The International Journal of Biochemistry & Cell Biology 59 (2015) 21–29

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Involvement of BK channel in differentiation of vascular smooth muscle cells induced by mechanical stretch Xue-Jiao Wan a , Hu-Cheng Zhao b , Ping Zhang a , Bo Huo c , Bao-Rong Shen a , Zhi-Qiang Yan a , Ying-Xin Qi a,∗ , Zong-Lai Jiang a a

Institute of Mechanobiology & Medical Engineering, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, China Lab of Biomechanics, Department of Engineering Mechanics, Tsinghua University, Beijing, China c School of Aerospace Engineering, Beijing Institute of Technology, Beijing, China b

a r t i c l e

i n f o

Article history: Received 7 August 2014 Received in revised form 13 November 2014 Accepted 25 November 2014 Available online 4 December 2014 Keywords: Vascular smooth muscle cells Differentiation Mechanical stretch BK channel Endoplasmic reticulum stress

a b s t r a c t The differentiation of vascular smooth muscle cells (VSMCs), which are exposed to mechanical stretch in vivo, plays an important role in vascular remodeling during hypertension. Here, we demonstrated the mechanobiological roles of large conductance calcium and voltage-activated potassium (BK) channels in this process. In comparison with 5% stretch (physiological), 15% stretch (pathological) induced the de-differentiation of VSMCs, resulting in significantly decreased expressions of VSMC markers, i.e., ␣-actin, calponin and SM22. The activity of BK channels, assessed by patch clamp recording, was significantly increased by 15% stretch and was accompanied by an increased alternative splicing of BK channel ␣-subunit at the stress axis-regulated exons (STREX). Furthermore, transfection of whole BK or STREXdeleted BK plasmids revealed that STREX was important for BK channels to sense mechanical stretch. Using thapsigargin (TG) which induces endoplasmic reticulum (ER) stress, and xbp1-targeted siRNA transfection which blocks ER stress, the results revealed that ER stress was contribute to stretch-induced alternative splicing of STREX. Our results suggested that during hypertension, pathological stretch may induce the ER stress in VSMCs, which affects the alternative splicing and activity of BK channels, and subsequently modulates VSMC differentiation. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Vascular smooth muscle cells (VSMCs) in mature vessels are highly differentiated cells whose principal functions are contraction and regulation of blood vessel tone-diameter (Owens et al., 2004). There is strong evidence that the de-differentiation of VSMCs, defined by phenotypic transformation, abnormal proliferation, migration and synthetic capacity, contributes to pathogenesis of vascular remodeling (Parizek et al., 2011). It had been revealed that pathologically increased mechanical stretch, caused by the repetitive deformation of cells as arterial wall rhythmically distends and relaxes with blood pressure (Pfisterer et al., 2012), plays an important role in vascular remodeling during hypertension (Hoefer et al., 2013). However, the mechanism by which

∗ Corresponding author at: Institute of Mechanobiology & Medical Engineering, School of Life Sciences and Biotechnology, P.O. Box 888, Shanghai Jiao Tong University, 800 Dong Chuan Road, Minhang, Shanghai 200240, China. Tel.: +86 21 34204863; fax: +86 21 34204118. E-mail address: [email protected] (Y.-X. Qi). http://dx.doi.org/10.1016/j.biocel.2014.11.011 1357-2725/© 2014 Elsevier Ltd. All rights reserved.

mechanical stretch modulates differentiation/de-differentiation of VSMCs remains to be elucidated. Contractile proteins, such as ␣-actin, SM22␣, calponin, and myosin heavy chain, have been shown to be specific differentiation markers and have the ability to maintain the contractility of VSMCs (Owens et al., 2004). Previous research found that 10% physiological stretch promotes VSMC differentiation through up-regulating of smooth muscle myogenesis (Qu et al., 2007), while pathological stretch induces VSMC de-differentiation with a concomitant downregulation of contractile protein expression (Owens et al., 2004). It has been revealed that the increase of intracellular calcium initiates VSMC contraction, differentiation and proliferation (Kudryavtseva et al., 2013), which suggests that calcium may be a crucial molecule, and involved in the stretch-induced VSMC differentiation. It has been proved that voltage gated calcium channels, including L-type and T-type, play important roles in VSMC differentiation via modulation of calcium influx (Kuhr et al., 2012; Kudryavtseva et al., 2013). The down-regulation of L-type calcium channels is accompanied with VSMC de-differentiation (Gollasch et al., 1998). T-type channels inactivation are faster than L-type channels, and Kuhr et al. (2012) proved that calcium influx through T-type

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channels is associated with VSMC proliferation and differentiation. Beside the calcium influx via calcium channel, the calcium homeostasis is also regulated by calcium releasing from endoplasmic reticulum (ER), the crucial intracellular calcium store (Galva et al., 2012). Further researches revealed that calcium influx via calcium channels and calcium releasing from ER are both regulated by complex mechanism, including large conductance calcium and voltage-activated potassium (BK) channels (Joseph et al., 2013). BK channels are predominantly expressed in VSMCs and play important roles in modulating vascular tone to regulate blood flow (Pang and Rusch, 2009). BK channel is composed of the ␣subunit that forms the pore structure, and the accessory ␤-subunit (Sausbier et al., 2005). Increased expression of the BK channel ␣subunit inhibits proliferation of endothelial cells under shear stress application, which suggests that BK channel may be mechanosensitive (Jia et al., 2013). It has been reported that BK channels may also participate in the modulation of VSMC differentiation (Long et al., 2009). However, the roles and mechanisms of BK channels in pathological stretch-induced VSMC de-differentiation are still unclear. It has been shown that alternative splicing at the C terminal of the BK channel ␣-subunit is a major determinant of BK channel function (Ma et al., 2007). Naruse et al. (2009) revealed that stress axis-regulated exons (STREX) located at the C terminal of the BK channel ␣-subunit are necessary for chick myocyte mechanosensitivity. Therefore, we hypothesized that the alternative splicing of STREX in BK channel may contribute to the pathological stretchinduced de-differentiation of VSMCs during hypertension. In the present study, to demonstrate the roles of the activity and mechano-sensitive alternative splicing of BK channel in VSMC differentiation modulated by mechanical stretch, VSMCs were subjected to 5% and 15% stretch in vitro to mimic physiological and pathological mechanical situations, respectively (Qi et al., 2010). The effects of different stretches on the expression and activity of BK channel, as well as on the differentiation and intracellular calcium levels in VSMCs, were also examined. Furthermore, the underlying mechanism of alternative splicing in response to the pathological stretch was studied. Studying the effects of BK channel on VSMC differentiation modulated by mechanical stimuli will help to understand the pathogenesis of hypertension.

