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。     

比索洛尔改善心力衰竭大鼠心功能的机制探讨

目的:观察比索洛尔对心力衰竭大鼠心功能的影响,并探讨其机制。方法:80只SD大鼠随机选取10只为正常对照组,10只作为假手术组,假手术组腹腔注射10 ml/kg的生理盐水。造模过程中有15只死亡,左心室短轴缩短率不达标5只,40只成功造模大鼠按随机数字表法分成4组:模型组(n=10)、比索洛尔组(n=10)、卡托普利组(n=10)和比索洛尔+卡托普利组(n=10)。观察各组大鼠心功能指标,酶联免疫吸附测定(ELISA)法检测血浆B型利钠肽水平,实时定量逆转录-聚合酶链式反应(RT-PCR)检测心肌miR-25-3p表达水平,免疫蛋白印迹试验(Western blot)检测心肌肌浆网钙ATP酶2a(SERCA2a)和受磷蛋白(PLB)表达水平,定磷法测定心肌SERCA2a活性。结果:与正常对照组比较,模型组大鼠心输出量、左心室短轴缩短率、左心室射血分数、心肌SERCA2a和PLB表达水平、SERCA2a/PLB比值和SERCA2a活性显著降低,血浆B型利钠肽水平和心肌miR-25-3p表达水平显著升高(P均0.01)。与模型组比较,比索洛尔组、卡托普利组和比索洛尔+卡托普利组大鼠心输出量、左心室短轴缩短率、左心室射血分数、心肌SERCA2a和PLB表达水平、SERCA2a/PLB比值和SERCA2a活性显著升高,血浆B型利钠肽水平和心肌miR-25-3p表达水平显著降低(P均0.05)。结论:比索洛尔可以改善心力衰竭大鼠心功能,机制可能与下调心肌miR-25-3p表达,提高SERCA2a和PLB表达,提升SERCA2a/PLB比值,增强SERCA2a活性有关。     

比索洛尔对心力衰竭大鼠心肌肌浆网钙ATP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ATP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,随机分为正常对照组、假手术组、模型组、比索洛尔组、卡托普利组和比索洛尔+卡托普利组,分别以蒸馏水、比索洛尔、卡托普利、比索洛尔+卡托普利连续灌胃35d。观察大鼠心功能指标,ELISA法检测血浆脑钠肽水平,茎环状引物实时定量PCR检测心肌miR-25-3p表达水平,Western blot检测心肌SERCA2a和PLB蛋白表达水平,定磷法测定心肌SERCA2a活性。结果:比索洛尔组、卡托普利组和比索洛尔+卡托普利组大鼠心功能指标明显高于模型组,血浆脑钠肽水平明显低于模型组,miR-25-3p表达水平明显低于模型组,SERCA2a和受磷蛋白(PLB)蛋白表达水平明显高于模型组(P0.01)。比索洛尔组和比索洛尔+卡托普利组SERCA2a/PLB比值和SERCA2a活性明显高于模型组(P0.01或P0.05),卡托普利组与模型组比较无明显差异。结论:比索洛尔可以改善心力衰竭大鼠心功能,机制可能与下调心肌miR-25-3p表达水平,提高SERCA2a、PLB表达水平,提升SERCA2a/PLB比值,增强SERCA2a活性有关。     

