新生大鼠右心室心肌细胞的原代培养及鉴定

目的:建立一种简便、重复性好的新生大鼠右心室心肌细胞原代培养方法,为右心疾病及肺循环右心相关研究提供可靠的细胞工具。方法:采用出生1～3 d无特定病原体级,SD新生大鼠12只,无菌操作开胸取心脏后,在体视显微镜下分离右心室,用胰蛋白酶与Ⅱ型胶原酶联合消化法消化右心室心肌组织,差速贴壁法纯化右心室心肌细胞。用0.4%台盼蓝染色检测右心室心肌细胞存活率,在倒置显微镜下观察右心室心肌细胞的生长特点并记录细胞搏动频率。利用cTnT对右心室心肌细胞进行免疫荧光染色鉴定细胞纯度。结果:采用胰蛋白酶及Ⅱ型胶原酶联合消化右心室心肌组织及差速贴壁法分离所得右心室心肌细胞存活率为(94.96±2.13)%。培养的心肌细胞24 h大部分贴壁并伸出伪足,少数细胞出现自发性搏动;48 h后细胞基本全部贴壁,伪足延长,出现局部同步化搏动;72 h后细胞伪足继续延长并形成细胞簇,搏动基本同步化;96 h后细胞连接成片,呈现完全同步化搏动,搏动频率为(121.2±10.7)次/min。用cTnT染色培养的细胞纯度达(96.71±2.66)%。结论:体视显微镜下分离右心室结合复合酶消化法能成功培养出纯度高、功能较好的新生SD大鼠右心室心肌细胞。     

促红细胞生成素对急性心肌梗死大鼠心脏的保护作用及抗凋亡信号机制

目的 探讨促红细胞生成素(EPO)对急性心肌梗死(AMI)大鼠心肌梗死面积、细胞凋亡和磷酸化Akt(p-Akt)蛋白表达的影响.方法 24只雄性SD大鼠,按随机数字表法分为假手术组、AMI组和EPO组(每组8只),用结扎左冠状动脉前降支方法制作大鼠AMI模型,EPO组同时给予5 000 U/kg重组人EPO(rhEPO)腹腔内注射.建模24 h后摘取心脏标本分别用TTC染色检测心肌梗死面积、TUNEL法检测心肌细胞凋亡、Western印迹分析检测心肌组织中p-Akt蛋白表达的变化.结果 与假手术组比较,AMI组和EPO组心肌梗死区/左心室质量比值均显著增加[(0.00±0.00)％ vs (21.73 ±4.14)％和(17.95±2.66)％,P均＜0.01],但与AMI组比较,EPO组心肌梗死区/左心室质量比值明显降低(P＜0.05);与假手术组比较,AMI组和EPO组心肌细胞凋亡指数均显著增加[(0.51 ±0.08)％ vs (29.62±3.02)％和(21.37±2.39)％,P均＜0.01],但与AMI组比较,EPO组心肌细胞凋亡指数显著减少(P＜0.01).p-Akt蛋白在三组心肌组织中均有表达,但与假手术组比较,AMI组心肌组织p-Akt蛋白表达显著减少,仅为假手术组的41％ (P ＜0.01);与AMI组比较,EPO组心肌组织p-Akt蛋白表达显著增多,比AMI组升高1.7倍(P＜0.01).结论 EPO干预通过明显缩小AMI大鼠的心肌梗死面积和显著减少心肌细胞凋亡发挥心脏保护作用,其机制可能与上调p-Akt蛋白表达激活Akt抗细胞凋亡信号通路有关.     

川芎嗪通过激活Akt/eNOS通路预防阿霉素致小鼠心脏毒性

目的研究川芎嗪对阿霉素诱导的小鼠心脏毒性的预防作用及其机制。方法建立阿霉素诱导的心脏毒性小鼠模型,将小鼠随机分为3组:对照组、阿霉素组(阿霉素15 mg/kg)、川芎嗪组[川芎嗪60 mg/(kg·d)+阿霉素15 mg/kg],心脏超声检测川芎嗪干预后阿霉素对小鼠心脏功能的影响,HE染色及Masson三色染色检测小鼠心脏组织学及胶原蛋白的变化,TUNEL染色检测小鼠心肌细胞凋亡情况,Western blot检测p-Akt、Akt、p-eNOS、eNOS及cleaved Caspase-3蛋白的表达。结果与阿霉素组相比,川芎嗪组左心室短轴缩短率(FS)和左心室射血分数(EF)显著升高(P0.01);川芎嗪显著降低小鼠心肌细胞凋亡(P0.01),明显抑制小鼠心肌纤维化和炎症细胞浸润(P0.01);与阿霉素组相比,川芎嗪组显著上调p-Akt和p-eNOS的表达(P0.05),显著下调cleaved Caspase-3的表达(P0.01)。结论川芎嗪通过激活Akt/eNOS通路抑制心肌细胞凋亡保护阿霉素诱导损伤心肌可能是川芎嗪预防阿霉素致心脏毒性的一个潜在机制。     

