Am J Physiol Cell Physiol 307: C373–C383, 2014. First published June 11, 2014; doi:10.1152/ajpcell.00115.2014.

Flow shear stress enhances intracellular Ca2⫹ signaling in pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension Shanshan Song,1,2 Aya Yamamura,3 Hisao Yamamura,4 Ramon J. Ayon,1,2 Kimberly A. Smith,1 Haiyang Tang,1,2 Ayako Makino,1,2 and Jason X.-J. Yuan1,2 1

Department of Medicine, University of Illinois at Chicago, Chicago, Illinois; 2Departments of Medicine and Physiology, University of Arizona College of Medicine, Tucson, Arizona; 3Kinjo Gakuin University School of Pharmacy, Nagoya, Japan; and 4Nagoya City University Graduate School of Pharmaceutical Sciences, Nagoya, Japan Submitted 10 April 2014; accepted in final form 10 June 2014

Song S, Yamamura A, Yamamura H, Ayon RJ, Smith KA, Tang H, Makino A, Yuan JX. Flow shear stress enhances intracellular Ca2⫹ signaling in pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension. Am J Physiol Cell Physiol 307: C373–C383, 2014. First published June 11, 2014; doi:10.1152/ajpcell.00115.2014.—An increase in cytosolic Ca2⫹ concentration ([Ca2⫹]cyt) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for pulmonary arterial medial hypertrophy in patients with idiopathic pulmonary arterial hypertension (IPAH). Vascular smooth muscle cells (SMC) sense the blood flow shear stress through interstitial fluid driven by pressure or direct exposure to blood flow in case of endothelial injury. Mechanical stimulus can increase [Ca2⫹]cyt. Here we report that flow shear stress raised [Ca2⫹]cyt in PASMC, while the shear stress-mediated rise in [Ca2⫹]cyt and the protein expression level of TRPM7 and TRPV4 channels were significantly greater in IPAH-PASMC than in normal PASMC. Blockade of TRPM7 by 2-APB or TRPV4 by Ruthenium red inhibited shear stress-induced rise in [Ca2⫹]cyt in normal and IPAH-PASMC, while activation of TRPM7 by bradykinin or TRPV4 by 4␣PDD induced greater increase in [Ca2⫹]cyt in IPAH-PASMC than in normal PASMC. The bradykinin-mediated activation of TRPM7 also led to a greater increase in [Mg2⫹]cyt in IPAH-PASMC than in normal PASMC. Knockdown of TRPM7 and TRPV4 by siRNA significantly attenuated the shear stress-mediated [Ca2⫹]cyt increases in normal and IPAH-PASMC. In conclusion, upregulated mechanosensitive channels (e.g., TRPM7, TRPV4, TRPC6) contribute to the enhanced [Ca2⫹]cyt increase induced by shear stress in PASMC from IPAH patients. Blockade of the mechanosensitive cation channels may represent a novel therapeutic approach for relieving elevated [Ca2⫹]cyt in PASMC and thereby inhibiting sustained pulmonary vasoconstriction and pulmonary vascular remodeling in patients with IPAH. mechanosensitive channel; TRPM7; TRPV4; shear stress PULMONARY CIRCULATION is a high-flow, low-resistance, and low-pressure circulatory system under physiological conditions that receives the entire cardiac output at all time (23). Endothelial cells (EC) are normally exposed directly to the high-flow blood flow through the lungs, which produces flow shear stress on the order of 10 dyn/cm2 (27, 39), while the underlying smooth muscle cells (SMC) experience significant mechanical stretch from the blood flow and intraluminal pressure. However, in cases of endothelial injury and denudation, which occur, for example, in cardiovascular intervention of angioplasty, vascular graft anastomoses, pulmonary arterial

Address for reprint requests and other correspondence: J. X.-J. Yuan, Dept. of Medicine, The Univ. of Arizona, P.O. Box 210202, 1295 North Martin Ave., Tucson, AZ 85721-0202 (e-mail: [email protected]). http://www.ajpcell.org

catheterization (especially during the measurement of pulmonary arterial wedge pressure), and atherosclerotic disease (5, 15, 33), SMC will be exposed to the flow shear stress directly. Another more subtle mechanism by which SMC are exposed to the shear stress is through the transmural interstitial flow driven by transvascular pressure difference (5, 32, 33, 39). Interstitial flow is influenced dramatically by the hemodynamic state. Starling’s law mentioned that interstitial flow shear stress on SMC is approximately proportional to blood pressure (5, 22, 32, 34). Accordingly, any increase in blood pressure is expected to cause a proportional increase of transmural flow shear stress on SMC. Increased blood flow leads to increases in hydraulic conductivity, which, in turn, results in elevated transmural flow and shear stress on SMC. In addition, the presence of the internal elastic lamina (IEL) with leaky fenestral pores underneath the intima alters greatly the boundary flow area around SMC (5, 16, 32, 39). The velocity of fluid outflowing from an individual pore could be 100-fold greater than superficial velocity in tunica media, resulting in significant uneven distribution of shear stress on SMC that lie just beneath the IEL. Although the superficial velocity of interstitial flow is rather slow (10⫺5–10⫺6 cm/s), the spaces of interstitial tissue are so small that shear stress on SMC is significant (5). The estimated shear stress on the order of 1 dyn/cm2 on systemic arterial SMC under physiological conditions has been shown to affect SMC function (11, 21, 28, 35, 39), and elevated shear stress on SMC plays a role in cell proliferation and migration that occur in diseases like atherosclerosis and intimal hyperplasia (35). Intracellular Ca2⫹ is a major signaling element in mediating pulmonary arterial SMC (PASMC) contraction, migration, and proliferation. An increase in cytosolic free Ca2⫹ concentration ([Ca2⫹]cyt) in PASMC is a critical trigger for pulmonary vasoconstriction and an important stimulus for pulmonary vascular media hypertrophy, two major causes of elevated pulmonary vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH) (46). Physical stimuli such as mechanical stimulation (shear stress, stretch, temperature) can induce Ca2⫹-influx in cells through mechanosensitive channels (5). Indeed, fluid flow has been shown to modulate Ca2⫹ dynamic in bone and endothelial cells, and physiological levels of shear stress increased [Ca2⫹]cyt in rat aorta SMC through a mechanism that involves Ca2⫹ entry through a gadolinium-sensitive pathway (32). Although effects of flow shear stress on vascular SMC have been described (42), the signaling sensing and transduction mechanisms are poorly understood. The transient receptor potential (TRP) channel superfamily is an emerging channel