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。     

补阳还五汤对舒张性心力衰竭大鼠心肌钙转运蛋白表达水平的影响

目的研究补阳还五汤对舒张性心力衰竭(DHF)大鼠心肌钙转运蛋白L型钙通道(LTCC)、兰碱尼受体(RyR2)、肌浆网上的钙泵(SERCA2a)和受磷蛋白(PLB)mRNA表达的影响,初步探寻补阳还五汤治疗DHF的作用机制和药物干预靶点。方法选择60只雄性SD大鼠,随机分为假手术组和造模组,造模组利用腹主动脉缩窄法建立DHF大鼠模型,将造模成功的大鼠随机分为模型组、中药组、西药组,每组7只。中药组给予补阳还五汤12.72 g/(kg·d)灌胃,西药组给予酒石酸美托洛尔0.004 5 g/(kg·d)灌胃,假手术组和模型组给予等量的去离子水灌胃,各组均每日灌胃1次。药物干预8周后,运用聚合酶链式反应法(PCR)检测LTCC、RyR2、SERCA2a和PLB的mRNA转录。结果各组LTCC mRNA表达水平比较差异无统计学意义(P0.05);与假手术组相比,模型组、中药组RyR2 mRNA表达降低(P0.05),而西药组RyR2 mRNA的表达与假手术组差异无统计学意义(P0.05),中药组、西药组与模型组组间相比差异无统计学意义(P0.05)。模型组、中药组、西药组SERCA2a和PLB mRNA表达与假手术组相比均降低(P0.05),西药组、中药组SERCA2a和PLB mRNA表达与模型组相比均升高(P0.05),中药组与西药组相比差异无统计学意义(P0.05)。结论补阳还五汤可明显提高DHF大鼠SERCA2a、PLB的mRNA转录,有可能通过作用于这两种蛋白改善DHF大鼠左室舒张功能。     

NADPH氧化酶在AngⅡ介导H9C2心肌细胞凋亡中的作用

目的分析血管紧张素Ⅱ(AngⅡ)介导的H9C2心肌细胞凋亡是否通过NADPH氧化酶/P38MAPK途径。方法体外培养H9C2心肌细胞,分组干预:(1)Control组:仅加细胞培养液;(2)AngⅡ组:在Control组的基础上加入AngⅡ;(3)apocynin组:在Control组的基础上加apocynin;(4)AngⅡ+apocynin组:在Control组的基础上加入AngⅡ以及apocynin(n=6)。干预24 h后测定NADPH氧化酶活性,TUNEL法检测细胞凋亡,Western Blot检测P38MARK及相关凋亡蛋白的表达水平。结果 Control组、apocynin组、AngⅡ组、Ang II+apocynin组凋亡比例分别为(12.20±1.18)%、(14.71±3.88)%、(62.33±4.79)%、(13.67±2.59)%。与Control组比较,AngⅡ组凋亡比例明显增加,而AngⅡ+apocynin组凋亡比例较AngⅡ组下降,差异有统计学意义(P均0.05)。与Control组比较,加入NADPH氧化酶抑制剂apocynin后细胞NADPH氧化酶活性降低;而仅加入AngⅡ的细胞NADPH氧化酶活性明显升高,差异有统计学意义(P0.05)。AngⅡ+apocynin组的NADPH氧化酶活性较AngⅡ组降低,差异有统计学意义(P0.05)。与正常细胞比较,AngⅡ作用后,P38MAPK表达增加,凋亡蛋白Bax表达增加,抗凋亡蛋白Bcl-2表达减少,差异有统计学意义(P均0.05)。Ang II+apocynin组较AngⅡ组P38MAPK表达降低,凋亡蛋白Bax表达减少,抗凋亡蛋白Bcl-2表达增加,差异有统计学意义(P均0.05)。结论血管紧张素II通过NADPH氧化酶/P38MAPK途径介导H9C2心肌细胞凋亡。     

糖尿病大鼠心肌钠钙交换蛋白与肌浆网钙泵mRNA的表达

大鼠腹腔注射链佐霉素制备实验性糖尿病大鼠模型.将大鼠成模后分别于4、6和8周时取心肌组织,进行病理学检查及计算心脏相对重量;应用RT-PCR方法扩增钠钙交换蛋白Ⅰ亚型(NCX1)及肌浆网钙泵2a亚型(SERCA2a)两种产物,计算其mRNA 相对数量的变化.发现糖尿病各组心脏湿重与体质量的比值均明显高于对照组,病理检查显示心肌细胞呈不同程度变性坏死.8周时,糖尿病组NCX1 和SERCA2a mRNA相对含量比对照组明显减少(P<0.05).提示在糖尿病大鼠心脏功能的减退与NCX1和SERCA2a的mRNA水平降低有一定关系.     

