TWIK-2 channel deficiency leads to pulmonary hypertension through a rho-kinase-mediated process

Abstract

TWIK-2 (KCNK6) is a member of the 2-pore domain (K2P) family of potassium channels, which are highly expressed in the vascular system. We tested the hypothesis that TWIK-2 deficiency leads to pulmonary hypertension. TWIK-2 knockout mice and their wildtype littermates at 8 weeks of age had similar mean right ventricular systolic pressures (24±3 and 21±3 mm Hg, respectively.) Significantly, by 20 weeks of age, the mean right ventricular systolic pressures in TWIK-2 knockout mice increased to 35±3 mm Hg (P≤0.036), whereas mean right ventricular systolic pressures in wildtype littermates remained at 22±3 mm Hg. Elevated mean right ventricular systolic pressures in the TWIK-2 knockout mice was accompanied by pulmonary vascular remodeling as determined by a 25% increase in the cross-sectional area of the vessels occupied by the vessel wall. Additionally, secondary branches of the pulmonary artery from 20-week-old TWIK-2 knockout mice showed an enhanced contractile response to U46619 (10(-6) moles/L), a thromboxane A2 mimetic, which was completely abolished with the Rho-kinase inhibitor, Y27632 (10(-6) and 10(-5) moles/L). Treatment of TWIK-2 knockout mice with the Rho-kinase inhibitor, fasudil, in the drinking water for 12 weeks, abolished the development of pulmonary hypertension and attenuated the vessel remodeling. We concluded that mice deficient in the TWIK-2 channel develop pulmonary hypertension between 8 and 20 weeks of age through a mechanism involving Rho-kinase. Our results suggest that downregulation of TWIK-2 in the pulmonary vasculature may be an underlying mechanism in the development of pulmonary hypertension.

Keywords: KCNK6; Rho-kinase; TWIK-2; potassium channel; pulmonary hypertension.

Figure 1

A: Mean right ventricular pressures in 8 and 20 week old TWIK-2 KO and their WT littermates (n=4 per group). *p ≤ 0.036 compared to WT at 20 weeks and TWIK-2 KO at 8 weeks. B: The percent of the cross-sectional area in lung vessels occupied by the vessel wall in TWIK-2 KO mice and their WT littermates at 20 weeks of age. This measure provides an index of wall thickness relative to the vessel size. * p= 0.001 compared to all other groups (n=4 per group). C: H&E stained sections (top panels) and immuno-stained sections of smooth muscle α-actin (bottom panels) from the lung of a 20 week old TWIK-2 KO and its WT littermates. The arrows in the top panels indicate the vessel wall. Red in the bottom panels denotes smooth muscle α-actin and blue represents cell nuclei.

Figure 2

Contractile responses to (A) phenylephrine, an α-adrenoreceptor agonist, (n=4 each group) or (B) U46619, a thromboxane A2 mimetic, in first order branches of the pulmonary artery obtained from 20 week old TWIK-2 KO and WT mice. * p=0.04 compared to 10⁻⁶ M U46619 in WT, n=8 for each group. C: Contractile responses to U46619 in first order branches of the pulmonary artery from 20 week old WT and TWIK-2 KO mice in the presence of 1 uM (10⁻⁶ moles/liter) and 10 uM (10⁻⁵ moles/liter) Y27632, an inhibitor of Rho-kinase. * p≤ 0.002 compared to all other groups, ** p=0.038 compared to KO with 1 uM Y27632.

Figure 3

A: Mean right ventricular systolic pressure in 20 week old TWIK-2 KO mice and their WT littermates. Mice were provided with either 1 mg/ml fasudil in the drinking water or drinking water alone (Control). (n= 3–4 per group) * p=0.007 and 0.02 compared to TWIK-2 KO with fasudil and WT control respectively. B: The percent of the cross-sectional area in lung vessels occupied by the vessel wall in TWIK-2 KO mice and their WT littermates at 20 weeks of age with and without 12 weeks of Fasudil treatment. This measure provides an index of wall thickness relative to the vessel size. * p<0.001 compared to WT Control and TWIK-2 KO mice treated with Fasudil, ** p=0.004 compared to WT Control.