Alteration of calcium signalling in cardiomyocyte induced by simulated microgravity and hypergravity

Abstract Objectives Cardiac Ca2+ signalling plays an essential role in regulating excitation‐contraction coupling and cardiac remodelling. However, the response of cardiomyocytes to simulated microgravity and hypergravity and the effects on Ca2+ signalling remain unknown. Here, we elucidate the mechanisms underlying the proliferation and remodelling of HL‐1 cardiomyocytes subjected to rotation‐simulated microgravity and 4G hypergravity. Materials and Methods The cardiomyocyte cell line HL‐1 was used in this study. A clinostat and centrifuge were used to study the effects of microgravity and hypergravity, respectively, on cells. Calcium signalling was detected with laser scanning confocal microscopy. Protein and mRNA levels were detected by Western blotting and real‐time PCR, respectively. Wheat germ agglutinin (WGA) staining was used to analyse cell size. Results Our data showed that spontaneous calcium oscillations and cytosolic calcium concentration are both increased in HL‐1 cells after simulated microgravity and 4G hypergravity. Increased cytosolic calcium leads to activation of calmodulin‐dependent protein kinase II/histone deacetylase 4 (CaMKII/HDAC4) signalling and upregulation of the foetal genes ANP and BNP, indicating cardiac remodelling. WGA staining indicated that cell size was decreased following rotation‐simulated microgravity and increased following 4G hypergravity. Moreover, HL‐1 cell proliferation was increased significantly under hypergravity but not rotation‐simulated microgravity. Conclusions Our study demonstrates for the first time that Ca2+/CaMKII/HDAC4 signalling plays a pivotal role in myocardial remodelling under rotation‐simulated microgravity and hypergravity.


| INTRODUC TI ON
Altered gravity conditions, such as micro-and hypergravity, have different effects on living beings at various levels of organization, including changing the biophysical properties of a single cell up to the level of the entire organism. [1][2][3][4][5] The human cardiovascular system has adapted to the 1G gravity on Earth. Changes in gravity can modulate the structure and morphology of the heart. Exposure to the microgravity environment of space leads to cardiac atrophy and a decline in cardiac function. Studies of head-down-tilt bed rest, shown to be a useful and reliable model for many of the physiological effects induced by human spaceflight, have demonstrated that the human heart atrophies at a rate of approximately 1% per week in the absence of countermeasures. 6,7 Goldstein et al 8 found that the cross-sectional area of myofibrils of papillary and ventricular muscles was decreased, and the myocardium had atrophied in rats after space flight. Hindlimb unloading (HU) of rodents has been used as a ground-based model to mimic the effects of microgravity. Our previous study demonstrated that the phosphorylation levels of histone deacetylase 4 (HDAC4) were increased in the hearts of mice after 28 days of HU-simulated microgravity. Phosphorylation of HDAC4 causes its relocalization to the cytoplasm and activation of myocyte enhancer factor 2 (MEF2) and cardiac remodelling genes, such as atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in cardiomyocytes. [9][10][11] Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) activates transcriptional regulators directly by phosphorylating HDAC4.
Intracellular Ca 2+ levels also play an important role in the regulation of cardiac remodelling. Ca 2+ functions through the Ca 2+ binding protein calmodulin (CaM) to activate CaMKII, which is activated by different pathological processes in the heart. This Ca 2+ -CaMKIIdependent gene regulation during cardiac remodelling suggests novel strategies for Ca 2+ -CaMKII-dependent "transcriptional therapies" to control cardiac gene expression and function. 9 In general, much less is known about the effects of hypergravity on the heart.
In one study, heart mass was significantly increased in hypergravity-exposed mice compared with a 1 G control group, 12 and our previous study indicated that hypergravity induced differentiation of bone marrow mesenchymal stem cells into cardiomyocytes. 13 However, with the exception of these studies, little is known of the mechanisms regulating the alteration of cardiomyocytes induced by hypergravity.
Ca 2+ is a highly versatile intracellular signal that regulates many different cellular processes. 14 Dynamic cardiac Ca 2+ signalling plays an essential role in regulating cardiac functions, including cardiac contraction, relaxation and remodelling. 15 Under conditions of microgravity, urinary calcium excretion is increased, intestinal calcium absorption is decreased, serum calcium is increased, and the overall calcium balance in the body is disrupted. 16,17 Microgravity can also lead to disrupted calcium homoeostasis in cardiovascular cells. 18  In vitro studies have demonstrated that space flight and simulated microgravity induce significant changes in gene expression patterns, [24][25][26] autophagy, 27 cell migration, 28,29 extracellular matrix composition 30 and the cytoskeleton. 31 The clinostat is widely used for space biology research, as it can simulate the effect of microgravity on cells. [32][33][34][35] Although HL-1 cells derive from atrial myocytes, they maintain the ability to contract and retain differentiated cardiac morphological, biochemical and electrophysiological properties. 36 HL-1 cells have thus proven useful as a model for studying contracting cardiomyocytes, because of their organized structure and ability to contract in culture 37 ; HL-1 cells have been used in many studies of myocardial remodelling. [38][39][40][41] Here, we report that calcium signalling plays a pivotal role in regulating gravity alteration-induced cardiac remodelling through the Ca 2+ /CaMKII/HDAC4 signalling pathway.

