miR‐137 and its target T‐type CaV3.1 channel modulate dedifferentiation and proliferation of cerebrovascular smooth muscle cells in simulated microgravity rats by regulating calcineurin/NFAT pathway

Abstract Objectives Postflight orthostatic intolerance has been regarded as a major adverse effect after microgravity exposure, in which cerebrovascular adaptation plays a critical role. Our previous finding suggested that dedifferentiation of vascular smooth muscle cells (VSMCs) might be one of the key contributors to cerebrovascular adaptation under simulated microgravity. This study was aimed to confirm this concept and elucidate the underlying mechanisms. Materials and Methods Sprague Dawley rats were subjected to 28‐day hindlimb‐unloading to simulate microgravity exposure. VSMC dedifferentiation was evaluated by ultrastructural analysis and contractile/synthetic maker detection. The role of T‐type CaV3.1 channel was revealed by assessing its blocking effects. MiR‐137 was identified as the upstream of CaV3.1 channel by luciferase assay and investigated by gain/loss‐of‐function approaches. Calcineurin/nuclear factor of activated T lymphocytes (NFAT) pathway, the downstream of CaV3.1 channel, was investigated by detecting calcineurin activity and NFAT nuclear translocation. Results Simulated microgravity induced the dedifferentiation and proliferation in rat cerebral VSMCs. T‐type CaV3.1 channel promoted the dedifferentiation and proliferation of VSMC. MiR‐137 and calcineurin/NFATc3 pathway were the upstream and downstream signalling of T‐type CaV3.1 channel in modulating the dedifferentiation and proliferation of VSMCs, respectively. Conclusions The present work demonstrated that miR‐137 and its target T‐type CaV3.1 channel modulate the dedifferentiation and proliferation of rat cerebral VSMCs under simulated microgravity by regulating calcineurin/NFATc3 pathway.


| INTRODUC TI ON
Postflight orthostatic intolerance, associated with high risk to astronaut's health and performance, has been considered as one of the major adverse effects when exposed to microgravity environment, and there are still no effective countermeasures. 1,2 It has been reported that multiple mechanisms are implicated in the occurrence of postflight orthostatic intolerance, including hypovolaemia, alterations in baroreceptor reflex, decreases in exercise tolerance and aerobic fitness, and cardiovascular dysfunction. 3,4 Recently, both human studies from spaceflights and head-down tilt bed tests and animal studies from tail-suspended hindlimb-unweighting rat model revealed that functional and structural adaptation in cerebral arteries including the augmented myogenic tone, the enhanced arterial reactivity, increased media thickness and the number of smooth muscle cell layers, could be one of the fundamental causes in the postflight orthostatic intolerance, but the underlying mechanisms remain to be clarified. 5,6 Vascular smooth muscle cell (VSMC) displays a remarkable plasticity in different phenotypes, which is crucial to maintain vascular function normally. 7 The majority of VSMCs are stable in differentiated contractile phenotype in physiological conditions. These differentiated contractile VSMCs could undergo a rapid shift to dedifferentiated synthetic phenotype when exposed to environmental stimuli, which known as dedifferentiation or phenotype switching. 8 During the process of dedifferentiation, VSMC lose the contractile ability, but restart the programme of cell growth, synthesis, proliferation, migration and secretion. It has been demonstrated that dedifferentiation of VSMC is characterized by reduced expression of contractile-specific proteins, such as smooth muscle α-actin (SM-α-actin), smooth muscle myosin heavy chain (SM-MHC), smoothelin, calponin and smooth muscle protein 22α (SM22α), and increased expression of some early-response genes, including c-Fos, osteopontin (OPN) and proliferating cell nuclear antigen (PCNA). 7,8 Dedifferentiation of VSMC is an initial and key incident in series of cardiovascular physiologies and pathologies, such as angiogenesis, pregnancy, injury repair, atherosclerosis, hypertension and artery stenosis. 9, 10 We previously observed that simulated microgravity could induce dedifferentiation and proliferation of VSMCs in the basilar arteries of rats, suggesting that it may be one of the key contributors to cerebrovascular adaptation when exposed to microgravity. 11 The intracellular Ca 2+ , tightly controlled by Ca 2+ channels and transporters, is an important messenger in VSMC dedifferentiation. 12 One of the most important components of Ca 2+ signalling in VSMC is the extracellular Ca 2+ influx through voltage-dependent Ca 2+ channels (VDCCs). The high-voltage activated L-type (large or long-lasting) and low-voltage activated T-type (tiny or transient) Ca 2+ channels are the two main types of VDCCs in VSMC. 12,13 L-type VDCCs has been reported to be significantly increased in differentiated contractile VSMCs and decreased in dedifferentiated synthetic VSMCs and suppressed the dedifferentiation of VSMC. 13,14 Our previous study demonstrated that simulated microgravity could upregulate the expression of L-type VDCCs in dedifferentiated synthetic VSMCs of rat cerebral arteries, 15 suggesting there are other Ca 2+ signal pathways responsible for the dedifferentiation of cerebral VSMCs under simulated microgravity. Recent studies revealed that T-type VDCCs were increased in dedifferentiated synthetic VSMCs. 8, 16 We also found that the expression of T-type VDCCs were upregulated in dedifferentiated cerebral VSMCs of simulate microgravity rats. However, the role of T-type VDCCs in modulating the dedifferentiation of VSMC has not been elucidated up to now.  17 Moreover, a series of specific microRNAs (miRNAs) have also been identified as the upstream signals of intracellular Ca 2+ signalling in VSMC dedifferentiation. 8,18 Interestingly, our research showed that simulated microgravity upregulated the expression of T-type VDCCs in a post-transcriptional way, indicating a potential role for miRNAs in it. In this study, we aim to investigate the role of T-type VDCCs in VSMC dedifferentiation and proliferation under simulated microgravity and its underlying mechanisms. suspension and hindlimbs-unloading to simulate the cardiovascular effects of microgravity as previously described. 15 The soleus/body weight radio was measured to assess simulated microgravity efficiency in tail-suspended (SUS) rats routinely.