2. Materials and methods 2.1. Cell culture SD rats were euthanized with sodium pentobarbital at 120 mg/kg, and then thoracic aorta was surgically removed. Primary VSMCs were cultured from the medial portion of thoracic aortas after the removal of adventitia and endothelium as described in previous study (Qi et al., 2010). VSMCs from at least four isolations and in passages 3–8 were used for experiments. Since there is no BK channel expression on natural HEK cells, HEK transfected with whole BK channel plasmids (HEK-Slo) or STREX-deleted BK channel plasmids (HEK-STREX delete) were used to explore the role of STREX splicing on BK activity (Ma et al., 2010). HEK cells were purchased from the Beijing Cancer Hospital and were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS). Cells were grown in 95% air and 5% CO2 at 37 ◦ C. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health, and the protocol was approved by the Animal Research Committee of Shanghai Jiao Tong University.

2.2. Mechanical stretch application For western blot and RT-PCR, FX-4000T Stretch Unit (Flexercell International, USA) was used to apply cyclic stretch. Briefly, VSMCs were seeded on collagen I-coated flexible silicone bottom plates (Flexercell International, USA) at a density of 3 × 105 cells per well (9.32 cm2 ). The cells were serum starved for 24 h prior to cyclic stretch application. The following mechanical parameters were used: stretch magnitudes of 5% and 15% respectively, at a constant loading duration of 24 h and a frequency of 1.25 Hz. For patch-clamp recording and calcium imaging, cells were subjected to mechanical stretch by horizontal stretch device as previously described (Ahmed et al., 2010). Briefly, 1 ml SYLGARD 184 (Dow Corning Corporation, USA) was poured onto the surface of a cover slip for 24–48 h at room temperature to form silicone gels with smooth surfaces, and these gels were then cut into slices for cell culture (Armbruster et al., 2009). VSMCs and HEK cells were trypsinized and seeded at a density of 3 × 105 cells per silicone gel slice, which was positioned onto flexible baseplates. Cells were serum starved for 24 h and then elongated by 5% or 15% as adjusted by the horizontal movement distance of the baseplates.

2.3. Plasmid transfection To study the role of BK channel in VSMC differentiation, the GFP and whole BK channel plasmids were co-transfected into VSMCs using X-TREME GENE HP DNA Transfection Reagent (Roche). Briefly, after the cells reached 70% confluence in 24-well plates, VSMCs were incubated with a transfection mixture including 1 ␮g GFP plasmid, 4 ␮g BK channel plasmid, 4 ␮l Transfection Reagent (Roche), and 100 ␮l Opti-MEM (Invitrogen). The cultures were brought to a final volume of 500 ␮l without serum and antibiotics. After transfection for 24 h, the cells were used for experiments. To study the role of STREX in BK channel subjected to mechanical stretch, GFP plasmid with whole BK channel or STREX-deleted plasmids were co-transfected into HEK cells (HEK-Slo or HEK-STREX delete) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Briefly, 4 ␮g BK channel plasmid and 1 ␮g GFP plasmid or 4 ␮g STREX-deleted plasmid and 1 ␮g GFP plasmid were mixed with 5 ␮l Lipofectamine 2000 (Invitrogen) diluted in 100 ␮l Opti-MEM (Invitrogen) without serum and antibiotics. The mixture was added onto the cells in 6-well plates to a final culture volume of 800 ␮l. After transfection for 24 h, the cells were trypsinized, seeded on silicone gels and allowed to attach for 24 h prior to mechanical stretch.

2.4. RNA interference VSMCs were seeded at a density of 2 × 105 cells per well on Flexercell plates and grown in 10% FBS/DMEM. After 24 h, the cells were transfected with siRNA targeting xbp1 using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. Briefly, 100 nmol siRNA and 5 ␮l Lipofectamine 2000 (Invitrogen) were diluted in Opti-MEM (Invitrogen) without serum and antibiotics. After mixing for 20 min at room temperature, the mixture was added dropwise to a final volume of 800 ␮l, and the cells were incubated at 37 ◦ C in a humidified incubator. After 24 h incubation, the interference mixture was removed, and complete culture medium was added. Non-silencing siRNA was used as a mock control. The sequences of the double stranded siRNA targeting xbp1 were 5 GCUG UUGC CUCU UCAG AUUT T-3 and 5 -AATC TGAA GAGG CAAC AGCT T-3 ; the sequences for the mock control were 5 -UUCU CCGA ACGU GUCA CGUT T-3 and 5 -ACGU GACA CGUU CGGA GAAT T-3 .

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2.5. Application of drugs

2.8. Western blot analysis

VSMCs were incubated with BK channel agonist NS1619 (100 ␮M) or antagonist IBTX (100 nM). To induce the ER stress, ER Ca2+ -ATPase inhibitor thapsigargin (TG, 10 ␮M) was used. All drugs were soluble in DMSO. The same amount of DMSO was added to the control group.

