Published online before print November 23, 2005, doi: 10.1161/01.RES.0000197785.51819.e8
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© 2006 American Heart Association, Inc.
Cellular Biology |
Microtubules Modulate the Stiffness of Cardiomyocytes Against Shear Stress
From the Department of Cardiovascular Medicine (S. Nishimura, M.K., H.Y., R.N.), Graduate School of Medicine, and The Institute of Environmental Studies (S. Nagai, J.-i.O., T.H., S.S.), Graduate School of Frontier Sciences, The University of Tokyo; and the Department of Physiology (Y.S.), Dental School, Tsurumi University, Japan.
Correspondence to Seiryo Sugiura, Institute of Environmental Studies, Graduate School of Frontier Sciences, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail sugiura{at}k.u-tokyo.ac.jp
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Abstract |
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Although microtubules are involved in various pathological conditions of the heart including hypertrophy and congestive heart failure, the mechanical role of microtubules in cardiomyocytes under such conditions is not well understood. In the present study, we measured multiple aspects of the mechanical properties of single cardiomyocytes, including tensile stiffness, transverse (indentation) stiffness, and shear stiffness in both transverse and longitudinal planes using carbon fiber–based systems and compared these parameters under control, microtubule depolymerized (colchicine treated), and microtubule hyperpolymerized (paclitaxel treated) conditions. From all of these measurements, we found that only the stiffness against shear in the longitudinal plane was modulated by the microtubule cytoskeleton. A simulation model of the myocyte in which microtubules serve as compression-resistant elements successfully reproduced the experimental results. In the complex strain field that living myocytes experience in the body, observed changes in shear stiffness may have a significant influence on the diastolic property of the diseased heart.
Key Words: cytoskeleton • microtubules • cardiomyocyte
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Introduction |
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In mammalian cells, microtubules and actin filaments constitute the major components of the cytoskeleton and participate in a variety of cellular processes, including organelle transport, cell division, and migration as well as maintenance or alteration of the cell morphology in response to mechanical stimuli transmitted from the surrounding extracellular matrix.1–3 However, in the case of postmitotic adult ventricular cardiomyocytes, the functional significance of microtubules may be somewhat different. Their relative content of microtubules (tubulin) is small compared with other types of cells4 but increases in various disease conditions, such as cardiac hypertrophy or heart failure, in which the heart is subjected to abnormally high loads.5,6
In this context, studies at the tissue (papillary muscle) and cellular levels have focused on the impact of microtubule proliferation on the contractile function of the myocardium, but the results obtained are controversial. The microtubule proliferation observed in hypertrophied hearts was associated with contractile dysfunction and pharmacological disruption of the microtubules by colchicines (COLs) normalized the contractile function.7,8 However, in the absence of the preceding hypertrophic proliferation of microtubules, COLs do not improve contractile function.9,10 The structural role of the microtubules has also been evaluated by recording the stress–strain relationship of single cardiomyocytes in the longitudinal direction, but the results failed to establish causality for the stiff passive properties observed in the diseased heart.11,12 Studies using magnetic twisting cytometry revealed increased cytoskeletal stiffness and viscosity in hypertrophied myocytes with a high microtubule density,13 but this methodology did not provide any information regarding the anisotropic properties of these polarized cells or allow evaluation of the reported parameters in the complex strain field that living myocytes experience in the body.
The current study investigated the role of microtubules in the cardiac adaptation process by evaluating the structural properties of single rat cardiomyocytes containing variable amounts of microtubules. In addition to ordinary measurements of the stress–strain relationship in the long axis of each myocyte, we also recorded the transverse stiffness and shear stiffness in both the longitudinal and transverse planes. It was found that microtubules modulate the stiffness of cardiomyocyte only against the shear stress in the longitudinal plane. This result can be taken to indicate that microtubules serve as compression-resistant elements as suggested by the cellular tensegrity model.1 Simulation study based on this model successfully reproduced the experimental findings.
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Materials and Methods |
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An expanded Materials and Methods section is provided in the online data supplement available at http://circres.ahajournals.org.
Isolation of Cardiomyocytes
Single ventricular myocytes were isolated from 7-week-old female Wistar rats as described previously.14 We also studied the myocytes from 10-week-old male cardiomyopathic hamsters (Bio TO-2 strain) and age-matched Syrian golden hamsters (Bio-Breeders Institute, Cambridge, Mass). All the experiments were performed at room temperature. All studies were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee.