0363-6143/14 Copyright © 2014 the American Physiological Society

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protein family and has been proposed to play a role in sensing environmental stimulus (6, 14, 29, 31). TRPM7, a member of the melastatin-related TRP channel family, which is ubiquitously expressed in various cell types and functions both as an ion channel and a kinase, is permeable to bivalent cations such as Ca2⫹ and Mg2⫹ (1, 13, 38, 41, 45). TRPM7 is considered to be an important regulator of Mg2⫹ homeostasis, as well as a shear stress-sensitive cation channel (26, 27). Previous studies reported that TRPM7 accumulates rapidly at the plasma membrane in vascular SMC in response to fluid flow shear stress at physiological levels (27). In addition to TRPM7, the vanilloidrelated TRP channel TRPV4 is also an important mechanically sensitive ion channel (e.g., to shear stress and stretch) functionally expressed on the plasma membrane in vascular SMC. TRPV4 has been studied extensively as a shear stress-sensing ion channel in vascular endothelial cells (8, 10, 12, 44). Ca2⫹ influx through endothelial TRPV4 channels in response to blood flow shear stress mediates flow-induced vasodilation (3, 9, 25). Thus, we inferred that TRPV4 (and TRPV7) channels might also be expressed in PASMC and play a role in sensing shear stress in PASMC. In this study, we examined and compared fluid flow shear stress-induced changes in [Ca2⫹]cyt and possible candidates for the shear stress-sensitive ion channels in PASMC isolated from normal subjects and IPAH patients. The data from this study indicated that 1) flow shear stress-mediated increase in [Ca2⫹]cyt in IPAH-PASMC was significantly greater than in normal PASMC; 2) pharmacological blockade of TRPM7 and TRPV4 significantly attenuated shear stress-mediated [Ca2⫹]cyt increases in IPAH-PASMC; and 3) the altered [Ca2⫹]cyt response magnitude to flow shear may be due to differential expression levels of mechanosensitive ion channels (TRPM7, TRPV4) in normal and IPAH-PASMC. These observations imply that in IPAH, increased shear stress from increased transmural interstitial flow will exert a greater increase in [Ca2⫹]cyt in PASMC, thereby causing sustained pulmonary vasoconstriction and excessive vascular remodeling. Thus, pharmacological blockade of mechanosensitive cation channels could be a potential strategy for developing novel clinical therapeutic approaches for pulmonary hypertension. MATERIALS AND METHODS

Cell culture. Human PASMC from normal subjects and IPAH patients were cultured in M199 medium (Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 25 mg/l of D-valine (Sigma-Aldrich, St. Louis, MO), 100 IU/ml penicillin and 100 ␮g/ml streptomycin (Sigma-Aldrich), and 20 ␮g/ml cell growth supplement (BD Biosciences, Franklin Lakes, NJ) in a humidified atmosphere at 37°C and 5% CO2. Use of human lung tissues and cells was approved by the University of Illinois at Chicago Institutional Review Board. The cells at passage 5-8 were used for the experiments. [Ca2⫹]cyt measurement. [Ca2⫹]cyt in PASMC was measured according to the previously described method (36, 50). Briefly, PASMC were grown to passage 5-8 at 50 – 60% confluence on 25-mmdiameter circular glass coverslips. The cells were loaded with 4 ␮M fura-2 acetoxymethyl ester (fura-2/AM; Invitrogen/Molecular Probes, Eugene, OR) in HEPES-buffered solution for 60 min at room temperature. PASMC loaded with fura-2/AM were placed in a recording chamber mounted on the stage of an inverted fluorescence microscope (Eclipse Ti-E; Nikon, Tokyo, Japan) equipped with an objective lens (S Plan Fluor ⫻20/0.45 ELWD; Nikon), an EM-CCD camera

(Evolve; Photometrics, Tucson, AZ), and NIS Elements software (version 3.2; Nikon), and superfused with an HEPES-buffered solution for 30 min to wash out extracellular residual fura-2/AM and allow sufficient time for intracellular esterase to convert fura-2/AM to fura-2. The fluorescence intensity emitted at 520 nm in cells excited by illumination at 340 nm and 380 nm was measured within a region of interest at the rate of one ratio every 2 s. The HEPES-buffered bath solution had an ionic composition (in mM) of 137 NaCl, 5.9 KCl, 2.2 CaCl2, 1.2 MgCl2, 14 D-glucose, and 10 HEPES. The pH was adjusted to 7.4 with 10 N NaOH. The Ca2⫹-free bath solution was prepared by replacing CaCl2 with equimolar MgCl2; 1 mM EGTA was added to chelate the residual Ca2⫹ in the solution. To determine [Ca2⫹]cyt from fura-2-fluorescence ratios, the intracellular minimal and maximal fluorescence ratios (Rmin and Rmax, respectively) were determined as previously described. To determine Rmin, fura-2-loaded PASMC were perfused with a solution containing (in mM): 137 NaCl, 5.9 KCl, 1.2 MgCl2, 14 D-glucose, 10 HEPES, and 5 EGTA with 2 mM ionomycin (pH 7.40). An intracellular Rmax value was determined by perfusing PASMC with a similar solution except that the extracellular concentration of CaCl2 was increased to 11 mM. The values of intracellular Rmin and Rmax were used to calculate [Ca2⫹]cyt according to the following formula: [Ca2⫹]cyt ⫽ Kd (R ⫺ Rmin)/[(Rmax ⫺ R)(Sf2/Sb2)], where Kd is the dissociation constant of fura-2, and Sf2 and Sb2 are the fluorescence intensities at ⬃510 nm of the Ca2⫹-free and Ca2⫹saturated indicator, respectively. All [Ca2⫹]cyt measurements were performed at 32°C to prevent uptake of fura-2 to intracellular organelles. Western blotting. Total protein was isolated from PASMC which were lysed in 1⫻ RIPA buffer (Bio-Rad). Protein was loaded on an 8% acrylamide gel, transferred to an Immobilon-P transfer membrane (Millipore, Bedford, MA), and immunoblotted with anti-TRPM7 (SAB2501067; 1:1,000; Sigma-Aldrich) and anti-TRPV4 monoclonal antibody (SAB2104216; 1:1,000; Sigma-Aldrich). Signals were detected using a Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific). The protein levels were normalized to ␤-actin (sc-81178, 1:1,000; Santa Cruz Biotechnology) and are expressed in arbitrary units. Transfection siRNA. PASMC were transiently transfected with control siRNA (10 ␮M, sc-37007; Santa Cruz Biotechnology), or TRPM7 siRNA (10 ␮M, sc-42662; Santa Cruz Biotechnology) or TRPV4 siRNA (10 ␮M, sc-64726; Santa Cruz Biotechnology) using Xfect siRNA transfection reagent protocol (Clontech Laboratories). [Ca2⫹]cyt measurement and Western blot experiments using siRNAtransfected cells were preformed 48 –72 h after transfection. Flow setup and calculation. A mini-pump (Control Company) connected through thin tubing to an 800 ␮m ID needle provided fluid flow at a rate of 1.9 ml/min. The pipette was positioned ⬃1 mm from the cell. The magnitude of shear stress (␶) generated by the fluid flow was calculated using the formula: ␶ ⫽ (␳V2/2)(0.664/公Rx) (27), where ␳ is the density of water (⫽1,025 kg/m3) and V is the fluid velocity, which is calculated by V ⫽ Q/A, where A ⫽ ␲d2/4 and is equal to 0.503 ⫻ 10⫺6 m for a pipette diameter of d ⫽ 800 ␮m and Q is the flow generated by the syringe pump measured in m3/s. Rx, the Reynolds number, was Rx ⫽ V ⫻ x/␯, where ␯ is the kinematic viscosity of the water (⬇1.139 ⫻ 106 m2/s) and x is the distance between pipette and cell (⫽ 1 mm). To determine whether flow was laminar, we also used the Reynolds number defined as Rh ⫽ Vh/v, where h, the fluid level in the dish (1 mm), was kept constant; v and V are the kinematic viscosity of the water and the fluid velocity, respectively. For the highest value of the flow used in our experiments, the Reynolds numbers were Rx ⫽ 27.5 and Rh ⫽ 627. Laminar flow occurs for Rx ⬍ 500 000 and Rh ⬍ 3,900. Drugs and chemicals. Ruthenium red (RR), diltiazem (Dilt), and lathanum (La3⫹) were prepared as concentrated stock solutions in distilled water. 2-Aminoethoxydiphenyl borate (2-APB) and bradykinin (BRK) were prepared as stock solutions in ethanol. 4␣-Phorbol 12,13-didecanoate (4␣PDD), 1-{2-(4-methoxyphenyl)-2-[3-(4-me-

