利用RNA干扰技术治疗慢性心衰大鼠的研究

目的利用RNA干扰（RNA interference，RNAi）技术抑制心衰大鼠受磷蛋白（phospho—lamban，PLN）mRNA的表达，改善心脏功能。方法构建表达PLN shRNA的重组腺相关病毒载体（rAAV2-phRi1及rAAV2-phRi2）；通过经腹心包穿刺术将rAAV2-phRi1及rAAV2-phRi2注入心衰大鼠心包内，基因导入后10d，30dRT—PCR法检测RNAi对心肌细胞PLN mRNA的抑制效应，并测定肌浆网Ca^2＋-ATP酶（sarcoplasmic reticulum Ca^2＋-ATPase，SERCA2a）活性，比较血流动力学的变化。结果rAAV2-phRi1及rAAV2-phRi2心包注射后，与心衰组比较，10d时PLN mRNA分别降低18．76％／16．36％，SERCA2a活性及血流动力学指标无明显改善；30d时rAAV2-phRi1及rAAV2-phRi2对PLN mRNA的抑制效率为85．32％／67．65％，SERCA2a活性增加104％／74％，血流动力学显著改善（P〈0．05），达到假手术组水平（P〉0．05）；rAAV2-phRi1的抑制效率，对心功能的改善显著优于rAAV2-phRi2（P〈0．05）。结论在体水平上，RNAi能够抑制心衰大鼠PLN mRNA表达，改善心脏功能，rAAV2-phRi1优于rAAV2-phRi2。     

绞股蓝总皂苷对阿霉素致心力衰竭大鼠心功能的影响

目的观察绞股蓝总皂苷(GP)对阿霉素(ADR)致心衰大鼠心肌ATPase活性和肌浆网Ca2+-ATPase(SERCA2a)的影响。方法 50只SD大鼠随机分为5组,正常对照组(NC),阿霉素组(ADR),绞股蓝总皂苷低、中、高剂量组(L-GP,MPGP and H-GP)。大鼠腹腔注射ADR2.5 mg·kg-1·d-1,隔日1次,共6次,复制心衰模型。绞股蓝总皂苷低、中、高剂量组分别给予绞股蓝总皂苷50、100和200 mg·kg-1·d-1灌胃。第7周末,采集大鼠心室内压,分析心功能;电镜观察心肌组织超微结构变化,同时测定心肌组织ATPase活性、琥珀酸脱氢酶(SDH)和SERCA2a活性;RT-PCR检测心肌SERCA2a mRNA表达。结果 (1)与NC组相比,ADR组大鼠LVSP和±dP/dtmax均明显下降,LVEDP上抬(P<0.05或P<0.01),心肌ATPase活性和SDH活性显著下降(P<0.01);SERCA2a活性和SERCA2a mRNA表达水平显著下降(P<0.01);电镜显示心肌细胞变性坏死;(2)与ADR组比较,给予中、高剂量GP后大鼠心功能均有改善,电镜下心肌细胞变性坏死程度及心肌细胞超微结构形态改变显著减轻,心肌ATPase活性、SDH活性和SERCA2a活性明显升高,SERCA2a mRNA表达水平增加(P<0.05或P<0.01)。结论绞股蓝总皂苷改善阿霉素致心衰大鼠心肌功能,其机制可能与升高心肌组织ATPase活性、SERCA2a活性有关。     

