慢性缺氧对幼鼠心肌CaM CaMKⅡ及细胞内Ca^(2+)活动的影响

Effects of chronic hypoxia on the expression of calmodulin and calcicum/calmodulin-dependent protein kinase Ⅱ and the calcium activity in myocardial cells in young rats

目的急性缺血缺氧可致心功能受损和心律失常,钙离子在其中起重要作用,慢性缺氧对心功能及心肌细胞内钙离子活动同样产生影响,但机制不同。该研究拟通过慢性缺氧动物模型,研究慢性缺氧对心肌细胞内钙调素(calmodulin,CaM)、钙/钙调素依赖性蛋白激酶Ⅱ(calcium/calmodulin-dependent protein kinase Ⅱ,CaMKⅡ)的表达及其对细胞内Ca2+活动的影响,深入了解慢性缺氧对心脏功能及电活动的影响机制。方法通过吸入低浓度含氧气体(FiO2:10%)建立慢性缺氧大鼠动物模型。在实验1周和3周时,应用RT-PCR,Western Blot方法分别检测对照组和慢性缺氧组动物心肌细胞内CaM和CaMKⅡγ、CaMKIIδmRNA和蛋白表达;分离并培养正常心肌细胞和缺氧3周细胞,应用激光共聚焦法分别检测两种心肌细胞内Ca2+活动,同时应用CaMKⅡ特异性抑制剂KN-62,观察CaMKII在慢性缺氧下对心肌细胞内Ca2+活动的影响。结果实验1周和3周时,慢性缺氧大鼠心肌细胞内CaM和CaMKIIγ、CaMKIIδ的mRNA和蛋白表达均较正常动物高(P<0.01);在缺氧1周和3周组动物间CaM和CaMKIIδ也存在差异(P<0.01),但CaMKⅡγ无差异(P>0.05)。激光共聚焦研究发现,慢性缺氧心肌细胞内Ca2+活动其钙波振幅虽和正常心肌细胞无差异(P>0.05),但钙波时程延长(P<0.01);应用KN-62后,慢性缺氧动物钙波振幅和时程改变较明显(P<0.01)。结论慢性缺氧可使大鼠心肌细胞内CaM和CaMKⅡ合成代偿性增加,保持大鼠心肌细胞内钙稳态,从而在一定时期内维持心功能稳定。但随缺氧时间延长,心功能可受损并可致心律失常。

Objective Calcium plays an important role in the impairment of heart function and arrhythmia under the condition of acute hypoxia, but the mechanism is different from that of chronic hypoxia. This study aimed to evaluate the effect of chronic hypoxia on the expression of calmodulin （ CaM ） and calcicum/calmodulin-dependent protein kinase Ⅱ （CaMKⅡ） and the calcium activity in myocardial cells through an animal model of chronic hypoxia in order to get a deeper sight into the mechanism. Methods A chronic hypoxia model of the rat was prepared by hypoxia exposure （FiO2 = 10% ）. The expression of mRNA and protein of CaM and CaMKⅡγ and CaMKⅡδ in myocardial cells were measured by RT-PCR and Western Blot in normal rats and hypoxia rats 1 and the 3 weeks after exposure. The cardiac cells of the rats from the control group and the 3-week hypoxia group were cultured. Then the intracellular calcium activity was detected using laser confocal equipment. The effect of CaMKⅡ on the calcium activity in myocardial cells was evaluated by the application of KN-62 （CaMKⅡ specific inhibitor）. Results The expression of CaM, CaMKⅡγ and CaMKⅡδ mRNA in myocardial tissues increased in hypoxia rats compared with that in normal controls （ P 〈 0.01 ）. The CaM and CaMKⅡδ mRNA expression was different between the 1-week and the 3-week hypoxia groups （ P 〈 0.01 ）. The laser confocal demonstrated that the amplitude of calcium wave in hypoxic myocardial cells was not different from that in normal controls, but the duration of calcium wave in hypoxic myocardial cells was longer than that in normal controls （ P 〈0.01 ）. After KN- 62 use, the amplitude of calcium wave decreased and the duration of calcium wave prolonged significantly. Conclusions The contents of CaM and CaMKⅡ in myocardial cells increased under condition of chronic hypoxia as a compensation to keep calcium homeostasis in a certain time. With more prolonged hypoxia time, abnormal electric activities of heart occurred and the heart function may be impaired.