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: 10650246
• 负责人: Leo Q. Wan
• 金额: $ 38.61万
• 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/10650246
• 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.

在超过八千分之一的活产中观察到偏侧性缺陷，并且具有重大的临床意义。 胚胎心脏开始时是沿着胚胎中线的直心管，随后 转变为C形心形环，可靠地朝向身体右侧。这个心脏c环是 被认为是左右（LR）不对称性破缺（也称为手性或 用手习惯）的人体器官。心脏循环的反向偏侧化常常导致严重的临床后果 后果，包括右位心、隔膜缺损、右心室双出口，甚至胎儿死亡 婴儿。越来越多的证据表明，不对称的心脏循环源自未知的组织—— 内在机制。最近，我们概括了微图案表面和中的手性形态发生 3D 水凝胶并证明心肌细胞在不对称成环之前具有明确的手性。蛋白质 激酶 C (PKC) 激活剂可以逆转细胞手性和心脏 c 循环。我们的理由是新的细胞 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对称性研究。我们建议结合使用微加工、 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 和不对称环，并确定心脏 c 环期间细胞手性的细胞骨架机制。 具体目标 1：识别调节心脏手性的成分和信号通路 通过卵外胚胎培养进行通量筛选和验证。我们将筛选PKC的抑制剂/激活剂 同种型、它们的下游效应子、可能的底物和小分子激酶库，决定了 作用机制，并用全胚胎离卵培养验证研究结果。 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 环 显微镜检查以及腹侧心肌上的细胞是否表现出内在的手性偏差。 具体目标 3：确定 c 环过程中心脏细胞手性的细胞骨架机制。我们将 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，并确认 卵外全胚胎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循环的生物物理学，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，并有可能开辟一个新的领域 研究：细胞手性，一种基本的细胞特性，定义组织发育中的对称性破缺。