慢性心衰大鼠心肌细胞钙调控异常及药物干预的研究
|
摘要
第一部分慢性心力衰竭与心肌细胞异常的钙循环
目的:
心肌细胞肌浆网的钙循环异常是心力衰竭发病的主要原因之一,但其确切机理和相关分子机制还不甚清楚,本实验以大鼠慢性心衰模型作为研究对象,在细胞和亚细胞水平对心肌细胞胞浆内钙瞬变、肌浆网内钙容量以及钙调控相关蛋白表达的变化进行了研究,从而更深入地了解心衰的分子机制。
方法:
(1)实验分组:28只雄性SD大鼠随机分为两组,假手术组(n=10)和心衰组(n=18);(2)慢性心衰模型的建立:心衰组大鼠行冠状动脉左前降支永久性结扎,假手术组大鼠除未结扎冠脉外,其余手术步骤均同心衰组。4周后分别观察各组动物存活率和心功能各项指标;(3)心肌细胞L-型钙电流、胞浆内钙瞬变和肌浆网内钙容量的测定:采用常规酶解法分离心室肌细胞,Fluo-3/AM负载心肌细胞,运用全细胞膜片钳-激光扫描共聚焦显微镜联机技术同步记录各组大鼠心肌细胞L-型钙电流和胞浆内钙瞬变(即钙诱导钙瞬变);利用咖啡因诱导钙瞬变的方法,间接记录肌浆网内钙容量(即咖啡因诱导钙瞬变)的变化;同时利用Fluo-5N/AM负载心肌细胞,直接记录肌浆网内钙容量的变化;(4)钙调控蛋白mRNA转录量的检测:提取各组大鼠心肌组织总RNA,采用Real-time quantitative RT-PCR方法检测钙调控蛋白mRNA的转录量;(5)钙调控蛋白表达的测定:将各组大鼠左室心肌组织的膜蛋白提取后,采用Western-blot法测定钙调控蛋白的表达水平,并进行蛋白表达半定量分析;(6)统计学分析:利用SPSS11.5统计软件进行数据处理,所有数据以均数±标准差( (X|—)±SD)表示,两组间均数的比较采用t检验,率的比较采用Fisher’s Exact检验。P<0.05为差异有统计学意义。
结果:
(1)心功能的比较:心衰组和假手术组存活率分别为83.3%和100%,两者相比差异无显著(P>0.05);与假手术组(n=10)相比,心衰组(n=15)左室终末舒张压(LVEDP)明显升高(8.3±0.42 mmHg VS 4.7±0.65 mmHg,P<0.01),心重体重比(HW/BW)明显增加(6.76±0.36 VS 3.33±0.41,P<0.01),左心室内压最大上升速率(dp/dt_(max))明显减小(2,140.41±118.38 mmHg/s VS 4,355.75±259.71 mmHg/s,P<0.01),左心室内压最大下降速率(dp/dt_(min))明显减小(-1,798.33±111.41 mmHg/s VS -2,531.22±414.66 mmHg/s,P<0.01);(2)心衰组大鼠(n=20)单个心肌细胞的I_(Ca·L)比假手术组(n=18)显著降低,且平均电流密度值亦明显减小(1.01±0.09 VS 2.18±0.1,P<0.01);(3)心衰组大鼠(n=12)CICR过程中的钙瞬变幅度比假手术组(n=10)明显下降,且钙瞬变峰值(ΔF/F_0)亦明显下降(12.2±0.56 VS 17.8±0.53,P<0.01);(4)心衰组大鼠(n=12)咖啡因诱导钙瞬变过程中钙瞬变幅度比假手术组(n=10)明显降低,且钙瞬变峰值(ΔF/F_0)亦明显下降(12.4±0.4 VS 33.2±1.9,P<0.01);(5)心衰组大鼠单个心肌细胞(n=12)Fluo-5N/AM染色后荧光强度比假手术组(n=10)显著降低,且荧光强度变化的平均值(ΔF/F_0)亦明显下降(26.6±1.87 VS 49.6±2.02,P<0.01);(6)与假手术组相比,心衰组大鼠心肌细胞RyR2的mRNA表达RyR2/GAPDH(0.063±0.002 VS 0.064±0.003 ,n=8 , P>0.05 );PLB的mRNA表达PLB/GAPDH (0.063±0.003 VS 0.058±0.002,n=8,P>0.05)均无显著差异;FKBP12.6、SERCA2a、NCX、Cav1.2的mRNA表达FKBP12.6/GAPDH ( 0.018±0.002 VS 0.042±0.002 , n=8 , P<0.01 )、SERCA2a/GAPDH(0.14±0.019 VS 0.28±0.016,n=8,P<0.01)、NCX/GAPDH(0.07±0.016 VS 0.12±0.019,n=8,P<0.01)及Cav1.2/GAPDH(0.01±0.0012 VS 0.026±0.0019,n=8,P<0.01)均明显降低;(7)与假手术组相比,心衰组大鼠心肌细胞RyR2/GAPDH(0.36±0.028 VS 0.39±0.03,n=8,P>0.05),PLB/GAPDH(0.5±0.02 VS 0.57±0.03,n=8,P>0.05)的表达均无显著差异,FKBP12.6/GAPDH(0.13±0.007 VS 0.9±0.05,n=8,P<0.01)、SERCA2a/GAPDH(0.74±0.02 VS 1.36±0.009,n=8,P<0.01)、NCX/GAPDH(0.18±0.03 VS 0.78±0.05,n=8,P<0.01)及DHPR/GAPDH(0.11±0.01 VS 1.2±0.05,n=8,P<0.01)的表达均明显降低。
结论:
慢性心衰大鼠心肌细胞内钙循环异常主要是由与钙释放和钙回摄相关的钙调控蛋白表达的异常所引起。具体如下:
(1)慢性心衰大鼠心肌细胞L-型钙通道(DHPR)表达下调,造成钙诱导钙释放(CICR)过程出现异常,最终导致心肌兴奋-收缩耦联(ECC)障碍,心肌收缩力下降。
(2)慢性心衰大鼠心肌细胞内FKBP12.6表达下调,使其对RyR2的稳定作用减弱,导致RyR2在心肌细胞舒张期泄漏过多的Ca~(2+),使肌浆网内钙容量降低,从而引起ECC过程中所需要的钙释放量减少,最终导致心肌收缩力降低。
(3)慢性心衰大鼠心肌细胞内钙泵(SERCA2a)表达下调,致ECC结束后钙回摄功能障碍,导致肌浆网内钙容量减少,最终造成心肌收缩力下降。
第二部分氧化苦参碱对慢性心衰大鼠心肌保护作用机制的研究
目的:
氧化苦参碱(Oxymatrine, OMT)是从豆科槐属植物苦豆子提取的氧化生物碱,属于四环的喹嗪啶类,具有抗炎、抗过敏、保肝、抗病毒及抗寄生虫等多方面药理作用,是临床上常用的治疗肝炎的药物。本实验以慢性心衰大鼠作为模型,在先前研究的基础上探讨氧化苦参碱对心肌细胞内钙离子,钙通道及钙调节相关蛋白的影响,以期获得其心脏保护作用的机制,并为此药在临床上的开发及应用提供基础理论支持。
方法:
(1)实验分组:60只雄性SD大鼠随机分为五组,假手术组(n=12)、心衰组(n=12)、低剂量OMT干预组(n=12)(25mg/kg, qd)、中剂量OMT干预组(n=12)(50mg/kg, qd)和高剂量OMT干预组(n=12)(100mg/kg, qd);(2)心衰模型的建立:心衰组和药物干预组大鼠行冠状动脉左前降支永久性结扎,假手术组大鼠除未结扎冠脉外,其余手术步骤均同心衰组。4周后分别检测各组动物存活率和心功能各项指标;(3)心肌细胞L-型钙电流、胞浆内钙瞬变和肌浆网内钙容量的测定:采用常规酶解法分离心室肌细胞,Fluo-3/AM负载心肌细胞,运用全细胞膜片钳-激光扫描共聚焦显微镜联机技术同步记录各组大鼠心肌细胞L-型钙电流和胞浆内钙瞬变;利用咖啡因诱导钙瞬变的方法,间接记录肌浆网内钙容量;(4)透射电子显微镜形态学观察:各组大鼠心室肌组织被切成1mm~3的小块后,经固定、脱水、包埋、修块、切片、染色等处理后于电镜下观察并拍照;(5)钙调控蛋白mRNA转录量的检测:提取各组大鼠心肌组织总RNA,采用Real-time quantitative RT-PCR方法检测钙调控蛋白mRNA的转录量;(6)钙调控蛋白表达的测定:将各组大鼠左室心肌组织的膜蛋白提取后,采用Western-blot法测定钙调控蛋白的表达水平,并进行蛋白表达半定量分析;(7)统计学分析:采用SPSS11.5统计软件进行数据处理,所有数据以均数±标准差( (X|—)±SD)表示,各组间均数的比较采用单因素方差分析(ANOVA),组间两两比较时用LSD法。P<0.05认为差异有统计学意义。
结果:
(1)各组大鼠心功能的比较:各组大鼠存活率均为100%。与假手术组相比,心衰组左室终末舒张压(LVEDP)明显上升(8.1±0.24 VS 3.7±0.49,n=12, P<0.01),心重体重比(HW/BW)明显增加(4.39±0.36 VS 2.41±0.06,n=12, P<0.01),左心室内压最大上升速率(dp/dt_(max))(2,458.85±124.05 mmHg/s VS 4,164.63±130.69 mmHg/s,n=12, P<0.01)、左心室内压最大下降速率(dp/dt_(min))(-1,594.85±214.22 mmHg/s VS -2,765.82±152.99 mmHg/s,n=12, P<0.01)均明显减小;与心衰组相比,中剂量和高剂量药物干预组大鼠的左室终末舒张压(LVEDP)明显下降(5.1±0.24 mmHg,4.6±0.34 mmHg VS 8.1±0.24 mmHg,n=12, P<0.01),心重体重比(HW/BW)明显降低(3.11±0.08,3.09±0.08 VS 4.39±0.36,n=12, P<0.05),左心室内压最大上升速率(dp/dt_(max))(3,827.77±212.73 mmHg/s,3,910.72±101.36 mmHg/s VS 2,458.85±124.05 mmHg/s,n=12, P<0.01)、左心室内压最大下降速率(dp/dt_(min))(-2,537.75±213.09 mmHg/s,-2,548.28±205.65 mmHg/s VS -1,594.85±214.22 mmHg/s,n=12, P<0.01)均明显升高;(2)与假手术组相比,心衰组大鼠单个心肌细胞的ICa·L显著降低,且平均电流密度值亦明显减小(1.89±0.19 VS 6.48±0.33,n=20,P<0.01);与心衰组比较,中剂量和高剂量药物干预组大鼠单个心肌细胞的I_(Ca·L)明显加强,且平均电流密度值亦明显增强(4.35±0.32,4.21±0.24 VS 1.89±0.19,n=20,P<0.01);(3)与假手术组相比,心衰组大鼠CICR过程中的钙瞬变幅度明显下降,且钙瞬变峰值(ΔF/F_0)亦明显下降(12.52±1.38 VS 41.37±2.44,n=12,P<0.01);与心衰组相比,中剂量和高剂量药物干预组大鼠CICR过程中的钙瞬变幅度明显升高,且钙瞬变峰值(ΔF/F_0)亦明显上升(26.82±1.02,30.07±0.91 VS 12.52±1.38,n=12,P<0.01);(4)各组大鼠心肌细胞SR钙容量的比较:与假手术组相比,心衰组大鼠咖啡因诱导钙瞬变过程中钙瞬变幅度明显降低,且钙瞬变峰值(ΔF/F_0)亦明显下降(17.05±0.61 VS 35.36±0.89,n=12,P<0.01);与心衰组比较,中剂量和高剂量药物干预组大鼠咖啡因诱导钙瞬变过程中钙瞬变幅度明显升高,且钙瞬变峰值(ΔF/F_0)亦明显上升(32.3±0.74,32.19±0.51 VS 17.05±0.61,n=12,P<0.01);(5)各组大鼠心室肌细胞超微结构显示:假手术组大鼠心肌细胞的肌小节完整,肌丝排列整齐,线粒体结构正常,嵴排列紧密,糖原颗粒丰富。心衰组大鼠部分心肌细胞肌丝溶解,并出现大小不等的空泡,线粒体肿胀,嵴断裂。低剂量药物干预组大鼠心肌细胞肌丝溶解有轻微恢复,但线粒体仍肿胀,嵴断裂。中剂量药物干预组大鼠心肌细胞肌丝溶解现象消失,肌丝排列整齐,紧密,清晰,线粒体结构基本完整,嵴排列紧密。高剂量药物干预组大鼠心肌细胞表现基本同中剂量组,且线粒体的电子密度增加;(6)与心衰组比较,中剂量和高剂量药物干预组大鼠心肌细胞SERCA2a、Cav1.2的mRNA表达SERCA2a/GAPDH(0.2±0.017,0.2±0.019 VS 0.12±0.014,n=8,P<0.01)、Cav1.2/GAPDH(0.02±0.002,0.02±0.002 VS 0.014±0.002,n=8,P<0.01)均明显升高;(7)与心衰组比较,中剂量和高剂量药物干预组大鼠心肌细胞SERCA2a/GAPDH(0.94±0.053,1.36±0.059 VS 0.25±0.032,n=8,P<0.01)、DHPR/GAPDH(0.3±0.027,0.28±0.019 VS 0.18±0.015,n=8,P<0.01)的表达均明显升高。
结论:
氧化苦参碱(OMT)对慢性心衰大鼠的心肌细胞具有保护作用,而这种保护作用表现在大鼠心功能的改善。具体如下:
(1)在氧化苦参碱(OMT)的干预下,慢性心衰大鼠心肌细胞L-型钙通道(DHPR)表达上调,促使L-型钙电流的增大和钙诱导钙释放(CICR)的恢复,最终使心肌收缩力增强,心功能得以改善。
(2)在氧化苦参碱(OMT)的干预下,慢性心衰大鼠心肌细胞内钙泵(SERCA2a)表达上调,促使心肌兴奋-收缩耦联(ECC)结束后钙回摄功能恢复,肌浆网内钙容量增加,最终促使心肌收缩力加强,心功能改善。
Objective:
Altered intracellular Ca~(2+) handling by the sarcoplasmic reticulum (SR) plays a crucial role in the pathogenesis of heart failure (HF). Despite extensive effort, the underlying causes of abnormal SR Ca~(2+) handling in HF are not clarified. To determine whether the diastolic SR Ca~(2+) leak along with reduced Ca~(2+) reuptake are required for decreased contractility, we investigated the cytosolic Ca~(2+) transients,SR Ca~(2+) content and assessed the expression of Ca~(2+) handling genes and proteins, using SD-rat model of chronic heart failure.
Methods:
28 male SD rats were randomly divided into two groups: sham-operated rats (n=10), heart failure rats (n=18). Rats were deeply anesthetized, following intubation and placement on a respirator, a left lateral thoracotomy was performed and the left anterior descending (LAD) coronary artery was permanently ligated. Sham-operated animal sunder went the same procedure without ligation of the LAD. Twenty-eight days after myocardial infarction (MI), survival rate, hemodynamic and heart weight of rats were assessed. Single cardiomyocytes of the rat heart were isolated by an enzymatic dissociation method. Patch-clamp and laser scanning confocal microscope synchronous recording system software was used to record transmembrane Ca~(2+) currents (I_(Ca·L)) and intracellular Ca~(2+) transients simultaneously. SR Ca~(2+) content can be measured by caffeine-induced Ca~(2+) transients (CCT) indirectly and can be measured by permeabilized myocytes with SR-entrapped Fluo-5N/AM directly. The total RNA was extracted from the homogenate and the mRNA levels of Ca~(2+) handling proteins were measured using real-time quantitative RT-PCR. The levels of Ca~(2+) handling proteins were determined by immunoblot analysis. SPSS11.5 software was used for the statistical analyses. All data are presented as mean±SD and t-test or Fisher’s Exact test was used for the statistical analyses. P-values of <0.05 were considered significant.
