心衰情况下心室肌细胞透壁电生理特异性的仿真研究

仿真研究心衰情况下心室肌细胞离子通道的变异对细胞电生理和透壁特异性的影响机制,以及心力衰竭下心室透壁复极化的改变与心律失常之间的关系.基于反映正常和衰竭人体心肌细胞离子通道透壁特异性的实验数据,建立离子通道水平的心肌细胞电生理数学模型,模拟仿真研究心衰情况下心室肌心外膜、中层、心内膜细胞电生理透壁特异性的变化.结果:仿真研究发现心衰导致透壁心室肌细胞电生理重建,透壁细胞的动作电位持续时间都有明显的延长,改变了动作电位的透壁特异性,进而减小了动作电位的透壁梯度,同时衰竭心脏中快步率时动作电位的比率依赖性会增加.模型研究认为这可能与心衰细胞中离子电流ICaL和Iks的透壁特异性的电生理重构有关.所发展的细胞模型不仅可以辅助细胞电生理实验分析研究,同时也是今后仿真研究心衰情况下心肌细胞兴奋-收缩耦联力学特性的重要基础.     

联合成像系统研究心肌细胞微区力学及细胞内钙离子变化

目的 使用联合成像系统在亚细胞水平研究心肌细胞微区力学性质及细胞内钙离子的快速变化,并探讨两者之间的关系.方法 应用原子力显微镜(AFM)和激光扫描共聚焦显微镜(LSCM)组合成的联合成像系统对急性分离的大鼠单个心室肌细胞微区力进行检测,同时测定细胞内钙离子浓度的变化,分析细胞微区力和细胞内钙离子浓度变化两者之间的相关性.结果 观察到心肌细胞内钙离子浓度的快速变化引起了钙波的传递.通过AFM微悬臂的偏转可以计算出细胞微区力,细胞微区力的大小与细胞内钙离子浓度呈正相关(r=0.701,P=0.003).结论 联合成像系统可以同步研究心肌细胞微区力学及细胞内分子快速变化,进而在亚细胞水平研究心脏疾病的发病机制.     

基于电生理-力学复合心脏模型的右心室三维运动分析

在原有电生理心脏模型基础上,我们采用有限元方法和复合材料理论建立了一个电生理-力学复合双心室模型,并基于该模型对右心室心壁在收缩期的三维运动进行仿真分析。模型建立过程中,考虑了心肌的纤维旋向、心电兴奋产生的心肌收缩力。心室壁位移、最小主应变(E 3)等用来表征心室的运动。仿真结果与加标记磁共振成像的实验结果基本一致。另外,仿真结果还表明,虽然心基部位移最大,但最大收缩力却发生在心尖部,这个结果用一般的动物实验或人体实验是难以得到的。因此,电生理-力学复合双心室模型对于今后进一步评价心脏功能具有重要意义。     

Changes of calcium release in myocardial sarcoplasmic reticulum during heart failure

目的:探讨慢性心力衰竭时心肌细胞肌浆网内钙离子释放的变化.方法:采用冠状动脉左前降支结扎法制备大鼠慢性心衰模型,酶解法分离成年大鼠心室肌细胞.用全细胞模式膜片钳技术和共聚焦显微镜钙成像技术同步实时记录心肌细胞的L-型钙电流以及胞质内的钙瞬变.结果:心衰组的钙诱导钙瞬变幅度小于对照组,并且心衰组的咖啡因诱导钙瞬变幅度也低于对照组.结论:心力衰竭的发生与心肌细胞内钙离子释放变化有着十分密切的关系.     

容量负荷与心室结构重建的研究进展

容量负荷即心脏的前负荷,是指心肌收缩之前遇到的负荷.在生理范围,适度前负荷增加,则心肌收缩力增强,每搏量增加;但若前负荷过度增加,则使心肌收缩力减弱,每搏量减少;前负荷增加持续存在,则心室腔代偿性扩张,即离心性心脏肥大,最终导致心力衰竭.有关心力衰竭的研究已有300余年的历史,但直到20世纪90年代才认识到心室重建是心衰发生发展过程中的共同特征.有关容量过负荷导致心室结构重建及心力衰竭的分子生物学机制目前还不是很清楚.     

