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.

Keywords: Ca2+; cardiac remodelling; hypergravity; microgravity; proliferation.

Figure 1

The clinostat developed and provided by the China Astronaut Research and Training Center. A, Clinostat device. B, Internal composition of the clinostat with cell culture flasks. The columnar bottle or cell culture flasks were filled with culture medium

Figure 2

Altered spontaneous calcium signalling of HL‐1 cells following rotation‐simulated microgravity or hypergravity. A, Resting [Ca²⁺]_i and [Ca²⁺] released from the endoplasmic reticulum (ER) of HL‐1 cells in the rotation‐simulated microgravity (MG) and control (Ctrl) groups, shown by imaging cellular Ca²⁺ signals with Fluo‐4 AM. B, Basal cytosolic Ca²⁺ levels ([Ca²⁺]_i) in HL‐1 cells in the presence or absence of MG (n = 74 [Ctrl] and n = 51 [MG]). Cells were pooled from three independent experiments. C, Ca²⁺ releases from the ER ([Ca²⁺]_ER release) in HL‐1 cells in the control and MG groups (n = 66 [Ctrl] and n = 50 [MG]). Cells were pooled from three independent experiments. D, Ca²⁺ transients in HL‐1 cells in the presence or absence of MG, detected by line scanning with a confocal microscope. Scale bar: 50 μm. E, Chart shows the frequency of spontaneous calcium oscillations, which were detected by frame scanning with a confocal microscope (n = 106 [Ctrl] and n = 124 [MG]). F, Measurement of [Ca²⁺]_i and [Ca²⁺]_ER release in the 4G hypergravity and control groups. G, [Ca²⁺]_i in HL‐1 cells in the presence or absence of 4G hypergravity (n = 29 [Ctrl] and n = 25 [4G]). Cells were pooled from three independent experiments. H, [Ca²⁺]_ER release in HL‐1 cells from the control and 4G hypergravity groups (n = 26 [Ctrl] and n = 22 [MG]). Cells were pooled from three independent experiments. I, Ca²⁺ transients in HL‐1 cells in the presence or absence of 4G hypergravity, detected by line scanning with a confocal microscope. Scale bar: 50 μm. J, Chart shows the frequency of spontaneous calcium oscillations, detected by frame scanning with a confocal microscope (n = 77 [Ctrl] and n = 76 [4G]). Representative results of three independent experiments are shown. Data are shown as mean ± standard error of the mean (SEM); unpaired Student's t test, **P < .01 and ***P < .001

Figure 3

Rotation‐simulated microgravity and hypergravity activated cardiomyocyte remodelling. A, Expression of CaMKII and its phosphorylation at Thr287 (p‐CaMKII), HDAC4 and its phosphorylation at Ser632 (p‐HDAC4) and α‐MHC in HL‐1 cells. B‐D, mRNA levels of ANP, BNP and α‐MHC in HL‐1 cells. E and F, Wheat germ agglutinin (WGA) staining was used to demarcate the boundaries of HL‐1 cells following rotation for 48 h. The cell area was analysed and quantified. Scale bar: 50 μm (n = 78 [Ctrl] and n = 151 [MG]). G, Expression of p‐CaMKII, p‐HDAC4 and α‐MHC following 4G hypergravity. H‐J, Analysis of ANP, BNP and α‐MHC mRNA levels following 4G hypergravity. K and L, WGA staining was used to demarcate the boundaries of HL‐1 cells following 4G centrifugation for 48 h. The cell area was analysed and quantified. Scale bar: 50 μm (n = 256 [Ctrl] and n = 125 [4G]). CaMKII, calcium/calmodulin‐dependent protein kinase II; HDAC4, histone deacetylase 4; α‐MHC, myosin heavy chain α. ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide. Representative results of three independent experiments are shown. Data are shown as mean ± SEM; unpaired Student's t test, *P < .05, **P < .01 and ***P < .001

Figure 4

Effects of rotation‐simulated 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

Figure 5

Schematic of the potential mechanism underlying the effects of gravity on myocardial remodelling. Alteration of microgravity induces dynamic changes in intracellular calcium signalling. The increase in [Ca²⁺]_i activates the CaMKII/HDAC4 signalling pathway and regulates myocardial remodelling. CaM, calmodulin; CaMKII, calmodulin‐dependent kinase type II; HDAC4, histone deacetylase 4; MEF2, myocyte enhancer factor 2; TF, transcription factor