Effects of Altered Gravity on the Cytoskeleton of ... - Springer Link

Microgravity Sci. Technol (2010) 22:45–52 DOI 10.1007/s12217-008-9103-7

Effects of Altered Gravity on the Cytoskeleton of Neonatal Rat Cardiocytes Fen Yang · Zhongquan Dai · Yingjun Tan · Yinghui Li

Received: 5 April 2008 / Accepted: 17 November 2008 / Published online: 24 December 2008 © Springer Science + Business Media B.V. 2008

Abstract As an intracellular load-bearing structure, the cytoskeleton is hypothesized to play a crucial role in gravity perception of the cell. Recent data show that the cytoskeleton, which includes actin microfilaments and microtubules, is involved in modulating both the electrical and the mechanical activities of the myocardium. The present study employed observation and quantified analyses of fluorescent images of cardiocytes under different gravity conditions. In acute gravitational change (micro- and hypergravity) induced by parabolic flight, we found disassembly of microtubules but enhanced polymerization of microfilaments, with rearrangement from G-actin to F-actin. In ground-based experiments, exposure of cardiocytes to 2×g hypergravity (centrifugation) led to increased width and number of actin fibers from 2 to 48 h, while microtubules showed no significant changes except polarization at 24 and 48 h. In contrast, exposure of cardiocytes to clinorotation led to disassembly of microtubules from 1 to 48 h, while microfilaments showed no significant changes except redistribution, which was accompanied by rounding of the cells (48 h). We assume that the sensitivity of microfilaments to hypergravity and that of microtubules to microgravity might contribute to the specific cytoskeletal changes

F. Yang · Z. Dai · Y. Tan · Y. Li (B) Laboratory of Space Cell and Molecular Biology, China Astronaut Research and Training Center, 1 Yuanmingyuan West Road, P.O. Box 5104, 100193 Beijing, China e-mail: [email protected] F. Yang e-mail: [email protected]

observed in parabolic flight. These findings indicate different sensitivity and responses of microfilaments and microtubules to different gravitational changes, which might be part of functional adaptations of the cardiocytes to altered gravitational environments. Keywords Microfilaments · Microtubules · Microgravity · Hypergravity

Introduction The eukaryotic cytoskeleton is composed of three basic types of filaments, microfilaments (MFs), microtubules (MTs), and intermediate filaments (IFs), together with hundreds of associated structural and signaling proteins. The cytoskeleton and associated proteins are interconnected and form a sensory network for mechanotransduction. Many types of cell have been tested under altered gravity conditions provided by spaceflight, parabolic flight, sounding-rocket flight and ground-based experiments such as simulated microgravity (clinorotation) and simulated hypergravity (centrifugation). In these experiments, the cytoskeleton components (MFs, MTs and IFs) were found to be redistributed and reorganized, which may mediate microgravity-induced cellular changes, including abnormal mitochondria distribution, increased apoptosis, arrested cell cycle and reduced signal transduction from focal contacts (Crawford-Young 2006; Kacena et al. 2004; Rosner et al. 2006; Searby et al. 2005). Much research on cardiovascular alterations in real or simulated microgravity has been performed at an integrated level, and some animal experiments have shown metabolic and structural changes in the myocardium

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during space flight (Goldstein et al. 1992; Philpott et al. 1990; Thomason et al. 1992), but little is known about how cardiocytes sense and respond to altered gravity. In the myocardium, the cytoskeleton and associated signaling molecules have been demonstrated to play an important role underlying the mechanisms that link biomechanical forces to the reaction of intracellular events, and they also mediate the hypertrophic and maladaptive responses of heart cells to mechanical stress (Domingos et al. 2002; Torsoni et al. 2003). The cytoskeleton itself has been implicated in modulating both the electrical activity (through ion channels and exchangers) and the mechanical (or contractile) activity of the heart (Calaghana et al. 2004). So, the cytoskeleton in cardiocytes is not only supporting structure, but also involved in crucial functions. To explore the structural and functional responses of cardiocytes to altered gravity, the cytoskeletal changes might provide some clues at cellular level. In the present study, we examined the cytoskeletal changes in cultured neonatal rat cardiac myocytes under conditions of acute gravitational change (microand hypergravity) induced by parabolic flight, and ground-based simulated microgravity (clinorotation) and hypergravity (centrifugation). By analyzing fluorescent images of microfilaments (F-actin) and microtubules (tubulin), we found that they are sensitive to gravitational changes. However, these two cytoskeletal components respond to different gravitational conditions with differential sensitivity, and their reorganization and redistribution occur in different patterns. We assume that the sensitivity of microfilaments to hypergravity and that of microtubules to microgravity might contribute to the enhanced polymerization of microfilaments and disassembly of microtubules observed in parabolic flight.

