KEYNOTE: Hemodynamic disruption in the proximal outflow tract generates complex congenital heart valve defects without genetic mutation

Abstract

Proper growth and division of the embryonic outflow tract (OFT) into balanced pulmonary and aortic outlets, complete with functioning semilunar valves is essential for robust delivery of oxygenated blood. Many clinically serious cardiac malformations arise later in fetal development, presumed due to growth and/or maturation deficiencies. The vast majority of these defects, including tetralogy of Fallot (ToF), persistent truncus arteriosus (PTA), and bicuspid aortic valve (BAV), occur spontaneously without specific genetic mutations. Local maldistribution of hemodynamic forces is a presumed alternative cause of OFT malformation. We have innovated non-invasive multiphoton microscopy guided femtosecond laser photoablation to create precise tissue microdefects to test genetically unbiased mechanisms of OFT malformation. We performed targeted ablations of the proximal parietal (pP) or septal (pS) cushions in vivo in HH24 chick embryos, analyzing both acute and chronic timepoints. We determined that specific cushion and defect size associated with the development of specific OFT malformations, with 50 mm pS ablations generating 80% BAV phenotypes and 100 mm pP ablations generating 75% ToF phenotype as quantified by Micro-CT. Focusing on ToF, local pP ablation caused accelerated compaction of this cushion and increased expansion of the contralateral pS cushion, resulting in reduced pulmonary outlet lumen size, larger pulmonary semilunar cushions, and larger overriding aortic outlet, together mimicking clinical ToF. Computational simulations identified that pP compaction and pS expansion was associated with local increases in surface unidirectional shear stress (USS) and oscillatory shear stress (respectively). Ex vivo OFT organ culture under defined flow patterns validated that local OSS induces profound cushion expansion via osmotic swelling via aquaporin and b-catenin/pSmad1/5-Yap mediated proliferation, while USS induced compaction via pSer-19 and reduction in Yap. These findings were then validated in vivo via spatial registration of biomechanical properties and protein expression. Taken together, these results establish that local hemodynamic perturbation is sufficient to generate robust clinical OFT malformations without genetic mutation. This informs genotype-phenotype mismatch and provides new techniques for studying mechanobiological mechanisms of malformation.