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 H(2)O(2) from the mitochondria of endothelial cells (ECs). How flow activates ECs to elicit the mitochondrial release of H(2)O(2) remains unclear. Here, we examined the role of the transient receptor potential vanilloid type 4 (TRPV4) channel, a mechanosensitive Ca(2+)-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 Ca(2+) influx) intracellular Ca(2+) 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 H(2)O(2)-metabolizing enzyme. Fluorescence ROS assays showed that 4α-PDD increased the production of mitochondrial superoxide in HCAECs. 4α-PDD also enhanced the production of H(2)O(2) 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 Ca(2+) 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.

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.

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.

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.

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.

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.

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.