Activation of endothelial TRPV4 channels mediates flow-induced dilation in human coronary arterioles: role of Ca²⁺ entry and mitochondrial ROS signaling

Abstract

In human coronary arterioles (HCAs) from patients with coronary artery disease, flow-induced dilation is mediated by a unique mechanism involving the release of H₂O₂ from the mitochondria of endothelial cells (ECs). How flow activates ECs to elicit the mitochondrial release of H₂O₂ remains unclear. Here, we examined the role of the transient receptor potential vanilloid type 4 (TRPV4) channel, a mechanosensitive Ca²⁺-permeable cation channel, in mediating ROS formation and flow-induced dilation in HCAs. Using RT-PCR, Western blot analysis, and immunohistochemical analysis, we detected the mRNA and protein expression of TRPV4 channels in ECs of HCAs and cultured human coronary artery ECs (HCAECs). In HCAECs, 4α-phorbol-12,13-didecanoate (4α-PDD), a selective TRPV4 agonist, markedly increased (via Ca²⁺ influx) intracellular Ca²⁺ concentration. In isolated HCAs, activation of TRPV4 channels by 4α-PDD resulted in a potent concentration-dependent dilation, and the dilation was inhibited by removal of the endothelium and by catalase, a H₂O₂-metabolizing enzyme. Fluorescence ROS assays showed that 4α-PDD increased the production of mitochondrial superoxide in HCAECs. 4α-PDD also enhanced the production of H₂O₂ and superoxide in HCAs. Finally, we found that flow-induced dilation of HCAs was markedly inhibited by different TRPV4 antagonists and TRPV4-specific small interfering RNA. In conclusion, the endothelial TRPV4 channel is critically involved in flow-mediated dilation of HCAs. TRPV4-mediated Ca²⁺ entry may be an important signaling event leading to the flow-induced release of mitochondrial ROS in HCAs. Elucidation of this novel TRPV4-ROS pathway may improve our understanding of the pathogenesis of coronary artery disease and/or other cardiovascular disorders.

shear stress generated by blood flow is an important physiological regulator of vascular tone. Flow or shear induces vasodilation in virtually every vascular bed studied, in both conduit arteries and resistance arteries (6, 15, 17, 18). Flow-induced dilation typically involves the release of the endothelial relaxing factors nitric oxide, prostacyclin, and/or cytochrome P-450 metabolites of arachidonic acid [e.g., epoxyeicosatrienoic acids (EETs)] (3). Our recent studies (20, 22, 26) indicated that, in human coronary arterioles (HCAs) from patients with coronary artery disease (CAD), flow induces vasodilation through a unique mechanism requiring the endothelial production of ROS, specifically H₂O₂ originating from the mitochondrial electron transport chain. The underlying mechanisms by which flow stimulates the release of mitochondrial H₂O₂ in coronary endothelial cells (ECs) remain largely unsolved.

The transient receptor potential (TRP) vanilloid type 4 (TRPV)4 channel, a Ca²⁺-permeable cation channel and a member of the TRP channel superfamily, is activated by chemical agonists, including the synthetic phorbol derivative 4α-phorbol-12,13-didecanoate (4α-PDD) (35) and arachidonic acid metabolites (e.g., EETs) (34, 36), as well as by mechanical stimuli, such as hypotonic cell swelling (19, 29), moderate warmth (>27°C) (11, 37), and membrane stretch (31). Recently, accumulating evidence has indicated that the TRPV4 channel is expressed in ECs and contributes to the regulation of vascular tone (41). In particular, activation of endothelial TRPV4 channels has been implicated in flow-mediated dilation in several animal vascular beds (12, 16, 23, 24). It is yet to be determined whether the TRPV4 channel plays a role in endothelium-dependent vasodilator responses in human vessels.

Given that an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) stimulates mitochondrial ROS generation in ECs, as well as in other mammalian cells (10, 40), we hypothesized that endothelial TRPV4-mediated Ca²⁺ entry may be a potential mechanism responsible for flow-induced mitochondrial ROS generation and vasodilation of HCAs. In this study, we examined TRPV4 expression and TRPV4-mediated Ca²⁺ responses in coronary ECs, TRPV4-mediated vasodilation and ROS production in HCAs, and the effect of TRPV4 blockade/knockdown on flow-induced dilation of HCAs. We also assessed mitochondrial ROS generation in coronary ECs in response to TRPV4 activation.

MATERIALS AND METHODS

Tissue acquisition.