细胞穿透肽PEP-1介导人铜,锌-超氧化物歧化酶转导入大鼠离体心脏及其对缺血再灌注损伤的保护作用

目的 采用离体心脏灌注模型,研究细胞穿透肽PEP-1介导入铜,锌-超氧化物歧化酶(Cu,Zn-SOD,SOD1)穿透心肌组织能力及其对心脏缺血再灌注损伤的保护作用.方法 构建的原核表达载体pET15b-SOD1和pET15b-PEP-1-SOD1,分别转化大肠杆菌,表达和纯化SOD1和PEP-1-SOD1融合蛋白.采用Langendorff灌流系统,大鼠离体心脏停灌30 min后复灌60 min建立缺血再灌注损伤模型.大鼠随机分为对照组,100μmol/L SOD1蛋白预处理组,25、50、100μmol/L PEP-1-SOD1蛋白预处理组.免疫荧光及Western blot检测PEP-1-SOD1的转导效果,超氧化物歧化酶(SOD)试剂盒检测转导的目的 蛋白的SOD活性.测定心肌组织丙二醛(MDA)含量和复灌后15 min内冠状动脉流出液肌酸激酶(CK)活性.脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测心肌细胞凋亡,复灌60 min时红四氮唑(TTC)染色测定心肌梗死面积.结果 免疫荧光及Western blot检测均显示PEP-1-SOD1以剂量依赖性方式转导入离体灌注的心肌组织内,SOD1蛋白预处理组心肌组织内未见到目的 蛋白.对照组,SOD1组和25、50、100 μmol/L PEP-1-SOD1处理组的SOD活性分别为(10.06±0.77),(10.59±0.71)和(32.29±1.42)、(43.16±1.16)、(55.14±1.59)U/mg蛋白,MDA含量分别为(1.48±0.19),(1.39±0.11)和(1.01±0.14)、(0.73±0.13)、(0.50±0.06)nmol/mg蛋白,CK活性分别为(1.73±0.58),(1.68±0.14)和(1.40±0.28)、(0.97±0.39)、(0.61±0.56)U/mg蛋白,心肌细胞凋亡率(AI)分别为(17.25±0.75)%,(16.63±1.07)%和(11.50±0.57)%、(6.50±0.63)%、(4.13±0.52)%,心肌梗死面积百分比分别为(55.13±2.18)%,(52.13±2.59)%和(33.88±2.06)%、(25.50±2.16)%、(15.38±1.14)%.与对照组和SOD1组比较,PEP-1-SOD13种剂量组的SOD活性、MDA含量、CK活性、心肌细胞凋亡率及心肌梗死面积百分比指标差异均有统计学意义(均P<0.01).说明PEP-1-SOD1蛋白预处理组SOD活性以剂量依赖性方式显著高于SOD1组,MDA含量、CK活性及心肌细胞凋亡率均明显低于SOD1组,心肌梗死面积小于SOD1组.结论 PEP-1肽介导SOD1蛋白能直接以天然活性的形式高效穿透进入离体心肌组织,转导的PEP-1-SOD1融合蛋白对心肌缺血再灌注损伤具有明显的保护作用.     

