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.     

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。     

腺相关病毒介导心肌肌浆网三磷酸腺苷酶基因治疗心力衰竭犬的安全性分析

目的研究心肌注射重组腺相关病毒(rAAV)_(2/1)介导心肌肌浆网Ca~(2+)-ATP酶(SERCA2a)基因治疗慢性心力衰竭犬的安全性。方法选取成年比格犬8只,随机分为对照组和SERCA2a基因转导组(转导组),每组4只。分别采用PCR、RT PCR和Western blot法从DNA、RNA和蛋白水平检测rAAV_(2/1)SERCA2a转导后在犬体内的分布;采用双夹心ELISA法检测转导组犬的血清。结果除注射rAAV_(2/1)的心肌区域外,未注射rAAV_(2/1)的心肌区域、主要脏器、骨骼肌和性腺中均未检测到rAAV_(2/1)-SERCA2a。对照组与转导组犬心肌、腰大肌、咀嚼肌和睾丸表现出相同的SERCA2a蛋白表达谱,未在其他组织中检测到。转导组犬血清内未检测到抗rAAV_(2/1)抗体。结论 rAAV_(2/1)介导的基因转导是相对安全的,未整合到心脏以外的其他组织,也未在犬体内引发体液免疫。     

翻译后修饰对SERCA2a的作用在心血管疾病中的研究进展

肌浆/内质网Ca2+ATP酶(SERCA)2a作为SERCA在心脏中表达的亚型,介导心肌细胞收缩后胞浆中Ca2+再回摄进入肌浆网中。肌浆网摄取Ca2+的速率决定了心肌舒张的速率,调节SERCA2a的活性可控制心脏的收缩性和舒张性,影响心脏功能。近年来,多项研究表明SERCA2a受到翻译后修饰(PTM)的调节,如类泛素化、乙酰化等,从而影响SERCA2a的活性而调节Ca2+水平。因此,SERCA2a可成为心血管疾病潜在的治疗靶标。     

长期运动对慢性心力衰竭大鼠左室重构的抑制作用

目的观察8 w运动对心力衰竭大鼠左室结构、功能和重构基因表达的影响,探讨运动改善左室重构的可能机制。方法 40只SD大鼠随机分为心力衰竭对照组(HC组,n=10)、心力衰竭运动组(HE组,n=10)、假手术对照组(SC组,n=10)和假手术运动组(SE组,n=10),心力衰竭模型采用冠状动脉结扎术。HE组和SE组进行8 w跑台运动,SC组和HC组在鼠笼内自由活动。超声心动图检测左室结构和功能;分离左室后行Masson染色法进行组织病理学观察并获得胶原容积分数(CVF);实时荧光定量PCR检测左室心肌心钠素(ANF)、α-肌球蛋白重链(α-MHC)、β-肌球蛋白重链(β-MHC)和肌质网Ca~(2+)-ATP酶(SERCA2a)mRNA表达量。结果 (1)与SC组比较,SE组大鼠左室出现离心性肥大,心功能提高,α-MHC和SERCA2a mRNA升高(P<0.05);(2)与SC组比较,HC组左室出现向心性肥大,心功能降低,CVF明显增加(P<0.05),心肌α-MHC和SERCA2a mRNA表达下调(P<0.05),ANF和β-MHC mRNA表达上调(P<0.05);(3)与HC组比较,HE组大鼠左室发生离心性肥大,心功能提高,CVF明显减少(P<0.05),心肌α-MHC和SERCA2a mRNA表达上调(P<0.05),ANF和β-MHC mRNA表达下调(P<0.05)。结论长期运动抑制心力衰竭大鼠左室重构并改善心功能,其机制与心肌纤维化程度减轻、胚胎基因表达下调、收缩蛋白表达上调有关。     

