Pulmonary hypertension in wild type mice and animals with genetic deficit in KCa2.3 and KCa3.1 channels

Abstract

Objective: In vascular biology, endothelial KCa2.3 and KCa3.1 channels contribute to arterial blood pressure regulation by producing membrane hyperpolarization and smooth muscle relaxation. The role of KCa2.3 and KCa3.1 channels in the pulmonary circulation is not fully established. Using mice with genetically encoded deficit of KCa2.3 and KCa3.1 channels, this study investigated the effect of loss of the channels in hypoxia-induced pulmonary hypertension.

Approach and result: Male wild type and KCa3.1-/-/KCa2.3T/T(+DOX) mice were exposed to chronic hypoxia for four weeks to induce pulmonary hypertension. The degree of pulmonary hypertension was evaluated by right ventricular pressure and assessment of right ventricular hypertrophy. Segments of pulmonary arteries were mounted in a wire myograph for functional studies and morphometric studies were performed on lung sections. Chronic hypoxia induced pulmonary hypertension, right ventricular hypertrophy, increased lung weight, and increased hematocrit levels in either genotype. The KCa3.1-/-/KCa2.3T/T(+DOX) mice developed structural alterations in the heart with increased right ventricular wall thickness as well as in pulmonary vessels with increased lumen size in partially- and fully-muscularized vessels and decreased wall area, not seen in wild type mice. Exposure to chronic hypoxia up-regulated the gene expression of the KCa2.3 channel by twofold in wild type mice and increased by 2.5-fold the relaxation evoked by the KCa2.3 and KCa3.1 channel activator NS309, whereas the acetylcholine-induced relaxation - sensitive to the combination of KCa2.3 and KCa3.1 channel blockers, apamin and charybdotoxin - was reduced by 2.5-fold in chronic hypoxic mice of either genotype.

Conclusion: Despite the deficits of the KCa2.3 and KCa3.1 channels failed to change hypoxia-induced pulmonary hypertension, the up-regulation of KCa2.3-gene expression and increased NS309-induced relaxation in wild-type mice point to a novel mechanism to counteract pulmonary hypertension and to a potential therapeutic utility of KCa2.3/KCa3.1 activators for the treatment of pulmonary hypertension.

Figure 1. Right ventricular systolic blood pressure and hypertrophy.

A) The effect of chronic hypoxia on right ventricular systolic blood pressure (RVSBP) in wild type and K_Ca3.1^−/−/K_Ca2.3^T/T(+Dox) mice. Values are means ±SEM, normoxic wild type (n = 8) and K_Ca3.1^−/−/K_Ca2.3^T/T(+Dox) mice (n = 7). B) Representative trace of right ventricular pressure measurements in normoxic wild type mice (top) and normoxic K_Ca3.1^−/−/K_Ca2.3^T/T(+Dox) mice (bottom). C) Hypoxia induced right ventricular hypertrophy as indicated by alterations of the weight ratio of right ventricle/left ventricle + septum, in wild type and K_Ca3.1^−/−/K_Ca2.3^T/T(+Dox) mice, n = 8. D) The effect of hypoxia on right ventricular wall thickness/heart weight (HW) in wild type and K_Ca3.1^−/−/K_Ca2.3^T/T(+Dox) mice. Values are mean ±SEM, n = 8. Data were analyzed by 2−way ANOVA and differences were considered significant when *P<0.05 vs. wild type, ^# P<0.05 vs. normoxia.

Figure 2. Lung mRNA expression levels of A) eNOS, K_Ca2.1, K_Ca2.2, K_Ca2.3, K_Ca3.1, and K_Ca1.1; B) α-Smooth muscle actin (α-SMA), collagen-1, and TGFβ.

Data are given as means ±SEM, n = 7–8. Data were analyzed by two-way ANOVA and differences were considered significant when *P<0.05 from wild type, ^# P<0.05 from normoxia. Statistical interaction (£) was observed in K_Ca2.3 expression (A). C–E) Genotyping: C:Gel electrophoresis shows that polymerase chain reacton (PCR) detected the K_Ca2.3-wild type allele (wild type (+)) and the tTA allele (T) in K_Ca2.3^T/+, and K_Ca2.3^T/T. D and E: PCR detected the targeted allele in K_Ca3.1^−/+ and in K_Ca3.1^−/− as well as the wild type allele in K_Ca3.1^+/− and K_Ca3.1^+/+. A DNA ladder was used to determine products sizes.

Figure 3. Images of mouse lung showing pulmonary vessels stained for von-Willebrand factor and α-smooth muscle actin from K_Ca3.1^−/−/ K_Ca2.3^T/T(+Dox) - and wild type mice under normoxic and hypoxic conditions, identifying non-muscularized vessels, partially-muscularized vessels, and fully-muscularized vessels.

Figure 4. Morphometric measurements.

A: Lumen diameter (µm) in vessels divided in groups of non-muscularized-, partially- and fully-muscularized vessels. B: Wall/lumen ratio divided in groups of non-muscularized-, partially- and fully-muscularized vessels. C: Number of non-muscularized-, partially- and muscularized vessels in each experimental group. D: Wall area of the vessels divided in groups of vessel diameter. * P<0.05 vs. wild type and ^# P<0.05 vs. normoxia. Statistical interaction was observed in Figure (B) for non-muscularized vessels.

Figure 5. Functional studies of acetylcholine-induced (3*10⁻⁷ M) relaxation (A–C) and NS309-induced (10⁻⁶ M) relaxation (D–F) in wild type and K_Ca3.1^−/−/K_Ca2.3^T/T(+Dox) mice.

A) Acetylcholine-induced relaxation. B) Acetylcholine-evoked relaxation in the presence of apamin (5*10⁻⁷ M) and ChTx (10⁻⁷ M). C) Acetylcholine-evoked relaxation in the presence of L-NNA (10^-4 M). D) NS309-induced relaxation. E) NS309-induced relaxation in the presence of apamin (5*10⁻⁷ M) and ChTx (10⁻⁷ M). F) NS309-evoked relaxation in the presence of L-NNA (10⁻⁴ M). Data were analyzed by 2-way ANOVA and * P<0.05 vs. wild type and ^# P<0.05 vs. normoxia; n = 6-7 per group.