Cells were gently washed twice with cold PBS, scraped into lysis buffer (0.15 M Tris, pH 6.8, 1.2% SDS, 15% mercaptoethanol), and incubated on ice as previously described (Qi et al., 2010). The lysates were centrifuged at 13,000 × g for 3 min, and the supernatant was boiled for 8 min and separated on 10% SDS-polyacrylamide gels. Antibodies against the BK ␣-subunit (rabbit anti-BK ␣-subunit, ABBIOTEC, 1:500), VSMC differentiation markers (mouse anti-␣actin, Sigma, 1:1000; mouse anti-calponin, Sigma, 1:1000 and rabbit anti-SM22, Sigma, 1:1000), and ER stress marker (rabbit antixbp1, Santa Cruz, 1:500) were used and incubated at 4 ◦ C overnight. The results were analyzed using the QuantityOne imaging software (Bio-Rad). The specific protein expression levels were normalized to GAPDH expression (Santa Cruz, 1:500).

2.6. Electrophysiology Single channel inside-out patch clamp and whole-cell techniques were performed as previously described (Zhao and Wang, 2010; Qi et al., 2005). Briefly, HEK-Slo and HEK-STREX delete cells were stretched for 24 h. GFP-positive cells were then identified using a fluorescence microscope and used for the patch-clamp experiments. For single channel recording analysis, the pipette solution contained 145 mM K-gluconate, 1 mM EGTA, 10 mM HEPES, and 5 mM glucose (pH 7.4). A bath solution of the same makeup was also prepared with various Ca2+ concentrations, as previously described (Zhao and Wang, 2010). Ca2+ concentration in the solution was adjusted to the desired level by adding the corresponding concentration of CaCl2 , which was calculated by using WinMAXC software (Stanford University). For whole cell recording, the pipette solution contained 145 mM KCl, 10 mM EGTA, 10 mM HEPES (pH 7.3 with Tris), and 1 mM CaCl2 . The bath solution, Hanks’ balanced salts solution (HBSS, Sigma), contained 1.3 mM CaCl2 , 0.8 mM MgSO4 , 5.4 mM KCl, 0.4 mM KH2 PO4 , 136.9 mM NaCl, 0.3 mM Na2 PO4 , 10 mM d-glucose, and 4.2 mM NaHCO3 (Qi et al., 2005). The pipette resistance varied between 2 and 5 M. All experiments were performed at 20–24 ◦ C. The currents were analyzed using CLAMP software (version 10.0; Axon Instruments, Foster City, California, USA), and the Po -voltage and Po -calcium curves were fitted using the Boltzmann equation and Hill equation, respectively (Zhao and Wang, 2010).

2.9. Real-time RT-PCR Total RNA was extracted using TRIzol reagent (Invitrogen), and cDNA was synthesized from 2 ␮g RNA of each sample using cDNA Synthesis Kit (Fermentas). Quanti-Tect SYBR PCR kit (Bio-Rad) and iCycler (Bio-Rad 582BR) were used for real-time (RT)-PCR analysis. The sequences of STREX primers were 5 -AGCC GAGC ATGT TGTT TTGA T-3 and 5 -ACGC ACAC GGCC TGAC A-3 ; and the sequences of GAPDH primers were 5 -GGCA GCCC AGAA CATC ATCC-3 and 5 -GCCA GCCC CAGC ATCA AAG-3 . Target gene expression was normalized to GAPDH expression in each sample. 2.10. Statistical analysis Electrophysiological curve fitting and calcium imaging were analyzed using the Origin 8.0 software. All data were presented as mean ± SD. Significant differences between two groups were performed using one-way ANOVA. Statistical evaluation of the significance was defined as p-value < 0.05. 3. Results

2.7. Calcium imaging In parallel with the electrophysiology experiment, intracellular calcium response was also observed in VSMCs after stretching for 24 h using a fluorescence measurement with an inverted microscope (Olympus IX-70) as previously described (Huo et al., 2010). The silicone gels containing the VSMCs were carefully placed into 24-well plates and rinsed twice with DMEM. Then, the gels were stained with 5 ␮M Fluo-4 AM (Invitrogen) in DMSO and 0.02% Pluronic F-127 (Invitrogen) for at least 30 min at 37 ◦ C. To evaluate the effects of the drugs on the VSMCs, fluorescence intensity after drug treatment was measured and normalized to the baseline fluorescence obtained before the drug addition. Randomly selected fields of cells were recorded for 10 min as time-lapse images with 3-s intervals. The fluorescence emission was long-pass filtered at 495 nm. In this study, 40 cells were selected per dish, and each experiment included three to five dishes. The data were analyzed as the relative fluorescence within a defined region of each cell using the METAMORPH imaging software (Huo et al., 2010). Changes of fluorescence intensity were associated with cytoplasmic calcium concentration. Calcium peak represents the increased cytosolic calcium fluorescence value, and baseline represents the mean fluorescence. Relative to mean fluorescence, two-fold increase, at least, in cytosolic calcium fluorescence is defined as an occurrence of calcium oscillation; the number of calcium oscillation in 1 min is defined as the frequency of calcium oscillation (Ren and Wu, 2012).