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。     

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。     

心力衰竭大鼠心肌β_3肾上腺素受体表达及其对钙离子相关调节蛋白的作用

目的研究心力衰竭(HF)大鼠心肌β3肾上腺素受体(beta3-adrenergic receptors,β3-AR)表达情况及对钙离子相关调节蛋白的作用。方法以正常大鼠作对照组,皮下注射生理盐水;实验组大鼠皮下注射异丙肾上腺素(Isoproterenol,ISO),常规饲养8w。造模成功后实验组随机分为3组,激动剂组给予尾静脉注射β3-AR激动剂BRL37344,抑制剂组给予尾静脉注射β3-AR抑制剂SR59230A,HF组给予生理盐水。用药8w后行心脏彩超、RT-PCR法检测心肌组织钙调蛋白(CaM)、钙调蛋白激酶Ⅱ(CaMKⅡ)、肌内质网Ca2+-ATP酶异构体2a(SERCA2a)的mRNA表达。结果①HF组较对照组β3-ARmRNA表达水平增高,CaM、CaMKⅡ、SERCA2amRNA表达水平减低(均P0.05)。②与HF组相比较,激动剂组β3-ARmRNA水平表达增加,CaM、CaMKⅡ、SERCA2a表达进一步减少(均P0.05)。③抑制剂组β3-ARmRNA表达水平较对照组减少,CaM、CaMKⅡ、SERCA2a较对照组减低,但较HF组增加(均P0.05)。结论β3-AR激动剂减低衰竭心肌CaM、CaMKⅡ、SERCA2a的mRNA表达,导致心功恶化,β3-AR抑制剂则相反,可延缓HF进展,预示β3-AR抑制剂在药物治疗HF中的应用前景。     

微电极阵列技术研究肌质网钙ATP酶过表达对大鼠衰竭心肌电活动的改善作用

目的 探索重组腺病毒(rAd)介导的肌质网Ca2+-ATP酶(SERCA2a)过表达对大鼠心肌梗死后心力衰竭心肌电活动节律和传导的改善作用,并探讨可能的电活动机制.方法 将26只成年雄性SD大鼠随机分为3组:假手术组(n=l0),空病毒对照组(rAd.β-gal组,n=8)和肌质网Ca2+-ATP酶(SERCA2a)转染组(rAd.SERCA2a组,n=8).假手术组仅开胸不结扎动脉,rAd.β-gal组和rAd.SERCA2a组分别进行左冠状动脉前降支结扎建立大鼠心肌梗死后心力衰竭动物模型,同时分别将携带β-gal和SERCA2a基因的重组腺病毒(rAd)导入衰竭心脏,术后2周超声心电图检测心脏舒张功能和收缩功能,心电图监测体表心电活动以及微电极阵列(MEA)技术监测离体心脏组织电活动情况.结果 rAd携带SERCA2a与β-gal基因均成功转入大鼠衰竭心脏.rAd.SERCA2a组可改善心功能,与假手术组相比心室舒张末期容积与心室收缩末期容积轻微增加[(0.41±0.13)cm2对(0.39±0.02)cm2,(0.08±0.02)cm2对(0.06±0.01)cm2,P＞0.05],左心室射血分数[(0.82±0.05)对(0.86±0.01),P＞0.05]和短轴缩短率[(46.6±2.32)％对(49.58±1.71)％,P＞0.05]无明显改变.与假手术组相比,rAd.β-gal组体表心电图QT间期延长[(111.02±7.42) ms对(94.7±1.55) ms,n=6,P＜0.05],室性早搏发生率达71.5％ (5/7),而rAd.SERCA2a组QT间期缩短[(81.45±4.97)ms对(94.7±1.55)ms,n=6,P＜0.05],室性早搏发生率达14.3％(1/7).MEA记录可发现rAd.SERCA2a组心率与假手术组相比差异无统计学意义[(435±31)次/min对(442 ±22)次/min,n=6,P＞0.05],与rAd.β-gal组相比,rAd.SERCA2a组最大场电位[(0.82±0.39)mV对(0.64±0.13) mV,n=6,P＜0.05]、最小场电位[(1.88±0.57) mV对(1.35±0.12) mV n=6,P＜0.05]、场电位时限[(124.17±21.08)ms对(113.23±12.02) ms n=6,P＜0.05]均延长;rAd.β-gal组梗死区与梗死对侧区心肌组织场电位时限差异有统计学意义[(60.36±2.08)ms对(103.24±7.35) ms,n=5,P＜0.05],并且60通道记录梗死区心肌组织场电位时限离散度大于rAd.SERCA2a组[(38.5ms±4.62)ms对(26.88±5.09) ms,n=5];rAd.SERCA2a组传导基本一致,使心肌梗死面心室肌组织电活动呈均一性传导.结论 SERCA2a转基因治疗可以显著改善心力衰竭大鼠的左心室收缩功能、舒张功能,同时可以降低心肌梗死后心力衰竭伴发心律失常的发生,改善心脏电活动的均一传导.MEA技术是一项检测心血管疾病动物模型心脏组织电生理节律和频率以及传导活动的理想技术.     

