肌浆网钙泵和磷酸受纳蛋白在舒张性心衰患者中的表达

目的　研究肌浆网钙泵和磷酸受纳蛋白在舒张性心衰 (DHF)患者心肌细胞内 Ca2 调节中的作用。方法　采用三电极直流等离子体原子发射光电直读光谱法测定心肌细胞内 Ca2 含量 ,采用免疫组化和 Western blot技术测定 DHF患者和正常对照组肌浆网钙泵 (Ca2 -ATPase)和磷酸受纳蛋白表达的变化。结果　 DHF患者心肌细胞内 Ca2 含量明显高于与正常组 (1 1 63.88± 568.67μg/ ml,2 4 7.75± 1 4 6.86μg/ml,P<0 .0 1 ) ;Ca2 - ATPase蛋白的相对含量明显低于对照组 (0 .76± 0 .1 1 ,1 .0 1± 0 .0 3,P<0 .0 5) ;磷酸受纳蛋白的相对含量无显著性差异(0 .98± 0 .0 4 ,1 .0 0± 0 .0 2 )。结论　 SR Ca2 - ATPase表达减低是导致心肌细胞内 Ca2 超负荷和 DHF发生的重要因素     

舒张性心力衰竭患者Ca2+调控蛋白基因转录和蛋白质表达的研究

目的　探讨心肌细胞内Ca2 超负荷及Ca2 调控蛋白在舒张性心力衰竭 (DHF)患者发生中的作用。方法　采用RT PCR和Westernblot技术测定DHF患者和正常对照组Ca2 调控蛋白mRNA转录和蛋白质表达的变化。结果　与正常组相比 ,DHF患者心肌细胞内Ca2 含量提高 3倍以上 (116 3.88± 5 6 8.6 7vs 2 47.75± 146 .86 ,P <0 .0 1) ;肌浆网 (SR)Ca2 ATPasemRNA水平减低 33.6 4%(0 .71± 0 .19vs 1.0 7± 0 .11,P <0 .0 5 ) ,细胞膜L型Ca2 通道mRNA水平减低 30 .19% (0 .74± 0 .2 0vs1.0 6± 0 .13,P <0 .0 5 ) ,SR磷酸受纳蛋白和兰尼碱受体、肌集钙蛋白的mRNA转录无显著性差异 (P >0 .0 5 ) ;Ca2 ATPase蛋白的相对含量明显低于对照组 (0 .76± 0 .11vs 1.0 1± 0 .0 3,P <0 .0 5 ) ;磷酸受纳蛋白的相对含量无显著性差异 (0 .98± 0 .0 4vs 1.0 0± 0 .0 2 ,P >0 .0 5 )。结论　SRCa2 ATPase基因转录和蛋白质表达减低以及细胞膜L型Ca2 通道基因转录减低是导致心肌细胞内Ca2 超负荷和DHF发生的主要分子机制之一     

心房颤动电重构心房肌Ca2+调控蛋白异常与解剖学改变关系的研究

目的探讨心房肌Ca2+调控蛋白(calcium handling proteins)在心房颤动(atrial fibrilation,AF)电重构中的作用及与解剖学结构改变的关系.方法测定10例慢性AF患者和6例对照组心房肌Ca2+含量和细胞膜L型Ca2+通道(L-type calcium channel)、肌浆网钙泵(sarcoplasmic reticulum calciumadenodinetriphosphatase,SR Ca2+-ATPase)、磷酸受纳蛋白(phospholamban)、兰尼碱受体(ryanodine receptor)和肌集钙蛋白(calsequestrin)的信使核糖核酸(messenger ribonucleic acid,mRNA)表达;测量AF患者左、右心房内径、二尖瓣口面积和肺动脉收缩压.结果与对照组比较,AF患者心肌细胞内Ca2+含量增加[(1 330±770)μg/ml vs(302±31)μg/ml,P<0.01];细胞膜L型Ca2+通道、SR Ca2+-ATP ase和兰尼碱受体mRNA下调[(0.65±0.30)v(0.97±0.19),P<0.01;(0.73±0.13)vs(1.10±0.11),P<0.001;(0.71±0.25)vs(0.90±0.13),P<0.05].SRCa2+-ATPasemRNA表达与左心房内径中度负相关(r=-0.56,P<0.05);与二尖瓣口面积正相关(r=0.70,P<0.05);细胞膜L型Ca2+通道mR-NA表达与二尖瓣口面积显著正相关(r=0.84,P<0.01).结论频率相关的细胞内Ca2+超负荷可能是AF电重构的始动因素,心房肌Ca2+调控蛋白异常是Ca2+超负荷的分子生物学机制,Ca+调控蛋白mRNA表达异常与左心房解剖学改变之间存在内在联系.     

