脱氢紫堇碱在心肌细胞钙调控中的作用

背景已有动物实验表明脱氢紫堇碱(DHC)可能通过阻止心肌细胞内钙超载,提高心肌细胞的自我保护作用,但还没有相关实验阐明DHC调控心肌细胞保护的分子机制.目的 探讨DHC对心肌细胞内Ca2+超负荷及Ca2+调控相关蛋白的表达变化.方法 采用逆转录聚合酶链反应(RT-PCR)方法 对心肌细胞Ca2+调控相关蛋白,包括钙三磷...     

心肌细胞内钙释放对心肌细胞PTEN表达的影响

目的探讨雷尼丁（Ryanodine,RY）介导的心肌细胞内钙释放对PTEN mRNA和蛋白表达影响。方法新生Wistar乳鼠心肌细胞原代培养。用不同浓度的RY（0.0110μmol/L）促进心肌细胞内钙释放,特异的钙敏感指示剂Fura-2/AM测定心肌细胞内钙浓度（[Ca2+]i）。逆转录聚合酶链式反应（RT-PCR）、Western blot分别检测PTEN mRNA和蛋白表达。结果RY在0.011μmol/L浓度范围内,能浓度依赖性地促进心肌细胞内钙释放,RY在10μmol/L时,心肌细胞内钙浓度低于RY在1μmol/L时的效应,但仍高于对照组;RY在0.011μmol/L浓度范围内能浓度依赖性促进心肌细胞PTEN mRNA和蛋白表达,RY在10μmol/时,心肌细胞PTEN mRNA和蛋白表达低于1μmol/L的效应,但仍高于对照组。结论RY介导的心肌细胞内钙释放可影响心肌细胞PTEN基因表达,其表达在RY处于0.011μmol/L范围内具有浓度依赖性。     

钙调神经磷酸酶信号通路与心肌细胞肥大

目的　探讨不同来源的细胞内Ca2 ([Ca2 ]i)在钙调神经磷酸酶 (CaN) 活化T细胞核因子 3 (NFAT3 )介导的心肌肥大中的作用。方法　分别用血管紧张素Ⅱ (AngⅡ )或雷尼丁刺激培养的大鼠心肌细胞外Ca2 跨膜内流或细胞内Ca2 释放 ,检测CaN、NFAT3、锌指转录因子 (GATA4)蛋白量、NFAT3定位以及氚 亮氨酸 (3H Leu)掺入量 ,环孢素A作为CaN特异抑制剂。结果　AngⅡ、雷尼丁刺激 1、3d ,心肌细胞CaN、NFAT3、GATA4蛋白表达及3H Leu掺入量较对照组明显增高(P值 <0 0 5或 <0 0 1)。AngⅡ和雷尼丁刺激第 1天 ,心肌细胞NFAT3表达由胞质转入胞核表达为主。环孢素A可抑制上述作用 ,与刺激组相比差异有显著性 (P <0 0 5或 <0 0 1)。结论　刺激心肌细胞Ca2 内流及Ca2 释放 ,均可激活CaN NFAT3信号通路。CaN NFAT3信号通路的激活与[Ca2 ]i增加有关 ,而与 [Ca2 ]i的来源无关。环孢素A能够抑制AngⅡ和雷尼丁介导的CaN NFAT 3 GATA 4表达的增加和蛋白质合成     

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。     

牛磺酸对大鼠培养心肌细胞钙运转功能的作用

以大鼠培养心肌细胞的脂质过氧化损伤模型，采用＾４５Ｃａ示踪技术，直接进行心肌肌浆网，线粒体钙转运动功能的测定，同时观察牛磺酸对心肌过氧化损伤的保护作用，结果表明，牛磺酸能显著改善心肌肌浆网，线粒体钙转运功能，对心肌细胞具有保护作用。     

