Mechanobiology of Cardiac Outflow Tract Morphogenesis

心脏流出道（OFT）正常生长、分隔和成熟为带瓣主动脉和肺动脉 出口对于出生后的氧合循环至关重要。1-2%的活产婴儿和高达30%的早产胎儿 死亡有先天性心脏缺陷，其中许多影响瓣膜间隔原始组织的重塑， 称为近端和远端流出缓冲垫。尽管人们花了很多努力来揭示早期OFT的遗传基础， 垫形成，这种理解并没有解释临床相关的阶段的增长，冷凝 并延伸成裂孔和隔膜。其中一个原因似乎是有条件和 通过遗传学方法很容易获得的集体信号机制。机械力（剪切力 应力、压力、张力）在OFT生长和重塑的复杂时期一直存在，但迄今为止， 没有研究调查这些关键的相互作用，特别是它们对OFT缺陷的贡献。我们认为 临床相关的OFT重塑由不适当的垫内分泌和/或间质感觉引起， 和/或响应于它们的局部机械环境，这又驱动不正确的信令程序。 布彻实验室开创了创新技术1）量化体内局部机械力， OFT区域并将它们与局部原位基因/蛋白质表达配准，2）非侵入性地可视化， 精确消融心内组织，而不会在体内造成附带损伤，以及3）直接测试机械生物学 离体内膜垫生长和重塑的机制。本提案中的初步数据 两个机械调节分子开关的证据，增强了OFT垫增殖和 分化，这激发了新的假设，局部机械感觉操作分子开关 以控制流出阀和隔膜的尺寸、形状和分层。目标1将实施创新的非 侵入性激光光消融形成的近端或远端垫的鸟类OFT，以产生遗传 无偏倚的临床相关流出道畸形。然后，我们将定量分析和登记他们的 血液动力学、形态学和表型变化。我们将进一步应用新的反卷积积分， sc-Seq和slide-seq以前所未有的时空分辨率揭示畸形的细胞过程， 并详细阐述了已知的和新发现的分子调控程序如何与局部机械 压力变化。目的2将测试机械转导操作分子的机械因果性 通过剪切应力模式在OFT缓冲内循环中切换。目标3将测试不同的操作 通过拉伸/压缩在垫间充质中进行机械生物学转换。使用高通量离体 器官培养这些研究的结果将大大促进我们对 机械调节和条件信号在流出道valovloseptal成熟，铺平了道路， 策略来操纵这些信号程序，以减少甚至挽救子宫内CHD的严重程度。

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.