[1]丁翔,仝识非,秦瑶,等.钙/钙调素依赖性蛋白激酶Ⅱ抑制剂对新生大鼠心房肌细胞钙超载的干预作用[J].陆军军医大学学报(原第三军医大学学报),2010,32(12):1275-1277.
 Ding Xiang,Tong Shifei,Qin Yao,et al.Interventional effect of inhibitor of calcium/calmodulin-dependent protein kinaseⅡon calcium overloading of atrial muscle cells in neonatal rats[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(12):1275-1277.
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钙/钙调素依赖性蛋白激酶Ⅱ抑制剂对新生大鼠心房肌细胞钙超载的干预作用(/HTML )
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陆军军医大学学报(原第三军医大学学报)[ISSN:1000-5404/CN:51-1095/R]

卷:
32卷
期数:
2010年第12期
页码:
1275-1277
栏目:
论著
出版日期:
2010-06-30

文章信息/Info

Title:
Interventional effect of inhibitor of calcium/calmodulin-dependent protein kinaseⅡon calcium overloading of atrial muscle cells in neonatal rats
作者:
丁翔仝识非秦瑶宋治远
第三军医大学西南医院心血管内科
Author(s):
Ding Xiang Tong Shifei Qin Yao Song Zhiyuan
Department of Cardiology, Southwest Hospital, Third Military Medical University, Chongqing, 400038, China
关键词:
钙-钙调素依赖性蛋白激酶2型肌细胞心脏大鼠
Keywords:
calcium-calmodulin-dependent protein kinase type 2 myocytes cardiac calcium rats
分类号:
R-332;R541.75;R453
文献标志码:
A
摘要:
目的    观察钙/钙调素依赖性蛋白激酶Ⅱ(calcium/calmodulin-dependent protein kinaseⅡ, CaMKⅡ)抑制剂KN93对新生大鼠心房肌细胞钙负荷的影响,并对细胞CaMKⅡ的表达变化进行检测。    方法    新生大鼠心房肌细胞原代培养96 h,应用钙离子导入剂ionomycin(1.0 μmol/L)建立心房肌细胞钙超载模型,并在KN93 3种浓度(0.25、0.5、1.0 μmol/L)的干预下,以钙离子指示剂Fluo-3 /AM负载心房肌细胞,激光共聚焦显微镜观察心房肌细胞内游离钙的变化;应用Western blot法检测CaMKⅡ表达的变化。    结果    ①细胞培养至第4天,免疫组织化学染色90%以上细胞α-肌动蛋白抗体阳性。②与对照组比较,钙离子导入剂ionomycin明显增加细胞内钙离子荧光值[(660.16±108.47) vs (376.12±57.57),P<0.01];KN93对细胞内钙离子荧光值无明显影响(P>0.05)。③预先加入3种不同浓度KN93可显著降低ionomycin导致的细胞内钙离子荧光强度的增加幅度(P<0.01),与对照组比较差异也有统计学意义(P<0.01)。④钙超载组细胞CaMKⅡ表达较对照组明显增加(P<0.01);而KN93对细胞CaMKⅡ表达影响不明显。⑤不同浓度KN93预处理后,钙超载组细胞CaMKⅡ的表达显著降低(P<0.01)。    结论    CaMKⅡ抑制剂KN93可降低ionomycin引发的大鼠心房肌细胞钙负荷,并下调细胞CaMKⅡ表达。
Abstract:
Objective    To investigate the effect of calcium/calmodulin-dependent protein kinaseⅡ (CaMKⅡ) inhibitor, KN93 on calcium overloading of atrial muscle cells in neonatal rats and detect the expression of CaMKⅡ.     Methods    The atrial muscle cells from neonatal rats were primarily cultured for 96 h and then divided into 6 group, control, calcium overloading group, KN93 group (0.5 μmol/L), low-, moderate- and high-dose of KN93+ calcium overloading group. A model of calcium overloading for atrial muscle cells was established by using calcium ionophore (ionomycin, 1.0 μmol/L). For the later 3 groups, KN93 at doses of 0.25, 0.5 and 1.0 μmol/L was added into the culture medium for 30 min followed by 1.0 μmol/L ionomycin treatment for another 30 min. The identification of α-actin was performed by immunofluorescence staining. In the present of Fluo-3/AM (an indicator of calcium), intracellular calcium and the expression of CaMKⅡ were detected under the intervention of KN93 with laser cofocal microscopy and Western blotting respectively.     Results    More than 90% of cultured cells were positive to αactin antibody. Compared with the control group, the fluorescence intensity of intracellular Ca2+ was increased significantly by ionomycin (660.16±108.47 vs 376.12±57.57, P<0.01), but KN93 alone had no effect on it (389.00±64.01, P<0.05). In presence of KN93 pretreatment (0.25, 0.5 and 1.0 μmol/L), the fluorescence intensity of intracellular Ca2+ induced by ionomycin was significantly decreased (558.00±102.56, 499.58±64.99, 494.25±78.77 vs 660.16±108.47, P<0.01). The expression of CaMKⅡ was increased in calcium overloading group compared with control (P<0.01) and KN93 alone had no effect on it. But KN93 pretreatment significantly reduced the expression of CaMKⅡ compared with in cells of calcium overloading group (P<0.01).     Conclusion    KN93, an inhibitor of CaMKⅡ, can reduce the calcium loading of atrial muscle cells induced by ionomycin, and downregulate the expression of CaMKⅡ.

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更新日期/Last Update: 2010-06-22