Fresh human right atrial appendages were obtained as discarded surgical specimens from patients undergoing cardiopulmonary bypass procedures, as previously described (25). After surgical removal, atrial tissues were immersed in an ice-cold cardioplegia solution and immediately transported to the laboratory. Demographic data were obtained from hospital records at the time of surgery and are shown in Table 1. All protocols were approved by the appropriate local Institutional Review Boards on the use of human subjects in research.

Table 1.

Patient demographics

	Number of Patients	Percentage of Patients
Sex
Male	34	57
Female	26	43
Age, yr (mean ± SD)	64 ± 14
Surgical procedure
Myocardial revascularization	29	48
Coronary artery bypass graft	4	7
Mitral valve replacement	23	38
Aortic valve replacement	18	30
Tricuspid valve replacement	11	18
Other	1	2
Underlying disease
Coronary artery disease	38	63
Hypertension	33	55
Hyperlipidemia	27	45
Diabetes mellitus	11	18
Atrial fibrillation	17	28
Congestive heart failure	8	13
Myocardial infarction	6	10
Tobacco use	13	22
None of the above	6	10

A total of 60 patients were studied.

Cell culture.

Human coronary artery ECs (HCAECs) and human coronary artery smooth muscle cells (SMCs) were obtained from Lonza (Walkersville, MD) and maintained in full growth medium (EGM-2 MV and SmGM-2 for ECs and SMCs, respectively) according to the manufacturer's instructions. Cells between passages 4 and 6 were used for experiments.

RNA extraction and RT-PCR.

Total RNA from cultured cells was extracted with TRIzol, and cDNA was synthesized using the iScript cDNA Synthesis kit (Bio-Rad). For some experiments, ECs and SMCs were enzymatically dissociated from isolated arterioles and selectively collected using a glass pipette, followed by cell disruption and cDNA synthesis as previously described (24). Gene-specific fragments were amplified by PCR with the following primers: TRPV4, forward 5′-ACAACAGCAAGATTGAGAACC-3′ and reverse 5′-ACCAGGACAGAGTAGATGAAG-3′ [for a 364-bp fragment (NM_021625)]; platelet-EC adhesion molecule (PECAM)-1, forward 5′-AGACAACCCCACTGAAGAC-3′ and reverse 5′-TCCAGACTCCACCACCTTAC-3′ [for a 154-bp fragment (NM_000442)]; and GAPDH, forward 5′-ATGGCAAATTCCATGGCACCGT-3′ and reverse 5′-TGCAGGAGGCATTGCTGATGAT-3′ [for a 300-bp fragment (NM_002046)].

Western blot analysis.

Vascular tissues or cells were homogenized in ice-cold lysis buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1% deoxycholic acid, 0.1% SDS, and 0.5% Nonidet P-40] supplemented with a protease inhibitor cocktail (Roche) and centrifuged at 12,000 g for 10 min at 4°C. Protein samples (10–20 μg) were subjected to 10% SDS-PAGE and transferred to nitrocellulose or polyvinylidene difluoride membranes. Membranes were blotted with a polyclonal antibody against the human TRPV4 channel (1:1,000 dilution, LS-A8583, MBL) or a monoclonal antibody against β-actin (1:10,000 dilution, ab6276, Abcam AC-15 clone) followed by peroxidase-conjugated secondary antibodies (1:5,000 dilution). Immunoreactive complexes were visualized using an ECL chemiluminescence detection system (Amersham).

Immunohistochemistry.

Freshly dissected coronary arteries and arterioles were fixed with 10% buffered zinc formalin, embedded into paraffin blocks, and cut into 4-μm sections. Tissue sections were deparaffinized in xylene and rehydrated through a graded series of ethanol and water. Sections were then treated with 3% H₂O₂ in PBS to block endogenous peroxidase followed by antigen retrieval by heating slides in citrate buffer (pH 6.0). After an additional block with 3% BSA in PBS, tissue sections were probed with a polyclonal antibody against the human TRPV4 channel (1:400 dilution, LS-A8583, MBL). For immunodetection, sections were incubated with a polyclonal goat anti-rabbit horseradish peroxidase-conjugated antibody (1:000 dilution, DAKO) and then with a peroxidase-substrate solution (DAKO Liquid DAB-Substrate Chromogen System, DAKO). Samples were then rinsed, counterstained with the nuclear dye hematoxylin (DAKO), and mounted. Negative controls were obtained by omitting the primary antibody.

Small interfering RNA transfection.