Materials and Methods Antibodies and Chemicals Polyclonal rabbit anti-β-tubulin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, California 95060, USA). Texas red (TR)-conjugated phalloidin and Alexa Fluor 488-conjugated DNase I were supplied by Molecular Probes, Invitrogen detection technologies (Eugene, OR 97402, USA). The 4 ,6-diamidino2-phenyindole (DAPI) was obtained from Roche Co. (Grenzacherstrasse 124, CH-4070 Basel, Switzerland). Cell culture products were supplied by Invitrogen, GIBCO Cell Culture Systems (Carlsbad, California 92008, USA). Serum was purchased from HyClone

Microgravity Sci. Technol (2010) 22:45–52

(Logan, UT 84321, USA). Unless stated otherwise, all chemicals were of reagent grade. Cell Culture Neonatal rat cardiac myocytes were prepared as described previously (Yonemochi et al. 2000). In brief, myocytes were isolated from 1- to 3-day-old Wistar rats and digested with 0.25% trypsin–0.02% EDTA in PBS (KH2 PO4 0.2 g/L, KCl 0.2 g/L, Na2 HPO4 ·12H2 O 2.9 g/L, NaCl 8.0 g/L, pH 7.4). The cells were resuspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), containing 2 mM glutamine, 50 U/ml penicillin, 50 μg/mL streptomycin, and 15 mM HEPES. The nonmyocardial cells were removed by differential attachment in 25 cm2 cell culture flasks applied twice for 45 min. The suspended myocytes were transferred at a density of 2 × 105 cells/mL to six-well plates containing glass coverslips and cultured at 37◦ C in a humidified atmosphere of 5% CO2 . Flight Experiments On the fifth day after seeding, the cultures were sealed into biocompatible polyethylene bags with two supporting bars (Dai et al. 2006). The medium was replaced by 2.2 mL of DMEM with 10% FBS, and the samples were then transferred to plastic boxes and positioned in an incubator at 37◦ C. The ground control samples were submitted to identical apparatus and timing conditions. The parabolic flight was performed during the 52nd parabolic flight campaign which was developed by Centre National d’Etudes Spatiales (CNES). The flight consisted of six groups of five parabolas. Each parabola lasted 3 min and included a 25 s hypergravity phase at 1.8–2.2×g (pull up), a 22 s microgravity phase at 0.03–0.05×g, and another 25 s hypergravity phase at 1.8–2.2×g (pull out). Four to 8 min of 1×g flight separated each group of five parabolas. The total flight duration was approximately 3 h. In the current CNES campaign, three parabolic flights were performed in 3 days. Within 30 min after the parabolic flight, the samples were fixed using 4% paraformaldehyde. Ground-Based Experiments For simulated microgravity and hypergravity experiments, cardiocytes were cultured in flasks with coverslips stuck onto the bottom On the fifth day after seeding, the flasks were filled with 10% FBS DMEM, the bubbles removed, and the flasks were then placed