压力超负荷性心肌肥厚大鼠心肌细胞内钙激活蛋白酶活性分布的研究

目的研究大鼠心肌肥厚时,钙激活蛋白酶(Calpain)在心肌细胞胞浆和细胞核的活性分布,并探讨细胞核钙摄取的改变,以进一步阐明心肌肥厚的发生机制.方法将100只健康雄性Wistar大鼠(150～200g)随机分为对照组(n＝50)和腹主动脉缩窄组(n＝50),制备腹主动脉缩窄大鼠心肌肥厚模型、差速离心和密度梯度离心提纯心肌细胞核,荧光法测酶活性,以45Ca2+测定细胞核摄取能力.结果与对照组相比,腹主动脉缩窄组大鼠左心室重量指数增加,伴有明显的血流动力学异常,有非常显著性差异(P＜0.01);其心肌细胞核Calpain活性亦增加40.78%,有显著性意义(P＜0.05);细胞浆Calpain活性下降21.71%,有非常显著性意义(P＜0.05).对照组心肌Calpain在细胞浆的活性显著高于在细胞核的活性,有非常显著性意义(P＜0.01),而腹主动脉缩窄组心肌Calpain在细胞核的活性与在细胞浆的活性无显著差异;细胞核45Ca2+摄入量也显著增加(较对照组高28%～97%,P＜0.01).结论肥厚心肌Calpain由细胞浆向细胞核转位、细胞核内Calpain活性增加,细胞核钙摄取能力增强,提示压力超负荷时Ca2+与Calpain调节的细胞核反应水平加强,可能在介导心肌肥厚的细胞核功能调控中起重要作用.     

虎杖苷对严重烫伤大鼠心肌细胞钙信号调控的作用

目的：观察虎杖苷（polydatin,PD）对烫伤大鼠心功能损害的钙信号调控,并探讨其对烫伤大鼠心肌细胞保护作用的机制。方法：28只成年SD大鼠随机分为4组,即假手术组、烫伤组、PD组及烫伤＋PD组,每组7只大鼠（n=7）。 各实验组使用Power lab系统进行实时心功能监测。手术后5 min通过静脉给予PD（10 mg/kg）,12 h后分离心室肌细胞,通过激光共聚焦显微镜观测PD对烫伤大鼠心室肌细胞钙火花发放频率、钙库水平、钙瞬变的作用。结果：与烫伤组比较,烫伤＋PD组大鼠左室收缩压、左心室内压力上升的最大速率（±dP/dtmax）均显著增加、钙瞬变和心肌收缩力明显增强、使烫伤引起的钙库容量下降基本恢复到正常水平,烫伤引起的高频钙火花发放显著抑制（均P〈0.01）,钙火花时程的增加也明显抑制（P〈0.05）。结论： PD可以显著抑制烫伤引起的心肌细胞肌浆网钙漏流,恢复肌浆网钙库容量水平,增强收缩期钙瞬变和心肌收缩力,从而改善烫伤引起的心功能障碍。     

甲状腺激素对心肌细胞钙调控蛋白表达的影响

从基因及分子水平上阐明甲状腺激素对心肌舒缩功能的影响作用及其机制,将为心力衰竭的治疗提供新的方法和奠定理论基础。本文就甲状腺激素对心钙调控蛋白表达的调控以及对心功能的影响作一综述。     

