兔收缩性和舒张性心衰模型的心功能及心肌钙调节相关蛋白表达和活性比较

Comparison of cardiac function and expression and activity of myocardial calcium regulatory proteins in rabbit systolic and diastolic heart failure models

本文旨在探讨兔收缩性心衰(systolic heart failure,SHF)和舒张性心衰(diastolic heart failure,DHF)模型之间的心功能及心肌钙调节相关蛋白表达和活性的差异。选取新西兰白兔,随机分为3组:DHF组(行腹主动脉缩窄术)、SHF组(行腹主动脉缩窄术联合主动脉瓣毁损术)、假手术组(行假手术)。通过超声心动图和血流动力学检查心功能的改变,RT-PCR检测肌浆网钙泵(SERCA2a)和受磷蛋白(PLB)mRNA水平的表达,Western blot检测SERCA2a、PLB、16位丝氨酸磷酸化-PLB(pSer-16-PLB)、蛋白激酶A(PKA)蛋白水平的表达,并确定PLB的磷酸化水平,无机磷酸根法检测肌浆网钙泵(SERCA2a)的活性,γ-32P掺入法检测PKA的活性。结果显示,与假手术组相比,DHF组左室壁厚度明显增加,舒张功能显著下降,左室舒张末压力显著升高;而SHF组左室腔明显增大,收缩功能显著下降,左室舒张末压力也显著升高(P<0.05或P<0.01)。与假手术组相比,DHF组和SHF组SERCA2a的表达均明显下降(P<0.05),PKA的表达和活性均显著升高(P<0.05或P<0.01),两心衰组之间无显著差异。SHF组总PLB、pSer-16-PLB蛋白表达量及其磷酸化水平和SERCA2a活性均较假手术组和DHF组明显降低(P<0.05或P<0.01);而与假手术组相比较,DHF组总PLB、pSer-16-PLB蛋白表达量无明显差异,但其PLB的磷酸化水平和SERCA2a活性明显升高(P<0.01)。以上结果表明本实验成功复制出两种不同的心衰模型,且两者的部分钙调节蛋白存在着表达和活性的差异。

The aim of the present study is to investigate the differences in cardiac function, and the expression and activity of calcium regulatory proteins between rabbit systolic heart failure （SHF） and diastolic heart failure （DHF） models. New Zealand white rabbits were randomly divided into three groups： sham operation （SO） group, DHF group （receiving abdominal aortic constriction） and SHF group （receiving aortic valve destruction and abdominal aortic constriction）. The cardiac function was detected by echocardiographic and hemodynamic assays. The mRNA expression levels of sarcoplasmic reticulum Ca（2＋） ATPase 2a （SERCA2a） and phospholamban （PLB） were evaluated by RT-PCR. The protein expression levels of SERCA2a, PLB, phosphoserine 16-PLB （pSer-16-PLB） and protein kinase A （PKA） were evaluated by Western blot, and the phosphorylation status of PLB was determined by the ratio of pSer-16-PLB protein level to that of PLB. The activity of SERCA2a was measured through inorganic phosphate. The activity of PKA was measured by gamma-（32）P ATP-binding assays. Compared with SO group, there were significantly increased ventricular wall thickness, raised left ventricular end diastolic pressure （LVEDP）, reduced diastolic function in DHF group （P〈0.05 or P〈0.01）, and significantly increased ventricular cavity size and LVEDP, reduced systolic function in SHF group （P〈0.05 or P〈0.01）. The expression levels of SERCA2a in DHF and SHF groups were lower than that in SO group （P〈0.05）, while the expression and activity of PKA in DHF and SHF groups were higher than that in SO group （P〈0.05 or P〈0.01）, and there was no significant difference between DHF and SHF groups. The expression levels of PLB and pSer-16-PLB as well as the phosphorylation status of PLB and activity of SERCA2a in SHF group were lower than those in DHF and SO groups respectively. Posing a contrast, the phosphorylation status of PLB and activity of SERCA2a in DHF group were higher than that in SO group （P〈0.05）. These results indicate that the SHF and DHF models were successfully established, and there are some differences in the expression and activity of calcium regulatory proteins between two models.