【作者】 米青；

【导师】 易岂建；

【作者基本信息】 重庆医科大学 ， 儿科学， 2008， 硕士

【摘要】 [研究背景]心力衰竭（heart failure,HF）,又称心功能不全,是指在正常静脉回流情况下,心脏的排血量不能满足机体代谢需要的一种复杂的临床综合征。近半个世纪以来,对HF发病机理及治疗研究取得很大进展。目前针对心衰患者给予了相对完善的治疗方案,但心衰的发病率和死亡率仍然呈现上升趋势。纽约心脏协会（New York Heart Association,NYHA）心功能分级Ⅲ-Ⅳ的患者两年死亡率高达50%。在儿科临床,HF是最常见的危重急症之一,也是儿科临床的重要死亡原因。因此进行HF发病机理及治疗药物的深入研究具有重要的理论价值和临床指导意义。心肌细胞舒缩功能的维持与胞内钙离子内环境稳态是密不可分的。兴奋-收缩偶联（excitation-contraction coupling,E-C coupling）过程中,细胞膜的去极化使膜上的L型钙通道(L-type Ca²⁺ channel ,LTCC)开放,少量的Ca²⁺内流。内流的Ca²⁺进一步激活与LTCC紧挨着的肌浆网（Sarcoplasmic reticulum ,SR）上的Ca²⁺释放通道-2型兰尼碱受体（type 2 ryanodine receptor,RyR2）,RyR2构型改变,通道开放,大量Ca²⁺经RyR2从SR释放出来,当心肌细胞内游离[Ca²⁺]达10-5mol/l时肌钙蛋白被激活,肌钙蛋白Ⅰ构象改变使肌球蛋白和肌动蛋白相互接触,引起细胞的收缩;舒张期开始于Ca²⁺从肌钙蛋白上的解离,大部分（70﹪）被SR上受到受磷蛋白（phospholamban ,PLB）磷酸化水平调控的Ca²⁺-ATP酶(Ca²⁺-ATPase,SERCA2a)重吸收到SR内,少部分（28﹪）通过胞膜上的Na+/Ca²⁺交换体泵出到细胞外,当胞浆内游离Ca²⁺浓度降低达10-7mol/L时,肌球蛋白和肌动蛋白分离,引起心肌细胞舒张,形成一个完整的心脏收缩-舒张过程。有研究提示心肌细胞膜上G-蛋白偶联受体（Gαq-coupled receptors ,G-R）和β-肾上腺素受体（?-adrenergic receptor,β-AR）信号通路的过度激活可导致SR膜上SERCA2a、RyR2及其相关蛋白- FKBP12.6（FK506 binding protein,FKBP12.6）结构和功能异常,使细胞内Ca²⁺转运失衡,导致心肌收缩和舒张功能异常。目前关于小儿HF时Ca²⁺转运的改变以及血管紧张素Ⅱ受体拮抗剂（AngiotensinⅡreceptor antagonist ,ARB）类药物对HF时RyR2和SERCA2a的调节尚未见研究报道。[目的]观察HF时SR膜PLB磷酸化状态,SERCA2a酶活性及Ca²⁺转运变化,并探讨ARB类（缬沙坦,Valsartan）对其干预作用。[方法] 5周龄Wistar幼鼠,腹主动脉-下腔静脉造瘘法建立心衰模型,术后8周随机分为2组:心衰组和缬沙坦治疗组,另设假手术对照组。缬沙坦灌胃给药,每日观察幼鼠的呼吸、皮毛颜色、活动量等发育情况,4周后超声检测幼鼠心功能:左室舒张末期内径（left ventricular internal dimension diastole ,LVIDd）、左室收缩末期内径（left ventricular internal dimension systole ,LVIDs）、左室舒张末期容积（left ventricular end-diastolic volume,LVEDV）、左室收缩末期容积leftventricular end-systolic volume,LVESV)、左室射血分数（left ventricular ejection fraction,LVEF）、左室短轴缩短率（left ventricular fractional shortening,LVFS）后处死大鼠,心尖组织行HE染色,观察形态学改变;左心室组织提取SR膜,Western blotting法测定PLB磷酸化水平;定磷法测定SERCA2a酶活性;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（n=15）比较,HF组（n=15）幼鼠LVIDd、LVIDs、LVEDV、LVESV均明显升高（P<0.01）, LVEF、LVFS均明显降低（P<0.01）,与HF组比较,缬沙坦治疗组（n=20）LVIDd、LVIDs、LVEDV、LVESV均明显降低（P<0.01）,LVFS、LVEF升高（P<0.01）;与假手术组比较,HF组体重（body weight,BW）降低（P<0.01）,左心室相对质量（left ventricular relative weight ,LVRW）、右心室相对质量（right ventricular relative weight,RVRW）均明显升高（P<0.01）,缬沙坦治疗组BW（P<0.01）升高,LVRW、RVRW降低（P<0.01）;光镜下HF组心肌细胞排列紊乱,水肿、变性、局灶坏死,缬沙坦治疗组的病理改变明显减轻而假手术组幼鼠未见HF组的病理改变;三组大鼠SR膜SERCA2a活性比较结果显示,HF组的SERCA2a活性较假手术组明显降低（P<0.01）,而经缬沙坦治疗后,酶活性接近假手术组（P>0.05）;与假手术组比较,HF组16位丝氨酸磷酸化-受磷蛋白（Serine Phosphate 16-PLB,Ser-16-PLB）蛋白量显著降低（P<0.01）,与HF组比较,缬沙坦治疗组Ser-16-PLB蛋白量明显升高（P<0.01）,接近假手术组水平;三组总PLB蛋白量差异无统计学意义（P>0.05）;若分别向含有三组SR的缓冲液加入三磷酸腺苷（adenosine triphosphate,ATP）（0.5mmol/L）后.,HF组的Ca²⁺的重吸收量同假手术组和缬沙坦治疗组比较,明显降低（P<0.01）;当分别向含有三组SR的缓冲液中加入thapsigargin（1μmol/L）后,HF组同另外两组比较出现明显的Ca²⁺渗漏（P<0.01）;而当thapsigargin （1μmol/L）和FKBP12.6抑制剂-FK506（30μmol/L）一起加入三组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HF组较单独加入thapsigargin（1μmol/L）时Ca²⁺渗漏仅轻微增多（P>0.05）。[结论] HF时心功能显著降低,心肌组织肥厚,心肌细胞SERCA2a活性下降,心肌PLB磷酸化水平显著降低,细胞Ca²⁺吸收量降低、渗漏明显。缬沙坦一方面通过抑制去甲肾上腺素（Norepinephrine,NE）的释放,阻止蛋白激酶A（Protein kinase A,PKA）对RyR2的过度磷酸化作用,恢复其与FKBP12.6的紧密结合,从而抑制Ca²⁺渗漏,另一方面提高PLB的磷酸化水平,恢复SERCA2a的Ca²⁺重吸收能力,提高心脏功能并有效抑制心室重塑。更多还原

