PDGF信号在心肌再生及心肌细胞增殖中的作用与机制研究

The hearts of 1-day-old mice can regenerate completely, after myocardial infarction or apical resection (15% ventricular myocardium resected), over 21 days. In contrast, 7-day-old mice are not able to fully regenerate with myocardial infarction or apex resection. Therefore, it is an ideal model to study the molecular mechanism of mammalian cardiac regeneration. We here found that the activation of PDGF signaling can promote myocardial regeneration by up-regulating the expression of the enhancer of zeste homologue 2 (Ezh2) according to RNA sequencing analysis results. The declining level of PDGFR-β in cardiomyocytes is accompanied by reduction of Ezh2, suggesting a key role of PDGF/EZH2 signaling in heart regeneration. We intend to take the experiments as follows: ① To analyze the expression regulation of EZH2 by PDGF signaling, we will detect the expression EZH2 by PCR and the activity of EZH2 by measuring the level of trimethylation of histone H3K27 (H3K27me3) in cardiomyocyte conditional knockout of PDGFR-β mice (PDGFR-βΔcm) and in the mice keeping PDGFR-β phosphorylating activated in cardiomyocytes with D849V mutation (PDGFRβD849Vcm). ② To evaluate the effects of EZH2 on myocardial regeneration of 1-day-old mice (EZH2Δcm) with myocardial infarction, we knock out EZH2 in cardiomyocytes conditionally. ③ To find out whether the downstream of PDGF is EZH2 in heart regeneration, we test the function of heart after myocardial infarction, in which mice, the PDGFR-β is activated and EZH2 is knocked out in cardiomyocytes (cmEzh2KO-PDGFRβD849V). ④ The primary PDGF signaling pathway regulating EZH2 will be studied in vitro by treating primary cardiomyocytes isolated from neonatal mouse hearts with the various related inhibitors. ⑤ To clarify the downstream molecular mechanism of PDGF/EZH2 in cardiac regeneration, we will take the EZH2 CHIP-Seq from cardiomyocytes with overexpressed PDGFR-β conditionally. Finally, we will elucidate the role of PDGF-EZH2 signaling in myocardial regeneration and cardiomyocyte proliferation, which offers a new theory of and a potential target for myocardial regeneration.

1天龄小鼠心梗后21天可完全再生（7天龄小鼠则无此能力），是研究哺乳动物心肌再生机制的理想模型。我们前期实验发现：激活PDGF信号能促进心肌再生同时上调EZH2表达；且心肌中EZH2与PDGFRβ存在协同表达；提示PDGF信号在心肌再生中扮演了重要角色。本项目拟①构建心肌细胞条件敲除和突变激活PDGFRβ小鼠，正反两方面分析PDGF信号对EZH2表达的调控作用。②构建心肌细胞条件敲除EZH2小鼠，1天龄制作心梗，分析EZH2对心肌再生是否必需。③PDGFRβ激活小鼠中同时敲除EZH2，心梗后功能学水平分析PDGF信号是否通过EZH2调控心肌再生。④分离原代心肌细胞，应用信号通路抑制剂分析PDGF信号调节EZH2表达及其活性的关键通路。⑤心肌细胞PDGFRβ激活后进行EZH2 ChIP-Seq实验，探讨PDGF/EZH2调控心肌细胞增殖的下游分子机制。通过如上研究，为揭示心肌再生提供新理论。

1天龄小鼠心梗后21天可完全再生（7天龄小鼠则无此能力），是研究哺乳动物心肌再生机制的理想模型。PDGFR-β作为一种酪氨酸醇受体，能够将细胞外的信号传导到细胞内并启动增殖、代谢、迁移等生物过程。但是对于PDGF信号通路在哺乳动物心脏再生中作用的研究甚少。本研究通过构建构建心肌细胞条件敲除和突变激活PDGFR-β小鼠、心肌细胞条件敲除EZH2小鼠并在PDGFR-β激活小鼠中同时敲除EZH2，发现PDGFR-β信号通路在增殖的心肌中表达升高，且PDGFR-β信号激活通过上调EZH2 促进心肌组织增殖和心肌再生。通过心肌细胞PDGFR-β激活后进行EZH2 ChIP-Seq实验，发现PDGFRβ信号通路调控EZH2促进心肌组织增殖再生是通过Akt/PI3K通路完成。在PDGF信号介导心肌再生的机制研究中，我们还发现骨髓来源的生长因子（Myeloid-derived growth factor，Mydgf）可能也参与了心肌再生的调控。Mydgf是一种新型骨髓来源的单核-巨噬细胞产生的旁分泌蛋白，在成年小鼠心肌梗死后修复起着重要作用。然而，Mydgf是否参与调控新生小鼠心脏再生尚无报道。本研究利用Mydgf敲除（Mydgf-KO）小鼠结合心尖切除（apical resection，AR）和成年心肌梗死（myocardial infarction，MI）手术，结果发现Mydgf参与AR术后心肌再生，Mydgf-KO阻碍新生小鼠心肌再生过程；Mydgf-KO加重成年小鼠MI后的心脏损伤，重组蛋白Mydgf治疗可提高成年MI后的心脏修复能力。综上所述，我们的研究结果表明，PDGFR-β和Mydgf参与新生小鼠心肌再生，为心脏再生调控机制提供新的依据与线索。相关研究成果在Cell Reports（IF=9.423）、Theranostics（IF=11.556）、Circulation（IF=29.690）、J Mol Cell Cardiol（IF=5.000）、Adv Sci（IF=16.806）、Cell Mol Immunol（IF=11.530）、J Cell Mol Med（IF=5.310）等SCI杂志发表论文12篇（影响因子为2021年数据）。