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目的观察美托洛尔对家兔心力衰竭(心衰)心肌细胞钙调控蛋白表达的影响,探讨其改善心衰的可能分子机制。方法 30只家兔随机分为3组,即假手术组(n=11)、心衰组(n=11)、美托洛尔干预组(n=8)。心衰组和美托洛尔干预组家兔先建立实验性主动脉瓣关闭不全,2周后行腹主动脉缩窄,利用心脏多普勒观察术前后家兔心脏功能的变化,共观察6周。采用常规酶解法分离心室肌细胞,经 Fluo-3/AM 负载后,利用激光共聚焦显微技术,观察咖啡因诱导的钙瞬变过程中细胞内钙浓度的动态变化。从左心室心肌组织提取膜蛋白后,采用 Western blot 法测定钙调控蛋白表达水平,并应用 UVIDoc 成像仪进行蛋白表达半定量分析。结果假手术组与心衰组射血分数分别为(72.6±5.0)%、(45.7±3.0)%(P<0.01),咖啡因诱导的钙瞬变幅度(FI)分别为43.5±6.2、16.0±3.5(P<0.01),峰值到达时间分别为(52.2±7.4)s、(129.8±14.5)s(P<0.01),兰尼碱受体表达量分别为0.203±0.021、0.106±0.007(P<0.01)及肌浆网钙泵与钠钙交换体(sERCA2a/NCX)表达量比值分别为1.96±0.12、1.22±0.23(P<0.01)。与心衰组相比,美托洛尔干预组射血分数值[(60.2±5.1)%,P<0.05]明显增加,钙瞬变幅度增加(32.8±5.4,P<0.05),峰值到达时间缩短(91.4±10.9)s,P<0.05],兰尼碱受体表达量(0.164±0.016,P<0.05)和 SERCA2a/NCX 表达量比值(1.68±0.17,P<0.05)增加。结论美托洛尔可延缓心衰心肌细胞钙调控蛋白表达的改变,进而改善钙瞬变,这可能是长期应用β受体阻滞剂改善心衰患者心功能的分子机制之一。
Objective To investigate the effect of metoprolol on the expression of calcium regulatory protein in cardiomyocytes of rabbits with heart failure (HF) and to explore the possible molecular mechanism of metoprolol in improving heart failure. Methods Thirty rabbits were randomly divided into three groups: sham operation group (n = 11), heart failure group (n = 11) and metoprolol intervention group (n = 8). Heart failure group and metoprolol intervention group rabbits to establish experimental aortic valve regurgitation, abdominal aortic constriction after 2 weeks, the use of heart Doppler observation of rabbit before and after the change of cardiac function, were observed for 6 weeks. The ventricular myocytes were isolated by conventional enzymatic method. After the cells were loaded with Fluo-3 / AM, the changes of intracellular Ca2 + concentration during Ca2 + -induced calcium transient were observed by laser confocal microscopy. After membrane protein was extracted from left ventricular myocardium, the expression level of calcium regulatory protein was determined by Western blot and the protein expression was semi-quantitatively analyzed by UVIDoc imager. Results The ejection fraction of sham-operation group and heart failure group were (72.6 ± 5.0)% and (45.7 ± 3.0)% respectively (P <0.01), and the caffeine-induced calcium transients were 43.5 ± 6.2 and 16.0 ± 3.5 (P <0.01) and peak arrival time were (52.2 ± 7.4) s and (129.8 ± 14.5) s respectively (P <0.01) .The expression of ryanodine receptor were 0.203 ± 0.021 and 0.106 ± 0.007 <0.01) and sACCA2a / NCX were 1.96 ± 0.12,1.22 ± 0.23 (P <0.01), respectively. Compared with heart failure group, the value of ejection fraction of metoprolol group [(60.2 ± 5.1)%, P <0.05] and the amplitude of calcium transient increase (32.8 ± 5.4, P <0.05) (91.4 ± 10.9) s, P <0.05]. The expression of ryanodine receptor (0.164 ± 0.016, P <0.05) and the ratio of SERCA2a / NCX expression (1.68 ± 0.17, P <0.05) increased. Conclusion Metoprolol can delay the alteration of calcium-regulated protein expression in cardiomyocytes with cardiac failure and improve calcium transients. This may be one of the molecular mechanisms that long-term use of β-blockers to improve cardiac function in patients with heart failure.