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thoxyphenyl)propoxy]ethyl}-1H-imidazole hydrochloride or SKF 96365 hydrochloride (SKF), and cyclopiazonic acid (CPA) were prepared as concentrated stock solutions in dimethyl sulfoxide (DMSO). All stock solutions (in water, ethanol, or DMSO) were aliquoted and kept frozen at ⫺20°C until use. On the day of experiments, aliquots of the stock solutions were diluted 1,000 –2,000 times in the HEPES-buffered bath solution to the final concentrations for each drug. The pH values of all solutions were measured after addition of the drugs and adjusted to 7.4. All drugs were from Sigma Chemical, unless otherwise indicated. Statistical analysis. Pooled data are shown as means ⫾ SE. Statistical significance between two groups was determined by Student’s t-test. Statistical significance among groups was determined by Scheffé’s test after one-way analysis of variance. Significant difference is expressed in the figures as P ⬍ 0.05. RESULTS

Flow shear stress enhances two-phase [Ca2⫹]cyt increases in PASMC from IPAH patients. Flow shear stress (by perfusion) caused a transient increase in [Ca2⫹]cyt followed by a sustained increase in [Ca2⫹]cyt in ⬃85% normal and IPAHPASMC. The fluid flow shear stress-mediated transient and sustained increases in [Ca2⫹]cyt were significantly enhanced in IPAH-PASMC compared with normal PASMC (Fig. 1A). As shown in Fig. 1, B and C, both transient and sustained increases in [Ca2⫹]cyt were significantly greater in IPAH-PASMC than in normal PASMC. To characterize pharmacological properties of the shear stress-mediated transient and sustained increases in [Ca2⫹]cyt in normal and IPAH-PASMC, we applied 50 ␮M SKF (Fig. 1A), a nonspecific TRP channel blocker (17, 26), which significantly attenuated the transient and sustained increases in [Ca2⫹]cyt in both normal and IPAH-PASMC (Fig. 1D), indicating that the shear stress-mediated Ca2⫹ influx is due, at least partially, to Ca2⫹ entry through SKF-sensitive TRP channels. We then removed extracellular Ca2⫹ and applied 10 ␮M CPA (24, 30) before or during perfusion of Ca2⫹-free (0Ca) solution (Fig. 1A). As shown in Fig. 1D, 0Ca perfusion significantly decreased or abolished transient and sustained increase in [Ca2⫹]cyt in both normal and IPAHPASMC as well as CPA-treated cells, indicating that the shear stress-mediated transient and sustained increase in [Ca2⫹]cyt is predominantly due to Ca2⫹ influx and Ca2⫹ release from CPA-sensitive stores. Flow shear stress induces enhanced oscillatory [Ca2⫹]cyt increases in PASMC from IPAH patients. In addition to the two-phase [Ca2⫹]cyt increase, fluid flow shear stress also elicited oscillatory [Ca2⫹]cyt increase in ⬃15% of normal and IPAH-PASMC (Fig. 2, A–C). The amplitude of the oscillatory [Ca2⫹]cyt increase in IPAH-PASMC was significantly greater than in normal PASMC (Fig. 2, A–D). To characterize the flow shear stress-mediated Ca2⫹ oscillation, we removed extracellular Ca2⫹ or applied CPA before or during 0Ca perfusion (Fig. 2, A and B). Perfusion with Ca2⫹-free solution abolished the oscillatory [Ca2⫹]cyt increase in both normal and IPAHPASMC as well as CPA-treated cells (Fig. 2, A and B), indicating that the shear stress-mediated oscillatory increase in [Ca2⫹]cyt is also predominantly due to Ca2⫹ influx and Ca2⫹ release from CPA- or inositol trisphosphate-sensitive stores. Pharmacological blockade of TRPM7 (and TRPC6) channels with 2-APB decreased shear stress-mediated [Ca2⫹]cyt increases in both normal and IPAH-PASMC. To define which subtypes of TRP channels might be involved in the enhanced