肌浆网/内质网钙调控在心血管疾病中的研究进展

肌浆网/内质网(SR/ER)是细胞内重要的钙储库及调控系统,主要通过Ca2+释放通道雷尼丁受体(RyR)和1,4,5-三磷酸肌醇受体(IP3R),以及Ca2+摄取蛋白肌浆网Ca2+-ATP酶(SERCA)和基质交感分子1(STIM1)等调节钙信号,维持细胞内钙稳态。研究发现,SR/ER钙平衡对细胞正常生理功能的发挥和信息传递非常重要,SR/ER钙稳态失调与缺血性心脏病、心肌肥厚、高血压及心力衰竭等心血管疾病的发生发展密切相关。本文就SR/ER钙调控在心血管疾病中的研究进展作一概述,重点阐述SR/ER钙离子通道和相关调控蛋白对细胞内钙信号的调节机制,以期为临床心血管疾病的治疗提供重要策略和潜在靶点。     

钙网蛋白介导缺氧预处理对心脏保护机制的研究

目的探讨缺氧预处理(HPC)对于心肌钙网蛋白表达与肌浆网钙稳态的影响及其信号转导机制。方法选择SD大鼠22只,随机分为假手术组6只,模型组8只,HPC组8只。复制SD大鼠HPC和心肌梗死模型,检测左心室压力最大上升速率和最大下降速率(±dp/dtmax)、TTC法测定心肌梗死面积,差速离心法制备心肌肌浆网并鉴定其纯度,以Millipore滤过法测定肌浆网Ca2+摄取活性和肌浆网Ca2+释放速率,Western blot检测钙网蛋白、p38丝裂原活化蛋白激酶和磷酸化p38丝裂原活化蛋白激酶水平。结果与假手术组比较,模型组+dp/dtmax和-dp/dtmax分别下降39%和46%(P<0.05);与模型组比较,HPC组分别升高43%和59%(P<0.05),肌浆网Ca2+摄取升高[(60.38±5.76)nmol Ca2+/(mg·min)vs(31.10±3.13)nmol Ca2+/(mg·min)],肌浆网Ca2+释放降低[(32.12±1.18)nmol Ca2+/(mg·15s)vs(39.61±1.16)nmol Ca2+/(mg·15s),P<0.05],钙网蛋白表达和p38丝裂原活化蛋白激酶水平明显升高(P<0.05)。结论 HPC通过p38丝裂原活化蛋白激酶途径上调钙网蛋白表达,改善心肌肌浆网Ca2+摄取和肌浆网Ca2+释放功能、减轻细胞内钙超载而保护缺血心肌。     

脱氢紫堇碱在心肌细胞钙调控中的作用

背景已有动物实验表明脱氢紫堇碱(DHC)可能通过阻止心肌细胞内钙超载,提高心肌细胞的自我保护作用,但还没有相关实验阐明DHC调控心肌细胞保护的分子机制.目的 探讨DHC对心肌细胞内Ca2+超负荷及Ca2+调控相关蛋白的表达变化.方法 采用逆转录聚合酶链反应(RT-PCR)方法 对心肌细胞Ca2+调控相关蛋白,包括钙三磷...     