in a 37◦ C incubator for at least 3 h to prevent the cells being affected by the closed environments. To perform clinorotation (simulated microgravity), the flasks were positioned on a clinostat (Institute of Biophysics, China Academy of Sciences) (Dai et al. 2006) and maintained under continuous rotation at 30 rpm for 1, 2, 4, 24, and 48 h. To perform centrifugation (simulated hypergravity), the flasks were positioned on a centrifuge (manufactured by our laboratory) and maintained at 134 rpm (2×g) for 1, 2, 4, 24, and 48 h. Control samples were placed in the same apparatus. Immunofluorescence After flight or ground-based experiments, cells were washed in PBS and fixed with 4% paraformaldehyde for 30 min followed by penetration with 0.1% Triton X-100 for 10 min. Nonspecific binding was blocked with 1% bovine serum albumin (BSA) for 30 min. For observation of cytoskeleton architecture, cells were incubated in turn with anti-β-tubulin antibody for 1.5 h, with FITC-labeled anti-rabbit IgG for 60 min, with TR-Phalloidin for 60 min, and with DAPI for 30 min. To observe the transformation between F-actin and G-actin, F- and G-actin were stained by TR-phalloidin and Alexa Fluor 488-DNase I, respectively. The coverslips were then mounted in Mowiol (Calbiochem, Meudon, France). Image Analyses At least three fields on each coverslip were randomly selected, and the fluorescent labels were visualized using an LSM 510 META (Carl Zeiss Inc., Oberkochen, Germany) confocal scanning laser system. The actin fiber width, number of actin fibers per cell, and the area of staining were determined using Image Pro Plus 5.0 software (MediaCybernetics Inc., Bethesda MD 208144411 USA) (Kacena et al. 2004). To analyze the height of the microtubular network, the image stacks were viewed from the top slice to the bottom slice using confocal software (Searby et al. 2005). Statistical Analyses Data were representative of three parabolic flights or separate ground-based experiments performed with three to four individual coverslips per group. Values were expressed by the mean±standard error of the mean (SE). Statistical evaluation was performed and figures produced using GraphPad Prism 4.0 (GraphPad Software Inc., San Diego, CA 92130, USA).

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Differences were compared using the t test (flight experiments) or one-way ANOVA (ground-based experiments), with significance set at p < 0.05.

Results Effects of Parabolic Flight on the Cytoskeleton of Cardiocytes Altered Organization of Microtubules and Microfilaments In this study, the effects of parabolic flight or simulated microgravity and hypergravity on the cytoarchitecture, including microfilaments (filamentous actin, F-actin), microtubules and nuclei, were visualized by multiple fluorescent staining. As shown in Fig. 1, microfilaments were stained red by Texas Red-conjugated phalloidin, which specifically binds F-actin, microtubules were stained green by specific anti-β-tubulin antibody, and nuclei were stained blue by DAPI, a fluorescent dye that binds specifically to DNA. The microfilaments in ground cells were located at the cell periphery or penetrated through the cytoplasm in linear form; the microtubules spanned large regions around the nucleus as tortuous thin filaments, and the nuclei were regularly shaped (Fig. 1a). The microfilaments in flight cells were more prominent and thick than those in ground cells (red in Fig. 1a). Quantitative analyses using Image Pro software showed that the average number of actin fibers per flight cell increased by 33.8%, and the average width of actin fibers increased by 74.3% (Fig. 1c). Not as round as those in ground cells, the nuclei in flight cells (blue in Fig. 1a) became spindly and were aligned parallel to the thick polarized microfilaments. However, there were no morphological changes related to apoptosis in the nuclei of flight cells. Microtubules (green in Fig. 1a) were uniform and continuous in ground cells, and some protrusions with long straight microtubules could be seen. In contrast, the microtubules in about 12% of flight cells were disrupted significantly (Fig. 1d). The microtubules distributed in perinuclear regions were decreased in flight cells, the straight microtubular filaments on the periphery were intermittent, and the protrusive edges became obtuse, with looped microtubules. The above findings indicate that the cytoskeleton components responded to gravitational changes in different ways: microtubules were disrupted but microfilaments showed enhanced polymerization.

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Shift in the F/G-actin Equilibrium in Favor of F-actin There is a dynamic equilibrium between filamentous (F) actin and globular monomeric (G) actin in the cell. To verify the enhanced polymerization of microfilaments (F-actin), we further observed the F/G-actin equilibrium by double fluorescence staining. As shown in Fig. 1b, F-actin was stained red by Texas-Red-

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phalloidin, and G-actin was stained green by Alexa Fluor 488-DNase I. When compared with ground cells, the flight cells demonstrated an increase of F-actin and a decrease of in G-actin. Quantitative analyses demonstrated a decrease of the area of G-actin staining in flight cells by 57.7% (Fig. 1c). Therefore, the F/Gactin equilibrium shifted to F-actin under conditions of parabolic flight.