| Isolation of cerebral VSMCs
Isolation of cerebral VSMCs was carried out as previously described. 15 Briefly, the cerebral arteries were carefully removed and placed in 4°C physiological salt solution (PSS). For electrophysiological using, cerebral arteries were digested for 18 minutes at F I G U R E 3 Comparisons of synthetic maker expressions in cerebral arteries of CON and SUS rats. A-H, Immunohistochemical staining for OPN and PCNA and quantitative analysis by the relative optical density, which was calculated by normalizing integrated optical density to vessel wall area (n = 6/group). I-J, The protein expression of OPN and PCNA was examined by Western blotting. GAPDH was used as loading control. Representative Western blots (I) and densitometry analysis (J) were shown (n = 10/group). K, mRNA levels of OPN and PCNA were assessed by qRT-PCR analysis. GAPDH was used for normalization (n = 10/group). Data were presented as means ± SEM. *P < .05 as compared with control (analysed by Student's t test) 37°C with solution containing 4 mg/mL papain (Biochrom), 2 mg/ mL dithioerythritol (Amresco), 1 mg/mL bovine serum albumin (MP Biomedicals) and 5 mmol/L taurine in PSS. Isolated VSMCs were stored at 4°C for use within 8 hours.

| Immunohistochemical staining
Basilar artery segments were dissected along with partial brain, embedded and sectioned. Immunohistochemical staining was then performed following protocols ZSGB-bio (ZSGB-bio) recommended. to vessel wall area. All image analyses were performed using Image Pro Plus 6.0 software.

| Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Briefly, cerebral arteries and A7r5 cells was mixed with RNAiso (Takara) and homogenized by grinding. After centrifugation, phase separation and precipitation, the resulting RNA pellet was dissolved in RNase-free water and stored at −80°C until further analysis. After reverse transcription by using Mir-X miRNA First-Strand Synthesis Kit (Takara), qRT-PCR was performed using a CFX96 (BIO-RAD) instrument and SYBR Premix Ex TaqTM (Takara) according to the manufacturer's protocol. The data were analysed via the ΔΔCt method.
The PCR primers used were listed in Table1.

| Electrophysiological recordings
Currents were recorded using the whole-cell patch-clamp technique in isolated cerebral arteries VSMCs as described previously. 19 Command-voltage protocols and data acquisition were performed with pCLAMP software (version 8.0, Axon Instruments). To account for differences in cell size, currents were normalized to Cm to obtain the current densities. Currents were filtered at 0.5 kHz and digitized at 4 kHz. Ba 2+ replaced Ca 2+ as charge carrier to increase unitary cur- to identify the properties of L-and T-type VDCC channels.