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shear stress-mediated [Ca2⫹]cyt increase in IPAH-PASMC, we examined the effect of 2-APB, a blocker of TRPM7 (19, 26, 41) and TRPC6 channels (40), on shear stress-mediated increase in [Ca2⫹]cyt in normal and IPAH-PASMC. As shown in Fig. 3A, application of 20 ␮M 2-APB before and during application of fluid shear stress (perfusion) significantly decreased the transient and sustained (or plateau) [Ca2⫹]cyt increases in normal and IPAH-PASMC (Fig. 3B). Bradykinin (BRK, 10 ␮M), a G protein-coupled receptor agonist that has been demonstrated to activate TRPM7 channels (4, 41), elicited significantly greater transient [Ca2⫹]cyt increase in IPAHPASMC than in normal PASMC, while blockade of TRPM7 channels with 2-APB significantly attenuated BRK-induced transient [Ca2⫹]cyt increase in IPAH-PASMC (Fig. 3, C and D). These data suggest that Ca2⫹ influx through the mechanosensitive channel TRPM7 plays a role in the shear stressinduced [Ca2⫹]cyt increase observed in both normal and IPAHPASMC. Ruthenium red decreased flow shear stress-mediated [Ca2⫹]cyt increases in normal and IPAH-PASMC. In addition to TRPM7, we also sought to examine whether TRPV4 channel activity is involved in shear stress-mediated [Ca2⫹]cyt increase in normal and IPAH-PASMC. Ruthenium red (RR, 1 ␮M), which can block TRPV4 channels (2, 25), significantly decreased shear stress-mediated transient and sustained increases in [Ca2⫹]cyt in normal and IPAH-PASMC (Fig. 4, A and B). RR abolished the transient increase in [Ca2⫹]cyt induced by flow shear stress (perfusion) in IPAH-PASMC, and markedly decreased the amplitude of the sustained (or plateau) phase of the flow shear stress-mediated increase in [Ca2⫹]cyt (Fig. 4, A and B, bottom). These results indicate that, in addition to TRPM7, TRPV4 may also act as a mechanosensitive ion channel responsible for the shear stress-mediated Ca2⫹ influx in PASMC. To further prove the involvement of TRPV4 in shear stressmediated Ca2⫹ influx in PASMC, we examined the effect of 4␣PDD, a selective TRPV4 channel agonist (2, 25), on shear stress-mediated increases in [Ca2⫹]cyt in normal and IPAHPASMC. As shown in Fig. 5, there was almost no increase in [Ca2⫹]cyt in response to 4␣PDD (10 ␮M) in normal PASMC (Fig. 5, A and B, left, and C). However, a significant [Ca2⫹]cyt increase was observed in most of IPAH-PASMC; ⬃99% of IPAH-PASMC demonstrated different response patterns of sustained or oscillatory [Ca2⫹]cyt increase in response to 4␣PDD (10 ␮M) (Fig. 5, A and B, middle, and C). Pretreatment of the cells with 1 ␮M RR significantly decreased the 4␣PDDinduced [Ca2⫹]cyt increase in IPAH-PASMC (Fig. 5, A and B, right, and C). As shown in Fig. 5C, pharmacological blockade of TRPV4 channels with RR caused an approximate 50% inhibition of 4␣PDD-mediated increase in [Ca2⫹]cyt in IPAHPASMC. These data suggest that, in addition to TRPM7, TRPV4 may also play a role in mediating shear stress-induced [Ca2⫹]cyt increase in IPAH-PASMC. Upregulated protein expression of mechanosensitive ion channels in PASMC from IPAH patients. The enhanced shear stress-mediated increases in [Ca2⫹]cyt in IPAH-PASMC were associated with significantly upregulated protein expression of TRPM7 and TRPV4 (Fig. 6A). In addition, TRPC6, one of the TRP canonical channels that are considered to be mechanosensitive cation channels (37), was also upregulated in IPAHPASMC compared with normal PASMC (Fig. 6B) (47– 49).

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Fig. 1. Flow shear stress-induced increase in cytosolic Ca2⫹ concentration ([Ca2⫹]cyt) is significantly enhanced in pulmonary arterial smooth muscle cells (PASMC) from idiopathic pulmonary arterial hypertension patients (IPAH) in comparison to PASMC from normal subjects (Normal). A: representative traces showing changes in [Ca2⫹]cyt in Normal (top) and IPAH (bottom) PASMC before and during application of flow shear stress (Perfusion) in the absence or presence of 50 ␮M SKF (a nonselective blocker of cation channels), in the absence of extracellular Ca2⫹ (0Ca), or in the presence of 10 ␮M cyclopiazonic acid (CPA, an inhibitor of SERCA) dissolved in 0Ca solution (0Ca ⫹ CPA). B: summarized data (means ⫾ SE) showing the amplitude of shear stress-induced transient (Trans) and plateau (Plateau) phases of the increase in [Ca2⫹]cyt in Normal and IPAH-PASMC (left). *P ⬍ 0.05 vs. Normal. C: amplitude distributions of shear stress-induced transient and plateau increases in [Ca2⫹]cyt in Normal (Nor, top) and IPAH (IPAH, bottom) PASMC. D: summarized data (means ⫾ SE) showing the amplitude of shear stress-induced transient and plateau increases in [Ca2⫹]cyt in Normal (Nor, top) and IPAH (IPAH, bottom) PASMC in the absence or presence of SKF (left), in the absence of extracellular Ca2⫹ (0Ca, middle), or in the presence of 10 ␮M CPA dissolved in 0Ca solution (0Ca ⫹ CPA, right). *P ⬍ 0.05 vs. control (Cont, left), 1.8 mM Ca2⫹ (1.8Ca, middle), and 0Ca (0Ca, right) in both normal (Nor) and IPAH (IPAH) PASMC.

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Fig. 2. Amplitude of shear stress-induced oscillatory increases in [Ca2⫹]cyt is significantly greater in IPAH-PASMC than in normal PASMC, and removal of extracellular Ca2⫹ abolishes the shear stressmediated Ca2⫹ oscillations. A: representative traces showing changes in [Ca2⫹]cyt in normal (Normal, top) and IPAH (IPAH, bottom) PASMC before and during application of flow shear (Perfusion) in the absence (0Ca) or presence (1.8Ca) of 1.8 mM extracellular Ca2⫹ or in the presence of 10 ␮M cyclopiazonic acid (CPA; an inhibitor of SERCA) dissolved in 0Ca solution (0Ca ⫹ CPA). B: summarized data (means ⫾ SE) showing the amplitude of shear stressmediated rises in [Ca2⫹]cyt in Normal (top) and IPAH (bottom) PASMC in the absence (0Ca) or presence (1.8Ca) of extracellular Ca2⫹ or in the presence of 10 ␮M CPA in 0Ca solution (0Ca ⫹ CPA). *P ⬍ 0.05 vs. 1.8Ca, #P ⬍ 0.05 vs. 0Ca. C: summarized data (means ⫾ SE) showing the amplitude (left) and frequency (right) of shear stressinduced oscillatory increases in [Ca2⫹]cyt in Normal and IPAH-PASMC. *P ⬍ 0.05 vs. Normal. D: histogram showing the amplitude (left) and frequency (right) distribution of shear stress-induced [Ca2⫹]cyt oscillations in Normal (top) and IPAH (bottom) PASMC.

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However, expression of Piezo 1 and Piezo 2, a novel family of mechanosensitive channels that have recently been identified in different cell types (7), was actually lower in IPAH-PASMC than in normal PASMC (data not shown). These data suggest that upregulated TRPM7, TRPV4, and TRPC6 are the cause for the enhanced shear-mediated increase in [Ca2⫹]cyt in IPAH-PASMC. TRPM7 and TRPV4 are necessary for shear stress-mediated [Ca2⫹]cyt increase in PASMC. To obtain direct evidence for involvement of TRPM7 and TRPV4 channels in shear stressmediated [Ca2⫹]cyt increase, we examined the effect of shear stress on IPAH-PASMC treated with TRPM7- or TRPV4specific siRNA. As shown in Fig. 7A, transfection with TRPM7- or TRPV4-siRNA significantly decreased TRPM7 and TRPV4 protein expression levels, respectively, in IPAHPASMC. The siRNA-mediated downregulation of TRPM7 and TRPV4 was associated with a significant inhibition in shear stress-mediated transient [Ca2⫹]cyt increase in IPAH-PASMC. Although there was a tendency for TRPM7 and TRPV4 knockdown to attenuate sustained [Ca2⫹]cyt increase in IPAHPASMC, this level of inhibition was not statistically significant (Fig. 7, B and C). It is possible that other Ca2⫹-permeable channels and Ca2⫹ transporters contribute to the sustained increase in [Ca2⫹]cyt. In addition, knockdown of TRPM7 and TRPV4 with siRNA also reduced the shear stress-mediated increase in [Ca2⫹]cyt in normal PASMC (Fig. 7, B and C), although the TRPM7- or TRPV4-siRNA-mediated inhibition was much smaller in normal PASMC than in IPAH-PASMC.

