Abstract 432: Loss of Primary Cilia PertErbBs Ventricular Maturation in the Developing Heart

Abstract

The embryonic heart requires cardiac neural crest cells (CNCC), which elaborate the sensory organelle called the primary cilium and assist in the development of the cardiac valves, the interventricular septum, and the outflow tract. Our laboratory has modeled congenital heart defects (CHD) resulting from CNCC-specific ciliary loss, using a Wnt1:Cre-2, Ift88 -targeted conditional elimination of primary cilia. Ift88 -homozygous mutants (MUT) displayed a variety of predicted CNCC-associated CHDs, but also revealed a novel disorganization of the ventricular endocardium, pronounced noncompaction of the ventricular myocardium, and perinatal lethality. Neuregulin-1 / ErbB signaling is a well-known key modulator of myocardial development as well as the compaction process of the ventricular myocardium. Flow cytometric analysis of embryonic day 12.5 (E12.5) whole hearts revealed that loss of cilia in CNCC of MUT hearts led to a 43% reduction in cardiomyocytes (CM), defined as VCAM-1-positive/CD31-negative cells (p<0.01), and a trend towards increased CD31-positive endocardial cell (EC) populations (34% increase; p = 0.09) when compared to control (CON) hearts. Analysis of cell surface expression of ErbB receptors revealed a slight decrease in ErbB2 and ErbB4 expression in VCAM-1-positive CM of MUT with no change in ErbB3 expression, and decreased ErbB4 expression in MUT EC. Immunofluorescence analysis of proliferation was quantified in various cardiac subpopulations, including cardiac Troponin-T-positive (cTnT+/CM) cells in the ventricular myocardium. While overall cell density at E12.5 did not differ between the MUT and CON (p=0.43), CM proliferation increased significantly in MUT (5.50%) when compared to CON (2.70%; p<0.01). Taken together, decreased ErbB4 expression in the expanding MUT EC population, combined with decreased CM numbers, could serve as a novel mechanistic explanation for the pronounced hypertrabeculation and noncompaction of the MUT phenotype seen at E14.5. Our results indicate that loss of CNCC primary cilia leads to ventricular noncompaction, with a shift in the relative proportion and dynamics of both CM and EC subpopulations, which is potentially attributable to impaired ErbB signaling of the developing endocardium.