Results:
(1) According to the experiment, the survival is 83.3% in HF group and 100% in sham group(P>0.05). There was a significant increase in the left ventricular end-diastolic pressure (LVEDP) in HF rats (8.3±0.42 mmHg, n=15) compared with sham rats (4.7±0.65 mmHg, n=10, P<0.01). There was a significant decrease in the maximal change of systolic pressure over time (dp/dt_(max)) in HF rats (2,140.41±118.38 mmHg/s, n=15) compared with control rats (4,355.75±259.71 mmHg/s, n=10, P<0.01) and there was a significant decrease in the maximum change in the rate of relaxation over time (dp/dt_(min)) in HF rats (-1,798.33±111.41 mmHg/s, n=15) compared with sham rats (-2,531.22±414.66 mmHg/s, n=10, P<0.01). Furthermore, there was a significant increase in heart weight (HW) divided by body weight (BW) in HF rats (6.76±0.36, n=15) compared with sham rats (3.33±0.41, n=10, P<0.01). (2) The amplitude of I_(Ca·L) was reduced in HF myocytes (1.01±0.09, n=20) compared with sham (2.18±0.1, n=18, P<0.01). Moreover the amplitude of Ca~(2+) transients was also reduced in HF group. There was a significant decrease in spatial averages (ΔF/F_0) of I_(Ca·L)-induced Ca~(2+) transients in HF (12.2±0.56, n=12) compared with sham (17.8±0.53, n=10, P<0.01). SR Ca~(2+) content can be measured by caffeine-induced Ca~(2+) transients (CCT) during diastolic phase. The amplitude of Ca~(2+) transients was reduced in HF group. Caffeine-induced Ca~(2+) transients (ΔF/F_0) was significantly decreased in HF (12.4±0.4, n=12) compared with sham (33.2±1.9, n=10, P<0.01). Isolated cardiomyocytes were loaded with Fluo-5N/AM and SR Ca~(2+) content can be measured directly. The result showed that the SR Ca~(2+) content was dramatically reduced in HF myocytes. There was a significant decrease in spatial averages (ΔF/F_0) in HF (26.6±1.87, n=12) compared with sham (49.6±2.02, n=10, P<0.01). (3) We found that the mRNA expression of RyR2 (0.063±0.002 VS 0.064±0.003,n=8, P>0.05) and PLB (0.063±0.003 VS 0.058±0.002,n=8, P>0.05) failed to alter in HF compared with sham group. The downregulation of FKBP12.6 (0.018±0.002 VS 0.042±0.002,n=8, P<0.01), SERCA2a (0.14±0.019 VS 0.28±0.016,n=8, P<0.01), NCX (0.07±0.016 VS 0.12±0.019,n=8, P<0.01) and Cav1.2 (0.01±0.0012 VS 0.026±0.0019,n=8, P<0.01) was established by RT-PCR to be significant relative to the sham, respectively. Compared with the sham rat, the expression of RyR2 (0.36±0.028 VS 0.39±0.03,n=8, P>0.05) and PLB (0.5±0.02 VS 0.57±0.03,n=8, P>0.05) failed to alter in HF rat, whereas FKBP12.6 (0.13±0.007 VS 0.9±0.05, n=8, P<0.01) content was significantly reduced in HF rat. Furthermore, the expression of SERCA2a (0.74±0.02 VS 1.36±0.009,n=8, P<0.01), NCX (0.18±0.03 VS 0.78±0.05,n=8, P<0.01) and DHPR (0.11±0.01 VS 1.2±0.05,n=8, P<0.01) were also reduced in HF rat.
Conclusion:
Diastolic SR Ca~(2+) leak (due to dissociation of FKBP12.6 from RyR2) along with reduced SR Ca~(2+) uptake (due to down-regulation of SERCA2a) and defective E-C coupling(due to down-regulation of DHPR)could contribute to the decreased contractility observed in failing hearts associated with reduced amplitude and slowed decay of the intracellular Ca~(2+) transients.
PartⅡProtective Effect of Oxymatrine on Chronic Rat Heart Failure
Objective:
Oxymatrine is one of the alkaloids extracted from Chinese herb Sophora japonica (Sophora flavescens Ait.) with activities of anti-inflammation, antivirus and protecting hepatocytes. However, the effect of oxymatrine on heart failure has not been known yet. In this study, the effect of oxymatrine on heart failure was investigated using SD-rat model of chronic heart failure.
Methods:
60 male SD rats were randomly divided into five groups: sham-operated rats (n=12), heart failure rats (n=12), high dose group (rats with MI and 100 mg/kg OMT treatment, qd), middle dose group (rats with MI and 50 mg/kg OMT treatment, qd), low dose group (rats with MI and 25 mg/kg OMT treatment, qd). Rats were deeply anesthetized, following intubation and placement on a respirator, a left lateral thoracotomy was performed and the left anterior descending (LAD) coronary artery was permanently ligated. Sham-operated animal sunder went the same procedure without ligation of the LAD. Twenty-eight days after MI, survival rate, hemodynamic and heart weight of rats were assessed. Single cardiomyocytes of the rat heart were isolated by an enzymatic dissociation method. Patch-clamp and laser scanning confocal microscope synchronous recording system software was used to record transmembrane Ca~(2+) currents (I_(Ca·L)) and intracellular Ca~(2+) transients simultaneously. SR Ca~(2+) content can be measured by caffeine-induced Ca~(2+) transients (CCT) indirectly. The ultrastructure of myocardial cells in left ventricle of rats was studied under transmission electron microscopy. The total RNA was extracted from the homogenate and the mRNA levels of Ca~(2+) handling proteins were measured using real-time quantitative RT-PCR. The levels of Ca~(2+) handling proteins were determined by immunoblot analysis. SPSS11.5 software was used for the statistical analyses. Data were expressed as mean±SD for all the experiments. Statistical analysis were made with ANOVA and followed by LSD test for individual comparisons of means. P-values of <0.05 were considered significant.
Results:
(1) According to the experiment, the survival is 100% in every group. There was a significant increase in the left ventricular end-diastolic pressure (LVEDP) in HF rats (8.1±0.24 mmHg) compared with sham rats (3.7±0.49 mmHg, n=12, P<0.01). There was a significant decrease in the maximal change of systolic pressure over time (dp/dt_(max)) in HF rats (2,458.85±124.05 mmHg/s) compared with sham rats (4,164.63±130.69 mmHg/s, n=12, P<0.01) and there was a significant decrease in the maximum change in the rate of relaxation over time (dp/dt_(min)) in HF rats (-1,594.85±214.22 mmHg/s) compared with sham rats (-2,765.82±152.99 mmHg/s, n=12, P<0.01). Furthermore, there was a significant increase in heart weight (HW) divided by body weight (BW) in HF rats (4.39±0.36) compared with sham rats (2.41±0.06, n=12, P<0.01). There was a significant decrease in LVEDP in middle (5.1±0.24 mmHg) and high dose group (4.6±0.34 mmHg) rats compared with HF rats (8.1±0.24 mmHg, n=12, P<0.01). There was a significant increase in dp/dt_(max) in middle (3,827.77±212.73 mmHg/s) and high dose group (3,910.72±101.36 mmHg/s) rats compared with HF rats (2,458.85±124.05 mmHg/s, n=12, P<0.01) and there was a significant increase in dp/dt_(min) in middle (-2,537.75±213.09 mmHg/s) and high dose group (-2,548.28±205.65 mmHg/s) rats compared with HF rats (-1,594.85±214.22 mmHg/s, n=12, P<0.01). Furthermore, there was a significant decrease in HW/BW in middle (3.11±0.08) and high dose group (3.09±0.08) rats compared with HF rats (4.39±0.36, n=12, P<0.01). (2) The amplitude of I_(Ca·L) was reduced in HF myocytes (1.89±0.19) compared with sham (6.48±0.33, n=20, P<0.01). The amplitude of I_(Ca·L) was enhanced in middle (4.35±0.32) and high dose group (4.21±0.24) myocytes compared with HF myocytes (1.89±0.19, n=20, P<0.01). Moreover the amplitude of Ca~(2+) transients was also reduced in HF group. There was a significant decrease in spatial averages (ΔF/F_0) of I_(Ca·L)-induced Ca~(2+) transients in HF (12.52±1.38) compared with sham (41.37±2.44, n=12, P<0.01). The amplitude of Ca~(2+) transients was also enhanced in middle and high dose group. There was a significant increase in spatial averages (ΔF/F_0) of I_(Ca·L)-induced Ca~(2+) transients in middle (26.82±1.02) and high dose group (30.07±0.91) compared with HF group (12.52±1.38,n=12, P<0.01). (3) SR Ca~(2+) content can be measured by CCT during diastolic phase. The amplitude of Ca~(2+) transients was reduced in HF group. Caffeine-induced Ca~(2+) transients (ΔF/F_0) was significantly decreased in HF (17.05±0.61) compared with sham (35.36±0.89, n=12, P<0.01). The amplitude of Ca~(2+) transients was enhanced in middle and high dose group compared with HF group. Caffeine-induced Ca~(2+) transients (ΔF/F_0) was significantly increased in middle (32.3±0.74) and high dose group (32.19±0.51) compared with HF group (17.05±0.61,n=12, P<0.01). (4) The ultrastructure of myocardial cells in left ventricle of rats was studied under TEM. In sham group, the myocardial cell appeared normal with intact sarcomeres, mitochondria contain tightly packed cristae. Numerous glycogen granules were present. In HF group, some parts of the myocardial cell appeared intermyofibrillar lysis and vesicles of varying size. Mitochondria were swollen, and mitochondrial cristae were separated. In group with treated by low dose of OMT, intermyofibrillar lysis slightly restored, but a few mitochondria were still swollen, and mitochondrial cristae were separated. In group with treated by middle dose of OMT, intermyofibrillar lysis disappeared, myofilaments were orderly, closely, and evenly arranged, and mitochondria contain tightly packed cristae. The ultrastructure of myocardial cells, treated by high dose of OMT, was similar with that of treated by middle dose of OMT, and electron density of some mitochondria increased. (5) We found that the mRNA express of SERCA2a was significantly increased in middle (0.2±0.017) and high (0.2±0.019) dose group compared with the HF group (0.12±0.014, n=8, P<0.01). Furthermore, the mRNA content of Cav1.2 was significantly increased in middle (0.02±0.002) and high dose group (0.02±0.002) compared with the HF rat (0.014±0.002, n=8, P<0.01). (6) The expression of SERCA2a was increased in middle (0.94±0.053) and high dose group (1.36±0.059) compared with HF group (0.25±0.032, n=8, P<0.01). The expression of DHPR was also increased in middle (0.3±0.027) and high dose group (0.28±0.019) compared with HF group (0.18±0.015, n=8, P<0.01).
Conclusion:
The present results suggest that oxymatrine improves heart failure by ameliorating the cardiac function, and that this amelioration is associated with upregulation of SERCA2a and DHPR. We suggest that OMT maybe a novel, effective therapeutic drug for the treatment of heart failure.
目的:
心肌细胞肌浆网的钙循环异常是心力衰竭发病的主要原因之一,但其确切机理和相关分子机制还不甚清楚,本实验以大鼠慢性心衰模型作为研究对象,在细胞和亚细胞水平对心肌细胞胞浆内钙瞬变、肌浆网内钙容量以及钙调控相关蛋白表达的变化进行了研究,从而更深入地了解心衰的分子机制。
方法:
(1)实验分组:28只雄性SD大鼠随机分为两组,假手术组(n=10)和心衰组(n=18);(2)慢性心衰模型的建立:心衰组大鼠行冠状动脉左前降支永久性结扎,假手术组大鼠除未结扎冠脉外,其余手术步骤均同心衰组。4周后分别观察各组动物存活率和心功能各项指标;(3)心肌细胞L-型钙电流、胞浆内钙瞬变和肌浆网内钙容量的测定:采用常规酶解法分离心室肌细胞,Fluo-3/AM负载心肌细胞,运用全细胞膜片钳-激光扫描共聚焦显微镜联机技术同步记录各组大鼠心肌细胞L-型钙电流和胞浆内钙瞬变(即钙诱导钙瞬变);利用咖啡因诱导钙瞬变的方法,间接记录肌浆网内钙容量(即咖啡因诱导钙瞬变)的变化;同时利用Fluo-5N/AM负载心肌细胞,直接记录肌浆网内钙容量的变化;(4)钙调控蛋白mRNA转录量的检测:提取各组大鼠心肌组织总RNA,采用Real-time quantitative RT-PCR方法检测钙调控蛋白mRNA的转录量;(5)钙调控蛋白表达的测定:将各组大鼠左室心肌组织的膜蛋白提取后,采用Western-blot法测定钙调控蛋白的表达水平,并进行蛋白表达半定量分析;(6)统计学分析:利用SPSS11.5统计软件进行数据处理,所有数据以均数±标准差( (X|—)±SD)表示,两组间均数的比较采用t检验,率的比较采用Fisher’s Exact检验。P<0.05为差异有统计学意义。
结果:
