Mutation of LRP1 in cardiac neural crest cells causes congenital heart defects by perturbing outflow lengthening

Abstract

The recent recovery of mutations in vesicular trafficking genes causing congenital heart disease (CHD) revealed an unexpected role for the endocytic pathway. We now show that mice with a C4232R missense mutation in Low density lipoprotein receptor related protein 1 (LRP1) exhibit atrioventricular septal defects with double outlet right ventricle. Lrp1m/m mice exhibit shortened outflow tracts (OFT) and dysmorphic hypocellular cushions with reduced proliferation and increased apoptosis. Lrp1m/m embryonic fibroblasts show decreased cell motility and focal adhesion turnover associated with retention of mutant LRP1 in endoplasmic reticulum and reduced LRP1 expression. Conditional deletion of Lrp1 in cardiac neural crest cells (CNC) replicates the full CHD phenotype. Cushion explants showed defective cell migration, with gene expression analysis indicating perturbation of Wnt and other signaling pathways. Thus, LRP1 function in CNCs is required for normal OFT development with other cell lineages along the CNC migratory path playing a supporting role.

Endocytic Protein Defects in the Neural Crest Cell Lineage and Its Pathway Are Associated with Congenital Heart Defects

“…Among the mutations involved in the endocytic trafficking proteins, a missense mutation in low-density lipoprotein receptor-related protein 1 (Lrp1), a member of the multifunctional low-density lipoprotein (LDL) receptor-related protein (LRP) family [24][25][26], was noted to result in CHDs [20,27]. LRP is the main receptor for the uptake of lipoproteins in vertebrate cells [26].…”

“…The α-chain of LRP1 contains various repeated motifs and the β-chain contains the C4232R mutation and two cytoplasmic NPxY sequences that serve as signals for endocytosis [24,25]. By using different endocytic trafficking markers and Western blot, the C4232R mutant protein had a substantial reduction in the 85 kDa LRP1 β-chain expression secondary to altering endocytic trafficking with endoplasmic reticulum retention of the mutant LRP1 C4232R protein [27]. LRP1 was originally described to mediate endocytosis of lipoproteins, but it is now also appreciated that LRP1 can bind dozens of other ligands.…”

“…Mice harboring the Lrp1 C4232R mutation exhibit homozygous lethality (Lrp1 m/m ) at E14.5-E15.5, with rare stillborn pups [27]. Cardiac assessments using fetal echocardiography and episcopic confocal microscopy (ECM) demonstrated that all Lrp1 m/m mutants have congenital heart defects.…”

“…Cardiac assessments using fetal echocardiography and episcopic confocal microscopy (ECM) demonstrated that all Lrp1 m/m mutants have congenital heart defects. The majority (85%) had the outflow tract malalignment defect of double outlet right ventricle (DORV) and an atrioventricular septal defect (AVSD) [27]; 9% had isolated DORV and 6% had isolated AVSD (Figure 1). A representative fetal echocardiogram and ECM demonstrated that the Lrp1 m/m mutant showed a flow across the ventricular septum (ventricular septal defect, VSD) and side-by-side great vessels, with the aorta positioned to the right of the pulmonary artery (Figure 1).…”

“…LRP1 is expressed in multiple cell lineages that are crucial during heart development, including neural crest, first heart field, second heart field, and mesenchymal cells of the developing endocardial cushion [27]. A floxed Lrp1 (Lrp1 f/f ) allele [29] with different Cre drivers to orchestrate conditional Lrp1 deletion in cell lineages associated with cardiac development was generated to investigate the pathomechanisms of DORV and AVSD.…”