Cell Mechanics Measurements
We characterized the mechanical properties of cardiomyocytes in 3 ways, ie, "tensile stiffness," "transverse stiffness," and "shear stiffness." The tensile stiffness was measured using a cell adhesive carbon fiber–based system as described previously.14,15 Briefly, a rod-shaped quiescent single cardiomyocyte was selected under a microscope and a pair of carbon fibers was attached to both ends using micromanipulators (Figure 1A). One fiber was compliant, whereas the other was thick and rigid and served as a mechanical anchor. The position of the compliant fiber was controlled by a piezoelectric translator (PZT; P-841.40; Physik Instrumente, Karlsruhe, Germany) by a personal computer, and the position of the free end (attached to the cell) was monitored by projecting its image onto a linear photodiode array (S3903; Hamamatsu Photonics, Hamamatsu City, Japan).14 The sarcomere length was simultaneously measured by real-time fast Fourier transform analysis of the striation pattern (IonOptix, Milton, Mass). To measure the tensile stiffness, the cell was stretched at a strain rate of 0.01/sec by pulling the attached carbon fibers, and the tensile stress–strain relationship was obtained.
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We used the same experimental setup to measure the transverse stiffness, with some modifications. We attached a latex microsphere (diameter: 5 µm; Polysciences, Warrington, Pa) to the side of the tip of the compliant carbon fiber, such that the microsphere could be pushed against the myocyte horizontally (Figure 3A). In each experiment, we selected a myocyte with a rectangular shape, placed it along the sidewall in a glass chamber, and performed an indentation test by moving the compliant fiber (2 µm/sec). Because the area of contact clearly increased during the experiment and this was difficult to quantify, we determined the effective transverse stiffness (Keff) by evaluating the slope of the force-indentation curve as the indentation (
) approached 016: 
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To evaluate the shear stiffness, we held the myocyte between the bottom coverglass and a small thin glass plate at the top. Small pieces of thin glass plates (thickness: 5 µm; Glass Flakes, REF-160; Nippon Sheet Glass, Tokyo, Japan) were precoated with laminin (Sigma, St Louis, Mo). Under a microscope, a selected single piece of an appropriate size to cover the whole cell was glued to the tip of the thin carbon fiber (Aronalpha; TOAGOSEI, Tokyo, Japan) in the experimental chamber consisting of a Plexiglas frame and a laminin-coated coverglass at the bottom. We gently attached the glass plate to the top surface of the cell (Figure 4A and 4B), and the myocyte was placed either parallel or perpendicular to the direction of the applied shear force. A 10% shear strain was applied by shifting the glass flake connected to a piezoelectric translator via the carbon fiber in 10 seconds in both the longitudinal and transverse plan. All the signals were recorded at 1 kHz by a personal computer (PowerLab/8SP; AD Instruments, Castle Hill, NSW, Australia).
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Altering the Polymerization State of Microtubules
To alter the polymerization state of microtubules, COL (Sigma) or paclitaxel (PAC) (Sigma) was added to Tyrode solution and incubated before the mechanics measurements.
Immunocytochemical Procedures
The microtubules were immunocytochemically labeled with a monoclonal antibody against tubulin after fixation, and actin was simultaneously stained with rhodamine phalloidin (Invitrogen). The cells were observed using a laser confocal microscopy (CSU21; Yokogawa-Denki, Musashino, Japan).
Data Analysis
Results are expressed as the mean±SEM. The statistical significance of the microtubule density for each mechanical property was assessed by ANOVA. If statistically significant differences were discovered, pairwise comparisons (Student’s t test) were performed. A probability value of less than 0.05 was considered statistically significant.
