卡托普利对缺氧/复氧乳鼠心室肌细胞游离钙的影响及其离子通道机制

目的：观察卡托普利晚期预处理对缺氧/复氧乳鼠心室肌细胞游离钙的影响及其离子通道机制。方法:建立培养乳鼠心肌细胞缺氧/复氧损伤模型。设正常对照组、缺氧/复氧组、缺氧预适应组和卡托普利组。经Flou-3/AM负载染色后，采用流式细胞分析技术，测定细胞内钙离子浓度(［Ca²⁺］i)；利用膜片钳技术，观察L-型钙通道和钠钙交换电流的变化。结果:（1）缺氧/复氧时，［Ca²⁺］i和Na⁺/Ca²⁺交换电流高于正常对照组（P<0.01），L-型钙电流（I_Ca-L）峰值下降，I-V曲线上移，半数失活电压(V_0.5)减小，ICa-L失活曲线左移。（2）晚期预处理和卡托普利使缺氧/复氧时［Ca²⁺］i低于缺氧/复氧组（P<0.01）；I_Ca-L增加，I-V曲线下移，V_0.5增大及稳态失活曲线右移；Na⁺/Ca²⁺ 交换电流减少；但［Ca²⁺］i和Na⁺/Ca²⁺交换电流高于对照组（P<0.05）。（3）卡托普利组与缺氧预适应组比较上述指标均无显著差异 。结论：心肌细胞缺氧/复氧，通过Na⁺/Ca²⁺交换电流的异常增加可引起［Ca²⁺］i的异常升高及其钙超载；卡托普利通过轻度增加Na⁺/Ca²⁺交换电流及其［Ca²⁺］i而触发晚期预处理，抑制后续缺氧/复氧引起的Na⁺/Ca²⁺交换电流及其［Ca²⁺］i的异常增加。     

苦豆碱对缺血再灌注引起的H9c2心肌细胞损伤和炎症应答的作用

目的:明确苦豆碱对缺血再灌注(I/R)引起的大鼠心肌H9c2细胞损伤和炎症应答的作用及其机制。方法:采用缺氧/复氧的方法体外模拟缺血再灌注(SI/R)心肌损伤,用不同浓度苦豆碱处理,MTT实验分析苦豆碱对H9c2细胞存活率的影响;流式细胞术检测细胞凋亡;试剂盒检测乳酸脱氢酶(LDH)和丙二醛(MDA)的水平及caspase-3的活性;ELISA分析多种炎性因子水平;同时用Western blot法分析苦豆碱对PI3K/AKT信号通路的影响。结果:苦豆碱可对抗SI/R抑制的H9c2细胞存活率,同时明显降低SI/R引起的LDH及MDA浓度的增加(P0.05)。此外,苦豆碱能够显著抑制SI/R触发的细胞凋亡(P0.05),同时降低SI/R处理后引起的caspase-3活性的增加,并上调Bcl-2/Bax比值(P0.05)。与SI/R组相比,苦豆碱处理可显著性降低H9c2心肌细胞炎性因子白细胞介素6、肿瘤坏死因子α和白细胞介素1β的浓度(P0.05)。此外,与SI/R组相比,苦豆碱处理后可显著增加p-PI3K和p-AKT的蛋白水平(P0.05);当用LY294002抑制PI3K/AKT通路后,苦豆碱促心肌细胞存活、抗凋亡及抗炎症的作用明显减弱(P0.05)。结论:苦豆碱可通过激活PI3K/AKT信号通路对抗心肌细胞缺血再灌注引起的损伤及炎症应答,提示其对心肌缺血再灌注损伤的防治具有潜在应用价值。     

Protective effects of novel single compound, Hirsutine on hypoxic neonatal rat cardiomyocytes

Uncaria rhynchophylla is a traditional Chinese herb that has been applied in China for treatment of ailments of the cardiovascular system, but little is known about its active constituents and effect in cardiomyocytes. In present study, we investigated the cardioprotective effect of 0.1 μΜ, 1 μΜ and 10 μΜ Hirsutine isolated from the methanolic extracts of Uncaria rhynchophylla by high performance liquid chromatography (HPLC) on neonatal rat cardiomyocytes treated with hypoxia to determine the mechanism underlying the protective effect with regard to cardiac anti-oxidant enzymes and apoptosis genes. Hirsutine significantly increased the viability of cardiomyocytes injured by hypoxia. Gene expression levels of proapoptotic genes (Bax, Fas and caspase-3) were significantly downregulated compared with the hypoxic control group (P < 0.05), whereas the expression level of Bcl-2 was upregulated following Hirsutine treatment (P < 0.05). Correspondingly, Hirsutine treatment increased Bcl-2 protein level and decreased Bax protein level. Assay investigating cardiac anti-oxidant enzymes provided further evidence for the protective effect of Hirsutine, as indicated by the induction of the anti-oxidant enzymes superoxide dismutase. The results of present study suggest that the mechanism of action of Hirsutine in hypoxic neonatal rat cardiomyocytes may be related to its anti-oxidant and anti-apoptotic properties. This may open an avenue for developing novel candidate compounds with cardioprotective effect from unique Chinese plant.     

