbeads tightly bound to the cytoskeleton. With changes of forcing amplitude, the storage modulus showed small but systematic nonlinearities, especially after treatment with a contractile agonist. In a dose-dependent manner, a large oscillatory shear applied from a few seconds up to 400s caused the cytoskeleton matrix to soften, a behavior comparable to physical rejuvenation observed in certain inert soft materials; the stiffness remained constant for as long as the large oscillatory shear was maintained, but suddenly fell with shear cessation. Stiffness then followed a slow scale-free recovery, a phenomenon comparable to physical aging. However, acetylated low-density lipoprotein acLDL-coated microbeads, which connect mainly to scavenger receptors, did not show similar out-of-equilibrium behaviors. Taken together, these data demonstrate in the cytoskeleton of the living cell behaviors with all the same signatures as that of soft inert condensed systems. This unexpected intersection of condensed matter physics and cytoskeletal biology suggests that trapping, intermittency, and approach to kinetic arrest represent central mesoscale features linking underlying molecular events to integrative cellular functions.

Cell prestress. I. Stiffness and prestress are closely associated in adherent contractile cells

NASA Technical Reports Server (NTRS)

Wang, Ning; Tolic-Norrelykke, Iva Marija; Chen, Jianxin; Mijailovich, Srboljub M.; Butler, James P.; Fredberg, Jeffrey J.; Stamenovic, Dimitrije; Ingber, D. E. (Principal Investigator)

2002-01-01

The tensegrity hypothesis holds that the cytoskeleton is a structure whose shape is stabilized predominantly by the tensile stresses borne by filamentous structures. Accordingly, cell stiffness must increase in proportion with the level of the tensile stress, which is called the prestress. Here we have tested that prediction in adherent human airway smooth muscle (HASM) cells. Traction microscopy was used to measure the distribution of contractile stresses arising at the interface between each cell and its substrate; this distribution is called the traction field. Because the traction field must be balanced by tensile stresses within the cell body, the prestress could be computed. Cell stiffness (G) was measured by oscillatory magnetic twisting cytometry. As the contractile state of the cell was modulated with graded concentrations of relaxing or contracting agonists (isoproterenol or histamine, respectively), the mean prestress ((t)) ranged from 350 to 1,900 Pa. Over that range, cell stiffness increased linearly with the prestress: G (Pa) = 0.18(t) + 92. While this association does not necessarily preclude other interpretations, it is the hallmark of systems that secure shape stability mainly through the prestress. Regardless of mechanism, these data establish a strong association between stiffness of HASM cells and the level of tensile stress within the cytoskeleton.

Additional in-series compliance reduces muscle force summation and alters the time course of force relaxation during fixed-end contractions.

PubMed

Mayfield, Dean L; Launikonis, Bradley S; Cresswell, Andrew G; Lichtwark, Glen A

2016-11-15

There are high mechanical demands placed on skeletal muscles in movements requiring rapid acceleration of the body or its limbs. Tendons are responsible for transmitting muscle forces, but, because of their elasticity, can manipulate the mechanics of the internal contractile apparatus. Shortening of the contractile apparatus against the stretch of tendon affects force generation according to known mechanical properties; however, the extent to which differences in tendon compliance alter force development in response to a burst of electrical impulses is unclear. To establish the influence of series compliance on force summation, we studied electrically evoked doublet contractions in the cane toad peroneus muscle in the presence and absence of a compliant artificial tendon. Additional series compliance reduced tetanic force by two-thirds, a finding predicted based on the force-length property of skeletal muscle. Doublet force and force-time integral expressed relative to the twitch were also reduced by additional series compliance. Active shortening over a larger range of the ascending limb of the force-length curve and at a higher velocity, leading to a progressive reduction in force-generating potential, could be responsible. Muscle-tendon interaction may also explain the accelerated time course of force relaxation in the presence of additional compliance. Our findings suggest that a compliant tendon limits force summation under constant-length conditions. However, high series compliance can be mechanically advantageous when a muscle-tendon unit is actively stretched, permitting muscle fibres to generate force almost isometrically, as shown during stretch-shorten cycles in locomotor activities. Restricting active shortening would likely favour rapid force development. © 2016. Published by The Company of Biologists Ltd.

Differentiation of Human Adipose Derived Stem Cells into Smooth Muscle Cells Is Modulated by CaMKIIγ

PubMed Central

Aji, Kaisaier; Maimaijiang, Munila; Aimaiti, Abudusaimi; Rexiati, Mulati; Azhati, Baihetiya; Tusong, Hamulati

2016-01-01

The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is known to participate in maintenance and switches of smooth muscle cell (SMC) phenotypes. However, which isoform of CaMKII is involved in differentiation of adult mesenchymal stem cells into contractile SMCs remains unclear. In the present study, we detected γ isoform of CaMKII in differentiation of human adipose derived stem cells (hASCs) into SMCs that resulted from treatment with TGF-β1 and BMP4 in combination for 7 days. The results showed that CaMKIIγ increased gradually during differentiation of hASCs as determined by real-time PCR and western blot analysis. The siRNA-mediated knockdown of CaMKIIγ decreased the protein levels and transcriptional levels of smooth muscle contractile markers (a-SMA, SM22a, calponin, and SM-MHC), while CaMKIIγ overexpression increases the transcriptional and protein levels of smooth muscle contractile markers. These results suggested that γ isoform of CaMKII plays a significant role in smooth muscle differentiation of hASCs. PMID:27493668

Hypotonic swelling promotes nitric oxide release in cardiac ventricular myocytes: impact on swelling-induced negative inotropic effect

PubMed Central

Gonano, Luis Alberto; Morell, Malena; Burgos, Juan Ignacio; Dulce, Raul Ariel; De Giusti, Verónica Celeste; Aiello, Ernesto Alejandro; Hare, Joshua Michael; Vila Petroff, Martin

2014-01-01

Aims Cardiomyocyte swelling occurs in multiple pathological situations and has been associated with contractile dysfunction, cell death, and enhanced propensity to arrhythmias. We investigate whether hypotonic swelling promotes nitric oxide (NO) release in cardiomyocytes, and whether it impacts on swelling-induced contractile dysfunction. Methods and results Superfusing rat cardiomyocytes with a hypotonic solution (HS; 217 mOsm), increased cell volume, reduced myocyte contraction and Ca2+ transient, and increased NO-sensitive 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM) fluorescence. When cells were exposed to HS + 2.5 mM of the NO synthase inhibitor l-NAME, cell swelling occurred in the absence of NO release. Swelling-induced NO release was also prevented by the nitric oxide synthase 1 (NOS1) inhibitor, nitroguanidine, and significantly reduced in NOS1 knockout mice. Additionally, colchicine (inhibitor of microtubule polymerization) prevented the increase in DAF-FM fluorescence induced by HS, indicating that microtubule integrity is necessary for swelling-induced NO release. The swelling-induced negative inotropic effect was exacerbated in the presence of either l-NAME, nitroguandine, the guanylate cyclase inhibitor, ODQ, or the PKG inhibitor, KT5823, suggesting that NOS1-derived NO provides contractile support via a cGMP/PKG-dependent mechanism. Indeed, ODQ reduced Ca2+ wave velocity and both ODQ and KT5823 reduced the HS-induced increment in ryanodine receptor (RyR2, Ser2808) phosphorylation, suggesting that in this context, cGMP/PKG may contribute to preserve contractile function by enhancing sarcoplasmic reticulum Ca2+ release. Conclusions Our findings suggest a novel mechanism for NO release in cardiomyocytes with putative pathophysiological relevance determined, at least in part, by its capability to reduce the extent of contractile dysfunction associated with hypotonic swelling. PMID:25344365

Pericyte contractility controls endothelial cell cycle progression and sprouting: insights into angiogenic switch mechanics.

PubMed

Durham, Jennifer T; Surks, Howard K; Dulmovits, Brian M; Herman, Ira M

2014-11-01

Microvascular stability and regulation of capillary tonus are regulated by pericytes and their interactions with endothelial cells (EC). While the RhoA/Rho kinase (ROCK) pathway has been implicated in modulation of pericyte contractility, in part via regulation of the myosin light chain phosphatase (MLCP), the mechanisms linking Rho GTPase activity with actomyosin-based contraction and the cytoskeleton are equivocal. Recently, the myosin phosphatase-RhoA-interacting protein (MRIP) was shown to mediate the RhoA/ROCK-directed MLCP inactivation in vascular smooth muscle. Here we report that MRIP directly interacts with the β-actin-specific capping protein βcap73. Furthermore, manipulation of MRIP expression influences pericyte contractility, with MRIP silencing inducing cytoskeletal remodeling and cellular hypertrophy. MRIP knockdown induces a repositioning of βcap73 from the leading edge to stress fibers; thus MRIP-silenced pericytes increase F-actin-driven cell spreading twofold. These hypertrophied and cytoskeleton-enriched pericytes demonstrate a 2.2-fold increase in contractility upon MRIP knockdown when cells are plated on a deformable substrate. In turn, silencing pericyte MRIP significantly affects EC cycle progression and angiogenic activation. When MRIP-silenced pericytes are cocultured with capillary EC, there is a 2.0-fold increase in EC cycle entry. Furthermore, in three-dimensional models of injury and repair, silencing pericyte MRIP results in a 1.6-fold elevation of total tube area due to EC network formation and increased angiogenic sprouting. The pivotal role of MRIP expression in governing pericyte contractile phenotype and endothelial growth should lend important new insights into how chemomechanical signaling pathways control the "angiogenic switch" and pathological angiogenic induction. Copyright © 2014 the American Physiological Society.

Comparison of human gastrocnemius forces predicted by Hill-type muscle models and estimated from ultrasound images

PubMed Central

Biewener, Andrew A.; Wakeling, James M.

2017-01-01

ABSTRACT Hill-type models are ubiquitous in the field of biomechanics, providing estimates of a muscle's force as a function of its activation state and its assumed force–length and force–velocity properties. However, despite their routine use, the accuracy with which Hill-type models predict the forces generated by muscles during submaximal, dynamic tasks remains largely unknown. This study compared human gastrocnemius forces predicted by Hill-type models with the forces estimated from ultrasound-based measures of tendon length changes and stiffness during cycling, over a range of loads and cadences. We tested both a traditional model, with one contractile element, and a differential model, with two contractile elements that accounted for independent contributions of slow and fast muscle fibres. Both models were driven by subject-specific, ultrasound-based measures of fascicle lengths, velocities and pennation angles and by activation patterns of slow and fast muscle fibres derived from surface electromyographic recordings. The models predicted, on average, 54% of the time-varying gastrocnemius forces estimated from the ultrasound-based methods. However, differences between predicted and estimated forces were smaller under low speed–high activation conditions, with models able to predict nearly 80% of the gastrocnemius force over a complete pedal cycle. Additionally, the predictions from the Hill-type muscle models tested here showed that a similar pattern of force production could be achieved for most conditions with and without accounting for the independent contributions of different muscle fibre types. PMID:28202584

Vascular smooth muscle cell contractile protein expression is increased through protein kinase G-dependent and -independent pathways by glucose-6-phosphate dehydrogenase inhibition and deficiency.

PubMed

Chettimada, Sukrutha; Joshi, Sachindra Raj; Dhagia, Vidhi; Aiezza, Alessandro; Lincoln, Thomas M; Gupte, Rakhee; Miano, Joseph M; Gupte, Sachin A

2016-10-01

Homeostatic control of vascular smooth muscle cell (VSMC) differentiation is critical for contractile activity and regulation of blood flow. Recently, we reported that precontracted blood vessels are relaxed and the phenotype of VSMC is regulated from a synthetic to contractile state by glucose-6-phosphate dehydrogenase (G6PD) inhibition. In the current study, we investigated whether the increase in the expression of VSMC contractile proteins by inhibition and knockdown of G6PD is mediated through a protein kinase G (PKG)-dependent pathway and whether it regulates blood pressure. We found that the expression of VSMC-restricted contractile proteins, myocardin (MYOCD), and miR-1 and miR-143 are increased by G6PD inhibition or knockdown. Importantly, RNA-sequence analysis of aortic tissue from G6PD-deficient mice revealed uniform increases in VSMC-restricted genes, particularly those regulated by the MYOCD-serum response factor (SRF) switch. Conversely, expression of Krüppel-like factor 4 (KLF4) is decreased by G6PD inhibition. Interestingly, the G6PD inhibition-induced expression of miR-1 and contractile proteins was blocked by Rp-β-phenyl-1,N 2 -etheno-8-bromo-guanosine-3',5'-cyclic monophosphorothioate, a PKG inhibitor. On the other hand, MYOCD and miR-143 levels are increased by G6PD inhibition through a PKG-independent manner. Furthermore, blood pressure was lower in the G6PD-deficient compared with wild-type mice. Therefore, our results suggest that the expression of VSMC contractile proteins induced by G6PD inhibition occurs via PKG1α-dependent and -independent pathways. Copyright © 2016 the American Physiological Society.

Activation of Toll-like receptor 3 increases mouse aortic vascular smooth muscle cell contractility through ERK1/2 pathway.

PubMed

Hardigan, Trevor; Spitler, Kathryn; Matsumoto, Takayuki; Carrillo-Sepulveda, Maria Alicia

2015-11-01

Activation of Toll-like receptor 3 (TLR3), a pattern recognition receptor of the innate immune system, is associated with vascular complications. However, whether activation of TLR3 alters vascular contractility is unknown. We, therefore, hypothesized that TLR3 activation augments vascular contractility and activates vascular smooth muscle cell (VSMC) contractile apparatus proteins. Male mice were treated with polyinosinic-polycytidylic acid (Poly I:C group, 14 days), a TLR3 agonist; control mice received saline (vehicle, 14 days). At the end of protocol, blood pressure was measured by tail cuff method. Aortas were isolated and assessed for contractility experiments using a wire myograph. Aortic protein content was used to determine phosphorylated/total interferon regulatory factor 3 (IRF3), a downstream target of TLR3 signaling, and ERK1/2 using Western blot. We investigated the TLR3/IRF3/ERK1/2 signaling pathway and contractile-related proteins such as phosphorylated/total myosin light chain (MLC) and caldesmon (CaD) in aortic VSMC primary cultures. Poly I:C-treated mice exhibited (vs. vehicle-treated mice) (1) elevated systolic blood pressure. Moreover, Poly I:C treatment (2) enhanced aortic phenylephrine-induced maximum contraction, which was suppressed by PD98059 (ERK1/2 inhibitor), and (3) increased aortic levels of phosphorylated IRF3 and ERK1/2. Stimulation of mouse aortic VSMCs with Poly I:C resulted in increased phosphorylation of IRF3, ERK1/2, MLC, and CaD. Inhibition of ERK1/2 abolished Poly I:C-mediated phosphorylation of MLC and CaD. Our data provide functional evidence for the role of TLR3 in vascular contractile events, suggesting TLR3 as a potential new therapeutic target in vascular dysfunction and regulation of blood pressure.

A chemo-mechanical free-energy-based approach to model durotaxis and extracellular stiffness-dependent contraction and polarization of cells.

PubMed

Shenoy, Vivek B; Wang, Hailong; Wang, Xiao

2016-02-06

We propose a chemo-mechanical model based on stress-dependent recruitment of myosin motors to describe how the contractility, polarization and strain in cells vary with the stiffness of their surroundings and their shape. A contractility tensor, which depends on the distribution of myosin motors, is introduced to describe the chemical free energy of the cell due to myosin recruitment. We explicitly include the contributions to the free energy that arise from mechanosensitive signalling pathways (such as the SFX, Rho-Rock and MLCK pathways) through chemo-mechanical coupling parameters. Taking the variations of the total free energy, which consists of the chemical and mechanical components, in accordance with the second law of thermodynamics provides equations for the temporal evolution of the active stress and the contractility tensor. Following this approach, we are able to recover the well-known Hill relation for active stresses, based on the fundamental principles of irreversible thermodynamics rather than phenomenology. We have numerically implemented our free energy-based approach to model spatial distribution of strain and contractility in (i) cells supported by flexible microposts, (ii) cells on two-dimensional substrates, and (iii) cells in three-dimensional matrices. We demonstrate how the polarization of the cells and the orientation of stress fibres can be deduced from the eigenvalues and eigenvectors of the contractility tensor. Our calculations suggest that the chemical free energy of the cell decreases with the stiffness of the extracellular environment as the cytoskeleton polarizes in response to stress-dependent recruitment of molecular motors. The mechanical energy, which includes the strain energy and motor potential energy, however, increases with stiffness, but the overall energy is lower for cells in stiffer environments. This provides a thermodynamic basis for durotaxis, whereby cells preferentially migrate towards stiffer regions of the extracellular environment. Our models also explain, from an energetic perspective, why the shape of the cells can change in response to stiffness of the surroundings. The effect of the stiffness of the nucleus on its shape and the orientation of the stress fibres is also studied for all the above geometries. Along with making testable predictions, we have estimated the magnitudes of the chemo-mechanical coupling parameters for myofibroblasts based on data reported in the literature.

A chemo-mechanical free-energy-based approach to model durotaxis and extracellular stiffness-dependent contraction and polarization of cells

PubMed Central

Shenoy, Vivek B.; Wang, Hailong; Wang, Xiao

2016-01-01

We propose a chemo-mechanical model based on stress-dependent recruitment of myosin motors to describe how the contractility, polarization and strain in cells vary with the stiffness of their surroundings and their shape. A contractility tensor, which depends on the distribution of myosin motors, is introduced to describe the chemical free energy of the cell due to myosin recruitment. We explicitly include the contributions to the free energy that arise from mechanosensitive signalling pathways (such as the SFX, Rho-Rock and MLCK pathways) through chemo-mechanical coupling parameters. Taking the variations of the total free energy, which consists of the chemical and mechanical components, in accordance with the second law of thermodynamics provides equations for the temporal evolution of the active stress and the contractility tensor. Following this approach, we are able to recover the well-known Hill relation for active stresses, based on the fundamental principles of irreversible thermodynamics rather than phenomenology. We have numerically implemented our free energy-based approach to model spatial distribution of strain and contractility in (i) cells supported by flexible microposts, (ii) cells on two-dimensional substrates, and (iii) cells in three-dimensional matrices. We demonstrate how the polarization of the cells and the orientation of stress fibres can be deduced from the eigenvalues and eigenvectors of the contractility tensor. Our calculations suggest that the chemical free energy of the cell decreases with the stiffness of the extracellular environment as the cytoskeleton polarizes in response to stress-dependent recruitment of molecular motors. The mechanical energy, which includes the strain energy and motor potential energy, however, increases with stiffness, but the overall energy is lower for cells in stiffer environments. This provides a thermodynamic basis for durotaxis, whereby cells preferentially migrate towards stiffer regions of the extracellular environment. Our models also explain, from an energetic perspective, why the shape of the cells can change in response to stiffness of the surroundings. The effect of the stiffness of the nucleus on its shape and the orientation of the stress fibres is also studied for all the above geometries. Along with making testable predictions, we have estimated the magnitudes of the chemo-mechanical coupling parameters for myofibroblasts based on data reported in the literature. PMID:26855753

Active turnover regulates pattern formation and stress transmission in disordered acto-myosin networks

NASA Astrophysics Data System (ADS)

McCall, Patrick; Stam, Samantha; Kovar, David; Gardel, Margaret

The shape and mechanics of animal cells are controlled by a dynamic, thin network of semiflexible actin filaments and myosin-II motor proteins called the actomyosin cortex. Motor-generated stresses in the cortex drive changes in cell shape during cell division and morphogenesis, while dynamic turnover of actin filaments dissipates stress. The relative effects that force generation, force dissipation, and disassembly and reassembly of material have on motion in these networks are unknown. We find that cross-linked actin networks in vitro contract under myosin-generated stresses, resulting in partial filament disassembly, the formation of asters, and clustering of myosin motors. We observe a rapid restoration of uniform polymer density in the presence of the assembly factors which catalyze network turnover through elongation of severed actin filaments. When severing is accelerated further by the addition of a severing protein, network contraction and motor clustering are dramatically suppressed. We test the relative effects of material regeneration and force transmission using image analysis, and conclude that the dominant mechanism for this effect is relatively short-lived stresses that do not propagate over considerable distance or push network deformation into the nonlinear contractile regime we have previously characterized. Our results present a framework to understand cytoskeletal active matter that are influenced by a complex interplay between stress generation, network reorganization, and polymer turnover.