3.1. Mechanical stretch-modulated VSMC differentiation, calcium oscillation and BK channel expression The expressions of BK ␣-subunit and VSMC differentiation markers, i.e., ␣-actin, calponin and SM22, were all significantly decreased in 15% stretch group compared with 5% stretch group (Fig. 1A). The frequency of calcium oscillation was significantly increased under 15% stretch in comparison with that under 5% stretch (Fig. 1B). The whole-cell current of BK channels was enhanced with a voltage increase in both 5% and 15% stretch groups, but this increase was much more significant in 15% than in 5% stretch group (Fig. 1C). I–V curves showed that the whole-cell current density in 15% stretch group was larger than that in 5% stretch group (Fig. 1D). In short, these results indicate that the pathological stretch (15%) induced the de-differentiation of VSMCs, and increased the activity of BK channels. It is hypothesized that BK channel may be involved in the stretch-modulated differentiation of VSMCs via the mediation of calcium oscillation, which may play an important role in vascular remodeling during hypertension. 3.2. BK activation-modulated calcium oscillation and the differentiation of VSMCs As shown in Fig. 2A, in comparison with DMSO control, the BK channel antagonist, IBTX, enhanced the frequency of calcium oscillation, whereas the BK channel agonist, NS1619, maintained

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Fig. 1. 15% stretch induced de-differentiation, BK channel expression, and intracellular calcium of VSMCs. (A) The expression levels of BK ␣-subunit, ␣-actin, calponin and SM22 were significantly decreased by 15% stretch. The left panel represents the western blot results from a typical study; the right histogram shows the densitometric quantification. *p < 0.05 vs. 5% stretch (n = 4). (B) Calcium oscillation was significantly increased under 15% stretch compared with 5% stretch, the frequency was significantly increased from 0.016 ± 0.009 Hz in 5% stretch to 0.033 ± 0.002 Hz in 15% stretch (n = 6). (C) and (D) Whole-cell BK currents of VSMCs were measured in the presence of 1 ␮M calcium. Compared with 5% stretch, the BK channel was activated by 15% stretch at a significantly greater current density (n = 4).

calcium homeostasis. The expression levels of VSMC differentiation markers were significantly increased by NS1619 treatment, but were repressed by IBTX (Fig. 2B). These results suggest that the altered activation of BK channels may regulate VSMC differentiation via modulating calcium oscillation. Furthermore, the undergoing mechanism of stretch-induced activity of BK channels was investigated.

3.3. STREX alternative splicing of the BK ˛-subunit was induced by 15% stretch STREX splicing on the BK channel was shown to modulate BK channel activity in chick myocytes (Naruse et al., 2009). Hence, STREX expression in different stretch groups was examined to identify whether STREX splicing in VSMCs was modulated by mechanical stretch. As shown in Fig. 3A, the mRNA levels of STREX

in 15% stretch group were significantly higher than those in 5% stretch group. Since there is no native BK channel in HEK cells, transfection of cloned BK channel into the HEK cells is used to detect the electrophysiological properties of cloned BK channel in patch clamp (Ma et al., 2010). To examine the effect of STREX alternative splicing on BK channel activity during mechanical stretch, HEK cells were transfected with plasmids encoding the whole BK channel (HEK-Slo) or the STREX-deleted BK channel (HEK-STREX delete) and were then subjected to different stretch applications. Whole-cell patch recordings showed that there was no detectable endogenous BK channel activity in the non-transfected HEK cells. In HEK-Slo cells, typical BK channel current was observed, which was activated by NS1619 and blocked by IBTX (Supplementary Fig. S1). Supplementary Fig. S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biocel.2014.11.011.

Fig. 2. BK activation affected calcium oscillation and VSMC differentiation. (A) Compared with the VSMCs treated with DMSO (a) and the BK opener NS1619 (b), the BK blocker IBTX (c) enhanced calcium oscillations under the static conditions. The frequency was 0.013 ± 0.007 Hz in control group, 0.010 ± 0.006 Hz in NS1619 group, and 0.026 ± 0.002 Hz in IBTX group (n = 6). (B) The expression levels of the VSMC differentiation markers were up-regulated by NS1619 and down-regulated by IBTX. *p < 0.05 vs. DMSO control, # p < 0.05 vs. NS1619 (n = 6).

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Fig. 3. STREX alternative splicing was mechano-sensitive and modulated BK channel activity. (A) STREX mRNA expression was significantly increased by 15% stretch compared with 5% stretch. *p < 0.05 vs. 5% stretch (n = 4). (B) The Po -calcium curve was fitted using the Hill equation. In HEK-Slo cells, the Kd value was 1.55 ± 0.02 mM and the Hill coefficient was 2.96 ± 0.08; in HEK-STREX delete cells, the Kd value was 2.43 ± 0.04 mM and the Hill coefficient was 1.83 ± 0.10. The differences between HEK-Slo cells and HEK-STREX delete cells were significant (n = 6). (C) According to the fits with Boltzmann equation, the Po -voltage curves of the HEK-Slo cells under the static, 5% stretch and 15% stretch conditions were significantly shifted to the left compared with those of HEK-STREX delete cells in the presence of 1 ␮M calcium, which suggested that the voltage for half-activation of BK channel between HEK-STREX delete cells and HEK-Slo cells were significantly different (n = 6). (D) Whole-cell currents of HEK-Slo cells were significantly enhanced by 15% stretch compared with that of the HEK-STREX delete cells in the presence of 1 ␮M calcium (n = 6). (E) The application of 15% stretch remarkably enhanced the current density compared with the static and 5% stretch conditions in HEK-Slo cells. In HEK-STREX delete cells, the current densities of the static, 5% stretch and 15% stretch groups were not significantly altered by stretch application (n = 6).

