Engineered Early Embryonic Cardiac Tissue

描述(申请人提供)：我们利用在心脏形态发生期间分离的胚胎大鼠心肌细胞，开发了一种名为EEECT的工程化早期胚胎心脏组织，以研究未成熟心肌细胞(CM)增殖和分化的调节，并生成具有最佳心脏修复性能的可移植组织。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获得了出生后的表型(减少了增殖和增加了力量产生)。初步数据显示，将EEECT植入损伤的成年大鼠左心室存活，并改善左心室的舒缩功能。在这个修订的R01-A2提案中，我们确定了调控EEECT CM增殖的分子途径，并测试了我们的范例，即EEECT是心脏修复的最佳富含CM的组织结构。具体目标1：确定从胎儿CM表型向出生后CM表型转变过程中调控EEECT CM增殖的分子途径。我们推测，CM的增殖和生长调控包括两个同步的过程：(1)CM的增殖通过p38丝裂原激活的蛋白激酶(P38MAPK)和Akt扩大心肌质量；(2)CM的肌节成熟和生长通过整合素连接的激酶(ILK)、p38MAPK和Akt实现。我们将使用我们的体外EEECT模型(由ED14胚胎大鼠心肌细胞产生)来确定(1)周期性机械负荷；(2)刺激或阻断ILK、p38MAPK和Akt；以及(3)甲状腺激素治疗对CM增殖和表型转换的影响。EEECT的治疗后特征将包括：(1)CM增殖、分化和凋亡的组织学测量；(2)生物力学特性；(3)蛋白质含量和激酶活性；(4)全基因组RNA转录表达模式的变化。具体目的2：确定EEECT植入受损成人心肌后的命运，以及EEECT对受体心肌功能恢复和重塑的作用。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缩功能的恢复；(3)形成血管。我们在心肌梗死后2周将GFP+转基因大鼠EEECT植入同种异体大鼠的LV。3天、2周和8周的结果评估包括左心室功能测定和细胞增殖、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，在体外促进EEECT CM增殖的治疗将增加体内EEECT CM的存活率和功能恢复。公共卫生相关性：我们建议的实验首先确定在利用组织培养和未成熟心肌细胞产生的工程化早期胚胎心脏组织(EEECT)中调节心肌细胞增殖和成熟的途径。然后，作为心脏修复和再生策略的一部分，我们评估将EEECT植入受损成人心脏(心肌梗死)表面的成功情况。

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.