Cellular Mechanics of Primary Human Cervical Fibroblasts: Influence of Progesterone and a Pro-inflammatory Cytokine.

PubMed

Shukla, Vasudha; Barnhouse, Victoria; Ackerman, William E; Summerfield, Taryn L; Powell, Heather M; Leight, Jennifer L; Kniss, Douglas A; Ghadiali, Samir N

2018-01-01

The leading cause of neonatal mortality, pre-term birth, is often caused by pre-mature ripening/opening of the uterine cervix. Although cervical fibroblasts play an important role in modulating the cervix's extracellular matrix (ECM) and mechanical properties, it is not known how hormones, i.e., progesterone, and pro-inflammatory insults alter fibroblast mechanics, fibroblast-ECM interactions and the resulting changes in tissue mechanics. Here we investigate how progesterone and a pro-inflammatory cytokine, IL-1β, alter the biomechanical properties of human cervical fibroblasts and the fibroblast-ECM interactions that govern tissue-scale mechanics. Primary human fibroblasts were isolated from non-pregnant cervix and treated with estrogen/progesterone, IL-1β or both. The resulting changes in ECM gene expression, matrix remodeling, traction force generation, cell-ECM adhesion and tissue contractility were monitored. Results indicate that IL-1β induces a significant reduction in traction force and ECM adhesion independent of pre-treatment with progesterone. These cell level effects altered tissue-scale mechanics where IL-1β inhibited the contraction of a collagen gel over 6 days. Interestingly, progesterone treatment alone did not modulate traction forces or gel contraction but did result in a dramatic increase in cell-ECM adhesion. Therefore, the protective effect of progesterone may be due to altered adhesion dynamics as opposed to altered ECM remodeling.

CuZnSOD gene deletion targeted to skeletal muscle leads to loss of contractile force but does not cause muscle atrophy in adult mice

PubMed Central

Zhang, Yiqiang; Davis, Carol; Sakellariou, George K.; Shi, Yun; Kayani, Anna C.; Pulliam, Daniel; Bhattacharya, Arunabh; Richardson, Arlan; Jackson, Malcolm J.; McArdle, Anne; Brooks, Susan V.; Van Remmen, Holly

2013-01-01

We have previously shown that deletion of CuZnSOD in mice (Sod1−/− mice) leads to accelerated loss of muscle mass and contractile force during aging. To dissect the relative roles of skeletal muscle and motor neurons in this process, we used a Cre-Lox targeted approach to establish a skeletal muscle-specific Sod1-knockout (mKO) mouse to determine whether muscle-specific CuZnSOD deletion is sufficient to cause muscle atrophy. Surprisingly, mKO mice maintain muscle masses at or above those of wild-type control mice up to 18 mo of age. In contrast, maximum isometric specific force measured in gastrocnemius muscle is significantly reduced in the mKO mice. We found no detectable increases in global measures of oxidative stress or ROS production, no reduction in mitochondrial ATP production, and no induction of adaptive stress responses in muscle from mKO mice. However, Akt-mTOR signaling is elevated and the number of muscle fibers with centrally located nuclei is increased in skeletal muscle from mKO mice, which suggests elevated regenerative pathways. Our data demonstrate that lack of CuZnSOD restricted to skeletal muscle does not lead to muscle atrophy but does cause muscle weakness in adult mice and suggest loss of CuZnSOD may potentiate muscle regenerative pathways.—Zhang, Y., Davis, C., Sakellariou, G.K., Shi, Y., Kayani, A.C., Pulliam, D., Bhattacharya, A., Richardson, A., Jackson, M.J., McArdle, A., Brooks, S.V., Van Remmen, H. CuZnSOD gene deletion targeted to skeletal muscle leads to loss of contractile force but does not cause muscle atrophy in adult mice. PMID:23729587

A post-MI power struggle: adaptations in cardiac power occur at the sarcomere level alongside MyBP-C and RLC phosphorylation.

PubMed

Toepfer, Christopher N; Sikkel, Markus B; Caorsi, Valentina; Vydyanath, Anupama; Torre, Iratxe; Copeland, O'Neal; Lyon, Alexander R; Marston, Steven B; Luther, Pradeep K; Macleod, Kenneth T; West, Timothy G; Ferenczi, Michael A

2016-08-01

Myocardial remodeling in response to chronic myocardial infarction (CMI) progresses through two phases, hypertrophic "compensation" and congestive "decompensation." Nothing is known about the ability of uninfarcted myocardium to produce force, velocity, and power during these clinical phases, even though adaptation in these regions likely drives progression of compensation. We hypothesized that enhanced cross-bridge-level contractility underlies mechanical compensation and is controlled in part by changes in the phosphorylation states of myosin regulatory proteins. We induced CMI in rats by left anterior descending coronary artery ligation. We then measured mechanical performance in permeabilized ventricular trabecula taken distant from the infarct zone and assayed myosin regulatory protein phosphorylation in each individual trabecula. During full activation, the compensated myocardium produced twice as much power and 31% greater isometric force compared with noninfarcted controls. Isometric force during submaximal activations was raised >2.4-fold, while power was 2-fold greater. Electron and confocal microscopy demonstrated that these mechanical changes were not a result of increased density of contractile protein and therefore not an effect of tissue hypertrophy. Hence, sarcomere-level contractile adaptations are key determinants of enhanced trabecular mechanics and of the overall cardiac compensatory response. Phosphorylation of myosin regulatory light chain (RLC) increased and remained elevated post-MI, while phosphorylation of myosin binding protein-C (MyBP-C) was initially depressed but then increased as the hearts became decompensated. These sensitivities to CMI are in accordance with phosphorylation-dependent regulatory roles for RLC and MyBP-C in crossbridge function and with compensatory adaptation in force and power that we observed in post-CMI trabeculae. Copyright © 2016 the American Physiological Society.

Thick Filament Length and Isoform Composition Determine Self-Organized Contractile Units in Actomyosin Bundles

PubMed Central

Thoresen, Todd; Lenz, Martin; Gardel, Margaret L.

2013-01-01

Diverse myosin II isoforms regulate contractility of actomyosin bundles in disparate physiological processes by variations in both motor mechanochemistry and the extent to which motors are clustered into thick filaments. Although the role of mechanochemistry is well appreciated, the extent to which thick filament length regulates actomyosin contractility is unknown. Here, we study the contractility of minimal actomyosin bundles formed in vitro by mixtures of F-actin and thick filaments of nonmuscle, smooth, and skeletal muscle myosin isoforms with varied length. Diverse myosin II isoforms guide the self-organization of distinct contractile units within in vitro bundles with shortening rates similar to those of in vivo myofibrils and stress fibers. The tendency to form contractile units increases with the thick filament length, resulting in a bundle shortening rate proportional to the length of constituent myosin thick filament. We develop a model that describes our data, providing a framework in which to understand how diverse myosin II isoforms regulate the contractile behaviors of disordered actomyosin bundles found in muscle and nonmuscle cells. These experiments provide insight into physiological processes that use dynamic regulation of thick filament length, such as smooth muscle contraction. PMID:23442916

In vitro contraction of cytokinetic ring depends on myosin II but not on actin dynamics.

PubMed

Mishra, Mithilesh; Kashiwazaki, Jun; Takagi, Tomoko; Srinivasan, Ramanujam; Huang, Yinyi; Balasubramanian, Mohan K; Mabuchi, Issei

2013-07-01

Cytokinesis in many eukaryotes involves the contraction of an actomyosin-based contractile ring. However, the detailed mechanism of contractile ring contraction is not fully understood. Here, we establish an experimental system to study contraction of the ring to completion in vitro. We show that the contractile ring of permeabilized fission yeast cells undergoes rapid contraction in an ATP- and myosin-II-dependent manner in the absence of other cytoplasmic constituents. Surprisingly, neither actin polymerization nor its disassembly is required for contraction of the contractile ring, although addition of exogenous actin-crosslinking proteins blocks ring contraction. Using contractile rings generated from fission yeast cytokinesis mutants, we show that not all proteins required for assembly of the ring are required for its contraction in vitro. Our work provides the beginnings of the definition of a minimal contraction-competent cytokinetic ring apparatus.

Tight junctions negatively regulate mechanical forces applied to adherens junctions in vertebrate epithelial tissue.

PubMed

Hatte, Guillaume; Prigent, Claude; Tassan, Jean-Pierre

2018-02-05

Epithelia are layers of polarised cells tightly bound to each other by adhesive contacts. Epithelia act as barriers between an organism and its external environment. Understanding how epithelia maintain their essential integrity while remaining sufficiently plastic to allow events such as cytokinesis to take place is a key biological problem. In vertebrates, the remodelling and reinforcement of adherens junctions maintains epithelial integrity during cytokinesis. The involvement of tight junctions in cell division, however, has remained unexplored. Here, we examine the role of tight junctions during cytokinesis in the epithelium of the Xenopus laevis embryo. Depletion of the tight junction-associated proteins ZO-1 and GEF-H1 leads to altered cytokinesis duration and contractile ring geometry. Using a tension biosensor, we show that cytokinesis defects originate from misregulation of tensile forces applied to adherens junctions. Our results reveal that tight junctions regulate mechanical tension applied to adherens junctions, which in turn impacts cytokinesis.This article has an associated First Person interview with the first author of the paper. © 2018. Published by The Company of Biologists Ltd.

Mechanisms of mechanical strain memory in airway smooth muscle.

PubMed

Kim, Hak Rim; Hai, Chi-Ming

2005-10-01

We evaluated the hypothesis that mechanical deformation of airway smooth muscle induces structural remodeling of airway smooth muscle cells, thereby modulating mechanical performance in subsequent contractions. This hypothesis implied that past experience of mechanical deformation was retained (or "memorized") as structural changes in airway smooth muscle cells, which modulated the cell's subsequent contractile responses. We termed this phenomenon mechanical strain memory. Preshortening has been found to induce attenuation of both force and isotonic shortening velocity in cholinergic receptor-activated airway smooth muscle. Rapid stretching of cholinergic receptor-activated airway smooth muscle from an initial length to a final length resulted in post-stretch force and myosin light chain phosphorylation that correlated significantly with initial length. Thus post-stretch muscle strips appeared to retain memory of the initial length prior to rapid stretch (mechanical strain memory). Cytoskeletal recruitment of actin- and integrin-binding proteins and Erk 1/2 MAPK appeared to be important mechanisms of mechanical strain memory. Sinusoidal length oscillation led to force attenuation during oscillation and in subsequent contractions in intact airway smooth muscle, and p38 MAPK appeared to be an important mechanism. In contrast, application of local mechanical strain to cultured airway smooth muscle cells induced local actin polymerization and cytoskeletal stiffening. It is conceivable that deep inspiration-induced bronchoprotection may be a manifestation of mechanical strain memory such that mechanical deformation from past breathing cycles modulated the mechanical performance of airway smooth muscle in subsequent cycles in a continuous and dynamic manner.

Putative β4-adrenoceptors in rat ventricle mediate increases in contractile force and cell Ca2+: comparison with atrial receptors and relationship to (−)-[3H]-CGP 12177 binding

PubMed Central

Sarsero, Doreen; Molenaar, Peter; Kaumann, Alberto J; Freestone, Nicholas S

1999-01-01

We identified putative β4-adrenoceptors by radioligand binding, measured increases in ventricular contractile force by (−)-CGP 12177 and (±)-cyanopindolol and demonstrated increased Ca2+ transients by (−)-CGP 12177 in rat cardiomyocytes.(−)-[3H]-CGP 12177 labelled 13–22 fmol mg−1 protein ventricular β1, β2-adrenoceptors (pKD ∼9.0) and 50–90 fmol mg−1 protein putative β4-adrenoceptors (pKD ∼7.3). The affinity values (pKi) for (β1,β2-) and putative β4-adrenoceptors, estimated from binding inhibition, were (−)-propranolol 8.4, 5.7; (−)-bupranolol 9.7, 5.8; (±)-cyanopindolol 10.0,7.4.In left ventricular papillary muscle, in the presence of 30 μM 3-isobutyl-1-methylxanthine, (−)-CGP 12177 and (±)-cyanopindolol caused positive inotropic effects, (pEC50, (−)-CGP 12177, 7.6; (±)-cyanopindolol, 7.0) which were antagonized by (−)-bupranolol (pKB 6.7–7.0) and (−)-CGP 20712A (pKB 6.3–6.6). The cardiostimulant effects of (−)-CGP 12177 in papillary muscle, left and right atrium were antagonized by (±)-cyanopindolol (pKP 7.0–7.4).(−)-CGP 12177 (1 μM) in the presence of 200 nM (−)-propranolol increased Ca2+ transient amplitude by 56% in atrial myocytes, but only caused a marginal increase in ventricular myocytes. In the presence of 1 μM 3-isobutyl-1-methylxanthine and 200 nM (−)-propranolol, 1 μM (−)-CGP 12177 caused a 73% increase in Ca2+ transient amplitude in ventricular myocytes. (−)-CGP 12177 elicited arrhythmic transients in some atrial and ventricular myocytes.Probably by preventing cyclic AMP hydrolysis, 3-isobutyl-1-methylxanthine facilitates the inotropic function of ventricular putative β4-adrenoceptors, suggesting coupling to Gs protein-adenylyl cyclase. The receptor-mediated increases in contractile force are related to increases of Ca2+ in atrial and ventricular myocytes. The agreement of binding affinities of agonists with cardiostimulant potencies is consistent with mediation through putative β4-adrenoceptors labelled with (−)-[3H]-CGP 12177. PMID:10602323

Comparison of human gastrocnemius forces predicted by Hill-type muscle models and estimated from ultrasound images.

PubMed

Dick, Taylor J M; Biewener, Andrew A; Wakeling, James M

2017-05-01

Hill-type models are ubiquitous in the field of biomechanics, providing estimates of a muscle's force as a function of its activation state and its assumed force-length and force-velocity properties. However, despite their routine use, the accuracy with which Hill-type models predict the forces generated by muscles during submaximal, dynamic tasks remains largely unknown. This study compared human gastrocnemius forces predicted by Hill-type models with the forces estimated from ultrasound-based measures of tendon length changes and stiffness during cycling, over a range of loads and cadences. We tested both a traditional model, with one contractile element, and a differential model, with two contractile elements that accounted for independent contributions of slow and fast muscle fibres. Both models were driven by subject-specific, ultrasound-based measures of fascicle lengths, velocities and pennation angles and by activation patterns of slow and fast muscle fibres derived from surface electromyographic recordings. The models predicted, on average, 54% of the time-varying gastrocnemius forces estimated from the ultrasound-based methods. However, differences between predicted and estimated forces were smaller under low speed-high activation conditions, with models able to predict nearly 80% of the gastrocnemius force over a complete pedal cycle. Additionally, the predictions from the Hill-type muscle models tested here showed that a similar pattern of force production could be achieved for most conditions with and without accounting for the independent contributions of different muscle fibre types. © 2017. Published by The Company of Biologists Ltd.

Diabetes attenuates urothelial modulation of detrusor contractility and spontaneous activity.

PubMed

Wang, Yi; Tar, Moses T; Fu, Shibo; Melman, Arnold; Davies, Kelvin P

2014-10-01

To investigate the effect of diabetes on urothelial modulation of bladder contractility. Bladder strips (urothelium intact or denuded) were prepared from 8-week-old streptozotocin-induced diabetic (n = 19) and non-diabetic control rats (n = 10). The effect of modulators of MaxiK (iberiotoxin and tetraethylammonium) and Kv7 (XE991 and retigabine) potassium channel activity were investigated for their effects on both carbachol-induced force generation and spontaneous contractile activity. In bladder strips from non-diabetic animals, the presence of the urothelium resulted in marked sensitivity to carbachol-induced force generation by modulators of MaxiK and Kv7 channel activity, whereas in the diabetic animal urothelial sensitivity to these agents was significantly diminished. Urothelial-intact bladder strips from non-diabetic animals were more sensitive to modulators of Kv7 activity in reducing the amplitude of spontaneous phasic contractions than urothelial-denuded bladder strips, whereas in diabetic animals the presence or absence of the urothelium did not alter the sensitivity to modulators of Kv7 activity. Spontaneous activity in the presence of tetraethylammonium was not affected by the urothelium in bladder strips from either diabetic or non-diabetic animals. The presence of the urothelium in bladders from non-diabetic animals modulates the activity of potassium blockers to affect bladder contractility, whereas in the diabetic bladder this effect is attenuated. These findings could help to explain the lack of success of pharmaceutical treatments targeting potassium channels to treat bladder pathology in patients with diseases imparing urothelial function. © 2014 The Japanese Urological Association.

Isolation and maintenance-free culture of contractile myotubes from Manduca sexta embryos.

PubMed

Baryshyan, Amanda L; Woods, William; Trimmer, Barry A; Kaplan, David L

2012-01-01

Skeletal muscle tissue engineering has the potential to treat tissue loss and degenerative diseases. However, these systems are also applicable for a variety of devices where actuation is needed, such as microelectromechanical systems (MEMS) and robotics. Most current efforts to generate muscle bioactuators are focused on using mammalian cells, which require exacting conditions for survival and function. In contrast, invertebrate cells are more environmentally robust, metabolically adaptable and relatively autonomous. Our hypothesis is that the use of invertebrate muscle cells will obviate many of the limitations encountered when mammalian cells are used for bioactuation. We focus on the tobacco hornworm, Manduca sexta, due to its easy availability, large size and well-characterized muscle contractile properties. Using isolated embryonic cells, we have developed culture conditions to grow and characterize contractile M. sexta muscles. The insect hormone 20-hydroxyecdysone was used to induce differentiation in the system, resulting in cells that stained positive for myosin, contract spontaneously for the duration of the culture, and do not require media changes over periods of more than a month. These cells proliferate under normal conditions, but the application of juvenile hormone induced further proliferation and inhibited differentiation. Cellular metabolism under normal and low glucose conditions was compared for C2C12 mouse and M. sexta myoblast cells. While differentiated C2C12 cells consumed glucose and produced lactate over one week as expected, M. sexta muscle did not consume significant glucose, and lactate production exceeded mammalian muscle production on a per cell basis. Contractile properties were evaluated using index of movement analysis, which demonstrated the potential of these cells to perform mechanical work. The ability of cultured M. sexta muscle to continuously function at ambient conditions without medium replenishment, combined with the interesting metabolic properties, suggests that this cell source is a promising candidate for further investigation toward bioactuator applications.

Contractile function recovery in severely injured gastrocnemius muscle of rats treated with either oleic or linoleic acid.