从钙离子调控改变探讨兔肾素血管紧张素系统阻断剂逆转甲状腺素促心肌肥厚的机制

目的 研究甲状腺素诱导的肥厚心肌细胞内钙调控变化及其机制,探讨肾素血管紧张素系统(RAS)阻断剂逆转甲状腺素促心肌肥厚的机制.方法 腹腔注射左旋甲状腺素(L-Thy)建立兔甲状腺素性心肌肥厚模型,经胸二维超声心动图测定室间隔厚度(IVS)、左心室内径(LV)及左心室后壁厚度(LVPW)后收集心肌标本,光镜及透射电镜下观察心肌细胞结构改变,Fluo3/AM荷载共聚焦显微镜检测细胞内钙离子浓度,逆转录聚合酶链半定量检测L型Ca2+通道(LCC)、兰尼碱受体(RyR)、肌浆网钙泵(SERCA)mRNA表达,对硝基苯磷酸酯(P-nitrophenyl phosphate,P-NPP)法检测SERCA活性,免疫组织化学法检测IP3R蛋白表达.结果 甲状腺素可诱导心肌肥厚、心肌细胞结构改变及纤维组织增生,甲状腺素组细胞内钙离子浓度明显高于对照组,分别为(576.2±41.7)nmol/L比(314.6±35.6)nmol/L(P<0.01);甲状腺素组RyR mRNA、SERCA mRNA及IP3R蛋白较对照组表达上调,分别为1.19±0.21比0.73±0.15(P<0.01)、1.01±0.08比0.76±0.09(P<0.01)、65.3±13.7比47.9±10.2(P<0.01);LCC mRNA表达和SERCA活力降低,分别为0.48±0.11比0.75±0.16(P<0.01)、(0.062±0.013)μmol·min-1·g-1比(0.133±0.022)μmol·min-1·g-1(P<0.01).咪达普利和缬沙坦干预可显著抑制甲状腺素诱导的心肌细胞肥厚,降低细胞内钙离子浓度,咪达普利组和缬沙坦组细胞内钙离子浓度分别为(376.4±32.5)nmol/L和(392.6±41.2)nmol/L均显著低于甲状腺素组(576.2±41.7)nmol/L(P<0.01);咪达普利和缬沙坦干预后LCC mRNA表达高于甲状腺素组,分别为0.68±0.14比0.48±0.11(P<0.01)、0.64±0.13比0.48±0.11(P<0.01);咪达普利和缬沙坦干预增加SERCA的活力,分别为(0.115±0.019)μmol·min-1·g-1比甲状腺素组(0.062±0.013)μmol·min-1·g-1(P<0.01)、(0.109±0.015)μmol·min-1·g-1比甲状腺素组(0.052±0.013)μmol·min-1·g-1(P<0.01),但对RyR mRNA、SERCA mRNA及IP3R蛋白表达无影响.结论 RAS和细胞内钙超载可能参与甲状腺索性心肌肥厚的发病机制,SERCA活力降低可能是导致甲状腺素性肥厚心肌细胞内钙超载的重要因素.咪达普利和缬沙坦可以改善L-Thy诱导的心肌重构和细胞内钙超载并对SERCA活力具有保护作用.     

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。     

比索洛尔对容量超负荷心力衰竭大鼠心室肌细胞钙调节蛋白的影响

目的观察容量超负荷心力衰竭大鼠心室肌细胞钙调节蛋白心肌浆网Ca2+-ATPase 2a(SERCA2a)、钠钙交换体1型(NCX1)的表达变化及富马酸比索洛尔干预对其的影响。方法成年雄性SD大鼠72只,随机分为对照组16只、假手术组16只、心力衰竭组20只和干预组20只,后2组采用腹主动脉-下腔静脉穿刺造瘘法制作容量超负荷心力衰竭模型。术后8周,干预组给予富马酸比索洛尔(1mg/kg,1次/d)灌胃,连续4周。术后12周后,采用RT-PCR和Western blot检测各组大鼠心室肌细胞SERCA2a、NCX1mRNA及蛋白表达。结果心力衰竭组较假手术组SERCA2amRNA和蛋白(0.57±0.15 vs 0.96±0.03,0.20±0.17 vs 0.85±0.19)表达明显降低、NCX1mRNA和蛋白(2.26±0.36 vs 0.99±0.24,1.66±0.14 vs 0.28±0.05)表达明显升高;干预组较心力衰竭组SERCA2amRNA和蛋白(1.15±0.12 vs 0.57±0.15,1.11±0.20 vs 0.20±0.17)表达明显升高、NCX1mRNA和蛋白(1.03±0.26 vs 2.26±0.36,0.32±0.06 vs 1.66±0.14)表达明显降低(P0.05,P0.01)。结论富马酸比索洛尔干预可部分逆转容量超负荷心力衰竭大鼠心室肌细胞钙调节蛋白表达的改变。     

