Multiscale Analysis of Extracellular Matrix Remodeling in the Failing Heart

Rationale: Cardiac extracellular matrix (ECM) comprises a dynamic molecular network providing structural support to heart tissue function. Understanding the impact of ECM remodeling on cardiac cells during heart failure (HF) is essential to prevent adverse ventricular remodeling and restore organ functionality in affected patients. Objectives: We aimed to (i) identify consistent modifications to cardiac ECM structure and mechanics that contribute to HF and (ii) determine the underlying molecular mechanisms. Methods and Results: We first performed decellularization of human and murine ECM (dECM) and then analyzed the pathological changes occurring in dECM during HF by atomic force (AFM), two-photon microscopy, high-resolution 3D image analysis and computational fluid dynamics (CFD) simulation. We then performed molecular and functional assays in patient-derived cardiac fibroblasts (CFs) based on YAP-TEAD mechanosensing activity and collagen contraction assays. The analysis of HF dECM resulting from ischemic (IHD) or dilated cardiomyopathy (DCM), as well as from mouse infarcted tissue, identified a common pattern of modifications in their 3D topography. As compared to healthy heart, HF ECM exhibited aligned, flat and compact fiber bundles, with reduced elasticity and organizational complexity. At the molecular level, RNA sequencing of HF CFs highlighted the overrepresentation of dysregulated genes involved in ECM organization, or being connected to TGFß1, Interleukin-1, TNF-alpha and BDNF signaling pathways. Functional tests performed on HF CFs pointed at mechanosensor YAP as a key player in ECM remodeling in the diseased heart via transcriptional activation of focal adhesion assembly. Finally, in vitro experiments clarified pathological cardiac ECM prevents cell homing, thus providing further hints to identify a possible window of action for cell therapy in cardiac diseases. Conclusions: Our multi-parametric approach has highlighted repercussions of ECM remodeling on cell homing, CF activation and focal adhesion protein expression via hyper-activated YAP signaling during HF. Key words: Decellularization, extracellular matrix, dilated cardiomyopathy, ischemic heart disease, YAP, dilated cardiomyopathy, mechanobiology. Overall design: RNA sequencing of DCM CFs from 2 patients (DCMA and DCMB) and 1 healthy CFs overexpressing YAP (YAP 8SA). 1 Healthy CFs were used as control or reference for differential gene expression analysis. The experiment was perfomed in triplicate.

研究依据：心脏细胞外基质（extracellular matrix, ECM）是一类动态分子网络，为心脏组织功能提供结构支撑。阐明心力衰竭（heart failure, HF）进程中细胞外基质重塑对心肌细胞的影响，对于预防不良心室重塑、恢复患者的器官功能至关重要。
研究目标：本研究旨在（1）明确参与心力衰竭发生的心脏细胞外基质结构与力学特性的一致性改变；（2）阐明其潜在的分子机制。
方法与结果：本研究首先对人类与小鼠的细胞外基质进行脱细胞处理，获得脱细胞细胞外基质（decellularized ECM, dECM）；随后通过原子力显微镜（atomic force microscopy, AFM）、双光子显微镜、高分辨率三维图像分析及计算流体动力学（computational fluid dynamics, CFD）模拟，分析心力衰竭过程中脱细胞细胞外基质的病理变化。随后基于YAP-TEAD机械感知活性及胶原收缩实验，对患者来源的心脏成纤维细胞（cardiac fibroblasts, CFs）开展分子与功能学检测。对缺血性心脏病（ischemic heart disease, IHD）、扩张型心肌病（dilated cardiomyopathy, DCM）所致心力衰竭的脱细胞细胞外基质，以及小鼠梗死组织的脱细胞细胞外基质进行分析，结果发现其三维形貌存在共同的改变模式。与健康心脏相比，心力衰竭组的细胞外基质表现为排列规整、扁平致密的纤维束，弹性与结构复杂度均有所降低。在分子层面，对心力衰竭患者来源的心脏成纤维细胞进行RNA测序的结果显示，参与细胞外基质组织、或与转化生长因子β1（TGF-β1）、白细胞介素-1（Interleukin-1）、肿瘤坏死因子-α（TNF-α）及脑源性神经营养因子（BDNF）信号通路相关的异常表达基因显著富集。针对心力衰竭患者来源的心脏成纤维细胞的功能实验表明，机械感受器YAP通过转录激活黏着斑组装，是患病心脏中细胞外基质重塑的关键调控因子。最后，体外实验证实病理性心脏细胞外基质会抑制细胞归巢，为明确心脏疾病细胞治疗的潜在干预窗口提供了新的线索。
结论：本研究采用多参数分析方法，阐明了心力衰竭过程中，细胞外基质重塑可通过过度激活的YAP信号通路，影响细胞归巢、心脏成纤维细胞活化及黏着斑蛋白表达。
关键词：脱细胞处理，细胞外基质，扩张型心肌病，缺血性心脏病，YAP，扩张型心肌病，机械生物学。
实验设计：对2例扩张型心肌病患者来源的心脏成纤维细胞（DCMA与DCMB）以及1株过表达YAP的健康心脏成纤维细胞（YAP 8SA）进行RNA测序；以1株健康心脏成纤维细胞作为差异基因表达分析的对照或参照样本。本实验设置3次重复。