PubMed

Abreu, Phablo; Pinheiro, Carlos H J; Vitzel, Kaio F; Vasconcelos, Diogo A A; Torres, Rosângela P; Fortes, Marco S; Marzuca-Nassr, Gabriel N; Mancini-Filho, Jorge; Hirabara, Sandro M; Curi, Rui

2016-11-01

What is the central question of this study? Oleic and linoleic acids modulate fibroblast proliferation and myogenic differentiation in vitro. However, their in vivo effects on muscle regeneration have not yet been examined. We investigated the effects of either oleic or linoleic acid on a well-established model of muscle regeneration after severe laceration. What is the main finding and its importance? We found that linoleic acid increases fibrous tissue deposition and impairs muscle regeneration and recovery of contractile function, whereas oleic acid has the opposite effects in severely injured gastrocnemius muscle, suggesting that linoleic acid has a harmful effect and oleic acid a potential therapeutic effect on muscle regeneration. Oleic and linoleic acids control fibroblast proliferation and myogenic differentiation in vitro; however, there was no study in skeletal muscle in vivo. The aim of this study was to evaluate the effects of either oleic or linoleic acid on the fibrous tissue content (collagen deposition) of muscle and recovery of contractile function in rat gastrocnemius muscle after being severely injured by laceration. Rats were supplemented with either oleic or linoleic acid for 4 weeks after laceration [0.44 g (kg body weight) -1 day -1 ]. Muscle injury led to an increase in oleic-to-stearic acid and palmitoleic-to-palmitic acid ratios, suggesting an increase in Δ 9 desaturase activity. Increased fibrous tissue deposition and reduced isotonic and tetanic specific forces and resistance to fatigue were observed in the injured muscle. Supplementation with linoleic acid increased the content of eicosadienoic (20:2, n-6) and arachidonic (20:4, n-6) acids, reduced muscle mass and fibre cross-sectional areas, increased fibrous tissue deposition and further reduced the isotonic and tetanic specific forces and resistance to fatigue induced by laceration. Supplementation with oleic acid increased the content of docosahexaenoic acid (22:6, n-3) and abolished the increase in fibrous tissue area and the decrease in isotonic and tetanic specific forces and resistance to fatigue induced by muscle injury. We concluded that supplementation with linoleic acid impairs muscle regeneration and increases fibrous tissue deposition, resulting in impaired recovery of contractile function. Oleic acid supplementation reduced fibrous tissue deposition and improved recovery of contractile function, attenuating the tissue damage caused by muscle injury. © 2016 The Authors. Experimental Physiology © 2016 The Physiological Society.

A thermodynamical model for stress-fiber organization in contractile cells.

PubMed

Foucard, Louis; Vernerey, Franck J

2012-01-02

Cell mechanical adaptivity to external stimuli is vital to many of its biological functions. A critical question is therefore to understand the formation and organization of the stress fibers from which emerge the cell's mechanical properties. By accounting for the mechanical aspects and the viscoelastic behavior of stress fibers, we here propose a thermodynamic model to predict the formation and orientation of stress fibers in contractile cells subjected to constant or cyclic stretch and different substrate stiffness. Our results demonstrate that the stress fibers viscoelastic behavior plays a crucial role in their formation and organization and shows good consistency with various experiments.

Contractile Skeletal Muscle Cells Cultured with a Conducting Soft Wire for Effective, Selective Stimulation.

PubMed

Nagamine, Kuniaki; Sato, Hirotaka; Kai, Hiroyuki; Kaji, Hirokazu; Kanzaki, Makoto; Nishizawa, Matsuhiko

2018-02-02

Contractile skeletal muscle cells were cultured so as to wrap around an electrode wire to enable their selective stimulation even when they were co-cultured with other electrically-excitable cells. Since the electrode wire was composed of the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) and polyurethane (PU), which is soft and highly capacitive (~10 mF cm -2 ), non-faradaic electrical stimulation with charge/discharge currents could be applied to the surrounding cells without causing significant damage even for longer periods (more than a week). The advantage of this new culture system was demonstrated in the study of chemotactic interaction of monocytes and skeletal muscle cells via myokines.

Measurements of the contact force from myenteric contractions on a solid bolus.

PubMed

Terry, Benjamin S; Schoen, Jonathan A; Rentschler, Mark E

2013-03-01

The development of robotic capsule endoscopes (RCEs) is one avenue presently investigated by multiple research groups to minimize invasiveness and enhance outcomes of enteroscopic procedures. Understanding the biomechanical response of the small bowel to RCEs is needed for design optimization of these devices. In previous work, the authors developed, characterized, and tested the migrating motor complex force sensor (MFS), a novel sensor for quantifying the contact forces per unit of axial length exerted by the myenteron on a solid bolus. This work is a continuation, in which the MFS is used to quantify the contractile strength in the small intestine proximal, middle, and distal regions of five live porcine models. The MFSs are surgically implanted in a generally anesthetized animal, and force data from 5 min of dwell time are analyzed. The mean myenteric contact force from all porcine models and locations within the bowel is 1.9 ± 1.0 N cm(-1). Examining the results based on the small bowel region shows a statistically significant strengthening trend in the contractile force from proximal to middle to distal with mean forces of 1.2 ± 0.5, 1.9 ± 0.9, and 2.3 ± 1.0 N cm(-1), respectively (mean ± one standard deviation). Quantification of the contact force against a solid bolus provides developers of RCEs with a valuable, experimentally derived parameter of the intraluminal environment.

Extracellular cell wall β(1,3)glucan is required to couple septation to actomyosin ring contraction.

PubMed

Muñoz, Javier; Cortés, Juan Carlos G; Sipiczki, Matthias; Ramos, Mariona; Clemente-Ramos, José Angel; Moreno, M Belén; Martins, Ivone M; Pérez, Pilar; Ribas, Juan Carlos

2013-10-28

Cytokinesis has been extensively studied in different models, but the role of the extracellular cell wall is less understood. Here we studied this process in fission yeast. The essential protein Bgs4 synthesizes the main cell wall β(1,3)glucan. We show that Bgs4-derived β(1,3)glucan is required for correct and stable actomyosin ring positioning in the cell middle, before the start of septum formation and anchorage to the cell wall. Consequently, β(1,3)glucan loss generated ring sliding, oblique positioned rings and septa, misdirected septum synthesis indicative of relaxed rings, and uncoupling between a fast ring and membrane ingression and slow septum synthesis, suggesting that cytokinesis can progress with defective septum pushing and/or ring pulling forces. Moreover, Bgs4-derived β(1,3)glucan is essential for secondary septum formation and correct primary septum completion. Therefore, our results show that extracellular β(1,3)glucan is required for cytokinesis to connect the cell wall with the plasma membrane and for contractile ring function, as proposed for the equivalent extracellular matrix in animal cells.

Extracellular cell wall β(1,3)glucan is required to couple septation to actomyosin ring contraction

PubMed Central

Muñoz, Javier; Cortés, Juan Carlos G.; Sipiczki, Matthias; Ramos, Mariona; Clemente-Ramos, José Angel; Moreno, M. Belén; Martins, Ivone M.; Pérez, Pilar

2013-01-01

Cytokinesis has been extensively studied in different models, but the role of the extracellular cell wall is less understood. Here we studied this process in fission yeast. The essential protein Bgs4 synthesizes the main cell wall β(1,3)glucan. We show that Bgs4-derived β(1,3)glucan is required for correct and stable actomyosin ring positioning in the cell middle, before the start of septum formation and anchorage to the cell wall. Consequently, β(1,3)glucan loss generated ring sliding, oblique positioned rings and septa, misdirected septum synthesis indicative of relaxed rings, and uncoupling between a fast ring and membrane ingression and slow septum synthesis, suggesting that cytokinesis can progress with defective septum pushing and/or ring pulling forces. Moreover, Bgs4-derived β(1,3)glucan is essential for secondary septum formation and correct primary septum completion. Therefore, our results show that extracellular β(1,3)glucan is required for cytokinesis to connect the cell wall with the plasma membrane and for contractile ring function, as proposed for the equivalent extracellular matrix in animal cells. PMID:24165938

Effect of Electromechanical Stimulation on the Maturation of Myotubes on Aligned Electrospun Fibers

PubMed Central

Liao, I-Chien; Liu, Jason B.; Bursac, Nenad; Leong, Kam W.

2009-01-01

Tissue engineering may provide an alternative to cell injection as a therapeutic solution for myocardial infarction. A tissue-engineered muscle patch may offer better host integration and higher functional performance. This study examined the differentiation of skeletal myoblasts on aligned electrospun polyurethane (PU) fibers and in the presence of electromechanical stimulation. Skeletal myoblasts cultured on aligned PU fibers showed more pronounced elongation, better alignment, higher level of transient receptor potential cation channel-1 (TRPC-1) expression, upregulation of contractile proteins and higher percentage of striated myotubes compared to those cultured on random PU fibers and film. The resulting tissue constructs generated tetanus forces of 1.1 mN with a 10-ms time to tetanus. Additional mechanical, electrical, or synchronized electromechanical stimuli applied to myoblasts cultured on PU fibers increased the percentage of striated myotubes from 70 to 85% under optimal stimulation conditions, which was accompanied by an upregulation of contractile proteins such as α-actinin and myosin heavy chain. In describing how electromechanical cues can be combined with topographical cue, this study helped move towards the goal of generating a biomimetic microenvironment for engineering of functional skeletal muscle. PMID:19774099

Compromised store-operated Ca2+ entry in aged skeletal muscle.

PubMed

Zhao, Xiaoli; Weisleder, Noah; Thornton, Angela; Oppong, Yaa; Campbell, Rachel; Ma, Jianjie; Brotto, Marco

2008-08-01

In aged skeletal muscle, changes to the composition and function of the contractile machinery cannot fully explain the observed decrease in the specific force produced by the contractile machinery that characterizes muscle weakness during aging. Since modification in extracellular Ca(2+) entry in aged nonexcitable and excitable cells has been recently identified, we evaluated the functional status of store-operated Ca(2+) entry (SOCE) in aged mouse skeletal muscle. Using Mn(2+) quenching of Fura-2 fluorescence and confocal-microscopic imaging of Ca(2+) movement from the transverse tubules, we determined that SOCE was severely compromised in muscle fibers isolated from aged mice (26-27 months) as compared with those from young (2-5 months) mice. While reduced SOCE in aged skeletal muscle does not appear to result from altered expression levels of STIM1 or reduced expression of mRNA for Orai, this reduction in SOCE is mirrored in fibers isolated from young mice null for mitsugumin-29, a synaptophysin-related protein that displays decreased expression in aged skeletal muscle. Our data suggest that decreased mitsugumin-29 expression and reduced SOCE may contribute to the diminished intracellular Ca(2+) homeostatic capacity generally associated with muscle aging.

Compromised store-operated Ca2+ entry in aged skeletal muscle

PubMed Central

Zhao, Xiaoli; Weisleder, Noah; Thornton, Angela; Oppong, Yaa; Campbell, Rachel; Ma, Jianjie; Brotto, Marco

2010-01-01

Summary In aged skeletal muscle, changes to the composition and function of the contractile machinery cannot fully explain the observed decrease in the specific force produced by the contractile machinery that characterizes muscle weakness during aging. Since modification in extracellular Ca2+ entry in aged nonexcitable and excitable cells has been recently identified, we evaluated the functional status of store-operated Ca2+ entry (SOCE) in aged mouse skeletal muscle. Using Mn2+ quenching of Fura-2 fluorescence and confocal-microscopic imaging of Ca2+ movement from the transverse tubules, we determined that SOCE was severely compromised in muscle fibers isolated from aged mice (26–27 months) as compared with those from young (2–5 months) mice. While reduced SOCE in aged skeletal muscle does not appear to result from altered expression levels of STIM1 or reduced expression of mRNA for Orai, this reduction in SOCE is mirrored in fibers isolated from young mice null for mitsugumin-29, a synaptophysin-related protein that displays decreased expression in aged skeletal muscle. Our data suggest that decreased mitsugumin-29 expression and reduced SOCE may contribute to the diminished intracellular Ca2+ homeostatic capacity generally associated with muscle aging. PMID:18505477

Activation of MRTF-A–dependent gene expression with a small molecule promotes myofibroblast differentiation and wound healing

PubMed Central

Velasquez, Lissette S.; Sutherland, Lillian B.; Liu, Zhenan; Grinnell, Frederick; Kamm, Kristine E.; Schneider, Jay W.; Olson, Eric N.; Small, Eric M.

2013-01-01

Myocardin-related transcription factors (MRTFs) regulate cellular contractility and motility by associating with serum response factor (SRF) and activating genes involved in cytoskeletal dynamics. We reported previously that MRTF-A contributes to pathological cardiac remodeling by promoting differentiation of fibroblasts to myofibroblasts following myocardial infarction. Here, we show that forced expression of MRTF-A in dermal fibroblasts stimulates contraction of a collagen matrix, whereas contractility of MRTF-A null fibroblasts is impaired under basal conditions and in response to TGF–β1 stimulation. We also identify an isoxazole ring-containing small molecule, previously shown to induce smooth muscle α-actin gene expression in cardiac progenitor cells, as an agonist of myofibroblast differentiation. Isoxazole stimulates myofibroblast differentiation via induction of MRTF-A–dependent gene expression. The MRTF-SRF signaling axis is activated in response to skin injury, and treatment of dermal wounds with isoxazole accelerates wound closure and suppresses the inflammatory response. These results reveal an important role for MRTF-SRF signaling in dermal myofibroblast differentiation and wound healing and suggest that targeting MRTFs pharmacologically may prove useful in treating diseases associated with inappropriate myofibroblast activity. PMID:24082095

A resilient formin-derived cortical actin meshwork in the rear drives actomyosin-based motility in 2D confinement

PubMed Central

Ramalingam, Nagendran; Franke, Christof; Jaschinski, Evelin; Winterhoff, Moritz; Lu, Yao; Brühmann, Stefan; Junemann, Alexander; Meier, Helena; Noegel, Angelika A.; Weber, Igor; Zhao, Hongxia; Merkel, Rudolf; Schleicher, Michael; Faix, Jan

2015-01-01

Cell migration is driven by the establishment of disparity between the cortical properties of the softer front and the more rigid rear allowing front extension and actomyosin-based rear contraction. However, how the cortical actin meshwork in the rear is generated remains elusive. Here we identify the mDia1-like formin A (ForA) from Dictyostelium discoideum that generates a subset of filaments as the basis of a resilient cortical actin sheath in the rear. Mechanical resistance of this actin compartment is accomplished by actin crosslinkers and IQGAP-related proteins, and is mandatory to withstand the increased contractile forces in response to mechanical stress by impeding unproductive blebbing in the rear, allowing efficient cell migration in two-dimensional-confined environments. Consistently, ForA supresses the formation of lateral protrusions, rapidly relocalizes to new prospective ends in repolarizing cells and is required for cortical integrity. Finally, we show that ForA utilizes the phosphoinositide gradients in polarized cells for subcellular targeting. PMID:26415699

Differential effects of culture senescence and mechanical stimulation on the proliferation and leiomyogenic differentiation of MSC from different sources: implications for engineering vascular grafts.

PubMed

Koobatian, Maxwell T; Liang, Mao-Shih; Swartz, Daniel D; Andreadis, Stelios T

2015-04-01

We examined the effects of senescence on the proliferation and leiomyogenic differentiation potential of mesenchymal stem cells (MSCs) isolated from bone marrow (BM-MSCs) or hair follicles (HF-MSCs). To this end, we compared ovine HF-MSCs and BM-MSCs in terms of their proliferation and differentiation potential to the smooth muscle cell lineage. We discovered that HF-MSCs are less susceptible to culture senescence compared with BM-MSCs. We hypothesized that application of mechanical forces may enhance the contractility and mechanical properties of vascular constructs prepared from senescent MSCs. Interestingly, HF-MSCs and BM-MSCs responded differently to changes in the mechanical microenvironment, suggesting that despite phenotypic similarities, MSCs from different anatomic locations may activate different pathways in response to the same microenvironmental factors. In turn, this may also suggest that cell-based tissue regeneration approaches may need to be tailored to the stem cell origin, donor age, and culture time for optimal results.

ROCK1 and 2 differentially regulate actomyosin organization to drive cell and synaptic polarity

PubMed Central

Badoual, Mathilde; Asmussen, Hannelore; Patel, Heather; Whitmore, Leanna; Horwitz, Alan Rick

2015-01-01

RhoGTPases organize the actin cytoskeleton to generate diverse polarities, from front–back polarity in migrating cells to dendritic spine morphology in neurons. For example, RhoA through its effector kinase, RhoA kinase (ROCK), activates myosin II to form actomyosin filament bundles and large adhesions that locally inhibit and thereby polarize Rac1-driven actin polymerization to the protrusions of migratory fibroblasts and the head of dendritic spines. We have found that the two ROCK isoforms, ROCK1 and ROCK2, differentially regulate distinct molecular pathways downstream of RhoA, and their coordinated activities drive polarity in both cell migration and synapse formation. In particular, ROCK1 forms the stable actomyosin filament bundles that initiate front–back and dendritic spine polarity. In contrast, ROCK2 regulates contractile force and Rac1 activity at the leading edge of migratory cells and the spine head of neurons; it also specifically regulates cofilin-mediated actin remodeling that underlies the maturation of adhesions and the postsynaptic density of dendritic spines. PMID:26169356

Prostacyclin primes pregnant human myometrium for an enhanced contractile response in parturition

PubMed Central

Fetalvero, Kristina M.; Zhang, Peisheng; Shyu, Maureen; Young, Benjamin T.; Hwa, John; Young, Roger C.; Martin, Kathleen A.

2008-01-01

An incomplete understanding of the molecular events that regulate the myometrial transition from the quiescent pregnant state to the active contractile state during labor has hindered the development of improved therapies for preterm labor. During myometrial activation, proteins that prime the smooth muscle for contraction are upregulated, allowing maximal responsiveness to contractile agonists and thereby producing strong phasic contractions. Upregulation of one such protein, COX-2, generates PGs that induce contractions. Intriguingly, the predominant myometrial PG produced just prior to labor is prostacyclin (PGI2), a smooth muscle relaxant. However, here we have shown that activation of PGI2 receptor (IP) upregulated the expression of several contractile proteins and the gap junction protein connexin 43 through cAMP/PKA signaling in human myometrial tissue in organ and cell culture. Functionally, these IP-dependent changes in gene expression promoted an enhanced contractile response to oxytocin in pregnant human myometrial tissue strips, which was inhibited by the IP antagonist RO3244794. Furthermore, contractile protein induction was dependent on the concentration and time of exposure to the PGI2 analog iloprost and was blocked by both RO3244794 and PKA knockdown. We therefore propose that PGI2-mediated upregulation of contractile proteins and connexin 43 is a critical step in myometrial activation, allowing for a maximal contractile response. Our observations have important implications regarding activation of the myometrium prior to the onset of labor. PMID:19033666

Yap1 Protein Regulates Vascular Smooth Muscle Cell Phenotypic Switch by Interaction with Myocardin*

PubMed Central

Xie, Changqing; Guo, Yanhong; Zhu, Tianqing; Zhang, Jifeng; Ma, Peter X.; Chen, Y. Eugene

2012-01-01

The Hippo-Yap (Yes-associated protein) signaling pathway has emerged as one of the critical pathways regulating cell proliferation, differentiation, and apoptosis in response to environmental and developmental cues. However, Yap1 roles in vascular smooth muscle cell (VSMC) biology have not been investigated. VSMCs undergo phenotypic switch, a process characterized by decreased gene expression of VSMC contractile markers and increased proliferation, migration, and matrix synthesis. The goals of the present studies were to investigate the relationship between Yap1 and VSMC phenotypic switch and to determine the molecular mechanisms by which Yap1 affects this essential process in VSMC biology. Results demonstrated that the expression of Yap1 was rapidly up-regulated by stimulation with PDGF-BB (a known inducer of phenotypic switch in VSMCs) and in the injured vessel wall. Knockdown of Yap1 impaired VSMC proliferation in vitro and enhanced the expression of VSMC contractile genes as well by increasing serum response factor binding to CArG-containing regions of VSMC-specific contractile genes within intact chromatin. Conversely, the interaction between serum response factor and its co-activator myocardin was reduced by overexpression of Yap1 in a dose-dependent manner. Taken together, these results indicate that down-regulation of Yap1 promotes VSMC contractile phenotype by both up-regulating myocardin expression and promoting the association of the serum response factor-myocardin complex with VSMC contractile gene promoters and suggest that the Yap1 signaling pathway is a central regulator of phenotypic switch of VSMCs. PMID:22411986

Cross-linkers both drive and brake cytoskeletal remodeling and furrowing in cytokinesis.

