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

项目摘要 心肌梗死（MI）可导致心肌细胞大量死亡。静止心肌成纤维细胞 (CFs)在心肌梗死后迅速激活。它们增殖并分化成心肌成纤维细胞（CMF）， 介导心脏纤维化我们最近发现，在更稳定的梗死瘢痕中，CMFs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，这表明了一种先进的水平， 纤维化心肌梗死后的心脏纤维化反应稳定了梗死心肌，这可以防止心肌梗死。 心脏破裂还会降低心室壁顺应性和传导性，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 增强CF的有益作用并抑制其有害作用的策略。我们发现， MI时，CFs中Runx 1的表达显著增加， 肌成纤维细胞和基质纤维细胞分化。我们的初步研究表明，Runx 1促进CF 增殖并抑制其向肌成纤维细胞的分化。此外，我们将Ccn 2确定为 Runx 1的潜在目标。CCN 2在细胞增殖和分化的调节中起着多种作用 通过与不同的辅因子相互作用。因此，我们假设Runx 1的功能是平衡 心肌梗死急性期CFs的增殖和肌成纤维细胞分化，并促进基质纤维细胞 通过直接靶向Ccn 2，在更稳定的梗死瘢痕中分化，这对于维持组织 但也促进了不利的重塑。在目标#1中，CF特异性他莫昔芬诱导型Runx 1敲除（KO） 小鼠和野生型小鼠将经受MI损伤。免疫组织化学染色，RNA-seq，和功能 本研究将采用免疫组化方法研究Runx 1在成肌纤维细胞增殖、分化和凋亡中的作用。 心肌梗死后CFs的基质纤维细胞分化。在目标#2中，Runx 1调节 Ccn 2的表达将使用CUT&RUN、ATAC-seq、Hi-C和co-IP与质谱联用来研究。 无偏方法还将识别Runx 1的其他直接和间接目标。在目标#3中，CF特定 将使用他莫昔芬诱导的Ccn 2 KO小鼠来比较Ccn 2 KO和Runx 1 KO对CF的影响 增殖和分化。Ccn 2在CF增殖调节中的需要和 还将研究Runx 1的分化。Ccn 2及其辅因子HB-EGF和HB-EGF之间的协同作用是通过调节Ccn 2的浓度来实现的。 TGFβ3对心脏成纤维细胞增殖和分化的影响也将进行研究。

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.