Fibrin defines tissue stiffness and biomechanical signalling in regenerating zebrafish hearts as revealed by high-resolution stiffness mapping

The infarction of the human heart leads to irreversible scar formation, which permanently impairs its mechanical properties and physiological functions. Interestingly, the zebrafish heart is capable of resolving scar tissue and regenerating injured myocardium, offering a unique opportunity to study the interplay between scar tissue and cardiac mechanical properties during regeneration. In this study, we developed a novel method combining atomic force microscope-based nanoindentation with confocal microscopy to generate an elasticity map of the zebrafish heart. This high-resolution mapping revealed distinct regions of stiffness within the injury site, despite the overall global mechanical properties remaining unaffected. Specifically, we identified a stiff area that is cell-poor and fibrin-rich, contrasting with the center of injury, which is as soft as the surrounding myocardium. Extensive whole transcriptome analyses uncovered several components of the coagulation and fibrinolysis cascade in the regenerating heart. Pharmacological inhibition of Serpine1, a regulator of fibrinolysis, demonstrated that reduced fibrin-mediated stiffness impacts the activation of biomechanical Hippo pathway signaling in adjacent endocardial cells. Our approach characterizes the mechanical properties of different regions in the zebrafish ventricle, highlighting fibrin as the stiffest element influencing biomechanical signaling. This suggests it may play a structural role in the regeneration process. The methodology presented here offers a detailed view of the biomechanical environment and mechanotransductive signaling in regenerating cardiac tissue, which is crucial for understanding regenerative mechanisms. Overall design: RNAseq profiling of apexes of zebrafish hearts. Unijured hearts or hearts upon 1.5, 3, 7 and 14 days post cryoinjury were taken.

人类心肌梗死会形成不可逆的瘢痕组织，永久损害其机械特性与生理功能。值得关注的是，斑马鱼心脏能够清除瘢痕组织并再生受损心肌组织，为研究再生过程中瘢痕组织与心脏机械特性间的相互作用提供了独特契机。本研究开发了一种全新方法：将基于原子力显微镜（atomic force microscope）的纳米压痕技术与共聚焦显微镜（confocal microscopy）相结合，以绘制斑马鱼心脏的弹性分布图。该高分辨率成像揭示了损伤区域内存在不同刚度的区域，尽管心脏整体的机械特性并未发生改变。具体而言，我们发现了一个细胞稀少且富含纤维蛋白的硬区域，与损伤中心形成鲜明对比——损伤中心的硬度与周围心肌组织相当。全面的全转录组分析在再生心脏中鉴定出了凝血与纤溶级联反应的多个组分。对纤溶调节因子丝氨酸蛋白酶抑制剂E1（Serpine1）进行药物抑制实验后证实：纤维蛋白介导的刚度降低会影响相邻心内膜细胞内生物力学Hippo通路（Hippo pathway）信号的激活。本研究方法表征了斑马鱼心室不同区域的机械特性，明确纤维蛋白是影响生物力学信号的最主要刚性组分，这提示纤维蛋白可能在再生过程中发挥结构性作用。本研究提出的方法能够详细展现再生心脏组织中的生物力学环境与机械转导信号通路，这对于解析再生机制至关重要。实验整体设计：对斑马鱼心脏心尖组织进行RNA测序（RNA-seq）转录组分析。采集未损伤心脏，以及冷冻损伤后1.5天、3天、7天和14天的心脏组织样本。