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1 Effects of parabolic flight on the cytoskeleton architecture of cardiocytes. a Confocal images of microfilaments and microtubules. F-actin was stained red by Texas-Red phalloidin, microtubules were stained green by specific anti-β-tubulin antibody, and nuclei were stained blue by DAPI. When compared with ground cells (left), the microfilaments in flight cells (right) were more prominent and thick, and the nuclei became spindly and parallel to the thick polarized microfilaments. There were no morphological changes related to apoptosis in these flight nuclei. As to microtubules, about 12% among total cells (approximately 150–180 cells) were disrupted significantly in flight cells: the microtubules distributed in perinuclear regions were decreased, the straight microtubular filaments in the periphery were intermittent, and the protrusive edges became obtuse with looped microtubules. Bar = 10 μm for all. b Confocal images showing a shift in the F/G-actin equilibrium in favor of F-actin. Ground (left) and flight (right) cardiocytes were stained in turn by Texas-Red phalloidin and Alexa Fluor 488-DNase I to detect F-actin (red) and G-actin (green) respectively. Compared with controls, flight cells demonstrated increased F-actin (top) versus decreased G-actin (middle). The merged images (bottom) show the rearrangement of G-actin into F-actin in parabolic flight. Bar = 10 μm for all. c Quantitative analyses of confocal images by Image Pro Plus 5.0 software. When compared with the 1×g control, the actin fiber width increased by 74.3%, the number of actin fibers per cell increased by 33.8%, and the area of G-actin staining decreased by 57.7%. d Quantified analyses of microtubular disruption. The number of cells with microtubular disassembly and the total cell number (about 150–180 cells in every group) were recorded and the ratios calculated. Among ground cardiocytes, about 1.3% was found to have microtubular disruption, whereas among flight cells, the ratio is at 12% (* p < 0.05, # p < 0.01, compared with ground cells)

Microfilaments

Effects of Simulated Microgravity and Hypergravity on the Cytoskeleton of Cardiocytes

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F-actin, which is the component of microfilaments, was labeled by Texas Red-conjugated phalloidin. Representative images are shown in Fig. 2a–d; the width and number of actin fibers were quantified as shown in Fig. 2e–f. Figure 2a shows the baseline control (0 h). In

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The parabolic flight was composed of a series of acute alternating changes of micro- (0.03–0.05×g) and hypergravity (1.8–2.2×g) during 30 parabolas, and the time of changing gravity is maximal 2 h. In order to understand the cytoskeletal changes in parabolic flight, we performed ground-based experiments at simulated microgravity (clinorotation) and hypergravity (2×g centrifugation) for 1, 2, 4, 24, and 48 h.

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Fig. 2 Effects of simulated microgravity and hypergravity on microfilaments of cardiocytes. F-actin was labeled by Texas-Red phalloidin. a Baseline control (0 h). b Clinorotation at 48 h. The cells became retracted and round, which was accompanied by redistribution of the actin fibers. c Centrifugation (2×g) at 4 h. d Centrifugation (2×g) at 48 h. The actin fibers became thicker and more prominent in simulated hypergravity. e The actin fiber width in centrifuged cells began to increase significantly at 2 h (37.3%), to peak at 4 h (47.2%), and the levels at 24 and 48 h were still similar to that at 4 h. f The number of actin fibers per cell showed no change until 4 h (24.7%), and as time went on the change was more significant at 48 h (60.9%) (* p < 0.05, # p < 0.01, compared with control cells). Bar = 10 μm for all

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clinorotation experiments, there was no evident change in the images of actin fibers, and image analyses showed no statistically significant changes in actin fiber width and the number of actin fibers per cell from 1 to 24 h (data not shown). At 48 h, there was redistribution of the actin fibers to cortical regions that had undergone clinorotation, which was in concordance with the retraction and rounding of the cells (Fig. 2b). In centrifugation experiments, the cells were accelerated towards the bottom of the cell culture flasks, and the acceleration level (2×g) was equivalent to that of the parabolic flight (1.8–2.2×g). The actin fibers were sensitive to hypergravity conditions and became thicker and more prominent (representative images at 2 and 48 h are shown in Fig. 2c and d respectively). As shown in Fig. 2e, the actin fiber width in centrifuged cells began to increase significantly at 2 h (37.3%), to peak at 4 h (47.2%), and the levels at 24 and 48 h were similar to those at 4 h. The centrifugation cultures also contained more fibers per cell. In Fig. 2f, the change did not appear until 4 h (24.7%), and the change was more significant at 48 h (60.9%). In summary, exposure of cardiocytes to 2×g hypergravity (centrifugation) led to enhanced polymerization of microfilaments (actin fibers) early in the experiment (increased actin fiber width at 2 h and increased actin fiber number at 4 h), which lasts to 48 h. In contrast, simulated microgravity (clinorotation) appeared not to have significant effects on microfilaments, except that redistribution, accompanied by rounding of the cells, was seen at 48 h. Therefore, it appears that the microfilaments of cardiocytes are more sensitive to simulated hypergravity than to simulated microgravity.