Two TRPV4-specific small interfering (si)RNA duplexes were designed and synthesized by Qiagen using the following target sequences: 1) 5′-CTGGGATTTGCCGGTGCTCAA-3′ [nucleotides 3194–3214 (NM_021625)] and 2) 5′-CTGGAGTGAATTCTCTCTTCA-3′ [nucleotides 1702–1722 (NM_021625)]. A nonsilencing scrambled siRNA was used as a control. The scrambled and TRPV4 siRNA duplexes were first tested on HCAECs for their efficacy and specificity. The two TRPV4 siRNA duplexes were found to be equally effective. For isolated vessels, only siRNA 1 was used. Coronary arterioles were transfected with the scrambled or TRPV4 siRNA duplex (100 nM) complexed with Lipofectamine 2000 (0.5 μg/ml, Invitrogen) for 4–6 h according to the manufacturer's instructions. Arterioles were incubated for additional 24–36 h in fresh medium at 37°C and assessed for TRPV4 protein knockdown using Western blot analysis. Transfected arterioles were examined for vasodilator responses as described below.

Measurement of [Ca²⁺]_i.

ECs were plated onto 35-mm glass-bottom petri dishes and grown to 60–70% confluence. Cells were loaded with fura-2 AM (5 μM, Molecular Probes) at room temperature for 30–60 min in HEPES buffer containing (in mM) 140 NaCl, 4.7 KCl, 1.6 CaCl₂, 1.17 MgSO₄, 1.18 NaH₂PO₄, 5.5 glucose, and 10 HEPES (pH 7.4). Cytosolic Ca²⁺ changes were measured using a fluorescence imaging system as we have previously reported (24). Fura-2 signals were calibrated, and [Ca²⁺]_i was calculated as previously described (39). All experiments were performed at 37°C.

Videomicroscopy.

Coronary arterioles (≈100–200 μm) were cannulated with two glass micropipettes for the continuous videomicroscopic measurement of internal diameters as previously described (20, 22). Vessels were pressurized to 60 mmHg and equilibrated for 1 h at 37°C in Krebs-physiological salt solution gassed with 21% O₂-5% CO₂. Vessels were preconstricted with endothelin-1 (5 × 10¹⁰–10⁻⁹ M) to 30–50% of the baseline internal diameter if the spontaneous myogenic tone was not sufficient to achieve the target reduction in diameter. Concentration-dependent responses to 4α-PDD (5 × 10⁻⁸–5 × 10⁻⁶ M), a selective TRPV4 activator, were examined in the absence or presence of catalase (500 U/ml). 4α-PDD was applied intraluminally with minimal flow generated under a 10-cmH₂O pressure gradient, and catalase was given both intraluminally and extraluminally for 15 min before 4α-PDD. Flow was produced by changing the heights of two syringe reservoirs in equal and opposite directions. Flow-induced dilation (20–100 cmH₂O) was examined before and after treatment with the TRPV4 blocker ruthenium red (1 μM), gadolinium III (100 μM) (33), or RN-1734 (20 μM) (32) or after transfection with scrambled or TRPV4 siRNA. Responses to bradykinin (10⁻¹⁰–10⁻⁶ M), an endothelium-dependent receptor agonist, or papaverine (10⁻⁴ M), an endothelium-independent vasodilator, were recorded as controls. In some vessels, the endothelium was removed with an air bolus. Disruption of endothelial function was confirmed by a markedly reduced dilation (<10%) to bradykinin (10⁻⁶ M). Vasodilator responses are expressed as percent relaxation relative to spontaneous tone or endothelin-1 preconstriction, with 100% relaxation representing the passive baseline diameter.

Detection of vascular ROS.

Production of H₂O₂ and superoxide in coronary arterioles was assessed by fluorescence microscopy using 2′,7′-dichlorodihydrofluorescein (DCFH) diacetate and dihydroethidium (DHE), respectively (21, 22). Briefly, two vessels dissected from each atrial tissue were perfused with vehicle or 4α-PDD (1 μM) under 10-cmH₂O pressure gradient. Another vessel was incubated with polyethylene glycol (PEG)-SOD (150 U/ml) and PEG-catalase (500 U/ml) to assess nonspecific fluorescence. Vessels were then incubated with DCFH (5 μM) and DHE (5 μM) in a light-protected chamber for 30 min at 37°C. Vessel segments were then rinsed and examined under a fluorescence microscope. Samples were excited at 488 and 585 nm, and emission was measured at 526 and 620 nm, respectively. Images were analyzed for fluorescence intensity within a user-defined region. Artifactual autofluorescent regions were manually excluded from analysis. Relative average fluorescence intensity was normalized for surface area and compared between control and experimental vessels.

Measurement of mitochondrial ROS.