氯沙坦对心力衰竭兔肌浆网钙调控蛋白的影响

目的探讨血管紧张素Ⅱ受体拮抗剂氯沙坦干预慢性心力衰竭对兔心肌肌浆网钙泵（SERCA2）、钙释放通道（RyR2）、受磷蛋白（PLB）基因表达的影响及意义。方法通过结扎兔冠状动脉前降支复制心肌梗死（心梗）模型,以氯沙坦进行干预。于心梗后8周比较观察左室结构、血流动力学的变化及SERCA2、RyR2、PLB基因的表达。结果与对照组相比,心梗组左室舒张末压（LVEDP）显著升高（P〈0．01）,左室压力上升和下降最大速度（＋dr,／dtmax、-dp／dtmax）显著降低（P〈0．01）;氯沙坦组LVEDP显著低于心梗组（P〈0．05）,＋dp／dtmax、-dp／dtmax显著高于心梗组（P〈0．05）。心梗组SERCA2、RyR2、PLBmRNA显著低于对照组（P〈0．01）,而氯沙坦组的上述三项显著高于心梗组（P〈0．05）。结论氯沙坦长期干预心力衰竭,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、RyR2、PLB的基因表达有关。     

Cardiac FKBP12.6 overexpression protects against triggered ventricular tachycardia in pressure overloaded mouse hearts

Alterations in RyR2 function have been proposed as a major pathophysiological mechanism of arrhythmias and heart failure (HF). Cardiac FKBP12.6 overexpression protects against myocardial infarction-induced HF and catecholamine-promoted ventricular arrhythmias. We tested the hypothesis that FKBP12.6 overexpression protects against maladaptive LVH and triggered ventricular arrhythmias following transverse aorta constriction (TAC) in the mouse. The TAC-associated mortality rate was significantly lower in male transgenic (DT) than in Ctr mice (p < 0.05). TAC-associated maladaptive hypertrophy was blunted in DT mice especially 1 month post-TAC and their SERCA2a/PLB ratio remained unchanged 1 and 2 months post-TAC. Two months after TAC, trains of 30 stimuli (burst pacing) performed following isoproterenol injection (0.2 mg/kg, ip), induced VT in 50% of the TAC-Ctr and in none of the TAC-DT mice (p = 0.022). The increase in myocyte shortening and Ca²⁺ spark frequency observed in sham-operated Ctr mice in response to 50 nM isoproterenol was reduced in DT mice, and abolished in TAC-DT mice. NCX1 function was reduced in Sham-DT and TAC-DT compared with Sham-Ctr and TAC-Ctr mice, respectively (p < 0.05 for the 2 comparisons). In mice killed after isoproterenol injection and burst pacing, RyR2 S2814 phosphorylation was decreased by 50% in TAC-DT versus TAC-Ctr mice (p < 0.05), with no change in RyR2 S2808 and PLB S16 and T17 phosphorylation. Cardiac FKBP12.6 overexpression in the mouse blunts pressure overload-induced maladaptive LV remodelling and protects against catecholamine-promoted burst pacing-induced ventricular tachycardia by decreasing cardiac sensitivity to adrenergic stress and RyR2 S2814 phosphorylation, and decreasing NCX1 activity.     

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.     