大鼠心肌梗死修复中肌纤维母细胞细胞视黄醇结合蛋白-1的表达

目的:维甲酸是维生素A的体内衍生物,通过受体介导的基因,包括基质金属蛋白酶基因表达改变,发挥多种生物学效能。维甲酸细胞内转运和代谢由细胞视黄醇结合蛋白-1(CRBP-1)调节。在组织修复过程中肉芽组织表达CRBP-1。为探讨心肌梗死后心室重构的机制,检测了大鼠心肌梗死模型中CRBP-1的表达。方法:通过结扎左冠状动脉前降支获得大鼠心肌梗死模型。80只成年雄性Wistar大鼠分为3组。假手术组10只大鼠,假手术后6天处死取心脏。心肌梗死组50只大鼠,每时间点10只,分别于术后3天、6天、15天、30天和45天处死大鼠取心脏。蛋白检测组20只心肌梗死大鼠,每时间点5只,分别于术后3天、6天、15天和45天处死大鼠取心脏标本用于蛋白检测。心脏从心尖至心底部分为四部分,经过固定和石蜡包埋做成切片。苏木素—伊红、Masson’s trichrome染色以及兔抗CRBP-1抗体和鼠抗α平滑肌(α-SM)肌动蛋白抗体经免疫组织化学染色。并应用Western印迹法检测CRBP-1和α-SM肌动蛋白。结果:心肌梗死组大鼠均获得了大面积心肌梗死,按左心室周长法计算,梗死面积从43.3%到58.1%[平均(48.8±3.6)%]。心肌梗死组各时间点心肌梗死面积差异均无显著性。心肌梗死组各时间点大鼠右心室重量和右心室重量/体重以及左心室横径长度和长度/体重均较假手术组显著增加(P均<0.05),提示心肌梗死后左心室重构明显。观察见病区心肌细胞坏死、胶原沉积以及左心室重构。免疫组织化学染色显示,术后3天和6天心肌梗死心内、心外膜区域均有纤维母细胞浸润和心肌细胞坏死以及CRBP-1表达。术后6天和术后15天,心肌梗死区域α-SM肌动蛋白阳性的肌纤维母细胞有CRBP-1表达,这种表达于术后30天逐渐减低。结论:大鼠心肌梗死模型中纤维母细胞短暂而迅速地表达CRBP-1,提示CRBP-1和维甲酸可能在心肌梗死后心室重构中起重要作用。     

体外心脏震波对大鼠心肌梗死后心肌凋亡的影响

目的研究体外心脏震波治疗(CSWT)对大鼠心肌梗死后(MI)心肌细胞凋亡及凋亡蛋白的影响。方法结扎大鼠左冠状动脉前降支建立大鼠急性心肌梗死模型,随机分为假手术(Sham)组(n=10)、MI组(n=10)及CSWT组(n=10)。CSWT治疗4 w后,取各组心肌样本进行TUNEL检测观察心肌细胞凋亡,Masson染色观察心肌纤维化,Western印迹检测Bax、Bcl-2和Caspase-3蛋白表达。结果与Sham组相比,MI和CSWT组心肌细胞凋亡指数及纤维化程度均增高,Bax和Caspase-3表达显著上调,Bcl-2表达明显下调(P0.05);而CSWT组较MI组,心肌细胞凋亡指数及纤维化程度均降低,Bax和Caspase-3表达下调,Bcl-2则表达上调(P0.05)。结论大鼠心肌梗死后心肌凋亡明显增加,体外心脏震波可抑制大鼠心肌梗死后细胞凋亡,减轻左心室纤维化。     

4-辛基衣康酸减轻脂多糖诱导的心肌损伤

目的 探讨4-辛基衣康酸(4-OI)对脂多糖(LPS)诱导的小鼠心肌损伤的影响。方法 采用6～8周雄性C57BL/6J小鼠，经腹腔注射4-OI(25 mg/kg)或等量生理盐水连续预处理3 d后，腹腔注射LPS(10 mg/kg)或等量生理盐水，12 h后行心脏超声检查。取心脏，行苏木精-伊红(HE)染色观察心肌病理变化；Western blot检测心肌组织中肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)、p65、pp65、谷胱甘肽过氧化物酶4(GPx4)、前列腺素内过氧化物合成酶2(PTGS2)和核转录因子红系2相关因子2(Nrf2)的蛋白表达水平。结果 心脏超声结果显示，LPS腹腔注射后，小鼠左心室明显扩张，收缩功能减低；与LPS组相比，4-OI组小鼠左心室收缩功能改善。HE染色示LPS腹腔注射后，小鼠心脏炎症细胞浸润明显，心肌细胞排列疏松，表现出类似于脓毒症诱导的心肌损伤的病理特点。4-OI组小鼠心脏的炎症细胞浸润程度和病理改变较LPS组低。Western blot示LPS腹腔注射后，小鼠心肌组织中Nrf2和GPx4的表达明显降低，IL-1β、TNF-α、PTGS2以...     