| Calcineurin phosphatase activity assay
The activity of calcineurin was determined using a calcineurin activity assay kit following the manufacturer's protocol (Genmed).
Calcineurin activity was assessed by measuring the absorbance value at 660 nm using the following formula: calcineurin activity (U/ mg prot) = [(testing tube OD value − control tube OD value)/standard tube OD value -standard blank tube OD value] × concentration of standard tube × dilution multiple of reaction system/sample protein concentration, the value in control group was set to one.

| EdU incorporation assay
Proliferation of A7r5 cells was determined by a 5-ethynyl-2-deoxyuridine (EdU) incorporation assay. EdU (RiboBio) was added at 100 μmol/L, and the cells were cultured for an additional 2 hours.
After the removal of EdU-containing medium, the cells were fixed,

| Statistical analysis
Data are expressed as means ± SEM. All experiments were performed at least in triplicates. Statistical analysis using Student's t test (two group comparison) or one-way ANOVA (multiple group comparison) was done with Graphpad Prism software (version 5.01). A P value <.05 was considered to be statistically significant.

| Chemicals and reagents
All buffers, chemicals and reagents were purchased from Sigma-Aldrich unless otherwise stated.

| General data
As shown in Table. 2, there was no significant difference in the final body weights between CON and SUS rats, indicating a normal growth rate during simulated microgravity. The ratio of soleus/body weight significantly decreased in SUS rats as compared with that in CON rats, which suggested the deconditioning effects of simulated microgravity.

| Simulated microgravity induced a dedifferentiation from contractile to synthetic phenotype in rat cerebral VSMCs
As shown in Figure 1, cerebral VSMCs in CON rats showed a typical

| Simulated microgravity activated T-type Ca V 3.1 channel in rat cerebral VSMCs
As shown in Figure 4A-B, the protein expressions of T-type Ca V 3.2 channel in cerebral arteries did not show significant change between CON and SUS rats. However, the protein ( Figure 4A-B) and mRNA ( Figure 4C) expressions of T-type Ca V 3.1 channel in cerebral arteries of SUS rat significantly increased compared with that of CON rats. As shown in Figure 4D, there was a significant difference between the HP =−80 mV and HP =−40 mV recordings, which indicated that T-type VDCCs currents could be recorded at a more hyperpolarized holding potential, consistent with other reports. 22 In the present work, T-type VDCCs currents in our experimental system ( Figure 4F). As compared with that of CON rats, the current densities of T-type VDCCs in cerebral VSMCs of SUS rats were significantly increased.
These results indicated that simulated microgravity increased the expression and function of T-type Ca V 3.1 channel in cerebral VSMCs.

| T-type Ca V 3.1 channel promoted VSMC dedifferentiation and proliferation
During the process of serum-induced dedifferentiation in cultured A7r5 cells, the mRNA expression of contractile marker (SM-MHC) significantly decreased, whereas synthetic marker (OPN) markedly increased in a time-dependent manner ( Figure 5A, B). Accordingly, the mRNA expression of T-type Ca V 3.1 was also continuously increased during this process ( Figure 5C). Interestingly, whether silencing T-type Ca V 3.1 channel by specific siRNA oligonucleotide ( Figure 5D, F, G) or blocking it by 5 μmol/L mibefradil (Figure 5E F I G U R E 6 T-type Ca V 3.1 channel promoted VSMC dedifferentiation by activating calcineurin-NFATc3 signalling pathway. A, Calcineurin phosphatase activity in cerebral arteries of CON and SUS rats (n = 6/group). B-C, Expression of cytoplasmic/nuclear NFATc1-c4 in cerebral arteries of CON and SUS rats were assessed by Western blotting. GAPDH was used as cytoplasmic protein loading control. Histone-H3 was used as nuclear protein loading control. Representative Western blots (B) and densitometry analysis (C) were shown (n = 6/group). D, Calcineurin phosphatase activity in A7r5 cells after 48 h siCa V 3.1 transfection (n = 3/group). E, Expression of cytoplasmic and nuclear NFATc3 in 48 h siCa V 3.1 transfected A7r5 cells were assessed by Western blotting. GAPDH/Histone-H3 was used as loading control (n = 3/ group). F, Calcineurin phosphatase activity in A7r5 cells after 48 h mibefradil treatment (n = 3/group). G, Expression of cytoplasmic and nuclear NFATc3 in 48 h mibefradil treated A7r5 cells were assessed by Western blotting. GAPDH/Histone-H3 was used as loading control (n = 3/group). Data were presented as mean ± SEM. *P < .05 vs control (analysed by Student's t test) | 13 of 18 ZHANG et Al.