0.1

0.2

0.3

0.4

0.5 -1

Frenquency (min )

These data indicate that TRPM7 and/or TRPV4 channels are necessary for the enhanced shear stress-mediated [Ca2⫹]cyt increase in IPAH-PASMC. Shear stress-induced [Mg2⫹]cyt increase is greater in IPAHPASMC than in normal PASMC. TRPM7 is a divalent cation permeable ion channel with a greater permeability (P) to Mg2⫹ than Ca2⫹ (13). We took advantage of this characteristic (PMg ⬎ PCa) of TRPM7 channels to further confirm the role of TRPM7 in the process of sensing fluid flow shear stress, and explore its role in maintaining cytosolic Mg2⫹ concentration ([Mg2⫹]cyt) homeostasis during shear stress stimulation. To further confirm the role of TRPM7 channels in the process of sensing fluid flow shear stress, we examined the effect of flow shear stress on [Mg2⫹]cyt in normal and IPAH-PASMC using Mag-fura-2 as the Mg2⫹ indicator. The possibility of Ca2⫹ bound to Mag-fura-2 was excluded before measuring [Mg2⫹]cyt through application of CPA (a SERCA inhibitor that causes an increase in [Ca2⫹]cyt due to Ca2⫹ leakage or mobilization from the sarcoplasmic reticulum to the cytosol) before application of fluid shear stress (perfusion). As shown in Fig. 8A, extracellular application of 10 ␮M CPA caused an increase in [Ca2⫹]cyt in normal and IPAH-PASMC superfused in Ca2⫹-free (0Ca) solution due apparently to Ca2⫹ mobilization or leak from the sarcoplasmic reticulum to the cytosol. However, shear stress (Perfus) failed to increase [Ca2⫹]cyt in normal and IPAH-PASMC bathed in 0Ca solution and pretreated with CPA (Fig. 8A).

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Normal

800

600

IPAH

800

600

600 5 min

400

400

400

400

200

200

200

200

0

Perfusion

0

Perfusion

0

Perfusion

0

Perfusion

2-APB

600

800

IPAH

600

800

D

IPAH

600 5 min

400

400

400

200

200

200

0

BRK

0

BRK

0

BRK

800 600

Normal 800 600

IPAH

Perfusion Perfusion +2-APB

800 600

Perfusion Perfusion +2-APB

400 200 0

* Plateau

400 200 0

* Trans

* Plateau

Nor-BRK

*

2+

Normal

Rise in [Ca ]cyt (nM)

800

2-APB

2+

[Ca ]cyt (nM)

C

B

IPAH

2+

600

800

Rise in [Ca ]cyt (nM)

Normal

2+

800

Rise in (Ca )cyt (nM)

2+

[Ca ]cyt (nM)

A

SHEAR STRESS AND STRETCH INDUCE Ca2⫹ INFLUX IN IPAH-PASMC

400

IPAH-BRK IPAH-BRK +2-APB

200 0

2-APB

Fig. 3. Pharmacological blockade of the mechanosensitive channel TRPM7 with 2-aminoethoxydiphenyl borate (2-APB) significantly attenuates the shear stress-induced increase in [Ca2⫹]cyt in both normal and IPAH-PASMC. A: representative traces showing changes in [Ca2⫹]cyt in normal (Normal) and IPAH (IPAH) PASMC before and during application of shear stress (Perfusion) in the absence or presence of the TRPM7 (and TRPC6) channel inhibitor 2-APB (20 ␮M). B: summarized data (means ⫾ SE) showing the amplitude of shear stress-induced transient (Trans) and plateau (Plateau) increases in [Ca2⫹]cyt in the absence or presence of 20 ␮M 2-APB in Normal (left) and IPAH (right) PASMC. *P ⬍ 0.05 vs. Perfusion. C: representative traces showing changes in [Ca2⫹]cyt before and during extracellular application of 10 ␮M bradykinin (BRK) in Normal and IPAH-PASMC or treated by 20 ␮M 2-APB before and during BRK stimulation in IPAH-PASMC. D: summarized data (means ⫾ SE) showing the amplitude of BRK-induced rises in [Ca2⫹]cyt with or without treatment by 20 ␮M 2-APB. *P ⬍ 0.05 vs. Normal PASMC and 2-APB-treated IPAH.

Using Mag-fura-2 to measure [Mg2⫹]cyt, we found that extracellular application of CPA did not induce any changes of fluorescence intensity of Mag-fura-2, indicating that, under theses condition, Mag-fura-2 was an indicator specific for Mg2⫹ (Fig. 8B) because CPA-mediated increase in [Ca2⫹]cyt (Fig. 8A) did not interfere with the changes in Mag-fura-2 fluorescence intensity. Application of shear stress (Perfus), however, caused an increase in [Mg2⫹]cyt (determined by the increase of Mag-fura-2 intensity), due to Mg2⫹ influx possibly through TRPM7 channels, in PASMC bathed in 0Ca solution and pretreated with 10 ␮M CPA. More importantly, the shear stress-mediated increase in [Mg2⫹]cyt was significantly greater in IPAH-PASMC than in normal PASMC (Fig. 8, B and C). The enhanced shear stress-mediated increase in [Mg2⫹]cyt resulting from Mg2⫹ influx (Fig. 8, B and C) was associated with upregulation of TRPM7 protein expression (Fig. 6) in IPAH-PASMC compared with normal PASMC. To obtain further evidence for the involvement of TRPM7 channel activity in shear stress-mediated [Ca2⫹]cyt increase, we examined the effect of BRK on [Mg2⫹]cyt in normal and IPAH-PASMC. We first examined whether BRK could significantly induce [Ca2⫹]cyt increase after depleting Ca2⫹ stores using a Ca2⫹-free solution. Figure 8D shows that almost no BRK-induced [Ca2⫹]cyt increase was observed in normal and IPAH-PASMC after treatment with 10 ␮M CPA in Ca2⫹-free solution. The BRK-induced response of [Mg2⫹]cyt was measured after applying CPA in Ca2⫹-free solution. Interestingly, BRK was shown to increase [Mg2⫹]cyt in both normal and IPAH-PASMC, with slightly greater elevations of [Mg2⫹]cyt in IPAH-PASMC (Fig. 8, E and F). In the presence of 2-APB, the BRK-induced increase in [Mg2⫹]cyt was attenuated (Fig. 8, E and F). These data indicate that 1) TRPM 7 indeed plays a role in sensing fluid flow shear stress, mediating not only Ca2⫹

influx but also Mg2⫹ influx, 2) shear stress-induced Mg2⫹ influx is greater in IPAH-PASMC than in normal PASMC, and 3) the enhanced shear stress-induced Mg2⫹ influx in IPAHPASMC is due, at least partially, to upregulated TRPM7 expression in IPAH-PASMC. DISCUSSION