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Microtubules Microtubules were visualized by specific staining of β-tubulin, which is the main structural protein in microtubules. Figure 3a show representative image of the baseline control (0 h). In clinorotation experiments, some disruptions in the filamentous network of microtubules were found as early as 1 h. As shown in Fig. 3b, in the disrupted regions, including the cell body (in the circle box) and protruding edges (in the square boxes), the filaments were discontinuous and the microtubular networks disappeared. The disruptions located at the end of the protrusions appeared to restrain the stretch of the cell and made its edge more obtuse and round. At 48 h, most cells became round without elongated protrusions (Fig. 3c). The number of cells with microtubular disassembly and the total cell number (approximately 150–180 cells in each group) were counted, and the ratios are shown in Fig. 3e. At the 1 h time point,

Fig. 3 Effects of simulated microgravity and hypergravity on the microtubules of cardiocytes. Microtubules were visualized by specific staining of β-tubulin. a Baseline control (0 h). b Microtubules in clinorotation at 4 h. The microtubular filaments were disrupted in some regions, including the cell body and protruding edges. The microtubular filaments were discontinuous and the networks disappeared. The disruptions located at the end of protrusions appeared to restrain the stretch of the cell and made its edge more obtuse and round. c Clinorotation at 48 h. Most cells became round without elongated protrusions. d Centrifugation (2×g) at 48 h. In centrifugation experiments, there were no evident structural changes in microtubules. At 24 and 48 h, microtubular filaments were straighter and more compact with prominent protrusions, which were coincident with polarization of the cells. e Quantified analyses of microtubules under simulated microgravity. The number of cells with microtubular disassembly and the total cell number (about 150–180 cells in every group) were recorded and the ratios calculated. At time 1 h, 1.3% of the cells were found to have microtubular disruption. The ratio increased as time went on and peaked at 11% at 24 h. Bar = 10 μm for all


1.3% of the cells showed microtubular disruption. The ratio increased as time went on and peaked at 11% at 24 h. There was a slight decrease at 48 h when compared with 24 h, which might have resulted from retraction of the cells and fewer protrusions. In addition, the microtubular disruption was distributed in limited regions at all time points and did not have a significant effect on the global structure of the cells. In the centrifugation experiments, there were no evident structural changes in microtubules, except that, at 24 and 48 h, microtubular filaments appeared to be straighter and more compact with prominent protrusions, which were coincident with the polarization of the cells (Fig. 3d). Image analyses of the microtubule height showed no changes at all the centrifugation time points. In summary, exposure of cardiocytes to simulated microgravity (clinorotation) leads to disassembly of microtubules at an early time point (1 h), which remains at 48 h, while 2×g hypergravity (centrifugation) appears not to have significant effects on microtubular structure at early time points (from 1 to 4 h); the polarized microtubules appeared at 24 and 48 h. So, it appears that the microtubules of cardiocytes are more sensitive to simulated microgravity than to simulated hypergravity.

Discussion In the present study we report parabolic flight-induced cytoskeletal changes in cultured cardiocytes. Groundbased simulation experiments were also carried out to give further understanding of the results of parabolic flight. In flight experiments, we found disassembly of microtubules but enhanced polymerization of microfilaments, with rearrangement from G-actin to F-actin. In ground-based experiments, we found that microtubules were more sensitive to simulated microgravity with structural disruptions as early as 1 h, whereas microfilaments were more sensitive to simulated hypergravity with increased width and number of actin fibers under centrifugation conditions as early as 2 h. It appears that the different level of sensitivity of microtubules and microfilaments to microgravity and hypergravity contributes to the specific cytoskeletal changes observed in parabolic flight. Many types of eukaryotic cells, such as osteoblasts (Hughes-Fulford et al. 2006; Kacena et al. 2004; Searby et al. 2005), neurocytes (Rosner et al. 2006; Uva et al. 2002), lymphocytes (Schatten et al. 2001) and MCF7 (Vassy et al. 2001), have been studied for their cytoskeletal changes under altered gravity conditions. Although cytoskeletal changes have been described in