| Calcineurin/NFATc3 was the downstream signalling of T-type Ca V 3.1 channel in VSMC dedifferentiation and proliferation
To investigate the downstream signalling of T-type Ca V 3.1 channel, Ca 2+ -dependent calcineurin/NFAT was examined in cerebral arteries of rats. As shown in Figure 6A, calcineurin phosphatase activity was significantly increased in cerebral arteries of SUS rats as compared with that in CON rats. Considering nuclear translocation is required to activate NFAT-dependent transcription, cytoplasmic and nuclear NFATc1-c4 protein were extracted and quantified. Results showed that there was no significant difference about both cytoplasmic and nuclear protein expression of NFATc1 and NFATc4 in cerebral arteries between SUS rats and CON rats ( Figure 6B, C). NFATc2 protein expression in cerebral arteries of SUS rats and CON rats was too low to be detected. Interestingly, NFATc3 in cerebral arteries of SUS rats markedly decreased in cytoplasmic fractions, whereas significantly increased in nuclear fractions as compared with that in CON rats ( Figure Figure 7A).

| miR-137 was a negative regulator in VSMC dedifferentiation and proliferation
By RT-PCR analysis, miR-137 was found to be the most significantly decreased miRNA among candidate miRNAs in cerebral arteries of SUS rats as compared with that in CON rats ( Figure 7B). More samples were used to further confirm that miR-137 was significantly downregulated in dedifferentiated cerebral VSMCs of SUS rats in vivo ( Figure 7C) and in serum-induced dedifferentiated A7r5 cells in vitro ( Figure 7D). As shown in Figure 7E

| miR-137 modulated VSMC dedifferentiation and proliferation by targeting T-type Ca V 3.1 channel
Dual-luciferase reporter assay system was carried out by co-trans- We found that downregulation of miR-137 is a characteristic in VSMC dedifferentiation and proliferation in vivo and in vitro (Figure 7 C, D). Therefore, miR-137 inhibitor was used to assess the function of miR-137/Ca V 3.1 in VSMC dedifferentiation and proliferation. As shown in Figure 8E, F and G, downregulation of miR-137 by inhibitor transfection significantly increased the T-type Ca V 3.1 channel protein expression, accompanied with the decreased contractile markers F I G U R E 7 Identification of miR-137 as a regulator in VSMC differentiation. A, Computational prediction of miRNAs, which inhibited Ca V 3.1. B, qRT-PCR was performed to examine the mRNA levels of eightt candidate miRNAs in cerebral VSMCs of SUS rat. Normalization was to level of U6 (n = 3/group). C, qRT-PCR was performed to examine mRNA levels of miR-137 in VSMCs of SUS rats in vivo. Normalization was to level of U6 (n = 10/group). D, qRT-PCR was performed to examine mRNA levels of miR-137 in serum stimulation induced dedifferentiated A7r5 cells in vitro. Normalization was to level of U6 (n = 3/group). E-F, Expression of cytoplasmic and nuclear NFATc3 in 48 h miR-137 mimic/inhibitor transfected A7r5 cells were assessed by Western blotting. GAPDH/Histone-H3 was used as loading control. Representative Western blots (E) and densitometry analysis (F) were shown (n = 3/group). G-H, Expression of contractile (SM-MHC, SM-αactin and SM22α) and synthetic marker (OPN and PCNA) were assessed by Western blotting after 48 h miR-137 mimic (G) or inhibitor (H) transfection in A7r5 cells, respectively. GAPDH was used as loading control (n = 3/group). I-J, A7r5 cell proliferation was assessed by an EdU incorporation assay after 48 h miR-137 mimic/inhibitor transfection (n = 3/group). Representative staining images (I) and analysis (J) were shown. Data were presented as mean ± SEM. *P < .05 vs control or mock, # P < .05 vs T0h control (0 h after serum stimulation) or mimic/