(1)心功能的比较:心衰组和假手术组存活率分别为83.3%和100%,两者相比差异无显著(P>0.05);与假手术组(n=10)相比,心衰组(n=15)左室终末舒张压(LVEDP)明显升高(8.3±0.42 mmHg VS 4.7±0.65 mmHg,P<0.01),心重体重比(HW/BW)明显增加(6.76±0.36 VS 3.33±0.41,P<0.01),左心室内压最大上升速率(dp/dt_(max))明显减小(2,140.41±118.38 mmHg/s VS 4,355.75±259.71 mmHg/s,P<0.01),左心室内压最大下降速率(dp/dt_(min))明显减小(-1,798.33±111.41 mmHg/s VS -2,531.22±414.66 mmHg/s,P<0.01);(2)心衰组大鼠(n=20)单个心肌细胞的I_(Ca·L)比假手术组(n=18)显著降低,且平均电流密度值亦明显减小(1.01±0.09 VS 2.18±0.1,P<0.01);(3)心衰组大鼠(n=12)CICR过程中的钙瞬变幅度比假手术组(n=10)明显下降,且钙瞬变峰值(ΔF/F_0)亦明显下降(12.2±0.56 VS 17.8±0.53,P<0.01);(4)心衰组大鼠(n=12)咖啡因诱导钙瞬变过程中钙瞬变幅度比假手术组(n=10)明显降低,且钙瞬变峰值(ΔF/F_0)亦明显下降(12.4±0.4 VS 33.2±1.9,P<0.01);(5)心衰组大鼠单个心肌细胞(n=12)Fluo-5N/AM染色后荧光强度比假手术组(n=10)显著降低,且荧光强度变化的平均值(ΔF/F_0)亦明显下降(26.6±1.87 VS 49.6±2.02,P<0.01);(6)与假手术组相比,心衰组大鼠心肌细胞RyR2的mRNA表达RyR2/GAPDH(0.063±0.002 VS 0.064±0.003 ,n=8 , P>0.05 );PLB的mRNA表达PLB/GAPDH (0.063±0.003 VS 0.058±0.002,n=8,P>0.05)均无显著差异;FKBP12.6、SERCA2a、NCX、Cav1.2的mRNA表达FKBP12.6/GAPDH ( 0.018±0.002 VS 0.042±0.002 , n=8 , P<0.01 )、SERCA2a/GAPDH(0.14±0.019 VS 0.28±0.016,n=8,P<0.01)、NCX/GAPDH(0.07±0.016 VS 0.12±0.019,n=8,P<0.01)及Cav1.2/GAPDH(0.01±0.0012 VS 0.026±0.0019,n=8,P<0.01)均明显降低;(7)与假手术组相比,心衰组大鼠心肌细胞RyR2/GAPDH(0.36±0.028 VS 0.39±0.03,n=8,P>0.05),PLB/GAPDH(0.5±0.02 VS 0.57±0.03,n=8,P>0.05)的表达均无显著差异,FKBP12.6/GAPDH(0.13±0.007 VS 0.9±0.05,n=8,P<0.01)、SERCA2a/GAPDH(0.74±0.02 VS 1.36±0.009,n=8,P<0.01)、NCX/GAPDH(0.18±0.03 VS 0.78±0.05,n=8,P<0.01)及DHPR/GAPDH(0.11±0.01 VS 1.2±0.05,n=8,P<0.01)的表达均明显降低。
结论:
慢性心衰大鼠心肌细胞内钙循环异常主要是由与钙释放和钙回摄相关的钙调控蛋白表达的异常所引起。具体如下:
(1)慢性心衰大鼠心肌细胞L-型钙通道(DHPR)表达下调,造成钙诱导钙释放(CICR)过程出现异常,最终导致心肌兴奋-收缩耦联(ECC)障碍,心肌收缩力下降。
(2)慢性心衰大鼠心肌细胞内FKBP12.6表达下调,使其对RyR2的稳定作用减弱,导致RyR2在心肌细胞舒张期泄漏过多的Ca~(2+),使肌浆网内钙容量降低,从而引起ECC过程中所需要的钙释放量减少,最终导致心肌收缩力降低。
(3)慢性心衰大鼠心肌细胞内钙泵(SERCA2a)表达下调,致ECC结束后钙回摄功能障碍,导致肌浆网内钙容量减少,最终造成心肌收缩力下降。
第二部分氧化苦参碱对慢性心衰大鼠心肌保护作用机制的研究
目的:
氧化苦参碱(Oxymatrine, OMT)是从豆科槐属植物苦豆子提取的氧化生物碱,属于四环的喹嗪啶类,具有抗炎、抗过敏、保肝、抗病毒及抗寄生虫等多方面药理作用,是临床上常用的治疗肝炎的药物。本实验以慢性心衰大鼠作为模型,在先前研究的基础上探讨氧化苦参碱对心肌细胞内钙离子,钙通道及钙调节相关蛋白的影响,以期获得其心脏保护作用的机制,并为此药在临床上的开发及应用提供基础理论支持。
方法:
(1)实验分组:60只雄性SD大鼠随机分为五组,假手术组(n=12)、心衰组(n=12)、低剂量OMT干预组(n=12)(25mg/kg, qd)、中剂量OMT干预组(n=12)(50mg/kg, qd)和高剂量OMT干预组(n=12)(100mg/kg, qd);(2)心衰模型的建立:心衰组和药物干预组大鼠行冠状动脉左前降支永久性结扎,假手术组大鼠除未结扎冠脉外,其余手术步骤均同心衰组。4周后分别检测各组动物存活率和心功能各项指标;(3)心肌细胞L-型钙电流、胞浆内钙瞬变和肌浆网内钙容量的测定:采用常规酶解法分离心室肌细胞,Fluo-3/AM负载心肌细胞,运用全细胞膜片钳-激光扫描共聚焦显微镜联机技术同步记录各组大鼠心肌细胞L-型钙电流和胞浆内钙瞬变;利用咖啡因诱导钙瞬变的方法,间接记录肌浆网内钙容量;(4)透射电子显微镜形态学观察:各组大鼠心室肌组织被切成1mm~3的小块后,经固定、脱水、包埋、修块、切片、染色等处理后于电镜下观察并拍照;(5)钙调控蛋白mRNA转录量的检测:提取各组大鼠心肌组织总RNA,采用Real-time quantitative RT-PCR方法检测钙调控蛋白mRNA的转录量;(6)钙调控蛋白表达的测定:将各组大鼠左室心肌组织的膜蛋白提取后,采用Western-blot法测定钙调控蛋白的表达水平,并进行蛋白表达半定量分析;(7)统计学分析:采用SPSS11.5统计软件进行数据处理,所有数据以均数±标准差( (X|—)±SD)表示,各组间均数的比较采用单因素方差分析(ANOVA),组间两两比较时用LSD法。P<0.05认为差异有统计学意义。
结果:
(1)各组大鼠心功能的比较:各组大鼠存活率均为100%。与假手术组相比,心衰组左室终末舒张压(LVEDP)明显上升(8.1±0.24 VS 3.7±0.49,n=12, P<0.01),心重体重比(HW/BW)明显增加(4.39±0.36 VS 2.41±0.06,n=12, P<0.01),左心室内压最大上升速率(dp/dt_(max))(2,458.85±124.05 mmHg/s VS 4,164.63±130.69 mmHg/s,n=12, P<0.01)、左心室内压最大下降速率(dp/dt_(min))(-1,594.85±214.22 mmHg/s VS -2,765.82±152.99 mmHg/s,n=12, P<0.01)均明显减小;与心衰组相比,中剂量和高剂量药物干预组大鼠的左室终末舒张压(LVEDP)明显下降(5.1±0.24 mmHg,4.6±0.34 mmHg VS 8.1±0.24 mmHg,n=12, P<0.01),心重体重比(HW/BW)明显降低(3.11±0.08,3.09±0.08 VS 4.39±0.36,n=12, P<0.05),左心室内压最大上升速率(dp/dt_(max))(3,827.77±212.73 mmHg/s,3,910.72±101.36 mmHg/s VS 2,458.85±124.05 mmHg/s,n=12, P<0.01)、左心室内压最大下降速率(dp/dt_(min))(-2,537.75±213.09 mmHg/s,-2,548.28±205.65 mmHg/s VS -1,594.85±214.22 mmHg/s,n=12, P<0.01)均明显升高;(2)与假手术组相比,心衰组大鼠单个心肌细胞的ICa·L显著降低,且平均电流密度值亦明显减小(1.89±0.19 VS 6.48±0.33,n=20,P<0.01);与心衰组比较,中剂量和高剂量药物干预组大鼠单个心肌细胞的I_(Ca·L)明显加强,且平均电流密度值亦明显增强(4.35±0.32,4.21±0.24 VS 1.89±0.19,n=20,P<0.01);(3)与假手术组相比,心衰组大鼠CICR过程中的钙瞬变幅度明显下降,且钙瞬变峰值(ΔF/F_0)亦明显下降(12.52±1.38 VS 41.37±2.44,n=12,P<0.01);与心衰组相比,中剂量和高剂量药物干预组大鼠CICR过程中的钙瞬变幅度明显升高,且钙瞬变峰值(ΔF/F_0)亦明显上升(26.82±1.02,30.07±0.91 VS 12.52±1.38,n=12,P<0.01);(4)各组大鼠心肌细胞SR钙容量的比较:与假手术组相比,心衰组大鼠咖啡因诱导钙瞬变过程中钙瞬变幅度明显降低,且钙瞬变峰值(ΔF/F_0)亦明显下降(17.05±0.61 VS 35.36±0.89,n=12,P<0.01);与心衰组比较,中剂量和高剂量药物干预组大鼠咖啡因诱导钙瞬变过程中钙瞬变幅度明显升高,且钙瞬变峰值(ΔF/F_0)亦明显上升(32.3±0.74,32.19±0.51 VS 17.05±0.61,n=12,P<0.01);(5)各组大鼠心室肌细胞超微结构显示:假手术组大鼠心肌细胞的肌小节完整,肌丝排列整齐,线粒体结构正常,嵴排列紧密,糖原颗粒丰富。心衰组大鼠部分心肌细胞肌丝溶解,并出现大小不等的空泡,线粒体肿胀,嵴断裂。低剂量药物干预组大鼠心肌细胞肌丝溶解有轻微恢复,但线粒体仍肿胀,嵴断裂。中剂量药物干预组大鼠心肌细胞肌丝溶解现象消失,肌丝排列整齐,紧密,清晰,线粒体结构基本完整,嵴排列紧密。高剂量药物干预组大鼠心肌细胞表现基本同中剂量组,且线粒体的电子密度增加;(6)与心衰组比较,中剂量和高剂量药物干预组大鼠心肌细胞SERCA2a、Cav1.2的mRNA表达SERCA2a/GAPDH(0.2±0.017,0.2±0.019 VS 0.12±0.014,n=8,P<0.01)、Cav1.2/GAPDH(0.02±0.002,0.02±0.002 VS 0.014±0.002,n=8,P<0.01)均明显升高;(7)与心衰组比较,中剂量和高剂量药物干预组大鼠心肌细胞SERCA2a/GAPDH(0.94±0.053,1.36±0.059 VS 0.25±0.032,n=8,P<0.01)、DHPR/GAPDH(0.3±0.027,0.28±0.019 VS 0.18±0.015,n=8,P<0.01)的表达均明显升高。
结论:
氧化苦参碱(OMT)对慢性心衰大鼠的心肌细胞具有保护作用,而这种保护作用表现在大鼠心功能的改善。具体如下:
(1)在氧化苦参碱(OMT)的干预下,慢性心衰大鼠心肌细胞L-型钙通道(DHPR)表达上调,促使L-型钙电流的增大和钙诱导钙释放(CICR)的恢复,最终使心肌收缩力增强,心功能得以改善。
(2)在氧化苦参碱(OMT)的干预下,慢性心衰大鼠心肌细胞内钙泵(SERCA2a)表达上调,促使心肌兴奋-收缩耦联(ECC)结束后钙回摄功能恢复,肌浆网内钙容量增加,最终促使心肌收缩力加强,心功能改善。
Objective:
Altered intracellular Ca~(2+) handling by the sarcoplasmic reticulum (SR) plays a crucial role in the pathogenesis of heart failure (HF). Despite extensive effort, the underlying causes of abnormal SR Ca~(2+) handling in HF are not clarified. To determine whether the diastolic SR Ca~(2+) leak along with reduced Ca~(2+) reuptake are required for decreased contractility, we investigated the cytosolic Ca~(2+) transients,SR Ca~(2+) content and assessed the expression of Ca~(2+) handling genes and proteins, using SD-rat model of chronic heart failure.
Methods:
28 male SD rats were randomly divided into two groups: sham-operated rats (n=10), heart failure rats (n=18). Rats were deeply anesthetized, following intubation and placement on a respirator, a left lateral thoracotomy was performed and the left anterior descending (LAD) coronary artery was permanently ligated. Sham-operated animal sunder went the same procedure without ligation of the LAD. Twenty-eight days after myocardial infarction (MI), survival rate, hemodynamic and heart weight of rats were assessed. Single cardiomyocytes of the rat heart were isolated by an enzymatic dissociation method. Patch-clamp and laser scanning confocal microscope synchronous recording system software was used to record transmembrane Ca~(2+) currents (I_(Ca·L)) and intracellular Ca~(2+) transients simultaneously. SR Ca~(2+) content can be measured by caffeine-induced Ca~(2+) transients (CCT) indirectly and can be measured by permeabilized myocytes with SR-entrapped Fluo-5N/AM directly. The total RNA was extracted from the homogenate and the mRNA levels of Ca~(2+) handling proteins were measured using real-time quantitative RT-PCR. The levels of Ca~(2+) handling proteins were determined by immunoblot analysis. SPSS11.5 software was used for the statistical analyses. All data are presented as mean±SD and t-test or Fisher’s Exact test was used for the statistical analyses. P-values of <0.05 were considered significant.
Results:
(1) According to the experiment, the survival is 83.3% in HF group and 100% in sham group(P>0.05). There was a significant increase in the left ventricular end-diastolic pressure (LVEDP) in HF rats (8.3±0.42 mmHg, n=15) compared with sham rats (4.7±0.65 mmHg, n=10, P<0.01). There was a significant decrease in the maximal change of systolic pressure over time (dp/dt_(max)) in HF rats (2,140.41±118.38 mmHg/s, n=15) compared with control rats (4,355.75±259.71 mmHg/s, n=10, P<0.01) and there was a significant decrease in the maximum change in the rate of relaxation over time (dp/dt_(min)) in HF rats (-1,798.33±111.41 mmHg/s, n=15) compared with sham rats (-2,531.22±414.66 mmHg/s, n=10, P<0.01). Furthermore, there was a significant increase in heart weight (HW) divided by body weight (BW) in HF rats (6.76±0.36, n=15) compared with sham rats (3.33±0.41, n=10, P<0.01). (2) The amplitude of I_(Ca·L) was reduced in HF myocytes (1.01±0.09, n=20) compared with sham (2.18±0.1, n=18, P<0.01). Moreover the amplitude of Ca~(2+) transients was also reduced in HF group. There was a significant decrease in spatial averages (ΔF/F_0) of I_(Ca·L)-induced Ca~(2+) transients in HF (12.2±0.56, n=12) compared with sham (17.8±0.53, n=10, P<0.01). SR Ca~(2+) content can be measured by caffeine-induced Ca~(2+) transients (CCT) during diastolic phase. The amplitude of Ca~(2+) transients was reduced in HF group. Caffeine-induced Ca~(2+) transients (ΔF/F_0) was significantly decreased in HF (12.4±0.4, n=12) compared with sham (33.2±1.9, n=10, P<0.01). Isolated cardiomyocytes were loaded with Fluo-5N/AM and SR Ca~(2+) content can be measured directly. The result showed that the SR Ca~(2+) content was dramatically reduced in HF myocytes. There was a significant decrease in spatial averages (ΔF/F_0) in HF (26.6±1.87, n=12) compared with sham (49.6±2.02, n=10, P<0.01). (3) We found that the mRNA expression of RyR2 (0.063±0.002 VS 0.064±0.003,n=8, P>0.05) and PLB (0.063±0.003 VS 0.058±0.002,n=8, P>0.05) failed to alter in HF compared with sham group. The downregulation of FKBP12.6 (0.018±0.002 VS 0.042±0.002,n=8, P<0.01), SERCA2a (0.14±0.019 VS 0.28±0.016,n=8, P<0.01), NCX (0.07±0.016 VS 0.12±0.019,n=8, P<0.01) and Cav1.2 (0.01±0.0012 VS 0.026±0.0019,n=8, P<0.01) was established by RT-PCR to be significant relative to the sham, respectively. Compared with the sham rat, the expression of RyR2 (0.36±0.028 VS 0.39±0.03,n=8, P>0.05) and PLB (0.5±0.02 VS 0.57±0.03,n=8, P>0.05) failed to alter in HF rat, whereas FKBP12.6 (0.13±0.007 VS 0.9±0.05, n=8, P<0.01) content was significantly reduced in HF rat. Furthermore, the expression of SERCA2a (0.74±0.02 VS 1.36±0.009,n=8, P<0.01), NCX (0.18±0.03 VS 0.78±0.05,n=8, P<0.01) and DHPR (0.11±0.01 VS 1.2±0.05,n=8, P<0.01) were also reduced in HF rat.
Conclusion:
Diastolic SR Ca~(2+) leak (due to dissociation of FKBP12.6 from RyR2) along with reduced SR Ca~(2+) uptake (due to down-regulation of SERCA2a) and defective E-C coupling(due to down-regulation of DHPR)could contribute to the decreased contractility observed in failing hearts associated with reduced amplitude and slowed decay of the intracellular Ca~(2+) transients.
PartⅡProtective Effect of Oxymatrine on Chronic Rat Heart Failure
Objective:
Oxymatrine is one of the alkaloids extracted from Chinese herb Sophora japonica (Sophora flavescens Ait.) with activities of anti-inflammation, antivirus and protecting hepatocytes. However, the effect of oxymatrine on heart failure has not been known yet. In this study, the effect of oxymatrine on heart failure was investigated using SD-rat model of chronic heart failure.
Methods:
60 male SD rats were randomly divided into five groups: sham-operated rats (n=12), heart failure rats (n=12), high dose group (rats with MI and 100 mg/kg OMT treatment, qd), middle dose group (rats with MI and 50 mg/kg OMT treatment, qd), low dose group (rats with MI and 25 mg/kg OMT treatment, qd). Rats were deeply anesthetized, following intubation and placement on a respirator, a left lateral thoracotomy was performed and the left anterior descending (LAD) coronary artery was permanently ligated. Sham-operated animal sunder went the same procedure without ligation of the LAD. Twenty-eight days after MI, survival rate, hemodynamic and heart weight of rats were assessed. Single cardiomyocytes of the rat heart were isolated by an enzymatic dissociation method. Patch-clamp and laser scanning confocal microscope synchronous recording system software was used to record transmembrane Ca~(2+) currents (I_(Ca·L)) and intracellular Ca~(2+) transients simultaneously. SR Ca~(2+) content can be measured by caffeine-induced Ca~(2+) transients (CCT) indirectly. The ultrastructure of myocardial cells in left ventricle of rats was studied under transmission electron microscopy. The total RNA was extracted from the homogenate and the mRNA levels of Ca~(2+) handling proteins were measured using real-time quantitative RT-PCR. The levels of Ca~(2+) handling proteins were determined by immunoblot analysis. SPSS11.5 software was used for the statistical analyses. Data were expressed as mean±SD for all the experiments. Statistical analysis were made with ANOVA and followed by LSD test for individual comparisons of means. P-values of <0.05 were considered significant.
