Role of ASIC1 in the development of chronic hypoxia-induced pulmonary hypertension

Abstract

Chronic hypoxia (CH) associated with respiratory disease results in elevated pulmonary vascular intracellular Ca(2+) concentration, which elicits enhanced vasoconstriction and promotes vascular arterial remodeling and thus has important implications in the development of pulmonary hypertension (PH). Store-operated Ca(2+) entry (SOCE) contributes to this elevated intracellular Ca(2+) concentration and has also been linked to acute hypoxic pulmonary vasoconstriction (HPV). Since our laboratory has recently demonstrated an important role for acid-sensing ion channel 1 (ASIC1) in mediating SOCE, we hypothesized that ASIC1 contributes to both HPV and the development of CH-induced PH. To test this hypothesis, we examined responses to acute hypoxia in isolated lungs and assessed the effects of CH on indexes of PH, arterial remodeling, and vasoconstrictor reactivity in wild-type (ASIC1(+/+)) and ASIC1 knockout (ASIC1(-/-)) mice. Restoration of ASIC1 expression in pulmonary arterial smooth muscle cells from ASIC1(-/-) mice rescued SOCE, confirming the requirement for ASIC1 in this response. HPV responses were blunted in lungs from ASIC1(-/-) mice. Both SOCE and receptor-mediated Ca(2+) entry, along with agonist-dependent vasoconstrictor responses, were diminished in small pulmonary arteries from control ASIC(-/-) mice compared with ASIC(+/+) mice. The effects of CH to augment receptor-mediated vasoconstrictor and SOCE responses in vessels from ASIC1(+/+) mice were not observed after CH in ASIC1(-/-) mice. In addition, ASIC1(-/-) mice exhibited diminished right ventricular systolic pressure, right ventricular hypertrophy, and arterial remodeling in response to CH compared with ASIC1(+/+) mice. Taken together, these data demonstrate an important role for ASIC1 in both HPV and the development of CH-induced PH.

Keywords: acid-sensing ion channels; capacitative Ca2+ entry; degenerin/epithelial Na+ channel; hypoxic pulmonary vasoconstriction; pulmonary vascular remodeling; receptor-mediated vasoconstriction; store-operated Ca2+ entry.

Fig. 1.

Restoration of acid-sensing ion channel 1 (ASIC1) in pulmonary arterial smooth muscle cells (PASMCs) from ASIC1 knockout (ASIC1^−/−) mice rescues store-operated Ca²⁺ entry (SOCE). A: RT-PCR for ASIC1 and β-actin in PASMCs from ASIC1^+/+ and ASIC1^−/− mice transfected with human (h)ASIC1. B: smooth muscle-22α (SM22α; green) and ASIC1 (red) immunofluorescence in PASMCs from ASIC1^+/+ and ASIC1^−/− mice. PASMCs from ASIC1^−/− mice were additionally transfected with hASIC1. C: summary data showing SOCE-induced changes (Δ) in intracellular Ca²⁺ concentration ([Ca²⁺]_i) [expressed as changes in the 340-to-380-nm fluorescence ratio (ΔF₃₄₀/F₃₈₀)] in PASMCs isolated from ASIC1^+/+ and ASIC1^−/− mice. PASMCs from ASIC1^−/− mice were additionally transfected with hASIC1. All experiments were performed in the presence of cyclopiazonic acid (CPA; 10 μM) and diltiazem (50 μM). Values are means ± SE; numbers of animals (n values) are indicated in bars. *P < 0.05 vs. PASMCs from ASIC1^+/+ mice; #P <0.05 vs. PASMCs from ASIC1^−/− mice.

Fig. 2.

Hypoxic pulmonary vasoconstriction is blunted in ASIC1^−/− mice. A and B: changes in pulmonary arterial resistance (in mmHg·ml⁻¹·min·kg) to hypoxia (A) and KCl (20–30 mM; B) in isolated lungs from ASIC1^+/+ and ASIC1^−/− mice. All experiments were conducted in the presence of N^G-nitro-l-arginine (300 μM) and meclofenamate (30 μM). Data are expressed as means ± SE; n = 5–7 animals/group. *P ≤ 0.05 vs. normoxic ventilation.