To examine the effect of STREX sequence on the calcium dependence of channel activity, the single-channel activities in the presence of 0.01 ␮M, 0.1 ␮M, 1 ␮M, 10 ␮M or 100 ␮M calcium were examined. Supplementary Fig. S2 shows that at +40 mV voltage, the open probability (Po ) of the BK channels in HEK-Slo cells significantly increased in comparison with that of the HEK-STREX delete cells. The Po -calcium curve was fitted using the Hill equation (Zhao and Wang, 2010), which showed the calcium concentration necessary to open half of the channels (Kd ). The Po -calcium curve of the HEK-STREX delete cells was shifted to the right in comparison with that of the HEK-Slo cells, and the slope was significantly increased in the HEK-Slo cells under condition of +40 mV membrane potential (Fig. 3B). These results suggested that the activity of BK channels was decreased in HEK-STREX delete cells compared with that in HEK-Slo cells. Supplementary Fig. S2 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biocel.2014.11.011. Inside-out patch clamp recording revealed that in the presence of 1 ␮M calcium, the Po of BK channels in HEK-Slo cells increased under 15% stretch; this increase was greater than that observed in HEK-STREX delete cells. The Po -voltage relationships were fitted using the Boltzmann equation (Zhao and Wang, 2010) to determine the half-activation voltage of the channel (Fig. 3C, Supplementary Fig. S3). The half-activation voltage in HEK-Slo cells was 0.87 ± 2.13 mV under static conditions and shifted to −13.08 ± 2.33 mV after 5% stretch and −27.30 ± 1.25 mV after 15% stretch. In HEK-STREX delete cells, the voltage was 1.61 ± 7.83 mV under static conditions and shifted to −3.61 ± 3.08 mV after 5% stretch and −20.68 ± 5.87 mV after 15% stretch. These results suggested that 15% stretch enhanced the activity of BK channels and the BK channel with STREX was more sensitive to the stretch application. Supplementary Fig. S3 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biocel.2014.11.011.

Whole-cell recordings were used to confirm the relationships between STREX and the voltage dependence of BK channel activation in HEK-Slo and HEK-STREX delete cells. Fig. 3D shows that whole-cell currents in HEK-Slo and HEK-STREX delete cells increased with depolarizing voltage steps in the presence of 1 ␮M calcium. Fig. 3E shows the mean current density versus voltage plot. In comparison with 5% stretch, 15% stretch significantly enhanced the whole-cell current density in the HEK-Slo cells, while the increase induced by 15% stretch in the HEK-STREX delete cells was very slight. These results revealed that pathological stretch induced the alternative splicing of STREX, which had a significant effect on BK channel activity. 3.4. ER stress induced alternative splicing and decreased expression of BK channel ˛-subunit under 15% stretch Since ER stress induce calcium overload which play important roles in the post-transcription process and alternative splicing (Xie, 2008), we hypothesized that ER stress may be associated with the pathological stretch-induced STREX splicing of BK channel. As shown in Fig. 4A, compared with 5% stretch, the protein expression of specific ER stress marker, xbp1, was significantly increased by 15% stretch, which suggested that the ER stress was trigged by the pathological stretch. The role of ER stress in the expression of the BK channel ␣-subunit was further elucidated. 3.5. ER stress induced STREX splicing and VSMC de-differentiation TG significantly increased the protein expression of xbp1 (Fig. 4B), which suggested that ER stress was induced (Schonthal, 2013). As shown in Fig. 5A, TG treatment repressed the expression of BK channel ␣-subunit, but increased STREX alternative splicing (Fig. 5B), which were consistent with the changes of BK channel

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channels with STREX were overexpressed in VSMCs. Fig. 6A shows that the BK ␣-subunit was significantly increased in VSMCs after transfection with the whole BK channel plasmid. The expression levels of VSMC differentiation markers, ␣-actin, calponin and SM22, were significantly higher in the BK channeloverexpressed group than that in the control group under the static, 5% stretch and 15% stretch conditions (Fig. 6B). The results suggest that the increased BK channel activity correlated with VSMC differentiation. 4. Discussion

Fig. 4. The ER stress was induced by 15% stretch and TG treatment. (A) The expression of the ER stress marker xbp1 was significantly increased following 15% stretch compared with 5% stretch. *p < 0.05 vs. 5% stretch (n = 4). (B) xbp1 expression was significantly increased following TG treatment. *p < 0.05 vs. DMSO control (n = 6).

under 15% stretch application. TG treatment also decreased the expression levels of VSMC differentiation markers in static (Fig. 5A). Xbp1 target siRNA transfection significantly down-regulated the protein expression of xbp1 (Fig. 5C), which suggested that ER stress was significantly repressed (Allagnat et al., 2010). The expression of BK ␣-subunit and VSMC differentiation markers, including ␣-actin, calponin and SM22 were significantly increased by xbp1 siRNA transfection in static (Fig. 5C). While the STREX alternative splicing was significantly decreased following xbp1 siRNA transfection in the static (Fig. 5D). These results suggested that STREX splicing were induced by ER stress, which affected BK channel activity and may subsequently induce the de-differentiation of VSMCs. 3.6. Expressions of VSMC differentiation markers were increased by the overexpression of BK channel To further identify the effects of increased BK channel activity on VSMC differentiation during mechanical stretch application, BK

This work presented the possible roles and mechanisms of BK channels in VSMC differentiation during hypertension. We found that BK channel was sensitive to mechanical stretch and was significantly activated by pathological stretch (15%) compared with normal stretch (5%), which was consistent with the observed results that the activity of BK channels were enhanced in the arteries of experimental hypertensive animals (Joseph et al., 2013). BK channel is a transmembrane protein which comprise the poreforming ␣-subunit, and the regulatory ␤-subunits. Borbouse et al. (2009) revealed that the activity of BK channel is impaired in a metabolic syndrome model, but the expression of the ␣-subunit was increased. In our study, we explored the underlying mechanical mechanism to explain the opposite effect on the expression and activation of BK channels induced by pathological stretch in VSMCs. Pathological stretch decreased the expression of the BK channel ␣subunit but increased the activity via ER stress-induced alternative splicing of STREX, which subsequently modulated BK activation and VSMC de-differentiation. Although, BK channel ␣-subunit has functions without ␤subunit (Brenner et al., 2000), the regulatory effect of ␤-subunit may play roles during hypertension. It has been proved that upexpression of BK ␤-subunits lead to a disruption of constriction and hypertension (Chang et al., 2006). However, Xu et al. (2011) reported that arterial pressure in BK ␤-subunits knock out mice

Fig. 5. The ER stress was involved in the STREX splicing of the BK channel and VSMC de-differentiation. (A) The expressions of BK channel ␣-subunit and the VSMC differentiation markers were significantly decreased in the TG treatment groups compared with the DMSO control in the static. *p < 0.05 vs. DMSO control (n = 6). (B) STREX mRNA expression in the TG treatment group was increased significantly compared with the DMSO control in the static. *p < 0.05 vs. DMSO control (n = 4). (C) The expression of ER stress marker xbp1 was significantly decreased by xbp1 siRNA transfection, while the expressions of BK channel ␣-subunit and the VSMC differentiation markers were significantly increased in the xbp1 siRNA treatment groups compared with the control groups in the static. *p < 0.05 vs. control transfected with the negative control siRNA (n = 4). (D) STREX mRNA expression was significantly decreased following xbp1 siRNA transfection in static, *p < 0.05 vs. control transfected with the negative control siRNA (n = 6).