大鼠心肌梗死后心肌中转化生长因子β诱导基因-22蛋白表达的变化规律及白细胞介素-6对其表达的诱导作用

目的:检测大鼠心肌梗死(心梗)后心肌组织中转化生长因子β诱导基因-22(TSC-22)蛋白水平表达的动态变化,并在细胞水平上研究白细胞介素-6(IL-6)是否对其蛋白表达有诱导作用.方法:雄性Sprague-Dawley大鼠,通过结扎左冠状动脉前降支制备心梗模型.分为心肌梗死模型组(心梗组)和假手术对照组(假手术组),于术后1天、3天、7天、2周、4周进行心脏标本取材,酶联免疫吸附(ELISA)法检测大鼠左心室心肌组织中TSC-22蛋白的表达.胰酶消化法分离培养乳鼠原代心肌细胞,随机分为心肌细胞对照组和心肌细胞实验组,心肌细胞对照组正常培养,心肌细胞实验组分别在2.5、5.0、10.0 ng/ml浓度的IL-6刺激下培养24 h.采用ELISA法、免疫细胞化学法和免疫印迹法(Western blot)等方法检测TSC-22蛋白表达.结果:ELISA法结果表明,大鼠心肌梗死1天、7天心梗组比假手术组左心室心肌组织中TSC-22蛋白含量显著升高,4周时则显著降低,差异均有统计学意义(P均＜0.05).心肌细胞实验的ELISA法结果表明,在10.0 ng/ml的IL-6刺激24 h后,心肌细胞实验组比心肌细胞对照组TSC-22蛋白表达水平显著升高,差异有统计学意义(P＜0.05),在2.5、5.0 ng/ml IL-6刺激24 h后,心肌细胞实验组与心肌细胞对照组比较差异无统计学意义;免疫细胞化学法和蛋白免疫印迹法结果显示,10.0 ng/ml的IL-6刺激24 h后,TSC-22在细胞核中的染色明显加深.结论:大鼠心梗后的急性期,如梗死1天、7天后,左心室心肌组织中TSC-22蛋白水平迅速升高.炎症细胞因子IL-6可能是其在心梗后上调的诱导因素之一.     

糖原合酶激酶-3β及DVL-1在大鼠心肌重塑中的表达

目的观察大鼠心肌重塑过程中心肌成纤维细胞DVL-1蛋白及糖原合酶激酶-3β(GSK-3β)蛋白表达变化.方法采用颈静脉输注去甲肾上腺素(NE)方法复制大鼠心肌重塑病理模型,用超声检测心脏结构和收缩功能.应用Western blot技术检测大鼠心肌成纤维细胞GSK-3β及DVL-1蛋白质的表达水平.结果与对照组比较,实验组大鼠左心室心肌Ⅰ/Ⅲ胶原蛋白比值(6.2±1.5 vs 2.3±0.7)明显升高(P＜0.01);实验组左心室成纤维细胞GSK-3β蛋白表达水平(0.48±0.03,1.4±0.2)显著降低(P＜0.01);实验组DVL-1蛋白质的表达水平(4.3±0.3 vs 1.2±0.2),显著升高(P＜0.01).结论 NE致大鼠心肌重塑过程中GSK-3β蛋白表达显著降低,DVL-1蛋白表达显著升高,可能与心肌纤维化有关.     

ADAMTS-4在心力衰竭患者心肌中的表达及意义

目的探讨含Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶-4(ADAMTS-4)在心力衰竭患者心肌中的表达及作用。方法收集2012年7月~2017年3月武汉大学人民医院终末期心力衰竭患者心脏移植后的病变心脏及配型不成功的正常供体心脏标本,心力衰竭患者17例,纳入心力衰竭组,男性12例,女性5例,年龄(46.5±9.7)岁;配型不成功的正常供体心脏8例,纳入对照组,均为男性,年龄(37.6±6.9)岁。心脏离体后立即分离样本,心肌组织采用HE和天狼星红染色,采用Real-time PCR及Western-blot技术检测ADAMTS-4在心肌组织中的表达水平。结果光镜下,对照组心肌细胞形态完整,胞浆均匀致密,细胞排列整齐,可见清晰的细胞核;心力衰竭组患者心肌细胞肿大,形态不规整,肌丝断裂,排列紊乱,可见畸形、增大的细胞核。天狼星红染色显示,心力衰竭组患者心肌组织胶原纤维含量较对照组增多,胶原纤维增粗、排列紊乱,胶原容积分数较对照组升高,(10.18±0.63)%vs.(2.56±0.25)%,差异具有统计学意义(P0.01)。与对照组相比,心力衰竭组患者心肌组织ADAMTS-4m RNA相对表达水平明显增高,(1.00±0.03)vs.(1.50±0.03),差异具有统计学意义(P0.01)。与对照组相比,心力衰竭组患者心肌组织ADAMTS-4蛋白表达水平明显升高,(1.00±0.06)vs.(2.01±0.09),差异具有统计学意义(P0.01)。结论含Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶-4可能通过参与心肌组织重构影响心力衰竭的发生。     

