蛋白磷酸酶1调控CaMKⅡδ可变剪接在心肌缺血再灌注损伤中的作用及机制研究

The mechanism of myocardial ischemia reperfusion injury is unknown. The pre mRNA of calcium/calmodulin-dependent protein kinase Ⅱδ (CaMKⅡδ) was able to be alternative spliced to produce variants, which was regulated by protein phosphatase 1 (PP1) and splicing factors. Our previous study suggested that splicing variants of the CaMKIIδisoform played an important role in myocardium hypertrophy. Our preliminary experiments showed that there was disorder on splicing variants of the CaMKIIδ isoform in myocardium after ischemia and reperfusion injury. There was interaction between PP1 and splicing factors such as ASF and SC35 in cardiomyocyte. PP1γ overexpression enhanced apoptosis in cardiomyocyte subjected to hypoxia-reoxygenation. Base on these results, we put forward the scientific hypothesis that PP1 is to alter phosphorylation level of splicing factor such as ASF and SC35, regulate alternative splicing of CaMKⅡδ, affect the expression, location and target protein of variants of the CaMKⅡδ isoform, induce calcium release, increase oxidative stress and apoptosis, and to promote myocardial ischemia reperfusion injury. In order to verify the hypothesis, the research will use CaMKⅡδ minigene and adenovirus transfection method, combined with GST pulldown and immunoprecipitation technique in cell hypoxia-reoxygenation and myocardial ischemia reperfusion injury. It may provide novel target for the treatment of myocardial ischemia reperfusion injury.

心肌缺血再灌注损伤机制不明。钙/钙调素依赖性蛋白激酶Ⅱδ（CaMKⅡδ）mRNA前体受蛋白磷酸酶1（PP1）和剪接因子调控，存在可变剪接产生变异体。我们既住研究证实CaMKⅡδ变异体在心肌肥厚中发挥重要作用。预实验发现：心肌缺血再灌注损伤中CaMKⅡδ可变剪接发生紊乱；心肌细胞中PP1与ASF和SC35等剪接因子存在相互作用；过表达PP1γ增加心肌细胞缺氧复氧后凋亡。在上述基础上，我们提出科学假说：PP1通过改变ASF和SC35等剪接因子的磷酸化水平，调节CaMKⅡδ可变剪接，影响心肌细胞中CaMKⅡδ变异体的表达、分布与靶蛋白，激活钙离子释放、增加氧化应激和调亡，促进心肌缺血再灌注损伤。本课题拟运用CaMKⅡδ迷你基因和腺病毒转染等手段，联合GST-pulldown、免疫共沉淀等技术，利用心肌细胞缺氧复氧及动物心肌缺血再灌注模型验证该假说，为心肌缺血再灌注损伤防治提供新靶点。

阐明PP1调控CaMKⅡδ可变剪接对心肌缺血再灌注损伤的调控作用及机制，我们的研究发现：1、心肌缺血再灌注损伤后心肌组织CaMKⅡδA、CaMKⅡδB基因表达下降，CaMKⅡδC表达增加;2、心肌组织中I1PP1表达明显增强，PP1表达水平明显抑制;3、心脏过表达I1PP1后减小缺血再灌注心肌梗死面积;4、心脏过表达I1PP1后降低心肌缺血再灌注后血清CK和LDH活力;5、心脏过表达I1PP1改善缺血再灌注后心肌病理结构;6、心脏过表达I1PP1后降低心肌缺血再灌注后PLB磷酸化水平，CaMKⅡδA、CaMKⅡδB基因表达，减少CaMKⅡδC表达;7、心脏过表达I1PP1后减弱DHE荧光强度，降低MDA水平，提高T-AOC;8、过表达I1PP1后改善缺血再灌注后心肌线粒体超微结构;9、心脏过表达I1PP1降低缺血再灌注后心肌线粒体中DRP1表达，增加OPA1表达;10、心肌细胞缺氧复氧后CaMKⅡδA、CaMKⅡδB变异体表达明显下降，CaMKⅡδC变异体表达明显增加;11、I1PP1重组腺病毒转染48 h后，心肌细胞中I1PP1表达明显增强，PP1表达水平受到明显抑制;12、H/R引起LDH释放增多， I1PP1过表达后可增加H/R损伤后ATP水平;13、I1PP1过表达抑制p-PLB表达升高，提高了CaMKⅡδA和CaMKⅡδB表达，降低了CaMKⅡδC表达;14、I1PP1过表达可减弱线粒体以及胞浆中ROS的产生;15、I1PP1过表达可减少H/R损伤后CaMKⅡ氧化和磷酸化;16、H/R损伤显著降低OPA1蛋白的表达，增加DRP1蛋白的表达，I1PP1过表达可恢复两种蛋白的表达失调;17、预先给予CaMKⅡ抑制剂KN93显著降低了心肌细胞中ROS的含量，尤其是线粒体中ROS的含量降低更为明显。I1PP1过表达减轻心肌细胞缺氧-复氧后CaMKⅡδ可变剪接紊乱，抑制CaMKⅡ磷酸化和氧化，减少ROS积聚，减轻缺氧-复氧损伤；过表达I1PP1调节CaMKⅡδ可变剪接可减轻心肌缺血再灌注损伤，其机制可能与保护线粒体功能、降低氧化应激有关。其作用及机制为防治心肌缺血再灌注损伤提供新思路和潜在生物学靶点。