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Fig. 6. Over-expression of the BK channel induced VSMC differentiation. (A) Expression of BK channel ␣-subunit was increased by transfection of the whole BK channel plasmid under the static, 5% stretch and 15% stretch conditions. *p < 0.05 vs. control transfected with pcDNA3.1 (n = 4). (B) The expression levels of the VSMC differentiation markers were significantly increased following transfection with the whole BK channel plasmid under the static, 5% stretch and 15% stretch conditions. *p < 0.05 vs. control transfected with pcDNA3.1 (n = 4).

is similar with that in wild-type. The roles of BK channel ␤subunits in the hypertension need to be further studied in the future. The ER stores intracellular calcium and plays important roles in controlling the alternative splicing of pre-mRNA transcripts (Xie, 2008). It had been reported that ER stress induces intracellular calcium accumulation, which causes vasoconstriction and contributes to vascular remodeling during hypertension (Dromparis et al., 2013). Our results showed that 15% stretch increased the intracellular calcium levels and enhanced the alternative splicing of STREX in the BK channel ␣-subunit. However the molecular mechanism of the alternative splicing on BK channel modulated by stretch is still unclear. Here, we speculated that ER stress may be involved in this process and subsequently induced VSMC de-differentiation. Our results showed that TG, the ER stress inducer, increased the post-translational splicing of BK channel ␣-subunit which were also induced by 15% stretch. Furthermore, the alternative splicing was decreased by the siRNA-mediated knockdown of xbp1, which is an important signaling during ER stress (Schonthal, 2013). It suggested that pathological stretch induced the splicing of BK channel may via the ER stress. It has been reported that stressors, such as altered environments and secreted hormones, trigger the STREX variants, which result in the functional diversity of BK channel (Xie, 2008). Taken together, the alternative splicing of the STREX in BK channel via the ER stress may be an important determinant of BK channel activity under different conditions. MacDonald et al. (2006) reported that the transcription of total BK channel increased with the down-regulation of STREX expression in the murine central nervous system. Ermolinsky et al. (2011) found that the mRNA level of STREX increased in rat neurons following status epilepticus, which is associated with the down-regulation of the BK channel ␣-subunit. In this study, we also found that STREX splicing in rat VSMCs was increased by pathological stretch, while the expression of BK channel ␣-subunit was decreased. Ma et al. (2007) revealed that STREX splicing affected the BK channel trafficking process. Chen et al. (2010) reported that the alternative splicing of the BK channel ␣-subunit was associated with BK channel localization as well. These results suggested that STREX alternative splicing modulates localization of BK channels which may participates in the regulation of activity and expression of BK channels. However, the molecular mechanism on the opposite changes of BK expression and STREX splicing still need further research in the future.

One function of BK channel is to regulate the level of intracellular calcium and maintain homeostasis (Hoshi et al., 2013; Joseph et al., 2013). In this work, calcium oscillation was induced by 15% stretch, along with BK channel activation. It has been proposed that the IBTX-mediated inhibition of BK channel results in VSMC depolarization and, thereby, increases the opening of the voltageactivated L-type calcium channels (Xu et al., 2012). In this study, the BK channel antagonist IBTX increased calcium oscillation and resulted in the down-regulation of VSMC differentiation markers. In contrast, BK channel agonist NS1619 significantly increased the expression of VSMC differentiation markers. It suggests that physiological stretch hyperpolarizes VSMCs, resulting in a correlated increase in BK channel activity, and negatively regulates intracellular calcium homeostasis. Calcium oscillation, which is altered by shear stress, is involved in osteoclast differentiation (Li et al., 2012). Our previous research demonstrated that certain levels of stretch are essential for maintaining VSMC differentiation (Qu et al., 2007). The present results showed that different stretch magnitudes induced inherent intracellular calcium accumulation, which suggests that pathological stretch induced VSMC de-differentiation may due to the activation of calcium signaling. It is reported that calcium signaling is important for a variety of VSMC functions, such as VSMC differentiation and vascular tone. However, it is still unknown how the changed calcium induces VSMC functions. Recent researches suggested that intracellular calcium signaling is crucial for transcription factor activity in VSMCs (Kudryavtseva et al., 2013). For example, cAMP response element-binding protein (CREB), which is activated during hypertension (Wellman et al., 2001), is phosphorylated by calcium/calmodulin-dependent protein kinase, prevented by decreasing intracellular calcium, and then modulates the expression of VSMC differentiation markers (Kudryavtseva et al., 2013). Intracellular calcium is regulated by the balance of calcium influx via calcium channel and calcium releasing from intracellular calcium stores. Two main types of voltage gated calcium channels, i.e. L-type and T-type, regulate calcium influx in VSMCs (Kuhr et al., 2012). The interplay of calcium channels and BK channels was studied in previous works. Stimulation of intracellular calcium via L-type calcium channels increases BK channel activity, which leads to compensatory closure of L-type calcium channels and vasodilation (Joseph et al., 2013). While other researcher reported that inhibition of T-type calcium channels had no significant effect on BK channel in human gliomas cells (Weaver et al., 2007).