Calcium sensitivity of force in human ventricular cardiomyocytes from donor and failing hearts

In failing human myocardium changes occur, in particular, in isoform composition and phosphorylation level of the troponin T (TnT) and troponin I (TnI) subunits of the actin filament and the myosin light chains (MLC-1 and -2), but it is unclear to what extent they influence cardiac performance. This overview concentrates on the relation between contractile function, contractile protein composition and phosphorylation levels in small biopsies from control (donor) hearts, from biopsies obtained during open heart surgery (NYHA Class I – IV) and from end-stage failing (explanted, NYHA class IV) hearts. Furthermore, attention is paid to the effect of the catalytic subunit of protein kinase A on isometric force development in single Triton-skinned human cardiomyocytes isolated from donor and end-stage failing left ventricular myocardium at different resting sarcomere lengths. A reduction in sarcomere length from 2.2 to 1.8 µm caused reductions in maximum isometric force by approximately 35 % both in donor and in failing cardiomyocytes. The midpoints of the calcium sensitivity curves (pCa ₅₀) of donor and end-stage failing hearts differed markedly at all sarcomere lengths (mean ?pCa ₅₀ = 0.22). Our findings indicate that 1) TnI phosphorylation contributes to the differences in calcium sensitivity between donor and end-stage failing hearts, 2) human ventricular myocardium is heterogeneous with respect of the phosphorylation of TnT, MLC-2 and the isoform distribution of MLC-1 and MLC-2, and 3) the Frank-Starling mechanism is preserved in end-stage failing myocardium.     

Force/shortening-frequency relationship in multicellular muscle strips and single cardiomyocytes of human failing and nonfailing hearts.

BACKGROUND: Force of contraction (FOC) frequency-dependently increases in multicellular muscle strip preparations of human nonfailing myocardium, whereas FOC declines in human failing myocardium with increasing stimulation frequency. We investigated whether these characteristics can be observed in single isolated myocytes. METHODS AND RESULTS: Isolated multicellular muscle strip preparations and single isolated cardiomyocytes of failing (heart transplants, dilative cardiomyopathy; n = 11) and nonfailing (donor hearts; n = 11) human hearts were studied. The changes in contraction amplitude (cell shortening in micrometers) at increasing frequency of stimulation (0.5-2 Hz) were continuously recorded with a 1-dimensional high-speed camera that detected the cell edges and measured their distance during contraction. The increase in stimulation frequency was associated with a significant decrease in FOC (2 v 0.5 Hz; 68% basal) and a decrease in cell shortening of human left ventricular cardiomyocytes from failing hearts (2 v 0.5 Hz; 65% basal). In contrast, in human nonfailing myocardium, contraction increased at increasing stimulation frequencies (2 v 0.5 Hz; FOC, 180% basal; cell shortening, 129% basal). CONCLUSIONS: The negative force-frequency relationship measured in multicellular preparations of failing human myocardium results from alterations at the single cell level.     

Effect of protein kinase A on calcium sensitivity of force and its sarcomere length dependence in human cardiomyocytes

OBJECTIVE: We investigated whether the Frank-Starling mechanism is absent or preserved in end-stage failing human myocardium and if phosphorylation of contractile proteins modulates its magnitude through the sarcomere length-dependence of calcium sensitivity of isometric force development. METHODS: The effect of phosphorylation of troponin I and C-protein by the catalytic subunit of protein kinase A (3 microg/ml; 40 min at 20 degrees C) was studied in single Triton-skinned human cardiomyocytes isolated from donor and end-stage failing left ventricular myocardium at sarcomere lengths measured at rest of 1.8, 2.0 and 2.2 microm. Isometric force development was studied at various free-calcium concentrations before and after protein kinase A incubation at 15 degrees C (pH 7.1). RESULTS: Maximal isometric tension at 2.2 microm amounted to 39.6+/-10.4 and 33.7+/-3.5 kN/m2 in donor and end-stage failing cardiomyocytes, respectively. The midpoints of the calcium sensitivity curves (pCa50) of donor and end-stage failing hearts differed markedly at all sarcomere lengths (mean delta pCa50=0.22). A reduction in sarcomere length from 2.2 to 1.8 microm caused reductions in maximum isometric force to 64% and 65% and in pCa50 by 0.10 and 0.08 pCa units in donor and failing cardiomyocytes, respectively. In donor tissue, the effect of protein kinase A treatment was rather small, while in end-stage failing myocardium it was much larger (delta pCa50=0.24) irrespective of sarcomere length. CONCLUSIONS: The data obtained indicate that the Frank-Starling mechanism is preserved in end-stage failing myocardium and suggest that sarcomere length dependence of calcium sensitivity and the effects of phosphorylation of troponin I and C-protein are independent.     

