Abstract
Normal blood flow is essential for proper heart formation during embryonic development, as abnormal hemodynamic load (blood pressure and shear stress) results in cardiac defects seen in congenital heart disease. However, the progressive detrimental remodeling processes that relate altered blood flow to cardiac defects remain unclear. Endothelial–mesenchymal cell transition is one of the many complex developmental events involved in transforming the early embryonic outflow tract into the aorta, pulmonary trunk, interventricular septum, and semilunar valves. This study elucidated the effects of increased hemodynamic load on endothelial–mesenchymal transition remodeling of the outflow tract cushions in vivo. Outflow tract banding was used to increase hemodynamic load in the chicken embryo heart between Hamburger and Hamilton stages 18 and 24. Increased hemodynamic load induced increased cell density in outflow tract cushions, fewer cells along the endocardial lining, endocardium junction disruption, and altered periostin expression as measured by confocal microscopy analysis. In addition, 3D focused ion beam scanning electron microscopy analysis determined that a portion of endocardial cells adopted a migratory shape after outflow tract banding that is more irregular, elongated, and with extensive cellular projections compared to normal cells. Proteomic mass-spectrometry analysis quantified altered protein composition after banding that is consistent with a more active stage of endothelial–mesenchymal transition. Outflow tract banding enhances the endothelial–mesenchymal transition phenotype during formation of the outflow tract cushions, suggesting that endothelial–mesenchymal transition is a critical developmental process that when disturbed by altered blood flow gives rise to cardiac malformation and defects.
Highlights
Congenital heart defects affect nearly 1% of newborns and are the leading cause of infant death in the United States (Hoffman and Kaplan, 2002; Yang et al, 2006)
F-actin staining with phalloidin at HH15, HH18, and HH24 in both the outflow and atrioventricular segments of control hearts displayed the progression of Endothelial mesenchymal transition (EMT) (Figure 3)
EMT had begun in the outflow tract by HH18 with a few strands of F-actin stain that extended from the center of the cushion endocardium, which coincided with a more disbursed configuration of F-actin staining along the entire length of the cushions in the atrioventricular canal
Summary
Congenital heart defects affect nearly 1% of newborns and are the leading cause of infant death in the United States (Hoffman and Kaplan, 2002; Yang et al, 2006). Blood flow dynamics play a critical role in regulating early heart development (Culver and Dickinson, 2010), as numerous studies have shown that surgically altered blood flow results in a spectrum of cardiac defects seen in human congenital heart disease (CHD) (Clark and Rosenquist, 1978; Clark et al, 1989; Hogers et al, 1997, 1999; Sedmera et al, 1999; Tobita et al, 2002; Hu et al, 2009; Midgett and Rugonyi, 2014). Endocardial cushions (localized thickenings of the wall) form in the outflow tract and atrioventricular canal, and eventually serve as primitive valves that block blood flow upon contraction of the myocardium. The outflow tract cushions are frequently studied because they are very sensitive to hemodynamic perturbation and later transform into the interventricular septum and semilunar valves, which are often involved in congenital heart defects (Hogers et al, 1997; Hove et al, 2003; Gittenberger-de Groot et al, 2005)
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