Simulation Model
Microtubules, cytoskeletal actin filaments, and desmin filaments (intermediate filaments) were modeled by truss elements (no. 1874) using the Young’s moduli and geometrical parameters listed in the supplemental Table.17–20 Young’s modulus of microtubule has been estimated by either applying bending force to microtubules18,21,22 or recording thermally induced shape fluctuations (statistical mechanics model).22,23 From the reported values ranging from 100 MPa to 1.2 GPa, we adopted an intermediate value of 500 MPa, which corresponds to a persistence length of 2200 µm. Myofibrils were modeled as solid elements (no. 21540), the material properties of which were characterized using the anisotropic hyperelasticity proposed by Humphrey et al for cardiac muscle tissue.24
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Results |
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Mean sarcomere length of studied rat cardiomyocytes was 1.93±0.08 µm (n=8) before the strain was applied. When tensile stress was applied to a myocyte by pulling a pair of carbon fibers attached to both ends of the cell (Figure 1 A), the simultaneously measured sarcomere length changed proportionally to the segment length, demonstrating that the stress was distributed homogeneously along the myocyte. From the stress–strain relationship obtained, the average stiffness value for control (CTRL) myocytes (n=8) under tensile stress was determined to be 6.02±0.80 mN/mm2 at 5% strain and 12.81±0.73 mN/mm2 at 10% strain (Figure 1B). Visualization of the microtubule density in CTRL myocytes by immunolabeling with an anti–ß-tubulin antibody (red) revealed a fine network spread over the entire cell surrounding myofibrils (blue), with occasional formation of loop-type structures (Figure 2 A). After COL treatment (1 µmol/L, 60 minutes), the mesh-like microtubule network had almost disappeared, whereas the number of microtubules apposed to myofibrils increased significantly after PAC treatment (10 µmol/L, 3 hour) (Figure 2A). These tendencies were confirmed by quantification of the red-light intensities in confocal images normalized to the cell area (Figure 2B). We also noted that these dynamic changes occurred preferentially in the longitudinal microtubules, consistent with previous immunolabeling light and electron microscopic studies.25 These drug-induced changes in the microtubule density did not have any effect on the tensile stiffness of the myocytes (Figure 1B).
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The transverse stiffness (Keff) was 11.6±1.6 nN/µm for CTRL myocytes (n=9). Keff tended to change in parallel with the microtubule density (Figure 3 B), but the difference did not reach statistical significance (P=0.19 for group comparisons by ANOVA).
We also evaluated the shear stiffness (shear stress/shear strain) in 2 directions, ie, the longitudinal and transverse planes, using a novel technique involving a small glass plate coupled with a carbon fiber (Figure 4 A and 4B). The myocyte was held between the bottom coverglass and a small thin glass plate attached onto the top surface. We applied shear stress by shifting the top glass plate connected to a piezoelectric translator via the carbon fiber. Fluorescent staining with a voltage-sensitive indicator (Di2-ANEPEQ) facilitated visualization of the area in contact with the glass plate, and we confirmed that this contact area did not change appreciably during the shear deformation. We confined our analysis to small deformations (linear range) by evaluating the slope at the origin. The shear stiffness values thus obtained for the longitudinal plane (4.57±0.20 kPa, n=15) was nearly double that for the transverse plane (2.94±0.27 kPa, n=13) in CTRL myocytes at 10% strain (Figure 4C and 4D). As in the case of the tensile stiffness, the shear stiffness in the transverse plane did not change significantly by the drug interventions (Figure 4D). However, in the longitudinal plane, hyperpolymerization induced by PAC treatment caused increased the shear stiffness by approximately 2-fold, whereas COL treatment decreased the value by approximately 50% (Figure 4C).
In some myocytes, we evaluated the contribution of cross-bridge formation by repeating these measurements in the calcium-free (Ca 0 mmol/L, EGTA 0.4 mmol/L) solution with 20 mmol/L of butane-dione monoxime (BDM). Inhibition of cross-bridge formation decreased the tensile stiffness by 27% at 10% strain (Figure 5 A). On the other hand, neither transverse stiffness (Figure 5B) nor shear stiffness in both longitudinal and transverse planes was altered by cross-bridge inhibition (Figure 5C).
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Because a previous study showed the effect of microtubules proliferation on the viscosity of the myocardial tissue in response to the tensile deformation,12 we also evaluated the viscous properties of cardiomyocyte by applying sinusoidal strain of varying frequencies. The elastic (storage) and viscous (loss) components of the stress/strain modulus were estimated using Fourier transform at the frequency between 1 and 10 Hz (corresponding to the strain rate of 0.1 to 1 sec–1) (Figure 6 A and 6B). Whereas the elastic moduli did not change appreciably over the examined frequency range, the viscous moduli were dependent on the strain rate. Neither COL nor Taxol treatment altered the relation between the elastic modulus and strain rate, but the PAC treatment increased the slope of the relation between the tensile strain rate and the viscous modulus significantly (COL: 9.4±0.5 mN/mm2 per second; CTRL: 11.0±0.8 mN/mm2 per second; PAC: 14.8±1.1 mN/mm2 per second; n=6 for each, P<0.05 PAC versus COL and CTRL) (Figure 6B). All of these results were consistent with the previous report.12
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In the case of shear in the longitudinal plane, elastic moduli were also independent of the frequency (Figure 6C), but the average value over the tested range (1.0 to 10 Hz corresponding to the shear rate of 0.5 to 5/sec) differed among the 3 groups (COL: 3.4±0.3 kPa; CTRL: 4.8±0.4 kPa; PAC: 8.4±0.6 kPa; n=6 for each, P<0.05 for group comparisons by ANOVA). Similar to the tensile stiffness measurement, the viscous moduli were dependent on the shear rate (Figure 5D), and the slope of the relation was significantly greater in PAC-treated myocytes (COL: 0.46±0.01 kPa per second; CTRL: 0.46±0.02 kPa per second; PAC: 0.53±0.013 kPa per second; n=6 for each, P<0.05 PAC versus COL and CTRL).