风湿性心脏病心肌细胞内肌浆网钙离子转运功能变化的机制

目的：探讨风湿性心脏病（风心病）慢性心力衰竭时心肌细胞内肌浆网Ca2 转运功能变化的机制。方法：采用Westernblot法测定19例风心病患者（风心病组）和6例意外脑死亡者（对照组）心肌肌浆网钙泵蛋白和兰尼碱受体含量,同时采用草酸盐易化的肌浆网Ca2 摄取法测定两组心肌肌浆网的Ca2 摄取功能,采用测定“裸露”心肌束的咖啡因敏感性方法测定风心病患者心肌肌浆网的Ca2 释放功能。结果：风心病组钙泵蛋白和兰尼碱受体相对含量比对照组分别显著减少23％（P＜0．01）和10％（P＜0．05）,心肌匀浆肌浆网Ca2 摄取量和摄取率也均显著低于对照组（P＜0．05～P＜0．01）,并且风心病患者钙泵蛋白相对含量变化与肌浆网Ca2 摄取率变化呈显著正相关（r=0．81,P＜0．01）,“裸露”心肌束的相对Ca2 释放速率变化也明显低于正常水平,且与兰尼碱受体相对含量变化密切相关（r=0．67,P＜0．05）。结论：风心病心肌细胞内肌浆网Ca2 摄取和释放功能下降的机制主要与肌浆网的Ca2 摄取和释放蛋白含量变化有关。     

心房颤动患者心房肌钙平衡调节蛋白基因的mRNA表达的研究

目的　通过对临床患者的心房肌钙平衡调节蛋白的mRNA表达水平的测定 ,揭示心房颤动 (房颤 )的发生机制。方法　采集风湿性心脏病窦性心律 (窦律 )组患者 (12例 )和房颤组患者 (12例 )的右心耳组织 ,应用逆转录 聚合酶链反应技术以GAPDH为内参照 ,测定心房肌钙平衡调节蛋白mRNA表达水平。结果　与窦律组相比 ,房颤组细胞膜L型钙通道的mRNA表达减低 44 6 2 % (0 2 5±0 16与 0 13± 0 13,P <0 0 5 ) ,肌浆网钙泵mRNA表达水平降低 2 6 2 2 % (0 38± 0 14与 0 2 8± 0 14,P<0 0 5 ) ,而磷酸受纳蛋白、ryanodine受体和细胞膜钠 钙交换的mRNA表达水平差异无显著性。结论房颤患者心房肌细胞膜L型钙通道和肌浆网钙泵mRNA表达水平降低 ,提示细胞内钙超载机制参与了房颤发生和维持。     

心房肌钙调蛋白基因表达与风湿性心脏病心房纤颤发病关系的研究

取10例风湿性心脏病（风心病）窦性心律（窦律）患者，10例心房纤颤（房颤）患者的右心耳组织，应用逆转录－聚合酶链反应技术，以GAPDH为内参照，测定心房肌钙调蛋白mRNA表达水平。结果显示，与窦律组相比，房颤患者细胞膜L型Ca^2 通道α1c亚基及肌浆网钙泵的mRNA表达减低，两组间有显著性差异（P＜0．05），而磷酸受钠蛋白、钙释放通道和细胞膜Na^ -Ca^2 交换的mRNA表达水平无显著性差异。提示细胞内钙超载机制参与了房颤发生和维持。     

阿霉素中毒心肌细胞钙离子调节功能的变化

本文采用Fura-2/AM荧光标记同位素~(45)Ca~(2+)负载示踪法测定了心肌细胞内钙浓度及肌浆网摄钙功能,探讨了阿霉素对心肌细胞内钙调节功能的影响,同时还观察了维拉帕米预处理10min后,阿霉素对心肌细胞内钙浓度及钙调节功能的影响.结果显示,阿霉素作用早期(10min)能够增加心肌细胞外钙的快相内流,使心肌细胞内游离钙浓度增高,肌浆网摄钙量也有轻度增加,阿霉素作用60min后,与对照组相比细胞内~(45)Ca~(2+)总量增加,而肌浆网对~(45)Ca~(2+)的摄取明显减低,细胞浆内游离钙浓度明显升高.维拉帕米预处理组,维拉帕米部分阻断了阿霉素对细胞内钙的调节作用.结果提示,阿霉素通过增加心肌细胞钙内流速度和抑制肌浆网摄钙量,导致心肌细胞钙过负荷,进而影响心肌细胞的收缩舒张功能.     

