NFATc3 is required for chronic hypoxia-induced pulmonary hypertension in adult and neonatal mice

Abstract

Pulmonary hypertension occurs with prolonged exposure to chronic hypoxia in both adults and neonates. The Ca(2+)-dependent transcription factor, nuclear factor of activated T cells isoform c3 (NFATc3), has been implicated in chronic hypoxia-induced pulmonary arterial remodeling in adult mice. Therefore, we hypothesized that NFATc3 is required for chronic hypoxia-induced pulmonary hypertension in adult and neonatal mice. The aim of this study was to determine whether 1) NFATc3 mediates chronic hypoxia-induced increases in right ventricular systolic pressure in adult mice; 2) NFATc3 is activated in neonatal mice exposed to chronic hypoxia; and 3) NFATc3 is involved in chronic hypoxia-induced right ventricular hypertrophy and pulmonary vascular remodeling in neonatal mice. Adult mice were exposed to hypobaric hypoxia for 2, 7, and 21 days. Neonatal mouse pups were exposed for 7 days to hypobaric chronic hypoxia within 2 days after delivery. Hypoxia-induced increases in right ventricular systolic pressure were absent in NFATc3 knockout adult mice. In neonatal mice, chronic hypoxia caused NFAT activation in whole lung and nuclear accumulation of NFATc3 in both pulmonary vascular smooth muscle and endothelial cells. In addition, heterozygous NFATc3 neonates showed less right ventricular hypertrophy and pulmonary artery wall thickness in response to chronic hypoxia than did wild-type neonates. Our results suggest that NFATc3 mediates pulmonary hypertension and vascular remodeling in both adult and neonatal mice.

Fig. 1.

Nuclear factor of activated T cells isoform c3 (NFATc3) is required for chronic hypoxia (CH)-induced increases in right ventricular (RV) systolic pressure (RVSP) and RV hypertrophy in adult mice. WT, NFATc3 wild type; HET, NFATc3 heterozygote; KO, NFATc3 knockout mice exposed to normoxia or CH for 21 days. A: RVSP. B: RV hypertrophy expressed as the %ratio of RV to left ventricle (LV) and septum (S) (%RV/LV+S). Values are means ± SE; n = no. of mice (see bars). *P < 0.05 vs. normoxia, #P < 0.05 vs. CH WT (two-way ANOVA followed by Bonferroni's post hoc test).

Fig. 2.

NFATc3 is required for early CH-induced pulmonary arterial smooth muscle (PASMC) proliferation in adult mice. NFATc3 KO mice were exposed to normoxia (N) or CH for 2, 7, and 21 days. Values are means ± SE of %5-ethynyl-2′-deoxyuridine (EdU)-positive PASMC; n = no. of mice (see bars). *P < 0.05 vs. N WT, #P < 0.05 vs. CH 2d WT (two-way ANOVA followed by Bonferroni's post hoc test). Twelve arteries/animal were analyzed.

Fig. 3.

CH activates NFATc3 in lungs from neonatal mice. A: CH increases luciferase activity in whole lungs from neonatal mice. Values are means ± SE; n = 7 mice/group. *P < 0.05, Student's t-test. RLU, relative light units. B: representative images of NFATc3 nuclear accumulation in endothelial cells (EC) and PASMC from pulmonary arteries of neonatal mice. Arrows indicate NFATc3 nuclear-positive cells. The white arrow depicts an EC, and the yellow arrow a PASMC. Red, NFATc3 staining; blue, α-actin staining; green, nuclear staining. α-Actin staining was used to distinguish PASMC from EC. Scale bar = 20 μm. C: summary showing CH-induced increase in %NFATc3 nuclear-positive EC and PASMC. Values are means ± SE; n = no. of arteries (see bars) from 7 mice/group. *P < 0.05, Student's t-test. 100 PASMC and 50 EC nuclei were counted per animal.

Fig. 4.

NFATc3 is required for CH-induced RV hypertrophy in neonatal mice. NFATc3 HET neonatal mice were exposed to normoxia or CH for 7 days. %RV/LV+S is expressed as means ± SE; n = no. of mice (see bars). *P < 0.05 vs. normoxia, #P < 0.05 vs. CH WT (two-way ANOVA followed by Bonferroni's post hoc test).

Fig. 5.

Greater pulmonary arterial wall thickness in CH neonatal mice requires NFATc3. NFATc3 HET neonatal mice were exposed to normoxia or CH for 7 days. A: representative images. Scale bar = 50 μm. B: summary of results. External diameter ranged from 25 to 60 μm. Values are means ± SE; n = no. of mice (see bars). *P < 0.05 vs. normoxia, #P < 0.05 vs. CH WT (two-way ANOVA followed by Bonferroni's post hoc test). Ten arteries per animal were analyzed.

Fig. 6.

NFATc3 is not required for CH-induced decreases in PASMC proliferation in neonatal mice. Incorporation of EdU in PASMC nuclei from NFATc3 WT and HET neonatal mice exposed to normoxia or CH for 7 days was expressed as %EdU-positive PASMC nuclei. Values are means ± SE; n = no. of mice (see bars). *P < 0.05 vs. normoxia (two-way ANOVA followed by Bonferroni's post hoc test). Twelve arteries per animal were analyzed.

Fig. 7.

CH does not induce pulmonary arterial cellular apoptosis in neonatal mice. A: representative images of activated caspase-3 detected by immunohistochemistry microscopy in lung sections from NFATc3 WT and HET neonatal mice exposed to normoxia or CH for 7 days. Activated caspase-3 was not detected in ECs or PASMC of any of the pulmonary arteries present in one section per animal in any of the experimental groups. Arrows depict positive activated caspase-3 cells, demonstrating apoptotic cells in lung parenchyma. Scale bar = 50 μm. B: representative images of a TUNEL-positive cell (depicted by arrow) present in lung parenchyma: first image, total nuclei were stained with DAPI; second image, positive TUNEL nuclei; third image, composite of DAPI and TUNEL channels; fourth image, DNAse-treated positive control. No TUNEL-positive cells were found in pulmonary arteries from the different experimental groups. All of the arteries present in three consecutive lung sections from four animals per group were analyzed.