PubMed

Descovich, Carlos Patino; Cortes, Daniel B; Ryan, Sean; Nash, Jazmine; Zhang, Li; Maddox, Paul S; Nedelec, Francois; Maddox, Amy Shaub

2018-03-01

Cell shape changes such as cytokinesis are driven by the actomyosin contractile cytoskeleton. The molecular rearrangements that bring about contractility in nonmuscle cells are currently debated. Specifically, both filament sliding by myosin motors, as well as cytoskeletal cross-linking by myosins and nonmotor cross-linkers, are thought to promote contractility. Here we examined how the abundance of motor and nonmotor cross-linkers affects the speed of cytokinetic furrowing. We built a minimal model to simulate contractile dynamics in the Caenorhabditis elegans zygote cytokinetic ring. This model predicted that intermediate levels of nonmotor cross-linkers are ideal for contractility; in vivo, intermediate levels of the scaffold protein anillin allowed maximal contraction speed. Our model also demonstrated a nonlinear relationship between the abundance of motor ensembles and contraction speed. In vivo, thorough depletion of nonmuscle myosin II delayed furrow initiation, slowed F-actin alignment, and reduced maximum contraction speed, but partial depletion allowed faster-than-expected kinetics. Thus, cytokinetic ring closure is promoted by moderate levels of both motor and nonmotor cross-linkers but attenuated by an over-abundance of motor and nonmotor cross-linkers. Together, our findings extend the growing appreciation for the roles of cross-linkers in cytokinesis and reveal that they not only drive but also brake cytoskeletal remodeling. © 2018 Descovich, Cortes, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Nanoscale architecture of the Schizosaccharomyces pombe contractile ring.

PubMed

McDonald, Nathan A; Lind, Abigail L; Smith, Sarah E; Li, Rong; Gould, Kathleen L

2017-09-15

The contractile ring is a complex molecular apparatus which physically divides many eukaryotic cells. Despite knowledge of its protein composition, the molecular architecture of the ring is not known. Here we have applied super-resolution microscopy and FRET to determine the nanoscale spatial organization of Schizosaccharomyces pombe contractile ring components relative to the plasma membrane. Similar to other membrane-tethered actin structures, we find proteins localize in specific layers relative to the membrane. The most membrane-proximal layer (0-80 nm) is composed of membrane-binding scaffolds, formin, and the tail of the essential myosin-II. An intermediate layer (80-160 nm) consists of a network of cytokinesis accessory proteins as well as multiple signaling components which influence cell division. Farthest from the membrane (160-350 nm) we find F-actin, the motor domains of myosins, and a major F-actin crosslinker. Circumferentially within the ring, multiple proteins proximal to the membrane form clusters of different sizes, while components farther from the membrane are uniformly distributed. This comprehensive organizational map provides a framework for understanding contractile ring function.

Nanoscale architecture of the Schizosaccharomyces pombe contractile ring

PubMed Central

McDonald, Nathan A; Lind, Abigail L; Smith, Sarah E; Li, Rong

2017-01-01

The contractile ring is a complex molecular apparatus which physically divides many eukaryotic cells. Despite knowledge of its protein composition, the molecular architecture of the ring is not known. Here we have applied super-resolution microscopy and FRET to determine the nanoscale spatial organization of Schizosaccharomyces pombe contractile ring components relative to the plasma membrane. Similar to other membrane-tethered actin structures, we find proteins localize in specific layers relative to the membrane. The most membrane-proximal layer (0–80 nm) is composed of membrane-binding scaffolds, formin, and the tail of the essential myosin-II. An intermediate layer (80–160 nm) consists of a network of cytokinesis accessory proteins as well as multiple signaling components which influence cell division. Farthest from the membrane (160–350 nm) we find F-actin, the motor domains of myosins, and a major F-actin crosslinker. Circumferentially within the ring, multiple proteins proximal to the membrane form clusters of different sizes, while components farther from the membrane are uniformly distributed. This comprehensive organizational map provides a framework for understanding contractile ring function. PMID:28914606

Role of the Z band in the mechanical properties of the heart.

PubMed

Goldstein, M A; Schroeter, J P; Michael, L H

1991-05-01

In striated muscle the mechanism of contraction involves the cooperative movement of contractile and elastic components. This review emphasizes a structural approach that describes the cellular and extracellular components with known anatomical, biochemical, and physical properties that make them candidates for these contractile and elastic components. Classical models of contractile and elastic elements and their underlying assumptions are presented. Mechanical properties of cardiac and skeletal muscle are compared and contrasted and then related to ultrastructure. Information from these approaches leads to the conclusion that the Z band is essential for muscle contraction. Our review of Z band structure shows the Z band at the interface where extracellular components meet the cell surface. The Z band is also the interface from cell surface to myofibril, from extra-myofibrillar to myofibril, and finally from sarcomere to sarcomere. Our studies of Z band in defined physiologic states show that this lattice is an integral part of the contractile elements and can function as an elastic component. The Z band is a complex dynamic lattice uniquely suited to play several roles in muscle contraction.

Structural and Functional Maturation of Cardiomyocytes Derived from Human Pluripotent Stem Cells

PubMed Central

Lundy, Scott D.; Zhu, Wei-Zhong

2013-01-01

Despite preclinical studies demonstrating the functional benefit of transplanting human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) into damaged myocardium, the ability of these immature cells to adopt a more adult-like cardiomyocyte (CM) phenotype remains uncertain. To address this issue, we tested the hypothesis that prolonged in vitro culture of human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived CMs would result in the maturation of their structural and contractile properties to a more adult-like phenotype. Compared to their early-stage counterparts (PSC-CMs after 20–40 days of in vitro differentiation and culture), late-stage hESC-CMs and hiPSC-CMs (80–120 days) showed dramatic differences in morphology, including increased cell size and anisotropy, greater myofibril density and alignment, sarcomeres visible by bright-field microscopy, and a 10-fold increase in the fraction of multinucleated CMs. Ultrastructural analysis confirmed improvements in the myofibrillar density, alignment, and morphology. We measured the contractile performance of late-stage hESC-CMs and hiPSC-CMs and noted a doubling in shortening magnitude with slowed contraction kinetics compared to the early-stage cells. We then examined changes in the calcium-handling properties of these matured CMs and found an increase in calcium release and reuptake rates with no change in the maximum amplitude. Finally, we performed electrophysiological assessments in hESC-CMs and found that late-stage myocytes have hyperpolarized maximum diastolic potentials, increased action potential amplitudes, and faster upstroke velocities. To correlate these functional changes with gene expression, we performed qPCR and found a robust induction of the key cardiac structural markers, including β-myosin heavy chain and connexin-43, in late-stage hESC-CMs and hiPSC-CMs. These findings suggest that PSC-CMs are capable of slowly maturing to more closely resemble the phenotype of adult CMs and may eventually possess the potential to regenerate the lost myocardium with robust de novo force-producing tissue. PMID:23461462

NADPH Oxidase 5 Is a Pro-Contractile Nox Isoform and a Point of Cross-Talk for Calcium and Redox Signaling-Implications in Vascular Function.

PubMed

Montezano, Augusto C; De Lucca Camargo, Livia; Persson, Patrik; Rios, Francisco J; Harvey, Adam P; Anagnostopoulou, Aikaterini; Palacios, Roberto; Gandara, Ana Caroline P; Alves-Lopes, Rheure; Neves, Karla B; Dulak-Lis, Maria; Holterman, Chet E; de Oliveira, Pedro Lagerblad; Graham, Delyth; Kennedy, Christopher; Touyz, Rhian M

2018-06-15

NADPH Oxidase 5 (Nox5) is a calcium-sensitive superoxide-generating Nox. It is present in lower forms and higher mammals, but not in rodents. Nox5 is expressed in vascular cells, but the functional significance remains elusive. Given that contraction is controlled by calcium and reactive oxygen species, both associated with Nox5, we questioned the role of Nox5 in pro-contractile signaling and vascular function. Transgenic mice expressing human Nox5 in a vascular smooth muscle cell-specific manner (Nox5 mice) and Rhodnius prolixus , an arthropod model that expresses Nox5 endogenoulsy, were studied. Reactive oxygen species generation was increased systemically and in the vasculature and heart in Nox5 mice. In Nox5-expressing mice, agonist-induced vasoconstriction was exaggerated and endothelium-dependent vasorelaxation was impaired. Vascular structural and mechanical properties were not influenced by Nox5. Vascular contractile responses in Nox5 mice were normalized by N -acetylcysteine and inhibitors of calcium channels, calmodulin, and endoplasmic reticulum ryanodine receptors, but not by GKT137831 (Nox1/4 inhibitor). At the cellular level, vascular changes in Nox5 mice were associated with increased vascular smooth muscle cell [Ca 2+ ] i , increased reactive oxygen species and nitrotyrosine levels, and hyperphosphorylation of pro-contractile signaling molecules MLC20 (myosin light chain 20) and MYPT1 (myosin phosphatase target subunit 1). Blood pressure was similar in wild-type and Nox5 mice. Nox5 did not amplify angiotensin II effects. In R. prolixus , gastrointestinal smooth muscle contraction was blunted by Nox5 silencing, but not by VAS2870 (Nox1/2/4 inhibitor). Nox5 is a pro-contractile Nox isoform important in redox-sensitive contraction. This involves calcium-calmodulin and endoplasmic reticulum-regulated mechanisms. Our findings define a novel function for vascular Nox5, linking calcium and reactive oxygen species to the pro-contractile molecular machinery in vascular smooth muscle cells. © 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Maturation of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in 3D collagen matrix: Effects of niche cell supplementation and mechanical stimulation.

PubMed

Zhang, W; Kong, C W; Tong, M H; Chooi, W H; Huang, N; Li, R A; Chan, B P

2017-02-01

Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) are regarded as a promising source for regenerative medicine, drug testing and disease modeling. Nevertheless, cardiomyocytes are immature in terms of their contractile structure, metabolism and electrophysiological properties. Here, we fabricate cardiac muscle strips by encapsulating hESC-CMs in collagen-based biomaterials. Supplementation of niche cells at 3% to the number of hESC-CMs enhance the maturation of the hESC-CMs in 3D tissue matrix. The benefits of adding mesenchymal stem cells (MSCs) are comparable to that of adding fibroblasts. These two cell types demonstrate similar effects in promoting the compaction and cell spreading, as well as expression of maturation markers at both gene and protein levels. Mechanical loading, particularly cyclic stretch, produces engineered cardiac tissues with higher maturity in terms of twitch force, elastic modulus, sarcomere length and molecular signature, when comparing to static stretch or non-stretched controls. The current study demonstrates that the application of niche cells and mechanical stretch both stimulate the maturation of hESC-CMs in 3D architecture. Our results therefore suggest that this 3D model can be used for in vitro cardiac maturation study. Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) are regarded as being a promising source of cells for regenerative medicine, drug testing and disease modeling. Nevertheless, cardiomyocytes are immature in terms of their contractile structure, metabolism and electrophysiological properties. In the current study, we have fabricated cardiac muscle strips by encapsulating hESC-CMs in collagen-based biomaterials and demonstrated that supplementation of mesenchymal niche cells as well as provision of mechanical loading particularly stretching have significantly promoted the maturation of the cardiomyocytes and hence improved the mechanical functional characteristics of the tissue strips. Specifically, with 3% niche cells including both fibroblasts and mesenchymal stem cells, a more mature hESC-CMs derived cardiac strip was resulted, in terms of compaction and spreading of cells, and upregulation of molecular signature in both gene and protein expression of maturation. Mechanical loading, particularly cyclic stretch, produces engineered cardiac tissues with higher maturity in terms of molecular signature markers and functional parameters including twitch force, elastic modulus and sarcomere length, when comparing with static stretch or non-stretched controls. The current study demonstrates that the application of niche cells and mechanical stretch both stimulate the maturation of hESC-CMs in 3D architecture, resulting in more mature cardiac strips. Our results contribute to bioengineering of functional heart tissue strips for drug screening and disease modeling. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

High-molecular-weight tropomyosins localize to the contractile rings of dividing CNS cells but are absent from malignant pediatric and adult CNS tumors.

PubMed

Hughes, Julie A I; Cooke-Yarborough, Claire M; Chadwick, Nigel C; Schevzov, Galina; Arbuckle, Susan M; Gunning, Peter; Weinberger, Ron P

2003-04-01

Tropomyosin has been implicated in the control of actin filament dynamics during cell migration, morphogenesis, and cytokinesis. In order to gain insight into the role of tropomyosins in cell division, we examined their expression in developing and neoplastic brain tissue. We found that the high-molecular-weight tropomyosins are downregulated at birth, which correlates with glial cell differentiation and withdrawal of most cells from the cell cycle. Expression of these isoforms was restricted to proliferative areas in the embryonic brain and was absent from the adult, where the majority of cells are quiescent. However, they were induced under conditions where glial cells became proliferative in response to injury. During cytokinesis, these tropomyosin isoforms were associated with the contractile ring. We also investigated tropomyosin expression in neoplastic CNS tissues. Low-grade astrocytic tumors expressed high-molecular-weight tropomyosins, while highly malignant CNS tumors of diverse origin did not (P

Contractile Ring-independent Localization of DdINCENP, a Protein Important for Spindle Stability and CytokinesisD⃞V⃞

PubMed Central

Chen, Qian; Li, Hui; De Lozanne, Arturo

2006-01-01

Dictyostelium DdINCENP is a chromosomal passenger protein associated with centromeres, the spindle midzone, and poles during mitosis and the cleavage furrow during cytokinesis. Disruption of the single DdINCENP gene revealed important roles for this protein in mitosis and cytokinesis. DdINCENP null cells lack a robust spindle midzone and are hypersensitive to microtubule-depolymerizing drugs, suggesting that their spindles may not be stable. Furthermore DdCP224, a protein homologous to the microtubule-stabilizing protein TOGp/XMAP215, was absent from the spindle midzone of DdINCENP null cells. Overexpression of DdCP224 rescued the weak spindle midzone defect of DdINCENP null cells. Although not required for the localization of the myosin II contractile ring and subsequent formation of a cleavage furrow, DdINCENP is important for the abscission of daughter cells at the end of cytokinesis. Finally, we show that the localization of DdINCENP at the cleavage furrow is modulated by myosin II but it occurs by a mechanism different from that controlling the formation of the contractile ring. PMID:16339076

Single-platelet nanomechanics measured by high-throughput cytometry

NASA Astrophysics Data System (ADS)

Myers, David R.; Qiu, Yongzhi; Fay, Meredith E.; Tennenbaum, Michael; Chester, Daniel; Cuadrado, Jonas; Sakurai, Yumiko; Baek, Jong; Tran, Reginald; Ciciliano, Jordan C.; Ahn, Byungwook; Mannino, Robert G.; Bunting, Silvia T.; Bennett, Carolyn; Briones, Michael; Fernandez-Nieves, Alberto; Smith, Michael L.; Brown, Ashley C.; Sulchek, Todd; Lam, Wilbur A.

2017-02-01

Haemostasis occurs at sites of vascular injury, where flowing blood forms a clot, a dynamic and heterogeneous fibrin-based biomaterial. Paramount in the clot's capability to stem haemorrhage are its changing mechanical properties, the major drivers of which are the contractile forces exerted by platelets against the fibrin scaffold. However, how platelets transduce microenvironmental cues to mediate contraction and alter clot mechanics is unknown. This is clinically relevant, as overly softened and stiffened clots are associated with bleeding and thrombotic disorders. Here, we report a high-throughput hydrogel-based platelet-contraction cytometer that quantifies single-platelet contraction forces in different clot microenvironments. We also show that platelets, via the Rho/ROCK pathway, synergistically couple mechanical and biochemical inputs to mediate contraction. Moreover, highly contractile platelet subpopulations present in healthy controls are conspicuously absent in a subset of patients with undiagnosed bleeding disorders, and therefore may function as a clinical diagnostic biophysical biomarker.

The Inhibitory Effect of Botulinum Toxin Type A on Rat Pyloric Smooth Muscle Contractile Response to Substance P In Vitro

PubMed Central

Shao, Yu-Feng; Xie, Jun-Fan; Ren, Yin-Xiang; Wang, Can; Kong, Xiang-Pan; Zong, Xiao-Jian; Fan, Lin-Lan; Hou, Yi-Ping

2015-01-01

A decrease in pyloric myoelectrical activity and pyloric substance P (SP) content following intrasphincteric injection of botulinum toxin type A (BTX-A) in free move rats have been demonstrated in our previous studies. The aim of the present study was to investigate the inhibitory effect of BTX-A on rat pyloric muscle contractile response to SP in vitro and the distributions of SP and neurokinin 1 receptor (NK1R) immunoreactive (IR) cells and fibers within pylorus. After treatment with atropine, BTX-A (10 U/mL), similar to [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-SP (APTL-SP, 1 μmol/L) which is an NK1R antagonist, decreased electric field stimulation (EFS)-induced contractile tension and frequency, whereas, subsequent administration of APTL-SP did not act on contractility. Incubation with BTX-A at 4 and 10 U/mL for 4 h respectively decreased SP (1 μmol/L)-induced contractions by 26.64% ± 5.12% and 74.92% ± 3.62%. SP-IR fibers and NK1R-IR cells both located within pylorus including mucosa and circular muscle layer. However, fewer SP-fibers were observed in pylorus treated with BTX-A (10 U/mL). In conclusion, BTX-A inhibits SP release from enteric terminals in pylorus and EFS-induced contractile responses when muscarinic cholinergic receptors are blocked by atropine. In addition, BTX-A concentration- and time-dependently directly inhibits SP-induced pyloric smooth muscle contractility. PMID:26501321

The Inhibitory Effect of Botulinum Toxin Type A on Rat Pyloric Smooth Muscle Contractile Response to Substance P In Vitro.

PubMed

Shao, Yu-Feng; Xie, Jun-Fan; Ren, Yin-Xiang; Wang, Can; Kong, Xiang-Pan; Zong, Xiao-Jian; Fan, Lin-Lan; Hou, Yi-Ping

2015-10-15

A decrease in pyloric myoelectrical activity and pyloric substance P (SP) content following intrasphincteric injection of botulinum toxin type A (BTX-A) in free move rats have been demonstrated in our previous studies. The aim of the present study was to investigate the inhibitory effect of BTX-A on rat pyloric muscle contractile response to SP in vitro and the distributions of SP and neurokinin 1 receptor (NK1R) immunoreactive (IR) cells and fibers within pylorus. After treatment with atropine, BTX-A (10 U/mL), similar to [D-Arg¹, D-Phe⁵, D-Trp(7,9), Leu(11)]-SP (APTL-SP, 1 μmol/L) which is an NK1R antagonist, decreased electric field stimulation (EFS)-induced contractile tension and frequency, whereas, subsequent administration of APTL-SP did not act on contractility. Incubation with BTX-A at 4 and 10 U/mL for 4 h respectively decreased SP (1 μmol/L)-induced contractions by 26.64% ± 5.12% and 74.92% ± 3.62%. SP-IR fibers and NK1R-IR cells both located within pylorus including mucosa and circular muscle layer. However, fewer SP-fibers were observed in pylorus treated with BTX-A (10 U/mL). In conclusion, BTX-A inhibits SP release from enteric terminals in pylorus and EFS-induced contractile responses when muscarinic cholinergic receptors are blocked by atropine. In addition, BTX-A concentration- and time-dependently directly inhibits SP-induced pyloric smooth muscle contractility.

Transversal Stiffness and Beta-Actin and Alpha-Actinin-4 Content of the M. Soleus Fibers in the Conditions of a 3-Day Reloading after 14-Day Gravitational Unloading

PubMed Central

Ogneva, I. V.

2011-01-01

The aim of the work was to analyze the structural changes in different parts of the sarcolemma and contractile apparatus of muscle fibers by measuring their transversal stiffness by atomic force microscopy in a three-day reloading after a 14-day gravity disuse, which was carried out by hind-limbs suspension. The object of the study was the soleus muscle of the Wistar rat. It was shown that after 14 days of disuse, there was a reduction of transversal stiffness of all points of the sarcolemma and contractile apparatus. Readaptation for 3 days leads to complete recovery of the values of the transversal stiffness of the sarcolemma and to partial value recovery of the contractile apparatus. The changes in transversal stiffness of sarcolemma correlate with beta-actin and alpha-actinin-4 in membrane protein fractions. PMID:21941432

Transversal stiffness and beta-actin and alpha-actinin-4 content of the M. soleus fibers in the conditions of a 3-day reloading after 14-day gravitational unloading.

