缬沙坦对心力衰竭时心肌钙调节蛋白作用的研究

Effect of valsartan on myocardial Ca^(2+) regulatory proteins in junior rat with heart failure

目的:研究缬沙坦对心力衰竭(HF)时肌浆网(SR)钙离子调节蛋白的保护作用。方法:采用腹主动脉-下腔静脉造瘘术建立幼鼠HF模型,术后8周随机分为2组:未治疗组和治疗组(管饲缬沙坦),另设假手术组。对各组幼鼠左室组织提取SR膜,Western blotting法测定受磷蛋白(PLB)磷酸化水平,荧光分光光度仪检测钙离子(Ca2+)的重吸收和渗漏。结果:与假手术组比较,未治疗组体重降低(P<0.01),左室相对质量(LVRW)、右室相对质量(RVRW)均明显升高(P<0.01),16位丝氨酸磷酸化-受磷蛋白(Ser-16-PLB)蛋白量显著降低(P<0.01);与未治疗组比较,治疗组体重(P<0.01)升高,LVRW、RVRW降低(P<0.01),Ser-16-PLB蛋白量明显升高(P<0.01),接近假手术组水平;3组总PLB蛋白量差异无统计学意义(P>0.05);若分别向含有3组SR的缓冲液加入ATP(0.5mmol/L)后,未治疗组的Ca2+的重吸收量同假手术组和治疗组比较,明显降低(P<0.01);当分别向含有3组SR的缓冲液中加入毒胡萝卜内酯(1μmol/L)后,未治疗组同另外2组比较出现明显的Ca2+渗漏(P<0.01);而当毒胡萝卜内酯(1μmol/L)和FKBP抑制剂FK506(30μmol/L)一起加入3组SR的缓冲液中,假手术组、治疗组出现明显的Ca2+渗漏(P<0.01),而未治疗组较单独加入毒胡萝卜内酯时Ca2+渗漏仅轻微增多(P>0.05)。结论:HF时心肌组织肥厚,细胞Ca2+重吸收量降低、渗漏明显,心肌PLB磷酸化水平显著降低。缬沙坦一方面抑制Ca2+渗漏,另一方面提高PLB的磷酸化水平,恢复Ca2+-ATP酶的Ca2+重吸收能力,提高心脏功能并有效抑制心室重塑。

Objective：To observe the changes of Ca^2＋ regulatory proteins in the sarcoplasmic reticulum（SR）. known as Ca^2＋-ATPase（SERCA2a） , phospholamban（PLB） in junior rat with heart failure （HF） and the protective effect of angiotensin Ⅱ receptor blockade（va-lsartan） on these proteins. Method： The animal model of congestive heart failure was established by fistulation of abdominal aorta, and inferior vena cava. Five weeks old rats were randomly divided into 3 groups： ① HF group without treatment （n= 20）; ②HF group treated with valsartan （n = 15）; ③Sham-operated group （n= 15）. Valstan was administered through direct gastric gavage after 8 weeks of operation, then the rats were killed 4 weeks later, SR was fractionated with velocity centrifugation. Ser-16-PLB and PLB were detected by immunohistochemical technique. The time course of Ca^2＋ uptake and leak were determined by fluorescent spectrophotometer. Result：Compared with the sham-operated group, the body weight decresed（P〈 0.01 ） white the left ventricular relative weigh （LVRW） and the right ventricular relative weigh （RVRW） were all significantly increased （P〈0.01） in HF group without treatment. In contrast, both of the LVRW and the RVRW were decreased （P〈0.01） in the group of HF treated with valsartan when compared with HF group. Mean protein level （detected by Western blotting） of Ser-16-PLB in HF group were significantly lower （P〈0.01） than that in sham-operated group, and it increased almost to the normal level in HF group after treated with valsartan. After adding ATP to the different buffer including different from SR three groups, Ca^2＋ uptake were significantly decreased（P〈0.01） in HF group, compaired with those in sham-operated group and group treated with valsartan After adding thapsigargin to the different buffer of those three groups,there were fewer Ca^2＋ leak in sham-operated group and group treated with valsartan （P〈0.01） ,while after adding FK506 and thapsigargin together to the buffer including SR of three groups, there were marked Ca^2＋ leak in sham-operated group and HF group treated with valsartan （P〈0.01） and no additional increase in Ca^2＋ leak in HF group compared with the value when only thapsigargin was added（P〉0.05）. Conclusion：There were less Ca^2＋ uptake and more Ca^2＋ leak in HF. Valsartan could not only improv Ca^2＋ uptake by increasing ser-16-PLB level, but also prerent protein A（PKA） from phosphorylating, rendering the SR less susceptible to Ca^2＋ leak.