Mitochondrial ROS production in ECs was measured using MitoSOX, a fluorescent probe specific for mitochondrial superoxide. Cells were incubated in HEPES buffer containing vehicle, 4α-PDD (2 μM), 4α-PDD plus ruthenium red (1 μM), or 4α-PDD plus Mito Vitamin E (1 μM, a mitochondria-targeted antioxidant) (5). MitoSOX (5 μM, Molecular Probes) was added and incubated for 30 min at 37°C. Cellular fluorescence was examined under a fluorescence microscope at an excitation/emission of 510/580 nm according to the manufacturer's instructions. Results were expressed as relative fluorescence intensity normalized to controls.

Data analysis.

Data are presented as mean ± SE. Significant differences between mean values were evaluated by a Student t-test or repeated-measures ANOVA followed by the Student-Newman-Keuls multiple-comparison test. P values of <0.05 were considered statistically significant.

RESULTS

TRPV4 expression in human coronary vascular cells.

ECs and SMCs were freshly isolated from HCAs and examined for TRPV4 mRNA expression by RT-PCR analysis. As shown in Fig. 1A, TRPV4 transcripts were detected in ECs but not in SMCs. As parallel controls, a PCR product for PECAM-1, an endothelial marker, was detected in ECs but not in SMCs, whereas amplification of GAPDH transcripts was found in both cell types. Similarly, TRPV4 transcripts were detected in primary cultures of HCAECs but not human coronary artery SMCs. Furthermore, TRPV4 protein was detected in HCAECs and HCAs by Western blot analysis (Fig. 1B). The TRPV4 antibody detected one primary band of ∼110 kDa in HCAECs and HCAs and a second band of ∼98 kDa in HCAECs and less frequently in HCAs (data not shown). The 110-kDa band presumably represents the glycosylated form of TRPV4 protein (1), whereas the 98-kDa band matches with the unprocessed precursor of human TRPV4 protein with a calculated molecular weight of 98 kDa. Cell type-specific expression of TRPV4 protein was also examined in coronary arterioles and small coronary arteries using immunohistochemistry (Fig. 1C). In tissue sections of both types of vessels, the endothelium demonstrated a strong staining for TRPV4. There was much less immunoreactivity in the underlying smooth muscle, especially for small coronary arteries, and virtually no staining in the adventitial layer. The control section without the primary antibody showed negative staining. Similar to small coronary arteries or arterioles, we also detected prominent staining for TRPV4 in the endothelium of large human coronary arteries (data not shown).

Fig. 1.

Transient receptor potential vanilloid type 4 (TRPV4) expression in human coronary vascular cells. A: RT-PCR analysis of TRPV4 mRNA expression in endothelial cells (ECs) and smooth muscle cells (SMCs) freshly isolated from human coronary arterioles (HCAs; top) as well as in cultured human coronary artery ECs (HCAECs) and human coronary artery (HCASMCs; bottom). Amplification of platelet-EC adhesion molecule (PECAM)-1, an EC marker, and GAPDH, a ubiquitous housekeeping protein, were performed in parallel as controls. B: Western blot analysis of TRPV4 protein in HCAECs and HCAs. C: immunohistochemical staining for TRPV4 in HCAs and human coronary small arteries. Control sections were similarly processed except that the primary antibody was omitted (no 1° Ab). Scale bar = 10 μm. All data are representative of 3–4 separate experiments.

TRPV4-mediated endothelial Ca²⁺ entry.

To examine whether TRPV4 channels are functional in coronary ECs, we examined the TRPV4-mediated Ca²⁺ response using a fura-2 assay. As shown in Fig. 2A, the addition of 4α-PDD (2 μM), a specific TRPV4 channel activator, elicited a marked increase in [Ca²⁺]_i in HCAECs (change in [Ca²⁺]_i: 253 ± 42 nM). This response was rapidly reversed by ruthenium red (1 μM, 55 ± 5 nM; Fig. 2B), a TRPV4 channel blocker, or by removing extracellular Ca²⁺ (20 ± 2 nM; Fig. 2D). Preincubation of cells with ruthenium red prevented 4α-PDD-induced Ca²⁺ responses but did not significantly affect the peak Ca²⁺ transient in response to ATP (1 mM), a receptor agonist that induces inositol 1,4,5-trisphosphate-mediated Ca²⁺ release from intracellular stores (Fig. 2C). Summary data are shown in Fig. 2E. ECs were more sensitive to the TRPV4 activator 4α-PDD at physiological (37°C) temperature compared with room temperature (data not shown), which is in accordance with the thermosensitive characteristics of TRPV4 channels.

Fig. 2.

