Understanding Cardiac C-Looping Using Microscale In Vitro Models

使用微型体外模型了解心脏 C 环

基本信息

批准号：
10448260
负责人：
Leo Q. Wan
金额：
$ 38.98万
依托单位：
RENSSELAER POLYTECHNIC INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-07 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10448260
关键词：
3-Dimensional Actins Animal Genetics Animal Model Anterior Apoptosis Biochemical Biological Assay Biomechanics Biophysical Process Biophysics Cadherins Cardiac Cardiac development Cell Differentiation process Cells Chemicals Chick Clinical Congenital Abnormality Crosslinker Cytoskeleton Defect Development Dextrocardia Double Outlet Right Ventricle Drosophila genus Embryo Embryonic Heart Event Exhibits Fetal Death Genetic Genital Genitalia Handedness Heart Hindgut Human Hydrogels In Vitro Infant Left Libraries Literature Live Birth Measures Microfabrication Molecular Morphogenesis Morphology Myocardium Myosin Type II Nature Nodal Organ Outcome Pharmaceutical Preparations Phenotype Phosphotransferases Population Property Protein Isoforms Protein Kinase C Research Role Rotation Scaffolding Protein Side Signal Pathway Signal Transduction Structure Surface System Technology Teratogens Tissues Traction Force Microscopy Translating Tube Vertebrates Visceral Work alpha Actinin base biomechanical model biophysical analysis congenital heart disorder crosslink cytokine directional cell embryo culture experimental study fascin high throughput screening in vitro Model inhibitor interest kinase inhibitor live cell imaging mechanical force mouse model novel screening septal defect small molecule small molecule inhibitor tool

项目摘要

Defects in laterality are observed in more than 1 in 8000 live births and have significant clinical implications. The embryonic heart starts as a straight cardiac tube along the midline of the embryo, which is subsequently transformed into a c-shaped heart loop reliably toward the right side of the body. This cardiac c-looping is considered as the earliest evident event of left-right (LR) asymmetry breaking (also called chirality or handedness) of a human organ. The inversed lateralization of cardiac looping often leads to severe clinical outcomes, including dextrocardia, septum defects, double outlet right ventricle, and even death of fetuses and infants. Accumulating evidence suggests that asymmetric cardiac looping derives from an unknown tissue- intrinsic mechanism. Recently, we have recapitulated chiral morphogenesis on micropatterned surfaces and in 3D hydrogels and demonstrated that cardiac cells have a definite chirality before asymmetric looping. Protein kinase C (PKC) activators can reverse both cell chirality and cardiac c looping. Our rationale is that novel cell chirality based high-throughput platforms, together with a better understanding of molecular mechanisms of cell chirality, can facilitate the LR symmetry research. We propose to use a combination of micro-fabrication, hydrogel technology, live-cell imaging, molecular assays, traction force microscopy, high-throughput screening, ex vivo culture, and genetic mouse models as tools to elucidate the biophysical and biochemical mechanisms. Our objectives are to determine biomolecular and biomechanical mechanisms of PKC regulated cell chirality and asymmetric looping and to identify cytoskeletal mechanisms of cell chirality during cardiac c-looping. SPECIFIC AIM 1: Identify components and signaling pathways that regulate cardiac chirality with high- throughput screening and validate with ex ovo embryo culture. We will screen inhibitors/activators of PKC isoforms, their downstream effectors, possible substrates, and a small-molecule kinase library, determine mechanisms of action, and validate the findings with the whole-embryo ex ovo culture. SPECIFIC AIM 2: Determine the biomechanical role of cell chirality in multicellular morphogenesis. We will examine whether chiral mechanical forces are sufficient to induce cardiac c-looping using traction force microscopy and whether the cells on ventral myocardium exhibit intrinsic chiral biases. SPECIFIC AIM 3: Determine cytoskeletal mechanisms in cardiac cell chirality during c-looping. We will analyze the chirality of actin dynamics of cardiac cells, observe its change under drugs of interest, and confirm the findings with ex ovo whole-embryo culture and genetic mouse models. If the project is successful, we will be able to establish a set of novel high-throughput platforms for studying the biophysics of asymmetric cardiac looping by measuring cell chirality, and further our understanding of congenital heart disease. Also, this proposed research is transformative, and potentially open a new field of research: cell chirality, a fundamental cellular property defining symmetry breaking in tissue development.

横向性缺陷在8000个活产中的超过1个以上，具有显着的临床意义。胚胎心脏沿着胚胎的中线开始是直型心脏管，随后是可靠地向身体右侧可靠地转变为C形心循环。这种心脏C循环是被认为是最早的左右事件（LR）不对称破坏（也称为手性或人体器官的手。心脏循环的偏侧化通常会导致严重的临床结局，包括右心脏，隔膜缺陷，双出口右心室，甚至是胎儿死亡和死亡婴儿。积累的证据表明，不对称心脏循环源自未知的组织内在机制。最近，我们概括了微图表上的手性形态发生和 3D水凝胶并证明心脏细胞在不对称循环之前具有明确的手性。蛋白质激酶C（PKC）活化剂可以逆转细胞手性和心脏C循环。我们的理由是那个新细胞基于手性的高通量平台，以及对分子机制的更好理解细胞手性，可以促进LR对称研究。我们建议使用微型制作的组合，水凝胶技术，活细胞成像，分子测定，牵引力显微镜，高通量筛选，离体培养和遗传小鼠模型作为阐明生物物理和生化机制的工具。我们的目标是确定PKC调节细胞手性的生物分子和生物力学机制和不对称的循环并确定心脏循环过程中细胞手性的细胞骨架机制。特定目的1：确定组件和信号通路，以高度调节心脏手性吞吐量筛选并用evo胚胎培养物进行验证。我们将筛选PKC的抑制剂/激活剂同工型，其下游效应子，可能的底物和小分子激酶库，确定作用机理，并用整个Evo培养物来验证发现。具体目标2：确定细胞手性在多细胞形态发生中的生物力学作用。我们将检查手性机械力是否足以使用牵引力诱导心脏C循环显微镜以及腹心肌上的细胞是否表现出内在的手性偏见。特定目标3：确定C循环过程中心脏细胞手性的细胞骨架机制。我们将分析心脏细胞肌动蛋白动力学的手性，观察其在感兴趣的药物下的变化，并确认具有外卵形全孔培养和遗传小鼠模型的发现。如果该项目成功，我们将能够建立一组新型的高通量平台，以研究该平台通过测量细胞手性的非对称心脏循环的生物物理学，并进一步了解先天性心脏病。此外，这项拟议的研究具有变革性，并有可能开放研究：细胞手性，一种基本的细胞特性，定义了组织发育中对称性破坏。