PKG-1α leucine zipper domain defect increases pulmonary vascular tone: implications in hypoxic pulmonary hypertension

Abstract

Pulmonary hypertension (PH) is a chronic disease characterized by a progressive increase in vasomotor tone, narrowing of the vasculature with structural remodeling, and increase in pulmonary vascular resistance. Current treatment strategies include nitric oxide therapy and methods to increase cGMP-mediated vasodilatation. cGMP-dependent protein kinases (PKG) are known mediators of nitric oxide- and cGMP-induced vasodilatation. Deletion of PKG-1 in mice has been shown to induce PH, however, the exact mechanisms by which loss of PKG-1 function leads to PH is not known. In a mouse model with a selective mutation in the NH2-terminus leucine zipper protein interaction domain of PKG-1α [leucine zipper mutant (LZM)], we found a progressive increase in right ventricular systolic pressure and right heart hypertrophy compared with wild-type (WT) mice and increased RhoA-GTPase activity in the lungs. When exposed to chronic hypoxia, LZM mice developed modestly enhanced right ventricular remodeling compared with WT mice. Tadalafil, a phosphodiesterase-5 inhibitor that increases cGMP levels, significantly attenuated hypoxia-induced cardiopulmonary remodeling in WT mice but had no effect in LZM mice. We conclude that a functional leucine zipper domain in PKG-1α is essential for maintenance of a low pulmonary vascular tone in normoxia and for cGMP-mediated beneficial effects of phosphodiesterase-5 inhibition in hypoxic cardiopulmonary remodeling.

Keywords: hypoxia; leucine zipper mutant guanosine 3′,5′-cyclic monophosphate-dependent protein kinase; phosphodiesterases; pulmonary hypertension and right ventricular hypertrophy.

Fig. 1.

cGMP-dependent protein kinases (PKG) 1 expression in the pulmonary vasculature of leucine zipper mutant (LZM) and wild-type (WT) mice. A: PKG-1 expression in lungs of LZM and WT mice. The LZM PKG-1α expression was probed by Western blotting in lung lysates of two LZM and two WT mice. Defective expression of PKG-1α protein is seen in LZM mice with an antibody specific to the NH₂-terminus of WT PKG-1α. Probing with antibody toward a common epitope to both isoforms of PKG-1 (PKG1com) and PKG-1β antibody showed similar protein expression between LZM and WT mice. Actin served as the loading control. Representative blot from three experiments is shown. B: PKG-1 expression and signaling in pulmonary artery smooth muscle cells (PASMC) of LZM and WT mice. PASMCs isolated as described under materials and methods were treated with 8-Bromo-guanosine 3′5′-cyclic monophosphate sodium salt (8-Br-cGMP), and lysates were analyzed by Western blotting using the specified antibodies. Representative blot from three experiments is shown. VASP, vasodilator-stimulated protein.

Fig. 2.

PKG-1α LZM mice exhibit increased right ventricular (RV) systolic pressures (SP) and right heart hypertrophy. A: RV systolic pressures (in mmHg) were measured in 7-mo-old LZM mice (solid bar) and WT littermates (open bar) as described in materials and methods. Data are expressed as means ± SE (3 males, 3 females in WT; 4 males, 3 females in LZM group). *P < 0.05. B: ratio of RV weight to that of left ventricle plus septum (LV + S) was obtained as a measure of right heart hypertrophy in 7-mo-old mice LZM and WT mice (n = 7; 4 males, 3 females). *P < 0.05 C: RV mass normalized for body weight (BW) in 7-mo-old LZM and WT mice (n = 7). *P < 0.05. D: representative hematoxylin and eosin (H&E)-stained sections from 7-mo-old WT and LZM mouse lungs are shown with arrows pointing to the pulmonary vessels. A, airway. Scale bar represents 100 μm. E: medial wall thickness of the pulmonary arterioles (<80 μm diameter) scored and determined from H&E-stained lung sections from 7-mo-old LZM and WT mice as described under materials and methods. Values are represented as percentage of ratio of wall area to vessel cross-sectional area. F: quantification of mean linear intercept in H&E-stained lung sections from 2-mo-old LZM and WT mice (n = 3) as described under materials and methods.

Fig. 3.

RhoA signaling pathway is increased in the pulmonary vasculature of LZM mice. A: lungs of 7-mo-old LZM mice show increased RhoA-GTPase activation in a pull-down assay. Extracts from perfused lungs were prepared in Rho lysis buffer and bound to Rhotekin beads before analyzing by Western blotting with a RhoA specific antibody (top) as described in materials and methods. Total lung extracts were resolved by SDS-PAGE and probed with RhoA antibody (bottom). Experiments were repeated three times. A representative blot from two 7-mo-old WT and LZM mice is shown. B: basal RhoA phosphorylation is reduced in PASMC from LZM mice. Lysates prepared from isolated PASMCs from LZM and WT mice were resolved by SDS-PAGE and probed with phospho-RhoA-Ser¹⁸⁸ antibody by immunoblotting. Total RhoA and actin levels were also assessed on gels. Experiments were repeated three times. Representative blot with cell lysates from two different WT and LZM mice is shown.

Fig. 4.

Development of pulmonary hypertension after chronic hypoxia exposure in 2-mo-old LZM and WT mice. A: RV systolic pressure in 2-mo-old LZM and WT mice in normoxia and after 2 wk hypoxia (10% O₂) exposure (n = 5–6/group). NS, not significant. *P < 0.01 vs. normoxia. B: ratio of RV weight to that of LV + S as a measure of RV hypertrophy in LZM and WT mice exposed to room air or chronic hypoxia (10% oxygen) for 2 wk; 3 males, 2 females in normoxia group; 4 males, 2 females in hypoxia group. *P < 0.01 vs. normoxia group and #P < 0.05 vs. WT hypoxia group. C: RV weight-to-body weight (BW) ratio in LZM and WT mice exposed to room air or chronic hypoxia (10% oxygen) for 2 wk. *P < 0.01 vs. normoxia group and #P < 0.05 vs. WT hypoxia group. D: hematocrits (HCT) were determined to follow development of polycythemia in LZM and WT mice exposed to room air or chronic hypoxia (10% oxygen) for 2 wk. *P < 0.05 vs. normoxia. Open bar, WT; solid bar, LZM.

Fig. 5.

Tadalafil inhibition of hypoxic pulmonary hypertension is absent in LZM mice. A: ratio of RV weight to that of LV + S as a measure of RV hypertrophy of LZM and WT mice treated with tadalafil or vehicle during exposure to chronic hypoxia (10% oxygen) for 2 wk or in room air as described under materials and methods; 3 males, 2 females in both hypoxia groups; 3 males, 3 females in WT/hypoxia/tadalafil group; 5 males, 3 females in LZM/hypoxia/tadalafil group. *P < 0.01 vs. normoxia, #P < 0.05 vs. WT hypoxia group, and &P < 0.05 vs. WT/hypoxia/tadalafil group. B: medial wall thickness of pulmonary arterioles (<80 μm diameter) from tadalafil- or vehicle-treated LZM and WT mice measured from H&E-stained lung sections to assess vessel remodeling in chronic hypoxia and room air. Values are represented as percentage of ratio of wall area to vessel cross-sectional area and plotted as means ± SE. *P < 0.05 and #P < 0.05 vs. WT hypoxia group. Open bar, WT; solid bar, LZM.