The role of the extracellular biophysical and biomechanical milieu in CHDs

描述（由申请人提供）：先天性心脏缺陷（CHD）是婴幼儿死亡的主要原因，患有先天性疝和相关CHD（特别是左心发育不全（LHH））的婴幼儿死亡率较高。主要假设是CHD是由导致左心血流改变的机械因素引起的，例如突出到胸腔中的内脏结构对左心的压迫。在轻度LHH中，左心室尺寸在出生和疝修补后趋于正常化，进一步暗示了机械负荷改变在某些形式的CHD中的作用。与正常心脏相比，LHH中心脏弹性蛋白原和前胶原基因表达降低以及左心室功能降低表明发生了与正常ECM组成和心肌机械性质的显著偏离。鉴于ECM特性可影响各种细胞功能，包括增殖、分化和表型，CHD进展中ECM的正常组成和硬度的任何偏离将对肌细胞产生显著影响，导致心肌发育和功能的深刻改变。此外，通过发育中的心脏的血流改变会影响心室壁的机械应变，研究表明机械应变的变化会显著影响细胞功能。我们假设通过模拟与LHH相关的ECM组成、硬度和/或机械应变的变化，我们将能够引导胚胎肌细胞朝向LHH表型，同时模拟健康的胚胎心脏环境将导致正常的肌细胞发育。目的一：利用杀螟松诱导大鼠胚胎先天性腹股沟疝，建立大鼠先天性心脏病模型。来自胚胎和胎儿阶段的健康和患病心脏将经历脱细胞化以获得心脏ECM，将测定心脏ECM以确定任何组成差异和机械刚度的改变。在这些相同的生命阶段，我们将表征健康和CHD心脏中的天然肌细胞增殖和成熟。然后，我们将培养从健康和CHD心室分离的胚胎/胎儿肌细胞，在合理改变的2D环境中，使用ECM涂覆的聚丙烯酰胺（PA）凝胶系统模拟健康和患病生物物理特性的不同组合，并评估刚度和组成是否协同或拮抗作用，以及“健康”生物物理线索是否可以驱动“患病”肌细胞向健康表型发展。在目标2中，我们将使胚胎和胎儿心肌细胞经受不同幅度和频率的机械应变，并评估来自健康和CHD心脏的心肌细胞的增殖、成熟和功能。这些研究是新颖的，并将代表第一个实验，以评估改变的生物物理和生物力学信号在子宫内生长期间心脏病理学发展的作用之一。

DESCRIPTION (provided by applicant): Congenital heart defects (CHDs) are the leading cause of death in infants and young children and those suffering from congenital diaphragmatic hernia and associated CHDs (particularly with left heart hypoplasia (LHH)), have high mortality. The dominant hypothesis is that the CHDs result from mechanical factors leading to altered blood flow in the left heart, such as compression of the left heart by visceral structures protruding into the thoracic cavity. In mild LHH, left ventricular dimensions tend to normalize after birth and hernia repair, further implicating the role of altered mechanical loads in certain forms of CHD. Decreased cardiac tropoelastin and procollagen gene expression and decreased left ventricular function in LHH compared to normal hearts suggest that significant deviations from normal ECM composition and mechanical properties of the myocardium occur. Given that ECM properties can affect various cell functions including proliferation, differentiation, and phenotype, any deviations from the normal composition and stiffness of the ECM in the progression of CHD would have a significant impact on the myocytes, resulting in profound alterations to the development and function of the myocardium. Additionally, altered flow through the developing heart will affect mechanical strain in the ventricular wall, and studies have demonstrated that changes in mechanical strain can significantly impact cell function. We hypothesize that by mimicking changes in ECM composition, stiffness, and/or mechanical strain associated with LHH, we will be able to guide embryonic myocytes towards the LHH phenotype while mimicking the healthy embryonic heart environment will lead to normal myocyte development. In Aim 1 we will use nitrofen-induced congenital diaphragmatic hernia in developing rat embryos to generate models of CHD. Healthy and diseased hearts from embryonic and fetal stages will undergo decellularization to obtain cardiac ECM which will be assayed to determine any compositional differences and alterations in mechanical stiffness. At these same life stages, we will characterize native myocyte proliferation and maturation in healthy and CHD hearts. We will then culture embryonic/fetal myocytes isolated from healthy and CHD ventricles in rationally altered 2D environments that mimic different combinations of healthy and diseased biophysical properties using an ECM-coated polyacrylamide (PA) gel system and assess whether stiffness and composition act synergistically or antagonistically, and whether "healthy" biophysical cues can drive "diseased" myocytes towards a healthy phenotype. In Aim 2, we will subject embryonic and fetal myocytes to mechanical strain of varying amplitude and frequency and assess the proliferation, maturation, and function of myocytes from healthy and CHD hearts. These studies are novel and will represent one of the first experiments to assess the role of altered biophysical and biomechanical signaling in the development of cardiac pathologies during growth in utero.