Results:
(1) According to the experiment, the survival is 100% in every group. There was a significant increase in the left ventricular end-diastolic pressure (LVEDP) in HF rats (8.1±0.24 mmHg) compared with sham rats (3.7±0.49 mmHg, n=12, P<0.01). There was a significant decrease in the maximal change of systolic pressure over time (dp/dt_(max)) in HF rats (2,458.85±124.05 mmHg/s) compared with sham rats (4,164.63±130.69 mmHg/s, n=12, P<0.01) and there was a significant decrease in the maximum change in the rate of relaxation over time (dp/dt_(min)) in HF rats (-1,594.85±214.22 mmHg/s) compared with sham rats (-2,765.82±152.99 mmHg/s, n=12, P<0.01). Furthermore, there was a significant increase in heart weight (HW) divided by body weight (BW) in HF rats (4.39±0.36) compared with sham rats (2.41±0.06, n=12, P<0.01). There was a significant decrease in LVEDP in middle (5.1±0.24 mmHg) and high dose group (4.6±0.34 mmHg) rats compared with HF rats (8.1±0.24 mmHg, n=12, P<0.01). There was a significant increase in dp/dt_(max) in middle (3,827.77±212.73 mmHg/s) and high dose group (3,910.72±101.36 mmHg/s) rats compared with HF rats (2,458.85±124.05 mmHg/s, n=12, P<0.01) and there was a significant increase in dp/dt_(min) in middle (-2,537.75±213.09 mmHg/s) and high dose group (-2,548.28±205.65 mmHg/s) rats compared with HF rats (-1,594.85±214.22 mmHg/s, n=12, P<0.01). Furthermore, there was a significant decrease in HW/BW in middle (3.11±0.08) and high dose group (3.09±0.08) rats compared with HF rats (4.39±0.36, n=12, P<0.01). (2) The amplitude of I_(Ca·L) was reduced in HF myocytes (1.89±0.19) compared with sham (6.48±0.33, n=20, P<0.01). The amplitude of I_(Ca·L) was enhanced in middle (4.35±0.32) and high dose group (4.21±0.24) myocytes compared with HF myocytes (1.89±0.19, n=20, P<0.01). Moreover the amplitude of Ca~(2+) transients was also reduced in HF group. There was a significant decrease in spatial averages (ΔF/F_0) of I_(Ca·L)-induced Ca~(2+) transients in HF (12.52±1.38) compared with sham (41.37±2.44, n=12, P<0.01). The amplitude of Ca~(2+) transients was also enhanced in middle and high dose group. There was a significant increase in spatial averages (ΔF/F_0) of I_(Ca·L)-induced Ca~(2+) transients in middle (26.82±1.02) and high dose group (30.07±0.91) compared with HF group (12.52±1.38,n=12, P<0.01). (3) SR Ca~(2+) content can be measured by CCT during diastolic phase. The amplitude of Ca~(2+) transients was reduced in HF group. Caffeine-induced Ca~(2+) transients (ΔF/F_0) was significantly decreased in HF (17.05±0.61) compared with sham (35.36±0.89, n=12, P<0.01). The amplitude of Ca~(2+) transients was enhanced in middle and high dose group compared with HF group. Caffeine-induced Ca~(2+) transients (ΔF/F_0) was significantly increased in middle (32.3±0.74) and high dose group (32.19±0.51) compared with HF group (17.05±0.61,n=12, P<0.01). (4) The ultrastructure of myocardial cells in left ventricle of rats was studied under TEM. In sham group, the myocardial cell appeared normal with intact sarcomeres, mitochondria contain tightly packed cristae. Numerous glycogen granules were present. In HF group, some parts of the myocardial cell appeared intermyofibrillar lysis and vesicles of varying size. Mitochondria were swollen, and mitochondrial cristae were separated. In group with treated by low dose of OMT, intermyofibrillar lysis slightly restored, but a few mitochondria were still swollen, and mitochondrial cristae were separated. In group with treated by middle dose of OMT, intermyofibrillar lysis disappeared, myofilaments were orderly, closely, and evenly arranged, and mitochondria contain tightly packed cristae. The ultrastructure of myocardial cells, treated by high dose of OMT, was similar with that of treated by middle dose of OMT, and electron density of some mitochondria increased. (5) We found that the mRNA express of SERCA2a was significantly increased in middle (0.2±0.017) and high (0.2±0.019) dose group compared with the HF group (0.12±0.014, n=8, P<0.01). Furthermore, the mRNA content of Cav1.2 was significantly increased in middle (0.02±0.002) and high dose group (0.02±0.002) compared with the HF rat (0.014±0.002, n=8, P<0.01). (6) The expression of SERCA2a was increased in middle (0.94±0.053) and high dose group (1.36±0.059) compared with HF group (0.25±0.032, n=8, P<0.01). The expression of DHPR was also increased in middle (0.3±0.027) and high dose group (0.28±0.019) compared with HF group (0.18±0.015, n=8, P<0.01).
Conclusion:
The present results suggest that oxymatrine improves heart failure by ameliorating the cardiac function, and that this amelioration is associated with upregulation of SERCA2a and DHPR. We suggest that OMT maybe a novel, effective therapeutic drug for the treatment of heart failure.
引文
[1] Majeed A, Williams J, de Lusignan S, et al. Management of heart failure in primary care after implementation of the National Service Framework for Coronary Heart Disease: a cross-sectional study. Public Health. 2005; 119 (2): 105-111.
[2] Thomas R. Shannon, Wilbur Y. W. Lew. Diastolic Release of Calcium From the Sarcoplasmic Reticulum. Journal of the American College of Cardiology. 2009; 53(21): 2006-2008.
[3] Campbell AK. In: Intracellular Calcium: Its Universal Role as Regulator, edited by Gutfreund H. London: Wiley, 1983.
[4] Carafoli E, and Klee C. (Editors). Calcium as a Cellular Regulator. Oxford, UK: Oxford Univ. Press, 1999.
[5] Huxley AF. In: Mineral Metabolism, edited by Comman CL, and Bronner F. New York: Academic, 1961.
[6] Hasenfuss G, Pieske B. Calcium cycling in congestive heart failure. J Mol Cell Cardiol. 2002; 34: 951-969.
[7]Yatani A, Shen Y T, Yan L, et al. Down regulation of the L-type Ca~(2+) channel, GRK2 and phosphorylated phospholamban: protective mechanisms for the denervated failing heart. Mol Cell Cardiol. 2006; 40 (5): 619-628.
[8] Wang S Q,Song L S,Lakatta E G. Ca~(2+) signalling between single L-type Ca~(2+) channels and ryanodine receptors in heart cells. Nature. 2001; 410: 592.
[9] PiacentinoⅢV, Weber C, Chen X W, et a1. Cell basis of abnormal calcium transients of failing human ventricular myocytes. Circ Res. 2003; 92: 651-658.
[10] Yano M, Ikada Y, Matsuzaki. Altered intracellular Ca~(2+) handling in heart failure. J Clin Invest. 2005; 115: 556-564.
[11] Pessah I.N., Waterhouse A.L., Casida J.E. The calcium–ryanodine receptor complex of skeletal and cardiac muscle. Biochem. Biophys. Res. Commun. 1985; 128(1): 449-456.
[12] Masafumi Yano, Takeshi Yamamoto, Shigeki Kobayashi, et al. Role of ryanodine receptor as a Ca~(2+) regulatory center in normal and failing hearts. Journal of Cardiology 2009; 53: 1-7.
[13] Hiroki Tateishi, Masafumi Yano, Mamoru Mochizuki, et a1. Defective domain -domain interactions within the ryanodine receptor as a critical cause of diastolic Ca~(2+) leak in failing hearts. Cardiovasc Res. 2009; 81(3): 536-545.
[14] Wehrens, X.H. et al. FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death. Cell. 2003; 113 (7): 829-840.
[15] Subeena Sood, Mihail G. Chelu, Ralph J. van Oort, et a1. Intracellular calcium leak due to FKBP12.6 deficiency in mice facilitates the inducibility of atrial fibrillation. Heart Rhythm. 2008; 5(7): 1047-1054.
[16] Marx SO, Reiken S, Hisamatsu Y, et al. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor ): defective regulation in failing hearts. Cell. 2000; 101: 365-376.
[17] Yano M, Kobayashi S, Kohno M, et al. FKBP12.6-mediated stabilization of calcium-release channel (ryanodine receptor) as a novel therapeutic strategy against heart failure. Circulation. 2003; 107: 477-484.
[18] Stange M, Xu L, Balshaw D, et al. Characterization of recombinant skeletal muscle (Ser-2843) and cardiac muscle (Ser-2809) ryanodine receptor phosphorylation mutants. J Biol Chem. 2003; 278(5): 51693-51702.
[19] MI Ya-fei, LI Xiao-ying, TANG Li-jiang, LU Xiao-chun, FU Zhi-qing and YE Wei -hua. Improvement in cardiac function after sarcoplasmic reticulum Ca~(2+)-ATPase gene transfer in a beagle heart failure model. Chinese Medical Journal 2009; 122(12): 142-1428.
[20] Schmidt U, Hajjar R J, Kim C S, et a1. Human heart failure: cAMP stimulation of SR Ca~(2+)-ATPase activity and phosphorylation level of phospholamban. Am J Physiol, 1999; 277: H474-480.
[21] K, Kranias EG. Phospholamban and Cardiac Contractility. Annals of Medicine (The Finnish Med. Soc. Duodecim). 2000; 32: 572-578.
[22] Yoshie Tanaka, Takeshi Honda, Kenji Matsuura, Yoshihiro Kimura, Makoto Inui. In Vitro Selection and Characterization of DNA Aptamers Specific for Phospholamban. J Pharmacology and Experimental Therapeutics. 2009; 329(1): 57-63.
[23] Hasenfuss G. Alterations of calcium regulatory proteins in heart failure.Cardiovasc Res. 1998; 37(2): 279-289.
[24] Louiza Belkacemi, Isabelle B′edard, Lucie Simoneau, Julie Lafond. Calcium channels, transporters and exchangers in placenta. Cell Calcium. 2005; 37: 1-8.
[25] Liu M, Liu XY, Cheng JF. Advance in the pharmacological research on matrine. Zhongguo Zhong Yao Za Zhi. 2003; 28: 801-804.
[26] Ma L,Wen S, Zhan Y, et al. Anticancer effects of the Chinese medicine matrine onmurine hepatocellular carcinoma cells. Planta Med 2008; 74: 245-251.
[27] Liu SX, Chiou GC. Effects of Chinese herbal products on mammalian retinal functions. J Ocul Pharmacol Ther. 1996; 12: 377-386.
[28] Jiang H, Hou C, Zhang S, et al. Matrine upregulates the cell cycle protein E2F-1 and triggers apoptosis via the mitochondrial pathway in K562 cells. Eur J Pharmacol. 2007; 559: 98-108.
[29] Azzam HS, Goertz C, Fritts M, et al. Natural products and chronic hepatitis C virus. Liver Int. 2007; 27: 17-25.
[30] Yamazaki M. The pharmacological studies on matrine and oxymatrine. Yakugaku Zasshi. 2000; 120: 1025-1033.
[31] Zhang MJ, Huang J. Recent research progress of anti-tumor mechanism matrine. Zhongguo Zhong Yao Za Zhi. 2004; 29: 115-118.
[32] Jiang, H., Meng, F., Li, J., et al. Anti-apoptosis effects of oxymatrine protect the liver from warm ischemia reperfusion injury in rats. World J. Surg. 2005; 29: 1397-1401.
[33] Zhao, J., Yu, S., Tong, L., et al. Oxymatrine attenuates intestinal ischemia/reperfusion injury in rats. Surg. Today. 2008; 38: 931-937.
[34] Zuzana K, Dmitry T, Serge V. Abnormal intrastore calcium signaling in chronic heart failure.Proc Nail Acad Sci USA. 2005; 102(39): 14104-14109.
[1] Fabiato A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol. 1983; 245(1): C1-C14.
[2] Bers DM. Cardiac excitation-contraction coupling. Nature. 2002; 415(6868): 198-205.
[3] Hasenfuss G and Pieske B. Aug Calcium cycling in congestive heart failure. J Mol Cell Cardiol. 2002, 34(8): 951-969.
[4] Sipido KR, Eisner D. Something old, something new: changing views on the cellular mechanisms of heart failure. Cardiovasc Res. 2005, 68(2):167-174.
[5]Mccall E, Ginsburg KS, Bassani RA, et al. Ca~(2+) flux, contractility, and excitation-contraction coupling in hypertrophic rat ventricular myocytes. Am J Physiol. 1998; 274: H1348-H1360.
[6]沈建新,王世强,程和平等. Thapsigargin对心肌细胞钙释放和肌浆网钙容量的时间效应.中山大学学报. 2004; 25(1): 19-23.
[7] Yano M, Ikada Y, Matsuzaki. Altered intracellular Ca~(2+) handling in heart failure. J Clin Invest. 2005; 115: 556-654.
[8] Lin L, Kim SC, Wang Y, Gupta S, Davis B, Simon SI, Torre-Amione G, Knowlton AA. HSP60 in heart failure: abnormal distribution and role in cardiac myocyte apoptosis. Am J Physiol Heart Circ Physiol. 2007; 293(4): H2238-H2247.
[9] Ohmoto-Sekine Y, Uemura H, Tamagawa M, Nakaya H. Inhibitory effects of aprindine on the delayed rectifier K~+ current and the muscarinic acetylcholine receptor-operated K~+ current in guinea-pig atrial cells. Br J Pharmacol. 1999 126(3): 751-761.
[10] Tytgat J. How to isolate cardiac myocytes. Cardiovasc Res. 1994; 28(2): 280-283.
[11] Mitra R,Morad M.A uniform enzymatic method for dissociation of myocytes from hearts and stomachs of vertebrates. Am J Physiol. 1985; 249: H1056-1060.
[12]曹春梅,张雄,陈莹莹等.不同用途的心室肌细胞的分离。浙江大学学报(医学版), 2003; 32(1): 51-55.
[13] Nakaya H, Tohse N, Takeda Y, Kanno M. Effects of MS-551, a new class antiarrhythmic drug, on action potential and membrane currents in rabbit ventricular myocytes. Br J Pharmacol. 1993, 109(1): 157-163.
[14] Sakmann B, Neher E. Single-Channel Recording, New York: Plenum. 1983.
[15] Qi M Xia HJ,Dai DZ, Dai Y. A novel endothelin receptor antagonist CPU0213 improves diabetic cardiac insufficiency attributed to up-regulation of the expression of FKBPl2.6, SERCA2a,and PLB in rats. J Cardiovascular Pharmacology. 2006; 47:729-735.
[16] Long-Sheng Song, et al. Beta-Adrenergic stimulation synchronizes intracellular Ca~(2+) release during excitation-contraction coupling in cardiac myocytes. Circ Res. 2001; 88(8): 794-801.
[17]唐晓鸿.心脉隆注射液药理作用和治疗心力衰竭临床研究进展.中国新药杂志. 2008; 6(17): 461-464.
[18]于秀琼,蔡琳.疾病管理和心力衰竭.心血管病学进展. 2007; 5(28):739-740.
[19] Thomas R. Shannon, Wilbur Y. W. Lew. Diastolic Release of Calcium From the Sarcoplasmic Reticulum. Journal of the American College of Cardiology. 2009; 53(21): 2006-2008.
[20] Janczewski AM, Spurgeon HA, Stern MD, Lakatta EG. Effects of sarcoplasmic reticulum Ca~(2+) load on the gain function of Ca~(2+) release by Ca~(2+) current in cardiac cells. Am J Physiol. 1995; 268: H916-920.