Fig. 3.

ASIC1 contributes to augmented SOCE after chronic hypoxia (CH) in isolated small pulmonary arteries. SOCE-induced changes in arterial wall [Ca²⁺]_i (ΔF₃₄₀/F₃₈₀; A), vasoconstriction (percent baseline diameter; B), and magnitude of Mn²⁺ quenching 10 min after MnCl₂ (500 μM) administration (C). All experiments were performed in the presence of CPA (10 μM) and diltiazem (50 μM). F, fluorescence intensity; F₀, baseline fluorescence intensity at time 0. Values are means ± SE; n values are indicated in bars. *P < 0.05 vs. the control group; #P < 0.05 vs. the corresponding ASIC1^+/+ artery.

Fig. 4.

ASIC1 contributes to enhanced receptor-mediated vasoconstriction after CH in small pulmonary arteries. A–F: vasoconstriction (percent baseline diameter; A, C, and E) and changes in arterial wall [Ca²⁺]_i (ΔF₃₄₀/F₃₈₀; B, D, and F) to UTP (10⁻⁷ to 3 × 10⁻⁴ M; A and B), endothelin-1 (ET-1; 10⁻¹¹ to 10⁻⁷ M; C and D), and depolarizing concentrations of KCl (10^−1.75 to 10^−1.00 M; E and F) in small pulmonary arteries from control and CH ASIC1^+/+ and ASIC1^−/− mice. Values are means ± SE; n = 5–7 animals/group. *P ≤ 0.05 vs. the control group; #P <0.05 vs. the corresponding ASIC1^+/+ artery.

Fig. 5.

ASIC1 protein expression in small pulmonary arteries. Smooth muscle α-actin (SM α-actin) and ASIC1 immunofluorescence in ASIC1^+/+ and ASIC1^−/− lung sections demonstrate the presence of ASIC1 in small pulmonary arteries (∼70–100 μm). Fluorescence images were digitally inverted using ImageJ to provide better contrast and visibility of immunofluorescence. The bottom images represent digitally zoomed fluorescence overlay images corresponding to the boxes in the images of smooth muscle α-actin (green; top) and ASIC1 (red; middle).

Fig. 6.

CH does not alter ASIC1 protein levels in small pulmonary arteries. A: representative Western blots of ASIC1 and GAPDH (20 μg protein/lane). B: summary data for Western blot analysis of ASIC1/GAPDH protein expression. Values are means ± SE; n values are indicated in bars.

Fig. 7.

ASIC1 contributes to CH-induced pulmonary hypertension. A: representative traces of transdiaphragmatic measurements of right ventricular (RV) pressure. B and C: summary data for RV systolic pressure (RVSP; B) and heart rate [HR; in beats/min (bpm); C] in anesthetized control and CH ASIC1^+/+ and ASIC1^−/− mice. D and E: ratio of RV to left ventricular plus septal (LV + S) heart weight (D) and hematocrit (in %; E) in ASIC1^+/+ and ASIC1^−/− mice exposed to control or CH conditions. Values are means ± SE; n = 8–13 animals/group. *P < 0.05 vs. the control group; # P < 0.05 vs. the corresponding ASIC1^+/+ mice.

Fig. 8.

ASIC1 contributes to small pulmonary artery remodeling after CH. A: representative bright-field (left) or smooth muscle α-actin immunofluorescence (right) images of lung sections from control and CH ASIC1^+/+ and ASIC1^−/− mice. Bottom images show higher-magnification images of ∼10-μm arteries from CH ASIC1^+/+ and ASIC1^−/− mice. The arrow bottom middle image indicates an alveolar duct smooth muscle cell. Fluorescence images were digitally inverted to provide better contrast and visibility of immunofluorescence. B: numbers of fully muscularized arteries according to arterial diameter (–, –, and –60 μm) from 20 random images/lung section. C: percent muscularization calculated as percent thresholded smooth muscle α-actin area divided by total arterial wall area. Values are means ± SE; n = 4 animals/group. *P ≤ 0.05 vs. the control group; #P <0.05 vs. corresponding ASIC1^+/+ mice.