| Rotation-simulated microgravity
To simulate the effects of microgravity, we used a two-dimensional (2D) clinostat, which was developed and provided by the China Astronaut Research and Training Center ( Figure 1A). HL-1 cells were incubated in 25 cm 2 cell culture flasks or plated on 25-mm glass coverslips and filled with culture medium ( Figure 1B). To avoid the influence of shear stress, all culture flasks were filled with medium to eliminate air bubbles and hermetically closed during rotation. The cells were rotated around a horizontal axis at a speed of 30 rpm, which resulted in randomization of the gravitational vector. It was equivalent to the microgravity of low earth orbit (about 0.01 g). The control group was cultured in the same manner as the experimental group, but without clinorotation. The 2D clinostat has been described previously. 42

| Hypergravity centrifuge
A hypergravity centrifuge was used to detect the effects of hypergravity on cells. The details of the procedure were the same as described above for rotation-simulated microgravity. The hypergravity centrifuge was continuously rotated under the 4G hypergravity condition at 37°C for 48 hours. The hypergravity centrifuge has been described previously. 13

| Measurement of intracellular Ca 2+
A calcium indicator was used to measure intracellular Ca 2+ levels and dynamic calcium signalling, as described previously. 43 Briefly,

| RNA extraction and real-time PCR
Total RNA from HL-1 cells was extracted with TRIzol Reagent (Invitrogen) as the manufacturer's instructions. RNA (0.5 μg) was reverse transcribed with PrimeScript RT reagent Kit (TaKaRa) according to the manufacturer's instructions. cDNA was used for detecting mRNA expression by quantitative PCR using SYBR ® Premix Ex TaqTMII Kit (TaKaRa). Primers used in this study were as follows:  Images were acquired using a confocal microscope (LSM 710; Zeiss). Cell size analysis was performed with ImageJ software (NIH).

| Statistical analysis
All quantitative data are presented as the mean ± standard error of the mean. Data were generated from three independent replicates.
Statistical differences among groups were analysed by one-way analysis of variance (ANOVA) with a post hoc test applied. All statistical analyses were performed with Prism software (version 6.0; GraphPad Software Inc). Statistical significance was evaluated using unpaired Student's t test or one-way ANOVA for multiple samples. Differences were considered significant at *P < .05, **P < .01, and ***P < .001.