[21] Trafford AW, Díaz ME, Negretti N, Eisner DA. Enhanced calcium current and decreased calcium efflux restore sarcoplasmic reticulum Ca~(2+) content following depletion. Circ Res. 1997; 81(4): 477-484.
[22] Fill M,Copello J A. Ryanodine receptor calcium release channels. Physiol Rev. 2002; 82(4): 893-922.
[23] Trafford AW, Díaz ME, Sibbring GC, Eisner DA. Modulation of CICR has no maintained effect on systolic Ca~(2+): simultaneous measurements of sarcoplasmic reticulum and sarcolemmal Ca~(2+) fluxes in rat ventricular myocytes. J Physiol. 2000; 522: 259-270.
[24] Jean PB, Julio L, et al. L-type Ca~(2+) current in ventricular cardiomyocytes. J Mole Cel Cardio. 2010, 48:26-36.
[25] Hiroki Tateishi, Masafumi Yano, Mamoru Mochizuki, et a1. Defective domain-domain interactions within the ryanodine receptor as a critical cause of diastolic Ca~(2+) leak in failing hearts. Cardiovasc Res. 2009; 81(3): 536-545.
[26] Bers DM. Macromolecular complexes regulating cardiac ryanodine receptor function. J Mol Cell Cardiol. 2004; 37: 417-429.
[27] Nicolaou P, Rodriguez P, Kranias EG, et al. Inducible expression of active protein phosphatase-1 inhibitor-1 enhances basal cardiac function and protects against ischemia/reperfusion injury. Circ Res. 2009, 104(8): 1012-1020.
[28] Yano M, Yamamoto T, Kobayashi S, Matsuzaki M. Role of ryanodine receptor as a Ca~(2+) regulatory center in normal and failing hearts. J Cardiol. 2009, 53:1-7.
[29] Stange M, Xu L, Balshaw D, Yamaguchi N, Meissner G. Characterization of recombinant skeletal muscle (Ser-2843) and cardiac muscle (Ser-2809) ryanodine receptorphosphorylation mutants. J Biol Chem. 2003; 278(5):51693-51702.
[30] Waggoner JR, Ginsburg KS, Mitton B, Haghighi K, Robbins J, Bers DM, Kranias EG. Phospholamban overexpression in rabbit ventricular myocytes does not alter sarcoplasmic reticulum Ca transport. HAm J Physiol Heart Circ Physiol. 2009, 296(3): 698-703.
[31] Yoshie Tanaka, Takeshi Honda, Kenji Matsuura, Yoshihiro Kimura, Makoto Inui. In Vitro Selection and Characterization of DNA Aptamers Specific for Phospholamban. J Pharmacology and Experimental Therapeutics. 2009; 329(1): 57-63.
[32] Reuter H, Seitz N. The dependence of calcium efflux from cardiac muscle on temperature and external ion composition. J Physiol (Lond). 1968; 195: 45-70.
[33] Graham J. Jeffs, Bruno P. Meloni, Anthony J. Bakker, Neville W. Knuckey. The role of the Na~+/Ca~(2+) exchanger (NCX) in neurons following ischaemia. Journal of Clinical Neuroscience. 2007; 14: 507-514.
[34] M.P. Blaustein,WJ. Lederer, Sodium/calcium exchange: its physiological implications. Physiol. Rev. 1999; 79: 763-854.
[35] Li ZP, Matsuoka S, Hryshko LV, Nicoll DA, Bersohn MM, Burke EP, Lifton RP, Philipson KD. Cloning of the NCX2 isoform of the plasma membrane Na~+/Ca~(2+) exchanger. J Biol Chem. 1994; 269: 17434-17439.
[36] Nicoll DA, Quednau BD, Qiu ZY, Xia YR, Lusis AJ, Philipson KD. Cloning of a third mammalian Na~+/Ca~(2+) exchanger, NCX3. J Biol Chem 1996; 271: 24914-24921.
[37] Mihra S, Sabbah HN, Rastogi S, et a1. Reduced sarcoplasmic reticulum Ca~(2+) uptake and increased Na~+-Ca~(2+) exchanger expression in left ventricle myocardiurn of dogs with progression of heart failure. Heart Vessels. 2005; 20: 23-32.
[1] Gy(?)rke, I. & Gy(?)rke, S. Regulation of the cardiac ryanodine receptor channel by luminal Ca~(2+) involves luminal Ca~(2+) sensing sites. Biophys. J. 1998; 75: 2801-2810
[2] Ching, L. L., Williams, A. J. & Sitsapesan, R. Evidence for Ca(2+) activation and inactivation sites on the luminal side of the cardiac ryanodine receptor complex. Circ. Res. 2000; 87: 201-206.
[3] Bers, D. M. in Excitation-Contraction Coupling and Cardiac Contractile Force (Kluwer Academic, Dordrecht, The Netherlands), 2nd Ed. 2001.
[4] Lindner, M., Erdmann, E. & Beuckelmann, D. J. Calcium content of the sarcoplasmic reticulum in isolated ventricular myocytes from patients with terminal heart failure. J. Mol. Cell. Cardiol. 1998; 30: 743-749.
[5] O'Rourke, B., Kass, D. A., Tomaselli, G. F., K??b, S., Tunin, R. & Marbán, E. Mechanisms of altered excitation-contraction coupling in canine tachycardia-induced heart failure, I: experimental studies. Circ. Res. 1999; 84: 562-570.
[6] Houser, S. R., Piacentino, V., III, & Weisser, J. Abnormalities of calcium cycling in the hypertrophied and failing heart. J. Mol. Cell. Cardiol. 2000; 32: 1595-1607.
[7] Hasenfuss, G. & Pieske, Calcium cycling in congestive heart failure B. J. Mol. Cell. Card iol. 2002; 34: 951-969.
[8] Houser, S. R. & Margulies, K. B. Is depressed myocyte contractility centrally involved in heart failure? Circ. Res. 2003; 92: 350-358.
[9] Liu M, Liu XY, Cheng JF. Advance in the pharmacological research on matrine. Zhongguo Zhong Yao Za Zhi. 2003; 28: 801-804.
[10] Ma L,Wen S, Zhan Y, He Y, Liu X, Jiang J. Anticancer effects of the Chinese medicine matrine on murine hepatocellular carcinoma cells. Planta Med 2008; 74: 245-251.
[11] Liu SX, Chiou GC. Effects of Chinese herbal products on mammalian retinal functions. J Ocul Pharmacol Ther. 1996; 12: 377-386.
[12] Jiang H, Hou C, Zhang S, Xie H, Zhou W, Jin Q. Matrine upregulates the cell cycle protein E2F-1 and triggers apoptosis via the mitochondrial pathway in K562 cells. Eur J Pharmacol. 2007; 559: 98-108.
[13] Azzam HS, Goertz C, Fritts M, Jonas WB. Natural products and chronic hepatitis C virus. Liver Int. 2007; 27: 17-25.
[14] Yamazaki M. The pharmacological studies on matrine and oxymatrine. YakugakuZasshi. 2000; 120: 1025-1033.
[15] Zhang MJ, Huang J. Recent research progress of anti-tumor mechanism matrine. Zhongguo Zhong Yao Za Zhi. 2004; 29: 115-118.
[16] Jiang, H., Meng, F., Li, J., Sun, X. Anti-apoptosis effects of oxymatrine protect the liver from warm ischemia reperfusion injury in rats. World J. Surg. 2005; 29: 1397-1401.
[17] Zhao, J., Yu, S., Tong, L., Zhang, F., Jiang, X., Pan, S., Jiang,H., Sun, X. Oxymatrine attenuates intestinal ischemia/reperfusion injury in rats. Surg. Today. 2008; 38: 931-937.
[18]李丽于,宏伟,李雪峰,吴宜艳.苦参碱对大鼠缺血再灌注心肌损伤的保护作用.中国误诊学杂志. 2009; 9(3): 529-530.
[19]杨钰萍,沈祥春,刘兴德,方泰惠,许立.氧化苦参碱对急性心肌梗死诱发实验性大鼠心肌重塑的影响.中国实验方剂学杂志. 2010; 16(6): 125-128.
[20] Campbell AK. In: Intracellular Calcium: Its Universal Role as Regulator, edited by Gutfreund H. London: Wiley, 1983.
[21] Carafoli E, and Klee C.. Calcium as a Cellular Regulator. Oxford, UK: Oxford Univ. Press, 1999.
[22] Huxley AF. In: Mineral Metabolism, edited by Comman CL, and Bronner F. New York: Academic, 1961.
[23] Michael G, Joan H B.β-Adrenergic receptor signaling in the heart: Role of CaMKII. J Mol Cell Cardiol. 2010; 48: 322-330.
[24] Vafiadaki E, Papalouka V, Arvanitis DA, Kranias EG, Sanoudou D. The role of SERCA2a/PLN complex, Ca~(2+) homeostasis, and anti-apoptotic proteins in determining cell fate. HP flugers Arch. 2009, 457(3):687-700.
[25] Zuzana K, Dmitry T,Serge V,et a1.Abnormal intrastore calcium signaling in chronic heart failure. Proc Nail Acad Sci USA. 2005; 102(39): 14104-14109.
[26] PiacentinoⅢV, Weber C, Chen X W, et a1. Cell basis of abnormal calcium transients of failing human ventricular myocytes. Circ Res. 2003; 92: 651-658.
[27]Engelhardt S, Hein L, Kranias E G, Lohse M J. Altered calcium handling is critically involved in the cardiotoxic effects of chronic beta-adrenergic stimulation.Circulation.2004; 109(9): 1154-1160.
[1] Mosterd A, Hoes AW. Clinical epidemiology of heart failure. Heart. 2007; 93: 1137-1146.
[2]于秀琼,蔡琳.疾病管理和心力衰竭.心血管病学进展. 2007; 5(28):739-740.
[3] Yano M, Ikada Y, Matsuzaki. Altered intracellular Ca~(2+) handling in heart failure. J Clin Invest. 2005; 115: 556-564.
[4] Wehrens XH, Lehnart SE, Marks AR. Intracellular calcium release channels and cardiac disease. Annu Rev Physiol. 2005; 67: 69-98.
[5] Jiang MT, Lokuta AJ, Farrell EF, et al. Abnormal Ca~(2+) release, but normal ryanodine receptors, in canine and human heart failure. Circ Res 2002; 91: 1015-22.
[6] Yamamoto T, Yano M, Kohno M, et al. Abnormal Ca~(2+) release from cardiac sarcoplasmic reticulum in tachycardia-induced heart failure. Cardiovasc Res. 1999; 44: 146 -55.
[7] Campbell AK. In: Intracellular Calcium: Its Universal Role as Regulator, edited by Gutfreund H. London: Wiley. 1983.
[8] Carafoli E, and Klee C. Calcium as a Cellular Regulator. Oxford, UK: Oxford Univ. Press, 1999.
[9] Huxley AF. In: Mineral Metabolism, edited by Comman CL, and Bronner F. New York: Academic, 1961.
[10]Yatani A, Shen Y T, Yan L, et al. Down regulation of the L-type Ca~(2+) channel, GRK2 and phosphorylated phospholamban: protective mechanisms for the denervated failing heart. Mol Cell Cardiol. 2006; 40 (5): 619-628.
[11] Wang S Q,Song L S,Lakatta E G. Ca~(2+) signalling between single L-type Ca~(2+) channels and ryanodine receptors in heart cells. Nature. 2001; 410: 592.
[12] Fabiato A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol. 1983; 245: C1-14.
[13] PiacentinoⅢV, Weber C, Chen X W, et a1. Cell basis of abnormal calcium transients of failing human ventricular myocytes. Circ Res. 2003; 92: 651-658.
[14] Franzini-Armstrong, C.. Studies of the triad. J Cell Biol. 1970; 47: 488-499.
[15] Campbell, K., Franzini-Armstrong, C., & Shamoo, A. Further characterisation of light and heavy sarcoplasmic reticulum vesicles. Identification of the‘sarcoplasmic reticulum feet’associated with heavy sarcoplasmic reticulum vesicles. Biochim Biophys Acta. 1980;602: 97-116.
[16] Tunwell RE, Wickenden C, Bertrand BM, et al. The human cardiac muscle ryanodine receptor-calcium release channel: identification, primary structure and topological analysis. Biochem J. 1996; 318: 477-487.
[17] Giannini G, Sorrentino V. Molecular structure and tissue distribution of ryanodine receptors calcium channels. Med Res Rev 1995; 15: 313-323.
[18] Franzini-Armstrong C, Protasi F, Ramesh V. Shape, size, and distribution of Ca~(2+) release units and couplons in skeletal and cardiac muscles. Biophys J. 1999; 77: 1528- 1539.
[19] Bers DM. Macromolecular complexes regulating cardiac ryanodine receptor function. J Mol Cell Cardiol. 2004; 37: 417-429.
[20] Lehnart SE, Wehrens XH, Marks AR.Calstabin deficiency, ryanodine receptors, and sudden cardiac death. Biochem Biophys Res Commun. 2004; 322(4): 1267-1279.
[21] Marx, S.O. et al. Coupled gating between cardiac calcium release channels (ryanodine receptors). Circ. Res. 2001; 88(11): 1151-1158.
[22] Marx SO, Reiken S, Hisamatsu Y, et al. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell. 2000; 101: 365-376.
[23] Yano M, Kobayashi S, Kohno M, Doi M, Tokuhisa T, Okuda S, Suetsugu M, Hisaoka T, Obayashi M, Ohkusa T, Kohno M, Matsuzaki M. FKBP12.6-mediated stabilization of calcium-release channel (ryanodine receptor) as a novel therapeutic strategy against heart failure. Circulation. 2003; 107: 477-484.
[24] Engelhardt S, Hein L, Kranias E G, Lohse M J. Altered calcium handling is critically involved in the cardiotoxic effects of chronic beta-adrenergic stimulation.Circulation.2004; 109(9): 1154-1160.
[25] Stange M, Xu L, Balshaw D, Yamaguchi N, Meissner G. Characterization of recombinant skeletal muscle (Ser-2843) and cardiac muscle (Ser-2809) ryanodine receptor phosphorylation mutants. J Biol Chem. 2003; 278(5): 51693-51702.
[26] Tripathy A, Xu L, Mann G, Meissner G. Calmodulin activation and inhibition of skeletal muscle Ca~(2+) release channel (ryanodine receptor). Biophys J. 1995; 69: 106-119.
[27] Balshaw DM, Xu L, Yamaguchi N, Pasek DA, Meissner G. Calmodulin binding and inhibition of cardiac muscle calcium release channel (ryanodine receptor). J Biol Chem. 2001; 276: 20144-20153.
[28] Yamaguchi N, Xu L, Pasek DA, Evans KE, Meissner G. Molecular basis of calmodulin binding to cardiac muscle Ca~(2+) release channel (ryanodine receptor). J Biol Chem. 2003; 278: 23480-23486.
[29] Yamaguchi N, Takahashi N, Xu L, Smithies O, Meissner G. Early cardiac hypertrophy in mice with impaired calmodulin regulation of cardiac muscle Ca~(2+) release channel. J Clin Invest. 2007; 117: 1344-1353.
[30] Mitchell RD, Simmerman HK, Jones LR. Ca~(2+) binding effects on protein conformation and protein interactions of canine cardiac calsequestrin. J Biol Chem. 1988; 263: 1376-1381.
[31] Gyorke S, Terentyev D. Modulation of ryanodine receptor by luminal calcium and accessory proteins in health and cardiac disease. Cardiovasc Res. 2008; 77: 245-255.
[32] Gyorke I, Hester N, Jones LR, Gyorke S. The role of calsequestrin, triadin, and junctin in conferring cardiac ryanodine receptor responsiveness to luminal calcium. Biophys J. 2004; 86: 2121-2128.
[33] Tracy J. Pritchard, Evangelia G. Kranias. Junctin and the histidine-rich Ca~(2+) binding protein:potential roles in heart failure and arrhythmogenesis. J Physiol. 2009; 587(13): 3125-3133.