腺相关病毒介导心肌肌浆网Ca2+-ATPase 2a基因转导治疗大鼠慢性心力衰竭

目的 研究腺相关病毒为载体的心肌肌浆网Ca^2＋ -ATPase 2a（sarcoplasmic reticulum Ca^2＋ -ATPase,SERCA2a）基因转导对慢性心力衰竭（HF）大鼠的治疗作用,并探讨其多种可能的机理.方法 采用腹主动脉缩窄术建立HF大鼠模型,应用经腹心包腔内注射术分别将生理盐水、携带eGFP基因和携带SERCA2a基因的重组腺相关病毒导入HF、HF＋EGFP和HF＋SERCA2a组大鼠心脏.于导入30天,检测各组大鼠的心脏功能、SERCA2a蛋白表达和活性;比较HF组和HF＋SERCA2a组大鼠心肌蛋白质组表达的差异;检测各组大鼠心肌肌球蛋白重链（MHC）亚型的表达.结果 HF大鼠心脏内转导入SERCA2a基因30天,心脏收缩和舒张功能达到对照组大鼠水平,并且HF＋SERCA2a组左室重/体重比值显著降低;SERCA2a蛋白表达和活性明显升高至对照组大鼠水平;多种能量代谢酶表达明显增加;α-MHC、β-MHC的表达以及α-MHC/β-MHC恢复至对照组大鼠水平.结论 以重组腺相关病毒2作为载体,SERCA2a基因转导可以增强衰竭心脏的SERCA2a功能,增加心脏能量代谢,纠正MHC亚型的异常表达;在临床方面表现为显著改善心脏收缩和舒张功能,可能能够减轻心脏肥厚等病理性结构改变.     

益气活血方对大鼠心肌梗死边缘区Sigma-1R、SERCA2a、IP3R mRNA表达的影响

目的观察益气活血方对大鼠心肌梗死边缘区Sigma-1R、SERCA2a、IP3R mRNA表达的影响。方法结扎SD大鼠心脏左冠状动脉前降支建立心肌梗死后心力衰竭模型(假手术组只穿线,不结扎),随机分为模型组、益气活血低剂量组(益低组)、益气活血高剂量组(益高组)、氟伏沙明组。益低组、益高组分别予以含生药量15 g/kg、30 g/kg的益气活血方,氟伏沙明组予以氟伏沙明(1mg/kg),模型组和假手术组大鼠予以蒸馏水,每日1次灌胃。8周后,进行超声心动图检测;心脏取材并计算心脏质量指数;RT-PCR法检测左心室心肌梗死边缘区Sigma-1R、SERCA2a、IP3R mRNA的表达。结果与假手术组相比,模型组大鼠左室舒张末内径(LVDd)、左室收缩末内径(LVDs)、左室舒张末期容积(LV Vo Ld)、左室收缩末期容积(LV Vo Ls)及心脏质量指数显著增加(P0.01),左室后壁舒张末厚度(LVPWTd)、左室后壁收缩末厚度(LVPWTs)、射血分数(EF)、短轴缩短率(FS)显著降低(P0.01),其心肌组织Sigma-1R、SERCA2a mRNA的表达降低(P0.05),IP3R mRNA表达增加(P0.01)。与模型组相比,各药物组大鼠心肌重构和心功能有不同程度改善(P0.01);益低组、益高组和氟伏沙明组大鼠的心脏质量指数均显著降低(P0.01);同时,各药物组Sigma-1R和益高组SERCA2a mRNA表达显著增加(P0.01),氟伏沙明组SERCA2a mRNA表达增加(P0.05),益低组SERCA2a mRNA表达无统计学意义,益低组、益高组IP3R mRNA表达降低(P0.05),氟伏沙明组IP3R mRNA表达明显降低(P0.01)。结论益气活血方药可显著改善心肌梗死后心力衰竭大鼠心脏重构和心脏功能,其机制与调节心肌梗死后心肌组织Sigma-1R及相关因子SERCA2a、IP3R mRNA的表达有关。     