TRPV4-mediated Ca²⁺ responses in coronary ECs. A: representative images (left) of HCAECs loaded with the fluorescent Ca²⁺ indicator fura-2. 4α-Phorbol-12,13-didecanoate (4α-PDD; 2 μM), a specific TRPV4 agonist, elicited a marked increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i). Right, a typical trace of the 4α-PDD-induced [Ca²⁺]_i increase. F₃₄₀/F₃₈₀, ratio of fluorescence at 340 nm to that at 380 nm. B–E: other representative traces. B: the 4α-PDD-induced [Ca²⁺]_i increase was rapidly reversed by ruthenium red (RuR; 1 μM), a TRPV4 channel blocker. C: preincubation of ECs with RuR blocked the Ca²⁺ response to 4α-PDD but not to ATP (1 mM), a receptor agonist that induces an inositol 1,4,5-trisphosphate-mediated Ca²⁺ release from intracellular stores. D: the 4α-PDD-induced [Ca²⁺]_i increase was immediately reversed by removing extracellular Ca²⁺. E: summarized data. n = 3–6 experiments (10–20 cells/each). *P < 0.05 vs. 4α-PDD.

TRPV4-mediated dilation and ROS production.

To determine whether TRPV4 activation affects vascular tone, HCAs were preconstricted with endothelin-1, and 4α-PDD (1 and 5 μM) was applied by intraluminal perfusion under a 10-cmH₂O pressure gradient. The relative low flow generated under this pressure gradient alone did not produce significant vasodilation. Perfusion of arterioles with 4α-PDD elicited a potent endothelium-dependent dilation in a concentration-related manner. The dilation to 5 μM 4α-PDD was 61 ± 9% and −20 ± 2% for endothelium-intact and -denuded vessels, respectively (Fig. 3A).

Fig. 3.

TRPV4-mediated vasodilation and ROS generation in coronary arterioles. A: the TRPV4 agonist 4α-PDD (1 and 5 μM) dilated HCAs in an endothelium-dependent manner. Arterioles with an intact endothelium (intact) or after endothelial removal (denuded) were cannulated, and 4α-PDD was perfused into the lumen of arterioles with relatively low flow (10-cmH₂O pressure gradient), which itself did not produce significant dilation. n = 5. *P < 0.05 vs. control. B: 4α-PDD-induced dilation was reduced after pretreatment of vessels with catalase (500 U/ml, intraluminal and extraluminal). n = 6. *P < 0.05 vs. control. C: 4α-PDD (1 μM) increased vascular superoxide and H₂O₂ production, as indicated by the enhanced fluorescence intensities of oxidative products of dihydroethidium (DHE) and 2′,7′-dichlorodihydrofluorescein (DCFH) compared with controls. Arterioles treated with polyethylene glycol (PEG)-SOD (150 U/ml) and PEG-catalase (500 U/ml) were used to assess nonspecific fluorescence. Scale bar = 100 μm. Left, representative images; right, summary of fluorescence intensity normalized to controls. n = 8. *P < 0.05 vs. control.

Our previous studies (20, 22, 26) have demonstrated that H₂O₂ is the primary relaxing factor released in HCAs in response to shear stress. Therefore, we next examined whether H₂O₂ is also involved in 4α-PDD-induced dilation. As shown in Fig. 3B, incubation of HCAs with catalase (500 U/ml) inhibited vasodilator responses to 4α-PDD (dilation at 5 μM: 38 ± 5%). To directly assess whether TRPV4 activation induces vascular ROS generation, we performed semiquantitative analysis of H₂O₂ and superoxide production using the fluorescent probes DCFH and DHE, respectively. Perfusion of coronary arterioles with 4α-PDD (1 μM) markedly increased the production of both H₂O₂ and superoxide compared with controls, with normalized fluorescence intensities of 2.0 ± 0.5 and 3.4 ± 0.7, respectively (Fig. 3C).

TRPV4 channels in flow-induced dilation of HCAs.

Fluid flow generated under 20- to 100-cmH₂O pressure gradients induced a potent graded dilation of HCAs (dilation at 100 cmH₂O: 85 ± 3%, n = 6; Fig. 4A). This flow-induced dilation was markedly inhibited (33 ± 4%, n = 6, P < 0.05 vs. control) by the TRPV4 antagonist ruthenium red (1 μM). Flow-mediated dilation of HCAs was also reduced by pretreatment of arterioles with 100 μM gadolinium III (51 ± 7% vs. 83 ± 5% of controls, n = 5, P < 0.05; Fig. 4B), another commonly used TRPV4 antagonist, or with 20 μM RN-1734 (21 ± 4% vs. 60 ± 3% of controls, n = 5, P < 0.05; Fig. 4C), a recently developed TRPV4 selective antagonist (32). In contrast, bradykinin-induced dilation was not affected by the TRPV4 antagonist RN-1734 (20 μM; Fig. 4D). As shown in Table 2, no significant effect on baseline vascular tone was observed for ruthenium red, gadolinium, or RN-1734. In addition, similar dilation to the smooth muscle relaxant papaverine (100 μM) was found in arterioles with or without pretreatment with these TRPV4 inhibitors (data not shown).

