Engineered Early Embryonic Cardiac Tissue

描述（由申请人提供）：我们使用在心脏形态发生期间分离的胚胎大鼠心脏细胞开发了工程化早期胚胎心脏组织，称为EEECT，以研究未成熟心肌细胞（CM）增殖和分化的调节，并产生具有心脏修复最佳特性的可植入组织。我们的EEECT结构保留了未成熟CM的独特增殖和收缩特性，包括对周期性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（增殖减少，力产生增加）。初步数据显示，EEECT植入到损伤的成年大鼠左心室存活，并改善左心室舒张和收缩功能。在这个修订的R 01-A2提案中，我们确定了调节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的组织构建体。具体目标1：确定从胎儿到出生后CM表型转变期间调节EEECT CM增殖的分子途径。我们假设CM增殖和生长的调节涉及两个同时发生的过程：（1）CM通过p38丝裂原活化蛋白激酶（p38 MAPK）和Akt增殖以扩大心肌质量;（2）CM肌节成熟和非增殖CM的生长通过整合素连接激酶（ILK），p38 MAPK和Akt。我们将使用我们的体外EEECT模型（由艾德14胚胎大鼠心脏细胞产生）来确定（1）周期性机械负荷;（2）ILK、p38 MAPK和Akt的刺激或阻断;和（3）甲状腺激素治疗对CM增殖和表型转变的影响。EEECT的治疗后表征将包括：（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;以及（4）RNA转录本表达模式的全基因组变化。具体目标二：确定EEECT植入受损成人心肌后的结局以及EEECT对受体心肌功能恢复和重塑的贡献。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）积极促进梗死心肌的舒张和收缩功能恢复;（3）血管化。心肌梗死后2周，我们将GFP+转基因大鼠EEECTs植入同系大鼠LV。第3天、第2周和第8周的结局测定包括LV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表征。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在使用组织培养和未成熟心脏细胞产生的工程化早期胚胎心脏组织（EEECT）内调节心肌细胞增殖和成熟的途径。然后，我们评估EEECT植入到受伤的成人心脏（心肌梗死）的表面作为心脏修复和再生策略的一部分的成功。

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.