PubMed

Ogneva, I V

2011-01-01

The aim of the work was to analyze the structural changes in different parts of the sarcolemma and contractile apparatus of muscle fibers by measuring their transversal stiffness by atomic force microscopy in a three-day reloading after a 14-day gravity disuse, which was carried out by hind-limbs suspension. The object of the study was the soleus muscle of the Wistar rat. It was shown that after 14 days of disuse, there was a reduction of transversal stiffness of all points of the sarcolemma and contractile apparatus. Readaptation for 3 days leads to complete recovery of the values of the transversal stiffness of the sarcolemma and to partial value recovery of the contractile apparatus. The changes in transversal stiffness of sarcolemma correlate with beta-actin and alpha-actinin-4 in membrane protein fractions.

Thick filament length and isoform composition determine self-organized contractile units in actomyosin bundles.

PubMed

Thoresen, Todd; Lenz, Martin; Gardel, Margaret L

2013-02-05

Diverse myosin II isoforms regulate contractility of actomyosin bundles in disparate physiological processes by variations in both motor mechanochemistry and the extent to which motors are clustered into thick filaments. Although the role of mechanochemistry is well appreciated, the extent to which thick filament length regulates actomyosin contractility is unknown. Here, we study the contractility of minimal actomyosin bundles formed in vitro by mixtures of F-actin and thick filaments of nonmuscle, smooth, and skeletal muscle myosin isoforms with varied length. Diverse myosin II isoforms guide the self-organization of distinct contractile units within in vitro bundles with shortening rates similar to those of in vivo myofibrils and stress fibers. The tendency to form contractile units increases with the thick filament length, resulting in a bundle shortening rate proportional to the length of constituent myosin thick filament. We develop a model that describes our data, providing a framework in which to understand how diverse myosin II isoforms regulate the contractile behaviors of disordered actomyosin bundles found in muscle and nonmuscle cells. These experiments provide insight into physiological processes that use dynamic regulation of thick filament length, such as smooth muscle contraction. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Pericellular Versican Regulates the Fibroblast-Myofibroblast Transition

PubMed Central

Hattori, Noriko; Carrino, David A.; Lauer, Mark E.; Vasanji, Amit; Wylie, James D.; Nelson, Courtney M.; Apte, Suneel S.

2011-01-01

The cell and its glycosaminoglycan-rich pericellular matrix (PCM) comprise a functional unit. Because modification of PCM influences cell behavior, we investigated molecular mechanisms that regulate PCM volume and composition. In fibroblasts and other cells, aggregates of hyaluronan and versican are found in the PCM. Dermal fibroblasts from Adamts5−/− mice, which lack a versican-degrading protease, ADAMTS5, had reduced versican proteolysis, increased PCM, altered cell shape, enhanced α-smooth muscle actin (SMA) expression and increased contractility within three-dimensional collagen gels. The myofibroblast-like phenotype was associated with activation of TGFβ signaling. We tested the hypothesis that fibroblast-myofibroblast transition in Adamts5−/− cells resulted from versican accumulation in PCM. First, we noted that versican overexpression in human dermal fibroblasts led to increased SMA expression, enhanced contractility, and increased Smad2 phosphorylation. In contrast, dermal fibroblasts from Vcan haploinsufficient (Vcanhdf/+) mice had reduced contractility relative to wild type fibroblasts. Using a genetic approach to directly test if myofibroblast transition in Adamts5−/− cells resulted from increased PCM versican content, we generated Adamts5−/−;Vcanhdf/+ mice and isolated their dermal fibroblasts for comparison with dermal fibroblasts from Adamts5−/− mice. In Adamts5−/− fibroblasts, Vcan haploinsufficiency or exogenous ADAMTS5 restored normal fibroblast contractility. These findings demonstrate that altering PCM versican content through proteolytic activity of ADAMTS5 profoundly influenced the dermal fibroblast phenotype and may regulate a phenotypic continuum between the fibroblast and its alter ego, the myofibroblast. We propose that a physiological function of ADAMTS5 in dermal fibroblasts is to maintain optimal versican content and PCM volume by continually trimming versican in hyaluronan-versican aggregates. PMID:21828051

Mechanical signaling and the cellular response to extracellular matrix in angiogenesis and cardiovascular physiology

NASA Technical Reports Server (NTRS)

Ingber, Donald E.

2002-01-01

Great advances have been made in the identification of the soluble angiogenic factors, insoluble extracellular matrix (ECM) molecules, and receptor signaling pathways that mediate control of angiogenesis--the growth of blood capillaries. This review focuses on work that explores how endothelial cells integrate these chemical signals with mechanical cues from their local tissue microenvironment so as to produce functional capillary networks that exhibit specialized form as well as function. These studies have revealed that ECM governs whether an endothelial cell will switch between growth, differentiation, motility, or apoptosis programs in response to a soluble stimulus based on its ability to mechanically resist cell tractional forces and thereby produce cell and cytoskeletal distortion. Transmembrane integrin receptors play a key role in this mechanochemical transduction process because they both organize a cytoskeletal signaling complex within the focal adhesion and preferentially focus mechanical forces on this site. Molecular filaments within the internal cytoskeleton--microfilaments, microtubules, and intermediate filaments--also contribute to the cell's structural and functional response to mechanical stress through their role as discrete support elements within a tensegrity-stabilized cytoskeletal array. Importantly, a similar form of mechanical control also has been shown to be involved in the regulation of contractility in vascular smooth muscle cells and cardiac myocytes. Thus, the mechanism by which cells perform mechanochemical transduction and the implications of these findings for morphogenetic control are discussed in the wider context of vascular development and cardiovascular physiology.

Functional, structural, and chemical changes in myosin associated with hydrogen peroxide treatment of skeletal muscle fibers.

PubMed

Prochniewicz, Ewa; Lowe, Dawn A; Spakowicz, Daniel J; Higgins, LeeAnn; O'Conor, Kate; Thompson, LaDora V; Ferrington, Deborah A; Thomas, David D

2008-02-01

To understand the molecular mechanism of oxidation-induced inhibition of muscle contractility, we have studied the effects of hydrogen peroxide on permeabilized rabbit psoas muscle fibers, focusing on changes in myosin purified from these fibers. Oxidation by 5 mM peroxide decreased fiber contractility (isometric force and shortening velocity) without significant changes in the enzymatic activity of myofibrils and isolated myosin. The inhibitory effects were reversed by treating fibers with dithiothreitol. Oxidation by 50 mM peroxide had a more pronounced and irreversible inhibitory effect on fiber contractility and also affected enzymatic activity of myofibrils, myosin, and actomyosin. Peroxide treatment also affected regulation of contractility, resulting in fiber activation in the absence of calcium. Electron paramagnetic resonance of spin-labeled myosin in muscle fibers showed that oxidation increased the fraction of myosin heads in the strong-binding structural state under relaxing conditions (low calcium) but had no effect under activating conditions (high calcium). This change in the distribution of structural states of myosin provides a plausible explanation for the observed changes in both contractile and regulatory functions. Mass spectroscopy analysis showed that 50 mM but not 5 mM peroxide induced oxidative modifications in both isoforms of the essential light chains and in the heavy chain of myosin subfragment 1 by targeting multiple methionine residues. We conclude that 1) inhibition of muscle fiber contractility via oxidation of myosin occurs at high but not low concentrations of peroxide and 2) the inhibitory effects of oxidation suggest a critical and previously unknown role of methionines in myosin function.

Post-exercise contractility, diastolic function, and pressure: Operator-independent sensor-based intelligent monitoring for heart failure telemedicine

PubMed Central

Bombardini, Tonino; Gemignani, Vincenzo; Bianchini, Elisabetta; Pasanisi, Emilio; Pratali, Lorenza; Pianelli, Mascia; Faita, Francesco; Giannoni, Massimo; Arpesella, Giorgio; Sicari, Rosa; Picano, Eugenio

2009-01-01

Background New sensors for intelligent remote monitoring of the heart should be developed. Recently, a cutaneous force-frequency relation recording system has been validated based on heart sound amplitude and timing variations at increasing heart rates. Aim To assess sensor-based post-exercise contractility, diastolic function and pressure in normal and diseased hearts as a model of a wireless telemedicine system. Methods We enrolled 150 patients and 22 controls referred for exercise-stress echocardiography, age 55 ± 18 years. The sensor was attached in the precordial region by an ECG electrode. Stress and recovery contractility were derived by first heart sound amplitude vibration changes; diastolic times were acquired continuously. Systemic pressure changes were quantitatively documented by second heart sound recording. Results Interpretable sensor recordings were obtained in all patients (feasibility = 100%). Post-exercise contractility overshoot (defined as increase > 10% of recovery contractility vs exercise value) was more frequent in patients than controls (27% vs 8%, p < 0.05). At 100 bpm stress heart rate, systolic/diastolic time ratio (normal, < 1) was > 1 in 20 patients and in none of the controls (p < 0.01); at recovery systolic/diastolic ratio was > 1 in only 3 patients (p < 0.01 vs stress). Post-exercise reduced arterial pressure was sensed. Conclusion Post-exercise contractility, diastolic time and pressure changes can be continuously measured by a cutaneous sensor. Heart disease affects not only exercise systolic performance, but also post-exercise recovery, diastolic time intervals and blood pressure changes – in our study, all of these were monitored by a non-invasive wearable sensor. PMID:19442285

Pharmacological action of DA-9701 on the motility of feline stomach circular smooth muscle.

PubMed

Nguyen, Thanh Thao; Song, Hyun Ju; Ko, Sung Kwon; Sohn, Uy Dong

2015-03-01

DA-9701, a new prokinetic agent for the treatment of functional dyspepsia, is formulated with Pharbitis semen and Corydalis tuber. This study wasconducted to determine the pharmacological action of DA-9701 and to identify the receptors involved in DA-9701 -induced contractile responsesin the feline gastric corporal, fundic and antral circular smooth muscle. Concentration-response curve to DA-9701 was established. The tissue trips were exposed to methylsergide, ketanserin, ondansetron, GR 113808, atropine and dopamine before administration of DA-9701. The contractile force was determined before and after administration of drugs by a polygraph.DA-9701 enhanced the spontaneous contractile amplitude of antrum, corpus and fundus. However, it did not change the spontaneous contractile frequency of antrum and corpus, but concentration-dependently reduced that of fundus. In the fundus, DA-9701 -induced tonic contractions were inhibited by dopamine, methylsergide, ketanserine, ondansetron or GR 113808 respectively, but not by atropine, indicating that the contractile responses are mediated by multiple receptors: 5-HT2, 5-HT3, 5-HT4, and dopamine receptors. In the corpus, DA-9701-induced contractions were blocked by atropine, dopamine or GR 113808, but not by methysergide, ketanserin or ondansetron, indicating that they are involved in receptors on both, smooth muscles and neurons: 5-HT4 and dopamine receptors. However, contractile responses to DA-9701 are mainly mediated by dopamine receptors in the antrum. These results suggest that DA-9701 has important roles in gastric accommodation by enhancing tonic activity of fundus, and in gastric emptying and gastrointestinal transit by phasic contractions of corpus and antrum mediated by multiple receptors.

An Approach for Improvement of Carbon Fiber Technique to Study Cardiac Cell Contractility

NASA Astrophysics Data System (ADS)

Myachina, T.; Khokhlova, A.; Antsygin, I.; Lookin, O.

2018-05-01

The technologies to study cardiac cell mechanics in near-physiological conditions are limited. Carbon fiber (CF) technique is a unique tool to study single cardiomyocyte contractility. However, the CF adhesion to a cell is limited and it is difficult to control CF sliding occurred due to inappropriate adhesion. In this study, we present a CF adhesion quality index – a linear coefficient (slope) derived from “end-diastolic cell length - end-diastolic sarcomere length” relationship. Potential applicability of this index is demonstrated on isolated rat and guinea pig ventricular cardiomyocytes. Further improvement of the approach may help to increase the quality of the experimental data obtained by CF technique.

Establishing laboratory standards for biological flight experiments

NASA Technical Reports Server (NTRS)

Young, Ronald B.; Moriarity, Debra M.

1989-01-01

The general objective of this research was to assess the effects of exposure to simulated microgravity on ultrastructural aspects of the contractile system in chicken skeletal muscle cells. This general objective had two specific experimental components: (1) the progression of changes in cell morphology, fusion, and patterns of contractile filament organization in muscle cell cultures grown in hollow fibers in the Clinostat were evaluated, with appropriate controls; (2) to initiate experiments in which muscle cells were grown on the surface of microcarrier beads. The ultimate objective of this second portion of the work is to determine if these beads can be rotated in a bioreactor and thereby obtain a more accurate approximation of the effects of simulated microgravity on differentiated muscle cells.

[Effect of dopamine and its antagonists on contractile activity of the lower esophageal sphincter and the stomach (author's transl)].

PubMed

Itoh, Z; Aizawa, I; Nakamura, T

1980-06-01

Effect of dopamine and its antagonists, domperidone and metoclopramide (MCP), on contractile activity of the lower esophageal sphincter (LES) and the stomach was studied in 5 healthy conscious dogs. Contractile activity was measured by means of chronically implanted force transducers. Contractile activity of the LES and the stomach was completely inhibited by an intravenous infusion of dopamine (10, 20 and 40 micrograms/kg-min) during the digestive and interdigestive state. Domperidone, when administered alone (0.5, 1.0 and 2.0 mg/kg), had no effect on contractile activity of the LES and the stomach during the both periods. Though deprived of any noticeable effect on the digestive contractions, MCP (0.25, 0.5 and 1.0 mg/kg) abolished the interdigestive contractions and produced characteristic contractions. Domperidone restored postprandial and interdigestive contractions to their initial stage before dopamine administration in a dose-related fashion. Dopamine-induced inhibition was antagonized by MCP during the digestive state, however, MCP had no effect on the interdigestive contractions that had been inhibited by dopamine. Since domperidone has no activity upon normal contractions of the gastrointestinal tract, it may be assumed that if domperidone alone has some influence upon gut motor activity or any improvement in clinical symptoms is seen after domperidone, a disorder of the dopaminergic system could be strongly suggested.

Cellular Motility--Experiments on Contractile and Motile Mechanisms in the Slime Mould, Physarum Polycephalum

ERIC Educational Resources Information Center

Holmes, R. P.; Stewart, P. R.

1977-01-01

Actin and myosin have now been demonstrated to be important constituents of many eukaryotic cells. Their role is primarily that of a contractile system underlying all aspects of cellular motility. Described here is a simple experimental system to demonstrate quantitatively aspects of motility and its regulation in a slime mold. (Author/MA)

Fatigability and Blood Flow in the Rat Gastrocnemius-Plantaris-Soleus after Hindlimb Suspension

NASA Technical Reports Server (NTRS)

McDonald, K. S.; Delp, M. D.; Fitts, R. H.

1992-01-01

The purpose of this study was to test the hypothesis that hindlimb suspension increases the fatigability of the soleus during intense contractile activity and determine whether the increased fatigue is associated with a reduced muscle blood flow. Cage-control (C) and 15-day hindlimb-suspended (HS) rats were anesthetized, and either the gastrocnemius-plantaris-soleus (G-P-S) muscle group or the soleus was stimulated (100 Hz, 100-ms trains at 120/min) for 10 min in situ. In the G-P-S preparation, blood flow was measured with radiolabeled microspheres before and at 2 and 10 min of contractile activity. The G-P-S fatigued markedly at this stimulation frequency, and the differences between C and HS animals were not significant until the 9th min of contractile activity. In contrast, the stimulation resulted in faster rates and significantly larger amounts of fatigue in the soleus from HS than from C animals. The atrophied soleus showed significant differences by I min of stimulation (C = 70 +/- 1% vs. HS = 57 +/- 2% of peak train force) and remained different at 10 min (C = 64 +/- 4% vs. HS = 45 +/- 2% peak train force). Relative blood flow to the soleus was similar between groups before and during contractile activity (rest: C = 20 +/- 3 vs. HS= 12 +/- 3; 2 min: C= 128 +/- 6 vs. HS = 118 +/- 4; 10 min: C = 123 +/- 11 vs. HS = 105 +/- 11 ml min(exp -1) 100 g(exp -1)). In conclusion, these results established that 15 days of HS increased the fatigability of the soleus, but the effect was not caused by a reduced muscle blood flow.

Fiber-type-specific sensitivities and phenotypic adaptations to dietary fat overload differentially impact fast- versus slow-twitch muscle contractile function in C57BL/6J mice.

PubMed

Ciapaite, Jolita; van den Berg, Sjoerd A; Houten, Sander M; Nicolay, Klaas; van Dijk, Ko Willems; Jeneson, Jeroen A

2015-02-01

High-fat diets (HFDs) have been shown to interfere with skeletal muscle energy metabolism and cause peripheral insulin resistance. However, understanding of HFD impact on skeletal muscle primary function, i.e., contractile performance, is limited. Male C57BL/6J mice were fed HFD containing lard (HFL) or palm oil (HFP), or low-fat diet (LFD) for 5weeks. Fast-twitch (FT) extensor digitorum longus (EDL) and slow-twitch (ST) soleus muscles were characterized with respect to contractile function and selected biochemical features. In FT EDL muscle, a 30%-50% increase in fatty acid (FA) content and doubling of long-chain acylcarnitine (C14-C18) content in response to HFL and HFP feeding were accompanied by increase in protein levels of peroxisome proliferator-activated receptor-γ coactivator-1α, mitochondrial oxidative phosphorylation complexes and acyl-CoA dehydrogenases involved in mitochondrial FA β-oxidation. Peak force of FT EDL twitch and tetanic contractions was unaltered, but the relaxation time (RT) of twitch contractions was 30% slower compared to LFD controls. The latter was caused by accumulation of lipid intermediates rather than changes in the expression levels of proteins involved in calcium handling. In ST soleus muscle, no evidence for lipid overload was found in any HFD group. However, particularly in HFP group, the peak force of twitch and tetanic contractions was reduced, but RT was faster than LFD controls. The latter was associated with a fast-to-slow shift in troponin T isoform expression. Taken together, these data highlight fiber-type-specific sensitivities and phenotypic adaptations to dietary lipid overload that differentially impact fast- versus slow-twitch skeletal muscle contractile function. Copyright © 2015 Elsevier Inc. All rights reserved.

From damage response to action potentials: early evolution of neural and contractile modules in stem eukaryotes.

PubMed

Brunet, Thibaut; Arendt, Detlev

2016-01-05

Eukaryotic cells convert external stimuli into membrane depolarization, which in turn triggers effector responses such as secretion and contraction. Here, we put forward an evolutionary hypothesis for the origin of the depolarization-contraction-secretion (DCS) coupling, the functional core of animal neuromuscular circuits. We propose that DCS coupling evolved in unicellular stem eukaryotes as part of an 'emergency response' to calcium influx upon membrane rupture. We detail how this initial response was subsequently modified into an ancient mechanosensory-effector arc, present in the last eukaryotic common ancestor, which enabled contractile amoeboid movement that is widespread in extant eukaryotes. Elaborating on calcium-triggered membrane depolarization, we reason that the first action potentials evolved alongside the membrane of sensory-motile cilia, with the first voltage-sensitive sodium/calcium channels (Nav/Cav) enabling a fast and coordinated response of the entire cilium to mechanosensory stimuli. From the cilium, action potentials then spread across the entire cell, enabling global cellular responses such as concerted contraction in several independent eukaryote lineages. In animals, this process led to the invention of mechanosensory contractile cells. These gave rise to mechanosensory receptor cells, neurons and muscle cells by division of labour and can be regarded as the founder cell type of the nervous system. © 2015 The Authors.

