高糖对H9C2心肌细胞系和乳大鼠心室肌细胞钙库操纵性钙内流及相关蛋白的影响

目的 探究高糖环境对大鼠胚胎心肌细胞系（H9C2）和乳大鼠心室肌细胞（neonatal rat ventricular myocytes，NRVMs）钙库操纵性钙内流（store operated calcium entry，SOCE）功能的影响，对基质相互作用分子1（stromal interaction molecule 1，STIM1）蛋白表达及其激活结构的作用，完善SOCE在糖尿病心肌病（diabetic cardiomyopathy，DCM）中的潜在致病机制。方法 将H9C2和NRVMs细胞分为2组，对照组（5.5 mmol/L葡萄糖）和高糖组（25 mmol/L葡萄糖），分别培养48 h后用激光共聚焦显微镜实时观察各组心肌细胞在毒胡萝卜素（thapsigargin，TG）刺激后Ca²⁺荧光强度值的变化；采用Western blotting技术检测各组细胞STIM1和钙释放激活钙通道调节分子1（calcium release-activated calcium channel modulator 1，Orai1）蛋白表达的变化；借助化学交联技术和免疫荧光法检测NRVMs的STIM1蛋白的聚集变化；免疫共沉淀技术观察NRVMs心肌细胞内源性STIM1和Orai1蛋白的直接相互作用。结果 与对照组相比，高糖组H9C2和NRVMs心肌细胞钙内流（Ca²⁺ influx）显著增加（P<0.05）；高糖组H9C2和NRVMs心肌细胞STIM1以及Orai1蛋白表达上调（P<0.01）；钙库排空后，高糖组NRVMs心肌细胞STIM1蛋白聚集体显著增多（P<0.01）。结论 体外高糖能诱导大鼠胚胎心肌细胞系H9C2及乳大鼠原代培养心室肌细胞NRVMs的STIM1和Orai1蛋白表达上调，促进STIM1蛋白激活形成聚集体和SOCE激活，Ca²⁺内流增多。该作用提示高糖增加的心肌细胞SOCE可能与糖尿病心肌肥大有关。

Effects of high glucose on store operated calcium entry and related protein expressions in H9C2 and neonatal rat ventricular cardiomyocytes

Objective To investigate the effects of high glucose on store operated calcium entry (SOCE) and the endogenous stromal interaction molecule 1 (STIM1) expression and its activated structure "puncta" in rat embryonic cardiomyocytes line (H9C2) and neonatal rat ventricular myocytes (NRVMs), which may be involved in the pathogenic mechanism of diabetic cardiomyopathy (DCM). Methods H9C2 and NRVMs cardiomyocytes were divided into two groups:normal glucose group (5.5 mmol/L) and high glucose group (25 mmol/L) treated and cultured for 48 h. The alteration of Ca²⁺ influx mediated by SOCE pathway were observed by laser confocal microscope after treated with thapsigargin (TG, 2 μmol/L) in H9C2 and NRVMs cells. The expression levels of STIM1 and calcium release-activated calcium channel modulator 1 (Orai1) were detected by Western blotting. The aggregated formation of STIM1 puncta in NRVMs was detected by chemical crosslinking and immuno-fluorescence staining. Direct interaction between endogenous STIM1 and Orai1 in NRVMs was observed by co-immunoprecipitation. Results Compared with control group, TG stimulated sarcoplasmic reticulum(SR) Ca²⁺ store depletion and induced more SOCE (P<0.05), and increased STIM1 and Orai1 expressions (P<0.01) in the H9C2 and NRVMs cells cultured with high glucose. While the interaction between endogenous STIM1/Orai1 was not altered between the two groups of cells, the aggregated formation of STIM1 puncta was significantly increased after SR Ca²⁺ store depletion by TG in NRVMs cells cultured with high glucose (P<0.01). Conclusion High glucose could up-regulate the expression levels of STIM1 and Orai1 proteins and promote the formation of STIM1 puncta, leading to an increased SOCE in H9C2 and NRVMs cardiomyocytes, which implicated a potential mechanism involved in diabetic cardiomyopathy.