Fig. 4.

Effect of TRPV4 inhibitors on flow-induced dilation of coronary arterioles. Incubation of coronary arterioles with the TRPV4 inhibitors RuR (1 μM; A), gadolinium III (100 μM; B), or RN-1734 (20 μM; C) attenuated dilatory responses to flow. RN-1734 (20 μM; D) had no significant effect on bradykinin-induced dilation. Luminal flow was generated by pressure gradients of 10–100 cmH₂O (A and B) or of 20–100 cmH₂O (C). n = 6 (A), 4 (B), 4 (C), and 4 (D). *P < 0.05 vs. control.

Table 2.

Characteristics of isolated coronary arterioles

	Internal Diameter, μm
	Passive	Active (without inhibitor)	Active (with inhibitor)	Endothelin-1	Number of Coronary Arterioles
Control	141 ± 5	133 ± 4		90 ± 4	6
Ruthenium red	141 ± 5		133 ± 4	91 ± 3	6
Control	131 ± 16	126 ± 17		85 ± 10	5
Gadolinium	131 ± 16		125 ± 9	88 ± 10	5
Control	135 ± 18	134 ± 17		83 ± 9	4
RN-1734	135 ± 18		131 ± 20	79 ± 12	4
Scrambled	148 ± 11	133 ± 6		97 ± 5	5
Transient receptor potential vanilloid type 4 small interfering RNA	153 ± 9	140 ± 9		86 ± 11	5

Values are means ± SE of flow-mediated dilation.

Since the above TRPV4 antagonists, especially ruthenium red and gadolinium, inhibit other TRP channels with varying efficacy (32, 33), we used the siRNA approach to further evaluate the role of TRPV4 channels in flow-induced dilation. Compared with the scrambled control, TRPV4 siRNA reduced the expression of TRPV4 protein in coronary arterioles to 45 ± 10% of control 24–36 h after transfection (Fig. 5A). A similar or greater reduction in the expression of TRPV4 protein and mRNA was observed in HCAECs, whereas TRPV4 siRNA has no significant effect on the expression of PECAM-1 or β-actin (24).

Fig. 5.

Effect of TRPV4 small interfering (si)RNA on flow-induced dilation of coronary arterioles. A: TRPV4-specific siRNA (100 nM) markedly reduced TRPV4 protein expression in coronary arterioles compared with scrambled siRNA (100 nM). The band density of TRPV4 protein was normalized to β-actin (an internal loading control) and the scrambled siRNA. n = 3–4. *P < 0.05 vs. control. The inset shows a representative immunoblot. B: TRPV4 siRNA inhibited flow-induced dilation of coronary arterioles. Luminal flow was generated by pressure gradients of 10–100 cmH₂O. n = 5. *P < 0.05 vs. control.

Coronary arterioles transfected with TRPV4 siRNA had a blunted vasodilatory response to flow (dilation at 100 cmH₂O: 40 ± 10% vs. 79 ± 7% of scrambled controls, n = 5, P < 0.05; Fig. 5B). Incubation of arterioles with TRPV4 siRNA had no significant effect on 100 μM papaverine-induced dilation (100 ± 1% vs. 99 ± 2% of controls, n = 4 for both). In separate experiments, we confirmed that 24–36 h of organ culture did not significantly change the magnitude of flow-induced dilation compared with freshly isolated arterioles (dilation at 20 and 100 cmH₂O: 27 ± 4% and 82 ± 4% vs. 22 ± 4% and 79 ± 2% of controls, n = 5 and 4, respectively).

TRPV4 agonist-induced mitochondrial ROS generation.

