Atrioventricular (AV) valves are formed in the AV canal (AVC) from endocardial cushions. The molecular mechanisms involved are poorly understood. Our new preliminary data indicate that AV valve maturation involves condensation at E18.5, postnatal elongation, and postnatal extracellular matrix (ECM) remodeling. The type IA receptor for bone morphogenetic proteins (BMP), Alk3, plays an important role in early heart formation but its function at later stages is unknown. To study the role of Alk3 during AVC maturation in mice, we used a unique Cre/lox system driven by a cGATA6 enhancer to inactivate Alk3 in cardiac myocytes of the AVC. Adult mice with AVC-targeted deletion of Alk3 (cGATA6-Cre/Alk3) displayed AV valves defects, disruption of the annulus fibrosus, and ventricular pre-excitation compatible with Ebstein's anomaly. Ebstein's anomaly is a rare congenital heart defect that occurs with a probability of 1:20,000 and represents about 40% of congenital malformations of the tricuspid valve2. The valvular defects in our mutant mice start around embryonic day (E) 14.5-E15.5, with the premature disappearance of the myocardial layer in the tricuspid mural leaflet, and defective periostin expression. The origin of the other valvular defects and of the disruption of the annulus fibrosus is still not understood. Here, we propose to use a systematic approach, combined with innovative techniques, to analyze these mice and to advance our understanding of AV valve development and get insights into the causes of Ebstein's anomaly. Accordingly, the Specific Aims are: ? ? Specific Aim 1: To study in vivo the role of Alk3 in AVC cardiac myocytes during AV valve maturation in cGATA6-Cre/Alk3 mice. Our hypothesis is that Alk3 signaling in AVC cardiomyocytes is necessary to regulate cell survival, condensation, elongation, and ECM remodeling during AV valve development. To test this hypothesis, cardiac myocytes in which Alk3 was inactivated will be genetically labeled. Apoptosis and cell proliferation will be analyzed in these cells. Nuclear density and leaflet length will be measured. Expression of ECM proteins at proximity of Alk3-null myocytes will be studied by immunofluorescence, with a particular focus on ECM proteins that confer stress-resistance to adult leaflets and/or that are critical for anchoring of the leaflets to the annulus fibrosus. ? ? Specific Aim 2: To study ex vivo the role of Alk3 in postnatal ECM expression and cellular adhesion using cultured postnatal mitral valves in a newly designed Miniature Tissue Culture System. Our hypothesis is that Alk3 signaling in AVC cardiomyocytes is necessary for ECM expression and for adhesion of the fibrosa and spongosia. To test our hypothesis, BMP signaling will be inhibited in cultured postnatal mitral valves and the effect on the expression of ECM proteins and of markers of cellular adhesion tested by immunofluorescence. ? ? ? ?
| Kruithof, Boudewijn P T; Fritz, David T; Liu, Yijun et al. (2011) An autonomous BMP2 regulatory element in mesenchymal cells. J Cell Biochem 112:666-74 |
| Kruithof, Boudewijn P T; Xu, Junwang; Fritz, David T et al. (2011) An in vivo map of bone morphogenetic protein 2 post-transcriptional repression in the heart. Genesis 49:841-50 |
| Lieber, Samuel C; Kruithof, Boudewijn P T; Aubry, Nadine et al. (2010) Design of a miniature tissue culture system to culture mouse heart valves. Ann Biomed Eng 38:674-82 |