肌浆网钙ATP酶基因转导对慢性心力衰竭犬心肌蛋白质组影响的初步研究

目的 分析心肌肌浆网Ca2+-ATP酶(sarcoplasmic reticulum Ca2+ ATPase 2a,SERCA2a)基因转导对慢性心力衰竭(HF)犬心肌蛋白质组的影响,探讨SERCA2a基因转导改善心功能的机制.方法 快速右心室起搏建立HF犬模型并随机分为HF组、HF+绿色荧光蛋白(enhanced green fluorescent pmtein,EGFP)组、HF+SERCA2a组.后两组分别向心肌内注射携带EGFP和SERCA2a基因的rAAV载体.于基因转导30 d时停止起搏后进行超声心动图和血流动力学检查并制备心室肌双向电泳蛋白样品和心肌双向电泳图谱,图像分析软件分析蛋白表达差异点,MALDI-TOF-MS数据库搜索鉴定蛋白质.结果 基因转导30 d时,HF+SERCA2a组犬的症状、超声心动图和血流动力学指标与HF+EGFP组相比有显著好转(P＜0.05);与对照组相比差异无统计学意义(P＞0.05).挑选SERCA2a基因转导后表达量发生明显改变的10个蛋白点进行分析,经质谱鉴定分别为心肌收缩相关蛋白、线粒体能量代谢酶类和应激相关蛋白.结论 以rAAV为载体介导SERCA2a基因转导能够改善HF犬心脏的收缩和舒张功能,其可能的机制是恢复了心肌收缩相关蛋白正常表型或正常表达量,增加了心肌能量的产生,改变了应激相关蛋白的表达.     

心力衰竭家兔心肌细胞钙调控蛋白表达的异常

目的探讨心力衰竭(心衰)心肌细胞钙调控蛋白表达异常的临床意义。方法 16只家兔随机分为两组,假手术组和心衰组各8只。通过超容量负荷联合压力负荷建立家兔心衰模型,利用心导管术和心脏多普勒观察手术前后家兔血流动力学及心脏结构和功能的变化。采用蛋白免疫印迹(Western blot)法测定心肌组织 L 型钙通道(LTCC)、肌浆网钙释放通道(RyR2)、肌浆网钙泵(SERCA2a)以及钠钙交换体(NCX)表达水平。结果家兔心衰组与假手术组相比,左室/体重比值、心率、左室舒张末压明显增加(P<0.01);左室短轴缩短率[(21.3±4.00)%与(36.5±1.36)%]和左室射血分数(0.45±0.07与0.70±0.02)降低(P<0.01);心肌组织 LTCC、RyR2表达下降(R_(LTCC/actin):0.287±0.029与0.624±0.009,R_(RyR2/actin):0.106±0.001与0.203±0.011,P<0.01);SERCA2a、NCX 表达增加(R_(NCX/actin:0.497±0.015与0.221±0.014,R_(SERCA2a/actin:0.611±0.036与0.433±0.008,P<0.01)。结论 LTCC 和 RyR2表达下调是心衰心肌收缩力降低的因素之一,而心衰早期 SERCA2a、NCX 表达增加可能有利于心肌舒缩功能的保持。     

抑制NADPH氧化酶对心力衰竭兔心肌钙调节蛋白的影响

目的 探讨抑制NADPH氧化酶对心力衰竭(心衰)兔心功能的影响及其机制.方法 通过超容最负荷联合压力负荷建立家兔心衰模型,以NADPH氧化酶特异性抑制剂apocynin给予干预.8周后,观察兔心脏结构和功能的变化,并检测心肌NADPH氧化酶活性以及心肌肌浆网钙泵(sarcoplasmic reticulum Ca~(2+) ATPase,SERCA2a)、雷诺定受体2(ryanodine receptor 2,RyR2)、受磷蛋白(phospholamban,PLB)和钠钙交换体(sodium-calcium exchanger,NCX)基因表达的变化.结果 心衰兔出现明显的心脏扩大和心功能异常,这些改变与心肌NADPH氧化酶活性增加、SERCA2a、RyR2和PLB mRNA表达降低以及NCX mRNA表达增加相关.apocynin显著抑制心衰兔心肌NADPH氧化酶活性(P<0.05),上调SERCA2a、RyR2和PLB mRNA表达(SERCA2a/GAPDH比值:0.63±0.11与0.34±0.08,RyR2/GAPDH比值:0.23±0.04与0.17±0.06,PLB/GAPDH比值:1.28±0.13与0.95±0.09,P<0.05),下调NCX mRNA表达(NCX/GAPDH比值:0.67±0.10与0.95±0.12,P<0.05),并改善心功能[左室射血分数(60.06±10.07)%与(38.87±3.31)%,左室短轴缩短率(30.12±6.56)%与(17.40±2.45)%,P<0.05].结论 抑制NADPH氧化酶能够调节心衰时心肌钙调节蛋白异常表达,这可能是其改善心衰时心功能的分子机制.     