From damage response to action potentials: early evolution of neural and contractile modules in stem eukaryotes

PubMed Central

Brunet, Thibaut; Arendt, Detlev

2016-01-01

Eukaryotic cells convert external stimuli into membrane depolarization, which in turn triggers effector responses such as secretion and contraction. Here, we put forward an evolutionary hypothesis for the origin of the depolarization–contraction–secretion (DCS) coupling, the functional core of animal neuromuscular circuits. We propose that DCS coupling evolved in unicellular stem eukaryotes as part of an ‘emergency response’ to calcium influx upon membrane rupture. We detail how this initial response was subsequently modified into an ancient mechanosensory–effector arc, present in the last eukaryotic common ancestor, which enabled contractile amoeboid movement that is widespread in extant eukaryotes. Elaborating on calcium-triggered membrane depolarization, we reason that the first action potentials evolved alongside the membrane of sensory-motile cilia, with the first voltage-sensitive sodium/calcium channels (Nav/Cav) enabling a fast and coordinated response of the entire cilium to mechanosensory stimuli. From the cilium, action potentials then spread across the entire cell, enabling global cellular responses such as concerted contraction in several independent eukaryote lineages. In animals, this process led to the invention of mechanosensory contractile cells. These gave rise to mechanosensory receptor cells, neurons and muscle cells by division of labour and can be regarded as the founder cell type of the nervous system. PMID:26598726

Dynamics of myosin II organization into contractile networks and fibers at the medial cell cortex

NASA Astrophysics Data System (ADS)

Nie, Wei

The cellular morphology of adhered cells depends crucially on the formation of a contractile meshwork of parallel and cross-linked stress fibers along the contacting surface. The motor activity and mini-filament assembly of non-muscle myosin II is an important component of cell-level cytoskeletal remodeling during mechanosensing. To monitor the dynamics of non-muscle myosin II, we used confocal microscopy to image cultured HeLa cells that stably express myosin regulatory light chain tagged with GFP (MRLC-GFP). MRLC-GFP was monitored in time-lapse movies at steady state and during the response of cells to varying concentrations of blebbistatin (which disrupts actomyosin stress fibers). Using image correlation spectroscopy analysis, we quantified the kinetics of disassembly and reassembly of actomyosin networks and compared to studies by other groups. This analysis suggested the following processes: myosin minifilament assembly and disassembly; aligning and contraction; myosin filament stabilization upon increasing contractile tension. Numerical simulations that include those processes capture some of the main features observed in the experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness. We discuss methods to monitor myosin reorganization using non-linear imaging methods.

Effects of stress fiber contractility on uniaxial stretch guiding mitosis orientation and stress fiber alignment.

PubMed

Zhao, Lei; Sang, Chen; Yang, Chun; Zhuang, Fengyuan

2011-09-02

It has been documented that mitosis orientation (MO) is guided by stress fibers (SFs), which are perpendicular to exogenous cyclic uniaxial stretch. However, the effect of mechanical forces on MO and the mechanism of stretch-induced SFs reorientation are not well elucidated to date. In the present study, we used murine 3T3 fibroblasts as a model, to investigate the effects of uniaxial stretch on SFO and MO utilizing custom-made stretch device. We found that cyclic uniaxial stretch induced both SFs and mitosis directions orienting perpendicularly to the stretch direction. The F-actin and myosin II blockages, which resulted in disoriented SFs and mitosis directions under uniaxial stretch, suggested a high correlation between SFO and MO. Y27632 (10 μM), ML7 (50 μM, or 75 μM), and blebbistatin (50 μM, or 75 μM) treatments resulted in SFO parallel to the principle stretch direction. Upon stimulating and inhibiting the phosphorylation of myosin light chain (p-MLC), we observed a monotonic proportion of SFO to the level of p-MLC. These results suggested that the level of cell contraction is crucial to the response of SFs, either perpendicular or parallel, to the external stretch. Showing the possible role of cell contractility in tuning SFO under external stretch, our experimental data are valuable to understand the predominant factor controlling SFO response to exogenous uniaxial stretch, and thus helpful for improving mechanical models. Copyright © 2011 Elsevier Ltd. All rights reserved.

Transcriptional and Hormonal Regulation of Gravitropism of Woody Stems in Populus.

PubMed

Gerttula, Suzanne; Zinkgraf, Matthew; Muday, Gloria K; Lewis, Daniel R; Ibatullin, Farid M; Brumer, Harry; Hart, Foster; Mansfield, Shawn D; Filkov, Vladimir; Groover, Andrew

2015-10-01

Angiosperm trees reorient their woody stems by asymmetrically producing a specialized xylem tissue, tension wood, which exerts a strong contractile force resulting in negative gravitropism of the stem. Here, we show, in Populus trees, that initial gravity perception and response occurs in specialized cells through sedimentation of starch-filled amyloplasts and relocalization of the auxin transport protein, PIN3. Gibberellic acid treatment stimulates the rate of tension wood formation and gravibending and enhances tissue-specific expression of an auxin-responsive reporter. Gravibending, maturation of contractile fibers, and gibberellic acid (GA) stimulation of tension wood formation are all sensitive to transcript levels of the Class I KNOX homeodomain transcription factor-encoding gene ARBORKNOX2 (ARK2). We generated genome-wide transcriptomes for trees in which gene expression was perturbed by gravistimulation, GA treatment, and modulation of ARK2 expression. These data were employed in computational analyses to model the transcriptional networks underlying wood formation, including identification and dissection of gene coexpression modules associated with wood phenotypes, GA response, and ARK2 binding to genes within modules. We propose a model for gravitropism in the woody stem in which the peripheral location of PIN3-expressing cells relative to the cambium results in auxin transport toward the cambium in the top of the stem, triggering tension wood formation, while transport away from the cambium in the bottom of the stem triggers opposite wood formation. © 2015 American Society of Plant Biologists. All rights reserved.

Morphology and contractile gene expression of adipose-derived mesenchymal stem cells in response to short-term cyclic uniaxial strain and TGF-β1.

PubMed

Rashidi, Neda; Tafazzoli-Shadpour, Mohammad; Haghighipour, Nooshin; Khani, Mohammad-Mehdi

2018-06-27

Previous studies have shown smooth muscle induction in adipose-derived mesenchymal stem cells (ASCs) caused by long-term cyclic stretch. Here we examined the capability of the short-term straining with time steps of 4, 8, 16 and 24 h alone or combined with TGF-β1 on smooth muscle induction of rabbit ASCs. Alterations in cell morphology were quantified through the cell shape index and orientation angle, and expression levels of α-SMA, SM22-α, h-caldesmon and calponin3 markers were examined using the real-time polymerase chain reaction (PCR) method. Moreover, F-actin cytoskeleton organization was observed by fluorescence staining. Mechanical strain either alone or combined with growth factor treatment caused significant up-regulation of both early and intermediate smooth muscle cells (SMCs) specific markers during the initial hours of stimulation peaking in 8 to 16 h. Furthermore, gradual alignment of cells perpendicular to the strain direction during loading time, and cell elongation resembling contractile SMC phenotype, together with alignment and reorganization of F-actin fibers were observed. Considering previously reported protein up-regulation in following days of straining, the effects of short-term cyclic stretch on smooth muscle induction of ASCs were revealed which can be helpful in achieving functional contractile SMCs through synergistic mechano-chemical regulation of ASCs as an appealing cell source for vascular tissue engineering.

Contractile ring stability in S. pombe depends on F-BAR protein Cdc15p and Bgs1p transport from the Golgi complex.

PubMed

Arasada, Rajesh; Pollard, Thomas D

2014-09-11

Cdc15p is known to contribute to cytokinesis in fission yeast; however, the protein is not required to assemble the contractile ring of actin and myosin, but it helps to anchor the ring to the plasma membrane. Cdc15p has a lipid-binding F-BAR domain, suggesting that it provides a physical link between the plasma membrane and contractile ring proteins. However, we find that a more important function of Cdc15p during cytokinesis is to help deliver a transmembrane enzyme, Bgs1p (also called Cps1p), from the Golgi apparatus to the plasma membrane, where it appears to anchor the contractile ring. Bgs1p synthesizes the cell wall in the cleavage furrow, but its enzyme activity is not required to anchor the contractile ring. We estimate that ∼ 2,000 Bgs1p molecules are required to anchor the ring. Without Bgs1p anchors, contractile rings slide along the plasma membrane, a phenomenon that depends on an unconventional type II myosin called Myp2p. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

The effects of space flight on the contractile apparatus of antigravity muscles: implications for aging and deconditioning.

PubMed

Baldwin, K M; Caiozzo, V J; Haddad, F; Baker, M J; Herrick, R E

1994-05-01

Previous studies have shown that the unloading of skeletal muscle, as occurring during exposure to space flight, exerts a profound effect on both the mass (cross sectional area) of skeletal muscle fibers and the relative expression of protein isoforms comprising the contractile system. Available information suggests that slow (type I) fibers, comprising chiefly the antigravity muscles of experimental animals, in addition to atrophying, undergo alterations in the type of myosin heavy chain (MHC) expressed such that faster isoforms become concomitantly expressed in a sub-population of slow fibers when insufficient force-bearing activity is maintained on the muscle. Consequently, these transformations in both mass and myosin heavy chain phenotype could exert a significant impact on the functional properties of skeletal muscle as manifest in the strength, contractile speed, and endurance scope of the muscle. To further explore these issues, a study was performed in which young adult male rats were exposed to zero gravity for six days, following which, the antigravity soleus muscle was examined for a) contractile properties, determined in situ and b) isomyosin expression, as studied using biochemical, molecular biology, and histochemical/immunohistochemical techniques.

The effects of space flight on the contractile apparatus of antigravity muscles: implications for aging and deconditioning

NASA Technical Reports Server (NTRS)

Baldwin, K. M.; Caiozzo, V. J.; Haddad, F.; Baker, M. J.; Herrick, R. E.

1994-01-01

Previous studies have shown that the unloading of skeletal muscle, as occurring during exposure to space flight, exerts a profound effect on both the mass (cross sectional area) of skeletal muscle fibers and the relative expression of protein isoforms comprising the contractile system. Available information suggests that slow (type I) fibers, comprising chiefly the antigravity muscles of experimental animals, in addition to atrophying, undergo alterations in the type of myosin heavy chain (MHC) expressed such that faster isoforms become concomitantly expressed in a sub-population of slow fibers when insufficient force-bearing activity is maintained on the muscle. Consequently, these transformations in both mass and myosin heavy chain phenotype could exert a significant impact on the functional properties of skeletal muscle as manifest in the strength, contractile speed, and endurance scope of the muscle. To further explore these issues, a study was performed in which young adult male rats were exposed to zero gravity for six days, following which, the antigravity soleus muscle was examined for a) contractile properties, determined in situ and b) isomyosin expression, as studied using biochemical, molecular biology, and histochemical/immunohistochemical techniques.

TNFα enhances force generation in airway smooth muscle

PubMed Central

Han, Young-Soo; Delmotte, Philippe

2017-01-01

Airway inflammation is a hallmark of asthma, triggering airway smooth muscle (ASM) hyperreactivity and airway remodeling. TNFα increases both agonist-induced cytosolic Ca2+ concentration ([Ca2+]cyt) and force in ASM. The effects of TNFα on ASM force may also be due to an increase in Ca2+ sensitivity, cytoskeletal remodeling, and/or changes in contractile protein content. We hypothesized that 24 h of exposure to TNFα increases ASM force by changing actin and myosin heavy chain (MyHC) content and/or polymerization. Porcine ASM strips were permeabilized with 10% Triton X-100, and force was measured in response to increasing concentrations of Ca2+ (pCa 9.0 to 4.0) in control and TNFα-treated groups. Relative phosphorylation of the regulatory myosin light chain (p-MLC) and total actin, MLC, and MyHC concentrations were quantified at pCa 9.0, 6.1, and 4.0. Actin polymerization was quantified by the ratio of filamentous to globular actin at pCa 9.0 and 4.0. For determination of total cross-bridge formation, isometric ATP hydrolysis rate at pCa 4.0 was measured using an enzyme-coupled NADH-linked fluorometric technique. Exposure to TNFα significantly increased force across the range of Ca2+ activation but did not affect the intrinsic Ca2+ sensitivity of force generation. The TNFα-induced increase in ASM force was associated with an increase in total actin, MLC, and MyHC content, as well as an increase in actin polymerization and an increase in maximum isometric ATP hydrolysis rate. The results of this study support our hypothesis that TNFα increases force generation in ASM by increasing the number of contractile units (actin-myosin content) contributing to force generation. PMID:28385814

The pathophysiological roles of COX-1 and COX-2 in the intestinal smooth muscle contractility under the anaphylactic condition.

PubMed

Kadowaki, Hiroko; Yamamoto, Takeshi; Kageyama-Yahara, Natsuko; Kurokawa, Nobuo; Kadowaki, Makoto

2008-04-01

Various inflammatory mediators released from antigen-activated mast cells are considered to play a key role in the pathogenesis of food allergy. The aim of the present study was to determine the mechanisms underlying the antigen-induced anaphylactic responses in the rat colons. Wistar rats were sensitized by intraperitoneal injection of ovalbumin (OVA). The contractilities of isolated proximal colons of the sensitized rats were studied in the organ bath. OVA challenges of sensitized tissues induced prolonged contractile responses. The antigen-induced contractions were greatly reduced by mast cell stabilizer doxantrazole (10 microM). However, the contractions were resistant to histamine H1 receptor antagonist and prostaglandin D2 receptor antagonist. In contrast, non-selective cyclooxygenase (COX) inhibitor indomethacin (1 microM) significantly reduced the contractions by 61.0%. Furthermore, selective COX-1 inhibitor FR122047 (10 microM) as well as selective COX-2 inhibitor NS-398 (10 microM) significantly inhibited the contractions by 50.1% and 50.3%, respectively. Nevertheless, the transcript levels of COX-2 as well as COX-1 were not upregulated by OVA in the proximal colons of the sensitized rats. The present results indicate that de novo arachidonic acid metabolites synthesis by constitutive COX-1 as well as constitutive COX-2 within mast cells contribute to the altered smooth muscle contractilities in the colons under the anaphylactic condition.

Bone Morphogenetic Protein 4 Promotes Vascular Smooth Muscle Contractility by Activating MicroRNA-21 (miR-21), which Down-regulates Expression of Family of Dedicator of Cytokinesis (DOCK) Proteins*

PubMed Central

Kang, Hara; Davis-Dusenbery, Brandi N.; Nguyen, Peter H.; Lal, Ashish; Lieberman, Judy; Van Aelst, Linda; Lagna, Giorgio; Hata, Akiko

2012-01-01

The bone morphogenetic protein 4 (BMP4) signaling pathway plays a critical role in the promotion and maintenance of the contractile phenotype in vascular smooth muscle cell (vSMC). Misexpression or inactivating mutations of the BMP receptor gene can lead to dedifferentiation of vSMC characterized by increased migration and proliferation that is linked to vascular proliferative disorders. Previously we demonstrated that vSMCs increase microRNA-21 (miR-21) biogenesis upon BMP4 treatment, which induces contractile gene expression by targeting programmed cell death 4 (PDCD4). To identify novel targets of miR-21 that are critical for induction of the contractile phenotype by BMP4, biotinylated miR-21 was expressed in vSMCs followed by an affinity purification of mRNAs associated with miR-21. Nearly all members of the dedicator of cytokinesis (DOCK) 180-related protein superfamily were identified as targets of miR-21. Down-regulation of DOCK4, -5, and -7 by miR-21 inhibited cell migration and promoted cytoskeletal organization by modulating an activity of small GTPase. Thus, this study uncovers a regulatory mechanism of the vSMC phenotype by the BMP4-miR-21 axis through DOCK family proteins. PMID:22158624

Influence of ovarian muscle contraction and oocyte growth on egg chamber elongation in Drosophila.

PubMed

Andersen, Darcy; Horne-Badovinac, Sally

2016-04-15

Organs are formed from multiple cell types that make distinct contributions to their shape. The Drosophila egg chamber provides a tractable model to dissect such contributions during morphogenesis. Egg chambers consist of 16 germ cells (GCs) surrounded by a somatic epithelium. Initially spherical, these structures elongate as they mature. This morphogenesis is thought to occur through a 'molecular corset' mechanism, whereby structural elements within the epithelium become circumferentially organized perpendicular to the elongation axis and resist the expansive growth of the GCs to promote elongation. Whether this epithelial organization provides the hypothesized constraining force has been difficult to discern, however, and a role for GC growth has not been demonstrated. Here, we provide evidence for this mechanism by altering the contractile activity of the tubular muscle sheath that surrounds developing egg chambers. Muscle hypo-contraction indirectly reduces GC growth and shortens the egg, which demonstrates the necessity of GC growth for elongation. Conversely, muscle hyper-contraction enhances the elongation program. Although this is an abnormal function for this muscle, this observation suggests that a corset-like force from the egg chamber's exterior could promote its lengthening. These findings highlight how physical contributions from several cell types are integrated to shape an organ. © 2016. Published by The Company of Biologists Ltd.

Influence of ovarian muscle contraction and oocyte growth on egg chamber elongation in Drosophila

PubMed Central

Andersen, Darcy; Horne-Badovinac, Sally

2016-01-01

Organs are formed from multiple cell types that make distinct contributions to their shape. The Drosophila egg chamber provides a tractable model to dissect such contributions during morphogenesis. Egg chambers consist of 16 germ cells (GCs) surrounded by a somatic epithelium. Initially spherical, these structures elongate as they mature. This morphogenesis is thought to occur through a ‘molecular corset’ mechanism, whereby structural elements within the epithelium become circumferentially organized perpendicular to the elongation axis and resist the expansive growth of the GCs to promote elongation. Whether this epithelial organization provides the hypothesized constraining force has been difficult to discern, however, and a role for GC growth has not been demonstrated. Here, we provide evidence for this mechanism by altering the contractile activity of the tubular muscle sheath that surrounds developing egg chambers. Muscle hypo-contraction indirectly reduces GC growth and shortens the egg, which demonstrates the necessity of GC growth for elongation. Conversely, muscle hyper-contraction enhances the elongation program. Although this is an abnormal function for this muscle, this observation suggests that a corset-like force from the egg chamber's exterior could promote its lengthening. These findings highlight how physical contributions from several cell types are integrated to shape an organ. PMID:26952985

Contractile reserve and calcium regulation are depressed in myocytes from chronically unloaded hearts

NASA Technical Reports Server (NTRS)

Ito, Kenta; Nakayama, Masaharu; Hasan, Faisal; Yan, Xinhua; Schneider, Michael D.; Lorell, Beverly H.

2003-01-01

BACKGROUND: Chronic cardiac unloading of the normal heart results in the reduction of left ventricular (LV) mass, but effects on myocyte contractile function are not known. METHODS AND RESULTS: Cardiac unloading and reduction in LV mass were induced by heterotopic heart transplantation to the abdominal aorta in isogenic rats. Contractility and [Ca(2+)](i) regulation in LV myocytes were studied at both 2 and 5 weeks after transplantation. Native in situ hearts from recipient animals were used as the controls for all experiments. Contractile function indices in myocytes from 2-week unloaded and native (control) hearts were similar under baseline conditions (0.5 Hz, 1.2 mmol/L [Ca(2+)](o), and 36 degrees C) and in response to stimulation with high [Ca(2+)](o) (range 2.5 to 4.0 mmol/L). In myocytes from 5-week unloaded hearts, there were no differences in fractional cell shortening and peak-systolic [Ca(2+)](i) at baseline; however, time to 50% relengthening and time to 50% decline in [Ca(2+)](i) were prolonged compared with controls. Severe defects in fractional cell shortening and peak-systolic [Ca(2+)](i) were elicited in myocytes from 5-week unloaded hearts in response to high [Ca(2+)](o). However, there were no differences in the contractile response to isoproterenol between myocytes from unloaded and native hearts. In 5-week unloaded hearts, but not in 2-week unloaded hearts, LV protein levels of phospholamban were increased (345% of native heart values). Protein levels of sarcoplasmic reticulum Ca(2+) ATPase and the Na(+)/Ca(2+) exchanger were not changed. CONCLUSIONS: Chronic unloading of the normal heart caused a time-dependent depression of myocyte contractile function, suggesting the potential for impaired performance in states associated with prolonged cardiac atrophy.