Diminished expression of sarcoplasmic reticulum Ca(2+)-ATPase and ryanodine sensitive Ca(2+)Channel mRNA in streptozotocin-induced diabetic rat heart

The diabetic heart has an abnormal intracellular calcium ([Ca(2+)]i) metabolism. However, the responsible molecular mechanisms are unclear. The present study aimed to investigate mRNAs expressed in the proteins which regulate heart [Ca(2+)]i metabolism in streptozotocin (STZ)-induced diabetic rats. Expression of sarcoplasmic reticulum Ca(2+)-adenosine triphosphatase (SR Ca(2+)-ATPase) mRNA was significantly less in the heart 3 weeks after STZ injection than that in the age-matched controls. Together with the down-regulation of SR Ca(2+)-ATPase, expression of ryanodine sensitive Ca(2+)channel (RYR) mRNA was also decreased 12 weeks after STZ injection. Insulin supplementation fully restored the decreased mRNAs expression of SR Ca(2+)-ATPase and RYR. The diminished expression and restoration with insulin supplementation of SR Ca(2+)-ATPase was further confirmed at the protein level. In contrast, expression of mRNAs coding the L-type Ca(2+)channel, Na(+)-Ca(2+)exchanger, or phospholamban were not affected 3 or 12 weeks after STZ injection. These results can be taken to indicate that the down-regulation of SR Ca(2+)-ATPase and RYR mRNAs is a possible underlying cause of cardiac dysfunction in STZ-induced diabetic rats.     

Reduced myocardial sarcoplasmic reticulum Ca(2+)-ATPase protein expression in compensated primary and secondary human cardiac hypertrophy.

Pathological intracellular calcium handling has been proposed to underlie the alterations of contractile behavior in hypertrophied myocardium. However, the myocardial protein expression of intracellular calcium transport proteins in compensated human left ventricular hypertrophy has not yet been studied. We investigated septal myocardial specimens of patients suffering from hypertrophic obstructive cardiomyopathy (n=14) or from acquired aortic valve stenosis (n=11) undergoing myectomy or aortic valve replacement, respectively. For comparison, we studied non-hypertrophied myocardium of six non-failing hearts which could not be transplanted for technical reasons. The myocardial density of the calcium release channel of the sarcoplasmic reticulum (SR) was determined by(3)H-ryanodine binding. Myocardial contents of SR Ca(2+)-ATPase, phospholamban, calsequestrin and Na(+)/Ca(2+)-exchanger were analysed by Western blot analysis. The myocardial SR calcium release channel density was not significantly different in hypertrophied and non-failing human myocardium. In both hypertrophic obstructive cardiomyopathy and in aortic valve stenosis, SR Ca(2+)-ATPase expression was reduced by about 30% compared to non-failing myocardium (P<0.05), whereas the expression of phospholamban, calsequestrin, and the Na(+)/Ca(2+)-exchanger was unchanged. The decrease of SR Ca(2+)-ATPase expression was still observable when related to its regulatory protein phospholamban or to the myosin content of the homogenates (P<0.05). Furthermore, the SR Ca(2+)-ATPase expression was inversely correlated to the septum thickness assessed by echocardiography, but not to age, cardiac index or outflow tract gradient. In primary as well as in secondary hypertrophied human myocardium, the expression of SR Ca(2+)-ATPase is reduced and inversely related to the degree of the hypertrophy. The diminished SR Ca(2+)-ATPase expression might result in reduced Ca(2+)reuptake into the SR and might contribute to altered contractile behavior in hypertrophied human myocardium.     

Regulation of myocardial Ca2+-ATPase and phospholamban mRNA expression in response to pressure overload and thyroid hormone.

