Abstract 1308: Activation of TRPV4 Channels Dilates Human Coronary Arterioles: Role of Ca2+ Influx and Mitochondrial Redox Signaling
We previously showed that mitochondria-derived hydrogen peroxide (H2O2) contributes to flow-mediated dilation (FMD) of human coronary arterioles (HCA) from patients with coronary disease. The signaling pathways by which flow induces mitochondrial H2O2 production remain largely unknown. Emerging evidence suggests the vanilloid subfamily of transient receptor potential channels, member 4 (TRPV4) is activated by shear stress leading to endothelial Ca2+-influx via this channel. We hypothesize that TPPV4-dependent Ca2+-influx induces mitochondrial H2O2 formation leading to vasodilatation, as part of the mechanism of FMD in HCA. Using immunoblots, TRPV4 protein was detected in HCA and human coronary endothelial cells. Using a Ca2+ assay with fura-2, 4α-phorbol-12,13-didecanoate (4αPDD, 1 μM), a selective TRPV4 opener, increased intracellular Ca2+ ([Ca2+]i) in endothelial cells (from 60±4 to 390±22 nM; n=3), a response that was modulated by temperature and blocked by removing extracellular Ca2+(124±12 nM) or by the TRPV4 blocker ruthenium red (RuR) (99±5 nM). RuR had no significant effect on [Ca2+]i elevation in response to ATP (1 mM), an intracellular Ca2+ store-mobilizing agonist. Measuring mitochondrial reactive oxygen species (ROS) with mitoSOX (mitochondrial targeted hydroethidine), we observed a 4αPDD-induced increase in mitochondrial ROS that was prevented by RuR, the mitochondrial targeted antioxidant MitoE, or by chelating intracellular Ca2+ with BAPTA (normalized values of 2.7±1.0, 0.7±0.2, 1.0±0.1, and 1.0±0.2, respectively; n=3). In isolated and perfused HCA, 4αPDD (50 nM-5 μM) elicits endothelium-dependent relaxations in a concentration-dependent manner (maximal relaxations of 61±9%; n=5). It is concluded that Ca2+ influx through TRPV4 channels stimulates mitochondrial ROS formation and elicits endothelium-dependent relaxation in HCA, implicating a novel mechanism of FMD in HCA. Elucidating of this signaling pathway may identify new pharmacological targets in the therapy of coronary disease.