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ER is intracellular calcium store, which play important roles in disruption of calcium homeostasis during hypertension. It has been shown that BK channels are in close proximity to ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs) in the ER of myocytes (Joseph et al., 2013). BK channels can be stimulated by the calcium sparks via RyRs (Wellman and Nelson, 2003). In basilar VSMCs, BK channels are stimulated by the calcium released from IP3 Rs dependently (Kim et al., 1998). As ER lumen is diffusible with the space between the inner and outer membranes of nuclear envelope lumen, the balance between the ER in cytoplasm and nucleoplasmic reticulum in nucleoplasm may be involved in the balance of calcium hemostasis (Galva et al., 2012). Although, all these researches suggested the potential regulatory effects of different molecules, including calcium channels, BK channels, RyRs, IP3 Rs, etc., on intracellular calcium hemostasis, the complexity of the networks are still far from clearly understood. The pathogenesis mechanisms of these molecules on VSMC differentiation, especially the mechanobiology mechanism during hypertension still need further research. In the present research, two types of in vitro mechanical stretch devices were used to demonstrate the role of mechanical stretch on BK channel function and VSMC differentiation. The Flexcell-4000T Strain Loading Unit has been widely used to study the mechanobiological mechanism of cyclic stretch on VSMC functions (Qu et al., 2007; Qi et al., 2010). However, this system cannot be applied to electrophysiology. Thus, the horizontal stretch method was used to demonstrate BK channel function (Ahmed et al., 2010). Using this method, VSMCs were seeded on silicone gels and simultaneously examined by patch and calcium imaging. Using these two stretching application systems, 5% and 15% in vitro stretch were used to mimic the physiological and pathological (hypertensive) mechanical stimuli of VSMCs in vivo, respectively, and to determine the potential mechanobiological mechanism by which BK channel affects VSMC differentiation. 5. Conclusions Our study revealed that the increased pathological stretch induces the ER stress in VSMCs, which increases the alternative splicing of the STREX and activity of BK channel. The mechanical response of BK channel and its effect on VSMC de-differentiation may be a potential mechanism of vascular remodeling in hypertension. Conflicts of interests None. Acknowledgments This research was supported by grants from the National Natural Science Foundation of China, Nos. 11232010, 11229202, 11222223 and 10732070. We thank Dr. Zhi Qi at Xia Men University, China for the gift of GFP, HEK-Slo, and HEK-STREX delete plasmids. References Ahmed WW, Kural MH, Saif TA. A novel platform for in situ investigation of cells and tissues under mechanical strain. Acta Biomater 2010;6:2979–90. Allagnat F, Christulia F, Ortis F, Pirot P, Lortz S, Lenzen S, et al. Sustained production of spliced X-box binding protein 1 (XBP1) induces pancreatic beta cell dysfunction and apoptosis. Diabetologia 2010;53:1120–30. Armbruster C, Schneider M, Schumann S, Gamerdinger K, Cuevas M, Rausch S, et al. Characteristics of highly flexible PDMS membranes for long-term mechanostimulation of biological tissue. J Biomed Mater Res B Appl Biomater 2009;91:700–5. Borbouse L, Dick GM, Asano S, Bender SB, Dincer UD, Payne GA, et al. Impaired function of coronary BK(Ca) channels in metabolic syndrome. Am J Physiol Heart Circ Physiol 2009;297:H1629–37.

