Körner Arlene, Mosqueira Matias, Hecker Markus, Ullrich Nina D
Division of Cardiovascular Physiology, Institute of Physiology and Pathophysiology, Heidelberg University, Heidelberg, Germany.
German Center for Cardiovascular Research (DZHK), Partner Site Heidelberg-Mannheim, Heidelberg, Germany.
Front Physiol. 2021 Aug 19;12:710619. doi: 10.3389/fphys.2021.710619. eCollection 2021.
Novel treatment strategies for cardiac tissue regeneration are heading for the use of engineered cardiac tissue made from induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Despite the proven cardiogenic phenotype of these cells, a significant lack of structural and functional properties of mature myocytes prevents safe integration into the diseased heart. To date, maturation processes of cardiomyocytes remain largely unknown but may comprise biophysical cues from the immediate cell environment. Mechanosensing is one critical ability of cells to react to environmental changes. Accordingly, the surrounding substrate stiffness, comprised of extracellular matrix (ECM), cells, and growth surface, critically influences the myocyte's physiology, as known from deleterious remodeling processes in fibrotic hearts. Conversely, the mechanical properties during culture of iPSC-CMs may impact on their structural and functional maturation. Here, we tested the hypothesis that the environmental stiffness influences structural and functional properties of iPSC-CMs and investigated the effect of different substrate stiffnesses on cell contractility, excitation-contraction (EC) coupling, and intercellular coupling. Culture surfaces with defined stiffnesses ranging from rigid glass with 25GPa to PDMS of physiological softness were coated with ECM proteins and seeded with murine iPSC-CMs. Using confocal imaging, cardiac protein expression was assessed. Ca handling and contractile properties were analyzed on different substrate stiffnesses. Intercellular coupling gap junctions was investigated by fluorescence recovery after photobleaching (FRAP). Our data revealed greater organization of L-type Ca channels and ryanodine receptors and increased EC-coupling gain, demonstrating structural and functional maturation in cells grown on soft surfaces. In addition, increased shortening and altered contraction dynamics revealed increased myofilament Ca sensitivity in phase-plane loops. Moreover, connexin 43 expression was significantly increased in iPSC-CMs grown on soft surfaces leading to improved intercellular coupling. Taken together, our results demonstrate that soft surfaces with stiffnesses in the physiological range improve the expression pattern and interaction of cardiac proteins relevant for EC-coupling. In parallel, soft substrates influence contractile properties and improve intercellular coupling in iPSC-CMs. We conclude that the mechanical stiffness of the cell environment plays an important role in driving iPSC-CMs toward further maturation by inducing adaptive responses.
心脏组织再生的新型治疗策略正朝着使用由诱导多能干细胞衍生的心肌细胞（iPSC-CMs）制成的工程化心脏组织发展。尽管这些细胞已被证实具有心脏发生表型，但成熟心肌细胞在结构和功能特性上的显著缺乏阻碍了其安全整合到患病心脏中。迄今为止，心肌细胞的成熟过程在很大程度上仍然未知，但可能包括来自紧邻细胞环境的生物物理线索。机械传感是细胞对环境变化做出反应的一种关键能力。因此，由细胞外基质（ECM）、细胞和生长表面组成的周围底物硬度会严重影响心肌细胞的生理功能，这在纤维化心脏的有害重塑过程中是已知的。相反，iPSC-CMs培养过程中的机械性能可能会影响其结构和功能成熟。在此，我们测试了以下假设：环境硬度会影响iPSC-CMs的结构和功能特性，并研究了不同底物硬度对细胞收缩性、兴奋-收缩（EC）偶联和细胞间偶联的影响。用ECM蛋白包被硬度从25GPa的刚性玻璃到生理柔软度的聚二甲基硅氧烷（PDMS）不等的具有确定硬度的培养表面，并接种小鼠iPSC-CMs。使用共聚焦成像评估心脏蛋白表达。在不同底物硬度上分析钙处理和收缩特性。通过光漂白后荧光恢复（FRAP）研究细胞间偶联间隙连接。我们的数据显示L型钙通道和兰尼碱受体的组织性更强，并且EC偶联增益增加，这表明在柔软表面上生长的细胞具有结构和功能成熟。此外，缩短增加和收缩动力学改变表明在相平面环中肌丝钙敏感性增加。而且，在柔软表面上生长的iPSC-CMs中连接蛋白43的表达显著增加，导致细胞间偶联得到改善。综上所述，我们的结果表明，生理范围内硬度的柔软表面可改善与EC偶联相关的心脏蛋白的表达模式和相互作用。同时，柔软底物会影响收缩特性并改善iPSC-CMs中的细胞间偶联。我们得出结论，细胞环境的机械硬度通过诱导适应性反应在驱动iPSC-CMs进一步成熟方面起着重要作用。
