Mechanobiology of Cardiac Outflow Tract Morphogenesis

心脏流出道(OFT)的正常生长、分隔和成熟进入带瓣的主动脉和肺 出口对于出生后的有氧循环是必不可少的。1-2%的活产儿和高达30%的早产儿 死亡患者有先天性心脏缺陷，其中许多会影响瓣膜间隔原始组织的重塑， 称为近端和远端流出气垫。尽管付出了很多努力来揭示早期OFT的遗传基础 垫层的形成，这种理解并没有解释临床上相关的生长、凝结阶段 并延伸到瓣膜和隔膜中。造成这种情况的一个原因似乎是条件和的支配地位 可由遗传方法很好地访问的集体信号机制。机械力(剪切力 应力、压力、张力)在OFT成长和重塑的这段复杂时期一直存在，但到目前为止 还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。我们相信 临床上相关的OFT重构是由不适当的缓冲心内膜和/或间质感觉引起的 和/或对其本地机械环境的响应，进而驱动不正确的信令程序。 布彻实验室开创了一项创新技术，用来量化体内局部的机械力 2)非侵入性可视化和原位基因/蛋白表达。 在活体内精确消融心内组织，无附带损伤；3)直接测试机械生物学 体外心内膜垫生长和重塑的机制。这份提案中的初步数据显示 证据表明两个机械调节的分子开关加强了OFT缓冲增殖和OFT缓冲增殖之间的关系 分化，这激发了新的假设，即局部机械感觉操作分子开关 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 侵入性激光消融形成的禽类OFT近端或远端垫层以创造遗传学 无偏见的临床相关流出道畸形。然后我们将对其进行量化分析和注册 血流动力学、形态和表型改变。我们将进一步应用新的反卷积积分 SC-Seq和Slide-Seq揭示了细胞畸形过程的前所未有的时空分辨率， 并阐述已知的和新发现的分子调控程序如何与局部机械 压力会发生变化。目标2将测试机械转导操作的分子的机制原因 OFT中的开关通过剪切应力模式缓冲心内膜。目标3将测试不同的操作 通过拉伸/压缩在缓冲间充质中的机械生物开关。在体外使用高通量 器官培养。这些研究的发现将从根本上促进我们对 流出道室间隔成熟中的机械调节和条件信号，为 操纵这类信号程序以减轻甚至挽救宫内冠心病严重程度的策略。

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.