As mentioned previously, flow-induced dilation in HCAs involves the release of H₂O₂, an endothelial relaxing factor primarily generated from the mitochondrial electron transport chain (22). We examined whether Ca²⁺ influx secondary to TRPV4 opening affects mitochondrial ROS production. The mitochondrial ROS production was examined using MitoSOX, a mitochondria-targeted fluorescent superoxide indicator. In preliminary experiments, we confirmed that MitoSOX fluorescence was localized in mitochondria by its colocalization with that of the mitochondria-specific probe MitoTracker green. As shown in Fig. 6, the TRPV4 agonist 4α-PDD (2 μM) increased mitochondrial ROS production in HCAECs. This increase was inhibited by ruthenium red (1 μM) and by Mito Vitamin E (1 μM), a mitochondria-targeted antioxidant, with the normalized fluorescence intensity was reduced from 2.3 ± 0.4 of 4α-PDD-treated cells to 0.9 ± 0.1 and 1.5 ± 0.1, respectively.

Fig. 6.

TRPV4 agonist-induced mitochondrial ROS production in coronary ECs. A: representative images of ECs loaded with MitoSOX, a fluorescent probe for mitochondrial superoxide. Activation of TRPV4 channels by 4α-PDD (2 μM) stimulated mitochondrial superoxide production, as indicated by the increased fluorescence intensity within the mitochondria compared with control cells. This response was markedly inhibited by RuR (1 μM), a TRPV4 blocker, and by Mito Vitamin E (MitoE; 1 μM), a mitochondria-targeted antioxidant. B: summary of fluorescence intensity normalized to controls. n = 5 experiments (10–20 cells/each). *P < 0.05 vs. control; #P < 0.05 vs. 4α-PDD.

DISCUSSION

This is the first study, to our knowledge, to examine the role of TRPV4 in the regulation of vascular tone in human vessels. Our findings indicate that TRPV4 channels play an essential role in mediating flow-induced dilation of HCAs. Furthermore, TRPV4-mediated Ca²⁺ influx may serve as a potential mechanism linking flow signal and mitochondrial ROS production in coronary ECs.

Previous studies have shown that flow increases [Ca²⁺]_i (via Ca²⁺ entry and/or Ca²⁺ release from internal stores) in vascular ECs (7, 13, 14) and that this Ca²⁺ increase is critical for the flow-induced synthesis and release of endothelial relaxing factors (2, 4). However, the molecular identity of the responsible Ca²⁺ entry channel(s) remains elusive. In a recent study, Kohler et al. (16) provided the first pharmacological evidence that TRPV4 channels are involved in flow-induced dilation of rat carotid arteries and skeletal muscle arterioles. Using two independently generated knockout mouse lines, several laboratories, including ours, have subsequently showed that TRPV4 activation contributes to flow-induced dilation in mouse carotid and small mesenteric arteries (12, 23, 24). In the present study, we found that flow-induced dilation in human coronary microvessels also requires the activation of endothelial TRPV4 channels. Collectively, these findings indicate that the activation of TRPV4 channels may serve as a conserved mechanism transducing flow or a shear stimulus to the endothelial Ca²⁺ response and subsequent vasodilation. In addition to ECs, TRPV4-mediated Ca²⁺ entry in response to flow has been shown in other tissues and cells, such as TRPV4-transfected HEK-293 cells (9, 24) and renal tubular ECs (30, 38).

In this study, TRPV4 activation increases mitochondrial ROS production in coronary ECs and induces ROS-dependent vasodilation in coronary arterioles. Therefore, TRPV4 channel-mediated Ca²⁺ influx may be a potential mechanistic link between the flow signal and mitochondrial ROS production in coronary ECs. This hypothesis is in line with a growing body of literature suggesting that mitochondria act as signaling organelles participating in various cellular functions, e.g., Ca²⁺ and ROS signaling, through proximity to other signaling machinery (8, 10, 40). Of note, we (21) have recently reported that endothelial cytoskeletal elements also play an important role in shear-induced mitochondrial ROS production and subsequent dilation of coronary arterioles. It is thus tempting to speculate that plasmalemmal TRPV4 channels are in close apposition with mitochondria in ECs, an association facilitated by the cytoskeleton, so that TRPV4-mediated Ca²⁺ influx may be selectively targeted to mitochondria, leading to subsequent mitochondrial ROS production. How the TRPV4-mediated Ca²⁺ increase is coupled to mitochondrial ROS production remains unclear. It is possible that in coronary ECs, an increase in mitochondrial Ca²⁺ concentration stimulates ROS generation in the mitochondria (8, 10, 40). Although uptake of Ca²⁺ or other cations depolarizes mitochondrial membrane potential, which can lead to reduced ROS formation, Ca²⁺ may increase mitochondrial ROS formation by other mechanisms, such as enhancement of electron flow into the respiratory chain, stimulation of nitric oxide production and consequent inhibition of complex IV, and activation of other ROS-generating enzymes, such as α-ketoglutarate dehydrogenase (8, 10, 40). Alternatively, a ROS-induced ROS release mechanism involving other ROS sources, such as NADPH oxidase (42), may also play a role in TRPV4-induced ROS production in ECs. Future studies are required to explore these possibilities.

