Administrative support to R01 HL148104: Understanding Cardiac C-Looping Using Microscale In Vitro Models

R01 HL148104 的行政支持：使用微型体外模型了解心脏 C 环

• 批准号: 10630645
• 负责人: Leo Q. Wan
• 金额: $ 6.78万
• 依托单位: RENSSELAER POLYTECHNIC INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10630645
• 关键词: Address; African American; Anterior; Biochemical; Biological Sciences; Biomechanics; Biophysical Process; Cardiac; Cardiac Myocytes; Cardiac development; Cells; Chick; Chick Embryo; Clinical; Collecting Tube; Defect; Development; Dextrocardia; Disease; Double Outlet Right Ventricle; Embryo; Embryonic Development; Embryonic Heart; Event; Fetal Death; Funding; Grant; Handedness; Health; Heart; Human; In Vitro; Individual; Infant; Institutes; Knowledge; Left; Location; Morphogenesis; Morphology; Myocardium; Myosin Type II; N-Cadherin; Organ; Outcome; Pathway interactions; Pattern; Pharmaceutical Preparations; Protein Kinase C; Proteins; Publications; Research; Research Support; Role; Side; Signal Transduction; Surface; Testing; Travel; Tube; United States National Institutes of Health; Vertebrates; WNT Signaling Pathway; base; cardiogenesis; cost; genetic approach; graduate student; human disease; immunocytochemistry; in vitro Model; planar cell polarity; septal defect; simulation; small molecule

During normal cardiac development of vertebrates, the embryonic heart starts as a straight tube along the midline of the embryo. It subsequently transforms into a c-shaped heart loop reliably toward the right side of the body. This cardiac c-looping is the earliest evident event of left-right asymmetry breaking (also called chirality or handedness) of a human organ. The inversed lateralization (i.e., toward the left side) of cardiac looping often leads to severe clinical outcomes, including dextrocardia, septum defects, double outlet right ventricle, and even death of fetuses and infants. In the past, we have been able to recapitulate multicellular chiral morphogenesis in vitro and shown that cardiac cells from chick hearts right before looping have an intrinsic rightward bias. Also, protein kinase C activators, newly identified regulators of cardiac cell chirality, can reverse chick cardiac c-looping. In the funded R01 grant (HL148104), we proposed to investigate how intrinsic cell chirality determines asymmetric cardiac looping biochemically and biomechanically. We now request a research supplement to address a Reviewers’ concern (relevance to human diseases) by using human cardiac cells and investigating the role of the planar cell polarity (PCP; also called noncanonical wnt) signaling, a pathway known for cardiac laterality defects. We hypothesize that PCP regulates the scale of multicellular chiral morphogenesis and cardiac c-looping. To test the hypothesis, we propose to evaluate the expression of PCP proteins and assess the role of PCP proteins in forming 2D in vitro multicellular chiral patterns and ex ovo looping hearts in chick embryonic development. Specifically, we will first assess the polarity and chirality of PCP markers in patterned human cardiac cells. We will pattern cardiomyocytes in 2D, examine the location and morphology of the PCP markers, and determine how PCP signals biomechanically regulate chiral morphogenesis. Then, we will determine the role of PCP signaling in chick cardiac c looping. With the chick ex ovo culture, we will evaluate the localization of PCP proteins during the looping stages, interfere with PCP signaling to examine the effects of PCP, and use cell vertex-based numerical simulation to assess the biomechanical role of PCP signaling in the asymmetric heart looping. Overall, the proposed research will delineate the role of PCP signaling in chiral multicellular morphogenesis and in early cardiac asymmetric looping.

在脊椎动物的正常心脏发育期间，胚胎心脏沿着沿着脊椎动物的直立管开始 胚胎的中线。随后，它可靠地转换为C形心态循环 身体。这种心脏C循环是左右不对称破坏的最早证据事件（也称为 人体器官的手性或惯用性。心脏的反向偏侧化（即左侧） 循环通常会导致严重的临床结果，包括右心脏，隔膜缺陷，双插座右 心室，甚至是胎儿和婴儿的死亡。过去，我们能够概括多细胞 手性形态发生在体外，并表明循环前的雏鸡心脏的心脏细胞具有 内在的右偏见。此外，蛋白激酶C激活剂是心脏细胞手性的新确定的调节剂，可以 反向雏鸡心脏C循环。在资助的R01赠款（HL148104）中，我们提出了研究固有的方式 细胞手性在生物力学上决定了不对称的心脏循环。我们现在要求 通过使用人类心脏的研究补充，以解决审稿人的关注（与人类疾病有关） 细胞并研究平面细胞极性（PCP;也称为非规范Wnt）信号的作用，A 以心脏侧向缺陷而闻名的途径。我们假设PCP调节多细胞的规模 手性形态发生和心脏C环。为了检验假设，我们建议评估 PCP蛋白质并评估PCP蛋白在形成2D体外多细胞手性模式和EVO的作用 在雏鸡胚胎发育中循环心脏。具体而言，我们将首先评估 图案化的人类心脏细胞中的PCP标记。我们将在2D中对心肌细胞进行模式，检查位置 PCP标记的形态和形态，并确定PCP信号如何通过生物力学调节手性 形态发生。然后，我们将确定PCP信号在雏鸡心脏C循环中的作用。与小鸡前 OVO培养，我们将评估循环阶段中PCP蛋白的定位，干扰PCP 信号传导检查PCP的效果，并使用基于细胞顶点的数值模拟来评估 PCP信号在不对称心脏循环中的生物力学作用。总体而言，拟议的研究将 描述PCP信号在手性多细胞形态发生和早期心脏不对称中的作用 循环。