[34] Beard NA, Casarotto MG, Wei L, Varsányi M, Laver DR, Dulhunty AF. Regulation of ryanodine receptors by calsequestrin: effect of high luminal Ca~(2+) and phosphorylation. Biophys J. 2005; 88: 3444-3454.
[35] Terentyev D, Nori A, Santoro M, Viatchenko-Karpinski S, Kubalova Z, Gyorke I, Terentyeva R, Vedamoorthyrao S, Blom NA, Valle G, Napolitano C, Williams SC, Volpe P, Priori SG, Gyorke S. Abnormal interactions of calsequestrin with the ryanodine receptor calcium release channel complex linked to exercise-induced sudden cardiac death. Circ Res. 2006; 98: 1151-1158.
[36] Oda T, Yano M, Yamamoto T, Tokuhisa T, Okuda S, Doi M, Ohkusa T, Ikeda Y, Kobayashi S, Ikemoto N, Matsuzaki M. Defective regulation of interdomain interactions within the ryanodine receptor plays a key role in the pathogenesis of heart failure. Circulation. 2005; 111: 3400-3410.
[37] Zaiain Herzberg A, MacLeun an DH, Periasanly. Characterization of rabbit saco (eodn) plasmic reticulum Ca~(2+)-ATPase gene. J BiDl Chem. 1990; 256: 4670-4677.
[38] Wu KD, LeeWS, Wey J, et al.Localiation and quantification of endoplasmlc reticulum Ca~(2+)-ATPaseisoformtranscripts. Am J Physiol. 1995; 269: C775-C784.
[39] Zhang P, Toyoshima C, Yonekura K, et al. Structure of the calcium pump from sarcoplasmic retieulum at 8-A resolution. Nature. 1998; 392: 835-839.
[40] Schmidt U, Hajjar RJ, Kim CS, et al. Human heart failure: cAMP stimulation of SR Ca~(2+)-ATPase activity and phosphorylation level of phospholambaa. Am J Physiol. 1999; 277: H474-H480.
[41]钟明,张运,张薇,等.舒张性心力衰竭兔Ca~(2+)调控蛋白mRNA和蛋白质的表达.中国病理生理杂志. 2002; 18: 124-127.
[42] J. Lytton, A. Zarain-Herzberg, M. Periasamy, D.H. MacLennan,Molecular cloning of the mammalian smooth muscle sarco(endo)plasmic reticulum Ca~(2+)-ATPase, J. Biol. Chem. 1989; 264: 7059-7065.
[43] Maclennan DH, Toyofuku T. Sites of regulatory interaction between calcium ATPase and phoepholamban. Basic Res Cardiol. l997; 92: l1-l5.
[44] Luo W, Gmpp IL, Hatter J, et al. Targeted ablation of the phospholamban gene is associated with markedly enhanced myocardial contractility and loss of beta-agonist stimulation. Circ Res. 1994; 75: 401-409.
[45] Díaz ME, Graham HK, Trafford AW. Enhanced sarcolemmal Ca~(2+) efflux reduces sarcoplasmic reticulum Ca~(2+) content and systolic Ca~(2+) in cardiac hypertrophy. Cardiovasc Res. 2004; 62:538-47.
[46] O'Rourke B, Kass DA, Tomaselli GF, Kaab S, Tunin R, Marban E. Mechanisms of altered excitation–contraction coupling in canine tachycardia-induced heart failure, I Experimental studies. Circ Res. 1999; 84: 562-570.
[47] Pieske B, Maier LS, Bers DM, Hasenfuss G. Ca~(2+) handling and sarcoplasmic reticulum Ca~(2+) content in isolated failing and nonfailing human myocardium. Circ Res 1999; 85: 38-46.
[48] Maclennan DH, Kranias EG. Phospholanlhan: a crucial regulator of cardiac contractility. Nat Rev Mol Cell Biol. 2003; 4(7): 566-577.
[49] Hoshijima M, lkeda Y, 1wanaga Y, el al. Chronic suppression of heart-failure progression by pseudophosphorylated mutant of phospholamhan via in vivo cardiac rAAV gene delivery. Nat Med. 2002; 8(8): 864-87l.
[50] Reuter H, Seitz N The dependence of calcium efflux from cardiac muscle on temperature and external ion composition. J Physiol (Lond). 1968; 195: 45-70.
[51] M.P. Blaustein,W.J. Lederer, Sodium/calcium exchange: its physiological implications. Physiol. Rev. 1999; 79: 763-854.
[52] Li ZP, Matsuoka S, Hryshko LV, Nicoll DA, Bersohn MM, Burke EP, Lifton RP, Philipson KD. Cloning of the NCX2 isoform of the plasma membrane Na~+/Ca~(2+) exchanger. J Biol Chem. 1994; 269: 17434-17439.
[53] Nicoll DA, Quednau BD, Qiu ZY, Xia YR, Lusis AJ, Philipson KD. Cloning of a third mammalian Na~+/Ca~(2+) exchanger, NCX3. J Biol Chem 1996; 271: 24914-24921.
[54] Santacruz-Toloza L, Ottolia M, Nicoll DA, Philipson KD. Functional analysis of a disulfide bond in the cardiac Na~+/Ca~(2+) exchanger. J Biol Chem. 2000; 275:182-188.
[55] Schwarz EM, Benzer S. Calx, a Na~+/Ca~(2+) exchanger gene of Drosophila melanogaster. Proc Natl Acad Sci. 1997; 94: 10249-10254.
[56] Nicoll DA, Hryshko LV, Matsuoka S, Frank JS, Philipson KD Mutation of amino acids residues in the putative transmembrane segments of the cardiac sarcolemmal Na~+/Ca~(2+) exchanger. J Biol Chem 1996; 271: 13385-13391.
[57] Iwamoto T, Uehara A, Imanaga I, Shigekawa M. The Na~+/Ca~(2+) exchanger NCX1 has oppositely oriented reentrant loop domains that contain conserved aspartic acids whose mutation alters its apparent Ca~(2+) affinity. J Biol Chem. 2000; 275: 38571-38580.
[58] Nicoll DA, Ottolia M, Lu LY, Lu YJ, Philipson KD. A new topological model of the cardiac sarcolemmal Na~+/Ca~(2+) exchanger. J Biol Chem. 1999; 274: 910-917.
[59] Qiu ZY, Nicoll DA, Philipson KD Helix packing of functionally important regions of the cardiac Na~+/Ca~(2+) exchanger. J Biol Chem. 2001; 276: 194-199.
[60] Schwarz EM, Benzer S Calx, a Na~+/Ca~(2+) exchanger gene of Drosophila melanogaster. Proc Natl Acad Sci 1997; 94: 10249-10254.
[61] Matsuoka S, Nicoll DA, Reilly RF, Hilgemann DW, Philipson KD Initial localization of regulatory regions of the cardiac sarcolemmal Na~+/Ca~(2+) exchanger. Proc Natl Acad Sci. 1993; 90: 3870-3874.
[62] Frank JS, Mottino G, Reid D, Molday RS, Philipson KD. Distribution of the Na~+/Ca~(2+) exchange protein in mammalian cardiac myocytes: an immunofluorescence and immunocolloidal gold-labeling study. J Cell Biol. 1992; 117: 337-345.
[63] Kieval RS, Bloch GE, Lindenmayer A, Ambesi A, Lederer WJ.Immunofluorescence localization of the Na~+/Ca~(2+) exchanger in heart cells. Am J Physiol 1992; 263: C545-C550.
[64] Scriven DRL, Dan P, Moore EDW. Distribution of proteins implicated in excitation -contraction coupling in rat ventricular myocytes. Biophys J 2000; 79: 2682-2691.
[65] Yang Z, Pascarel C, Steele DS, Komukai K, Brette F, Orchard CH. Na~+/Ca~(2+) exchange activity is localized in the Ttubulesof rat ventricular myocytes. Circ Res. 2002;91: 315-322.
[66] Iwamoto T. Forefront of Na~+/Ca~(2+) exchanger studies: molecular pharmacology of Na~+/Ca~(2+) exchange inhibitors. J Pharmacol Sci. 2004; 96: 27-32.
[67] Hobai IA , O’Rourke B. The potential of Na~+/Ca~(2+) Exchange blockers in the treatment of cardiac disease. Expert Opin Investig Drugs. 2004; 13: 653-664.
[68] Leblanc N, Hume JR. Sodium current induced release of calcium from cardiac sarcoplasmic reticulum. Science 1990; 248: 372-376.
[69] Levi AJ, SpitzerKW, Kohmoto O, Bridge JH. Depolarization-induced Ca~(2+) entry via Na~+/Ca~(2+) exchange triggers SR release in guinea pig cardiac myocytes. Am J Physiol. 1994; 266: H1422-1433.
[70] Litwin SE, Li J, Bridge JH. Na~+/Ca~(2+) exchange and the trigger for sarcoplasmic reticulum Ca~(2+) release: studies in adult rabbit ventricular myocytes. Biophys J. 1998; 75: 359-371.
[71] Wasserstrom JA,Vites AM. The role of Na~+/Ca~(2+) exchange in activation of excitation -contraction coupling in rat ventricular myocytes. J Physiol 1996; 493: 529-542.
[72] J Bridge J H B, Smolley J R, Spitzer K W. The relationship between charge movements associated with ICa and INa- Ca~(2+) in cardiac myocyts. Science. 1990; 248: 376 -378.
[73] J Nuss H B,Houser S R.Sodium-calcium exchange-mediated contractions in feline ventricular myocytes. Am J Physiol. 1992; 263: Hll6l-Hl169.
[74] Goldhaber J I, Lamp S T, Walter DO. et a1. Local regulation of the threshold for calcium sparks in rat ventricular myocytes: Role of sodium-calcium exchange. J Physiol. 1999; 520: 431-438.
[75] Schafer C,Ladilov Y,Inserte J,et a1. Role of reverse mode of the sodium-calcium exchanger in reoxygenation-induced cardiomyoeyte injury. Cardiovasc Res. 2001; 51: 241-250.
[76] Satoh H, Ginsburg K S, Qing K, et a1. KB-R7943 block of Ca~(2+) influx via Na~+/Ca~(2+) exchange does not alter twitches or glycoside inotropy but prevent Ca~(2+) overload in rat ventricular myocytes. Circulation. 2000; 101: 1441-1446.
[77] Reinecke H, Studer R, Vetter R, et a1. Na~+/Ca~(2+) exchange activity in patients with end stage heart failure. Cardio.vas Res. 1996: 31: 48-54.
[78] Arai M, Alpert N R, Maclennan D H. et a1. A1terations in sarcoplasmic reticulum gene expression in human heart failure: A possible mechanism for alterations in systolicand diastolic properties of the failing myocardium. Circ Res. 1993; 72: 463-469.
[79] Hasenfuss G, Schillinger W, Preu~s M. et a1. Relationshipbetween Na~+/Ca~(2+) exchanger protein levels and diastolic function of failing human myocardium. Circulation. 1999; 99: 641-648.
[80] Bers D M. Calcium fluxes involved in control of cardiac myocyte contraction. Circ Res. 2000; 87: 275-281.
[2] Thomas R. Shannon, Wilbur Y. W. Lew. Diastolic Release of Calcium From the Sarcoplasmic Reticulum. Journal of the American College of Cardiology. 2009; 53(21): 2006-2008.
[3] Campbell AK. In: Intracellular Calcium: Its Universal Role as Regulator, edited by Gutfreund H. London: Wiley, 1983.
[4] Carafoli E, and Klee C. (Editors). Calcium as a Cellular Regulator. Oxford, UK: Oxford Univ. Press, 1999.
[5] Huxley AF. In: Mineral Metabolism, edited by Comman CL, and Bronner F. New York: Academic, 1961.
[6] Hasenfuss G, Pieske B. Calcium cycling in congestive heart failure. J Mol Cell Cardiol. 2002; 34: 951-969.
[7]Yatani A, Shen Y T, Yan L, et al. Down regulation of the L-type Ca~(2+) channel, GRK2 and phosphorylated phospholamban: protective mechanisms for the denervated failing heart. Mol Cell Cardiol. 2006; 40 (5): 619-628.
[8] Wang S Q,Song L S,Lakatta E G. Ca~(2+) signalling between single L-type Ca~(2+) channels and ryanodine receptors in heart cells. Nature. 2001; 410: 592.
[9] PiacentinoⅢV, Weber C, Chen X W, et a1. Cell basis of abnormal calcium transients of failing human ventricular myocytes. Circ Res. 2003; 92: 651-658.
[10] Yano M, Ikada Y, Matsuzaki. Altered intracellular Ca~(2+) handling in heart failure. J Clin Invest. 2005; 115: 556-564.
[11] Pessah I.N., Waterhouse A.L., Casida J.E. The calcium–ryanodine receptor complex of skeletal and cardiac muscle. Biochem. Biophys. Res. Commun. 1985; 128(1): 449-456.
[12] Masafumi Yano, Takeshi Yamamoto, Shigeki Kobayashi, et al. Role of ryanodine receptor as a Ca~(2+) regulatory center in normal and failing hearts. Journal of Cardiology 2009; 53: 1-7.
[13] Hiroki Tateishi, Masafumi Yano, Mamoru Mochizuki, et a1. Defective domain -domain interactions within the ryanodine receptor as a critical cause of diastolic Ca~(2+) leak in failing hearts. Cardiovasc Res. 2009; 81(3): 536-545.
[14] Wehrens, X.H. et al. FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death. Cell. 2003; 113 (7): 829-840.
[15] Subeena Sood, Mihail G. Chelu, Ralph J. van Oort, et a1. Intracellular calcium leak due to FKBP12.6 deficiency in mice facilitates the inducibility of atrial fibrillation. Heart Rhythm. 2008; 5(7): 1047-1054.
[16] Marx SO, Reiken S, Hisamatsu Y, et al. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor ): defective regulation in failing hearts. Cell. 2000; 101: 365-376.
[17] Yano M, Kobayashi S, Kohno M, et al. FKBP12.6-mediated stabilization of calcium-release channel (ryanodine receptor) as a novel therapeutic strategy against heart failure. Circulation. 2003; 107: 477-484.
[18] Stange M, Xu L, Balshaw D, et al. Characterization of recombinant skeletal muscle (Ser-2843) and cardiac muscle (Ser-2809) ryanodine receptor phosphorylation mutants. J Biol Chem. 2003; 278(5): 51693-51702.
[19] MI Ya-fei, LI Xiao-ying, TANG Li-jiang, LU Xiao-chun, FU Zhi-qing and YE Wei -hua. Improvement in cardiac function after sarcoplasmic reticulum Ca~(2+)-ATPase gene transfer in a beagle heart failure model. Chinese Medical Journal 2009; 122(12): 142-1428.
[20] Schmidt U, Hajjar R J, Kim C S, et a1. Human heart failure: cAMP stimulation of SR Ca~(2+)-ATPase activity and phosphorylation level of phospholamban. Am J Physiol, 1999; 277: H474-480.
[21] K, Kranias EG. Phospholamban and Cardiac Contractility. Annals of Medicine (The Finnish Med. Soc. Duodecim). 2000; 32: 572-578.
[22] Yoshie Tanaka, Takeshi Honda, Kenji Matsuura, Yoshihiro Kimura, Makoto Inui. In Vitro Selection and Characterization of DNA Aptamers Specific for Phospholamban. J Pharmacology and Experimental Therapeutics. 2009; 329(1): 57-63.
[23] Hasenfuss G. Alterations of calcium regulatory proteins in heart failure.Cardiovasc Res. 1998; 37(2): 279-289.
[24] Louiza Belkacemi, Isabelle B′edard, Lucie Simoneau, Julie Lafond. Calcium channels, transporters and exchangers in placenta. Cell Calcium. 2005; 37: 1-8.
[25] Liu M, Liu XY, Cheng JF. Advance in the pharmacological research on matrine. Zhongguo Zhong Yao Za Zhi. 2003; 28: 801-804.
[26] Ma L,Wen S, Zhan Y, et al. Anticancer effects of the Chinese medicine matrine onmurine hepatocellular carcinoma cells. Planta Med 2008; 74: 245-251.