比索洛尔对心力衰竭大鼠心肌肌浆网钙三磷酸腺苷酶2a活性的影响

目的观察比索洛尔对心力衰竭大鼠心肌肌浆网钙三磷酸腺苷酶2a(SERCA2a)活性的影响。方法采用阿霉素腹腔注射法建立大鼠心力衰竭模型,随机分为假手术组、模型组和比索洛尔组,分别以蒸馏水或比索洛尔连续灌胃35d。心脏超声检测大鼠心功能指标,酶联免疫吸附试验(ELISA)检测血浆脑钠尿肽水平,实时定量聚合酶链反应检测心肌miR-25-3p表达水平,Western blot检测心肌SERCA2a和受磷蛋白表达水平,定磷法测定心肌SERCA2a活性。结果与假手术组比较,模型组大鼠心输出量、左心室短轴缩短率(FS)、左心室射血分数(LVEF)、心肌SERCA2a和受磷蛋白表达水平、SERCA2a与受磷蛋白比值和SERCA2a活性降低[SERCA2a活性(0.029±0.025)比(0.088±0.062)U/mg pro;P0.01],血浆脑钠尿肽和心肌miR-25-3p表达水平升高。与模型组比较,比索洛尔组大鼠心输出量、FS、LVEF、心肌SERCA2a和受磷蛋白表达水平、SERCA2a与受磷蛋白比值和SERCA2a活性升高[SERCA2a活性(0.072±0.063)比(0.029±0.025)U/mg pro;P0.05],血浆脑钠尿肽和心肌miR-25-3p表达水平降低。结论比索洛尔可以改善心力衰竭大鼠心功能,下调心肌miR-25-3p表达水平,提高SERCA2a与受磷蛋白比值,增强SERCA2a活性。     

缬沙坦对心力衰竭家兔心肌肌浆网钙调控相关蛋白基因的影响

目的探讨家兔慢性心力衰竭(心衰)时心肌肌浆网钙泵(SERCA2)、受磷蛋白(PLB)基因表达的改变及血管紧张素Ⅱ受体拮抗剂缬沙坦长期干预的意义。方法18只家兔随机分为3组,假手术组、心衰组和缬沙坦组,每组6只。通过超容量负荷联合压力负荷建立家兔心衰模型,术后7周观察左心室结构、血流动力学的变化及SERCA2、PLB基因的表达。结果与假手术组比较,心衰组左心室重量指数、心率、左心室舒张末压显著升高(P<0.05),左心室缩短率及LVEF明显降低(P<0.05);与心衰组比较,缬沙坦组左心室重量指数、心率、左心室舒张末压显著降低(P<0.05),左心室缩短率及左心室射血分数明显升高(P<0.05);心衰组SERCA2、PLBmRNA显著低于假手术组(P<0.05),缬沙坦组SERCA2、PLBmRNA显著高于心衰组(P<0.05)。结论缬沙坦长期干预心衰,能够改善心脏舒缩功能,可能与其上调肌浆网的钙调控蛋白SERCA2、PLB基因表达有关。     