培哚普利对心力衰竭大鼠心肌细胞钙瞬变及其调控蛋白的影响

目的研究培哚普利在心力衰竭(心衰)治疗中对心肌细胞的收缩特性、钙瞬变及其调控蛋白的影响。方法通过缩窄雄性Wistar大鼠的腹主动脉,制成压力负荷增高性心衰模型。随机分成培哚普利治疗组(培哚普利组,3mg·kg-1·d-1)、心衰对照组和假手术对照组(假手术组)。12周后分离左室心肌细胞,测定单个心肌细胞在电刺激时的缩短分数、钙瞬变以及钠-钙交换蛋白(NCX1)、内质网Ca2+-ATPase(SERCA2)、受磷蛋白等钙调控蛋白的转录与翻译水平。结果心肌细胞的缩短分数(FS)和[Ca2+]imax在心衰对照组均明显低于假手术组[FS(%):7.51±1.15与13.21±1.49,[Ca2+]imax(nmol/L):330.85±50.05与498.16±14.07;均为P<0.01]。给予培哚普利治疗后,培哚普利组的FS和[Ca2+]imax分别为(10.89±1.18)%和(488.72±22.27)nmol/L,明显高于心衰对照组(均为P<0.01)。在心衰对照组,NCX1和受磷蛋白的mRNA水平显著高于假手术组,而SERCA2的mRNA却明显低于假手术组。在培哚普利组,其NCX1和SERCA2的mRNA水平则介于心衰对照组和假手术组之间,差异有统计学意义(均为P<0.05)。在心衰对照组及培哚普利组,NCX1的蛋白表达量分别是假手术组的(1.141±0.047)倍(P<0.01)和(1.074±0.081)倍(P=0.018),SERCA2的表达量则是假手术组的(0.803±0.100)倍(P<0.01)和(0.893±0.084)倍(P=0.003)。结论培哚普利抗心衰治疗能够减轻心肌细胞的钙瞬变及其调控蛋白的异常变化,使心衰中单个心肌细胞的收缩特性得到保护。     

Adenoviral gene transfer of mutant phospholamban rescues contractile dysfunction in failing rabbit myocytes with relatively preserved SERCA function

In heart failure (HF) a main factor in reduced contractility is reduced SR Ca2+ content and reversed force-frequency response (FFR), ie, from positive to negative. Our arrhythmogenic rabbit HF model exhibits decreased contractility mainly due to an increase in Na/Ca exchange (NCX) activity (with only modest decrease in SR Ca2+-ATPase (SERCA) function), similar to many end-stage HF patients. Here we test whether phospholamban (PLB) inhibition using a dominant-negative mutant PLB adenovirus (K3E/R14E, AdPLB-dn, with beta-galactosidase adenovirus as control) could enhance SERCA function and restore Ca2+ transients and positive FFR in ventricular myocytes from these HF rabbits. HF myocytes infected with AdPLB-dn (versus control) had enhanced Ca2+ transient amplitude (2.0+/-0.1 versus 1.6+/-0.05 F/Fo at 0.5 Hz, P<0.05) and had a positive FFR, whereas acutely isolated HF myocytes or those infected with Adbetagal had negative FFR. Ca2+ transients declined faster in AdPLB-dn versus Adbetagal myocytes (RT50%: 317+/-29 versus 551+/-90 ms at 0.5 Hz, P<0.05) and had an increased SR Ca2+ load (3.5+/-0.3 versus 2.6+/-0.2 F/Fo at 0.5 Hz, P<0.05), indicative of increased SERCA function. Furthermore, this restoration of function was not due to changes in NCX or SERCA expression. Thus, increasing SERCA activity in failing myocytes by AdPLB-dn gene transfer reversed the contractile dysfunction (and restored positive FFR) by increasing SR Ca2+ load. This approach could enhance contractile function in failing hearts of various etiologies, even here where reduced SERCA activity is not the main dysfunction.     

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.     