Beta 1- and beta 2-adrenergic-receptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective beta 1-receptor down-regulation in heart failure

We used radioligand binding techniques and measurement of beta-agonist-mediated positive inotropic responses in isolated cardiac tissue to examine beta-adrenergic-receptor subpopulations in nonfailing and failing human left and right ventricular myocardium. In tissue derived from 48 human hearts the receptor subtypes identified in nonfailing ventricle by radioligand binding were beta 1 (77%) and beta 2 (23%), with no evidence of an "atypical" beta-adrenergic receptor. In failing left ventricle the beta 1:beta 2 ratio was markedly different, i.e., 60:38. This decrease in the beta 1 proportion and increase in the beta 2 proportion in the failing ventricles were due to a 62%, "selective" down-regulation of the beta 1 subpopulation, with little or no change in beta 2 receptors. In muscle bath experiments in isolated trabeculae derived from nonfailing and failing right ventricles, both beta 1- and beta 2-adrenergic receptors were coupled to a positive inotropic response. In nonfailing myocardium, beta 1 responses predominated, as the selective beta 1 agonist denopamine produced a response that was 66% of the total contractile response of isoproterenol. In heart failure the beta 1 component was markedly decreased, while the beta 2 component was not significantly diminished. Moreover, in heart failure the beta 2 component increased in prominence, as the contractile response to the selective beta 2 agonist zinterol increased from a minority (39%) to a majority (60%) of the total response generated by isoproterenol. We conclude that failing human ventricular myocardium contains a relatively high proportion of beta 2 receptors, due to selective down-regulation of beta 1 receptors. As a result, in the failing human heart the beta 2-receptor subpopulation is a relatively important mediator of inotropic support in response to nonselective beta-agonist stimulation and is available for inotropic stimulation by selective beta 2 agonists.     

Force/shortening[ndash ]frequency relationship in multicellular muscle strips and single cardiomyocytes of human failing and nonfailing hearts

Background: Force of contraction (FOC) frequency-dependently increases in multicellular muscle strip preparations of human nonfailing myocardium, whereas FOC declines in human failing myocardium with increasing stimulation frequency. We investigated whether these characteristics can be observed in single isolated myocytes. Methods and Results: Isolated multicellular muscle strip preparations and single isolated cardiomyocytes of failing (heart transplants, dilative cardiomyopathy; N = 11) and nonfailing (donor hearts; N = 11) human hearts were studied. The changes in contraction amplitude (cell shortening in micrometers) at increasing frequency of stimulation (0.5[ndash ]2 Hz) were continuously recorded with a 1-dimensional high-speed camera that detected the cell edges and measured their distance during contraction. The increase in stimulation frequency was associated with a significant decrease in FOC (2 v 0.5 Hz; 68% basal) and a decrease in cell shortening of human left ventricular cardiomyocytes from failing hearts (2 v 0.5 Hz; 65% basal). In contrast, in human nonfailing myocardium, contraction increased at increasing stimulation frequencies (2 v 0.5 Hz; FOC, 180% basal; cell shortening, 129% basal). Conclusions: The negative force-frequency relationship measured in multicellular preparations of failing human myocardium results from alterations at the single cell level.     

