Members of the cellular communication network family (CCN) of matricellular proteins, like CCN1, have long been implicated in the regulation of cellular processes underlying wound healing, tissue fibrogenesis, and collagen dynamics. While many studies suggest antifibrotic actions for CCN1 in the adult heart through the promotion of myofibroblast senescence, they largely relied on exogenous supplementation strategies in models of cardiac injury where its expression is already induced—which may confound interpretation of its function in this process. The objective of this study was to interrogate the role of the endogenous protein on fibroblast function, collagen structural dynamics, and its associated impact on cardiac fibrosis after myocardial infarction (MI). Here, we employed CCN1 loss-of-function methodologies, including both siRNA-mediated depletion and fibroblast-specific knockout mice to assess the role of the endogenous protein on cardiac fibroblast fibrotic signaling, and its involvement in acute scar formation after MI. depletion of CCN1 reduced cardiac fibroblast senescence and proliferation. Although depletion of CCN1 decreased the expression of collagen processing and stabilization enzymes (, P4HA1, PLOD1, and PLOD2), it did not inhibit myofibroblast induction or type I collagen synthesis. Alone, fibroblast-specific removal of CCN1 did not negatively impact ventricular performance or myocardial collagen content but did contribute to disorganization of collagen fibrils and increased matrix compliance. Similarly, ablated animals subjected to MI showed no discernible alterations in cardiac structure or function one week after permanent coronary artery ligation, but exhibited marked increases in incidence of mortality and cardiac rupture. Consistent with our findings that CCN1 depletion does not assuage myofibroblast conversion or type I collagen synthesis knockout animals revealed no measurable differences in collagen scar width or mass compared to controls; however, detailed structural analyses via SHG and TEM of scar regions revealed marked alterations in their scar collagen topography—exhibiting changes in numerous macro- and micro-level collagen architectural attributes. Specifically, knockout mice displayed heightened ECM structural complexity in post-MI scar regions, including diminished local alignment and heightened tortuosity of collagen fibers, as well as reduced organizational coherency, packing, and size of collagen fibrils. Associated with these changes in ECM topography with the loss of CCN1 were reductions in fibroblast-matrix interactions, as evidenced by reduced fibroblast nuclear and cellular deformation and reduced focal-adhesion formation ; findings that ultimately suggest CCN1’s ability to influence fibroblast-led collagen alignment may in part be credited to its capacity to augment fibroblast-matrix interactions. These findings underscore the pivotal role of endogenous CCN1 in the scar formation process occurring after MI, directing the appropriate arrangement of the extracellular matrix's collagenous components in the maturing scar—shaping the mechanical properties that support its structural stability. While this suggests an adaptive role for CCN1 in regulating collagen structural attributes crucial for supporting scar integrity post MI, the long-term protracted expression of CCN1 holds maladaptive implications, potentially diminishing collagen structural complexity and compliance in non-infarct regions.

中文翻译：

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基质细胞蛋白 CCN1 促进心肌梗死后胶原蛋白排列和瘢痕完整性


基质细胞蛋白的细胞通讯网络家族 （CCN） 成员，如 CCN1，长期以来一直与伤口愈合、组织纤维化和胶原蛋白动力学等细胞过程的调节有关。虽然许多研究表明 CCN1 通过促进肌成纤维细胞衰老在成人心脏中具有抗纤维化作用，但它们在很大程度上依赖于心脏损伤模型中的外源性补充策略，其中其表达已经被诱导——这可能会混淆对其在此过程中功能的解释。本研究的目的是询问内源性蛋白对成纤维细胞功能、胶原结构动力学及其对心肌梗死 （MI） 后心脏纤维化的相关影响。在这里，我们采用了 CCN1 功能丧失方法，包括 siRNA 介导的耗竭和成纤维细胞特异性敲除小鼠，以评估内源性蛋白对心脏成纤维细胞纤维化信号传导的作用，以及其参与 MI 后急性瘢痕形成。CCN1 的耗竭减少了心脏成纤维细胞的衰老和增殖。尽管 CCN1 的耗竭降低了胶原蛋白加工和稳定酶 （P4HA1、PLOD1 和 PLOD2） 的表达，但它并没有抑制肌成纤维细胞诱导或 I 型胶原蛋白合成。单独使用成纤维细胞特异性去除 CCN1 不会对心室功能或心肌胶原含量产生负面影响，但确实有助于胶原纤维的紊乱和基质顺应性增加。同样，接受 MI 的消融动物在永久冠状动脉结扎后 1 周没有表现出心脏结构或功能的明显改变，但死亡率和心脏破裂的发生率显着增加。 与我们的研究结果一致，即 CCN1 耗竭不会缓解肌成纤维细胞转化或 I 型胶原合成，敲除动物与对照组相比，胶原疤痕宽度或质量没有可测量的差异;然而，通过 SHG 和 TEM 对疤痕区域进行的详细结构分析显示，其疤痕胶原蛋白地形发生了显着变化——表现出许多宏观和微观水平胶原蛋白结构属性的变化。具体来说，敲除小鼠在 MI 后瘢痕区域表现出更高的 ECM 结构复杂性，包括胶原纤维的局部排列减少和迂曲度增加，以及胶原纤维的组织连贯性、堆积和大小降低。与 CCN1 丢失的 ECM 地形变化相关的是成纤维细胞-基质相互作用的减少，成纤维细胞核和细胞变形减少以及病灶粘附形成减少证明了这一点;最终表明 CCN1 影响成纤维细胞主导的胶原蛋白排列的能力的研究结果可能部分归功于其增强成纤维细胞-基质相互作用的能力。这些发现强调了内源性 CCN1 在 MI 后发生的瘢痕形成过程中的关键作用，指导细胞外基质的胶原成分在成熟瘢痕中的适当排列——塑造支持其结构稳定性的机械特性。虽然这表明 CCN1 在调节胶原蛋白结构属性中的适应性作用对于支持 MI 后疤痕完整性至关重要，但 CCN1 的长期表达具有适应不良的影响，可能会降低非梗死区域的胶原蛋白结构复杂性和顺应性。