Although not pursued in this study, the mechanisms by which flow activates endothelial TRPV4 channels in HCAs may involve endogenous activators such as arachidonic acid and/or its cytochrome P-450 metabolites (e.g., EETs) (34, 36), as has been suggested for flow-induced dilation of mouse carotid arteries (12, 23). In isolated HCAs, both arachidonic acid and EETs are potent vasodilators (27), and inhibition of cytochrome P-450 enzyme markedly reduces flow-induced vasodilation (28). It remains to be determined in future studies whether flow induces the release of arachidonic acid and/or its metabolites and whether these endogenous compounds activate endothelial TRPV4, leading to flow-induced dilation of HCAs.

TRPV4 channels have been found to be expressed in vascular SMCs of several vascular beds, such as rat cerebral and pulmonary arteries (41). Using immunohistochemical analysis, we also found positive staining of TRPV4 protein in SMCs of human coronary arteries and HCAs, although at a much lower level than that in ECs. These results seem not to be in accordance with RT-PCR data showing that TRPV4 mRNA transcripts are absent in freshly isolated or cultured coronary SMCs. Arniges et al. (1) recently identified five splice variants of human TRPV4 channels and grouped them into two classes: group I (TRPV4-A and TRPV4-D) and group II (TRPV4-B, TRPV4-C, and TRPV4-E). We could not detect PCR amplification products of TRPV4 mRNA in coronary SMCs when the primers as shown in Fig. 1 were selective for group I isoforms. However, PCR amplification products were detected when primers for all isoforms were used (data not shown). These results suggest that human coronary SMCs express other isoforms of TRPV4, most likely group II TRPV4 channels. Intriguingly, group II TRPV4 channels, which lack parts of the ankyrin domains, are unable to oligomerize and thus are retained intracellularly, mainly in the endoplasmic reticulum (1). Therefore, the functional significance of TRPV4 channels in coronary SMCs remains to be characterized. Since no significant dilatory responses (small constriction instead) to the TRPV4 agonist 4α-PDD were observed in denuded coronary arterioles (Fig. 3A), the potential contribution of smooth muscle TRPV4 to the overall flow-mediated dilation would be minimal in this vascular bed.

Due to technique difficulties, we were unable to culture ECs from HCAs. For those experiments shown in Figs. 2 and 6, we instead used macrovascular ECs (i.e., HCAECs), which were derived from large coronary arteries and were typically from donors free of cardiovascular diseases. Therefore, we cannot exclude the possibility that observations made in large coronary ECs may differ from those in arteriolar ECs or that ECs from healthy subjects respond differently from those cells from patients who, as in this study, are not completely free of cardiovascular diseases. However, a previous study (21) has shown that, similar to that observed in HCAs, shear induces mitochondrial ROS production in HCAECs.

In summary, the present study provides evidence that the endothelial TRPV4 channel is importantly involved in flow-mediated dilation of HCAs. TRPV4 channels may represent a critical but previously unidentified component of the redox signaling pathway linking the flow stimulus with the mitochondrial release of ROS and subsequent dilation of HCAs. Flow-induced dilation in the human coronary microcirculation is mediated by the release of H₂O₂ from the mitochondria of ECs, a unique mechanism of vasodilation that is more prominent in the presence of CAD and other cardiovascular diseases or risk factors (20, 22, 26). Therefore, studies on TRPV4-mediated redox signaling (especially in the human heart) will not only enhance our understanding of coronary blood flow regulation in health and disease but also help to identify new therapeutic options for the treatment of CAD and/or other cardiovascular pathologies.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants R01-HL-096647 (to D. X. Zhang) and R01-HL-080704 and R01-HL-094971 (to D. D. Gutterman) and by American Heart Association Grant 0830042N (to D. X. Zhang).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: A.H.B., S.A.M., X.Z., N.S.Z., and R.L. performed experiments; A.H.B., S.A.M., X.Z., and N.S.Z. analyzed data; A.H.B., S.A.M., X.Z., N.S.Z., R.L., D.D.G., and D.X.Z. edited and revised manuscript; A.H.B., S.A.M., X.Z., N.S.Z., R.L., D.D.G., and D.X.Z. approved final version of manuscript; D.D.G. and D.X.Z. conception and design of research; D.D.G. and D.X.Z. interpreted results of experiments; D.X.Z. drafted manuscript.