We have shown in this study that 1) flow shear stress induces increases in [Ca2⫹]cyt in human PASMC with different patterns (transient, sustained, and oscillatory increases); 2) the shear stress increases in [Ca2⫹]cyt were significantly enhanced in IPAH-PASMC in comparison to normal PASMC; 3) pharmacological blockade of TRPM7 channels with 2-APB or TRPV4 channels with Ruthenium red significantly attenuates the shear stress-mediated [Ca2⫹]cyt increase in IPAH-PASMC; 4) protein and mRNA expression of TRPM7, TRPV4, and TRPC6 was all significantly upregulated in IPAH-PASMC compared with normal PASMC; and 5) knockdown of TRPM7 or TRPV4 channels with specific siRNA for TRPM7 or TRPV4 significantly attenuates the shear stress-mediated increase in [Ca2⫹]cyt in IPAH-PASMC. These data indicate that PASMC isolated from IPAH patients are hyperresponsive to mechanical stimuli (e.g., fluid flow shear stress) compared with normal PASMC. Since TRPM7, TRPV4, and TRPC6 are all mechanosensitive channels (in addition to their sensitivity to ligands and receptor-operated signaling cascades), our observations imply that upregulated mechanosensitive channels (e.g., TRPM7, TRPV4, and TRPC6) in IPAH-PASMC may contribute to the development and progression of IPAH by inducing sustained pulmonary vasoconstriction, enhancing arterial myogenic tone, and promoting vascular wall thickening as ob-

AJP-Cell Physiol • doi:10.1152/ajpcell.00115.2014 • www.ajpcell.org

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600

Normal

400

B

600

400 5 min

200

200

0

0

Perfusion

Perfusion

Perfusion-induced 2+ Rise in [Ca ]cyt (nM)

2+

[Ca ]cyt (nM)

A

300

Cont RR

250 200 150 100 50 0

elastic lamina (IEL) leads to altered distribution of transmural flow field on SMC, which results in greater transmural flow shear stress on SMC just bordering subendothelial intima close to the pores or fenestrae (5, 32, 39). All of these factors mentioned above become much more critical in IPAH when augmented hydrostatic pressure significantly increases transmural flow, which would

*

A

Plateau

200

200

0

Perfusion

0

5 min

Perfusion RR

250 200 150

0

(Averaged)

IPAH

600

(Averaged)

IPAH (Averaged)

400

400

400

200

200

200

5 min

*

100 50

600

Normal

2+

400

Cont RR

[Ca ]cyt (nM)

400

300

600

B

* Trans

Plateau

0

4αPDD

4αPDD

600

(Individual)

0

4αPDD

600

RR (Individual)

(Individual)

400

400

400

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0 600

0 600

0 600

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0 600

0 600

0 600

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2+

C

0

4αPDD-induced 2+ Rise in [Ca ]cyt (nM)

served in patients with IPAH and animals with experimental PAH. Vascular SMC, when compared with endothelial cells (EC), are not a common study object for shear stress because SMC are usually considered to lie underneath the endothelium and not exposed to fluid/blood flow shear stress. Vascular SMC can be directly exposed to blood flow shear stress, however, in cases of endothelial injury or denudation (5, 15, 33), or be indirectly subjected to shear stress through transmural interstitial flow driven by the increased transmural pressure difference, which happens at physiological conditions and imposes shear stress on SMC on the order of 1 dyn/cm2 (5, 32, 33). Since transmural interstitial flow shear stress is approximately proportional to blood pressure, according to Starling’s law (5, 22, 32, 34), an increase in blood flow or intraluminal pressure will induce a proportional transmural flow shear stress on vascular SMC. Due to the upregulation of mechanosensitive cation channels (e.g., TRPM7, TRPV4, TRPC6, and others) in IPAH-PASMC, it is thus likely that augmentation of the flow shear stress-mediated increase in [Ca2⫹]cyt in IPAH-PASMC may be an important contributor to the development and progression of sustained pulmonary vasoconstriction, increased myogenic tone (and wall stiffness) of pulmonary arteries, and thickened arterial wall due to increased cell proliferation and migration. Another reason that makes transmural flow shear stress an important factor in IPAH is that the small interstitial space in pulmonary arterial tissue makes the transmural flow shear stress on SMC significant (the small space makes much higher hydrostatic pressure than in bigger space), even at the physiological level. Additionally, a system of pores or fenestrae on the internal

[Ca ]cyt (nM)

2+

Fig. 4. Pharmacological blockade of the mechanosensitive channel TRPV4 with Ruthenium red (RR) significantly attenuates the shear stress-induced increase in [Ca2⫹]cyt in both normal and IPAH-PASMC. A: representative traces showing changes in [Ca2⫹]cyt in normal (Normal, top) and IPAH (IPAH, bottom) PASMC before and during application of shear stress (Perfusion) in the absence or presence of the TRPV4 channel inhibitor RR (1 ␮M). B: summarized data (means ⫾ SE) showing the amplitude of shear stress-induced transient (Trans) and plateau (Plateau) increases of [Ca2⫹]cyt in the absence (Cont) or presence (RR) of 1 ␮M RR in Normal (top) and IPAH (bottom) PASMC. *P ⬍ 0.05 vs. Cont.

0 600

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IPAH

[Ca ]cyt (nM)

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Perfusion-induced 2+ Rise in [Ca ]cyt (nM)

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4αPDD

4αPDD

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4αPDD RR

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150 100 50 0

Nor

Cont

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IPAH

Fig. 5. Activation of TRPV4 channels with 4␣-phorbol 12,13-didecanoate (4␣PDD) causes a greater increase in [Ca2⫹]cyt in IPAH-PASMC than in normal PASMC. A and B: averaged records obtained from all cells (Averaged; A) and representative records obtained from selected individual cells (Individual; B) showing changes in [Ca2⫹]cyt before and during extracellular application of 10 ␮M 4␣PDD (a selective agonist of TRPV4 channels) in normal PASMC (Normal, left), IPAH-PASMC (middle), and IPAH-PASMC pretreated with 1 ␮M Ruthenium red (RR, a blocker of TRPV4 channels, right). C: summarized data (means ⫾ SE) showing the amplitude of 4␣PDD-induced increases in [Ca2⫹]cyt in normal (Nor) and IPAH-PASMC with (RR) or without (Cont) treatment with 1 ␮M RR. *P ⬍ 0.05 vs. Nor PASMC and Cont-IPAH-PASMC.