To study the roles of microtubules in pathological conditions, measurements were performed on myocytes from cardiomyopathic (CMP) hamster (Bio-TO2 strain), a well-known hereditary animal model of congestive heart failure26,27 using Syrian hamsters as CTRL. The CMP myocytes showed increased level of microtubules proliferation, which was normalized after COL treatment (Figure 7 A). Tensile stiffness at 10% strain did not differ between the 2 groups (Figure 7B), but longitudinal shear stiffness was increased in CMP. Furthermore, this increase in shear stiffness was normalized by COL treatment (Figure 7C).
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Discussion |
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In the present study, we measured cellular stiffness as well as stiffness against shear deformation in both the longitudinal and transverse planes. From all of these measurements, we found that changes in the microtubule density induced by drug interventions had marked influences on the shear stiffness in the longitudinal plane. In animal model of heart failure, we also demonstrated that the elevated level of microtubule proliferation in pathological condition was associated with an increase in shear stiffness in longitudinal plane, which was normalized by COL treatment.
Because microtubules have been implicated in many pathological conditions of the heart, such as cardiac hypertrophy, heart failure, and ischemia,5,6,28 many researchers have studied their roles in determining the mechanical properties.11–13,29,30 These studies by applying either stretch or anisoosmotic stress to the myocyte or muscle preparations found no significant change in the passive stiffness of the myocardium11,12,29,30 but only found an effect on viscosity of the microtubule proliferation. Our measurement using carbon fiber technique confirmed these findings. Quantitative comparison of the viscosity with previous studies is difficult because various indices of viscosity have been used.12,13 However, in a similar study applying cyclic stretch to the rat papillary muscle,12 Yamamoto et al reported approximately 1.7 fold increase (estimated from their Figure 7) in the slope of the relation between viscous constant (the area of the hysteresis loop) and the strain rate by PAC treatment. The increase in slope identified in this study (&1.35) is a little smaller, but the use of different index of viscosity may account for this discrepancy.
Although the effect on the tensile stiffness has not yet been definitely identified, a few studies have suggested the mechanical role of microtubules against shear strain. Tagawa et al,13 using magnetic twisting cytometry, showed a 100% increase in the stiffness and a 300% increase in the viscosity after microtubule proliferation induced by pressure overload hypertrophy. Recently, Lammerding et al31 measured local cell stiffness and reported anisotropy in the material properties of adult mouse cardiomyocytes. Their index of local stiffness differed by a factor of 2 between the longitudinal and transverse directions. We also found that, at the baseline, the cellular shear stiffness was anisotropic in nature (also differed by factor of 2), probably reflecting the preferential distribution of the microtubule density in the longitudinal direction.25 Although the data in these studies13,31 were obtained by applying rotational shear locally using magnetic twisting cytometry, thus not translated into cellular stiffness measured in this study in a straightforward manner, they can be taken to support the present finding,
The change in cross-bridge state induced by BDM did not affect the shear stiffness. This may be a surprising result if the shear stiffness of the intact myocytes measured in this study directly reflects the property of myofibril. However, as Palmer and Ross have shown in isolated rat cardiomyocytes,32 the lateral coupling between domains of sarcomeres (myofibril) is loose, and these domains slip in response to externally applied force as if they were solid bodies connected by strings. Therefore, we consider that the shear stiffness measured in the intact myocytes mainly reflects the property of cytoskeleton connecting the myofibrils. Similarly, our index of lateral stiffness derived from the initial phase of contact might not probe the small BDM-induced change in myofibrillar stiffness of the resting myocyte.