PTEN抑制钙/钙调神经磷酸酶信号通路、负性调控血管紧张素Ⅱ所致心肌细胞肥大

目的 观察PTEN过度表达对血管紧张素Ⅱ（AngⅡ）刺激所致钙／钙调神经磷酸酶（Ca^2＋／CaN）信号通路激活的影响,探讨PTEN负性调控心肌肥厚的作用机制。方法通过携带野生型PTEN基因的腺病毒（Ad-PTEN）感染构建过度表达PTEN的原代培养心肌细胞模型,用AngⅡ作为促心肌肥厚刺激剂,Fura-2／AM比率荧光成像系统检测细胞内Ca^2＋浓度（[Ca^2＋]i）,逆转录聚合酶链反应检测受感染细胞内心房利钠因子（ANF）、β-肌球蛋白重链（B-MHC）与CaNAβ的mRNA表达,Western blot检测CaNAβ的蛋白表达,同时测定CaN活性。结果 Ad-PTEN感染后,心肌细胞内过度表达PTEN的mRNA和蛋白。PTEN的过度表达能够明显抑制AngⅡ刺激所致的心肌细胞肥大标志基因表达。AngⅡ刺激使[Ca^2＋]i、CaNAβ mRNA与蛋白表达以及CaN活性明显增高,PTEN过度表达能够明显抑制AngⅡ引起的[Ca^2＋]i、CaNAβ mRNA与蛋白表达以及CaN活性增高。结论 PTEN过度表达可能通过抑制Ca^2＋／CaN信号通路,负性调控AngⅡ刺激所致的心肌细胞肥大。     

糖尿病人细胞内高钙及其对血管病变的影响

糖尿病可以引起许多组织器官的细胞内高钙，其形成原因可能与细胞膜上的钙通道，钙泵和Ｎａ＾＋－Ｋ＾＋－ＡＴＰ泵的功能改变有关。本文对这方面的研究概况作简要综述。     

去甲肾上腺素对缺氧心肌细胞钙通道的影响

目的 :研究去甲肾上腺素 ( NE)对缺氧心肌细胞钙通道的影响。方法 :运用全细胞膜片钳记录技术检测 NE对缺氧豚鼠心肌细胞钙通道电流 ( ICa)的影响。结果 :NE( 1× 10 - 8m ol/ L)可明显增加缺氧及缺氧 /再灌注心肌细胞 ICa( P <0 .0 1,<0 .0 5 ) ,使缺氧心肌细胞 ICa电流 -电压曲线下移 ;在缺氧状态及缺氧 /再灌注状态下 ,NE对心肌细胞 ICa的作用差异无显著性意义 ( P >0 .0 5 )。结论 :NE增加心肌细胞 ICa为其致缺血及缺血 /再灌注性心律失常机制之一     

牛磺酸对大鼠培养心肌细胞钙转运功能的作用

以大鼠培养心肌细胞的脂质过氧化损伤模型，采用４５Ｃａ示踪技术，直接进行心肌肌浆网、线粒体钙转运功能的测定，同时观察牛磺酸对心肌过氧化损伤的保护作用。结果表明，牛磺酸能显著改善心肌肌浆网、线粒体钙转运功能，对心肌细胞具有保护作用     

The role of protein kinase C in the regulation of cell growth and in signalling to the cell nucleus

The protein kinase C (PKC) family of serine/threonine kinases consists of at least 11 mammalian isoforms, which show slight differences in their molecular structure and enzymatic properties. PKC isoforms are involved in a wide variety of intracellular signalling events and play an important role in tumour promotion and cell growth control in general. Studies of expression levels in cancer cells and studies using overexpression of single isoforms or expression of dominant negative isoforms reveal that, depending on the cellular background, PKC isoforms can either promote or inhibit cell growth. To understand the role of PKC isoforms in growth control, it is essential to understand how PKC functions in the intracellular signalling cascades towards the cell nucleus. Recent work has shown that PKC isoforms can act either in the cytoplasm, and cause nuclear effects indirectly by triggering signalling pathways directed towards the cell nucleus, or, after translocation and activation, can themselves act in the cell nucleus. Received: 8 January 1999 / Accepted: 12 July 1999     

Therapeutic effect of diltiazem on myocardial cell injury during the calcium paradox

The calcium paradox induced massive structural damage to the sarcolemma and gap junctions. Treatment of these hearts with diltiazem, a calcium channel blocking agent, significantly prevented cell separation and rupture, gap junction alterations, and preserved tissue high energy phosphate content. It is suggested that the calcium paradoxical injury may be prevented with calcium channel blocking agents by maintaining the structural integrity of junctional sarcolemma and its components.     