| Rotation-simulated microgravity inducted altered spontaneous calcium signalling
To

| Altered spontaneous calcium signalling was triggered by hypergravity
Astronauts experience hypergravity when travelling in space. We analysed intracellular calcium signalling in HL-1 cells following centrifugation under the 4G hypergravity treatment. Basal cytosolic Ca 2+ and Ca 2+ released from the ER were measured ( Figure 2F).
[Ca 2+ ] i was increased significantly in HL-1 cells under the 4G treatment ( Figure 2G). In addition, more calcium was released from the ER in HL-1 cells under 4G hypergravity, suggesting that ER Ca 2+ stores are higher under conditions of hypergravity ( Figure 2H). Using the line scan mode of the confocal microscope, we determined that calcium transients were increased in cells exposed to 4G hypergravity ( Figure 2I); moreover, the frame scan mode showed more spontaneous calcium oscillations in these cells ( Figure 2J). These observations indicate that intracellular and ER calcium levels are both increased markedly under 4G hypergravity.

| Rotation-simulated microgravity induced cardiomyocyte atrophy
The changes in cytosolic calcium concentration caused by calcium oscillations can encode complex and diverse signals, enabling calcium ions to regulate specific downstream pathways. 14 To explore the effect of calcium oscillations on cardiomyocyte remodelling following clinostat-simulated microgravity, changes in signalling associated with cardiomyocyte remodelling, and in embryonic gene expression, were assessed. Western blotting revealed that the phosphorylation of CaMKIIδ (Thr287) and HDAC4 (Ser632) in HL-1 cells was increased significantly following rotation-simulated microgravity ( Figure 3A). Cell size was measured after 48 hours of rotation in the clinostat.
WGA staining showed demarcation of cell boundaries, and cell size analysis revealed that HL-1 cells were significantly smaller and atrophied after 48 hours of clinostat rotation ( Figure 3E-F); these effects could be prevented by treatment with siRNA-CaMKII ( Figure S1B).
These results demonstrate that rotation-simulated microgravity can lead to cardiomyocyte atrophy. and HDAC4 (Ser632) was increased significantly after hypergravity ( Figure 3G); these increases were inhibited by treatment with siRNA-CaMKII ( Figure S2A), demonstrating that the CaMKII/HDAC4 pathway was activated by hypergravity in cardiac myocytes. qPCR analysis showed that expression of the foetal genes ANP and BNP was increased significantly, indicating myocardial remodelling ( Figure 3H,I).

| Hypergravity induced cardiomyocyte hypertrophy
Expression of α-MHC was also increased after 48 hours of hypergravity ( Figure 3G,J), further suggesting that hypergravity resulted in the activation of signalling associated with cardiomyocyte remodelling.
To uncover the effects of hypergravity on cardiomyocytes, WGA staining was performed. HL-1 cell size was increased significantly after 48 hours of hypergravity ( Figure 3K,L), which could be prevented by siRNA-CaMKII ( Figure S2B). Thus, the CaMKII/HDAC4 pathway is also involved in hypergravity-induced cardiac myocyte hypertrophy.

| Rotation-simulated microgravity did not affect the proliferation of HL-1 cells
To determine the influence of microgravity on the proliferation of HL-1 cells, cell count, Western blotting and qPCR analyses were performed to assess changes in proliferation-related markers following rotation-simulated microgravity. As shown in Figure 4A, compared with the control group, the cell number did not change in the microgravity group after 48 hours of rotation. The qPCR results showed that expression of the cell cycle marker genes PCNA, CyclinD1 and C-fos did not change following rotation ( Figure 4B-D). We also analysed changes in the phosphorylation of mammalian target of rapamycin (mTOR), which is involved in protein synthesis. The relative levels of phosphorylated mTOR (p-mTOR)/mTOR and PCNA were also unchanged ( Figure 4E), indicating that rotation-simulated microgravity did not affect HL-1 cell proliferation.

| Hypergravity increased HL-1 cell proliferation
As shown in Figure 4F, the cell number was increased significantly in the hypergravity group ( Figure 4F). qPCR analysis showed that expression levels of the cell cycle marker genes PCNA, CyclinD1 and C-fos were also significantly increased following hypergravity treatment ( Figure 4G-I). We also analysed changes in mTOR phosphorylation and PCNA protein levels. The relative level of p-mTOR/mTOR was increased significantly, indicating increased protein synthesis ( Figure 4J). These results suggest that hypergravity increases the proliferation of HL-1 cells.  48 In this study, 48-hour 4G hypergravity and rotation-simulated microgravity were the altered gravity conditions.