TNFα对单个大鼠心室肌细胞L型钙电流的影响

目的探讨TNFα对心肌细胞L型钙电流的影响。方法分离大鼠单个心室肌细胞,用全细胞膜片钳方法检测TNFα对单个心室肌细胞L型钙电流的影响。结果 TNFα使单个大鼠心室肌细胞L型钙电流持续增强,促进细胞外钙离子持续向细胞内流动。结论 TNFα与感染性休克心脏收缩性改变相关。     

腹主动脉缩窄大鼠心力衰竭时心室肌细胞电生理失稳态及Ito的变化

目的探讨腹主动脉缩窄大鼠心力衰竭时电生理失稳态和心室肌细胞Ito的变化。方法采用腹主动脉缩窄法建立心力衰竭大鼠模型,术后16周检测心脏彩超和左室舒张末压,左室舒张末压≥15mmHg大鼠归入心力衰竭组,另设假结扎对照组。在术后16周测定两纽大鼠电生理指标。采用酶解法获得单个大鼠。室肌细胞并以标准全细胞膜片钳技术记录Ito结果心力衰竭大鼠与假结扎对照组大鼠比较左室舒张末压．心率和动脉血压均明显增加,校正QT间期和心室有效不应期则明显延长,而心室有效不应期的延长与大鼠左室舒张末压呈正相关。心力衰竭组细胞心室肌细胞膜电容与假结扎对照组相比明显增大。而该组心室肌细胞的Ito峰值及密度较假结扎对照组明显降低。两组大鼠。室肌细胞Ito激活、失活和复活动力学特征均无显著差异。结论腹主动脉缩窄大鼠心力衰竭时存在明显电生理失稳态变化,可能与心室肌细胞膜Ito的降低有关。     

心脏收缩力调节对慢性心力衰竭兔心室肌电重构的影响

目的:观察心脏收缩力调节(cardiac contractility modulation, CCM)对慢性心力衰竭兔心室肌电重构的影响并探讨其可能的机制。方法:30只新西兰大白兔随机分为3组:假手术组、心衰组(采用升主动脉根部套扎法建立兔慢性心力衰竭模型)和心衰+CCM刺激组(模型制作成功后给予4周的CCM治疗)。电生理记录仪测定QTc和心室有效不应期(ventricular effective refrective period, VERP);采用RT-qPCR和Western blot检测心室肌Kv1.4、Kv4.3和缝隙连接蛋白43 (connexin 43, Cx43)的mRNA和蛋白的表达水平。结果:(1)实验第12周末,心衰兔QTc显著延长(P<0.05);实验第16周末,与心衰组相比,心衰+CCM组经4周CCM刺激后QTc显著缩短(P<0.05)。实验第16周末,与假手术组相比,心衰组、心衰+CCM组VERP显著延长(P<0.05);与心衰组相比,CCM可缩短心衰模型兔的VERP(P<0.05)。(2)与假手术组相比,心衰组心肌组织中Kv1.4、Kv4.3和Cx43的mRNA水平显著下降(P<0.05)。与心衰组相比,CCM显著上调心衰兔心肌组织中Kv1.4、Kv4.3和Cx43的mRNA水平(P<0.05)。(3)与假手术组相比,心衰组心肌组织中Kv1.4、Kv4.3和Cx43的蛋白表达显著下降(P<0.05)。与心衰组相比,CCM可上调心衰兔心肌组织中Kv1.4、Kv4.3和Cx43的蛋白表达水平(P<0.05)。结论:心脏收缩力调节可改善慢性心力衰竭兔心室肌电重构,其潜在机制可能与Kv1.4、Kv4.3和Cx43的mRNA和蛋白表达上调有关。     

钙离子运转障碍与心力衰竭

钙离子运转障碍与心力衰竭南京医学院动脉粥样硬化研究中心（南京２１００２９）朱宇，蔡海江心力衰竭是临床上较为常见的一种病理过程。由于钙离子在心肌细胞内的正常运转对于心脏的兴奋一收缩偶联起十分重要的作用，因而人们对于钙离子运转障碍在心力衰竭发病机制中的作...     

