The function of Runx1 in cardiac fibroblasts and post-myocardial infarction healing

项目摘要 心肌梗死(MI)导致心肌细胞大量死亡。静止期心脏成纤维细胞 (Cf)在心肌梗死后迅速激活。它们增殖并分化为心肌成纤维细胞(CMF)， 调节心脏纤维化。我们最近发现，在更稳定的梗死区，瘢痕CMF进一步分化为 基质纤维细胞，一种类似于部分分化的软骨细胞的状态，表明 纤维化症。心肌梗死后的纤维化反应稳定了梗塞的心肌，这可能防止 心脏破裂还会降低室壁的顺应性和传导性，甚至干扰再生 努力。了解损伤后CF活动的调节机制将有助于治疗的发展 加强CFS的有益作用和抑制其有害影响的策略。我们发现作为回应， 心肌梗死后CFs中矮小相关转录因子1(RUNX1)的表达明显增加。 肌成纤维细胞与基质纤维细胞分化。我们的初步研究表明，RUNX1促进了CF 并抑制其在体外向肌成纤维细胞分化。此外，我们确定Ccn2是一种 RUNX1的潜在目标。Ccn2在细胞增殖和分化调控中发挥多种作用 通过与不同的辅因子相互作用。因此，我们假设RUNX1的功能是平衡 心肌梗死急性期CFs增殖及肌成纤维细胞分化促进基质纤维细胞 通过直接靶向Ccn2分化更稳定的梗死瘢痕，这对维持组织至关重要 正直，但也会促进不利的重塑。在AIM#1中，CF特异的他莫昔芬诱导的RUNX1基因敲除(KO) 小鼠和野生型小鼠将受到心肌梗死的损伤。免疫组织化学染色、rna-seq和功能性 本实验将用来研究RUNX1在细胞增殖、肌成纤维细胞分化和分化中的作用。 心肌梗死后CFs的基质纤维细胞分化。在目标#2中，RUNX1通过其调节 Ccn2的表达将使用Cut&Run、ATAC-Seq、Hi-C和co-IP与质谱学相耦合的方法进行研究。 这种不偏不倚的做法还将确定项目1的其他直接和间接目标。在Aim#3中，特定于CF 采用三苯氧胺诱导的Ccn2 KO小鼠，比较Ccn2 KO和RUNX1 KO对CFs的影响 心肌梗死后细胞增殖分化。Ccn2在调控CFs增殖和分化中的作用 还将研究RUNX1的差异。Ccn2及其辅因子Hb-EGF和Ccn2的协同作用 转化生长因子β-3对心脏成纤维细胞增殖和分化的影响也将被研究。

Project Summary Myocardial infarction (MI) induces the massive death of cardiomyocytes. Quiescent cardiac fibroblasts (CFs) are rapidly activated after MI. They proliferate and differentiate into cardiac myofibroblasts (CMFs), mediating cardiac fibrosis. We recently found that in more stabilized infarct scar CMFs further differentiate into matrifibrocytes, a state resembling partially differentiated chondrocytes, which indicated an advanced level of fibrosis. The post-MI fibrotic response in the heart stabilizes the infarcted myocardium, which may prevent cardiac rupture but also reduce ventricle wall compliance and conductivity, and even interfere with regeneration efforts. Understanding the mechanisms regulating post-injury CF activities will help the development of treatment strategies that enhance the beneficial effect of CFs and inhibit their deleterious effects. We found that in response to MI the expression of runt-related transcription factor 1 (Runx1) in CFs increased significantly during both myofibroblast and matrifibrocyte differentiation. Our preliminary study showed that Runx1 promoted CF proliferation and inhibited their differentiation into myofibroblasts in vitro. In addition, we identified Ccn2 as a potential target of Runx1. Ccn2 plays multiple roles in the regulation of cell proliferation and differentiation through interacting with different cofactors. Thus, we hypothesize that Runx1 functions to balance the proliferation and myofibroblast differentiation of CFs during the acute phase of MI and promote matrifibrocyte differentiation in more stabilized infarct scars through direct targeting Ccn2, which is critical for maintaining tissue integrity but also promotes adverse remodeling. In Aim #1, CF-specific tamoxifen-inducible Runx1 knockout (KO) mice and wild type mice will be subjected to MI injury. Immunohistochemical staining, RNA-seq, and functional assay will be employed to study the function of Runx1 in the proliferation, myofibroblast differentiation, and matrifibrocyte differentiation of CFs after MI. In Aim #2, the mechanism through which Runx1 regulates the expression of Ccn2 will be studied using CUT&RUN, ATAC-seq, Hi-C, and co-IP coupled to mass spectrometry. The unbiased approach will also identify other direct and indirect targets of Runx1. In Aim #3, CF-specific tamoxifen-inducible Ccn2 KO mice will be employed to compare the effect of Ccn2 KO and Runx1 KO on CF proliferation and differentiation after MI. The requirement of Ccn2 in the regulation of CF proliferation and differentiation by Runx1 will also be studied. The synergistic effect between Ccn2 and its cofactors, HB-EGF and TGFβ3, on cardiac fibroblast proliferation and differentiation will be studied as well.