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different ways in different types of cell, one consistent conclusion in these experiments is that the microfilaments and microtubules are sensitive to altered gravity. At the same time, some studies about the cells such as human SH-SY5Y neuroblastoma cells (Rosner et al. 2006), MCF-7 cells (Vassy et al. 2001) and embryonic chicken osteoblasts (Kacena et al. 2004) demonstrate that microfilaments and microtubules respond to microgravity and hypergravity in different fashions. In our initial work, we found clinorotation-induced redistribution of microfilaments and microtubules in neonatal rat cardiocytes, which is accompanied with the retraction and rounding of the cells (Xiong et al. 2003). Many models have been used to study cardiac structure and function in vitro. Although the neonatal cardiocytes have their own specific structure and function which are different from that of adult cardiocytes, isolated neonatal rat primary cardiocytes have been the most widely used models to study cardiac biology in vitro, especially in the field of cell and molecular biology. In the present study, we further confirmed that microfilaments and microtubules in neonatal rat cardiocytes respond to altered gravity by sensitive and specific fashions. The cytoskeleton of cardiocytes responded to acute gravitational changes (micro- and hypergravity) in parabolic flight with enhanced polymerization of actin fibers but disassembly of microtubules. In ground-based experiments, the cytoskeleton of cardiocytes was sensitive to simulated microgravity (clinorotation) and hypergravity (centrifugation) with some early responses at 2–4 h, although the type of reaction was different in microfilaments and microtubules. The microfilaments of cardiocytes in parabolic flight and centrifugation (2–4 h) showed similar changes with increased width and number of actin fibers, whereas the microtubules in parabolic flight and clinorotation (1–4 h) showed similar changes with microtubular disruption in some regions of the cell body and protruding edges. On the contrary, the microfilaments in clinorotation and the microtubules in centrifugation (2×g) did not show significant changes at early time points (from 1 to 4 h). We therefore assume that the sensitivity of microfilaments to hypergravity and the sensitivity of microtubules to microgravity might lead to the specific cytoskeletal changes observed in parabolic flight. Similarly, spaceflight experiments carried out on Shenzhou6 spacecraft by our laboratory demonstrated that the microtubules in neonatal rat cardiocytes were depolymerized significantly while the microfilaments were still well organized (Yang et al. 2008). All in all, in view of our findings and some other studies, it appears that microtubular organization is more sensitive to microgravity than to hypergravity, whereas the dynamics of

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microfilaments are more sensitive to hypergravity than to microgravity. The cytoskeleton, including actin microfilaments and microtubules, is important for the normal electrical and mechanical activities of the heart, such as the functions of ion channels, membrane exchangers, mechanical activity and [Ca2+]i , action potential duration and β-adrenergic signaling (reviewed by Calaghana et al. 2004). If the cytoskeleton in cardiocytes is substantially affected by sustained microgravity in spaceflight, it may lead to alterations in electrical and mechanical activities of the heart. Our working group carried out spaceflight experiments on Shenzhou-6 spacecraft and found time-dependent disassembly of microtubules but unchanged microfilaments in flight cardiocytes, which was accompanied by decreased beating and secretary functions of cardiocytes (Yang et al. 2008). In view of our flight and ground-based experiments, the cytoskeleton of cardiocytes is sensitive to altered gravity and demonstrates specific changes, but more work is needed to explore the role of the cytoskeleton in the functional changes of cardiocytes. Such researches may lead to new insights into the mechanisms underlying cardiovascular disorders that occur in spaceflight. Acknowledgements We are grateful to Mrs Guillemette Gauquelin-Koch and Mr. Jean-Baptiste behar of Centre National d’Etudes Spatiales (CNES), Mr. Gilbert Gasset and Mrs Brigitte Eche of Groupement Scientifique en Biologie et Médecine Spatiales, Université Paul- Sabatier, France for the assistance of the parabolic flight experiment in the present work. This study was supported by a grant from Advanced Space Medico-Engineering Research Project of China (SJ200706), grants from National Natural Science Foundation of China (30570452, 30600759, 30771104 and 30671076), and a grant from National Basic Research Program of China (973 Program No.460 2006CB705704).

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