Brenner R, Perez GJ, Bonev AD, Eckman DM, Kosek JC, Wiler SW, et al. Vasoregulation by the beta1 subunit of the calcium-activated potassium channel. Nature 2000;407:870–6. Chang T, Wu L, Wang R. Altered expression of BK channel beta1 subunit in vascular tissues from spontaneously hypertensive rats. Am J Hypertens 2006;19:678–85. Chen L, Jeffries O, Rowe IC, Liang Z, Knaus HG, Ruth P, et al. Membrane trafficking of large conductance calcium-activated potassium channels is regulated by alternative splicing of a transplantable, acidic trafficking motif in the RCK1-RCK2 linker. J Biol Chem 2010;285:23265–75. Dromparis P, Paulin R, Stenson TH, Haromy A, Sutendra G, Michelakis ED. Attenuating endoplasmic reticulum stress as a novel therapeutic strategy in pulmonary hypertension. Circulation 2013;127:115–25. Ermolinsky BS, Skinner F, Garcia I, Arshadmansab MF, Otalora LF, Zarei MM, et al. Upregulation of STREX splice variant of the large conductance Ca2+ -activated potassium (BK) channel in a rat model of mesial temporal lobe epilepsy. Neurosci Res 2011;69:73–80. Galva C, Artigas P, Gatto C. Nuclear Na+ /K+ -ATPase plays an active role in nucleoplasmic Ca2+ homeostasis. J Cell Sci 2012;125:6137–47. Gollasch M, Haase H, Ried C, Lindschau C, Morano I, Luft FC, et al. L-type calcium channel expression depends on the differentiated state of vascular smooth muscle cells. FASEB J 1998;12:593–601. Hoefer IE, den Adel B, Daemen MJ. Biomechanical factors as triggers of vascular growth. Cardiovasc Res 2013;99:276–83. Hoshi T, Pantazis A, Olcese R. Transduction of voltage and Ca2+ signals by Slo1 BK channels. Physiology 2013;28:172–89. Huo B, Lu XL, Guo XE. Intercellular calcium wave propagation in linear and circuitlike bone cell networks. Philos Trans A Math Phys Eng Sci 2010;368:617–33. Jia X, Yang J, Song W, Li P, Wang X, Guan C, et al. Involvement of large conductance Ca(2+)-activated K(+) channel in laminar shear stress-induced inhibition of vascular smooth muscle cell proliferation. Pflugers Arch 2013;465:221–32. Joseph BK, Thakali KM, Moore CL, Rhee SW. Ion channel remodeling in vascular smooth muscle during hypertension: implications for novel therapeutic approaches. Pharmacol Res 2013;70:126–38. Kim CJ, Weir BK, Macdonald RL, Zhang H. Erythrocyte lysate releases Ca2+ from IP3-sensitive stores and activates Ca(2+)-dependent K+ channels in rat basilar smooth muscle cells. Neurol Res 1998;20:23–30. Kudryavtseva O, Aalkjaer C, Matchkov VV. Vascular smooth muscle cell phenotype is defined by Ca2+ -dependent transcription factors. FEBS J 2013;280:5488–99. Kuhr FK, Smith KA, Song MY, Levitan I, Yuan JX. New mechanisms of pulmonary arterial hypertension: role of Ca2+ signaling. Am J Physiol Heart Circ Physiol 2012;302:H1546–62. Li P, Hu M, Sun S, Zhang Y, Gao Y, Long M, et al. Fluid flow-induced calcium response in early or late differentiated osteoclasts. Ann Biomed Eng 2012;40: 1874–83. Long X, Tharp DL, Georger MA, Slivano OJ, Lee MY, Wamhoff BR, et al. The smooth muscle cell-restricted KCNMB1 ion channel subunit is a direct transcriptional target of serum response factor and myocardin. J Biol Chem 2009;284: 33671–82. MacDonald SH, Ruth P, Knaus HG, Shipston MJ. Increased large conductance calciumactivated potassium (BK) channel expression accompanied by STREX variant downregulation in the developing mouse CNS. BMC Dev Biol 2006;6:37. Ma D, Nakata T, Zhang G, Hoshi T, Li M, Shikano S. Differential trafficking of carboxyl isoforms of Ca2+ -gated (Slo1) potassium channels. FEBS Lett 2007;581: 1000–8. Ma YG, Dong L, Ye XL, Deng CL, Cheng JH, Liu WC, et al. Activation of cloned BK(Ca) channels in nitric oxide-induced apoptosis of HEK293 cells. Apoptosis 2010;15:426–38. Naruse K, Tang QY, Sokabe M. Stress-axis regulated exon (STREX) in the C terminus of BK(Ca) channels is responsible for the stretch sensitivity. Biochem Biophys Res Commun 2009;385:634–9. Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 2004;84: 767–801. Pang L, Rusch NJ. High-conductance, Ca(2+)-activated K+ channels: altered expression profiles in aging and cardiovascular disease. Mol Interv 2009;9:230–3. Parizek M, Novotna K, Bacakova L. The role of smooth muscle cells in vessel wall pathophysiology and reconstruction using bioactive synthetic polymers. Physiol Res 2011;60:419–37. Pfisterer L, Feldner A, Hecker M, Korff T. Hypertension impairs myocardin function: a novel mechanism facilitating arterial remodelling. Cardiovasc Res 2012;96:120–9. Qi YX, Qu MJ, Yan ZQ, Zhao D, Jiang XH, Shen BR, et al. Cyclic strain modulates migration and proliferation of vascular smooth muscle cells via Rho-GDIalpha, Rac1, and p38 pathway. J Cell Biochem 2010;109:906–14. Qi Z, Chi S, Su X, Naruse K, Sokabe M. Activation of a mechanosensitive BK channel by membrane stress created with amphipaths. Mol Membr Biol 2005;22: 519–27. Qu MJ, Liu B, Wang HQ, Yan ZQ, Shen BR, Jiang ZL. Frequency-dependent phenotype modulation of vascular smooth muscle cells under cyclic mechanical strain. J Vasc Res 2007;44:345–53. Ren J, Wu JH. 17beta-estradiol rapidly activates calcium release from intracellular stores via the GPR30 pathway and MAPK phosphorylation in osteocyte-like MLO-Y4 cells. Calcif Tissue Int 2012;90:411–9. Sausbier M, Arntz C, Bucurenciu I, Zhao H, Zhou XB, Sausbier U, et al. Elevated blood pressure linked to primary hyperaldosteronism and impaired vasodilation in BK channel-deficient mice. Circulation 2005;112:60–8.

X.-J. Wan et al. / The International Journal of Biochemistry & Cell Biology 59 (2015) 21–29 Schonthal AH. Pharmacological targeting of endoplasmic reticulum stress signaling in cancer. Biochem Pharmacol 2013;85:653–66. Weaver AK, Olsen ML, McFerrin MB, Sontheimer H. BK channels are linked to inositol 1,4,5-triphosphate receptors via lipid rafts: a novel mechanism for coupling [Ca(2+)](i) to ion channel activation. J Biol Chem 2007;282:31558–68. Wellman GC, Cartin L, Eckman DM, Stevenson AS, Saundry CM, Lederer WJ, et al. Membrane depolarization, elevated Ca(2+) entry, and gene expression in cerebral arteries of hypertensive rats. Am J Physiol Heart Circ Physiol 2001;281:H2559–67. Wellman GC, Nelson MT. Signaling between SR and plasmalemma in smooth muscle: sparks and the activation of Ca2+ -sensitive ion channels. Cell Calcium 2003;34:211–29.

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Xie J. Control of alternative pre-mRNA splicing by Ca(++) signals. Biochim Biophys Acta 2008;1779:438–52. Xu H, Garver H, Galligan JJ, Fink GD. Large-conductance Ca2+ -activated K+ channel beta1-subunit knockout mice are not hypertensive. Am J Physiol Heart Circ Physiol 2011;300:H476–85. Xu H, Kandlikar SS, Westcott EB, Fink GD, Galligan JJ. Requirement for functional BK channels in maintaining oscillation in venomotor tone revealed by species differences in expression of the beta1 accessory subunits. J Cardiovasc Pharmacol 2012;59:29–36. Zhao HC, Wang F. Exercise training changes the gating properties of largeconductance Ca2+ -activated K+ channels in rat thoracic aorta smooth muscle cells. J Biomech 2010;43:263–7.