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1

2

3

4

TRPV4 β-actin TRPM7 β-actin

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B

TRPC6 β-actin

100 42

exert significant increased pressure and shear stress on PASMC lying in the limited and small medial space. There arises another question, however, that pulmonary vascular remodeling or vascular wall thickening in patients with IPAH indicates the existence of extracellular matrix hyperplasia, which might block the pore or fenestral pore on the IEL (5, 32, 39), resulting in decreased transmural flow and attenuated shear stress on SMC. It has been IPAH-PASMC

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[Ca ]cyt (nM)

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IPAH

suggested that extracellular matrix filling with the fenestral pore is much less dense than elastin fibers that comprise the IEL and that, therefore, transmural water flow can be funneled through the pore system as it enters the SMC layer or the media to exert shear stress (5). Our previous study showed that mRNA and protein expression level of the canonical TRP (TRPC) channels (e.g., TRPC6

90

TRPV4 β-actin

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C

Normal Control

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Cont-siR

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siR-TRPM7

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100

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kDa TRPM7 β-actin

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IPAH-PASMC

kDa

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Perfusion

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Perfusion

0

Perfusion

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siR-TRPV4

5 min

Perfusion

Perfusion-induced 2+ Rise in [Ca ]cyt (nM)

A

TRPC6 Protein Level (Fold Increase)

Fig. 6. Upregulated protein expression of the mechano- kDa 1 90 sensitive channels TRPV4, TRPM7, and TRPC6 in 42 PASMC from IPAH patients compared with PASMC from normal subjects. A: Western blot analysis of TRPV4 and 150 TRPV4 in two normal (Nor) PASMC samples and four 42 IPAH-PASMC samples (left). ␤-Actin was used as a control. Summarized data (means ⫾ SE) show expression levels of TRPM7 and TRPV4 in Nor and IPAH-PASMC (right). *P ⬍ 0.05 vs. Nor. B: Western blot analysis of TRPC6 from one normal PASMC sample and two IPAHPASMC samples (left), along with the summarized data (means ⫾ SE, right), showing significant upregulation of TRPC6 in IPAH-PASMC compared with normal PASMC. kDa *P ⬍ 0.05 vs. Nor.

IPAH

3.0

TRPM7 Protein Level (Fold Increase)

Nor

TRPV4 Protein Level (Fold Increase)

A

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200

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Control Cont-siR siR-TRPV4

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400

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Plateau

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Trans

Plateau

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[Ca ]cyt (nM)

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500

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500

siR-TRPM7

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400

400

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300

300

300

300

200

200

200

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100

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Perfusion

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Perfusion-induced 2+ Rise in [Ca ]cyt (nM)

IPAH 400

400

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Trans

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Plateau

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Fig. 7. Downregulation of TRPM7 or TRPV4 with siRNA significantly attenuates shear stress-mediated increases in [Ca2⫹]cyt in PASMC from IPAH patients. A: Western blot analysis of TRPM7 and TRPV4 in IPAH-PASMC (Control), IPAH-PASMC treated with scrambled siRNA (Cont-siR), and IPAH-PASMC treated with siRNA for TRPM7 (left) or TRPV4 (right). ␤-Actin was used as a control. B: representative traces showing changes in [Ca2⫹]cyt during application of flow shear stress (Perfusion) in normal (top) and IPAH (top) PASMC (Control), as well as normal and IPAH-PASMC treated with scrambled siRNA (Cont-siR) and siRNA for TRPM7 (siR-TRPM7) or TRPV4 (siR-TRPV4). C: summarized data (means ⫾ SE) showing the amplitude of shear stress-induced transient (Trans) and plateau (Plateau) phase increases of [Ca2⫹]cyt in normal PASMC (top, Control) and IPAH-PASMC (top, Control), as well as normal and IPAH-PASMC treated with Cont-siRNA, siR-TRPM7, or siR-TRPV4. *P ⬍ 0.05 vs. Control and Cont-siR. The results shown indicate that TRPM7 and TRPV4 are necessary for shear stress-induced increase in [Ca2⫹]cyt in normal and IPAH-PASMC. AJP-Cell Physiol • doi:10.1152/ajpcell.00115.2014 • www.ajpcell.org

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0.3

2+

0.3

CPA Perfus

0Ca

0Ca

Normal

IPAH

0.3

0.3

0.2

0.2

0.1

0.4

0Ca CPA BRK

E

2+

0.4

CPA Perfus

[Mg ]cyt (Ratio)

2+

[Ca ]cyt (Ratio)

D

0.2

0.2

0.1

0Ca CPA

BRK

0.30

Normal

0.30

0.27

0.27

0.24

0.24

0.21

0.21

0.18

0.18

0.15

0.15

CPA Perfus

Normal

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CPA Perfus

0.20

IPAH

0.20

0.18

0.18

0.18

0.16

0.16

0.16

0.14

0.14

0.14

0.12

0.12

0.12

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0Ca CPA BRK

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*

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Nor

0Ca

0Ca 0.20

C

IPAH

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0Ca CPA BRK

0.10

F

IPAH

IPAH

2+

B

IPAH

Rise in [Mg ]cyt (Ratio)

0.4

Perfusion-induced 2+ Rise in [Mg ]cyt (Ratio)

Normal

[Mg ]cyt (Ratio)

0.4

2+

[Ca ]cyt (Ratio)

A

5 min

0Ca

0.05

Nor-BRK

0.04

IPAH-BRK IPAH-BRK

0.03 0.02 0.01

+2-APB

*

0.00

CPA BRK+ 2-APB

Fig. 8. Shear stress-mediated increase in [Ca2⫹]cyt in normal and IPAH-PASMC is due to Ca2⫹ influx through nonselective cation channels. A: representative traces showing changes of [Ca2⫹]cyt in normal and IPAH-PASMC treated with 10 ␮M cyclopiazonic acid (CPA, a SERCA inhibitor) before application of shear stress (Perfus) in Ca2⫹-free solution (0Ca). In the absence of extracellular Ca2⫹, shear stress (perfusion) fails to induce increases in [Ca2⫹]cyt in both normal and IPAH-PASMC in which intracellularly stored Ca2⫹ is depleted by CPA. B: representative traces showing changes in [Mg2⫹]cyt for normal and IPAH-PASMC treated with 10 ␮M CPA before application of shear stress (Perfus) in 0Ca solution. C: summarized data (means ⫾ SE) showing the shear stress-mediated increases in [Mg2⫹]cyt for normal (Nor) and IPAH-PASMC. *P ⬍ 0.05 vs. Nor. D: representative traces showing changes in [Ca2⫹]cyt for Normal and IPAH-PASMC treated by 10 ␮M CPA (an inhibitor of SERCA) before BRK stimulation in Ca2⫹-free (0Ca) solution. E: representative traces showing changes in [Mg2⫹]cyt for Normal and IPAH-PASMC treated with CPA before BRK stimulation in Ca2⫹-free (0Ca) solution. IPAH-PASMC were also treated with 2-APB during BRK stimulation in 0Ca solution. F: summarized data (means ⫾ SE) showing the amplitude of BRK-induced rise in [Mg2⫹]cyt with or without 2-APB treatment in normal (Nor) or IPAH-PASMC. *P ⬍ 0.05 vs. Nor-BRK and IPAH-BRK ⫹ 2-APB.

