溶血磷脂酸信号对出生后早期心肌细胞增殖及心肌损伤修复的调控研究

Lysophosphatidic acid (LPA) is a bioactive lipid mediator. Our previous study has found that LPA induced proliferation of postnatal cardiomyocytes in vitro and in vivo. In the preliminary experiment, we found that proliferation of cardiomyocytes decreased in LPA receptor 3 (LPA3) knockout mice compared to wild type during first week but not on E14.5. Further, Lats was not involved in activating YAP induced by LPA. These findings make us hypothesize that LPA signaling is important in promoting normal cardiomyocyte proliferation after birth but not embryonic heart and the phosphatase but not kinase of Hippo pathway regulates this specific effect. Thus the present study plans to by using LPA1 and LPA3 global and condition knockout mice, ①confirm the specific effect of LPA promoting cardiomyocyte proliferation on postnatal but not embryonic heart and clarify the subtype of LPA receptors mediating the specific effect; ②observe the affection of LPA signaling regulating cardiomyocyte proliferation during first week after birth on the fully-functional heart in adult; ③demonstrate the beneficial role of LPA signaling in heart regeneration and repair in response to injury using animal model of myocardial infarction; ④explore the mechanism of LPA activating YAP specifically on postnatal but not embryonic period by RNA- Seq and siRNA interference. The aim of this study is to propose a new concept and mechanism about postnatal heart development and offer the new strategy for effective heart repair by understanding the mechanistic basis of heart development.

溶血磷脂酸（LPA）是一种内源性脂类信号分子。我们在细胞和整体实验发现LPA信号促进出生后心肌细胞增殖。预实验显示LPA受体3亚型（LPA3）敲除小鼠心肌细胞增殖数量在出生后1周低于野生鼠，而胚胎期却与野生鼠相近；还发现LPA激活YAP促心肌细胞增殖并不通过Lats激酶途径。推测LPA信号可能特异调控出生后心肌细胞增殖而不参与胚胎期增殖，其信号是通过Hippo通路的酯酶系统。本研究利用LPA1和LPA3整体和条件敲除小鼠，①阐明LPA信号特异调节出生后而非胚胎期心肌细胞增殖，明确调控的LPA受体亚型；②研究这种增殖对成年心肌细胞数量和心功能的影响；③借助心梗模型论证LPA信号对心肌损伤的修复；④以转录组分析和siRNA技术，明确LPA通过Hippo-酯酶途径特异调节出生后心肌细胞增殖及网络调控系统。本研究可能对出生后心脏发育成熟提出一个全新的理念和机制，为内源性心肌损伤再生修复提供新策略。

溶血磷脂酸（lysophosphatidic acid, LPA）是一类内源性磷脂信号分子，通过其特异性G蛋白偶联受体（LPA1-LPA6）发挥生物学功能。我们前期研究发现LPA信号可以促进出生后心肌细胞增殖，但其对心肌增殖的调控机制和意义不明。本研究首先利用两种LPA受体基因敲除小鼠（LPA1-/-和LPA3-/-）发现LPA3-/- 小鼠pH3 和 Ki67 阳性心肌细胞比率在胚胎期 14.5与出生后 1 天，较野生型小鼠无显著差异，而在出生后4天、7天，pH3和Ki67阳性心肌细胞比率较野生鼠显著下降，而且成年心肌细胞数量明显减少，而LPA1-/-则在各发育期较野生鼠心肌细胞数量均无显著变化，证实LPA3介导的LPA信号为出生后早期而非胚胎期心肌增殖中所需。然后利用心梗模型发现新生LPA3-/-小鼠较野生鼠心梗后心功能明显恶化，心梗面积显著增加，且pH3、 Ki67 和 Brdu 阳性心肌细胞显著减少。而利用腺相关病毒 9（AAV9）在心肌特异过表达 LPA3则发现LPA3 过表达可以促进新生小鼠心梗后心功能恢复、减小心梗面积，并且增加增殖心肌细胞数量，提示LPA3介导的心肌增殖促进新生小鼠心梗后心肌再生。随后，我们进一步探索了LPA-LPA3 信号的促增殖作用是否对成年心肌损伤后修复有保护作用，结果显示心梗后8周心功能明显改善，同时心梗面积明显减小，增殖心肌细胞数量增加，谱系追踪显示LPA3的过表达确实促进新的心肌细胞的形成。由此阐明了心肌特异过表达LPA3促进成年小鼠心梗后心功能恢复和心肌细胞增殖。最后，通过体外培养心肌细胞进一步证明了LPA通过 LPA3促进心肌细胞增殖，并利用RNA-seq 对 LPA 刺激的大鼠原代乳鼠心肌进行转录组分析，发现差异基因主要富集在Hippo-Yap、TGF-β、MAPK/ERK、PI3K/Akt信号通路。进一步的验证实验显示在体外培养系统中Hippo-Yap、BMP、PI3K/Akt、MAPK信号通路参与了 LPA 诱导的心肌细胞增殖。但是，在小鼠心梗后心肌组织中我们只发现了ERK信号的变化，由此推测LPA3介导的LPA信号通过激活ERK促进心肌增殖。本研究不仅发现了LPA信号在出生后心肌细胞增殖中的关键调节作用，并且发现特异性激活LPA3可以通过促进心肌增殖而有利于损伤后心肌修复过程，从而为心肌再生治疗提供了新的靶点。