Structural remodeling and functional maturation of stem cell-derived cardiomyocytes in novel 3D microprinted scaffolds.

干细胞来源的心肌细胞为心肌修复带来了新的希望，并可能取代病变的心肌细胞来补偿衰竭心脏的功能损失。在目前的分化程度下，这些细胞呈现出相当不成熟的表型，与成年心肌细胞相比，细胞结构不明确，肌丝排列紊乱，钙处理效率低下，自发收缩活动，强烈的不成熟标志，导致钙信号脆弱和不稳定，有利于这些细胞中不希望的电活动的发展。过早的天然肌细胞的结构和功能缺陷限制了它们成功融入宿主心肌，因此在这些细胞被用于细胞治疗方法之前，需要新的策略来促进它们的成熟。最大的挑战是确定触发进一步心源性成熟的干细胞衍生心肌细胞的具体要求和参数。在这里，我们提出假设，心肌细胞形状影响功能，从而决定心脏特征。与成人心肌细胞的形状类似，我们设计了用于单细胞培养的新型矩形3D支架，以评估特定细胞形状和几何形状对心肌细胞功能的影响。在初步实验中，在长方体支架中重塑干细胞来源的心肌细胞诱导了肌丝的显著结构重组，并提高了钙处理的效率。基于这些令人兴奋的新数据，我们提出了两种因果关联的实验策略，以深入了解特定形状的细胞生长过程中发生的结构和功能变化。在第一个目标中，我们研究了导致结构重塑的确切触发机制，并试图回答是否需要与支架壁相互作用（3D的立方体生长）或仅仅是具有精确长轴形成的矩形细胞重新定向（2D生长）来启动观察到的强重塑过程。由外而内的信号分析可以进一步深入了解细胞结构和心脏特异性微结构域（如t小管和嵌入盘）形成的遗传控制。项目提案的第二部分旨在阐明通过结构重塑导致Ca处理增强的机制。特别是，将测试细胞微结构的变化影响钙内流和钙释放途径的相互作用的假设，钙内流和钙释放途径对干细胞来源的心肌细胞的兴奋-收缩耦合起着关键的控制作用。总的来说，这个项目的主要目标是确定导致稳定和有效的Ca信号传导的机制，以降低这些新型心肌细胞中致心律失常的电活动的风险，并使它们更适合未来的临床应用。

Stem cell-derived cardiomyocytes raise new hope for myocardial repair and may serve to replace diseased cardiomyocytes to compensate for functional loss in the failing heart. At the current degree of differentiation, these cells present a rather immature phenotype, characterized by undefined cellular structure and disordered myofilament arrangement compared to adult cardiomyocytes, inefficient Ca handling and spontaneous contractile activity, strong hallmarks of immaturity, which lead to fragile and instable Ca signaling and favor the development of undesired electrical activities in these cells. The structural and functional deficiencies reminiscent of premature native myocytes limit successful integration into host myocardium and call for new strategies to enhance maturation before these cells may be used in cell therapeutic approaches. The great challenge is to identify the specific requirements and parameters of stem cell-derived cardiomyocytes that trigger further cardiogenic maturation. Here, we propose the hypothesis that myocyte shape influences function, thereby determining cardiac features. In analogy to the shape of adult cardiomyocytes, we have designed novel rectangular-shaped 3D scaffolds for single cell culture to evaluate the influence of a particular cell shape and geometry on cardiomyocyte function. In preliminary experiments, reshaping stem cell-derived cardiomyocytes in cuboid scaffolds induced significant structural reorganization of the myofilaments and enhanced the efficiency of Ca handling. Based on these exciting new data, we propose two causally linked experimental strategies to provide deep insight into the structural and functional changes occurring upon cell growth in specific shapes. In the first aim, we investigate the exact trigger mechanisms that lead to structural remodeling and try to answer to the question whether interaction with scaffold walls (cuboidal growth in 3D) or just cellular reorientation in a rectangular shape with precise long axis formation (growth in 2D) are required to initiate the observed strong remodeling processes. Outside-in signaling analysis may provide further insight into the genetic control of cell architecture and formation of cardiac-specific microdomains, such as t-tubules and intercalated disks. The second part of the project proposal aims to elucidate the mechanisms that lead to enhanced Ca handling by structural remodeling. In particular the hypothesis will be tested that changes in cell microarchitecture influence the interaction of the Ca influx and Ca release pathways that critically control excitation-contraction coupling in stem cell-derived cardiomyocytes. Overall, the major goal of this project is to define the mechanisms that lead to stable and efficient Ca signaling in order to reduce the risk of arrhythmogenic electrical activity in these novel cardiomyocytes and to make them better suited for future clinical applications.