急性心肌梗死大鼠SERCA2a mRNA表达与心肌损伤、心律失常、虚实辨证的相关性分析

目的分析急性心肌梗死大鼠肌浆网Ca~(2+)-ATP酶信使RNA(SERCA2a mRNA)表达与心肌组织形态学,心律失常及虚实辨证评分的相关性。方法将40只SD大鼠分为假手术组、模型组、心痛方组、硫氮卓酮组。采用结扎法制备心肌梗死模型,采用RT-PCR法测定SERCA2a mRNA表达,心肌细胞HE染色观察形态学改变,心电图检测心律失常,虚实辨证评分进行虚实辨证。结果心肌损伤程度与SERCA2a mRNA表达呈负相关(r=-0.780,P=0.00);未发生心律失常组SERCA2a mRNA表达明显增多(P0.00);SERCA2a mRNA表达与虚实辨证评分呈正相关(r=0.969,P=0.01)。结论 SERCA2a mRNA对心肌组织具有保护作用,SERCA2a mRNA表达与心律失常、虚实辨证具有相关性。     

过表达肌浆网钙ATP酶治疗实验性慢性缺血性心力衰竭

目的 研究肌浆网钙ATP酶2a(SERCA2a)基因过表达对慢性缺血性心力衰竭(心衰)心功能及心肌内质网应激(ERS)相关凋亡的影响.方法采用ameroid环束扎小型猪前降支制备慢性缺血性心衰模型.开胸心肌内注射重组腺相关病毒以过表达SERCA2a或对照报告基因绿色荧光蛋白.60 d后检测血流动力学、SERCA2a的表达和活性、心肌凋亡及ERS标志蛋白-分子伴侣GRp78、凋亡蛋白caspase-12的表达.结果基因转导后60 d,与心衰对照及报告基因组相比,转基因组SERCA2a蛋白表达和活性显著增高,心功能参数改善,心肌凋亡指数降低,伴GRP78和活化caspase12表达下降.结论过表达SERCA2a可改善慢性缺血性心衰的心脏功能,其机制可能涉及减轻ERS相关的心肌细胞凋亡.

Abstract：

Objective Chronic myocardial ischemia (CMI) has become the most importat cause of heart failure (HF) all over the world. The aim of the current study was to investigate the effects of Sarcoendoplasmic reticulum calcium ATPaee 2a (SERCA2a) gene transfer on cardiac function and endoplasmic reticulum stress (ERS) associated myocardial apoptosis in a minipig HF animal model induced by CMI. Methods HF was induced in minipigs by implantation of ameroid constrictor in the initial segment of left anterior descending (LAD) branch of coronary artery. After confirmation of myocardial perfusion defects and cardiac function impairment by myocardial perfusion imaging and echocardiography, animals were divided into 4 groups (n =4 each): HF group, HF + enhanced green fluorescent protein (EGFP) group,HF + SERCA2a group, and shamed animals as control group. A total amount of 1×1012 v.g. Of rAAV1EGFP or rAAV1-SERCA2a were injected intramyocardially to each animal of HF + EGFP and HF +SERCA2a groups. Sixty days after gene transfer, protein level and activity of SERCA2a were examined,cardiac functions and changes of serum inflammatory and neuro-hormonal factors were determined. Apoptotic index of the ischemic myocardium, protein levels of ER stress marker glucose regulated protein 78 ( GRP 78) and ER stress specific apoptotic marker caspase-12 were also assayed. Results At the study end,echocardiographic and hemodynamic measurements indicated a significant improvement of both cardiac systolic and diastolic function in HF + SERCA2a group compared with HF/HF + EGFP groups [LVEF (60.2±8.6)%vs (44.2±7.1)% and (46.8±6.7)%, Ev/Ay 1.28±0.24 vs 0.77 ±0.17 and 0.80±0.21, +dp/dtmax(2713.9 ±434.0) mm Hg/s ( 1 mm Hg =0.133 kPa) vs (1892.3 ±434.2) mm Hg/s and (1931.2±397.4)mm Hg/s, -dp/dtmax (1422.1±334.4) mm Hg/s vs (848.3±308.3) mm Hg/s and (849.5±278.3)mm Hg/s, P＜0.05], along with increase in both SERCA2a protein level (1.13±0.26 vs 0.73 ±0.17 and 0.64±0.18, P＜0.05) and activity [(16.2±5.5) IU/ml vs (7.9±3.1) IU/ml and (7.5 ±2.8)IU/ml, P ＜0.05] compared with HF/HF + EGFP groups. Serum concentrations of inflammatory factor tumor necrotic factor α [(382.3±114.4) ng/L vs (732.3±201.4) ng/L and (689.8±192 5) ng/L, P＜0. 05], neural-hormonal factors brain natriuretic peptide [(142.6±45.3) ng/L vs (422.3±113.6) ng/L and(393.7 ±103.3)ng/L, P＜0.01], endothelin-1 [(111.4 ±37.5)ng/L vs (193.5 ±54.3)ng/L and (201.0±72.1)ng/L,P＜0.05] and angiotensin Ⅱ[(189.7±65.2)μg/L vs (538.3 ± 135.2) μg/L and ( 525.5±144.1)μg/L, P＜0.01] were also significantly decreased in HF + SERCA2a group compared with HF/HF + EGFP groups. The apoptotic index [(12.71±4.11)% vs(23.22±7.23) % and (24.31±6.38)%, P＜0.05], protein levels of GRP78 (1.27±0.33 vs 3.23±1.14 and 4.18±1.13, P＜0.05)and protein level ratios of cleaved caspase-12 to total caspase-12[(4.62±1.93)% vs (9.71±2.70)% and (10.14±2.81)%, P＜0.05] were also significantly reduced in the ischemic myocardium of HF+SERCA2a group compared with the HF/HF + EGFP groups. Conclusion Overexpression of SERCA2a significantly improved cardiac systolic and diastolic function in this HF model partly through attenuation of ER stress related myocardial apoptosis, suggesting its therapeutic potential for CM1 related heart failure.     