| D ISCUSS I ON
Gravity or microgravity has a profound effect on the mechanical distribution of fluid within the cardiovascular system. 3 There is a hydrostatic pressure gradient from the head to feet in human upright posture due to 1 G gravity. When exposed to microgravity, the hydrostatic gradients are lost throughout the vasculature, which induced a cephalad shift in fluid distribution from the lower part of the body towards the upper body. 6 It is considered that arterial pressures around the heart level maintain relatively unchanged, but the peripheral vasculature exhibited an increased transmural pressure in the upper body and a decreased transmural pressure in the lower body during the microgravity. 3,6 The cerebral arteries would undergo adjustments due to adaptation to cerebral hypertension, which protects the down-stream microcirculation in the brain during spaceflight. However, it also contribute to postflight orthostatic intolerance. 11 It has been reported that a 28-day simulated microgravity could induce a functional adaptation including enhanced myogenic tone and vasoconstrictor reactivity in middle cerebral arteries of rats. 6,11 In addition, there was a structural adaptation including thicker wall, narrower lumen and a migration of proliferated VSMCs towards the subendothelial layer in the basilar arteries of simulated microgravity rats. 6,11 Studies demonstrated that elevated transmural pressures or mechanical stretches could act directly on VSMCs and are regarded as one of the crucial factors to activate the VSMC dedifferentiation and proliferation. 8,23 For example, spontaneous hypertension induced a partial dedifferentiation from contractile to synthetic phenotype in aortic VSMCs of rats. 24 We wonder whether the elevated transmural pressure of cerebral arteries under simulated microgravity could act on cerebral  The present work showed that T-type VDCCs currents in cerebral VSMCs of simulated microgravity rats were significantly increased,  (NFAT3) and NFAT5. 17 We found that simulated microgravity only increased nuclear translocation of NFATc3 among NFAT members that expressed in VSMC ( Figure 6B, 6). In addition, silencing or blocking of Ca V 3.1 inhibited activity of calcineurin/NFATc3 pathway ( Figure 6D-G) and suppressed VSMC dedifferentiation and proliferation ( Figure 5D-G). These results suggested that Ca V 3.  8,37 It has also been reported that miR-145 could control the expression of L-type Ca V 1.2 channel by targeting CamKIIδ and subsequently regulate the stretch-induced dedifferentiation in smooth muscle cells. 18,26 The present study showed that T-type Ca V 3. Moreover, we identified that miR-137 could directly bind to 3'UTR of T-type Ca V 3.1 and downregulate its expression by using dual-luciferase report assay and the gain-and loss-of-function approaches ( Figure 8A-D). The studies about miR-137 showed that it is related to the regulation of cell proliferation and differentiation in neural cells, embryonic stem cells and various human cancer cells. [38][39][40] For example, miR-137 has been reported to target cell division cycle 42 to decrease proliferation, invasion and G0/G1 cell cycle progression in colorectal cancer cells. 40 However, little is known about the role of miR-137 in cardiovascular disease. In the present work, we found that inhibition of miR-137 markedly promoted NFATc3 nuclear translocation, cell dedifferentiation and proliferation of cultured VSMCs, whereas the opposite effect was obtained through the overexpression of miR-137 ( Figure 7F, H). Finally, we demonstrated that blockage of T-type Ca V 3.1 channels by mibefradil could alleviate the dedifferentiation and proliferation of VSMC induced by miR-137 downregulation ( Figure 8E-G). These results validated that miR-137/Ca V 3.1 axis was involved in the VSMC dedifferentiation and proliferation.
As summarized in Figure 9, we demonstrated that miR-137 and its target T-type Ca V 3.1 channel modulate dedifferentiation and proliferation of cerebral VSMCs in simulated microgravity rats by regulating calcineurin/NFATc3 pathway. This finding provides a novel mechanism of microgravity-induced cerebrovascular adaptation and contributes to developing novel approaches as effective countermeasures against microgravity exposure. In particular, our study also has implications for VSMC dedifferentiation and proliferation in general conditions.

ACK N OWLED G EM ENTS
This work was supported by the National Natural Science No. 31270904.

CO N FLI C T O F I NTE R E S T
The authors declare no competing financial 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 from the corresponding author upon reasonable request.