Muscle Contraction.

PubMed

Sweeney, H Lee; Hammers, David W

2018-02-01

SUMMARYMuscle cells are designed to generate force and movement. There are three types of mammalian muscles-skeletal, cardiac, and smooth. Skeletal muscles are attached to bones and move them relative to each other. Cardiac muscle comprises the heart, which pumps blood through the vasculature. Skeletal and cardiac muscles are known as striated muscles, because the filaments of actin and myosin that power their contraction are organized into repeating arrays, called sarcomeres, that have a striated microscopic appearance. Smooth muscle does not contain sarcomeres but uses the contraction of filaments of actin and myosin to constrict blood vessels and move the contents of hollow organs in the body. Here, we review the principal molecular organization of the three types of muscle and their contractile regulation through signaling mechanisms and discuss their major structural and functional similarities that hint at the possible evolutionary relationships between the cell types. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Force-dependent breaching of the basement membrane.

PubMed

Chang, Tammy T; Thakar, Dhruv; Weaver, Valerie M

2017-01-01

Clinically, non-invasive carcinomas are confined to the epithelial side of the basement membrane and are classified as benign, whereas invasive cancers invade through the basement membrane and thereby acquire the potential to metastasize. Recent findings suggest that, in addition to protease-mediated degradation and chemotaxis-stimulated migration, basement membrane invasion by malignant cells is significantly influenced by the stiffness of the associated interstitial extracellular matrix and the contractility of the tumor cells that is dictated in part by their oncogenic genotype. In this review, we highlight recent findings that illustrate unifying molecular mechanisms whereby these physical cues contribute to tissue fibrosis and malignancy in three epithelial organs: breast, pancreas, and liver. We also discuss the clinical implications of these findings and the biological properties and clinical challenges linked to the unique biology of each of these organs. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

Targeted killing of myofibroblasts by biosurfactant di-rhamnolipid suggests a therapy against scar formation

PubMed Central

Shen, Chong; Jiang, Lifang; Shao, Huawei; You, Chuangang; Zhang, Guoliang; Ding, Sitong; Bian, Tingwei; Han, Chunmao; Meng, Qin

2016-01-01

Pathological myofibroblasts are often involved in skin scarring via generating contractile force and over-expressing collagen fibers, but no compound has been found to inhibit the myofibroblasts without showing severe toxicity to surrounding physiological cells. Here we report that di-rhamnolipid, a biosurfactant secreted by Pseudomonas aeruginosa, showed potent effects on scar therapy via a unique mechanism of targeted killing the myofibroblasts. In cell culture, the fibroblasts-derived myofibroblasts were more sensitive to di-rhamnolipid toxicity than fibroblasts at a concentration-dependent manner, and could be completely inhibited of their specific functions including α-SMA expression and collagen secretion/contraction. The anti-fibrotic function of di-rhamnolipid was further verified in rabbit ear hypertrophic scar models by presenting the significant reduction of scar elevation index, type I collagen fibers and α-SMA expression. In this regard, di-rhamnolipid treatment could be suggested as a therapy against skin scarring. PMID:27901027

New Insights into the Roles of Acidocalcisomes and the Contractile Vacuole Complex in Osmoregulation in Protists

PubMed Central

Docampo, Roberto; Jimenez, Veronica; Lander, Noelia; Li, Zhu-Hong; Niyogi, Sayantanee

2013-01-01

While free-living protists are usually subjected to hyposmotic environments, parasitic protists are also in contact with hyperosmotic habitats. Recent work in one of these parasites, Trypanosoma cruzi, has revealed that its contractile vacuole complex, which usually collects and expels excess water as a mechanism of regulatory volume decrease after hyposmotic stress, has also a role in cell shrinking when the cells are submitted to hyperosmotic stress. Trypanosomes also have an acidic calcium store rich in polyphosphate (polyP), named the acidocalcisome, which is involved in their response to osmotic stress. Here, we review newly emerging insights on the role of acidocalcisomes and the contractile vacuole complex in the cellular response to hyposmotic and hyperosmotic stresses. We also review the current state of knowledge on the composition of these organelles and their other roles in calcium homeostasis and protein trafficking. PMID:23890380

New insights into roles of acidocalcisomes and contractile vacuole complex in osmoregulation in protists.

PubMed

Docampo, Roberto; Jimenez, Veronica; Lander, Noelia; Li, Zhu-Hong; Niyogi, Sayantanee

2013-01-01

While free-living protists are usually subjected to hyposmotic environments, parasitic protists are also in contact with hyperosmotic habitats. Recent work in one of these parasites, Trypanosoma cruzi, has revealed that its contractile vacuole complex, which usually collects and expels excess water as a mechanism of regulatory volume decrease after hyposmotic stress, has also a role in cell shrinking when the cells are submitted to hyperosmotic stress. Trypanosomes also have an acidic calcium store rich in polyphosphate (polyP), named the acidocalcisome, which is involved in their response to osmotic stress. Here, we review newly emerging insights on the role of acidocalcisomes and the contractile vacuole complex in the cellular response to hyposmotic and hyperosmotic stresses. We also review the current state of knowledge on the composition of these organelles and their other roles in calcium homeostasis and protein trafficking. © 2013, Elsevier Inc. All Rights Reserved.

Differential basal-to-apical accessibility of lamin A/C epitopes in the nuclear lamina regulated by changes in cytoskeletal tension.

PubMed

Ihalainen, Teemu O; Aires, Lina; Herzog, Florian A; Schwartlander, Ruth; Moeller, Jens; Vogel, Viola

2015-12-01

Nuclear lamins play central roles at the intersection between cytoplasmic signalling and nuclear events. Here, we show that at least two N- and C-terminal lamin epitopes are not accessible at the basal side of the nuclear envelope under environmental conditions known to upregulate cell contractility. The conformational epitope on the Ig-domain of A-type lamins is more buried in the basal than apical nuclear envelope of human mesenchymal stem cells undergoing osteogenesis (but not adipogenesis), and in fibroblasts adhering to rigid (but not soft) polyacrylamide hydrogels. This structural polarization of the lamina is promoted by compressive forces, emerges during cell spreading, and requires lamin A/C multimerization, intact nucleoskeleton-cytoskeleton linkages (LINC), and apical-actin stress-fibre assembly. Notably, the identified Ig-epitope overlaps with emerin, DNA and histone binding sites, and comprises various laminopathy mutation sites. Our findings should help decipher how the physical properties of cellular microenvironments regulate nuclear events.

Differential basal-to-apical accessibility of lamin A/C epitopes in the nuclear lamina regulated by changes in cytoskeletal tension

NASA Astrophysics Data System (ADS)

Ihalainen, Teemu O.; Aires, Lina; Herzog, Florian A.; Schwartlander, Ruth; Moeller, Jens; Vogel, Viola

2015-12-01

Nuclear lamins play central roles at the intersection between cytoplasmic signalling and nuclear events. Here, we show that at least two N- and C-terminal lamin epitopes are not accessible at the basal side of the nuclear envelope under environmental conditions known to upregulate cell contractility. The conformational epitope on the Ig-domain of A-type lamins is more buried in the basal than apical nuclear envelope of human mesenchymal stem cells undergoing osteogenesis (but not adipogenesis), and in fibroblasts adhering to rigid (but not soft) polyacrylamide hydrogels. This structural polarization of the lamina is promoted by compressive forces, emerges during cell spreading, and requires lamin A/C multimerization, intact nucleoskeleton-cytoskeleton linkages (LINC), and apical-actin stress-fibre assembly. Notably, the identified Ig-epitope overlaps with emerin, DNA and histone binding sites, and comprises various laminopathy mutation sites. Our findings should help decipher how the physical properties of cellular microenvironments regulate nuclear events.

β2-Adrenergic stimulation enhances Ca2+ release and contractile properties of skeletal muscles, and counteracts exercise-induced reductions in Na+–K+-ATPase Vmax in trained men

PubMed Central

Hostrup, M; Kalsen, A; Ørtenblad, N; Juel, C; Mørch, K; Rzeppa, S; Karlsson, S; Backer, V; Bangsbo, J

2014-01-01

The aim of the present study was to examine the effect of β2-adrenergic stimulation on skeletal muscle contractile properties, sarcoplasmic reticulum (SR) rates of Ca2+ release and uptake, and Na+–K+-ATPase activity before and after fatiguing exercise in trained men. The study consisted of two experiments (EXP1, n = 10 males, EXP2, n = 20 males), where β2-adrenoceptor agonist (terbutaline) or placebo was randomly administered in double-blinded crossover designs. In EXP1, maximal voluntary isometric contraction (MVC) of m. quadriceps was measured, followed by exercise to fatigue at 120% of maximal oxygen uptake (). A muscle biopsy was taken after MVC (non-fatigue) and at time of fatigue. In EXP2, contractile properties of m. quadriceps were measured with electrical stimulations before (non-fatigue) and after two fatiguing 45 s sprints. Non-fatigued MVCs were 6 ± 3 and 6 ± 2% higher (P < 0.05) with terbutaline than placebo in EXP1 and EXP2, respectively. Furthermore, peak twitch force was 11 ± 7% higher (P < 0.01) with terbutaline than placebo at non-fatigue. After sprints, MVC declined (P < 0.05) to the same levels with terbutaline as placebo, whereas peak twitch force was lower (P < 0.05) and half-relaxation time was prolonged (P < 0.05) with terbutaline. Rates of SR Ca2+ release and uptake at 400 nm [Ca2+] were 15 ± 5 and 14 ± 5% (P < 0.05) higher, respectively, with terbutaline than placebo at non-fatigue, but declined (P < 0.05) to similar levels at time of fatigue. Na+–K+-ATPase activity was unaffected by terbutaline compared with placebo at non-fatigue, but terbutaline counteracted exercise-induced reductions in maximum rate of activity (Vmax) at time of fatigue. In conclusion, increased contractile force induced by β2-adrenergic stimulation is associated with enhanced rate of Ca2+ release in humans. While β2-adrenergic stimulation elicits positive inotropic and lusitropic effects on non-fatigued m. quadriceps, these effects are blunted when muscles fatigue. PMID:25344552

Orientation and length of mammalian skeletal myocytes in response to a unidirectional stretch

NASA Technical Reports Server (NTRS)

Collinsworth, A. M.; Torgan, C. E.; Nagda, S. N.; Rajalingam, R. J.; Kraus, W. E.; Truskey, G. A.

2000-01-01

Effects of mechanical forces exerted on mammalian skeletal muscle cells during development were studied using an in vitro model to unidirectionally stretch cultured C2C12 cells grown on silastic membrane. Previous models to date have not studied these responses of the mammalian system specifically. The silastic membrane upon which these cells were grown exhibited linear strain behavior over the range of 3.6-14.6% strain, with a Poisson's ratio of approximately 0.5. To mimic murine in utero long bone growth, cell substrates were stretched at an average strain rate of 2.36%/day for 4 days or 1.77%/day for 6 days with an overall membrane strain of 9.5% and 10.6%, respectively. Both control and stretched fibers stained positively for the contractile protein, alpha-actinin, demonstrating muscle fiber development. An effect of stretch on orientation and length of myofibers was observed. At both strain rates, stretched fibers aligned at a smaller angle relative to the direction of stretch and were significantly longer compared to randomly oriented control fibers. There was no effect of duration of stretch on orientation or length, suggesting the cellular responses are independent of strain rate for the range tested. These results demonstrate that, under conditions simulating mammalian long bone growth, cultured myocytes respond to mechanical forces by lengthening and orienting along the direction of stretch.

Spreading out Muscle Mass within a Hill-Type Model: A Computer Simulation Study

PubMed Central

Günther, Michael; Röhrle, Oliver; Haeufle, Daniel F. B.; Schmitt, Syn

2012-01-01

It is state of the art that muscle contraction dynamics is adequately described by a hyperbolic relation between muscle force and contraction velocity (Hill relation), thereby neglecting muscle internal mass inertia (first-order dynamics). Accordingly, the vast majority of modelling approaches also neglect muscle internal inertia. Assuming that such first-order contraction dynamics yet interacts with muscle internal mass distribution, this study investigates two questions: (i) what is the time scale on which the muscle responds to a force step? (ii) How does this response scale with muscle design parameters? Thereto, we simulated accelerated contractions of alternating sequences of Hill-type contractile elements and point masses. We found that in a typical small muscle the force levels off after about 0.2 ms, contraction velocity after about 0.5 ms. In an upscaled version representing bigger mammals' muscles, the force levels off after about 20 ms, and the theoretically expected maximum contraction velocity is not reached. We conclude (i) that it may be indispensable to introduce second-order contributions into muscle models to understand high-frequency muscle responses, particularly in bigger muscles. Additionally, (ii) constructing more elaborate measuring devices seems to be worthwhile to distinguish viscoelastic and inertia properties in rapid contractile responses of muscles. PMID:23227110

Atomic force microscopic imaging of Acanthamoeba castellanii and Balamuthia mandrillaris trophozoites and cysts.

PubMed

Aqeel, Yousuf; Siddiqui, Ruqaiyyah; Ateeq, Muhammad; Raza Shah, Muhammad; Kulsoom, Huma; Khan, Naveed Ahmed

2015-01-01

Light microscopy and electron microscopy have been successfully used in the study of microbes, as well as free-living protists. Unlike light microscopy, which enables us to observe living organisms or the electron microscope which provides a two-dimensional image, atomic force microscopy provides a three-dimensional surface profile. Here, we observed two free-living amoebae, Acanthamoeba castellanii and Balamuthia mandrillaris under the phase contrast inverted microscope, transmission electron microscope and atomic force microscope. Although light microscopy was of lower magnification, it revealed functional biology of live amoebae such as motility and osmoregulation using contractile vacuoles of the trophozoite stage, but it is of limited value in defining the cyst stage. In contrast, transmission electron microscopy showed significantly greater magnification and resolution to reveal the ultra-structural features of trophozoites and cysts including intracellular organelles and cyst wall characteristics but it only produced a snapshot in time of a dead amoeba cell. Atomic force microscopy produced three-dimensional images providing detailed topographic description of shape and surface, phase imaging measuring boundary stiffness, and amplitude measurements including width, height and length of A. castellanii and B. mandrillaris trophozoites and cysts. These results demonstrate the importance of the application of various microscopic methods in the biological and structural characterization of the whole cell, ultra-structural features, as well as surface components and cytoskeleton of protist pathogens. © 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists.

Contraction of gut smooth muscle cells assessed by fluorescence imaging.

PubMed

Tokita, Yohei; Akiho, Hirotada; Nakamura, Kazuhiko; Ihara, Eikichi; Yamamoto, Masahiro

2015-03-01

Here we discuss the development of a novel cell imaging system for the evaluation of smooth muscle cell (SMC) contraction. SMCs were isolated from the circular and longitudinal muscular layers of mouse small intestine by enzymatic digestion. SMCs were stimulated by test agents, thereafter fixed in acrolein. Actin in fixed SMCs was stained with phalloidin and cell length was determined by measuring diameter at the large end of phalloidin-stained strings within the cells. The contractile response was taken as the decrease in the average length of a population of stimulated-SMCs. Various mediators and chemically identified compounds of daikenchuto (DKT), pharmaceutical-grade traditional Japanese prokinetics, were examined. Verification of the integrity of SMC morphology by phalloidin and DAPI staining and semi-automatic measurement of cell length using an imaging analyzer was a reliable method by which to quantify the contractile response. Serotonin, substance P, prostaglandin E2 and histamine induced SMC contraction in concentration-dependent manner. Two components of DKT, hydroxy-α-sanshool and hydroxy-β-sanshool, induced contraction of SMCs. We established a novel cell imaging technique to evaluate SMC contractility. This method may facilitate investigation into SMC activity and its role in gastrointestinal motility, and may assist in the discovery of new prokinetic agents. Copyright © 2015 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

Muscle dysfunction in a zebrafish model of Duchenne muscular dystrophy.

PubMed

Widrick, Jeffrey J; Alexander, Matthew S; Sanchez, Benjamin; Gibbs, Devin E; Kawahara, Genri; Beggs, Alan H; Kunkel, Louis M

2016-11-01

Sapje zebrafish lack the protein dystrophin and are the smallest vertebrate model of Duchenne muscular dystrophy (DMD). Their small size makes them ideal for large-scale drug discovery screens. However, the extent that sapje mimic the muscle dysfunction of higher vertebrate models of DMD is unclear. We used an optical birefringence assay to differentiate affected dystrophic sapje larvae from their unaffected siblings and then studied trunk muscle contractility at 4-7 days postfertilization. Preparation cross-sectional area (CSA) was similar for affected and unaffected larvae, yet tetanic forces of affected preparations were only 30-60% of normal. ANCOVA indicated that the linear relationship observed between tetanic force and CSA for unaffected preparations was absent in the affected population. Consequently, the average force/CSA of affected larvae was depressed 30-70%. Disproportionate reductions in twitch vs. tetanic force, and a slowing of twitch tension development and relaxation, indicated that the myofibrillar disorganization evident in the birefringence assay could not explain the entire force loss. Single eccentric contractions, in which activated preparations were lengthened 5-10%, resulted in tetanic force deficits in both groups of larvae. However, deficits of affected preparations were three- to fivefold greater at all strains and ages, even after accounting for any recovery. Based on these functional assessments, we conclude that the sapje mutant zebrafish is a phenotypically severe model of DMD. The severe contractile deficits of sapje larvae represent novel physiological endpoints for therapeutic drug screening. Copyright © 2016 the American Physiological Society.

Cortical Flow-Driven Shapes of Nonadherent Cells.

PubMed

Callan-Jones, A C; Ruprecht, V; Wieser, S; Heisenberg, C P; Voituriez, R

2016-01-15

Nonadherent polarized cells have been observed to have a pearlike, elongated shape. Using a minimal model that describes the cell cortex as a thin layer of contractile active gel, we show that the anisotropy of active stresses, controlled by cortical viscosity and filament ordering, can account for this morphology. The predicted shapes can be determined from the flow pattern only; they prove to be independent of the mechanism at the origin of the cortical flow, and are only weakly sensitive to the cytoplasmic rheology. In the case of actin flows resulting from a contractile instability, we propose a phase diagram of three-dimensional cell shapes that encompasses nonpolarized spherical, elongated, as well as oblate shapes, all of which have been observed in experiment.

Static mechanical strain induces capillary endothelial cell cycle re-entry and sprouting.

PubMed

Zeiger, A S; Liu, F D; Durham, J T; Jagielska, A; Mahmoodian, R; Van Vliet, K J; Herman, I M

2016-08-16

Vascular endothelial cells are known to respond to a range of biochemical and time-varying mechanical cues that can promote blood vessel sprouting termed angiogenesis. It is less understood how these cells respond to sustained (i.e., static) mechanical cues such as the deformation generated by other contractile vascular cells, cues which can change with age and disease state. Here we demonstrate that static tensile strain of 10%, consistent with that exerted by contractile microvascular pericytes, can directly and rapidly induce cell cycle re-entry in growth-arrested microvascular endothelial cell monolayers. S-phase entry in response to this strain correlates with absence of nuclear p27, a cyclin-dependent kinase inhibitor. Furthermore, this modest strain promotes sprouting of endothelial cells, suggesting a novel mechanical 'angiogenic switch'. These findings suggest that static tensile strain can directly stimulate pathological angiogenesis, implying that pericyte absence or death is not necessarily required of endothelial cell re-activation.