[27] Liu SX, Chiou GC. Effects of Chinese herbal products on mammalian retinal functions. J Ocul Pharmacol Ther. 1996; 12: 377-386.
[28] Jiang H, Hou C, Zhang S, et al. Matrine upregulates the cell cycle protein E2F-1 and triggers apoptosis via the mitochondrial pathway in K562 cells. Eur J Pharmacol. 2007; 559: 98-108.
[29] Azzam HS, Goertz C, Fritts M, et al. Natural products and chronic hepatitis C virus. Liver Int. 2007; 27: 17-25.
[30] Yamazaki M. The pharmacological studies on matrine and oxymatrine. Yakugaku Zasshi. 2000; 120: 1025-1033.
[31] Zhang MJ, Huang J. Recent research progress of anti-tumor mechanism matrine. Zhongguo Zhong Yao Za Zhi. 2004; 29: 115-118.
[32] Jiang, H., Meng, F., Li, J., et al. Anti-apoptosis effects of oxymatrine protect the liver from warm ischemia reperfusion injury in rats. World J. Surg. 2005; 29: 1397-1401.
[33] Zhao, J., Yu, S., Tong, L., et al. Oxymatrine attenuates intestinal ischemia/reperfusion injury in rats. Surg. Today. 2008; 38: 931-937.
[34] Zuzana K, Dmitry T, Serge V. Abnormal intrastore calcium signaling in chronic heart failure.Proc Nail Acad Sci USA. 2005; 102(39): 14104-14109.
[1] Fabiato A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol. 1983; 245(1): C1-C14.
[2] Bers DM. Cardiac excitation-contraction coupling. Nature. 2002; 415(6868): 198-205.
[3] Hasenfuss G and Pieske B. Aug Calcium cycling in congestive heart failure. J Mol Cell Cardiol. 2002, 34(8): 951-969.
[4] Sipido KR, Eisner D. Something old, something new: changing views on the cellular mechanisms of heart failure. Cardiovasc Res. 2005, 68(2):167-174.
[5]Mccall E, Ginsburg KS, Bassani RA, et al. Ca~(2+) flux, contractility, and excitation-contraction coupling in hypertrophic rat ventricular myocytes. Am J Physiol. 1998; 274: H1348-H1360.
[6]沈建新,王世强,程和平等. Thapsigargin对心肌细胞钙释放和肌浆网钙容量的时间效应.中山大学学报. 2004; 25(1): 19-23.
[7] Yano M, Ikada Y, Matsuzaki. Altered intracellular Ca~(2+) handling in heart failure. J Clin Invest. 2005; 115: 556-654.
[8] Lin L, Kim SC, Wang Y, Gupta S, Davis B, Simon SI, Torre-Amione G, Knowlton AA. HSP60 in heart failure: abnormal distribution and role in cardiac myocyte apoptosis. Am J Physiol Heart Circ Physiol. 2007; 293(4): H2238-H2247.
[9] Ohmoto-Sekine Y, Uemura H, Tamagawa M, Nakaya H. Inhibitory effects of aprindine on the delayed rectifier K~+ current and the muscarinic acetylcholine receptor-operated K~+ current in guinea-pig atrial cells. Br J Pharmacol. 1999 126(3): 751-761.
[10] Tytgat J. How to isolate cardiac myocytes. Cardiovasc Res. 1994; 28(2): 280-283.
[11] Mitra R,Morad M.A uniform enzymatic method for dissociation of myocytes from hearts and stomachs of vertebrates. Am J Physiol. 1985; 249: H1056-1060.
[12]曹春梅,张雄,陈莹莹等.不同用途的心室肌细胞的分离。浙江大学学报(医学版), 2003; 32(1): 51-55.
[13] Nakaya H, Tohse N, Takeda Y, Kanno M. Effects of MS-551, a new class antiarrhythmic drug, on action potential and membrane currents in rabbit ventricular myocytes. Br J Pharmacol. 1993, 109(1): 157-163.
[14] Sakmann B, Neher E. Single-Channel Recording, New York: Plenum. 1983.
[15] Qi M Xia HJ,Dai DZ, Dai Y. A novel endothelin receptor antagonist CPU0213 improves diabetic cardiac insufficiency attributed to up-regulation of the expression of FKBPl2.6, SERCA2a,and PLB in rats. J Cardiovascular Pharmacology. 2006; 47:729-735.
[16] Long-Sheng Song, et al. Beta-Adrenergic stimulation synchronizes intracellular Ca~(2+) release during excitation-contraction coupling in cardiac myocytes. Circ Res. 2001; 88(8): 794-801.
[17]唐晓鸿.心脉隆注射液药理作用和治疗心力衰竭临床研究进展.中国新药杂志. 2008; 6(17): 461-464.
[18]于秀琼,蔡琳.疾病管理和心力衰竭.心血管病学进展. 2007; 5(28):739-740.
[19] Thomas R. Shannon, Wilbur Y. W. Lew. Diastolic Release of Calcium From the Sarcoplasmic Reticulum. Journal of the American College of Cardiology. 2009; 53(21): 2006-2008.
[20] Janczewski AM, Spurgeon HA, Stern MD, Lakatta EG. Effects of sarcoplasmic reticulum Ca~(2+) load on the gain function of Ca~(2+) release by Ca~(2+) current in cardiac cells. Am J Physiol. 1995; 268: H916-920.
[21] Trafford AW, Díaz ME, Negretti N, Eisner DA. Enhanced calcium current and decreased calcium efflux restore sarcoplasmic reticulum Ca~(2+) content following depletion. Circ Res. 1997; 81(4): 477-484.
[22] Fill M,Copello J A. Ryanodine receptor calcium release channels. Physiol Rev. 2002; 82(4): 893-922.
[23] Trafford AW, Díaz ME, Sibbring GC, Eisner DA. Modulation of CICR has no maintained effect on systolic Ca~(2+): simultaneous measurements of sarcoplasmic reticulum and sarcolemmal Ca~(2+) fluxes in rat ventricular myocytes. J Physiol. 2000; 522: 259-270.
[24] Jean PB, Julio L, et al. L-type Ca~(2+) current in ventricular cardiomyocytes. J Mole Cel Cardio. 2010, 48:26-36.
[25] Hiroki Tateishi, Masafumi Yano, Mamoru Mochizuki, et a1. Defective domain-domain interactions within the ryanodine receptor as a critical cause of diastolic Ca~(2+) leak in failing hearts. Cardiovasc Res. 2009; 81(3): 536-545.
[26] Bers DM. Macromolecular complexes regulating cardiac ryanodine receptor function. J Mol Cell Cardiol. 2004; 37: 417-429.
[27] Nicolaou P, Rodriguez P, Kranias EG, et al. Inducible expression of active protein phosphatase-1 inhibitor-1 enhances basal cardiac function and protects against ischemia/reperfusion injury. Circ Res. 2009, 104(8): 1012-1020.
[28] Yano M, Yamamoto T, Kobayashi S, Matsuzaki M. Role of ryanodine receptor as a Ca~(2+) regulatory center in normal and failing hearts. J Cardiol. 2009, 53:1-7.
[29] Stange M, Xu L, Balshaw D, Yamaguchi N, Meissner G. Characterization of recombinant skeletal muscle (Ser-2843) and cardiac muscle (Ser-2809) ryanodine receptorphosphorylation mutants. J Biol Chem. 2003; 278(5):51693-51702.
[30] Waggoner JR, Ginsburg KS, Mitton B, Haghighi K, Robbins J, Bers DM, Kranias EG. Phospholamban overexpression in rabbit ventricular myocytes does not alter sarcoplasmic reticulum Ca transport. HAm J Physiol Heart Circ Physiol. 2009, 296(3): 698-703.
[31] Yoshie Tanaka, Takeshi Honda, Kenji Matsuura, Yoshihiro Kimura, Makoto Inui. In Vitro Selection and Characterization of DNA Aptamers Specific for Phospholamban. J Pharmacology and Experimental Therapeutics. 2009; 329(1): 57-63.
[32] Reuter H, Seitz N. The dependence of calcium efflux from cardiac muscle on temperature and external ion composition. J Physiol (Lond). 1968; 195: 45-70.
[33] Graham J. Jeffs, Bruno P. Meloni, Anthony J. Bakker, Neville W. Knuckey. The role of the Na~+/Ca~(2+) exchanger (NCX) in neurons following ischaemia. Journal of Clinical Neuroscience. 2007; 14: 507-514.
[34] M.P. Blaustein,WJ. Lederer, Sodium/calcium exchange: its physiological implications. Physiol. Rev. 1999; 79: 763-854.
[35] Li ZP, Matsuoka S, Hryshko LV, Nicoll DA, Bersohn MM, Burke EP, Lifton RP, Philipson KD. Cloning of the NCX2 isoform of the plasma membrane Na~+/Ca~(2+) exchanger. J Biol Chem. 1994; 269: 17434-17439.
[36] Nicoll DA, Quednau BD, Qiu ZY, Xia YR, Lusis AJ, Philipson KD. Cloning of a third mammalian Na~+/Ca~(2+) exchanger, NCX3. J Biol Chem 1996; 271: 24914-24921.
[37] Mihra S, Sabbah HN, Rastogi S, et a1. Reduced sarcoplasmic reticulum Ca~(2+) uptake and increased Na~+-Ca~(2+) exchanger expression in left ventricle myocardiurn of dogs with progression of heart failure. Heart Vessels. 2005; 20: 23-32.
[1] Gy(?)rke, I. & Gy(?)rke, S. Regulation of the cardiac ryanodine receptor channel by luminal Ca~(2+) involves luminal Ca~(2+) sensing sites. Biophys. J. 1998; 75: 2801-2810
[2] Ching, L. L., Williams, A. J. & Sitsapesan, R. Evidence for Ca(2+) activation and inactivation sites on the luminal side of the cardiac ryanodine receptor complex. Circ. Res. 2000; 87: 201-206.
[3] Bers, D. M. in Excitation-Contraction Coupling and Cardiac Contractile Force (Kluwer Academic, Dordrecht, The Netherlands), 2nd Ed. 2001.
[4] Lindner, M., Erdmann, E. & Beuckelmann, D. J. Calcium content of the sarcoplasmic reticulum in isolated ventricular myocytes from patients with terminal heart failure. J. Mol. Cell. Cardiol. 1998; 30: 743-749.
[5] O'Rourke, B., Kass, D. A., Tomaselli, G. F., K??b, S., Tunin, R. & Marbán, E. Mechanisms of altered excitation-contraction coupling in canine tachycardia-induced heart failure, I: experimental studies. Circ. Res. 1999; 84: 562-570.
[6] Houser, S. R., Piacentino, V., III, & Weisser, J. Abnormalities of calcium cycling in the hypertrophied and failing heart. J. Mol. Cell. Cardiol. 2000; 32: 1595-1607.
[7] Hasenfuss, G. & Pieske, Calcium cycling in congestive heart failure B. J. Mol. Cell. Card iol. 2002; 34: 951-969.
[8] Houser, S. R. & Margulies, K. B. Is depressed myocyte contractility centrally involved in heart failure? Circ. Res. 2003; 92: 350-358.
[9] Liu M, Liu XY, Cheng JF. Advance in the pharmacological research on matrine. Zhongguo Zhong Yao Za Zhi. 2003; 28: 801-804.
[10] Ma L,Wen S, Zhan Y, He Y, Liu X, Jiang J. Anticancer effects of the Chinese medicine matrine on murine hepatocellular carcinoma cells. Planta Med 2008; 74: 245-251.
[11] Liu SX, Chiou GC. Effects of Chinese herbal products on mammalian retinal functions. J Ocul Pharmacol Ther. 1996; 12: 377-386.
[12] Jiang H, Hou C, Zhang S, Xie H, Zhou W, Jin Q. Matrine upregulates the cell cycle protein E2F-1 and triggers apoptosis via the mitochondrial pathway in K562 cells. Eur J Pharmacol. 2007; 559: 98-108.
[13] Azzam HS, Goertz C, Fritts M, Jonas WB. Natural products and chronic hepatitis C virus. Liver Int. 2007; 27: 17-25.
[14] Yamazaki M. The pharmacological studies on matrine and oxymatrine. YakugakuZasshi. 2000; 120: 1025-1033.
[15] Zhang MJ, Huang J. Recent research progress of anti-tumor mechanism matrine. Zhongguo Zhong Yao Za Zhi. 2004; 29: 115-118.
[16] Jiang, H., Meng, F., Li, J., Sun, X. Anti-apoptosis effects of oxymatrine protect the liver from warm ischemia reperfusion injury in rats. World J. Surg. 2005; 29: 1397-1401.
[17] Zhao, J., Yu, S., Tong, L., Zhang, F., Jiang, X., Pan, S., Jiang,H., Sun, X. Oxymatrine attenuates intestinal ischemia/reperfusion injury in rats. Surg. Today. 2008; 38: 931-937.
[18]李丽于,宏伟,李雪峰,吴宜艳.苦参碱对大鼠缺血再灌注心肌损伤的保护作用.中国误诊学杂志. 2009; 9(3): 529-530.
[19]杨钰萍,沈祥春,刘兴德,方泰惠,许立.氧化苦参碱对急性心肌梗死诱发实验性大鼠心肌重塑的影响.中国实验方剂学杂志. 2010; 16(6): 125-128.
[20] Campbell AK. In: Intracellular Calcium: Its Universal Role as Regulator, edited by Gutfreund H. London: Wiley, 1983.
[21] Carafoli E, and Klee C.. Calcium as a Cellular Regulator. Oxford, UK: Oxford Univ. Press, 1999.
[22] Huxley AF. In: Mineral Metabolism, edited by Comman CL, and Bronner F. New York: Academic, 1961.
[23] Michael G, Joan H B.β-Adrenergic receptor signaling in the heart: Role of CaMKII. J Mol Cell Cardiol. 2010; 48: 322-330.
[24] Vafiadaki E, Papalouka V, Arvanitis DA, Kranias EG, Sanoudou D. The role of SERCA2a/PLN complex, Ca~(2+) homeostasis, and anti-apoptotic proteins in determining cell fate. HP flugers Arch. 2009, 457(3):687-700.
[25] Zuzana K, Dmitry T,Serge V,et a1.Abnormal intrastore calcium signaling in chronic heart failure. Proc Nail Acad Sci USA. 2005; 102(39): 14104-14109.
[26] PiacentinoⅢV, Weber C, Chen X W, et a1. Cell basis of abnormal calcium transients of failing human ventricular myocytes. Circ Res. 2003; 92: 651-658.
[27]Engelhardt S, Hein L, Kranias E G, Lohse M J. Altered calcium handling is critically involved in the cardiotoxic effects of chronic beta-adrenergic stimulation.Circulation.2004; 109(9): 1154-1160.
[1] Mosterd A, Hoes AW. Clinical epidemiology of heart failure. Heart. 2007; 93: 1137-1146.
[2]于秀琼,蔡琳.疾病管理和心力衰竭.心血管病学进展. 2007; 5(28):739-740.
[3] Yano M, Ikada Y, Matsuzaki. Altered intracellular Ca~(2+) handling in heart failure. J Clin Invest. 2005; 115: 556-564.
[4] Wehrens XH, Lehnart SE, Marks AR. Intracellular calcium release channels and cardiac disease. Annu Rev Physiol. 2005; 67: 69-98.
[5] Jiang MT, Lokuta AJ, Farrell EF, et al. Abnormal Ca~(2+) release, but normal ryanodine receptors, in canine and human heart failure. Circ Res 2002; 91: 1015-22.
[6] Yamamoto T, Yano M, Kohno M, et al. Abnormal Ca~(2+) release from cardiac sarcoplasmic reticulum in tachycardia-induced heart failure. Cardiovasc Res. 1999; 44: 146 -55.
[7] Campbell AK. In: Intracellular Calcium: Its Universal Role as Regulator, edited by Gutfreund H. London: Wiley. 1983.
[8] Carafoli E, and Klee C. Calcium as a Cellular Regulator. Oxford, UK: Oxford Univ. Press, 1999.
[9] Huxley AF. In: Mineral Metabolism, edited by Comman CL, and Bronner F. New York: Academic, 1961.