The dipeptidyl peptidase-4 inhibitor-sitagliptin modulates calcium dysregulation,inflammation, and PPARs in hypertensive cardiomyocytes

Background

Hypertension induces cardiac dysfunction, calcium (Ca^2 +) dysregulation, and arrhythmogenesis. Dipeptidyl peptidase (DPP)-4 inhibitors, an antidiabetic agent with anti-inflammation and anti-hypertension potential, may regulate peroxisome proliferator-activated receptors (PPARs)-α, -γ, and -δ and Ca^2 + homeostasis.

Objective

The purpose of this study was to investigate whether DPP-4 inhibitor, sitagliptin, can modulate PPARs and Ca^2 + handling proteins in hypertensive hearts.

Methods

A Western blot analysis was used to evaluate protein expressions of myocardial PPAR isoforms, tumor necrosis factor (TNF)-α, interleukin (IL)-6, sarcoplasmic reticulum ATPase (SERCA2a), Na⁺–Ca^2 + exchanger (NCX), ryanodine receptor (RyR), voltage-dependent Ca^2 + (CaV1.2), slow-voltage potassium currents (Kvs), angiotensin II type 1 receptor (AT1R), and receptor of advanced glycated end-products (RAGE) from Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHR), and SHR treated with sitagliptin (10 mg/kg for 4 weeks). Conventional microelectrodes were used to record action potentials (APs) in the ventricular myocytes from each group.

Results

Compared to the control group, SHR had lower cardiac PPAR-α and PPAR-δ protein expressions, but had greater cardiac PPAR-γ levels, and TNF-α, IL-6, RAGE, and AT1R protein expressions, which were ameliorated in the sitagliptin-treated SHR. SHR had prolonged QT interval and AP duration with less SERCA2a and RyR, and greater CaV1.2 expressions, which were also attenuated in sitagliptin-treated SHR.

Conclusions

Sitagliptin significantly changed the cardiac electrophysiological characteristics and Ca^2 + regulation, which may have been caused by its effects on cardiac PPARs, proinflammatory cytokines, and AT1R.     

粉防己碱对缺氧和复氧损伤心室肌细胞内Ca2+超载的作用

目的 :研究粉防己碱 (Tet)对培养大鼠心室肌细胞缺氧和复氧损伤时细胞内 Ca2 超载的作用。方法 :采用荧光探针 Fura- 2 / AM结合计算机图像处理技术测定单个心室肌细胞内 Ca2 浓度。结果 :对照组心室肌细胞在缺氧和复氧过程中出现 2次细胞内 Ca2 浓度明显上升 ,维拉帕米 (Ver)处理组 (10μmol/ L )心室肌细胞出现 2次细胞内 Ca2 浓度轻度上升 ;Tet处理组 (30 0μmol/ L )心室肌细胞未出现细胞内 Ca2 浓度上升。两处理组分别与对照组比较均有极显著性差异 (P<0 .0 1) ,两处理组间比较亦有显著性差异 (P<0 .0 5 )。结论 :Tet对缺氧和复氧损伤心室肌细胞内 Ca2 超载有较强的阻抑作用     

