Identification of biophysical and paracrine factors governing electrical integration of cardiomyocytes into a functional syncytium

心肌细胞的合胞组织需要通过含有连接蛋白的间隙连接（GJ）进行密切的细胞-细胞接触。后者的构成通常在发育和患病的心脏中发生改变，引起心肌传导的生理和病理变化。控制gj组装的因素没有很好的定义，但可能包括生物物理刺激和生长因子。在体内和体外标准单层培养中评估gj组装和功能的机制，由于前者的生理复杂性和后者模型中心肌细胞成熟程度较低而受到混淆。在工程心脏组织（EHT）中，心肌细胞重新获得典型的杆状形态，并形成含有GJs的明显规则的端到端接触，本质上产生了各向异性组织的合胞体。因此，我们将使用EHT作为心肌发育的简化模型，以确定机械、电和旁分泌刺激对心肌细胞合胞组织的作用。此外，我们假设增强的合胞排列将改善EHT的收缩性能、电稳定性和体内整合。在确定大鼠EHT中gj的构成后，我们将利用一种新的基于胚胎干细胞（ESC）的组织工程概念和稳定表达的钙和电压传感器蛋白来详细了解gj的组装、维持和功能机制。基因工程esc - eht的高分辨率光学成像将允许对电建模和传导特性进行时间过程分析，以及在体外直接识别因素-原因关系。我们的最终目标是生成人类钙/电压传感器esc - eht，用于体外药物筛选和体内心脏修复。

Syncytial organization of cardiomyocytes requires intimate cell-cell-contacts through connexin-containing gap junctions (GJ). Constitution of the latter is commonly altered in developing and diseased hearts causing physiological and pathological changes of myocardial conduction. Factors controlling GJ-assembly are not well defined, but likely include biophysical stimuli and growth factors. Assessing mechanisms of GJ-assembly and -function in vivo and in standard monolayer cultures in vitro is confounded by physiological complexity of the former and low cardiomyocyte maturation in the latter model. In engineered heart tissue (EHT) myocytes regain a characteristic rod-shaped morphology and form apparently regular end-to-end contacts containing GJs, essentially generating an anisotropically organized syncytium. Thus, we will use EHT as a simplified model of heart muscle development to identify the role of mechanical, electrical, and paracrine stimuli on syncytial organization of cardiomyocytes. In addition, we hypothesize that enhanced syncytial arrangement will improve contractile performance, electrical stability, and in vivo integration of EHT. After identification of GJ-constitution in rat EHT, we will take advantage of a novel embryonic stem cell (ESC)-based tissue engineering concept and of stably expressed calcium- as well as voltage-sensor proteins to gain detailed insight into mechanisms of GJ-assembly, -maintenance, and -function. High resolution optical imaging of genetically engineered ESC-EHTs will allow time-course analyses of electrical modelling and conduction properties as well as direct identification of factor-cause relationships in vitro. We ultimately aim at generating human calcium/voltage-sensor ESC-EHTs for potential applications in drug screening in vitro and cardiac repair in vivo.