发育期心脏感应环境胁迫的Nkx2.5蛋白剪切机制研究

Congenital heart abnormalities will cause embryonic lethality, miscarriage and birth defects, severely deteriorating fetal and neonatal health. Environmental stress contributes to the generation of heart defects, but the precise mechanism remains obscure. Nkx2.5 is a crucial transcriptional factor regulating cardiac development and function, and its mutation or dysfunction is associated congenital heart diseases. A preliminary study revealed that environmental stress can trigger the protein cleavage of Nkx2.5, and this effect can be antagonized by caspase inhibitors. We speculate that Nkx2.5 is a substrate of caspase enzymes and cleavage fragments containing truncated domains block the transcriptional function of full-length Nkx2.5 via dominant interference. Due to the presence of endogenous caspase activity playing a non-apoptotic role during normal embryonic development, exogenous environmental stress will further superimpose caspase activities to initiate the cleavage of Nkx2.5, resulting in developmental retardation or heart malformations. Therefore, Nkx2.5 may be a sensitive molecule in response to environmental stress during heart development. This study will elucidate the molecular mechanism of Nkx2.5 cleavage and its effect on cardiac differentiation, cell survival, and electrophysiological function. Thereby, we will offer a novel method to reveal the sensitive cells during the critical stage of heart development by detecting truncated Nkx2.5, and provide new concepts to the prevention of congenital heart diseases.

心脏发育异常将导致死胎、流产及出生缺陷，危害胎儿及新生儿健康。环境干扰是引发心脏发育异常的重要原因，具体机制不清。Nkx2.5是调控心脏发育的关键转录因子，它的突变或功能异常与心脏发育障碍关系密切。本项目初步研究发现，环境胁迫将导致Nkx2.5蛋白发生剪切，该效应可被caspase抑制剂所拮抗。推测Nkx2.5是某一caspase蛋白酶的下游底物，其剪切片段对全长型蛋白的转录功能具有干扰作用。由于正常胚胎发育需要内源性caspase发挥非凋亡性作用，外源环境胁迫将引发caspase活性叠加性增高，启动Nkx2.5蛋白剪切，引致胚胎心脏发育中止或发育畸形。Nkx2.5可能是发育期心肌细胞感应环境胁迫并产生效应的重要分子。本项目将初步阐释Nkx2.5蛋白剪切的生化机制及对心肌分化、存活的影响，为先天性心脏病的防治及妊娠保健提供新的理论依据。

心脏发育异常将导致死胎、流产及出生缺陷，危害胎儿及新生儿健康。环境干扰是引发心脏发育异常的重要原因，具体机制不清。本研究发现，在心肌分化过程中，存在Caspase-3/8/9以及凋亡、自噬、内质网应激等细胞应激基因的阶段特异性表达，提示不同的Caspase酶参与了不同阶段的心肌分化过程。环境干扰物引发的氧化应激通过激活p38和JNK通路，正向促进心肌分化标志基因的表达，但是抑制细胞增殖。抑制Caspase会降低心肌分化基因表达，但是在环境干扰物诱导的Caspase上调过程中，抑制Caspase会增强心肌分化基因表达。这提示：Caspase参与心肌正常分化，但外源刺激诱导的Caspase活性叠加性增高反而是有害的。本项目研究发现，环境胁迫将导致Nkx2.5蛋白发生剪切，深入研究显示Nkx2.5蛋白剪切与溶酶体破裂相关，在酸性条件下酶切效率最高，并受氧化应激、Caspase活化、自噬等因素影响。但Nkx2.5具体的剪切位点及剪切酶等生化机制仍需进一步研究。本研究还发现p38和JNK通路直接增强Nkx2.5蛋白稳定性和功能活性，但是超过阈值引发自噬的条件下间接促进Nkx2.5降解。总之，本研究阐释了Caspase、p38和JNK等应激信号，对心肌分化存在阶段特异性和剂量依赖性的调控，超量活化和发育异常相关。