Understanding Cardiac C-Looping Using Microscale In Vitro Models

在8000例活产婴儿中观察到1例以上的偏侧性缺陷，并具有重要的临床意义。 胚胎心脏开始为沿着胚胎中线沿着的直心管，随后在胚胎的心脏中形成心脏。 转变成一个C形的心脏环，可靠地朝向身体的右侧。这个心脏C形环 被认为是最早的左右（LR）不对称性破坏（也称为手性或 （1）人体器官。心脏成环的反向偏侧化常常导致严重的临床症状， 结果，包括心绞痛，间隔缺损，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来源于一种未知的组织- 内在机制最近，我们已经概括了微图案化表面上的手性形态发生， 3D水凝胶，并证明了心脏细胞在不对称循环之前具有明确的手性。蛋白 激酶C（PKC）激活剂可以逆转细胞手性和心脏C环。我们的理论是， 基于手性的高通量平台，以及更好地理解 细胞的手性，可以方便LR对称性的研究。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物化学机制的工具。 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 和不对称成环，并鉴定心脏C-成环过程中细胞手性的细胞骨架机制。 具体目标1：确定高水平的心脏手性调控的组分和信号通路。 通量筛选和卵外胚胎培养验证。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 的作用机制，并验证与全胚胎卵外培养的结果。 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 显微镜和腹侧心肌细胞是否表现出内在的手性偏见。 特定目的3：确定c-环形成过程中心肌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，观察其在感兴趣的药物作用下的变化， 卵外全胚胎培养和遗传小鼠模型的研究结果。 如果该项目成功，我们将能够建立一套新的高通量平台，用于研究 通过测量细胞手性来研究不对称心脏循环的生物物理学，并进一步了解 先天性心脏病此外，这项拟议的研究是变革性的，并可能开辟一个新的领域， 研究：细胞手性，一个基本的细胞特性，定义了组织发育中的对称性破缺。

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.