[10]Yatani A, Shen Y T, Yan L, et al. Down regulation of the L-type Ca~(2+) channel, GRK2 and phosphorylated phospholamban: protective mechanisms for the denervated failing heart. Mol Cell Cardiol. 2006; 40 (5): 619-628.
[11] Wang S Q,Song L S,Lakatta E G. Ca~(2+) signalling between single L-type Ca~(2+) channels and ryanodine receptors in heart cells. Nature. 2001; 410: 592.
[12] Fabiato A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol. 1983; 245: C1-14.
[13] PiacentinoⅢV, Weber C, Chen X W, et a1. Cell basis of abnormal calcium transients of failing human ventricular myocytes. Circ Res. 2003; 92: 651-658.
[14] Franzini-Armstrong, C.. Studies of the triad. J Cell Biol. 1970; 47: 488-499.
[15] Campbell, K., Franzini-Armstrong, C., & Shamoo, A. Further characterisation of light and heavy sarcoplasmic reticulum vesicles. Identification of the‘sarcoplasmic reticulum feet’associated with heavy sarcoplasmic reticulum vesicles. Biochim Biophys Acta. 1980;602: 97-116.
[16] Tunwell RE, Wickenden C, Bertrand BM, et al. The human cardiac muscle ryanodine receptor-calcium release channel: identification, primary structure and topological analysis. Biochem J. 1996; 318: 477-487.
[17] Giannini G, Sorrentino V. Molecular structure and tissue distribution of ryanodine receptors calcium channels. Med Res Rev 1995; 15: 313-323.
[18] Franzini-Armstrong C, Protasi F, Ramesh V. Shape, size, and distribution of Ca~(2+) release units and couplons in skeletal and cardiac muscles. Biophys J. 1999; 77: 1528- 1539.
[19] Bers DM. Macromolecular complexes regulating cardiac ryanodine receptor function. J Mol Cell Cardiol. 2004; 37: 417-429.
[20] Lehnart SE, Wehrens XH, Marks AR.Calstabin deficiency, ryanodine receptors, and sudden cardiac death. Biochem Biophys Res Commun. 2004; 322(4): 1267-1279.
[21] Marx, S.O. et al. Coupled gating between cardiac calcium release channels (ryanodine receptors). Circ. Res. 2001; 88(11): 1151-1158.
[22] Marx SO, Reiken S, Hisamatsu Y, et al. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell. 2000; 101: 365-376.
[23] Yano M, Kobayashi S, Kohno M, Doi M, Tokuhisa T, Okuda S, Suetsugu M, Hisaoka T, Obayashi M, Ohkusa T, Kohno M, Matsuzaki M. FKBP12.6-mediated stabilization of calcium-release channel (ryanodine receptor) as a novel therapeutic strategy against heart failure. Circulation. 2003; 107: 477-484.
[24] Engelhardt S, Hein L, Kranias E G, Lohse M J. Altered calcium handling is critically involved in the cardiotoxic effects of chronic beta-adrenergic stimulation.Circulation.2004; 109(9): 1154-1160.
[25] Stange M, Xu L, Balshaw D, Yamaguchi N, Meissner G. Characterization of recombinant skeletal muscle (Ser-2843) and cardiac muscle (Ser-2809) ryanodine receptor phosphorylation mutants. J Biol Chem. 2003; 278(5): 51693-51702.
[26] Tripathy A, Xu L, Mann G, Meissner G. Calmodulin activation and inhibition of skeletal muscle Ca~(2+) release channel (ryanodine receptor). Biophys J. 1995; 69: 106-119.
[27] Balshaw DM, Xu L, Yamaguchi N, Pasek DA, Meissner G. Calmodulin binding and inhibition of cardiac muscle calcium release channel (ryanodine receptor). J Biol Chem. 2001; 276: 20144-20153.
[28] Yamaguchi N, Xu L, Pasek DA, Evans KE, Meissner G. Molecular basis of calmodulin binding to cardiac muscle Ca~(2+) release channel (ryanodine receptor). J Biol Chem. 2003; 278: 23480-23486.
[29] Yamaguchi N, Takahashi N, Xu L, Smithies O, Meissner G. Early cardiac hypertrophy in mice with impaired calmodulin regulation of cardiac muscle Ca~(2+) release channel. J Clin Invest. 2007; 117: 1344-1353.
[30] Mitchell RD, Simmerman HK, Jones LR. Ca~(2+) binding effects on protein conformation and protein interactions of canine cardiac calsequestrin. J Biol Chem. 1988; 263: 1376-1381.
[31] Gyorke S, Terentyev D. Modulation of ryanodine receptor by luminal calcium and accessory proteins in health and cardiac disease. Cardiovasc Res. 2008; 77: 245-255.
[32] Gyorke I, Hester N, Jones LR, Gyorke S. The role of calsequestrin, triadin, and junctin in conferring cardiac ryanodine receptor responsiveness to luminal calcium. Biophys J. 2004; 86: 2121-2128.
[33] Tracy J. Pritchard, Evangelia G. Kranias. Junctin and the histidine-rich Ca~(2+) binding protein:potential roles in heart failure and arrhythmogenesis. J Physiol. 2009; 587(13): 3125-3133.
[34] Beard NA, Casarotto MG, Wei L, Varsányi M, Laver DR, Dulhunty AF. Regulation of ryanodine receptors by calsequestrin: effect of high luminal Ca~(2+) and phosphorylation. Biophys J. 2005; 88: 3444-3454.
[35] Terentyev D, Nori A, Santoro M, Viatchenko-Karpinski S, Kubalova Z, Gyorke I, Terentyeva R, Vedamoorthyrao S, Blom NA, Valle G, Napolitano C, Williams SC, Volpe P, Priori SG, Gyorke S. Abnormal interactions of calsequestrin with the ryanodine receptor calcium release channel complex linked to exercise-induced sudden cardiac death. Circ Res. 2006; 98: 1151-1158.
[36] Oda T, Yano M, Yamamoto T, Tokuhisa T, Okuda S, Doi M, Ohkusa T, Ikeda Y, Kobayashi S, Ikemoto N, Matsuzaki M. Defective regulation of interdomain interactions within the ryanodine receptor plays a key role in the pathogenesis of heart failure. Circulation. 2005; 111: 3400-3410.
[37] Zaiain Herzberg A, MacLeun an DH, Periasanly. Characterization of rabbit saco (eodn) plasmic reticulum Ca~(2+)-ATPase gene. J BiDl Chem. 1990; 256: 4670-4677.
[38] Wu KD, LeeWS, Wey J, et al.Localiation and quantification of endoplasmlc reticulum Ca~(2+)-ATPaseisoformtranscripts. Am J Physiol. 1995; 269: C775-C784.
[39] Zhang P, Toyoshima C, Yonekura K, et al. Structure of the calcium pump from sarcoplasmic retieulum at 8-A resolution. Nature. 1998; 392: 835-839.
[40] Schmidt U, Hajjar RJ, Kim CS, et al. Human heart failure: cAMP stimulation of SR Ca~(2+)-ATPase activity and phosphorylation level of phospholambaa. Am J Physiol. 1999; 277: H474-H480.
[41]钟明,张运,张薇,等.舒张性心力衰竭兔Ca~(2+)调控蛋白mRNA和蛋白质的表达.中国病理生理杂志. 2002; 18: 124-127.
[42] J. Lytton, A. Zarain-Herzberg, M. Periasamy, D.H. MacLennan,Molecular cloning of the mammalian smooth muscle sarco(endo)plasmic reticulum Ca~(2+)-ATPase, J. Biol. Chem. 1989; 264: 7059-7065.
[43] Maclennan DH, Toyofuku T. Sites of regulatory interaction between calcium ATPase and phoepholamban. Basic Res Cardiol. l997; 92: l1-l5.
[44] Luo W, Gmpp IL, Hatter J, et al. Targeted ablation of the phospholamban gene is associated with markedly enhanced myocardial contractility and loss of beta-agonist stimulation. Circ Res. 1994; 75: 401-409.
[45] Díaz ME, Graham HK, Trafford AW. Enhanced sarcolemmal Ca~(2+) efflux reduces sarcoplasmic reticulum Ca~(2+) content and systolic Ca~(2+) in cardiac hypertrophy. Cardiovasc Res. 2004; 62:538-47.
[46] O'Rourke B, Kass DA, Tomaselli GF, Kaab S, Tunin R, Marban E. Mechanisms of altered excitation–contraction coupling in canine tachycardia-induced heart failure, I Experimental studies. Circ Res. 1999; 84: 562-570.
[47] Pieske B, Maier LS, Bers DM, Hasenfuss G. Ca~(2+) handling and sarcoplasmic reticulum Ca~(2+) content in isolated failing and nonfailing human myocardium. Circ Res 1999; 85: 38-46.
[48] Maclennan DH, Kranias EG. Phospholanlhan: a crucial regulator of cardiac contractility. Nat Rev Mol Cell Biol. 2003; 4(7): 566-577.
[49] Hoshijima M, lkeda Y, 1wanaga Y, el al. Chronic suppression of heart-failure progression by pseudophosphorylated mutant of phospholamhan via in vivo cardiac rAAV gene delivery. Nat Med. 2002; 8(8): 864-87l.
[50] Reuter H, Seitz N The dependence of calcium efflux from cardiac muscle on temperature and external ion composition. J Physiol (Lond). 1968; 195: 45-70.
[51] M.P. Blaustein,W.J. Lederer, Sodium/calcium exchange: its physiological implications. Physiol. Rev. 1999; 79: 763-854.
[52] Li ZP, Matsuoka S, Hryshko LV, Nicoll DA, Bersohn MM, Burke EP, Lifton RP, Philipson KD. Cloning of the NCX2 isoform of the plasma membrane Na~+/Ca~(2+) exchanger. J Biol Chem. 1994; 269: 17434-17439.
[53] Nicoll DA, Quednau BD, Qiu ZY, Xia YR, Lusis AJ, Philipson KD. Cloning of a third mammalian Na~+/Ca~(2+) exchanger, NCX3. J Biol Chem 1996; 271: 24914-24921.
[54] Santacruz-Toloza L, Ottolia M, Nicoll DA, Philipson KD. Functional analysis of a disulfide bond in the cardiac Na~+/Ca~(2+) exchanger. J Biol Chem. 2000; 275:182-188.
[55] Schwarz EM, Benzer S. Calx, a Na~+/Ca~(2+) exchanger gene of Drosophila melanogaster. Proc Natl Acad Sci. 1997; 94: 10249-10254.
[56] Nicoll DA, Hryshko LV, Matsuoka S, Frank JS, Philipson KD Mutation of amino acids residues in the putative transmembrane segments of the cardiac sarcolemmal Na~+/Ca~(2+) exchanger. J Biol Chem 1996; 271: 13385-13391.
[57] Iwamoto T, Uehara A, Imanaga I, Shigekawa M. The Na~+/Ca~(2+) exchanger NCX1 has oppositely oriented reentrant loop domains that contain conserved aspartic acids whose mutation alters its apparent Ca~(2+) affinity. J Biol Chem. 2000; 275: 38571-38580.
[58] Nicoll DA, Ottolia M, Lu LY, Lu YJ, Philipson KD. A new topological model of the cardiac sarcolemmal Na~+/Ca~(2+) exchanger. J Biol Chem. 1999; 274: 910-917.
[59] Qiu ZY, Nicoll DA, Philipson KD Helix packing of functionally important regions of the cardiac Na~+/Ca~(2+) exchanger. J Biol Chem. 2001; 276: 194-199.
[60] Schwarz EM, Benzer S Calx, a Na~+/Ca~(2+) exchanger gene of Drosophila melanogaster. Proc Natl Acad Sci 1997; 94: 10249-10254.
[61] Matsuoka S, Nicoll DA, Reilly RF, Hilgemann DW, Philipson KD Initial localization of regulatory regions of the cardiac sarcolemmal Na~+/Ca~(2+) exchanger. Proc Natl Acad Sci. 1993; 90: 3870-3874.
[62] Frank JS, Mottino G, Reid D, Molday RS, Philipson KD. Distribution of the Na~+/Ca~(2+) exchange protein in mammalian cardiac myocytes: an immunofluorescence and immunocolloidal gold-labeling study. J Cell Biol. 1992; 117: 337-345.
[63] Kieval RS, Bloch GE, Lindenmayer A, Ambesi A, Lederer WJ.Immunofluorescence localization of the Na~+/Ca~(2+) exchanger in heart cells. Am J Physiol 1992; 263: C545-C550.
[64] Scriven DRL, Dan P, Moore EDW. Distribution of proteins implicated in excitation -contraction coupling in rat ventricular myocytes. Biophys J 2000; 79: 2682-2691.
[65] Yang Z, Pascarel C, Steele DS, Komukai K, Brette F, Orchard CH. Na~+/Ca~(2+) exchange activity is localized in the Ttubulesof rat ventricular myocytes. Circ Res. 2002;91: 315-322.
[66] Iwamoto T. Forefront of Na~+/Ca~(2+) exchanger studies: molecular pharmacology of Na~+/Ca~(2+) exchange inhibitors. J Pharmacol Sci. 2004; 96: 27-32.
[67] Hobai IA , O’Rourke B. The potential of Na~+/Ca~(2+) Exchange blockers in the treatment of cardiac disease. Expert Opin Investig Drugs. 2004; 13: 653-664.
[68] Leblanc N, Hume JR. Sodium current induced release of calcium from cardiac sarcoplasmic reticulum. Science 1990; 248: 372-376.
[69] Levi AJ, SpitzerKW, Kohmoto O, Bridge JH. Depolarization-induced Ca~(2+) entry via Na~+/Ca~(2+) exchange triggers SR release in guinea pig cardiac myocytes. Am J Physiol. 1994; 266: H1422-1433.
[70] Litwin SE, Li J, Bridge JH. Na~+/Ca~(2+) exchange and the trigger for sarcoplasmic reticulum Ca~(2+) release: studies in adult rabbit ventricular myocytes. Biophys J. 1998; 75: 359-371.
[71] Wasserstrom JA,Vites AM. The role of Na~+/Ca~(2+) exchange in activation of excitation -contraction coupling in rat ventricular myocytes. J Physiol 1996; 493: 529-542.
[72] J Bridge J H B, Smolley J R, Spitzer K W. The relationship between charge movements associated with ICa and INa- Ca~(2+) in cardiac myocyts. Science. 1990; 248: 376 -378.
[73] J Nuss H B,Houser S R.Sodium-calcium exchange-mediated contractions in feline ventricular myocytes. Am J Physiol. 1992; 263: Hll6l-Hl169.
[74] Goldhaber J I, Lamp S T, Walter DO. et a1. Local regulation of the threshold for calcium sparks in rat ventricular myocytes: Role of sodium-calcium exchange. J Physiol. 1999; 520: 431-438.
[75] Schafer C,Ladilov Y,Inserte J,et a1. Role of reverse mode of the sodium-calcium exchanger in reoxygenation-induced cardiomyoeyte injury. Cardiovasc Res. 2001; 51: 241-250.
[76] Satoh H, Ginsburg K S, Qing K, et a1. KB-R7943 block of Ca~(2+) influx via Na~+/Ca~(2+) exchange does not alter twitches or glycoside inotropy but prevent Ca~(2+) overload in rat ventricular myocytes. Circulation. 2000; 101: 1441-1446.
[77] Reinecke H, Studer R, Vetter R, et a1. Na~+/Ca~(2+) exchange activity in patients with end stage heart failure. Cardio.vas Res. 1996: 31: 48-54.
[78] Arai M, Alpert N R, Maclennan D H. et a1. A1terations in sarcoplasmic reticulum gene expression in human heart failure: A possible mechanism for alterations in systolicand diastolic properties of the failing myocardium. Circ Res. 1993; 72: 463-469.
[79] Hasenfuss G, Schillinger W, Preu~s M. et a1. Relationshipbetween Na~+/Ca~(2+) exchanger protein levels and diastolic function of failing human myocardium. Circulation. 1999; 99: 641-648.
[80] Bers D M. Calcium fluxes involved in control of cardiac myocyte contraction. Circ Res. 2000; 87: 275-281.