抑制CaMKII减轻线粒体氧化应激可改善离体心脏缺血再灌注损伤

Inhibition of CaMKII alleviates myocardial ischemia-reperfusion injury by reducing mitochondrial oxidative stress in isolated perfused rat heart

目的探讨钙/钙调蛋白依赖的蛋白激酶Ⅱ(CaMKII)在离体心脏缺血再灌注(IR)损伤中的作用和可能机制。方法采用大鼠离体心脏缺血再灌注(IR 45 min/120 min)模型,将40只大鼠随机分为对照组(Control)、KN-93药物对照组(2.5μmol/L KN-93)、IR组和CaMKII特异性抑制剂KN-93干预缺血再灌注组(KN-93+IR),以左室心脏功能、冠脉流出液中乳酸脱氢酶(LDH)活性和心肌肌钙蛋白(cTnI)含量、心肌梗死面积评价心脏损伤程度,Western-blot检测CaMKII磷酸化(p-CaMKII)、CaMKII氧化(ox-CaMKII)和PLN磷酸化(p-PLN)蛋白的表达,试剂盒检测线粒体超氧化物歧化酶(SOD)的活性和丙二醛(MDA)的含量。结果与Control组相比,KN-93组各项指标均无明显变化;IR组心脏功能、线粒体SOD的活性降低(P<0.01),而心肌梗死面积、冠脉流出液中LDH活性和cTnI含量、p-CaMKII、ox-CaMKII和p-PLN的表达、线粒体MDA的含量均明显升高(P<0.01);KN-93干预IR组可明显改善心脏功能(P<0.01),增加线粒体SOD活性,减小心肌梗死面积、LDH活性、cTnI含量、p-CaMKII、ox-CaMKII和p-PLN的表达以及线粒体MDA含量(P<0.01)。结论 CaMKII参与离体心脏缺血再灌注损伤,抑制CaMKII可通过降低线粒体氧化应激进而减轻离体心脏缺血再灌注损伤。

Objective To investigate the role of calcium/calmodulin-dependent protein kinase II（CaMKII） in myocardial ischemia-reperfusion（IR） injury in isolated perfused rat heart and explore the underlying mechanisms.Methods An ischemiareperfusion（IR） model was prepared using isolated rat hearts perfused with Krebs-Henseleit solution were randomly divided into control group,2.5 μmol/L KN-93 group,IR（induced by ischemia for 45 min followed by reperfusion for 120 min） group and KN-93＋IR group.The myocardial performance was evaluated by assessing the left ventricular pressure.Lactate dehydrogenase（LDH） activity and cTnI content in the coronary flow and the infarct size were determined to evaluate the myocardial injury.The phosphorylation of CaMKII（p-CaMKII） and PLN（p-PLN） and oxidation of CaMKII（ox-CaMKII） were measured with Western blotting.The activity of mitochondrial superoxide dismutase（SOD） and the content of malondialdehyde（MDA） were determined using ELISA.Results Compared with the control group,KN-93 treatment at2.5 μmol/L produced no significant effects on cardiac function or performance in rat hearts without IR injury.Myocardial IR injury significantly decreased myocardial performance and mitochondrial SOD activity in the perfused hearts（P〈0.01） and caused significantly increased infarct size,LDH activity,cTnI content,expressions of p-CaMKII,ox-CaMKII and p-PLN,and also increased mitochondrial MDA content（P〈0.01）.KN-93 treatment at 2.5 μmol/L administered before ischemia and before reperfusion markedly attenuated such changes induced by ischemia and reperfusion（P〈0.01）.Conclusion CaMKII participates in myocardial IR injury in isolated rat heart,and inhibiting CaMKII can alleviate myocardial injury by relieving mitochondrial oxidation stress.