氯沙坦对心肌梗死后左心室肌球蛋白重链基因影响

目的 观察血管紧张素Ⅱ受体阻断药氯沙坦对急性心肌梗死后左心室心肌肌球蛋白重链（myosin heavy chain,MHC）基因表达的影响.方法 Sprague-Dawley大鼠24只随机分为3组,正常对照组,急性心肌梗死组,氯沙坦急性心肌梗死组,每组8只.急性心肌梗死造模后第2日开始灌胃给药,口服氯沙坦日剂量3 mg/kg,6周后测量左心室重量、进行心肌肌球蛋白重链基因分析.结果 急性心肌梗死组大鼠心脏重量指数明显增加,氯沙坦治疗后心脏重量指数明显下降（P＜0.01）;Northern plot发现,急性心肌梗死组大鼠心肌细胞的α-MHC mRNA表达量明显下降,而β-MHC mRNA的表达量则显著上升;而氯沙坦治疗组大鼠心肌的α-MHC mRNA表达较急性心肌梗死组增加,α-MHC mRNA表达明显降低（P＜0.01）.结论 氯沙坦能减轻梗死心肌肥厚,改善梗死心肌心室重构,机制与其调节心肌MHC的基因表达有关.     

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.     

Modification of myosin gene expression by imidapril in failing heart due to myocardial infarction

The beneficial effects of imidapril, an angiotensin converting enzyme (ACE) inhibitor were investigated on changes in myofibrillar ATPase as well as myosin heavy chain (MHC) content and gene expression due to myocardial infarction (MI). Three weeks after occluding the left coronary artery, rats were treated with or without imidapril (1 mg/kg/day), for 4 weeks. The infarcted hearts exhibited depressed rates of left ventricular (LV) pressure development (57+/-2.4% reduction) and pressure decay (55.5+/-1.6% reduction). LV myofibrillar Ca(2+) ATPase activity, unlike that in the right ventricle (RV), was decreased in the infarcted animals compared with controls (6.8+/-0.4 vs 10.3+/-0.6 micromol Pi/mg/hr). MHC alpha-isoform contents were decreased by 47 and 41% whereas those of MHC beta-isoform were increased by 823 and 1200% in the LV and RV due to MI, respectively. MHC alpha-isoform mRNA levels were decreased by 55 and 35% whereas those for MHC beta-isoform were increased by 50 and 30% in the infarcted LV and RV, respectively. Imidapril treatment partially prevented the changes due to MI in LV function (rate of pressure development, 24+/-2.3% reduction and rate of pressure decay, 14+/-1.8% reduction), myofibrillar Ca(2+) ATPase activity (8.2+/-0.7 micromol Pi/mg/hr), MHC protein content (alpha-MHC, 24% reduction and beta-MHC, 525% increase) and MHC gene expression (alpha-MHC, 18% reduction and beta-MHC, 15% increase). The results suggest that the beneficial effects of ACE inhibition on the failing heart are associated with improvements in myofibrillar ATPase activities as well as prevention of changes in MHC isozyme protein contents and their gene expression.     

RNA干扰治疗慢性心力衰竭大鼠的实验研究

目的利用RNA干扰(RNAi)技术抑制心力衰竭(心衰)大鼠受磷蛋白(PLN)mRNA的表达,改善心脏功能。方法选用SD大鼠40只,对照组7只;其余33只造心衰模型,存活21只,随机分为心衰组、rAAV2-phRi1组,rAAV2-phRi2组,每组7只;rAAV2-phRi导入后30天测定血流动力学,逆转录聚合酶链反应检测心肌PLN mRNA,定磷法测定肌浆网Ca2+-ATP酶(SERCA2a)活性。结果rAAV2-phRi导入30天时,与心衰组比较,rAAV2-phRi1及rAAV2-phRi2对PLN mRNA的抑制效率为85.32%和67.65%,SERCA2a活性增加了104%和74%,血流动力学显著改善(P<0.05),达到对照组水平(P>0.05);rAAV2-phRi1的抑制效率、对心功能的改善显著优于rAAV2-phRi2。结论体内水平上,RNAi能够抑制心衰大鼠PLN mRNA表达,改善心脏功能。     

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.     