Myocardial gene expression in heart failure patients treated with cardiac resynchronization therapy responders versus nonresponders.

OBJECTIVES: We studied whether functional improvement after cardiac resynchronization therapy (CRT) is associated with reversal of the heart failure (HF) gene program. BACKGROUND: Cardiac resynchronization therapy improves exercise tolerance and survival in patients with advanced congestive HF and dyssynchrony. METHODS: Twenty-four patients referred for CRT underwent left ventricular (LV) endomyocardial biopsies immediately before CRT implantation (baseline). In addition, 17 of them underwent LV endomyocardial biopsy procurement 4 months later (follow-up). In 6 control patients with normal LV function, LV biopsies were obtained at the time of coronary artery bypass grafting. The LV messenger ribonucleic acid (mRNA) levels of contractile and calcium regulatory genes were measured by quantitative real time polymerase chain reaction and normalized for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The HF patients showing an improvement in New York Heart Association (NYHA) functional class by >1 score and a relative increase in LV ejection fraction > or =25% at 4 months after CRT were considered as responders. RESULTS: The HF patients were characterized by lower LV mRNA levels of alpha-myosin heavy chain (alpha-MHC), beta-myosin heavy chain (beta-MHC), sarcoplasmic reticulum calcium ATPase 2alpha (SERCA), phospholamban (PLN), and higher brain natriuretic peptide (BNP) mRNA levels as compared with control subjects. Responders to CRT (n = 11) showed an increase in LVEF (p < 0.001), a decrease in left ventricular end-diastolic diameter (p = 0.003), and NYHA functional class (p = 0.002), and a reduction in N-terminal proBNP levels (p = 0.032) as compared with baseline. This was associated with an increase in mRNA levels of alpha-MHC (p = 0.035), SERCA (p = 0.032), a decrease in BNP mRNA levels (p = 0.002), and an increase in the ratio of alpha-/beta-MHC (p = 0.018) and SERCA/PLN (p = 0.012). No significant changes in molecular profile were observed in nonresponders. CONCLUSIONS: In HF patients with electromechanical cardiac dyssynchrony, functional improvement related to CRT is associated with favorable changes in established molecular markers of HF, including genes that regulate contractile function and pathologic hypertrophy.     

