CaMKⅡδ在有氧运动调控高血压心肌细胞凋亡中的作用

Role of CaMKⅡδ in the regulation of cardiomyocyte apoptosis in hypertension induced by aerobic exercise

目的:探讨Ⅱ型钙/钙调素依赖性蛋白激酶δ亚型(calcium/calmodulin-dependent protein kinaseⅡδ,Ca MKⅡδ)在有氧运动调控自发性高血压大鼠心肌细胞凋亡中的作用。方法:雄性12周龄自发性高血压大鼠和Wistar-Kyoto大鼠,随机分为正常安静组(WKY-SED)、正常有氧运动组(WKY-EX)、高血压安静组(SHR-SED)、高血压有氧运动组(SHR-EX)。经12周中等强度跑台运动(坡度0°,20m/min,60min/d,5d/w)后,测定大鼠尾动脉血压;Millar压力-容积系统测定在体心功能;Masson染色分析心肌胶原容积分数(CVF%);caspase 3活性检测与TUNEL染色观察心肌细胞凋亡;Western Blot检测Ca MKⅡδ、ox-Ca MKⅡδ、IκBα、NF-κB P65、HSP70、Bax、Bcl-2的蛋白表达。结果:12周有氧运动后,SHR-EX组较SHR-SED组心率、血压显著降低(P<0.05),心功能左室压力最大上升速率(+dp/dtmax)、射血分数(EF)与收缩末期压力-容积关系曲线(ESPVR)斜率(Ees)显著升高(P<0.05),心室压力最大下降速率(-dp/dtmax)的绝对值与每搏输出量(SV)显著升高(P<0.01),收缩末期压力(ESP)、舒张末期压力(EDP)与有效动脉弹性(Ea)显著下降(P<0.01),等容舒张常数(Tau)显著降低(P<0.05)。Masson染色结果显示SHR-EX组心肌组织纤维化减少,CVF%显著降低(P<0.01)。WKY-EX组较WKY-SED组caspase 3显著下降(P<0.05)。SHR-EX组较SHR-SED组caspase 3显著降低(P<0.05),TUNEL阳性颗粒数量显著降低(P<0.01)。Western Blot结果显示WKY-EX组较WKY-SED组NF-κB P65与Bax的蛋白表达、Bax/Bcl-2比值显著下降(P<0.05),IκBα与HSP70蛋白表达显著升高(P<0.01),Bcl-2蛋白表达显著上升(P<0.05);SHR-EX组较SHR-SED组ox-Ca MKⅡδ、NF-κB P65与Bax蛋白表达显著降低(P<0.05),Bax/Bcl-2比值显著降低(P<0.01),IκBα与Bcl-2蛋白表达显著上升(P<0.05),HSP70蛋白表达显著升高(P<0.01)。结论:有氧运动通过下调SHR心肌ox-Ca MKⅡδ抑制心肌细胞凋亡,同时激活抗凋亡相关信号通路改善心功能,可能是运动诱导高血压心脏重塑的机制之一。

Objective:To explore the role of calcium/calmodulin-dependent protein kinase Ⅱδ(CaMKⅡδ)in the regulation of cardiomyocyte apoptosis in spontaneous hypertensive rat(SHR)by aerobic exercise.Method:12-week-old male SHR and WKY rats were randomly divided into normal control group(WKYSED),normal aerobic exercise group(WKY-EX),hypertensive control group(SHR-SED)and hypertensive aerobic exercise group(SHR-EX).After 12 weeks of treadmill running(slope 0°,20 m/min,60 min/d,5 d/w),the blood pressure of the tail artery was measured.The Millar pressure-volume system was used to measure the heart function of the rats in vivo.Analysis of myocardial collagen volume fraction was made by Masson staining.Detection of caspase 3 activity and TUNEL staining were used to observe the apoptosis of cardiac myocytes.Western Blot was used to measure the protein expression of CaMKⅡδ,ox-CaMKⅡδ,IκBα,NF-κB P65,HSP70,Bax and Bcl-2 in rat myocardial tissue.Result:After 12 weeks of aerobic exercise,the heart rate and blood pressure in the SHR-EX group were significantly lower than those in the SHR-SED group(P<0.05).Compared with the SHR-SED group,the maximum rising rate of left ventricular pressure(+dp/dtmax),ejection fraction(EF)and slope Ees of end-systolic pressure-volume relationship curve(ESPVR)in the SHR-EX group were significantly increased(P<0.05),and the absolute value of the maximum decreasing rate of ventricular pressure(-dp/dtmax)and stroke volume(SV)were significantly increased(P<0.01);ventricular end-systolic pressure(ESP),end-diastolic pressure(EDP)and effective arterial elasticity(Ea)were significantly decreased(P<0.01);isovolumic diastolic constant(Tau)was significantly reduced(P<0.05).Compared with the SHR-SED group,the myocardial tissue fibrosis in the SHR-EX group was reduced,and the CVF% decreased significantly(P<0.01).Compared with WKY-SED group,the number of myocardial caspase 3 in WKY-EX group decreased significantly(P<0.05).Compared with the SHR-SED group,the number of myocardial caspase 3 in the SHR-EX group was significantly reduced(P<0.05),and the number of TUNEL-positive particles was significantly reduced(P<0.01).The results of Western Blot showed that the expressions of NF-κB P65 and Bax protein and the ratio of Bax/Bcl-2 in the WKY-EX group were significantly lower than those in the WKY-SED group(P<0.05),while the expressions of IκBαand HSP70 protein were significantly increased(P<0.01).The expression of Bcl-2 protein increased significantly(P<0.05).Compared with the SHR-SED group,the expressions of ox-CaMKIIδ,NF-κB P65 and Bax protein in the SHR-EX group were significantly decreased(P<0.05).The ratio of Bax/Bcl-2 was significantly decreased(P<0.01).The expressions of IκBαand Bcl-2 protein were significantly increased(P<0.05),and the expression of HSP70 protein was significantly increased(P<0.01).Conclusion:Aerobic exercise inhibits cardiomyocyte apoptosis by down-regulating SHR myocardium ox-CaMKⅡδ,while activating anti-apoptosis-related signal pathways,thus improves cardiac function,which may be one of the mechanisms of exercise-induced heart remodeling in hypertension.