Uniformity of calcium channel number and isometric contraction in human right and left ventricular myocardium

Summary We compared contractile performance in trabeculae carneae (n=25) from non-failing right and left ventricles (n=25) of brain dead organ donors without known cardiovascular disease and measured connective tissue content in trabeculae carneae from both non-failing and failing human hearts. Peak twitch force and time-course of contraction were not different between muscles taken from right or left ventricles. Peak twitch force was 13.9±3 vs. 13.7±2.7 mN/mm² for right and left ventricular trabeculae carneae, respectively in 2.5 mM [Ca²⁺]₀ at a 0.33 Hz stimulation frequency. Time to peak tension (405±21 vs. 405±12 ms), time to 50% relaxation from peak contractile response (277±21 vs. 278±14.6 ms) and time to 80% relaxation (428±29 vs. 433±22) were not different between right and left ventricular trabeculae carneae. Calcium channel number determined by [³H]PN200-100 dihydropyridine-radioligand binding assay was also not different (56.2±6.5 fmol/mg protein vs. 58.6±8.4 fmol/mg protein for right and left heart preparations, respectively). However, in myocardium obtained from ischemic hearts the left ventricle showed a reduced number of calcium channels compared to the right ventricle (55.3±3.8 vs. 36.6±3.9 fmol/mg protein for right and left ventricle, respectively p=0.027). No differences were noted in the number of DHP receptor binding sites between right and left ventricular myocardium from patients with idiopathic dilated cardiomyopathy (51.4±7.6 fmol/mg protein vs. 61.8±6.5 fmol/mg protein respectively). Our data indicate that calcium channel number is similar for non-failing left and right human ventricle. Contractile response to changes in [Ca²⁺]₀ and frequency were similar for trabeculae carneae from the left and right ventricles of non-failing human hearts. Studies involving calcium channel activation or inhibition in ischemic human myocardium, where there may be differences in calcium channel number and/or function are warranted. Whether changes in calcium channel number have biological consequences on contractile function remains to be determined. Importantly, careful studies of calcium channel function underin vivo conditions are warranted.Work supported in part by a grant from Glaxo Inc to JKG and the Institute for the Study of the Treatments of Cardiovascular Diseases, Cardiovascular Drug Development and Marketing Consultants, and HL-39091 and HL-36797 to JKG. HL 31117 to JPM. EJG is a fellow of the Stanley J. Sarnoff Society of Fellows for Research in Cardiovascular Science. JKG is an Established Investigator of the American Heart Association.     

Guanylyl cyclase (GC)-A and GC-B activities in ventricles and cardiomyocytes from failed and non-failed human hearts: GC-A is inactive in the failed cardiomyocyte

Cardiomyocytes release atrial natriuretic peptide (ANP) and B-type natriuretic peptide to stimulate processes that compensate for the failing heart by activating guanylyl cyclase (GC)-A. C-type natriuretic peptide is also elevated in the failing heart and inhibits cardiac remodeling by activating the homologous receptor, GC-B. We previously reported that GC-A is the most active membrane GC in normal mouse ventricles while GC-B is the most active membrane GC in failing ventricles due to increased GC-B and decreased GC-A activities. Here, we examined ANP and CNP-specific GC activity in membranes obtained from non-failing and failing human left ventricles and in membranes from matched cardiomyocyte-enriched pellet preparations. Similar to our findings in the murine study, we found that CNP-dependent GC activity was about half of the ANP-dependent GC activity in the non-failing ventricular and was increased in the failing ventricle. ANP and CNP increased GC activity 9- and 5-fold in non-failing ventricles, respectively. In contrast to the mouse study, in failing human ventricles, ANP-dependent activity was unchanged compared to non-failing values whereas CNP-dependent activity increased 35% (p=0.005). Compared with ventricular membranes, basal GC activity was reduced an order of magnitude in membranes derived from myocyte-enriched pellets from non-failing ventricles. ANP increased GC activity 2.4-fold but CNP only increased GC activity 1.3-fold. In contrast, neither ANP nor CNP increased GC activity in equivalent preparations from failing ventricles. We conclude that: 1) GC-B activity is increased in non-myocytes from failing human ventricles, possibly as a result of increased fibrosis, 2) human ventricular cardiomyocytes express low levels of GC-A and much lower levels or possibly no GC-B, and 3) GC-A in cardiomyocytes from failing human hearts is refractory to ANP stimulation.     

