Abstract：
Cardiac fibrosis occurs following insults to the myocardium and is characterized by the abnormal accumulation of non-compliant extracellular matrix (ECM), which compromises cardiomyocyte contractile activity and eventually leads to heart failure. This phenomenon is driven by the activation of cardiac fibroblasts (cFbs) to myofibroblasts and results in changes in ECM biochemical, structural and mechanical properties. The lack of predictive in vitro models of heart fibrosis has so far hampered the search for innovative treatments, as most of the cellular-based in vitro reductionist models do not take into account the leading role of ECM cues in driving the progression of the pathology. Here, we devised a single-step decellularization protocol to obtain and thoroughly characterize the biochemical and micro-mechanical properties of the ECM secreted by activated cFbs differentiated from human induced pluripotent stem cells (iPSCs). We activated iPSC-derived cFbs to the myofibroblast phenotype by tuning basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 (TGF-β1) signalling and confirmed that activated cells acquired key features of myofibroblast phenotype, like SMAD2/3 nuclear shuttling, the formation of aligned alpha-smooth muscle actin (α-SMA)-rich stress fibres and increased focal adhesions (FAs) assembly. Next, we used Mass Spectrometry, nanoindentation, scanning electron and confocal microscopy to unveil the characteristic composition and the visco-elastic properties of the abundant, collagen-rich ECM deposited by cardiac myofibroblasts in vitro. Finally, we demonstrated that the fibrotic ECM activates mechanosensitive pathways in iPSC-derived cardiomyocytes, impacting on their shape, sarcomere assembly, phenotype, and calcium handling properties. We thus propose human bio-inspired decellularized matrices as animal-free, isogenic cardiomyocyte culture substrates recapitulating key pathophysiological changes occurring at the cellular level during cardiac fibrosis.
摘要：
心脏纤维化发生在心肌损伤后，其特征是非顺应性细胞外基质（ECM）的异常积累，这会损害心肌细胞的收缩活动，最终导致心力衰竭。这种现象是由心脏成纤维细胞（cFbs）激活为肌成纤维细胞，并导致ECM生化变化，结构和机械性能。迄今为止，缺乏预测心脏纤维化的体外模型阻碍了对创新治疗方法的探索，因为大多数基于细胞的体外还原模型没有考虑ECM线索在驱动病理进展中的主导作用。这里,我们设计了单步脱细胞方案,以获得并全面表征由人诱导多能干细胞(iPSCs)分化的活化cFbs分泌的ECM的生化和微机械特性.我们通过调节碱性成纤维细胞生长因子（bFGF）和转化生长因子β1（TGF-β1）信号，将iPSC衍生的cFbs激活为肌成纤维细胞表型，并证实激活的细胞获得了肌成纤维细胞表型的关键特征。像SMAD2/3核穿梭，形成对齐的α-平滑肌肌动蛋白（α-SMA）-丰富的应力纤维和增加的粘着斑（FAs）组装。接下来,我们用了质谱,纳米压痕，扫描电子和共聚焦显微镜来揭示丰富的特征成分和粘弹性，心肌成纤维细胞在体外沉积的富含胶原蛋白的ECM。最后,我们证明了纤维化ECM激活iPSC衍生的心肌细胞中的机械敏感性途径，影响它们的形状，sarcomere组件，表型,和钙处理性能。因此，我们提出了人类生物启发的脱细胞基质作为无动物，等基因心肌细胞培养基质概括了心脏纤维化过程中在细胞水平上发生的关键病理生理变化。