3D cardiac μ tissues within a microfluidic device with real-time contractile stress readout

PubMed Central

Aung, Aereas; Bhullar, Ivneet Singh; Theprungsirikul, Jomkuan; Davey, Shruti Krishna; Lim, Han Liang; Chiu, Yu-Jui; Ma, Xuanyi; Dewan, Sukriti; Lo, Yu-Hwa; McCulloch, Andrew; Varghese, Shyni

2015-01-01

We present the development of three-dimensional (3D) cardiac microtissues within a microfluidic device with the ability to quantify real-time contractile stress measurements in situ. Using a 3D patterning technology that allows for the precise spatial distribution of cells within the device, we created an array of 3D cardiac microtissues from neonatal mouse cardiomyocytes. We integrated the 3D micropatterning technology with microfluidics to achieve perfused cell-laden structures. The cells were encapsulated within a degradable gelatin methacrylate hydrogel, which was sandwiched between two polyacrylamide hydrogels. The polyacrylamide hydrogels were used as “stress sensors” to acquire the contractile stresses generated by the beating cardiac cells. The cardiac-specific response of the engineered 3D system was examined by exposing it to epinephrine, an adrenergic neurotransmitter known to increase the magnitude and frequency of cardiac contractions. In response to exogenous epinephrine the engineered cardiac tissues exhibited an increased beating frequency and stress magnitude. Such cost-effective and easy-to-adapt 3D cardiac systems with real-time functional readout could be an attractive technological platform for drug discovery and development. PMID:26588203

Decrease of airway smooth muscle contractility induced by simulated breathing maneuvers is not simply proportional to strain.

PubMed

Pascoe, Chris D; Seow, Chun Y; Paré, Peter D; Bossé, Ynuk

2013-02-01

The lung is a dynamic organ and the oscillating stress applied to the airway wall during breathing maneuvers can decrease airway smooth muscle (ASM) contractility. However, it is unclear whether it is the stress or the attendant strain that is responsible for the decline of ASM force associated with breathing maneuvers, and whether tone can prevent the decline of force by attenuating the strain. To investigate these questions, ovine tracheal strips were subjected to oscillating stress that simulates breathing maneuvers, and the resulting strain and decline of force were measured in the absence or presence of different levels of tone elicited by acetylcholine. In relaxed ASM, high stress simulating 20 cm H(2)O-transpulmonary pressure excursions strained ASM strips by 20.7% and decreased force by 17.1%. When stress oscillations were initiated during measurement of ACh concentration-response curves, tone almost abrogated strain at an ACh concentration of 10(-6) M (1.1%) but the decline of force was not affected (18.9%). When stress oscillations were initiated after ACh-induced contraction had reached its maximal force, strain was almost abrogated at an ACh concentration of 10(-6) M (0.9%) and the decline of force was attenuated (10.1%). However, even at the highest ACh concentration (10(-4) M), substantial decline of force (6.1%) was still observed despite very small strain (0.7%). As expected, the results indicate that tone attenuated the strain experienced by ASM during breathing maneuver simulations. More surprisingly, the reduction of strain induced by tone was not proportional to its effect on the decline of force induced by simulated breathing maneuvers.

The role of microtubules in contractile ring function.

PubMed

Conrad, A H; Paulsen, A Q; Conrad, G W

1992-05-01

During cytokinesis, a cortical contractile ring forms around a cell, constricts to a stable tight neck and terminates in separation of the daughter cells. At first cleavage, Ilyanassa obsoleta embryos form two contractile rings simultaneously. The cleavage furrow (CF), in the animal hemisphere between the spindle poles, constricts to a stable tight neck and separates the daughter cells. The third polar lobe constriction (PLC-3), in the vegetal hemisphere below the spindle, constricts to a transient tight neck, but then relaxes, allowing the polar lobe cytoplasm to merge with one daughter cell. Eggs exposed to taxol, a drug that stabilizes microtubules, before the CF or the PLC-3 develop, fail to form CFs, but form stabilized tight PLCs. Eggs exposed to taxol at the time of PLC-3 formation develop varied numbers of constriction rings in their animal hemispheres and one PLC in their vegetal hemisphere, none of which relax. Eggs exposed to taxol after PLC-3 initiation form stabilized tight CFs and PLCs. At maximum constriction, control embryos display immunolocalization of nonextractable alpha-tubulin in their CFs, but not in their PLCs, and reveal, via electron microscopy, many microtubules extending through their CFs, but not through their PLCs. Embryos which form stabilized tightly constricted CFs and PLCs in the presence of taxol display immunolocalization of nonextractable alpha-tubulin in both constrictions and show many polymerized microtubules extending through both CFs and PLCs. These results suggest that the extension of microtubules through a tight contractile ring may be important for stabilizing that constriction and facilitating subsequent cytokinesis.

The role of microtubules in contractile ring function

NASA Technical Reports Server (NTRS)

Conrad, A. H.; Paulsen, A. Q.; Conrad, G. W.; Spooner, B. S. (Principal Investigator)

1992-01-01

During cytokinesis, a cortical contractile ring forms around a cell, constricts to a stable tight neck and terminates in separation of the daughter cells. At first cleavage, Ilyanassa obsoleta embryos form two contractile rings simultaneously. The cleavage furrow (CF), in the animal hemisphere between the spindle poles, constricts to a stable tight neck and separates the daughter cells. The third polar lobe constriction (PLC-3), in the vegetal hemisphere below the spindle, constricts to a transient tight neck, but then relaxes, allowing the polar lobe cytoplasm to merge with one daughter cell. Eggs exposed to taxol, a drug that stabilizes microtubules, before the CF or the PLC-3 develop, fail to form CFs, but form stabilized tight PLCs. Eggs exposed to taxol at the time of PLC-3 formation develop varied numbers of constriction rings in their animal hemispheres and one PLC in their vegetal hemisphere, none of which relax. Eggs exposed to taxol after PLC-3 initiation form stabilized tight CFs and PLCs. At maximum constriction, control embryos display immunolocalization of nonextractable alpha-tubulin in their CFs, but not in their PLCs, and reveal, via electron microscopy, many microtubules extending through their CFs, but not through their PLCs. Embryos which form stabilized tightly constricted CFs and PLCs in the presence of taxol display immunolocalization of nonextractable alpha-tubulin in both constrictions and show many polymerized microtubules extending through both CFs and PLCs. These results suggest that the extension of microtubules through a tight contractile ring may be important for stabilizing that constriction and facilitating subsequent cytokinesis.

Changes in cardiac contractility during graded exercise are greater in subjects with smaller body mass index, and greater in men than women: analyses using wave intensity and force-frequency relations.

PubMed

Tanaka, Midori; Sugawara, Motoaki; Niki, Kiyomi; Ogasawara, Yasuo

2018-06-15

Estimation of the contractility of the left ventricle during exercise is an important part of the rehabilitation protocol. It is known that cardiac contractility increases with an increase in heart rate. This phenomenon is called the force-frequency relation (FFR). Using wave intensity, we aimed to evaluate FFR noninvasively during graded exercise. We enrolled 83 healthy subjects. Using ultrasonic diagnostic equipment, we measured wave intensity (WD), which was defined in terms of blood velocity and arterial diameter, in the carotid artery and heart rate (HR) before and during bicycle ergometer exercise. FFRs were constructed by plotting the maximum value of WD (WD 1 ) against HR. We analyzed the variation among FFR responses of individual subjects. WD 1 increased linearly with an increase in HR during exercise. The average slope of the FFR was 1.0 ± 0.5 m/s 3  bpm. The slope of FFR decreased with an increase in body mass index (BMI). The slopes of FFRs were steeper in men than women, although there were no differences in BMI between men and women. The FFR was obtained noninvasively by carotid arterial wave intensity (WD 1 ) and graded exercise. The slope of the FFR decreased with an increase in BMI, and was steeper in men than women.

The contractile adaption to preload depends on the amount of afterload

PubMed Central

Schotola, Hanna; Sossalla, Samuel T.; Renner, André; Gummert, Jan; Danner, Bernhard C.; Schott, Peter

2017-01-01

Abstract Aims The Frank–Starling mechanism (rapid response (RR)) and the secondary slow response (SR) are known to contribute to increases contractile performance. The contractility of the heart muscle is influenced by pre‐load and after‐load. Because of the effect of pre‐load vs. after‐load on these mechanisms in not completely understood, we studied the effect in isolated muscle strips. Methods and results Progressive stretch lead to an increase in shortening/force development under isotonic (only pre‐load) and isometric conditions (pre‐ and after‐load). Muscle length with maximal function was reached earlier under isotonic (L max‐isotonic) compared with isometric conditions (L max‐isometric) in nonfailing rabbit, in human atrial and in failing ventricular muscles. Also, SR after stretch from slack to L max‐isotonic was comparable under isotonic and isometric conditions (human: isotonic 10 ± 4%, isometric 10 ± 4%). Moreover, a switch from isotonic to isometric conditions at L max‐isometric showed no SR proving independence of after‐load. To further analyse the degree of SR on the total contractile performance at higher pre‐load muscles were stretched from slack to 98% L max‐isometric under isotonic conditions. Thereby, the SR was 60 ± 9% in rabbit and 51 ± 14% in human muscle strips. Conclusions This work shows that the acute contractile response largely depends on the degree and type of mechanical load. Increased filling of the heart elevates pre‐load and prolongs the isotonic part of contraction. The reduction in shortening at higher levels of pre‐load is thereby partially compensated by the pre‐load‐induced SR. After‐load shifts the contractile curve to a better ‘myofilament function’ by probably influencing thin fibers and calcium sensitivity, but has no effect on the SR. PMID:29154423

Insulin-like Growth Factor-I and Slow, Bi-directional Perfusion Enhance the Formation of Tissue-Engineered Cardiac Grafts

PubMed Central

Cheng, Mingyu; Moretti, Matteo; Engelmayr, George C.

2009-01-01

Biochemical and mechanical signals enabling cardiac regeneration can be elucidated using in vitro tissue-engineering models. We hypothesized that insulin-like growth factor-I (IGF) and slow, bi-directional perfusion could act independently and interactively to enhance the survival, differentiation, and contractile performance of tissue-engineered cardiac grafts. Heart cells were cultured on three-dimensional porous scaffolds in medium with or without supplemental IGF and in the presence or absence of slow, bi-directional perfusion that enhanced transport and provided shear stress. Structural, molecular, and electrophysiologic properties of the resulting grafts were quantified on culture day 8. IGF had independent, beneficial effects on apoptosis (p < 0.01), cellular viability (p < 0.01), contractile amplitude (p < 0.01), and excitation threshold (p < 0.01). Perfusion independently affected the four aforementioned parameters and also increased amounts of cardiac troponin-I (p < 0.01), connexin-43 (p < 0.05), and total protein (p < 0.01) in the grafts. Interactive effects of IGF and perfusion on apoptosis were also present (p < 0.01). Myofibrillogenesis and spontaneous contractility were present only in grafts cultured with perfusion, although contractility was inducible by electrical field stimulation of grafts from all groups. Our findings demonstrate that multi-factorial stimulation of tissue-engineered cardiac grafts using IGF and perfusion resulted in independent and interactive effects on heart cell survival, differentiation, and contractility. PMID:18759675

Molecular organization of cytokinesis nodes and contractile rings by super-resolution fluorescence microscopy of live fission yeast

PubMed Central

Laplante, Caroline; Huang, Fang; Tebbs, Irene R.; Bewersdorf, Joerg; Pollard, Thomas D.

2016-01-01

Cytokinesis in animals, fungi, and amoebas depends on the constriction of a contractile ring built from a common set of conserved proteins. Many fundamental questions remain about how these proteins organize to generate the necessary tension for cytokinesis. Using quantitative high-speed fluorescence photoactivation localization microscopy (FPALM), we probed this question in live fission yeast cells at unprecedented resolution. We show that nodes, protein assembly precursors to the contractile ring, are discrete structural units with stoichiometric ratios and distinct distributions of constituent proteins. Anillin Mid1p, Fes/CIP4 homology-Bin/amphiphysin/Rvs (F-BAR) Cdc15p, IQ motif containing GTPase-activating protein (IQGAP) Rng2p, and formin Cdc12p form the base of the node that anchors the ends of myosin II tails to the plasma membrane, with myosin II heads extending into the cytoplasm. This general node organization persists in the contractile ring where nodes move bidirectionally during constriction. We observed the dynamics of the actin network during cytokinesis, starting with the extension of short actin strands from nodes, which sometimes connected neighboring nodes. Later in cytokinesis, a broad network of thick bundles coalesced into a tight ring around the equator of the cell. The actin ring was ∼125 nm wide and ∼125 nm thick. These observations establish the organization of the proteins in the functional units of a cytokinetic contractile ring. PMID:27647921

Smooth muscle-protein translocation and tissue function.

PubMed

Eddinger, Thomas J

2014-09-01

Smooth muscle (SM) tissue is a complex organization of multiple cell types and is regulated by numerous signaling molecules (neurotransmitters, hormones, cytokines, etc.). SM contractile function can be regulated via expression and distribution of the contractile and cytoskeletal proteins, and activation of any of the second messenger pathways that regulate them. Spatial-temporal changes in the contractile, cytoskeletal or regulatory components of SM cells (SMCs) have been proposed to alter SM contractile activity. Ca(2+) sensitization/desensitization can occur as a result of changes at any of these levels, and specific pathways have been identified at all of these levels. Understanding when and how proteins can translocate within the cytoplasm, or to-and-from the plasmalemma and the cytoplasm to alter contractile activity is critical. Numerous studies have reported translocation of proteins associated with the adherens junction and G protein-coupled receptor activation pathways in isolated SMC systems. Specific examples of translocation of vinculin to and from the adherens junction and protein kinase C (PKC) and 17 kDa PKC-potentiated inhibitor of myosin light chain phosphatase (CPI-17) to and from the plasmalemma in isolated SMC systems but not in intact SM tissues are discussed. Using both isolated SMC systems and SM tissues in parallel to pursue these studies will advance our understanding of both the role and mechanism of these pathways as well as their possible significance for Ca(2+) sensitization in intact SM tissues and organ systems. © 2014 Wiley Periodicals, Inc.

Airways in smooth muscle α-actin null mice experience a compensatory mechanism that modulates their contractile response.

PubMed

Shardonofsky, Felix R; Moore, Joan; Schwartz, Robert J; Boriek, Aladin M

2012-03-01

We hypothesized that ablation of smooth muscle α-actin (SM α-A), a contractile-cytoskeletal protein expressed in airway smooth muscle (ASM) cells, abolishes ASM shortening capacity and decreases lung stiffness. In both SM α-A knockout and wild-type (WT) mice, airway resistance (Raw) determined by the forced oscillation technique rose in response to intravenous methacholine (Mch). However, the slope of Raw (cmH(2)O·ml(-1)·s) vs. log(2) Mch dose (μg·kg(-1)·min(-1)) was lower (P = 0.007) in mutant (0.54 ± 0.14) than in WT mice (1.23 ± 0.19). RT-PCR analysis performed on lung tissues confirmed that mutant mice lacked SM α-A mRNA and showed that these mice had robust expressions of both SM γ-A mRNA and skeletal muscle (SKM) α-A mRNA, which were not expressed in WT mice, and an enhanced SM22 mRNA expression relative to that in WT mice. Compared with corresponding spontaneously breathing mice, mechanical ventilation-induced lung mechanical strain increased the expression of SM α-A mRNA in WT lungs; in mutant mice, it augmented the expressions of SM γ-A mRNA and SM22 mRNA and did not alter that of SKM α-A mRNA. In mutant mice, the expression of SM γ-A mRNA in the lung during spontaneous breathing and its enhanced expression following mechanical ventilation are consistent with the likely possibility that in the absence of SM α-A, SM γ-A underwent polymerization and interacted with smooth muscle myosin to produce ASM shortening during cholinergic stimulation. Thus our data are consistent with ASM in mutant mice experiencing compensatory mechanisms that modulated its contractile muscle capacity.

Chronic sustained hypoxia-induced redox remodeling causes contractile dysfunction in mouse sternohyoid muscle

PubMed Central

Lewis, Philip; Sheehan, David; Soares, Renata; Varela Coelho, Ana; O'Halloran, Ken D.

2015-01-01

Chronic sustained hypoxia (CH) induces structural and functional adaptations in respiratory muscles of animal models, however the underlying molecular mechanisms are unclear. This study explores the putative role of CH-induced redox remodeling in a translational mouse model, with a focus on the sternohyoid—a representative upper airway dilator muscle involved in the control of pharyngeal airway caliber. We hypothesized that exposure to CH induces redox disturbance in mouse sternohyoid muscle in a time-dependent manner affecting metabolic capacity and contractile performance. C57Bl6/J mice were exposed to normoxia or normobaric CH (FiO2 = 0.1) for 1, 3, or 6 weeks. A second cohort of animals was exposed to CH for 6 weeks with and without antioxidant supplementation (tempol or N-acetyl cysteine in the drinking water). Following CH exposure, we performed 2D redox proteomics with mass spectrometry, metabolic enzyme activity assays, and cell-signaling assays. Additionally, we assessed isotonic contractile and endurance properties ex vivo. Temporal changes in protein oxidation and glycolytic enzyme activities were observed. Redox modulation of sternohyoid muscle proteins key to contraction, metabolism and cellular homeostasis was identified. There was no change in redox-sensitive proteasome activity or HIF-1α content, but CH decreased phospho-JNK content independent of antioxidant supplementation. CH was detrimental to sternohyoid force- and power-generating capacity and this was prevented by chronic antioxidant supplementation. We conclude that CH causes upper airway dilator muscle dysfunction due to redox modulation of proteins key to function and homeostasis. Such changes could serve to further disrupt respiratory homeostasis in diseases characterized by CH such as chronic obstructive pulmonary disease. Antioxidants may have potential use as an adjunctive therapy in hypoxic respiratory disease. PMID:25941492

Cytoskeletal mechanics in pressure-overload cardiac hypertrophy

NASA Technical Reports Server (NTRS)

Tagawa, H.; Wang, N.; Narishige, T.; Ingber, D. E.; Zile, M. R.; Cooper, G. 4th

1997-01-01

We have shown that the cellular contractile dysfunction characteristic of pressure-overload cardiac hypertrophy results not from an abnormality intrinsic to the myofilament portion of the cardiocyte cytoskeleton but rather from an increased density of the microtubule component of the extramyofilament portion of the cardiocyte cytoskeleton. To determine how, in physical terms, this increased microtubule density mechanically overloads the contractile apparatus at the cellular level, we measured cytoskeletal stiffness and apparent viscosity in isolated cardiocytes via magnetic twisting cytometry, a technique by which magnetically induced force is applied directly to the cytoskeleton through integrin-coupled ferromagnetic beads coated with Arg-Gly-Asp (RGD) peptide. Measurements were made in two groups of cardiocytes from cats with right ventricular (RV) hypertrophy induced by pulmonary artery banding: (1) those from the pressure-overloaded RV and (2) those from the normally loaded same-animal control left ventricle (LV). Cytoskeletal stiffness increased almost twofold, from 8.53 +/- 0.77 dyne/cm2 in the normally loaded LV cardiocytes to 16.46 +/- 1.32 dyne/cm2 in the hypertrophied RV cardiocytes. Cytoskeletal apparent viscosity increased almost fourfold, from 20.97 +/- 1.92 poise in the normally loaded LV cardiocytes to 87.85 +/- 6.95 poise in the hypertrophied RV cardiocytes. In addition to these baseline data showing differing stiffness and, especially, apparent viscosity in the two groups of cardiocytes, microtubule depolymerization by colchicine was found to return both the stiffness and the apparent viscosity of the pressure overload-hypertrophied RV cells fully to normal. Conversely, microtubule hyperpolymerization by taxol increased the stiffness and apparent viscosity values of normally loaded LV cardiocytes to the abnormal values given above for pressure-hypertrophied RV cardiocytes. Thus, increased microtubule density constitutes primarily a viscous load on the cardiocyte contractile apparatus in pressure-overload cardiac hypertrophy.