Ameliorated stress related proteins are associated with improved cardiac function by sarcoplasmic reticulum calcium ATPase gene transfer in heart failure

Background

Previous studies showed that overexpression of sarco-endoplasmic reticulum calcium ATPase (SERCA2a) in a variety of heart failure (HF) models was associated with greatly enhanced cardiac performance. However, it still undefined the effect of SERCA2a overexpression on the systemic inflammatory response and neuro-hormonal factors.

Methods

A rapid right ventricular pacing model of experimental HF was used in beagles. Then the animals underwent recombinant adeno-associated virus 1 (rAAV1) mediated gene transfection by direct intra-myocardium injection. HF animals were randomized to receive the SERCA2a gene, enhanced green fluorescent protein (control) gene, or equivalent phosphate buffered saline. Thirty days after gene delivery, the cardiac function was evaluated by echocardiographic testing. The protein level of SERCA2a was measured by western blotting. The proteomic analysis of left ventricular (LV) sample was determined using two-dimensional (2-D) gel electrophoresis and MALDI-TOF-MS. The serum levels of the systemic inflammatory and neuro-hormonal factors were assayed using radioimmunoassay kits.

Results

The cardiac function improved after SERCA- 2a gene transfer due to the significantly increased SERCA2a protein level. Beagles treated with SERCA2a had significantly decreased serum levels of the inflammatory markers (interleukin-6 and tumor necrosis factor-α) and neuro-hormonal factors (brain natriuretic peptide, endothelin-1 and angiotensin II) compared with HF animals. The myocardial proteomic analysis showed that haptoglobin heavy chain, heat shock protein (alpha-crystallin-related, B6) were down-regulated, and galectin-1 was up-regulated in SERCA2a group compared with HF group, companied by up-regulated contractile proteins and NADH dehydrogenase.

Conclusions

These findings demonstrate that regional intramyocardial injections of rAAV1-SERCA2a vectors may improve global LV function, correlating with reverse activation of the systemic inflammatory, excessive neuroendocrine factors and the stress-associated myocardial proteins, suggesting that the beneficial effects of SERCA2a gene transfer may involve the attenuation of stress-associated reaction.     

Cardiac-specific overexpression of sarcolipin in phospholamban null mice impairs myocyte function that is restored by phosphorylation

Sarcolipin (SLN) inhibits the cardiac sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2a) by direct binding and is superinhibitory if it binds as a binary complex with phospholamban (PLN). To demonstrate whether overexpression of SLN in the heart might impair cardiac function directly, transgenic (TG) mice with cardiac-specific overexpression of NF-SLN (SLN tagged at its N terminus with the FLAG epitope) were generated on a phospholamban (PLN) null (PLN KO) background. In NF-SLN TG/PLN KO cardiac microsomes, the apparent affinity of SERCA2a for Ca2+ was decreased compared with non-TG littermate PLN KO hearts. Analyses of isolated NF-SLN/PLN KO cardiomyocytes revealed impaired cardiac contractility, reduced calcium transient peak amplitude, and slower decay kinetics compared to PLN KO animals. In these cardiomyocytes, isoproterenol restored calcium dynamics to the levels seen in PLN KO. Invasive hemodynamic and echocardiographic analyses of NF-SLN/PLN KO mouse cardiac muscle in vivo showed no direct effects of NF-SLN overexpression when compared to PLN KO mice. A possible mechanism for the lack of effects in the whole heart may be a responsiveness to phosphorylation because we determined that NF-SLN can be phosphorylated in cardiomyocytes in response to isoproterenol, and we provide evidence that serine/threonine kinase 16 is a kinase that can phosphorylate NF-SLN. Site-directed mutagenesis showed that SLN Thr-5 is the target site for this kinase. These data show that overexpression of NF-SLN can inhibit SERCA2a in the absence of PLN and that the inhibition of SERCA2a is correlated with impairment of contractility and calcium cycling in cardiomyocytes.     