Low triiodothyronine (T3) or reverse triiodothyronine (rT3) syndrome modifies gene expression in rats with congestive heart failure

Heart failure (HF) is frequently associated with euthyroid "sick" syndrome (low T3 and elevated rT3). We investigated if altered thyroid hormone in HF could affect expression of the TH receptor (TRalpha1), and alpha and beta myosin heavy chains (alpha-MHC, beta-MHC). HF was provoked in rats by aortic stenosis. We showed that rT3 generated from liver and kidney deiodination significantly increased and T3 decreased in HF; there was significantly higher TRalpha1 expression, no alpha-MHC expression, but beta-MHC expression. Changes in TRalpha could be compensating for low T3 from HF.     

Dronedarone administration prevents body weight gain and increases tolerance of the heart to ischemic stress: a possible involvement of thyroid hormone receptor alpha1.

Hypothyroid heart displays a phenotype of cardioprotection against ischemia and this study investigated whether administration of dronedarone, an amiodarone-like compound that has been shown to preferentially antagonize thyroid hormone binding to thyroid hormone receptor alpha1 (TRalpha1), results in a similar effect. Dronedarone was given in Wistar rats (90 mg/kg, once daily (od) for 2 weeks) (DRON), while untreated animals served as controls (CONT). Hypothyroidism (HYPO) was induced by propylthiouracil administration. Isolated rat hearts were perfused in Langendorff mode and subjected to 20 minutes of zero-flow global ischemia (I) followed by 45 minutes of reperfusion (R). 3,5,3' Triiodothyronine remained unchanged while body weight and food intake were reduced. alpha-Myosin heavy chain (alpha-MHC) decreased in DRON while beta-myosin heavy chain (beta-MHC) and sarcoplasmic reticulum Ca2+ adenosine triphosphatase (ATPase) expression (SERCA) was similar to CONT. In HYPO, alpha-MHC and SERCA were decreased while beta-MHC was increased. Myocardial glycogen content was increased in both DRON and HYPO. In DRON, resting heart rate and contractility were reduced and ischemic contracture was significantly suppressed while postischemic left ventricular end-diastolic pressure and lactate dehydrogenase release (IU/L min) after I/R were significantly decreased. In conclusion, dronedarone treatment results in cardioprotection by selectively mimicking hypothyroidism. This is accompanied by a reduction in body weight because of the suppression of food intake. TRs might prove novel pharmacologic targets for the treatment of cardiovascular illnesses.     

Reversal of chronic molecular and cellular abnormalities due to heart failure by passive mechanical ventricular containment

Passive mechanical containment of failing left ventricle (LV) with the Acorn Cardiac Support Device (CSD) was shown to prevent progressive LV dilation in dogs with heart failure (HF) and increase ejection fraction. To examine possible mechanisms for improved LV function with the CSD, we examined the effect of CSD therapy on the expression of cardiac stretch response proteins, myocyte hypertrophy, sarcoplasmic reticulum Ca2+-ATPase activity and uptake, and mRNA gene expression for myosin heavy chain (MHC) isoforms. HF was produced in 12 dogs by intracoronary microembolization. Six dogs were implanted with the CSD and 6 served as concurrent controls. LV tissue from 6 normal dogs was used for comparison. Compared with normal dogs, untreated HF dogs showed reduced cardiomyocyte contraction and relaxation, upregulation of stretch response proteins (p21ras, c-fos, and p38 alpha/beta mitogen-activated protein kinase), increased myocyte hypertrophy, reduced SERCA2a activity with unchanged affinity for calcium, reduced proportion of mRNA gene expression for alpha-MHC, and increased proportion of beta-MHC. Therapy with the CSD was associated with improved cardiomyocyte contraction and relaxation, downregulation of stretch response proteins, attenuation of cardiomyocyte hypertrophy, increased affinity of the pump for calcium, and restoration of alpha- and beta-MHC isoforms ratio. The results suggest that preventing LV dilation and stretch with the CSD promotes downregulation of stretch response proteins, attenuates myocyte hypertrophy and improves SR calcium cycling. These data offer possible mechanisms for improvement of LV function after CSD therapy.