应用心肌特异基因敲除小鼠探讨presenilin 1在心力衰竭中的功能与机制

The cellular mechanisms underlying heart failure involve a complex of signaling molecules and pathways. The effects and mechanism of PSEN1 in heart failure have not been completely solved yet. It has been reported that PSEN1 knockout mice display severely developmental defects of several organs, such as brain, vascular, muscle, and heart, leading to embryonic lethality. Recently, we have obtained the PSEN1 loxP mice, which could be crossed with tissue-specific Cre transgenic mice or tamoxifen-inducable Cre transgenic mice, to knock out PSEN1 in specific tissues or at specific developmental stages. In this project, we would use these mice to identify that PSEN1 mutation is involved in the process of heart failure, and to clarify the cellular basis of these functions in heart failure is related with cardiomyocytes, not in relationship with cardiac neural crest cells or vascular-related cells linages. Secondly, we would investigate the definitive facts of PSEN1 mutation in heart failure using microscopy and histology analysis. Addtionally, we would also further study the exact way of PSEN1 mutation in calcium-overload regulation. Finally, we would clarify the molecular mechanism of PSEN1 mutation in heart failure using molecular biology analysis. We believe that this project would provide new knowledge about molecular mechanism of heart failure.

慢性心衰的发病率和死亡率逐年升高，严重影响人类的健康状况。慢性心衰是一系列分子异常表达的结果，我们前期研究发现：PSEN1整胚基因敲除小鼠表现出明显的心脏发育异常，以致胚胎期死亡。本课题组已获得时间可调节的心脏特异性PSEN1基因敲除小鼠，本课题将应用这一小鼠探讨PSEN1在心力衰竭中的作用与机制，以确定：PSEN1基因突变对心力衰竭的作用及与心肌细胞的表达与功能失调的关系。本课题将通过PSEN1心肌特异敲除小鼠，探讨PSEN1基因突变参与心力衰竭的细胞学基础。通过组织学、显微、超微结构观察确定PSEN1基因突变引起心力衰竭时，其心肌肌丝、线粒体、内质网等超微结构改变；通过电生理和荧光技术阐明PSEN1基因突变影响心肌钙超载的具体方式、心肌细胞的钙离子平衡如何发生改变及是否影响心肌细胞凋亡；通过分子生物学技术确定PSEN1基因突变引发心力衰竭的分子基础。

PSEN1是一种多次跨膜蛋白，广泛表达在各种不同组织中。本项目发现PSEN1在平滑肌细胞和心肌细胞等心血管相关细胞系中均有表达。（一）在PDGFbb刺激的平滑肌细胞中表达降低；血管平滑肌特异敲除PSEN1导致血管弹力纤维排列紊乱，收缩压和舒张压都出现升高。进一步研究发现PSEN1 敲除在衰老过程中引起血管炎症。通过构建血管损伤模型，比较了动脉血管的狭窄程度，结果显示PSEN1敲除能够明显促进动脉缩窄所致的平滑肌重塑过程。通过导丝损伤模型建立了股动脉血管重塑模型，发现12周龄小鼠在PSEN1敲除组的血管重塑明显高于野生型对照组。进一步检测发现PSEN1敲除能够促进ERK、AKT的磷酸化水平；明显升高TEAD4的表达，而不影响YAP1的表达；升高SMAD2的蛋白表达水平和磷酸化水平；促进了CTNNB和Axin1的表达。（二）在缺血诱发的慢性心衰中，PSEN1表达降低。敲除PSEN1能够降低心脏的射血分数和左室短轴缩短率；能够导致细胞内RyR2表达增加，ATP2a2表达减少。在分离的成年心肌细胞中，PSEN1能够导致心肌细胞在静息期胞浆Ca2+浓度增加，肌浆网Ca2+浓度降低。改善心肌细胞的钙离子调控能够明显改善PSEN1敲除导致的心功能降低。这些结果表明，PSEN1敲除影响钙离子从肌浆网释放到细胞质的过程，导致的Ca2+稳态异常，这可能对衰老相关心脏病的治疗具有重要意义。