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（CHDs）是婴儿和幼儿死亡的主要原因，患有先天性膈疝和相关CHDs（特别是左心发育不全（LHH））的儿童死亡率很高。主要的假设是，冠心病是由导致左心血流改变的机械因素引起的，例如，突出胸腔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复后趋于正常化，进一步暗示机械负荷改变在某些形式的冠心病中的作用。与正常心脏相比，LHH的心肌弹力蛋白和前胶原基因表达下降，左心室功能下降，这表明心肌的ECM组成和力学性能明显偏离正常。考虑到ECM的特性可以影响细胞的多种功能，包括增殖、分化和表型，在冠心病的进展过程中，ECM的正常组成和硬度的任何偏离都会对心肌细胞产生重大影响，导致心肌的发育和功能发生深刻的改变。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成分、硬度和/或机械应变的变化，我们将能够引导胚胎肌细胞向LHH表型发展，同时模拟健康的胚胎心脏环境将导致正常的肌细胞发育。在目的1中，我们将在发育中的大鼠胚胎中使用硝芬诱导的先天性膈疝来产生冠心病模型。健康和患病的心脏从胚胎和胎儿阶段将进行脱细胞，以获得心脏ECM，将测定任何成分的差异和机械刚度的变化。在这些相同的生命阶段，我们将描述健康和冠心病心脏的天然心肌细胞增殖和成熟。然后，我们将使用ecm包被的聚丙烯酰胺（PA）凝胶系统，在合理改变的2D环境中培养从健康和冠心病心室分离的胚胎/胎儿肌细胞，模拟健康和患病生物物理特性的不同组合，并评估硬度和成分是协同作用还是拮抗作用，以及“健康”的生物物理提示是否可以驱动“患病”的肌细胞走向健康的表型。在目的2中，我们将使胚胎和胎儿肌细胞受到不同振幅和频率的机械应变，并评估来自健康和冠心病心脏的肌细胞的增殖、成熟和功能。这些研究是新颖的，将是评估子宫内生长过程中改变的生物物理和生物力学信号在心脏病理发展中的作用的首批实验之一。

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.