美托洛尔对家兔心力衰竭心肌细胞钙调控蛋白表达的影响

目的观察美托洛尔对家兔心力衰竭（心衰）心肌细胞钙调控蛋白表达的影响,探讨其改善心衰的可能分子机制。方法30只家兔随机分为3组,即假手术组（n=11）、心衰组（n=11）、美托洛尔干预组（n=8）。心衰组和美托洛尔干预组家兔先建立实验性主动脉瓣关闭不全,2周后行腹主动脉缩窄,利用心脏多普勒观察术前后家兔心脏功能的变化,共观察6周。采用常规酶解法分离心室肌细胞,经Fluo-3／AM负载后,利用激光共聚焦显微技术,观察咖啡因诱导的钙瞬变过程中细胞内钙浓度的动态变化。从左心室心肌组织提取膜蛋白后,采用Western blot法测定钙调控蛋白表达水平,并应用UVIDoc成像仪进行蛋白表达半定量分析。结果假手术组与心衰组射血分数分别为（72．6±5．0）％、（45．7±3．0）％（P〈0．01）,咖啡因诱导的钙瞬变幅度（FI）分别为43．5±6．2、16．0±3．5（P〈0．01）,峰值到达时间分别为（52．2±7．4）s、（129．8±14．5）s（P〈0．01）,兰尼碱受体表达量分别为0．203±0．021、0．106±0．007（P〈0．01）及肌浆网钙泵与钠钙交换体（SERCA2a／NCX）表达量比值分别为1．96±0．12、1．22±0．23（P〈0．01）。与心衰组相比,美托洛尔干预组射血分数值[（60．2±5．1）％,P〈0．05]明显增加,钙瞬变幅度增加（32．8±5．4,P〈0．05）,峰值到达时间缩短（91．4±10．9）s,P〈0．05],兰尼碱受体表达量（0．164±0．016,P〈0．05）和SERCA2a／NCX表达量比值（1．68±0．17,P〈0．05）增加。结论美托洛尔可延缓心衰心肌细胞钙调控蛋白表达的改变,进而改善钙瞬变,这可能是长期应用β受体阻滞剂改善心衰患者心功能的分子机制之一。     

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。     

Reduced sarcoplasmic reticulum Ca2+ uptake and increased Na+–Ca2+ exchanger expression in left ventricle myocardium of dogs with progression of heart failure

Alteration of intracellular Ca²⁺ homeostasis in failing cardiomyocytes is associated with changes in regulatory proteins located in the sarcoplasmic reticulum (SR) and sarcolemma, which participate in Ca²⁺ fluxes across the membrane during the cardiac cycle. These regulatory proteins include Ca²⁺-ATPase (SERCA 2A), phospholamban (PLB), ryanodine-sensitive Ca²⁺ release channels (RR), and the sarcolemmal Na⁺–Ca²⁺ exchanger (NCX). Although their status is known in failed myocardium, it is poorly understood during the progression of heart failure (HF), particularly in large animals. We studied the left ventricular (LV) myocardium of six dogs with moderate HF and six with severe HF produced by multiple intracoronary microembolizations, compared with six normal dogs (NL). Oxalate-dependent SR Ca²⁺ uptake and expression of SERCA 2A, PLB, phosphorylated PLB at serine 16 (PLB-Ser) and threonine 17 (PLB-Thr), RR, and NCX were determined. Percent LV ejection fraction declined by 47% compared with NL (34.1% ± 1% vs 64% ± 2%) in dogs with moderate HF (HF-2W) 2 weeks after the last embolization and by 42% (20.5% ± 1% vs 34.1% ± 1%) in dogs with severe HF (HF-4M) at 4 months compared with HF-2W. Left ventricular pressure during isovolumic contraction (+dP/dt, mmHg/s) and relaxation (–dP/dt, mmHg/s) was significantly reduced in severe compared with moderate HF. Oxalate-dependent SR Ca²⁺ uptake (nmol ⁴⁵Ca²⁺ accumulated/min per milligram noncollagen protein) declined by 25% (21.3 ± 1 vs 28.5 ± 2) in HF-2W and 49% in HF-4M. Protein expression of SERCA 2A and PLB decreased by 67% and 35%, respectively, in HF-2W compared with NL, whereas SERCA 2A expression increased by 167% and PLB decreased by 40% in HF-4M compared with HF-2W. However, SERCA 2A protein was still significantly lower in HF-4M compared with NL. PLB-Ser and PLB-Thr increased significantly in HF-2W but decreased in HF-4M compared with NL. Similar changes in mRNA encoding PLB and SERCA 2A were observed in dogs with moderate and severe HF. The RR protein level declined in dogs with moderate and severe HF, whereas NCX protein did not change with moderate HF but increased with sever HF. These results suggest that the regulatory proteins responsible for Ca²⁺ uptake, Ca²⁺ release, and Na⁺–Ca²⁺ exchange are critically associated with the deterioration of LV function during the progression of HF.