Smyd2单甲基化Grp78-K585通过阻遏Camk2内质网应激促心肌缺血再灌注损伤的机制研究

心肌缺血再灌注损伤（MIRI）为缺血性心脏病难治的关键成因，而缺血/复灌（I/R）致心肌细胞（CM）胁迫应激的早期精细机制仍知之甚少。前期小鼠与酶活实验发现，I/R-CM胞质甲基转移酶Smyd2可迅速转位内质网（ER）腔，并伴应激蛋白Grp78第585位赖氨酸上单甲基化修饰（K585me1）增加，而敲除Smyd2后该现象消失及CM钙超载与细胞凋亡减少。那么，该甲基化修饰是否通过促进ER钙调失衡介导MIRI？临床早期I/R-CM样本分析表明，SMYD2与GRP78集中分布于ER内，且与胞核外钙调蛋白激酶CAMKⅡ共定位。依此，申请人提出科学假说，即CM-ER内Smyd2催化Grp78-K585me1导致Camk2过度自磷酸化以促MIRI。本项目拟采用小鼠Smyd2心脏条件性敲除模型、蛋白质翻译后修饰组学、钙瞬变示踪等技术予以验证，为拓展相关信号轴成员为缺血性心脏病治疗潜在新靶点提供科学依据。

Myocardial ischemia-reperfusion injury (MIRI) is the key cause of refractory ischemic heart disease, and the early precise mechanism of ischemia/reperfusion (I/R) induced cardiomyocyte (CM) stress is still poorly understood. Previously, the mouse and enzyme activity experiments have found that I/R-CM cytoplasmic methyltransferase Smyd2 might rapidly translocate into the cavity of endoplasmic reticulum (ER) with an increase of mono-methylation modification on the 585th lysine of the stress protein Grp78, which could be subsided by genetic knockout of Smyd2. Plus, Smyd2 knockout could result in the decrease of CM calcium overload and apoptosis. Therefore, does this methylation modification mediate MIRI by promoting ER calcium imbalance? The analysis of I/R-CM samples in the early clinical stage showed that SMYD2 and GRP78 were concentrated in the ER and surprisingly co-localized with extra-nuclear calmodulin kinase CAMKⅡ. Accordingly, the applicant puts forward the scientific hypothesis that Smyd2 in CM-ER catalyzes Grp78-K585me1 to cause the hyper-autophosphorylation of Camk2 to promote MIRI. In this project, the cardiac Smyd2 conditional knockout mouse model, protein post-translational modification omics, calcium transient tracing and other technologies are to be used to verify this hypothesis, so as to provide scientific evidences for expanding the related signal chain members as potential novel targets for the treatment of ischemic heart disease.