Towards repairing congenital heart defects: The effects of fetal cardiac extracel

描述(申请人提供)：先天性心脏病(CHD)是活产婴儿死亡的主要原因(1，2)。左心发育不全综合征(HLHS)是一种罕见的冠心病，需要几次重大的重建手术(3)。然而，重建的心脏不能复制正常的解剖和功能，许多患者终生遭受严重的继发性并发症(4)。心脏组织工程学在治疗HLHS方面特别有希望，因为它可以创造出可以与孩子一起生长的活的功能性心脏组织。然而，在体外创造功能性心脏组织仍然是一个主要的挑战，因为成熟的心肌细胞(CM)大多是非增殖的(5)。虽然胚胎和胎儿CMS高度增殖，可以恢复受损或患病心脏的功能(6，7)，但出生后CM增殖减少和心脏再生能力丧失的原因仍很不清楚。阐明促进CM增殖的因素将极大地影响组织工程学和再生治疗儿童冠心病的方法。细胞外基质(ECM)调节多种细胞功能，包括增殖(8)，而且有证据表明，ECM的组成和心脏的硬度在发育和成熟过程中发生变化(9-12)。该项目的假设是，重现胎儿ECM环境将促进CMS的增殖；具体地说，预计促进CM增殖的胎儿心脏ECM的特征是在成人心脏中丢失或消失的信号。这一假说将在体外进行系统的测试，以确定细胞外基质成分和硬度在2D和3D培养中的单独和联合影响，在体内，作为未来细胞外基质成分研究的临床转化的第一步，天然胎鼠和成年大鼠心脏将被脱细胞以获得细胞外基质，然后溶解并吸附到组织培养皿中。将原代新生大鼠CMS接种到心脏ECM包被的基质上，并检测其增殖情况。同时，还将确定衬底刚度的影响。脱细胞心脏和天然心脏将接受机械测试，以确定细胞外基质的硬度。聚丙烯酰胺(PAAM)凝胶的硬度将模仿胎儿和成人的心脏。在这些研究中，I型胶原将在胎儿和成人刚性凝胶中以相同的配体密度使用，以分离成分和刚性，以研究细胞外基质成分和硬度对CM增殖的联合影响，溶解的心脏细胞外基质将被加入到PAAM凝胶中。可导致最高CM增殖的ECM/刚性组合将用于指导可注射ECM生物材料的设计。3D凝胶将在体外进行表征和优化，并将揭示2D和3D培养对CM增殖的不同影响。体外细胞存活率、细胞浸润和增殖能力最强的ECM凝胶将在体内测试其对新生大鼠CM增殖的刺激作用。这项工作的结果将通过开发和表征基于ECM的生物材料来促进CM的增殖和心脏组织的再生，从而对未来的心脏组织工程方法产生重大影响。 公共卫生相关性：该项目的长期目标是为患有先天性心脏病的儿童开发新的心脏组织。本项目将研究胎儿心脏基质对新生儿心肌细胞(心脏细胞)生长的影响。未来，胚胎动物心脏基质可能会被用来在实验室中生长新的心脏组织，或者可以被植入心脏，以促进人类新组织的生长。

DESCRIPTION (provided by applicant): Congenital heart defects (CHD) are the leading cause of mortality in live-born infants (1, 2). Hypoplastic Left Heart Syndrome (HLHS) is a rare CHD that requires several major reconstructive surgeries (3). However, the reconstructed heart does not replicate normal anatomy and function, and many patients suffer from serious secondary complications throughout life (4). Cardiac tissue engineering is especially promising for treating HLHS by creating living functional heart tissue that can grow with the child. However, creating functional heart tissue in vitro remains a major challenge, as mature cardiomyocytes (CMs) are mostly non-proliferative (5). While embryonic and fetal CMs are highly proliferative and can restore function in damaged or diseased hearts (6, 7), the causes of decreased CM proliferation and loss of cardiac regenerative capacity after birth are still largely unknown. Elucidating factor that promote CM proliferation will greatly impact tissue engineering and regenerative approaches to treating CHD in children. The extracellular matrix (ECM) modulates a variety of cell functions, including proliferation (8), and there is evidence that ECM composition and stiffness of the heart change during development and maturation (9-12). The hypothesis of this project is that recapitulation of the fetal ECM environment will promote the proliferation of CMs; specifically, it is expected that the features of fetal cardiac ECM that promote CM proliferation are the cues that are lost or diminished in the adult heart. The hypothesis will be systematically tested in vitro, to determine the individual and combined effects of ECM composition and stiffness in 2D and 3D culture, and in vivo, as an initial step towards future clinical translation For ECM composition studies, native fetal and adult rat hearts will be decellularized to obtain ECM and then solubilized and adsorbed onto tissue culture dishes. Primary neonatal rat CMs will be seeded onto cardiac ECM-coated substrates and assayed for proliferation. In parallel, the effects of substrate stiffness will be determined. Decellularized and native hearts will undergo mechanical testing to determine the stiffness of the ECM. Polyacrylamide (PAAm) gels will be made with stiffnesses that mimic fetal and adult hearts. In these studies, Collagen I will be used at the same ligand density in fetal vs. adult stiffness gels to decouple composition from stiffness To investigate the combined effects of ECM composition and stiffness on CM proliferation, solubilized cardiac ECM will be incorporated into PAAm gels. The ECM/stiffness combination that results in highest CM proliferation will be used to guide the design of an injectable ECM-based biomaterial. The 3D gel will be characterized and optimized in vitro and will reveal whether 2D vs. 3D culture has different effects on CM proliferation. The ECM gel which results in greatest cell viability, infiltration, and proliferation in vitro will be tested in vivo for itseffects on stimulating CM proliferation in neonatal rats. The results of this work will significantly impact future cardiac tissue engineering approaches through the development and characterization of ECM-based biomaterials that promote CM proliferation and regeneration of cardiac tissue. PUBLIC HEALTH RELEVANCE: The long-term goal of this project is to develop new heart tissue for children suffering from congenital heart defects. This project will investigate the effets of fetal heart matrix on neonatal cardiomyocyte (heart cell) growth. Fetal animal heart matrix could potentially be used in the future to grow new heart tissue in the lab or could be implanted in the heart to promote new tissue growth in humans.