Abstract P056: Voltage-gated Chloride Channel 6 Regulates Intracellular Calcium Signaling In Vascular Smooth Muscle Cells And Prevents Arterial Stiffening

Abstract

Hypertension is a global health problem that increases the risk of stroke, heart failure, and kidney disease. GWAS have linked the CLCN6 gene to blood pressure regulation. CLCN6 encodes the voltage-sensitive chloride channel 6 (ClC-6), an intracellular protein of little-known function. Due to its high expression in vascular smooth muscle cells (VSMCs) and its localization in the Golgi, we hypothesized that ClC-6 is involved in Ca 2+ uptake in the Golgi and that reducing Golgi Ca 2+ levels changes VSMC function and therefore blood pressure, potentially by altering protein trafficking or cell proliferation. Using a knock-out of Clcn6 on the Dahl salt-sensitive rat background, we determined that KO rats have impaired vasodilation in response to 10 μM acetylcholine in mesenteric arteries (82 ± 6% vs 41 ± 13%, N=6 rats, n ≥ 9 vessels, p<0.01). Two-Photon microscopy in mesenteric arteries determined that KO VSMCs continue to release intracellular Ca 2+ in response to acetylcholine at a greater frequency than WT. Additionally, arterial stiffness, determined by pulse-wave velocity (PWV) measurements, was greater in KO rats at baseline (4.5 ± 0.3 vs. 3.6 ± 0.2 mm/msec, N≥6 rats, p<0.05); however, after 3 weeks on a 4% NaCl diet to develop hypertension, the WT rat PWVs increased significantly (4.6 ± 0.1 vs. 3.6 ± 0.2 mm/sec, N=6, p<0.05) whereas the KO rat PWVs did not change (4.6 ± 0.1 vs. 4.5 ± 0.3 mm/msec, N=6, p=0.91). To assess the effect of ClC-6 on Golgi Ca 2+ , confocal microscopy with an organelle-specific Ca 2+ dye and pharmacological agents was used to measure Ca 2+ . SR Ca 2+ stores were depleted with thapsigargin + ATP, followed by release of Golgi Ca 2+ stores by emetine. Total Golgi calcium levels were determined by area under the curve measurements of recorded fluorescence changes. Emetine-sensitive Golgi Ca 2+ stores were significantly reduced in KO rat VSMCs compared to WT. In conclusion, these results suggest that ClC-6 regulates Golgi Ca 2+ levels, and alteration to these stores results in cellular changes that slow or prevent arterial stiffness during the development of hypertension. Future studies will assess vessel fibrosis, cell proliferation, and apoptosis to elucidate the underlying molecular mechanisms impacting vessel stiffness and Ca 2+ signaling.