NCA对受磷蛋白敲除小鼠心肌兴奋-收缩偶联的作用

 目的: 硝酰基(HNO)在轻微增加细胞内钙的基础上可以显著增加心肌肌丝对钙离子的反应性。本研究中,我们应用崭新的HNO供体乙酸1-亚硝基环己酯(NCA)来观察HNO对受磷蛋白敲除(PLB-KO)小鼠心室梳状肌的作用。方法: 小鼠右心室的完整梳状肌被连接在张力换能器与刺激电极之间,肌小节长度设定在2.2~2.3μm之间,K-H液表面灌流后,Fura-2经玻璃微电极负载进行离子透入法检测[Ca²⁺]_i,同时测定心肌收缩张力的变化。结果: PLB-KO小鼠心室梳状肌比野生型(WT)小鼠具有更高的钙瞬变及收缩力,同时展示负性收缩力-收缩频率相关性(FFR)。NCA(2.5μmol/L)在不同浓度细胞外钙([Ca²⁺]_o)条件下增加PLB-KO及WT小鼠心肌收缩力,但并不影响PLB-KO小鼠的负性FFR。稳态条件下2组小鼠去肌膜梳状肌的收缩力-钙离子相关性无显著性差异,NCA则增加去肌膜梳状肌的钙离子的反应性。结论: NCA提供的HNO通过增加PLB-KO及WT小鼠心肌肌丝对钙离子的反应性而增强心肌收缩力;心肌细胞内钙瞬变的增加伴随收缩力的增强表明HNO可改善钙离子活性,进一步证实HNO作为正性肌力药物的作用效果。     

Myocytes positive for in situ markers for DNA breaks in human hearts which are hypertrophic,but neither failed nor dilated: a manifestation of cardiac hypertrophy rather than failure

The significance of DNA breaks reported in failing hearts is controversial, although they may suggest myocyte apoptosis and may thus be responsible for the progression of heart failure. This study attempted to check the validity of the in situ markers for DNA breaks for detecting myocyte death and to evaluate separately two factors, failure or hypertrophy, crucial for DNA breaks in pathological human hearts. In the autopsy study, myocytes showed positivity for in situ nick end-labelling (TUNEL) and of Taq and Pfu polymerase-based in situ ligation assays not only in dilated cardiomyopathy (DCM, n = 9) with failure, but also in hypertrophic cardiomyopathy (HCM, n = 8) and hypertensive heart disease (HHD, n = 4) without failure. There was a significant correlation between each in situ marker and heart weight. The incidence of TUNEL-positive myocytes always exceeded that seen in in situ ligation assays. In addition, there were significant correlations between the in situ markers and the expression of the proliferating cell nuclear antigen (PCNA) and of the spliceosome component of 35 kD (SC-35). Similarly, in the left ventricular biopsy study using 23 DCM, 21 HCM, 11 HHD, and 13 non-hypertrophic hearts, the incidence of the in situ markers showed significant correlations with the left ventricular mass index and myocyte size, but not with cardiac function and dilatation. Positivity of myocytes for in situ markers for DNA breaks, such as TUNEL and in situ ligation assays, may be an epiphenomenon accompanying cardiac hypertrophy, but not myocyte death in pathological human hearts.     

Identification and distribution of atrial natriuretic polypeptide in ventricular myocardium of humans with myocardial infarction

The expression of atrial natriuretic polypeptide (ANP) in the ventricles of human hearts with myocardial infarction (MI) was studied immunohistochemically. Immunoreactive myocytes were identified in the ventricular tissues of all of 16 hearts with old MI (both with and without heart failure) and in all five hearts with subacute MI, but not in any of the eight hearts without MI nor in the five with acute MI. In the nonfailing hearts with MI, ANP positive myocytes surrounded the areas of infarction, and were also seen in the subendocardium of the infarcted segment. In the failing hearts with MI, ANP expression was noted in the whole ventricular subendocardial region, in addition to the border of infarcts. The sites of ANP expression corresponded well to those of marked stress attributable to tissue shrinkage or fibrosis due to MI, haemodynamic overload, or both. It thus appears that ANP expression is augmented in human hearts with MI regardless of the presence or absence of heart failure, and it is suggested that regional mechanical stress on the ventricular myocardium, as well as haemodynamic overload, may be very closely associated with ventricular ANP expression.     