Cholinergic regulation of calcium sensitivity in cardiac muscle

The range of calcium concentrations over which activation of cardiac contractile proteins occurs (calcium sensitivity) is regulated by a cAMP controlled phosphorylation of the inhibitory subunit of troponin (TNI) [6, 8]. Phosphorylation of TNI increases the concentration of calcium required for activating myofibrillar ATPase [8] and for generating active tension in hyperpermeable fibers [6]. As TNI goes from a completely dephosphorylated to a completely phosphorylated state, there is a decrease in calcium sensitivity that, in hyperpermeable fibers, may produce as much as a five fold increase in the concentration of calcium required for 50% of maximum activation ([Ca]₅₀) [6]. In both hyperpermeable [6] and intact [2, 11] cells, catecholamines appear to promote cAMP production and TNI phosphorylation. Dephosphorylation of TNI is promoted by cGMP in the hyperpermeable trabecula [6] and by acetylcholine in the perfused heart [2]. Therefore calcium sensitivity may be regulated not only by catecholamine control of protein kinase but in addition by cholinergic control of phosphatase through guanylate cyclase and cGMP. In the present study, the effects of muscarinic cholinergic stimulation on the calcium sensitivity of the contractile system have been examined in the hyperpermeable rat trabecula. Exposure to 10 μm methacholine increases calcium sensitivity in all fibers that initially require 3.3 μm Ca²⁺ or greater for 50% activation. The extent of the change is inversely and linearly related to the initial calcium sensitivity. The effect of methacholine on calcium sensitivity is prevented by 10 μm atropine, implicating the muscarinic receptor in the action of the cholinergic agent.     

亚硝酸盐对培养心肌细胞的直接作用

应用亚硝酸钠作用于培养的心肌细胞，观察到心肌细胞发生了超微形态结构改变；细胞内α－羟丁酸脱氢酶漏出增加（Ｐ＜０．０５～０．０１），３Ｈ—ＴｄＲ掺入变化呈双向性：小剂量组（０．００１～０．１×１０－６ｍｏｌ／Ｌ）呈现促进作用（Ｐ＜０．０５～０．０１）；大剂量组（１．０×１０－６ｍｏｌ／Ｌ）则表现为抑制效应（Ｐ＜０．０５）。细胞周期分析结果：小剂量组（０．０１×１０－６ｍｏｌ／Ｌ）Ｇ０Ｇ１期细胞减少４．１３％，Ｓ期增长３．９５％，Ｇ２＋Ｍ期增长０．３３％，与对照组比较，无显著性差异（Ｐ＞０．０５）。大剂量组（１、０×１０－６ｍｏｌ／Ｌ）Ｇ０Ｇ１期细胞增加９．１４％，Ｓ期减少１２．４６％，Ｇ２＋Ｍ期增加４．０６％，其中Ｓ期的改变达到显著差异（Ｐ＜０．０５）；细胞及培养液中脂质过氧化物含量增加（Ｐ＜０．０１）。表明亚硝酸盐能引起培养心肌细胞的直接损伤，其机理与脂质过氧化作用有关。     

Effects of nerve growth factor on the action potential duration and repolariz-ing currents in a rabbit model of myocardial infarction

Objective To investigate the effect of nerve growth factor (NGF) on the action potential and potassium currents of non-infarcted myocardium in the myocardial infarcted rabbit model. Methods Rabbits with occlusion of the left anterior descending coronary artery were prepared and allowed to recover for eight weeks (healed myocardial infarction, HMI). During ligation surgery of the left coronary artery, a polyethylene tube was placed near the left stellate ganglion in the subcutis of the neck for the purpose of administering NGF 400 U/d for eight weeks (HMI + NGF group). Cardiomyocytes were isolated from regions of the non-infarcted left ventricular wall and the action potentials and ion currents in these cells were recorded using whole-cell patch clamps. Results Compared with HMI and control cardiomyocytes, significant prolongation of APD₅₀ or APD₉₀ (Action potential duration (APD) measured at 50% and 90% of repolarization) in HMI + NGF cardiomyocytes was found. The results showed that the 4-aminopyridine sensitive transient outward potas?sium current (I_to), the rapidly activated omponent of delayed rectifier potassium current (I_Kr), the slowly activated component of delayed rectifier potassium current (I_Ks), and the L-type calcium current (I_CaL) were significantly altered in NGF + HMI cardiomyocytes compared with HMI and control cells. Conclusions Our results suggest that NGF treatment significantly prolongs APD in HMI cardiomyocytes and that a decrease in outward potassium currents and an increase of inward Ca²⁺ current are likely the underlying mechanism of action.