Engineered Early Embryonic Cardiac Tissue

描述（由申请人提供）：我们开发了一种工程早期胚胎心脏组织，称为EEECT，使用在心脏形态发生期间分离的胚胎大鼠心脏细胞，以研究未成熟心肌细胞（CM）增殖和分化的调节，并产生具有最佳心脏修复性能的可植入组织。我们的EEECT结构保留了未成熟CM独特的增殖和收缩特性，包括对循环机械拉伸的增殖反应。随着培养时间的延长，EEECT获得了产后表型（增殖减少，产力增加）。初步结果表明，在成年大鼠损伤左室植入EEECT后，其存活并能改善左室舒张和收缩功能。在这个修订后的R01-A2提案中，我们确定了调节EEECT CM增殖的分子途径，并验证了我们的范式，即EEECT是心脏修复的最佳富含CM的组织结构。特异性目的1：确定从胎儿到出生后CM表型转变过程中调节EEECT CM增殖的分子途径。我们假设CM增殖和生长的调控涉及两个并行过程：(1)CM增殖通过p38丝裂原活化蛋白激酶（p38MAPK）和Akt扩大心肌质量；(2)通过整合素连接激酶（ILK）、p38MAPK和Akt介导非增殖性CM肉瘤成熟和生长。我们将使用体外EEECT模型（由ED 14胚胎大鼠心脏细胞产生）来确定(1)循环机械负荷的影响；(2)刺激或阻断ILK、p38MAPK和Akt；(3)甲状腺激素治疗对CM增殖及表型转变的影响。EEECT治疗后的特征将包括：(1)CM增殖、分化和凋亡的组织学测量；(2)生物力学性能；(3)蛋白质含量和激酶活性；(4) RNA转录物表达模式的全基因组变化。特异性目的2：确定EEECT植入损伤的成人心肌后的命运，以及EEECT对受体心肌功能恢复和重构的贡献。我们假设植入的EEECT(1)显示持续的CM增殖和有限的细胞死亡；(2)积极促进梗死心肌舒张、收缩功能恢复；(3)血管化。我们在心肌梗死后2周将GFP+转基因大鼠EEECTs植入同基因大鼠LV。3天、2周和8周的结果分析包括左室功能分析和细胞增殖、细胞表型、细胞-细胞偶联和血管形成的组织学特征。我们进一步假设，增加EEECT CM增殖的体外治疗将增加体内EEECT CM的存活和功能恢复。公共卫生相关性：我们提出的实验首先确定了在使用组织培养和未成熟心脏细胞产生的工程早期胚胎心脏组织（EEECT）中调节心肌细胞增殖和成熟的途径。然后，我们评估了作为心脏修复和再生策略的一部分，在受伤的成人心脏（心肌梗死）表面植入EEECT的成功。

DESCRIPTION (provided by applicant): We developed an Engineered Early Embryonic Cardiac Tissue, termed EEECT, using embryonic rat cardiac cells isolated during the period of cardiac morphogenesis, in order to investigate the regulation of immature cardiomyocyte (CM) proliferation and differentiation and to generate implantable tissues with optimal properties for cardiac repair. Our EEECT construct preserves the unique proliferative and contractile properties of immature CM, including a proliferative response to cyclic mechanical stretch. With prolonged culture EEECT acquires a post-natal phenotype (reduced proliferation and increased force production). Preliminary data show that EEECT implanted onto injured adult rat LV survive and improve LV diastolic and systolic function. In this revised R01-A2 proposal we identify molecular pathways that regulate EEECT CM proliferation and test our paradigm that EEECT is an optimal CM-rich tissue construct for cardiac repair. Specific Aim 1: Define molecular pathways that regulate EEECT CM proliferation during the transition from fetal to post-natal CM phenotype. We hypothesize that the regulation of CM proliferation and growth involves 2 concurrent processes: (1) CM proliferation to expand myocardial mass via p38 mitogen-activatedprotein kinase (p38MAPK) and Akt; and (2) CM sarcomere maturation and growth of non-proliferating CM via integrin-linked kinase (ILK), p38MAPK, and Akt. We will use our in vitro EEECT model (generated from ED 14 embryonic rat cardiac cells) to define the impact of (1) cyclic mechanical loading; (2) stimulation or blockade of ILK, p38MAPK, and Akt; and (3) thyroid hormone treatment on CM proliferation and the phenotypic transition. Post-treatment characterization of EEECT will include: (1) histologic measures of CM proliferation, differentiation, and apoptosis; (2) biomechanical properties; (3) protein content and kinase activities; and (4) genome-wide changes in RNA transcript expression patterns. Specific Aim 2: Determine the fate of EEECT following implantation onto injured adult myocardium and the contribution of EEECT to recipient myocardial functional recovery and remodeling. We hypothesize implanted EEECT (1) display sustained CM proliferation and limited cell death; (2) positively contribute to the diastolic and systolic functional recovery of infarcted myocardium; and (3) become vascularized. We implant GFP+ transgenic rat EEECTs onto syngenic rat LV 2 weeks following myocardial infarction. Outcome assays at 3 days, 2 weeks, and 8 weeks include LV functional assay and histologic characterization of cell proliferation, cell phenotype, cell-cell coupling, and vascularization. We further hypothesize that in vitro treatments that increase EEECT CM proliferation will increase in vivo EEECT CM survival and functional recovery. PUBLIC HEALTH RELEVANCE: Our proposed experiments first identify pathways that regulate cardiomyocyte proliferation and maturation within an Engineered Early Embryonic Cardiac Tissue (EEECT) generated using tissue culture and immature cardiac cells. We then evaluate the success of EEECT implantation onto the surface of the injured adult heart (myocardial infarction) as part of a cardiac repair and regeneration strategy.