【Abstract】 [Background] Heart failure （HF） is a complicated clinical syndrome that leads to a disturbance of the normal pumping of blood to the peripheral organs to meet the metabolic demands of the body. For half a century, experts have been studying much about HF on pathogenesis and medicines, but the pathogenesis of chronic HF has still not been completely understood and the morbility and mortality of HF still increased. Patients with severe congestive heart failure （CHF）, cardiac functionⅢ-Ⅳclass of NYHA, two-year mortality reached 50%. In pediatrics, HF is one of most common serious emergency cases and often result in death.So it’s necessary to further explore the pathogenesis and look for effective medicine in children with HF.The homeostasis of Calcium(Ca²⁺) ions in cardiac muscle cell is important to maitain cardiac function.In cardiac excitation-contraction（E-C） coupling, a small amount of Ca²⁺ first enters through the L-type Ca²⁺ channel（LTCC） during membrane depolarization. This Ca²⁺ influx triggers a large-scale Ca²⁺ release through the Ca²⁺ release channel of the sarcoplasmic reticulum（SR）, referred to as the ryanodine receptor（RyR）, when the level of Ca²⁺ in cardiac muscle cell reaches 10-5mol/l, it binds to the troponin C within the myofilaments, which induces activation of the myofilaments and consequent muscle contraction. Relaxation is initiated by dissociation of Ca²⁺ from tropnin C, followed by its reuptake into the SR through phospholamban-regulated（PLB-regulated） Ca²⁺-ATPase （SERCA2a） （70﹪） and subsequent trans-sarcolemmal Ca²⁺ removal through the Na+/ Ca²⁺ exchanger（NCX）（28﹪）. When the content of Ca²⁺ decreases to 10-7mol/l, myosin and actin separates, which resulting in the relaxation of cardiac muscle cell at last.Several lines of evdience have accumulated demonstrating that changes in activiy and express profile of Ca²⁺ handling proteins,in particular the SERCA2a are thought to cause an overall reduction in the amount of SR- Ca²⁺ available for contraction. In addition, abnormal Ca²⁺-leakage through the RyR2, has been demonstrated as a possible trigger for the development of heart failure.In those studies of heart failure, Ca²⁺-leak was found to be induced by the dissociation of FK506 binding protein （FKBP12.6） from the RyR2 as a result of the hyperphorylation of the RyR2.Data from our laboratory and others suggested that ?-adrenergic receptor （?-AR） blockade can correct the defective interaction of FKBP12.6 and RyR2 which is triggered by the protein kinase A （PKA）-mediated hyperphosphorylation of RyR2. Indeed this correction leads to a prevention of Ca²⁺-leak from SR. AngiotensinⅡreceptor blocker（ARB） not only prevents hypertrophy and/or interstitial fibrosis in patients with HF, but also attenuates the downregulation of SERCA2a and improves intracellular Ca²⁺-handling.However, it donesn’t know whether it can increase the affinity between Ca²⁺ and SERCA2a and correct the defective interaction of FKBP12.6 and RyR2. There are less reports about change of Ca²⁺ homeostasis in pediatric HF and the effect of ARB on SERCA2a and RyR2 so far.[Objective] To investigate the changes of Ca²⁺ regulatory proteins of SR, known as SERCA2a、PLB、RyR2 in junior rat with HF and the protective effect of angiotensinⅡreceptor blockade（Valsartan） on these proteins.