and TRPC3) was significantly higher in IPAH-PASMC than in normal control PASMC; the upregulated TRPC6 channel was involved in enhanced cell proliferation of IPAH-PASMC (47). Here we reported that the melastatin-related TRP channel TRPM7 and the vanilloid-related TRP channel TRPV4 also expressed higher levels in IPAH-PASMC than in normal PASMC. At least 10 TRP channels, TRPC1/5/6, TRPM3/7, TRPV1/2/4, TRPA1, and TRPP2, exhibit mechanosensitivity as revealed by many investigators (17). TRPM7, also termed as chanzyme, is expressed ubiquitously in many cell and tissue types including vascular SMC. Because of its unique structure, TRPM7 comprises an ion channel pore that is fused to a kinase domain at the COOH terminus (1, 13, 38, 41, 45). TRPM7 channel was reported to accumulate quickly in the plasma membrane in response to physiological levels of fluid flow; endogenous native TRPM7 current was significantly increased in vascular SMC stimulated by fluid shear stress (27). Because of its high permeability to Ca2⫹, TRPM7 may be an important and critical contributor to the increased [Ca2⫹]cyt shown in PASMC from patients with IPAH (27) and animals with experimental pulmonary hypertension (43). Activation of upregulated TRPM7 channels in PASMC might play an important role in pathological response to vessel wall injury or increased hydrostatic pressure in IPAH. Here we reported that pharmacological blockage or knockdown expression of TRPM7 channels significantly inhibited shear stress-induced increase in [Ca2⫹]cyt in IPAH-PASMC, implying its potential role in pathological response to mechanical stimuli in IPAH. Another extensively studied mechanosensitive TRP channel, TRPV4, which has been much explored on endothelial cells, is

also a Ca2⫹-permeable cation channel (2, 25). Here we found that TRPV4 channel is expressed in both normal and IPAHPASMC, but protein expression level of TRPV4 was significantly higher in IPAH-PASMC than in normal PASMC. Pharmacological blockade (with Ruthenium red) or knockdown (with siRNA) of TRPV4 channels significantly attenuated shear stress-induced increase in [Ca2⫹]cyt in PASMC isolated from IPAH patients. These data indicate that 1) TRPV4 is involved in forming mechanosensitive cation channels in PASMC and plays an important role in sensing mechanical stress and 2) upregulated expression of TRPV4 channels in IPAH-PASMC may contribute to the enhanced shear stressmediated increase in [Ca2⫹]cyt and ultimately increased pulmonary vasoconstriction and vascular remodeling. In addition to the increased expression of TRPM7 and TRPV4, we have also observed upregulation of TRPC6 channel expression in PASMC from IPAH patients in comparison to PASMC from normal subjects and patients without pulmonary hypertension (Fig. 6) (47– 49). As mentioned earlier, TRPC6 is also considered to be an important member of mechanosensitive ion channels (17). The results from this study imply that upregulated homotetramers and/or heterotetramers formed by TRPM7, TRPV4, and/or TRPC6 may all contribute to the enhanced shear stress-mediated increase in [Ca2⫹]cyt in PASMC from IPAH patients. One potential limitation of this study is the use of cultured PASMC, which may have dedifferentiated compared with freshly isolated PASMC. The expression level of smooth muscle cell markers, such as ␣-actin, myosin heavy chain, and caldesmon, strongly decreases and it has been shown that

AJP-Cell Physiol • doi:10.1152/ajpcell.00115.2014 • www.ajpcell.org

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muscarinic receptors are absent in cultured cells that exhibit a proliferative phenotype. Thus, caution should be used when drawing conclusions from experiments using cultured PASMC as the study subject, as they have different molecular features compared with freshly isolated cells. More studies using freshly isolated human or animal tissue are needed to further prove and clarify the conclusions from this study. Additionally, a study showed that altering the culture condition could preserve the differentiated state of SMC (18), which would be a better way to study cultured SMC when freshly isolated SMC are not suitable. Another limitation of this study is the static culture condition for investigating the effect of flow shear stress on PASMC, and future studies should be more focused on mimicking the dynamic growing environment of PASMC in vivo. In conclusion, the augmented mechanosensitivity of PASMC from IPAH patients to fluid flow shear stress is associated with an enhanced increase in [Ca2⫹]cyt that may contribute to enhanced PASMC contraction, migration, and proliferation. The data presented here suggest that upregulated expression of TRPM7, TRPV4, and TRPC6 channels in PASMC from IPAH patients appears to be, at least in part, the cause of the enhanced increase in [Ca2⫹]cyt induced by mechanical stimuli (e.g., flow shear stress and stretch). The enhanced mechanosensitive rise in [Ca2⫹]cyt in PASMC from IPAH patients would contribute to increased pulmonary vascular myogenic tone, sustained pulmonary vasoconstriction, and pulmonary vascular medial hypertrophy. Pharmacological blockade of TRPM7, TRPV4, and TRPC6 channels and/or siRNA-driven downregulation of these channels’ expression may be a novel therapeutic approach for IPAH patients who manifest significant abnormal hemodynamics and do not respond to conventional drug therapy. GRANTS This work was supported in part by grants from the National Heart, Lung and Blood Institute of the National Institutes of Health (HL-115014, HL066012, and HL-098053). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS S.S., A.Y., H.Y., R.J.A., H.T., A.M., and J.X.-J.Y. conception and design of research; S.S., A.Y., H.Y., H.T., and J.X.-J.Y. performed experiments; S.S., A.Y., H.Y., R.J.A., K.A.S., A.M., and J.X.-J.Y. analyzed data; S.S., A.Y., H.Y., R.J.A., K.A.S., H.T., A.M., and J.X.-J.Y. interpreted results of experiments; S.S., A.Y., H.Y., R.J.A., K.A.S., and J.X.-J.Y. prepared figures; S.S., R.J.A., K.A.S., A.M., and J.X.-J.Y. drafted manuscript; S.S., A.Y., H.Y., R.J.A., K.A.S., H.T., A.M., and J.X.-J.Y. edited and revised manuscript; S.S., A.Y., H.Y., R.J.A., K.A.S., H.T., A.M., and J.X.-J.Y. approved final version of manuscript. REFERENCES 1. Aarts M, Iihara K, Wei WL, Xiong ZG, Arundine M, Cerwinski W, MacDonald JF, Tymianski M. A key role for TRPM7 channels in anoxic neuronal death. Cell 115: 863–877, 2003. 2. Bubolz AH, Mendoza SA, Zheng X, Zinkevich NS, Li R, Gutterman DD, Zhang DX. Activation of endothelial TRPV4 channels mediates flow-induced dilation in human coronary arterioles: role of Ca2⫹ entry and mitochondrial ROS signaling. Am J Physiol Heart Circ Physiol 302: H634 –H642, 2012. 3. Busse R, Fleming I. Regulation of endothelium-derived vasoactive autacoid production by hemodynamic forces. Trends Pharmacol Sci 24: 24 –29, 2003.

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