Why do microtubules modulate only the stiffness against shear strain without changing the tensile stiffness? Gittes et al23 measured the flexural rigidity of microtubules and actin filaments to find that the rigidity of microtubules is 3 orders of magnitude greater than that of actin filament. Because the estimated tensile stiffness of a single microtubule was much greater than that of the longitudinal stiffness of the cell, they concluded that, to accommodate strain, microtubules cannot be continuous throughout the length of the cell and that sliding must occur between the filaments. Similar reasoning can be applied to the cardiomyocyte in which microtubule structure has no effect on the tensile stiffness but does affect the viscosity. On the other hand, to modulate the stiffness against shear applied either locally13 or globally (in the present study), microtubule cytoskeleton must be linked, at least weakly, and anchored to the sarcolemma. We considered that crosslinking with other compliant cytoskeletal structure, eg, actin filament, microtubules can give such mechanical properties to the cardiomyocytes. That is, tensile strain is absorbed by the compliant actin network and the microtubules serve as beams to resist compression when shear stress was applied to the myocytes. The basic idea was similar to the cellular tensegrity model, in which compression-resistant elements (microtubules) support the cell against compression generated by the surrounding tensed cable network to form a structure for mechanotransduction.1,33
We developed a simulation model to evaluate this hypothesis. We used the finite element method to model myofilaments, desmin intermediate filaments, cytoskeletal actin filaments, and microtubules as distinct structures with their respective material properties reported in the literature (supplemental Table). Because the constitutive equation of the myofibril is not available currently, we modified and used the constitutive equation of the myocardial tissue proposed by Humphrey et al.24 Titin, the major determinant of the passive tensile stiffness of cardiac myocyte at shorter sarcomere length,29 was not modeled as a distinct element, but included in the myofibril. In addition, the following assumptions were made: (1) actin only bears tension and cannot resist compression; (2) microtubules are elastic and preferentially orientated in the longitudinal direction; (3) actin filaments and microtubules are connected to form the cytoskeleton and are anchored to the sarcolemma (outermost elements) in a discrete fashion; and (4) myofilaments are interconnected transversely by desmin intermediate filaments at the Z-line (Figure 8A and 8B). Because we applied a prestretch (1%) to the actin filaments, all the microtubules were in a compressed state under the control condition (coded in blue to green in Figure 8C). Next, we simulated the effects of tensile stress and shear stress using this model. When we applied a stretch (5%) to the cell, the microtubules became rearranged, but no change in the strain status of the microtubules was observed (Figure 8D). On the other hand, application of a shear (10%) induced high compression of some of the microtubules (green to red in Figure 6E). In accordance with the experimental results, in response to a 70% reduction in the number of microtubules (181 to 52), tensile stiffness (T) and shear stiffness in the transverse plane (S(T)) did not change appreciably, whereas the shear stiffness in the longitudinal plane (S(L)) clearly decreased (Figure 6F). Furthermore, we repeated the calculations under different conditions. (1) To examine whether prestress affect these parameters, we repeated the calculation under the condition of zero prestress. (2) Young’s modulus of microtubule was raised to 1.2 GPa corresponding to a persistence length of 5200 µm.23 The changes in the final result were modest in both cases (shown in the online data supplement).
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Shear deformation of the cardiac tissue has seldom attracted the interest of researchers, probably because the muscle has been regarded as a linear force generator from the conventional physiological point of view. In reality, however, each cardiomyocyte being stretched and contracted in the complex force field of the ventricular wall undergoes significant shear deformation. Omens et al measured the 3D strain in the isolated arrested canine left ventricle and found that the shear strain reached 0.05 to 0.1 at the endocardium when a 15 mm Hg of intraventricular pressure was applied.34 Dokos et al35 measured the shear properties of passive myocardial tissue to find highly anisotropic nature of myocardium reflecting the alignment of myocytes as well as their laminar structure. Also in beating human left ventricle, release of shear deformation has been demonstrated to play a critical role in relaxation.36 Furthermore, anisotropy in the shear stiffness has also been suggested to play an important role in cardiac function.37 The present data clearly showed the importance of microtubules in determining such mechanical properties and could establish a link between the constitutive properties of each myocyte and the whole ventricle. In addition, shear stress may also serve to transmit mechanical signals to the nucleus via the microtubule network during the development of cardiac hypertrophy.
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Acknowledgments |
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This study was supported by The Vehicle Racing Commemorative Foundation (to S.S.), The Nakatani Electronic Measuring Technology Association of Japan (to S.S.), and Core Research for Evolutional Science and Technology of the Japan Science and Technology Agency (to T.H. and J.-i.O.). We thank C. Miyazawa for excellent technical assistance.
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Footnotes |
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Original received August 1, 2005; revision received November 15, 2005; accepted November 16, 2005.
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