General chemicals and reagents, including acetylcholine (ACh), sodium nitroprusside (SNP), N^G-nitro-l-arginine methyl ester (L-NAME), phenylephrine (PE), retigabine, linopirdine, and ethyl alcohol, were purchased from Merck (Gyeonggi-Do, South Korea). Dimethyl sulfoxide (DMSO) was purchased from Mentos Biotechnology (Gyeonggi-Do, South Korea). Retigabine (50 mM stock) was made in DMSO, whereas linopirdine (10 mM stock) was dissolved in ethyl alcohol. Vehicle control experiments were also performed.

All experimental protocols were approved by the Animal Care and Use Committee of Incheon National University (INU 2017-11). Wild-type C57 BL/6 mice, between 10 and 15 weeks old, were used in this study. The mice were housed in a temperature-, humidity-, and light-controlled (12 hour light/dark cycle) animal facility and given free access to standard mouse chow and water. All mice were anesthetized by an intraperitoneal injection of 2.5% tribromoethanol (0.01 mL/g of body weight).

Under anesthesia, thoracic aortas were cut into 2 mm rings, isometrically mounted into a pin myography system (Model 620; Danish Myo Technology, Aarhus, Denmark), and maintained in physiological saline solution (PSS) in an atmosphere of 95% O₂ and 5% CO₂ at 37°C for the entire myography experiment. Continuous recordings of changes in the tension were acquired by using a PowerLab system (AD instruments, Colorado Springs, CO, USA). After a 30 min equilibration period, the aortas were placed under a pretension of 15 mN and equilibrated for a further 30 min. All aortas were stimulated with the cumulative addition of high K⁺ (50 and 100 mM KCl) to check the viability of the aortic rings. Based on previous studies^23,24, the aortic rings were pre-contracted with 1 μM PE for 15 min to achieve a stable level of basal constriction to determine the subsequent vasorelaxation response. After stable constriction was induced, the concentration-response curves were acquired by the cumulative addition of the Kv7 activator, retigabine (1-50 μM). The degree of vasorelaxation at each concentration was measured and expressed as the percentage of the force generated in response to PE. In another experiment, linopirdine (10 μM) or an inhibitor of nitric oxide (NO) synthase, L-NAME (100 μM), was pre-incubated for 20 min to verify that they blocked Kv7 channel activation or nitric oxide production. Subsequently, retigabine (1-50 μM) was used in the pre-incubated blood vessels to confirm whether the vasodilation response occurred. In other experiments, after pre-contraction of the vessels with 1 μM PE, 1 nM-10 μM ACh or 1 nM-10 μM SNP-induced vasorelaxation was assessed to examine the vascular response.

All data were presented as the mean ± standard error of the mean. To compare the means of two groups, the unpaired t-test was used. Two-way ANOVA followed by Bonferroni’s post-hoc test was performed to determine the significance between groups. All statistical analyses were computed by using GraphPad Prism (version 6.05, La Jolla, CA, USA). The significance of differences was accepted at P-values of <0.05, unless otherwise stated.

We first examined whether an endothelial-dependent vasodilator, ACh, and an endothelial-independent vasodilator, SNP, induced vasorelaxation in the aorta of male mice. Vasorelaxation by ACh (1 nM - 10 μM) or SNP (1 nM - 10 μM) was induced in the aorta in a dose-dependent manner, suggesting that both the endothelial and smooth muscle cells in the aorta were not impaired by the dissection procedures (Fig. 1).

The experiment was undertaken to assess whether the Kv7 channel activator, retigabine, induced vasorelaxation through the endothelial-derived relaxing molecule NO in aortic segments. Pre-incubation with an NO synthase inhibitor, L-NAME (100 μM, 20 min), completely abolished ACh-induced vasorelaxation, whereas pre-incubation with L-NAME did not block retigabine-induced vasorelaxation, which suggested that retigabine caused vasorelaxation via the activation of smooth muscle cells, rather than endothelial cells, in the aorta (Fig. 2).

We also tested whether a non-selective Kv7 channel inhibitor, linopirdine, abolished retigabine-induced vasorelaxation. Pre-incubation with linopirdine (10 μM, 20 min) reduced retigabine-induced vasorelaxation (1-50 μM) in the aorta, compared with vehicle-treated samples (Fig. 3).

Previous studies have suggested that the Kv7 channel blocker, linopirdine, exerts a depolarizing effect on vascular smooth muscle cells and causes constriction in the cerebral, renal, and coronary arteries^21,25,26. Experiments were performed to test whether the Kv7 channel was involved at rest condition in the mouse aortas. Pre-application of the Kv7 channel blocker, linopirdine (10 μM), resulted in a greater contractile response in mouse aorta compared with vehicle (Fig. 4).