Effects of Ca2+-Sensitizers in Permeabilized Cardiac Myocytes from Donor and End-Stage Failing Human Hearts

During heart failure, alterations occur in contractile protein expression and phosphorylation, which may influence the effects of Ca2+ -sensitizers. To quantify the magnitude of these effects, isometric force was studied in mechanically isolated Triton-skinned myocytes from end-stage failing and non-failing donor hearts under control conditions (pH 7.2; no added inorganic phosphate (Pi)) and under mimicked ischemic conditions (pH 6.5; 10 mM Pi). Two different Ca2+ -sensitizers were used: EMD 53998 (10 microM), which exerts its influence through the actin-myosin interaction, and OR-1896 (10 microM) (the active metabolite of levosimendan), which affects the Ca2+ -sensory function of the thin filaments. The maximal force (Po) measured at saturating Ca2+ concentration and the resting force (Prest) determined in the virtual absence of Ca2+ (pCa 9) did not differ between the failing and non-failing myocytes, but the Ca2+ concentration required to induce the half-maximal force under control conditions was significantly lower in the failing than in the non-failing myocytes (DeltapCa50=0.15). This difference in Ca2+ -sensitivity, however, was abolished during mimicked ischemia. EMD 53998 increased Po and Prest by approximately 15% of Po and greatly enhanced the Ca2+ -sensitivity (DeltapCa50 > 0.25) of force production. OR-1896 did not affect Po and Prest, and provoked a small, but significant Ca2+ -sensitization (DeltapCa50 approximately 0.1). All of these effects were comparable in the donor and failing myocytes, but, in contrast with OR-1896, EMD 53998 considerably diminished the difference in the Ca2+ -sensitivities between the failing and non-failing myocytes. The action of Ca2+ -sensitizers under mimicked ischemic conditions was impaired to a similar degree in the donor and the failing myocytes. Our results indicate that the Ca2+ -activation of the myofibrillar system is altered in end-stage human heart failure. This modulates the effects of Ca2+ -sensitizers both under control and under mimicked ischemic conditions.     

Increased contribution of alpha 1- vs. beta-adrenoceptor-mediated inotropic response in rats with congestive heart failure

AIM: In failing myocardium the mechanical response to beta-adrenoceptor stimulation is attenuated. Alternative signalling systems might provide inotropic support when the beta-adrenoceptor system is dysfunctioning. Accordingly, the inotropic responses to alpha 1- and beta-adrenoceptor stimulation by the endogenous adrenoceptor agonist noradrenaline in non-failing and failing rat hearts were compared. METHODS: Chronic heart failure was induced in male Wistar rats by coronary artery ligation. Corresponding sham groups were prepared. After 6 weeks, papillary muscles from non-failing and failing hearts were isolated. Receptor binding studies were performed in the corresponding myocardium. The alpha 1-adrenoceptor-mediated inotropic response was not changed while the beta-adrenoceptor-mediated response was substantially reduced in failing compared with non-failing myocardium. RESULTS: No change in potency for the agonists was observed at the alpha 1-adrenoceptors, while an increased potency for the agonists at the beta-adrenoceptors was found during heart failure. The lusitropic response to beta-adrenoceptor stimulation was intact during heart failure. No over all change in affinity or number of either adrenoceptor type was observed in receptor binding studies. The alpha 1-adrenoceptor-mediated inotropic response became dominating compared with the beta-adrenoceptor-mediated one in failing rat myocardium in contrast to the dominating role of the latter in non-failing myocardium. The attenuation of the beta-adrenoceptor-mediated inotropic response in rat failing myocardium was not because of a reduced number of receptors. CONCLUSION: Increasing contractility through stimulation of alpha 1-adrenoceptors in situ by the endogenous agonist may be an alternative way of inotropic support during heart failure and even more so during beta-adrenoceptor blockade.     