Heterogeneous transmural gene expression of calcium-handling proteins and natriuretic peptides in the failing human heart.

OBJECTIVE: Human heart failure is associated with a disturbed intracellular calcium (Ca2+) homeostasis. In this regard, ventricular wall stress is considered to be a determinant for expression of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a). In the present study, we analyzed the transmural protein and/or mRNA levels of SERCA2a, other Ca(2+)-handling proteins, and of atrial and brain natriuretic peptides (ANP and BNP) in the human heart. METHODS: Subepicardial (epi), midmyocardial (mid), and subendocardial (endo) sections of the left ventricular free wall from end-stage failing (n = 17) and nonfailing (n = 5) human hearts were analyzed by Western blot for immunoreactive protein levels of SERCA2a, phospholamban (PLN), and calsequestrin (CS). Subepi- and subendocardial sections were analyzed by Northern blot for steady-state mRNA levels of SERCA2a, Na(+)-Ca2+ exchanger (NCX1), ANP, and BNP. RESULTS: SERCA2a protein and mRNA levels were reduced by 40 +/- 5% (P < 0.01) and 25 +/- 7% (P < 0.05) in endo compared to epi in the failing heart and by 27 +/- 14% and 16 +/- 12% (non-significant) in the nonfailing heart, respectively. PLN protein levels were reduced by 23 +/- 6% (P < 0.05) in endo compared to epi in the failing heart and by 17 +/- 25% (non-significant) in the nonfailing heart, whereas CS protein levels and NCX1 mRNA levels were similar across the left ventricular wall. Strikingly, in the failing heart, both BNP and ANP mRNA levels were upregulated predominantly in endo. CONCLUSIONS: In the failing human heart, SERCA2a and PLN, as well as natriuretic peptides but not CS and NCX1 are differentially expressed across the left ventricular wall, implicating (1) different susceptibility of subendocardium and subepicardium to factors affecting expression of these proteins and (2) differences in regulation of the distinct calcium-cycling proteins.     

Cardiac-specific overexpression of sarcolipin inhibits sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2a) activity and impairs cardiac function in mice

Sarcolipin (SLN) inhibits the cardiac sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA2a) by direct binding and is superinhibitory if it binds through phospholamban (PLN). To determine whether overexpression of SLN in the heart might impair cardiac function, transgenic (TG) mice were generated with cardiac-specific overexpression of NF-SLN (SLN tagged at its N terminus with the FLAG epitope). The level of NF-SLN expression (the NF-SLN/PLN expression ratio) was equivalent to that which induces profound superinhibition when coexpressed with PLN and SERCA2a in HEK-293 cells. In TG hearts, the apparent affinity of SERCA2a for Ca(2+) was decreased compared with non-TG littermate control hearts. Invasive hemodynamic and echocardiographic analyses revealed impaired cardiac contractility and ventricular hypertrophy in TG mice. Basal PLN phosphorylation was reduced. In isolated papillary muscle subjected to isometric tension, peak amplitudes of Ca(2+) transients and peak tensions were reduced, whereas decay times of Ca(2+) transients and relaxation times of tension were increased in TG mice. Isoproterenol largely restored contractility in papillary muscle and stimulated PLN phosphorylation to wild-type levels in intact hearts. No compensatory changes in expression of SERCA2a, PLN, ryanodine receptor, and calsequestrin were observed in TG hearts. Coimmunoprecipitation indicated that overexpressed NF-SLN was bound to both SERCA2a and PLN, forming a ternary complex. These data suggest that NF-SLN overexpression inhibits SERCA2a through stabilization of SERCA2a-PLN interaction in the absence of PLN phosphorylation and through the inhibition of PLN phosphorylation. Inhibition of SERCA2a impairs contractility and calcium cycling, but responsiveness to beta-adrenergic agonists may prevent progression to heart failure.