RIPK3介导的CaMKⅡδ可变剪接对糖尿病心肌病心肌坏死性凋亡的调控作用

The mechanism of diabetic cardiomyopathy is not known well. The pre mRNA of calcium/calmodulin-dependent protein kinase Ⅱδ (CaMKⅡδ) was able to be alternatively spliced to produce variants by splicing factors. Our previous study suggested that splicing variants of the CaMKⅡδ isoform played an important role in myocardial hypertrophy and myocardial ischemia/reperfusion injury. Our preliminary experiments showed that hyperglycemia induced a change on the expression of variants of the CaMKⅡδ, an increase in the expression of receptor interacting protein kinase 3 (RIPK3) and an existence of necroptosis in cardiomyocytes. Compared with the wild type diabetic mice, the cardiac function was improved and the disorder of CaMKⅡδ alternative splicing was attenuated in RIPK3-/- diabetic mice. There was colocalization between RIPK3 and splicing factors such as ASF and SC35 in cardiomyocytes. In the project, isolated cardiomyocytes, RIPK3-/- diabetic mice and db/db mice were applied, combined with siRNA interference and adenovirus transfection methods, to verify the scientific hypothesis that hyperglycemia may increase RIPK3 expression and phosphorylation, regulate phosphorylation of splicing factors such as ASF and SC35, enhance CaMKⅡδ alternative splicing to change the expression of variants of the CaMKⅡδ in cardiomyocytes, induce myocardial necroptosis, and promote diabetic cardiomyopathy. It is beneficial to provide a novel mechanism of diabetic cardiomyopathy.

糖尿病心肌病机制仍不完全清楚。钙/钙调素依赖性蛋白激酶Ⅱδ（CaMKⅡδ）mRNA前体在剪接因子作用下发生可变剪接产生变异体。我们前期研究证实CaMKⅡδ变异体在心肌肥大、缺血再灌注损伤中发挥重要作用。预实验发现：高糖诱导心肌细胞CaMKⅡδ变异体表达改变、受体相互作用蛋白激酶3（RIPK3）表达增加，产生坏死性凋亡；与野生型糖尿病小鼠相比，RIPK3-/-糖尿病小鼠心功能改善、心肌CaMKⅡδ可变剪接紊乱减轻；心肌细胞RIPK3与剪接因子ASF、SC35存在共定位。本项目拟采用心肌细胞模型、RIPK3-/-小鼠糖尿病模型、db/db小鼠，联合siRNA干扰或腺病毒转染，验证科学假说：高糖提高RIPK3表达与磷酸化，调节剪接因子ASF和SC35磷酸化，介导CaMKⅡδ可变剪接，改变心肌CaMKⅡδ变异体表达，诱发心肌坏死性凋亡，促进糖尿病心肌病进展。本研究有利于揭示糖尿病心肌病新机制。

目的 糖尿病心肌病（diabetic cardiomyopathy，DCM）是独立于冠状动脉粥样硬化、高血压和其他心血管危险因素以外的心脏结构和功能的异常。坏死性凋亡（necroptosis）是今年来发现的新型细胞死亡方式，其形态学上不同于凋亡和坏死，受体相互作用蛋白激酶3（receptor interacting protein kinase 3，RIPK3）在坏死性凋亡的信号通路中起核心作用。阐明RIPK3在糖尿病心肌病中的具体作用及机制，为糖尿病心肌病的治疗提供新策略。.方法 提取1-3天的Sprague-Dawley（SD）乳大鼠心肌细胞或培养H9C2心肌细胞，转染RIPK3 siRNA后，使用高糖（25.5 mmol/L）培养基继续培养24 h，F-12培养基作为对照，F-12培养基中加入20 mmol/L甘露醇排除渗透压影响，使用荧光定量PCR（quantitative real-time PCR，RT-PCR）检测心肌细胞中钙调素依赖性蛋白激酶Ⅱ（Ca2+/calmodulin-dependent protein kinase，CaMKⅡ）δA、CaMKⅡδB和CaMKⅡδC三种变异体的表达水平，蛋白印迹（western blot）及免疫荧光测定心肌细胞RIPK3表达情况，测定乳酸脱氢酶（lactate dehydrogenase，LDH）释放量和腺苷三磷酸（adenosine triphosphate，ATP）含量，以评估细胞损伤和能量代谢。检测CaMKⅡ磷酸化（p-CaMKⅡ）和氧化（ox-CaMKⅡ）水平。MitoSOX和JC-1染色用于评估细胞氧化应激和线粒体膜电位。野生型及RIPK3基因敲除小鼠腹腔注射60 mg/kg/d 链脲佐菌素（streptozotocin，STZ），连续5 d，对照组小鼠腹腔注射等量柠檬酸盐缓冲液，继续喂养12 w后，小鼠尾静脉注射100 μL蛋白磷酸酶1抑制剂1（inhibitor 1 of protein phosphotase 1，I1PP1）（1×1011 PFU/mL）基因重组腺病毒，对照组小鼠尾静脉注射100 μL载体（vector）对照腺病毒，2 w后western blot测定心肌组织中I1PP1和蛋白磷酸酶1（protein phosphatase 1，PP1）水平。利用心脏超声测定小鼠射血分数（