植物雌激素α-ZAL和17βE_2对大鼠单个心室肌细胞缩舒功能和胞内钙瞬时变化的影响

目的:比较植物雌激素α-ZAL和17βE2对大鼠心肌细胞缩舒和胞内钙瞬变的影响。方法:从成年雌性大鼠心脏分离单个心肌细胞,应用美国Ionoptix视频跟踪计算机图像分析系统在0.5Hz的电刺激下测定离体单个心肌细胞的收缩参数,其中包括最大收缩幅度(PS)、最大收缩幅度时间(TPS)、90%舒张幅度时间(TR90)和最大收缩速率(+dL/dttmax)及最大舒张速率(-dL/dttmax);同时用Fura2/AM钙荧光指示剂测定胞内钙浓度的变化。结果:10-9~10-5mol/L17βE2能使PS产生浓度依赖性的增加,最大增加35%,高浓度的17βE2对TPS有显著影响(P<0.05),能够使ΔFFI最大增加25%;而10-9~10-5mol/Lα-ZAL无论是心肌细胞缩舒功能还是胞内钙含量均无明显变化。且α-ZAL和17βE2对心室肌细胞静息状态下胞内钙浓度和胞内钙的荧光衰减率均无显著影响。结论:α-ZAL对心室肌细胞的作用与17βE2有很大不同,17βE2对心肌细胞缩舒有直接刺激作用,增强心肌收缩可能是通过胞内钙释放与胞外钙内流所介导;而α-ZAL无这种作用,它可能是通过抑制胞外钙内流和其抗氧化反应达到保护心室肌细胞的作用。     

Protein phosphatase 2A affects myofilament contractility in non-failing but not in failing human myocardium

Protein phosphatase (PP) type 2A is a multifunctional serine/threonine phosphatase that is involved in cardiac excitation-contraction coupling. The PP2A core enzyme is a dimer, consisting of a catalytic C and a scaffolding A subunit, which is targeted to several cardiac proteins by a regulatory B subunit. At present, it is controversial whether PP2A and its subunits play a critical role in end-stage human heart failure. Here we report that the application of purified PP2AC significantly increased the Ca2+-sensitivity (ΔpCa50=0.05±0.01) of the contractile apparatus in isolated skinned myocytes of non-failing (NF) hearts. A higher phosphorylation of troponin I (cTnI) was found at protein kinase A sites (Ser23/24) in NF compared to failing myocardium. The basal Ca2+-responsiveness of myofilaments was enhanced in myocytes of ischemic (ICM, ΔpCa50=0.10±0.03) and dilated (DCM, ΔpCa50=0.06±0.04) cardiomyopathy compared to NF. However, in contrast to NF myocytes the treatment with PP2AC did not shift force-pCa relationships in failing myocytes. The higher basal Ca2+-sensitivity in failing myocytes coincided with a reduced protein expression of PP2AC in left ventricular tissue from patients suffering from ICM and DCM (by 50 and 56% compared to NF, respectively). However, PP2A activity was unchanged in failing hearts despite an increase of both total PP and PP1 activity. The expression of PP2AB56α was also decreased by 51 and 62% in ICM and DCM compared to NF, respectively. The phosphorylation of cTnI at Ser23/24 was reduced by 66 and 49% in ICM and DCM compared to NF hearts, respectively. Our results demonstrate that PP2A increases myofilament Ca2+-sensitivity in NF human hearts, most likely via cTnI dephosphorylation. This effect is not present in failing hearts, probably due to the lower baseline cTnI phosphorylation in failing compared to non-failing hearts.     

Increased contribution of α1‐ vs. β‐adrenoceptor‐mediated inotropic response in rats with congestive heart failure

Aim: In failing myocardium the mechanical response to β‐adrenoceptor stimulation is attenuated. Alternative signalling systems might provide inotropic support when the β‐adrenoceptor system is dysfunctioning. Accordingly, the inotropic responses to α₁‐ and β‐adrenoceptor stimulation by the endogenous adrenoceptor agonist noradrenaline in non‐failing and failing rat hearts were compared. Methods: Chronic heart failure was induced in male Wistar rats by coronary artery ligation. Corresponding sham groups were prepared. After 6 weeks, papillary muscles from non‐failing and failing hearts were isolated. Receptor binding studies were performed in the corresponding myocardium. The α₁‐adrenoceptor‐mediated inotropic response was not changed while the β‐adrenoceptor‐mediated response was substantially reduced in failing compared with non‐failing myocardium. Results: No change in potency for the agonists was observed at the α₁‐adrenoceptors, while an increased potency for the agonists at the β‐adrenoceptors was found during heart failure. The lusitropic response to β‐adrenoceptor stimulation was intact during heart failure. No over all change in affinity or number of either adrenoceptor type was observed in receptor binding studies. The α₁‐adrenoceptor‐mediated inotropic response became dominating compared with the β‐adrenoceptor‐mediated one in failing rat myocardium in contrast to the dominating role of the latter in non‐failing myocardium. The attenuation of the β‐adrenoceptor‐mediated inotropic response in rat failing myocardium was not because of a reduced number of receptors. Conclusion: Increasing contractility through stimulation of α₁‐adrenoceptors in situ by the endogenous agonist may be an alternative way of inotropic support during heart failure and even more so during β‐adrenoceptor blockade.     