Ultrasonic backscatter and collagen in normal ventricular myocardium

Integrated ultrasonic backscatter has been related to collagen deposition in fibrotic myocardium. The purpose of our study was to measure the integrated ultrasonic backscatter in the right and left ventricles of 10 normal freshly excised canine hearts and five normal formalin-fixed human hearts. A 2.25 MHz, 50% fractional bandwidth transducer was positioned at the transducer focal distance from the epicardium. The radio frequency backscatter signal, excluding specular reflections, was digitized, squared, and integrated to yield the integrated ultrasonic backscatter (in decibels down from a 100% reflector). The segment of myocardium corresponding to the integrated ultrasonic backscatter sample volume was excised and assayed for hydroxyproline, a marker for collagen. A second purpose of our study was to evaluate the influence of fixation with formalin on the backscatter. Regional integrated ultrasonic backscatter was therefore measured in 10 freshly excised canine left ventricles, which were fixed in 10% formalin for 2 weeks. Integrated ultrasonic backscatter measurements were then repeated. In freshly excised canine hearts, the integrated ultrasonic backscatter from right ventricle was higher than that from left ventricle (-60.4 +/- 1.6 [SEM] vs -66.9 +/- 1.0 dB; p less than .001). The collagen content of right ventricle was also higher than that of left ventricle (4.40 +/- 0.26 [SEM] vs 3.58 +/- 0.13 micrograms/mg dry weight; p less than .005). Similar results were obtained in human hearts. There were no correlations between integrated ultrasonic backscatter and collagen content (r = .28 and .32 for dogs and humans, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced expression and activity of xanthine oxidoreductase in the failing heart

The molecular basis for heart failure is unknown, but oxidative stress is associated with the pathogenesis of the disease. We tested the hypothesis that the activity of xanthine oxidoreductase (XOR), a free-radical generating enzyme, increases in hypertrophied and failing heart. We studied XOR in two rat models: (1) The monocrotaline-induced right ventricular hypertrophy and failure model; (2) coronary artery ligation induced heart failure, with left ventricular failure and compensatory right ventricular hypertrophy at different stages at 3 and 8 weeks post-infarction, respectively. XOR activity was measured at 30 degrees C and the reaction products were analysed by HPLC. In both models XOR activity in hypertrophic and control ventricles was similar. In the monocrotaline model, the hearts showed enhanced XOR activity in the failing right ventricle (65+/-5 mU/g w/w), as compared to that in the unaffected left ventricle (47+/-3 mU/g P<0.05, n=6-7). In the coronary ligation model, XOR activities did not differ at 3 and 8 weeks. In the infarcted left ventricle, XOR activity increased from 29.4+/-1.4 mU/g (n=6) in sham-operated rats, to 48+/-3 and 80+/-6 mU/g (n=8 P<0.05 v sham) in the viable and infarcted parts of failing rat hearts, respectively. With affinity-purified polyclonal antibody, XOR was localized in CD68+ inflammatory cells of which the number increased more in the failing than in sham-operated hearts. Our results show that the expression of functional XOR is elevated in failing but not in hypertrophic ventricles, suggesting its potential role in the transition from cardiac hypertrophy into failure.     

S-adenosylhomocysteine hydrolase activity in human myocardium.

OBJECTIVE: Measurement of S-adenosylhomocysteine (SAH) accumulation in the heart reflects the concentration of free cytosolic adenosine and is thus a sensitive indicator of regional myocardial ischaemia. To evaluate the possibility of applying this method in combination with 11C-SAH positron emission tomography (PET) to patients with ischaemic heart disease the activity of SAH hydrolase in human heart muscle and its regional distribution were studied. METHODS: Myocardium from patients with dilated cardiomyopathy (n = 4), hypertrophic obstructive cardiomyopathy (HOCM, n = 6), and mitral stenosis (n = 3) was analysed. Additional studies were performed in myocardium, isolated cardiomyocytes, and endothelial cells from dog and guinea pig hearts. Enzyme activity in synthetic and hydrolytic direction including kinetic data (Vmax, KM values, pH dependency) was measured in the cytosolic fraction of myocardial tissue and cell extracts, using high performance liquid chromatography and photometric methods, respectively. RESULTS: Rates of SAH synthesis (Vmax) in the left ventricle in dilated cardiomyopathy, mitral stenosis, and HOCM were 0.8 (SEM 0.1), 1.0(0.2), and 1.7(0.1) nmol.min-1.mg-1 protein respectively. KM values for DL-homocysteine, adenosine, and SAH in HOCM were 187, 2.2, and 3.4 microM, respectively. Enzyme activity was homogeneously distributed among right and left atria, right and left ventricles, and septum. Additional studies in homogenated muscle and isolated cardiomyocytes of guinea pig and canine hearts showed activities similar to man. CONCLUSIONS: (1) SAH hydrolase activity in the human heart is quantitatively comparable to that found in other mammals but certain myocardial diseases may go along with changes in SAH hydrolase activity; (2) the kinetic properties, absolute amounts, and homogeneous distribution of the enzyme may permit the non-invasive determination of free adenosine in the human heart by PET.