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.

在脊椎动物正常的心脏发育期间，胚胎心脏开始时是一根直管，沿着 胚胎的中线。随后，它会变成一个c形的心环，可靠地朝向 身体。这种心脏c-环是左-右不对称破裂的最早明显事件(也称为 手性或手性)人体器官。心脏的反向偏侧化(即向左侧) 瓣环的形成往往会导致严重的临床后果，包括右位心、室间隔缺损、右双出口。 脑室，甚至胎儿和婴儿的死亡。在过去，我们已经能够概括多细胞 体外手性形态发生实验表明，从鸡心脏中提取的心肌细胞在环化前有一种 固有的向右倾斜。此外，蛋白激酶C激活物，新发现的心肌细胞手性调节因子，可以 反转小鸡心脏c型环。在资助的R01拨款(HL148104)中，我们建议研究如何内在 细胞手性从生物化学和生物力学的角度决定了心脏的不对称环路。我们现在请求一个 通过使用人类心脏来解决审查者的关切(与人类疾病相关)的研究补充 并研究平面细胞极性(PCP；也称为非规范WNT)信号的作用，a 因心脏偏侧缺陷而闻名的通路。我们假设PCP调节多细胞的比例 手性形态发生与心脏c-环。为了检验这一假设，我们建议评估 PCP蛋白，并评估PCP蛋白在体外形成2D多细胞手性模式和体外实验中的作用 雏鸡胚胎发育中的心脏环状。具体地说，我们将首先评估极性和手性 图案化人类心肌细胞中的PCP标记物。我们将在2D中对心肌细胞进行建模，检查位置 和PCP标志物的形态，并确定PCP信号如何生物力学调节手性 形态发生。然后，我们将确定PCP信号在鸡心脏循环中的作用。和前女友在一起 卵子培养，我们将评估PCP蛋白在环化阶段的定位，干扰PCP 信令以检查PCP的影响，并使用基于单元顶点的数值模拟来评估 PCP信号在心脏不对称环路中的生物力学作用。总的来说，拟议的研究将 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。