Mg^2＋对离体灌流内皮素的大鼠心脏功能的影响

本实验在离体大鼠心脏灌流模型上,观察到10~(-9)mol/L内皮素灌流引起心肌挛缩,心功能降低,冠脉灌流量减少,以及心肌Ca~(2 )聚积,Mg~(2 )丧失等表现。灌流液含低镁(0.12mmol/L)时内皮素对心脏的上述作用显著增强,而在含高镁(4.8mmol/L)时明显减弱。Mg~(2 )影响内皮素对心脏作用的机制可能与其抑制心肌Ca~(2 )内流有关。实验结果提示避免缺镁,适当补充Mg~(2 )对于防治伴有循环内皮素水平增加的疾病或病理过程中的心脏损伤可能具有临床意义。     

MMP-7、MMP-10和TIMP-4在心力衰竭心室重构中的表达

目的:应用细胞因子抗体芯片技术筛选与心力衰竭心室重构关系密切的基质蛋白酶。方法:从本院的心脏病组织库中挑选6例病理诊断明确和各方面资料比较齐全的致心律失常性右室心肌病引起心力衰竭的心脏病标本(来源于心脏移植的受体),与年龄、性别和种族等因素相匹配的正常对照心脏组织(来源于心脏移植的供体)进行细胞因子抗体芯片分析,筛选在致心律失常性右室心肌病引起的心力衰竭中差异表达的基质蛋白酶,并应用酶联免疫分析和免疫组织化学的方法加以验证。结果:在所筛选的17种基质金属蛋白酶中,只有MMP-7和MMP-10在致心律失常性右室心肌病引起心力衰竭中高表达,而在4种基质金属蛋白酶内源性组织抑制剂中,只有TIMP-4 低表达。经酶联免疫分析和免疫组织化学的方法证实,不仅在致心律失常性右室心肌病引起心力衰竭的心肌,在缺血性心肌病和扩张性心肌病引起的心力衰竭心肌中也发现MMP-7和MMP-10 的高表达及TIMP-4的低表达。结论:心肌组织中MMP-7和MMP-10的高表达及TIMP-4的低表达在不同心肌病引起的心力衰竭心室重构分子机制中可能发挥重要的作用。     

Extracellular matrix profiles in the progression to heart failure. European Young Physiologists Symposium Keynote Lecture-Bratislava 2007

The myocardial extracellular matrix (ECM), which preserves the geometry and integrity of the myocardium, is a dynamic structure whose component proteins are maintained by a finely controlled homeostatic balance between deposition and degradation. One of the key targets in cardiology is the elucidation of the molecular mechanisms which mediate pathological remodelling of this matrix causing the transition from compensatory hypertrophy to congestive decompensated heart failure. In response to injury or increased workload, cardiac remodelling including myocyte hypertrophy, develops as the heart attempts to compensate for increased wall stresses. Persistence of these stresses over extended time periods leads to disruption of ECM homeostasis resulting in irreversible maladaptive cardiac remodelling, ventricular dilatation and finally heart failure. ECM remodelling is regulated by the matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs). Clinical studies and experimental models of cardiac disease states have reported alterations in the balance between the MMPs and TIMPs in the failing heart and crucially at intermediate time points in the progression to failure. This article reviews the recent clinical, genetic and experimental approaches employed to compare ECM, MMP and TIMP profiles in healthy, compensated and failing hearts and identifies common themes in the perturbation of ECM homeostasis in the transition to heart failure.