| D ISCUSS I ON
The heart undergoes continual remodelling in response to fluctuations in functional demand. Pathological hemodynamic overloading (eg, hypertension and myocardial infarction) 49,50 and unloading (eg, prolonged bed rest and ventricular assist devices) 51 induce pathological hypertrophy and atrophy, respectively. Although cardiac atrophy has distinct phenotypes compared with hypertrophy, it leads to a strikingly similar decline of cardiac function and upregulation of cardiac remodelling marker genes. 52 Moreover, altered gravity affects the structure and morphology of heart tissue. 12 As a consequence of hypergravity, heart mass was significantly increased in mice 12 and Well-characterized signalling molecules that regulate cardiac remodelling include CaMKII and HDAC4. 11,54 CaMKII activation, and its ability to regulate class II histone deacetylases such as HDAC4 and their nuclear shuttling, represents a critical Ca 2+ -dependent signalling circuit involved in cardiac hypertrophy and heart failure. 55 We previously reported that both simulated microgravity and pressure overload (transverse aortic constriction) induced phosphorylation of HDAC4 and led to cardiac remodelling in mice. 11 signals regulate contraction and also a host of other cellular processes including gene regulation, cellular growth and death. 58,59 Recent studies have uncovered that the magnitude and temporal signature of Ca 2+ signals is critical, as is the cellular localization of these signals. 60 Two types of Ca 2+ channels, the voltage-gated L-type Ca 2+ channels, which control Ca 2+ influx elicited by action Besides, cell proliferation plays a major role in maintaining cardiomyocyte homoeostasis. 65 Though adult cardiomyocyte has lost the ability to entry cell cycle, cardiomyocyte from embryonic and neonatal mammal is capable of proliferating. 66 Prior experiments have shown that externally applied forces result in increased proliferation in an E-cadherin force-dependent manner. 67 In this study, we demonstrated that simulated microgravity and hypergravity can influence the cardiomyocyte proliferation in different extent.
Rotation-simulated microgravity did not affect the proliferation of HL-1; however, the cell proliferation marker genes, PCAN and C-fos increased in hypergravity, and the level of p-mTOR/mTOR increased too. This study indicated that hypergravity increased the proliferation and protein synthesis of HL-1.

F I G U R E 4
Effects of rotationsimulated microgravity and hypergravity on HL-1 cell proliferation. A, Analysis of cell number following microgravity. B-D, mRNA levels of PCNA, C-fos and CyclinD1 were analysed by qPCR. E, Expression levels of mammalian target of rapamycin (mTOR), phosphorylated mTOR at Ser1248 (p-mTOR) and PCNA in HL-1 cells. F, Analysis of cell number following exposure to 4G hypergravity. G-I, mRNA levels of PCNA, C-fos and CyclinD1 were analysed after 4G centrifugation for 48 h. J, Expression of p-mTOR and PCNA in HL-1 cells treated with 4G hypergravity.
Representative results of three independent experiments are shown. Data are shown as mean ± SEM; unpaired Student's t test, *P < .05 and **P < .01 We have reported for the first time that gravity induced changes in calcium signalling in HL-1 cardiomyocytes ( Figure 5); furthermore, these changes altered the response of HL-1 cells to proliferation and remodelling. Consistent with previous reports, our study indicates that simulated microgravity leads to cardiomyocyte atrophy and that hypergravity promotes the proliferation of cardiomyocytes. Few studies have focused on the molecular mechanisms through which changes in gravity induce phenotypic alterations of cardiomyocytes.
Here, we show that calcium signalling was increased to a greater by hypergravity compared with microgravity, which may underlie the difference in extent of cardiomyocyte remodelling between these gravity states.

ACK N OWLED G EM ENTS
We would like to thank HP Cheng from Peking University for the intra-

CO N FLI C T O F I NTE R E S T
The authors declare no commercial or financial conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author (yingxianli@aliyun.com).