The secondary heart field is a new site of calcineurin/Nfatc1 signaling for semilunar valve development

Abstract

Semilunar valve malformations are common human congenital heart defects. Bicuspid aortic valves occur in 2-3% of the population, and pulmonic valve stenosis constitutes 10% of all congenital heart disease in adults (Brickner et al., 2000) [1]. Semilunar valve defects cause valve regurgitation, stenosis, or calcification, leading to endocarditis or congestive heart failure. These complications often require prolonged medical treatment or surgical intervention. Despite the medical importance of valve disease, the regulatory pathways governing semilunar valve development are not entirely clear. In this report we investigated the spatiotemporal role of calcineurin/Nfatc1 signaling in semilunar valve development. We generated conditional knockout mice with calcineurin gene disrupted in various tissues during semilunar valve development. Our studies showed that calcineurin/Nfatc1 pathway signals in the secondary heart field (SHF) but not in the outflow tract myocardium or neural crest cells to regulate semilunar valve morphogenesis. Without SHF calcineurin/Nfatc1 signaling, the conal endocardial cushions-the site of prospective semilunar valve formation--first develop and then regress due to apoptosis, resulting in a striking phenotype with complete absence of the aortic and pulmonic valves, severe valve regurgitation, and perinatal lethality. This role of calcineurin/Nfatc1 signaling in the SHF is different from the requirement of calcineurin/Nfatc1 in the endocardium for semilunar valve formation (Chang et al., 2004) [2], indicating that calcineurin/Nfatc1 signals in multiple tissues to organize semilunar valve development. Also, our studies suggest distinct mechanisms of calcineurin/Nfat signaling for semilunar and atrioventricular valve morphogenesis. Therefore, we demonstrate a novel developmental mechanism in which calcineurin signals through Nfatc1 in the secondary heart field to promote semilunar valve morphogenesis, revealing a new supportive role of the secondary heart field for semilunar valve formation.

Fig. 1. Calcineurin is essential in SHF for semilunar valve development

(A-D) Immunostaining of Cnb1 (brown) in control (Ctrl) and Mef2cCre;Cnb1^f/f embryos at E11.5-12.0. The dashed lines in (B) and (D) mark the junctions between myocardium and cushion mesenchyme. myo: myocardium. RV: right ventricle. epi: epicardium. (E, F) Gross morphology of E17.5 control and Mef2cCre;Cnb1^f/f embryos. (G-J) Hematoxylin-eosin staining of semilunar valves in control and Mef2cCre;Cnb1^f/f mice at E17.5. PA: pulmonary artery. Ao: Aorta. RV: right ventricle. (K, L) Alcian blue staining (blue) of the pulmonary valve in control and Mef2cCre;Cnb1^f/f mice at E17.5. Red: nuclear red counterstain.

Fig. 2. Cushion regression and severe regurgitation in mice lacking SHF Cnb1

(A, B) Doppler in utero echocardiography of control (upper panels) and Mef2cCre;Cnb1^f/f (lower panels) mice at E17.5. Orange color indicates blood flow towards the probe on the abdominal surface, and blue color shows blood flow moving away from the probe. Arrows: turbulent flows. Arrowheads: regurgitant flows. (C-H) Hematoxylin-eosin staining of the pulmonary valve in control (C, E, G) and Mef2cCre;Cnb1^f/f (D, F, H) at different gestational ages. The dashed lines mark the junctions between myocardium and cushion mesenchyme. Ao: aorta. RV: right ventricle. PA: pulmonary artery.

Fig. 3. Loss of SHF calcineurin increases OFT cushion apoptosis

(A, B, D, E) 5-bromo-2’-deoxyuridine (BrdU) staining of control and Mef2cCre;Cnb1^f/f pulmonary valve at E12.5 (A, B) and E13.5 (D, E) after 6-hour BrdU labeling. The dashed lines marked the junctions between myocardium and cushion mesenchyme. RV: right ventricle. (C, F) Quantitation of BrdU-positive cell percentage (left panels) and total cell density (right panels) in mesenchymal regions in control and Mef2cCre;Cnb1^f/f mice. (G, H) TUNEL staining of control and Mef2cCre;Cnb1^f/f pulmonary valve at E14.5. Dashed line marked the junction between mesenchymal cells and myocardium. (I) Quantitation of TUNEL-positive cell percentage in mesenchymal regions in control and Mef2cCre;Cnb1^f/f mice.

Fig. 4. Calcineurin functions in SHF myocardial progenitors

(A, B) Hematoxylin-eosin staining of control and Isl1Cre;Cnb1 ^f/f pulmonary valves at E14.5. Ao: aorta. RV: right ventricle. PA: pulmonary artery. (C, D) Hematoxylin-eosin staining of control and Sm22αCre;Cnb1 ^f/f pulmonary valves at E15.5. (E, F) Hematoxylin-eosin staining of control and Wnt1Cre;Cnb1 ^f/f pulmonary valves at E16.5.

Fig. 5. Calcineurin signals through Nfatc1 in SHF

(A, B) Hematoxylin-eosin staining of control and Mef2cCre;Nfatc1 ^f/f pulmonary valves at E16.5. Ao: aorta. RV: right ventricle. PA: pulmonary artery. (C) Spatiotemporal windows of calcineurin/Nfat signaling during heart valve development. SHF: secondary heart field.