Mechanobiology of Cardiac Outflow Tract Morphogenesis

Proper growth, septation, and maturation of the cardiac outflow tract (OFT) into valved aortic and pulmonary
outlets are essential for oxygenated circulation after birth. 1-2% of live births and up to 30% of pre-term fetal
deaths have congenital heart defects, many of which affect the remodeling of the valvuloseptal primordial tissues,
called the proximal and distal outflow cushions. Despite much effort uncovering the genetic basis of early OFT
cushion formation, this understanding has not explained the clinically relevant phases of growth, condensation
and elongation into valves and septa. One reason for this appears to be the domination of conditional and
collective signaling mechanisms that are well accessible by genetic approaches. Mechanical forces (shear
stress, pressure, tension) are ever present during this complex period of OFT growth and remodeling, but to date
no studies have investigated these key interactions, especially for their contributions to OFT defects. We believe
that clinically relevant OFT remodeling arise from improper cushion endocardial and/or mesenchymal sensation
of and/or response to their local mechanical environment, which in turn drives the incorrect signaling programs.
The Butcher lab has pioneered innovative technology 1) to quantify local in vivo mechanical forces within this
OFT region and register them with local in situ gene/protein expression, 2) to not-invasively visualize and
precisely ablate intracardiac tissues without collateral damage in vivo, and 3) to directly test mechanobiological
mechanisms of endocardial cushion growth and remodeling ex vivo. The preliminary data in this proposal present
evidence of two mechanoregulated molecular switches that potentiate between OFT cushion proliferation and
differentiation, which motivates the novel hypothesis that local mechanosensaton operates molecular switches
to control sizing, shape, and stratification of the outflow valves and septa. Aim 1 will implement innovative non-
invasive laser photoablations of the formed proximal or distal cushions of the avian OFT to create genetically
unbiased clinically relevant outflow tract malformations. We will then quantitatively analyze and register their
hemodynamic, morphological and phenotypic changes. We will further apply novel deconvolution integration of
sc-Seq and slide-seq to reveal unprecedented spatio-temporal resolution of the cellular course of malformation,
and elaborate how known and newly discovered molecular regulatory programs associate with local mechanical
stress changes. Aim 2 will test the mechanistic causailty of the mechanotransduction operated molecular
switches in the OFT cushion endocardium via shear stress patterns. Aim 3 will test the operation of different
mechanobiogical switches in cushion mesenchyme via tension/compression. using high throughput ex vivo
organ cultures. The findings from these studies will substantally advance our understanding of
mechanoregulation and conditional signaling in outflow tract valuvloseptal maturation, paving the way for
strategies to manipulate such signaling programs to reduce or even rescue CHD severity in utero.

Grant Number: 5R01HL160028-04
NIH Institute/Center: NIH
Principal Investigator: Jonathan Butcher