The sarcoplasmic reticulum (SR) and the contractile protein myosin play an important role in myocardial performance. Both of these systems exhibit plasticity--i.e., quantitative and/or qualitative reorganization during development and in response to stress. Recent studies indicate that SR Ca2+ uptake function is altered in adaptive cardiac hypertrophy and failure. The molecular basis (genetic and phenotypic) for these changes is not understood. In an effort to determine the underlying causes of these changes, we characterized the rabbit cardiac Ca2+-ATPase phenotype by molecular cloning and ribonuclease A mapping analysis. Our results show that the heart muscle expresses only the slow-twitch SR Ca2+-ATPase isoform. Second, we quantitated the steady-state mRNA levels of two major SR Ca2+ regulatory proteins, the Ca2+-ATPase and phospholamban, to see whether changes in mRNA content might provide insight into the basis for functional modification in the SR of hypertrophied hearts. In response to pressure overload hypertrophy, the relative level of the slow-twitch/cardiac SR Ca2+-ATPase mRNA was decreased to 34% of control at 1 week. The relative Ca2+-ATPase mRNA level increased to 167% of control after 3 days of treatment with thyroid hormone. In contrast, in hypothyroid animals, the relative Ca2+-ATPase mRNA level decreased to 51% of control at 2 weeks. The relative level of phospholamban mRNA was decreased to 36% in 1-week pressure overload. Hyperthyroidism induced a decrease to 61% in the phospholamban mRNA level after 3 days of treatment, while hypothyroidism had virtually no effect on phospholamban mRNA levels. These data indicate that the expression of SR Ca2+-ATPase and phospholamban mRNA may not be coordinately regulated during myocardial adaptation to different physiological conditions.     

应用单相动作电位研究房颤模型心房电重构的发生机制

目的应用单相动作电位(MAP)技术检测心房颤动模型电生理参数改变的特点,探讨房颤电重构发生的分子机制。方法健康杂种犬17只,随机分为房颤组(n=11)和对照组(n=6)。房颤组安装固定频率起搏器,以350~430次/min的频率快速心房起搏,对照组行假手术。于起搏前和8周后测量右房有效不应期(AERP)和单相动作电位,测量单相动作电位振幅(MAPA)、单相动作电位时限(MAPD)、复极90%时动作电位时限(MAPD90)、复极50%时动作电位时限(MAPD50)、复极90%动作电位时限与复极50%动作电位时限之差(MAPD90-50)。原子发射光电直读光谱法测定心房肌组织Ca2+含量。RT-PCR法检测心房肌浆网Ca2+-ATP酶和L型钙通道mRNA转录水平。结果对照组MAP时相明显。房颤组MAP形态发生改变。与对照组相比,房颤组MAPA有下降趋势;MAPD,MAPD90,MAPD50,MAPD90-50分别缩短13.79%,19.65%,13.59%和31.25%。MAPD,MAPD90,MAPD90-50与右房心肌Ca2+呈显著负相关;MAPD90-50与L型钙通道显著正相关。上述指标与肌浆网钙ATP酶均无相关性。结论 MAP是研究房颤电重构的可靠手段,心房肌细胞膜L型钙通道和肌浆网Ca2+-ATP酶mRNA表达改变可能是电重构的分子机制之一     

Thyroid hormone regulation of calcium cycling proteins.

Alterations in thyroid hormone levels have a profound impact on myocardial contractility, speed of relaxation, cardiac output, and heart rate. The mechanisms for these changes include altered expression of several key proteins, involved in the regulation of intracellular calcium homeostasis. Most notably, increases in thyroid hormone and the coordinated increases in cardiac contractile parameters are marked by increases in the levels of the sarcoplasmic reticulum (SR) Ca2+-adenosine triphosphatase (ATPase) and decreases in its inhibitor, phospholamban. These changes at the protein level result in enhanced SR calcium transport and myocyte calcium cycling, leading to increases in the force and rates of contraction as well as relaxation rates at the organ level. However, decreases in thyroid hormone levels are associated with opposite alterations in these two proteins, leading to reduced myocyte calcium handling capacity and lower cardiac contractility. Furthermore, changes in the relative ratio of phospholamban/Ca2+-ATPase correlate with changes in the affinity of the SR Ca2+-transport system and relaxation rates in beating hearts. These findings suggest that thyroid hormone directly regulates SR protein levels and thus, cardiac function.     

Effects of A1 adenosine receptor stimulation on the expression of genes involved in calcium homeostasis

