NFATc3 mediates chronic hypoxia-induced pulmonary arterial remodeling with alpha-actin up-regulation

Abstract

Physiological responses to chronic hypoxia include polycythemia, pulmonary arterial remodeling, and vasoconstriction. Chronic hypoxia causes pulmonary arterial hypertension leading to right ventricular hypertrophy and heart failure. During pulmonary hypertension, pulmonary arteries exhibit increased expression of smooth muscle-alpha-actin and -myosin heavy chain. NFATc3 (nuclear factor of activated T cells isoform c3), which is aCa(2+)-dependent transcription factor, has been recently linked to smooth muscle phenotypic maintenance through the regulation of the expression of alpha-actin. The aim of this study was to determine if: (a) NFATc3 is expressed in murine pulmonary arteries, (b) hypoxia induces NFAT activation, (c) NFATc3 mediates the up-regulation of alpha-actin during chronic hypoxia, and (d) NFATc3 is involved in chronic hypoxia-induced pulmonary vascular remodeling. NFATc3 transcript and protein were found in pulmonary arteries. NFAT-luciferase reporter mice were exposed to normoxia (630 torr) or hypoxia (380 torr) for 2, 7, or 21 days. Exposure to hypoxia elicited a significant increase in luciferase activity and pulmonary arterial smooth muscle nuclear NFATc3 localization, demonstrating NFAT activation. Hypoxia induced up-regulation of alpha-actin and was prevented by the calcineurin/NFAT inhibitor, cyclosporin A (25 mg/kg/day s.c.). In addition, NFATc3 knock-out mice did not showed increased alpha-actin levels and arterial wall thickness after hypoxia. These results strongly suggest that NFATc3 plays a role in the chronic hypoxia-induced vascular changes that underlie pulmonary hypertension.

Fig. 1

NFATc3 is expressed in mouse pulmonary arteries. A. RT-PCR analysis of NFATc3 expression in isolated intrapulmonary arteries from 9xNFAT-luc mice. Lane 1 shows amplification of NFATc3. Amplification of cyclophilin (control) from pulmonary arteries is shown in lane 2. Lane 3 corresponds to a control without the reverse transcription step. The experiment was repeated at least three times and the same bands were detected in pulmonary arteries from all the mouse strains used in this study. B. Western analysis of NFATc3 protein expression in thymus (C57Bl6), pulmonary arteries (9xNFAT-luc), and human Burkitt’s lymphoma-derived Ramos cells (Santa Cruz). Thymus and Ramos cells were used as positive control.

Fig. 2

NFAT is activated by CH in pulmonary arteries. A. Pulmonary arteries were isolated from 9xNFAT-luc mice exposed to normoxia (N), or to 2, 7 or 21 days of CH. RLU. μg⁻¹= luciferase activity normalized by μg of total protein. *p<0.05 vs. N; ^#p<0.05 vs. 2 days, n=5 animals. B. Pulmonary arteries were isolated from 9xNFAT-luc/NFATc3 WT, HET and KO mice exposed to 2 days of CH. Luciferase activity was normalized by μg of total protein and expressed as percent change from N WT (average=1.36±0.19). *p<0.01 vs. N WT, #p<0.05 vs. CH WT and HET, n=4–8 animals.

Fig. 3

NFATc3 nuclear accumulation increases in pulmonary arterial smooth muscle cells after CH. A. Representative images showing cytosolic localization of NFATc3 under normoxic (N) conditions and nuclear localization following exposure to CH for 2, 7, or 21 days. Lung sections from 9xNFAT-luc mice were co-stained with the DNA-binding dye SYTOX (green), anti-NFATc3 (red) and anti-SM-α-actin (blue). Pulmonary arterial smooth muscle nuclear co-localization of NFATc3 is shown in white. Arrows indicate examples of NFATc3 positive nuclei. B. Summary of effects of CH on NFATc3 nuclear accumulation over time. *p<0.05 vs. N, #p<0.05 vs. 7 and 21 days, n=14 images from at least 3 animals/group (4 arteries/animal). C. Summary of effects of CsA on CH-induced NFATc3 nuclear accumulation. C57B6 mice were treated with vehicle (V) or CsA (25mg/Kg/day) and exposed to N or 7 days of CH. Lung sections were stained as in A. *p<0.05 vs. N V, ^#p<0.05 vs. CH V, n= 6 images from at least 3 animals/group (2 arteries/animal).

Fig. 4

Calcineurin/NFATc3 mediates CH-induced RV hypertrophy but not polycythemia. A. Percent change in RV/T in vehicle (V)- and CsA-treated and WT and KO mice exposed to 7 and 21 days of CH, respectively, normalized to normoxia (N) values. *p<0.01 vs. V or WT. n= see columns. B. Hematocrit (%) in vehicle (V)- and CsA-treated and WT and KO mice exposed to 7 and 21 days of CH, respectively, normalized to normoxia (N) values. *p<0.01 vs. N, n= see columns.

Fig. 5

Calcineurin/NFAT mediates CH-induced up-regulation of SM-α-actin in pulmonary arteries. A. Western blot analysis of SM-α-actin protein expression in isolated pulmonary arteries from C57B6 mice treated with vehicle (V) or CsA and exposed to normoxia (N) or CH for 7 days. SM-α-actin band densities were normalized to total protein loaded per lane as determined by Coomasie blue or Sypro Red II staining of the membrane. AU= arbitrary units. *p<0.05 vs. N V, #p<0.05 vs. CH V, n=6 animals, B. Summary of qRT-PCR analysis of SM-α-actin transcript expression in isolated pulmonary arteries from the same animals described in A. Threshold crossing value (CT) was calculated using well factor background subtraction, and gene expression normalized to 18S for each sample. *p<0.05 vs. N V, n=5 animals.

Fig. 6

NFATc3 mediates CH-induced up-regulation of SM-α-actin in pulmonary arteries. A. Western blot analysis of SM-α-actin protein expression in isolated pulmonary arteries from WT and NFATc3 KO mice exposed to normoxia (N) or CH for 21 days. SM-α-actin band densities were normalized to total protein loaded per lane as determined by Coomasie blue or Sypro Red II staining of the membrane. AU= arbitrary units. *p<0.05 vs. N WT, #p<0.05 vs. CH WT, n=4 animals. B. Western blot analysis of β-actin protein expression in isolated pulmonary arteries from the same animals described in A. Band densities were normalized to total protein loaded per lane as determined by Sypro Red II staining of the membrane. n=4 animals.

Fig. 7

NFATc3 mediates CH-induced increases in pulmonary arterial wall thickness. A. Representative cross sections from normoxic (N) and CH (21 days) WT and KO mice. Arrows point internal and external elastic laminae. B. Percent arterial wall thickness of arteries with diameter of 231–310 μm. *p<0.001. Arteries n= see columns, number of mice 3–4.