美托洛尔下调CaMKⅡδC—p38通路对异丙肾上腺素诱导心力衰竭的保护机制

The protective effects of metoprolol in Cardiomyopathy produced by isoproterenol are associated with attenuation of the CaMKⅡδC and p38 MAPK

目的研究美托洛尔下调CaMKⅡδC-p38通路对异丙肾上腺素诱导心力衰竭的保护机制。方法40只SD大鼠随机分成四组,正常对照组(Control)、异丙肾上腺组(Iso)、异丙肾上腺+美托洛尔组(Iso+Meto)和美托洛尔组(Meto),每组10只,所有动物均自由进食进水。(1)Iso组大鼠背部皮下注射Iso 5 mg/(kg·d),连续10 d;对照组背部皮下注射相同体积的生理盐水;Iso+Meto组大鼠背部皮下注射Iso 5 mg/(kg·d),连续10 d,在背部皮下注射Iso前1天开始Meto 10 mg/(kg·d)灌胃,连续4周;Meto组给予10 mg/(kg·d),连续4周灌胃;(2)所有大鼠饲养4周后,采用Millar P-V Loop导管经颈动脉插管至左心室,使用Powerlab生理记录系统测量血流动力学相关指标;统计各组大鼠心脏重量和心脏重量/体重比值;(3)TUNEL法和Caspase-3活性检测心肌细胞凋亡;(4)ELISA分析CAMKⅡ活性;(5)Western blot检测CaMKⅡδ、p-CaMKⅡδ、CaMKⅡδC和MAPKs家族(p-38、JNK、ERK)、和凋亡相关基因Bcl-2/Bax的蛋白表达水平。结果40只SD大鼠实验过程精神状态好,进食进水正常,无呼吸困难及水肿。(1)Iso和Meto干预SD大鼠心脏重塑和血流动力学指标有显著改变,与Control组相比,Iso组心脏重量和心脏重量指数明显增加(P<0.05);而Iso+Meto组心脏重量和心脏重量指数明显低于Iso组(P<0.05);大鼠体重、肝重和肺重四组间也无明显差异。大鼠血流动力学指标心率(HR)、平均动脉血压(MBP)和左室舒张末压(LVEDP)Iso组明显高于Control组;但左室压力变化速率(LV±dp/dt max) Iso组明显低于Control组(P<0.05);而Iso+Meto组HR、MBP和LVEDP明显低于Iso组(P<0.05),但Iso+Meto组±dp/dtmax明显高于Iso组(P<0.05);(2)Iso组SD大鼠心肌细胞TUNEL阳性细胞数和Caspase-3活性明显高于Control组(P<0.05);Western杂交分析检测Iso组抗凋亡蛋白bcl-2表达明显低于Control组(P<0.05),而促抗凋亡蛋白Bax表达明显高于Control组;(3)CaMKⅡ活性分析结果显示Iso组明显高于Control组(P<0.05)。说明Iso诱导了明显的心肌细胞凋亡,并且心肌细胞凋亡和CaMKⅡ活性明显正相关;(4)CaMKⅡδ异构体、MAPKs家族在β1-AR持久兴奋的SD大鼠心肌表达情况,我们用Western blot方法分析结果显示Iso组CaMKⅡδC和p-CaMKⅡδ、p38 MAPK、JNK蛋白表达明显高于Control组(P<0.05),但与Control组比较,Iso组ERK蛋白表达明显减少(P<0.05)。与Iso组相比,β1-AR阻滞剂Meto可减少CaMKⅡδC和p-CaMKⅡδ、p38 MAPK、JNK蛋白表达,增加ERK蛋白表达(P<0.05)。结论(1)持久兴奋β1-AR激活CaMKⅡδC-p38通路诱导心肌细胞凋亡,导致心肌重塑、心力衰竭;(2)β1-AR阻断剂美托洛尔可阻断β1-AR持久激活CaMKⅡδC—p38导致心肌细胞凋亡途径,对心脏有保护效应。

Objectives To investigate the protective effects of metoprolol in Cardiomyopathy are associated with attenuation of the CaMKIIδC and p38 MAPK. Methods 40 male SD rats were randomly divid- ed into 4 groups： 1 ） Control group, 2） Isoproterenol group,Iso 5 mg/（kg · d）, for 10d; 3） Isoproterenolplus Metoprolol group, Iso 5 rag/（ kg · d）, for 10d ＋ Meto 10 mg/（ kg · d ）, for 4weeks and 4 ） Metoprolol group, Meto 10 mg/（ kg · d）, for 4 weeks. Heart function was measured by catheterization. Myocyte apoptosis was determined by TUNEL and caspase-3 activity, CaMKII activity was measured by ELISA kit, while CaMKIIδC, MAPKs family （ p-38, JNK, ERK）, and Bcl-2/Bax were measured by Western blot. Results Isoproterenol produced progressive LV dilation, systolic dysfunction, increased in heart weight and myocyte apoptosis. These changes were associated with increased phosphorylation of CaMKIIδC, p38MAPK, JNK and reduced phosphorylation of ERK. Metoprolol administration to cardiomyopathy produced by isoprot- erenol exhibited activation of the ERK pathway and attenuated the phosphorylation of CaMKIIδC, p38 MAPK and JNK. Metoprolol administration also increased Bcl-2/Bax and decreased cell death. Conclusions The cardiac apoptotic effects of sustained β1 -adrenergic stimulation are associated with the increased phosphorylation of CaMKIIδ, p38, JNK and activation of the CaMKIδC. Metoprolol exert cardioprotective effects to cardiomyopathy produced by isoproterenol via attenuation the phosphorylation of CaMKIIδ, p38, JNK and CaMKIIδC, increased the phosphorylation of ERK, and reduced cell death.