The long term objective of this research is to obtain a complete understanding of the genetic and molecular mechanisms underlying two hereditary forms of congenital heart disease: Conotruncal defects (CTDs) and patent ductus arteriosus (PDA). These will be studied in embryos and fetuses of unique lines that are accurate models of the human defects. In the CTD line, the following specific hypotheses will be tested: 1. Abnormal development of the conotruncal septum, producing ventricular septal defect, tetralogy of Fallot, or persistent truncus arteriosus, is associated with a generalized failure of growth of right ventricular myocardium in early embryos; 2) There is abnormal expression of myosin ventricular light chains in the affected myocardium; 3) A single major gene mutation underlies the CTD line defect and this gene may have identity with or be linked to myosin gene loci. In the PDA line, the following hypotheses will be tested: 1) The failure of intimal thickening in the ductus arteriosus of affected PDA line fetuses is associated with a genetically-determined block in the accumulation of hyaluronic acid (HA) in the subintimal region; 2) Ductus arteriosus (DA) specific protein molecules recently identified in endothelial and smooth muscle cells of the normal lamb DA are present in normal dog DA but one or both is absent in the hereditary PDA line dogs. Methods to be used include quantitative morphometry and BrdU-labelling of mitotic cells of the myocardium and cushion tissue of the embryonic heart, analysis of the distribution of myosin light chains in embryonic and fetal myocardium using protein gel electrophoresis (1D, 2D PAGE) and antibodies against these proteins, DNA Southern blot restriction fragment length polymorphism (RFLP) studies of linkage of the CTD gene to myosin genes and other genes. PDA studies include immunohistochemical and biochemical analysis of HA in the fetal and neonatal ductus arteriosus during intimal thickening, cell cultures analysis of radiolabeled proteins of DA, pulmonary artery, and aortic cells by SDS PAGE and autoradiography, and use of monospecific antibodies to study the distribution of the ductus-specific proteins in situ.
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