Understanding Cardiac C-Looping Using Microscale In Vitro Models

使用微型体外模型了解心脏 C 环

• 批准号: 10838024
• 负责人: Leo Q. Wan
• 金额: $ 8.08万
• 依托单位: RENSSELAER POLYTECHNIC INSTITUTE
• 依托单位国家: 美国
• 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/10838024
• 关键词: 3-Dimensional; Actins; Animal Genetics; Animal Model; Anterior; Apical; Apoptosis; Biochemical; Biological Assay; Biomechanics; Biophysical Process; Biophysics; Cadherins; Cardiac; 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; Pattern; Pharmaceutical Preparations; Phenotype; Phosphotransferases; Population; Property; Protein Isoforms; Protein Kinase C; Research; Role; Rotation; Scaffolding Protein; Shapes; Side; Signal Pathway; Signal Transduction; Structure; Surface; System; Technology; Teratogens; Tissues; Traction Force Microscopy; Translating; Tube; Vertebrates; Visceral; Work; alpha Actinin; biomechanical model; biophysical analysis; cardiogenesis; 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

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.

每8000名活产儿中就有一人观察到偏侧缺陷，这具有重要的临床意义。 胚胎心脏开始时是一根沿着胚胎中线的直心管，随后 变成了一个可靠地朝向身体右侧的C形心环。这个心脏的c-环是 被认为是左-右(LR)不对称断裂(也称为手性或手性)的最早明显事件 (用手)指人体器官。心脏环的倒置偏侧化常常导致严重的临床 预后，包括右位心、间隔缺陷、右室双出口，甚至胎儿死亡和 婴儿。越来越多的证据表明，不对称心跳起源于一种未知的组织- 内在机制。最近，我们概述了微图案化表面上的手性形态发生 3D水凝胶，证明心肌细胞在不对称环前有一定的手性。蛋白 蛋白激酶C(PKC)激活剂可以逆转细胞手性和心脏循环。我们的理论基础是那个新奇的细胞 基于手性的高通量平台，以及更好地理解 细胞手性，可以方便LR对称性的研究。我们建议使用微制造的组合， 水凝胶技术，活细胞成像，分子分析，牵引力显微镜，高通量筛选， 体外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。 和不对称环，并确定在心脏c-环过程中细胞手性的细胞骨架机制。 特定目标1：识别高密度脂蛋白调节心脏手性的成分和信号通路 通过体外胚胎培养进行吞吐量筛选和验证。我们将筛选PKC的抑制剂/激活剂 异构体、它们的下游效应物、可能的底物和小分子激酶库，决定了 作用机制，并用全胚胎体外培养验证研究结果。 特定目的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-环。 显微镜和腹侧心肌上的细胞是否表现出固有的手性偏向。 具体目的3：确定c-环过程中心肌细胞手性的细胞骨架机制。我们会 分析心肌细胞肌动蛋白动力学的手性，观察其在感兴趣药物作用下的变化，并证实 这一发现与体外全胚胎培养和遗传小鼠模型有关。 如果项目成功，我们将能够建立一套新颖的高通量平台来研究 通过测量细胞手性来研究不对称心脏环路的生物物理学，并进一步加深我们对 先天性心脏病。此外，这项拟议的研究具有变革性，并可能打开一个新的领域 研究：细胞手性，一种定义组织发育中对称性破坏的基本细胞属性。

项目成果

Cell jamming regulates epithelial chiral morphogenesis.

细胞干扰调节上皮手性形态发生。

• DOI: 10.1016/j.jbiomech.2023.111435
• 发表时间: 2023
• 期刊: Journal of biomechanics
• 影响因子: 2.4
• 作者: Rahman,Tasnif;Peters,Frank;Wan,LeoQ
• 通讯作者: Wan,LeoQ

Cell Chirality as a Novel Measure for Cytotoxicity.

• DOI: 10.1002/adbi.202101088
• 发表时间: 2022-01
• 期刊: ADVANCED BIOLOGY
• 影响因子: 3.7
• 作者: Zhang, Haokang;Wan, Leo Q.
• 通讯作者: Wan, Leo Q.

A Micropatterning Assay for Measuring Cell Chirality.

• DOI: 10.3791/63105
• 发表时间: 2022-03-11
• 期刊: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
• 影响因子: 1.2
• 作者: Zhang, Haokang;Ronaldson-Bouchard, Kacey;Vunjak-Novakovic, Gordana;Wan, Leo Q.
• 通讯作者: Wan, Leo Q.