We investigated whether A(1) adenosine receptor stimulation affects expression of genes involved in calcium homeostasis, including sarcolemmal L-type Ca(2+) channel, Na(+)/Ca(2+) exchanger, sarcoplasmic reticulum (SR) Ca(2+)-ATPase, phospholamban, or ryanodine receptor. Three models of A(1) stimulation were used: i) an acute model, i.e. isolated perfused rat hearts treated for 120 min with 15 nM R-phenylisopropyladenosine (R-PIA), an A(1) receptor agonist; ii) a subacute model, i.e. rats treated with 1.5 mg/kg R-PIA e.v. and sacrificed after 24 h; iii) a transgenic model, i.e. mice overexpressing A(1) adenosine receptors. In all models gene expression was determined by RT-PCR, and oxalate-supported Ca(2+) uptake, representing SR Ca(2+) uptake, was measured in the crude homogenate. Significant increase in the expression of the phospholamban gene was observed in each model of A(1) stimulation, while the expression of the other four genes was not significantly modified. In the acute model, SR Ca(2+) uptake was unaffected, however in the subacute and transgenic models uptake rate was significantly reduced. In parallel experiments, hearts obtained from the subacute model demonstrated a significant reduction in irreversible tissue injury from 30 min of ischemia and 120 min of reperfusion. Increased resistance to ischemia has already been reported also in our transgenic model. In conclusion, A(1) adenosine receptor stimulation up-regulates phospholamban gene expression, which leads within 24 h to a reduced rate of SR Ca(2+) uptake. Changes in Ca(2+) homeostasis might contribute to the delayed cardioprotective effect of adenosine.     

短期快速心房起搏钙平衡调节蛋白基因表达变化的研究

为观察家兔短期快速心房起搏所致心房肌电重构及其对钙平衡调节蛋白mRNA表达水平的影响 ,探讨心房肌电重构的发生机制 ,取新西兰大耳白兔 36只 ,随机分为 6组 ,经颈内静脉切开置入电极导管 ,以最快的心房 1∶1起搏的频率于右房行快速心房起搏 ,监测起搏前后心房有效不应期 (AERP)的变化 ,按分组分别于起搏 0 .5 ,1,2 ,4 ,8h后终止起搏 ,取右房组织 ,行半定量逆转录 聚合酶链式反应测定钙平衡调节蛋白基因表达的相对水平。结果 :快速心房起搏后AERP缩短 ,起搏后 0 .5h内AERP变化速率最大 ,最小值出现在快速心房起搏后 8h ,在 0 .5h后的整个短期起搏过程中变化不大 ;0 .5h至 8h的快速心房起搏使肌浆网钙泵和L型Ca2 + 通道基因表达水平逐渐下调 ,至起搏后 4h较起搏前有显著性差异 ;Na+ Ca2 + 交换的基因表达水平上调 2 3.71% ,但无统计学意义。磷酸受纳蛋白、Ryanodine受体的基因表达水平无明显变化。结论 :心房肌电重构的电生理变化发生于快速心房起搏0 .5h ,起搏 4h可引起钙平衡调节蛋白基因表达的变化 ,推测心房肌电重构的形成有细胞内钙超载机制的参与。     

Gene expression of proteins influencing the calcium homeostasis in patients with persistent and paroxysmal atrial fibrillation.

OBJECTIVE: Persistent atrial fibrillation (AF) results in an impairment of atrial function. In order to elucidate the mechanism behind this phenomenon, we investigated the gene expression of proteins influencing calcium handling. METHODS: Right atrial appendages were obtained from eight patients with paroxysmal AF, ten with persistent AF (> 8 months) and 18 matched controls in sinus rhythm. All controls underwent coronary artery bypass grafting, whereas most AF patients underwent Cox's MAZE surgery (n = 12). All patients had a normal left ventricular function. Total RNA was isolated and reversely transcribed into cDNA. In a semi-quantitative polymerase chain reaction the cDNA of interest and of glyceraldehyde-3-phosphate dehydrogenase were coamplified and separated by ethidium bromide-stained gel electrophoresis. Slot blot analysis was performed to study protein expression. RESULTS: L-type calcium channel alpha 1 and sarcoplasmic reticulum Ca(2+)-ATPase mRNA (-57%, p = 0.01 and -28%, p = 0.04, respectively) and protein contents (-43%, p = 0.02 and -28%, p = 0.04, respectively) were reduced in patients with persistent AF compared to the controls. mRNA contents of phospholamban, ryanodine receptor type 2 and sodium/calcium exchanger were comparable. No changes were observed in patients with paroxysmal AF. CONCLUSIONS: Alterations in gene expression of proteins involved in the calcium homeostasis occur only in patients with long-term persistent AF. In the absence of underlying heart disease, the changes are rather secondary than primary to AF.