[Methods] 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=15）; （2） HF group treated with Valsartan （n=20）; （3） Sham-operated group （n=15）. Valstan was administered through direct gastric gavage after 8 weeks of operation, The high frequency ultrasound was performed after 4 weeks of treatment ,then the rats were killed, the atria were removed, the right and left ventricles were dissected, separated, and weighed. Some part of tissues were used to assess Cardiac histological changes after hematoxylin and eosin（HE） staining;SR was fractionated with velocity centrifugation, Serine Phosphate 16-PLB （Ser-16-PLB） and PLB were detected by immunohistochemical technique;Enzyme activity of SERCA2a in SR was detected with ultraviolet spectrophotometer（UV）. the time course of Ca²⁺ uptake and leak were determined by fluorescent spectrophotometer.[Results] Compared with the sham-opreated group, left ventricular internal dimension diastole （LVIDd）、left ventricular internal dimension systole（LVIDs）、left ventricular end-diastolic volume（LVEDV）、left ventricular end-systolic volume（LVESV）、left ventricular ejection fraction（LVEF）、left ventricular fractional shortening（LVFS）were all significantly increased （P<0.01）, LVEF、LVFS were decreased （P<0.01） in HF group.LVIDd、LVIDs、LVEDV、LVESVwere all prominently decresed （P<0.01） ,LVEF、LVFS were inreased（P<0.01） in group of HF treated with Valsartan;Compared with the sham-operated group, the body weight decresed（P<0.01）,left ventricular relative weigh（LVRW） and right ventricular relative weigh （RVRW） were all significantly increased（P<0.01）in HF group without treatment.LVRWand RVRW were decreased （P<0.01）in the group of HF treated with Valsartan when compared with that of HF group.Mean protein level （detected by Western blotting） of Ser-16-PLB in HF group were significantly lower（P<0.01） than that of sham-operated group, and it increased almost to normal level in HF group after treated with Valsartan.Enzyme activity of SERCA2a in HF group without treatment were significantly lower （P<0.01） than that of other groups.After adding ATP to the buffer including SR of three groups ,Ca²⁺ uptake were significantly decreased（P<0.01） in HF group,compaired with that of sham-operated group and group treated with Valsartan;After adding thapsigargin to the buffer of three groups,there were fewer Ca²⁺ leak in sham-poerated 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 markedly Ca²⁺ leak in sham-operated group and HF group treated with Valsartan （p<0.01）,but there was no additional increase in Ca²⁺ leak in HF group compared with the value when only thapsigargin was added（P>0.05）.[Conclusion] Heart function and enzyme activity of SERCA2a decrease in junior rat with heart failure, there are less Ca²⁺ uptake and more Ca²⁺ leak in HF. Valsartan not only improved Ca²⁺ uptake by increasing ser-16-PLB level, but also prevented protein kinase A（PKA） from hyperphosphorylating, rendering the SR less susceptible to Ca²⁺ leak,contributing to the recovery of contractility and relaxation of the cadiomyocyte.更多还原