Hypoxia exacerbates Ca-handling disturbances induced by very low density lipoproteins (VLDL) in neonatal rat cardiomyocytes

It is known that myocardium suffers serious alterations under ischemic conditions such as lipid overloading and electrophysiological alterations. However, it is unknown whether intracellular lipid accumulation and calcium dysfunction share common pathophysiological mechanisms under ischemia. The aims of this study were 1) to analyze the effect of normal and high doses of very low density lipoproteins (VLDL) on lipid content and calcium handling; 2) to investigate whether hypoxia modulates the effect of high VLDL doses; and 3) to identify potentially underlying mechanisms in cardiomyocytes. For this purpose, neonatal rat ventricular myocytes cultures were prepared from hearts of 3-4-day-old rats. High doses of VLDL that induced cholesteryl ester (CE) and triglyceride (TG) accumulation strongly reduced sarco(endo)plasmic reticulum Ca ATPase-2 (SERCA-2) expression, calcium transient amplitude and sarcoplasmic reticulum (SR) calcium loading. Interestingly, hypoxia, by upregulating VLDL-receptor expression (4.5-fold at 16 h) increased CE (1.5-fold) and TG (3-fold) cardiomyocyte content and exacerbated the negative effect of VLDL on SERCA-2 expression. Functionally, the hypoxic exacerbation of VLDL-mediated SERCA-2 downregulation was translated into a stronger decrease in calcium transient amplitude and SR calcium loading in myocytes exposed simultaneously to hypoxia and high VLDL. In conclusion, high VLDL doses alter calcium handling in cardiomyocytes and SERCA-2 play a pivotal role in the hypoxic exacerbation of VLDL-mediated effects on cardiac calcium handling. Potentiation of VLDL's effects under hypoxia is explained, at least in part, by hypoxic upregulation of the expression of VLDL-receptor.     

从钙超载角度探讨通脉养心丸心肌保护作用的机制

目的从抑制钙超载的角度探讨通脉养心丸抗细胞凋亡保护心肌的作用机制。方法采用大鼠心肌细胞原代培养方法获得正常心肌细胞并建立缺氧损伤细胞模型,采用FLIPR Calcium 4试剂盒检测药物对胞浆内钙离子浓度变化的影响;Annexin V/PI双染法检测心肌细胞总凋亡率。结果缺氧损伤组心肌细胞胞浆内钙离子浓度显著升高(P<0.01),通脉养心丸各剂量组变化不显著;再次加药后,通脉养心丸各剂量组胞浆内钙离子浓度均显著低于缺氧损伤组(P<0.01)。缺氧损伤组心肌细胞凋亡率显著升高(P<0.01),通脉养心丸4 g/kg、8 g/kg剂量组可显著降低心肌细胞凋亡率(P<0.01)。结论抑制心肌细胞钙超载是通脉养心丸抗心肌细胞凋亡保护心肌的作用机制之一。     

针灸对心肌细胞钙超载保护机制的研究进展

钙离子(Ca~(2+))作为细胞内普遍存在的第二信使,其相关的信号转导通路参与了机体内多种生理活动和病理变化的信号转导,具有广泛和重要的生理学作用,但其具体机制尚未被完全阐明。目前在心肌损伤的机制研究中,钙超载学说已得到医学学术界的公认,越来越多的研究证明钙超载是引发心肌细胞损伤的重要原因之一。笔者查阅了近10年国内外相关的文献资料发现,针灸对心肌细胞钙超载的保护机制主要有细胞膜Ca~(2+)转运途径、肌浆网膜Ca~(2+)转运途径及线粒体途径(即能量代谢途径)等。     

细胞外液低钾对小鼠心室肌细胞跨膜电位影响的定量分析

目的 研究不同程度细胞外低钾对心肌细胞跨膜电位的效应,阐明低钾对心肌细胞电生理特性的详细影响。方法 分离C57BL/6J小鼠的左心室乳头肌,采用标准玻璃微电极胞内记录技术记录心室肌细胞的跨膜电位,观察细胞外液K⁺浓度由正常的5.4mmol/L分别降为3、2、1和0mmol/L时,心室肌细胞跨膜电位各参数的变化。结果 低钾对心肌细胞的静息电位(RP)有双向影响:细胞外K⁺浓度降为3mmol/L时,RP显著增大(超极化)(P=0.000),而细胞外K⁺浓度降为2、1和0mmol/L时,细胞RP先显著增大后显著减小(P=0.000),这些结果异于传统观点。当细胞外液K⁺浓度为3mmol/L时,动作电位振幅(APA)和0期最大除极速度(Vmax)均增大,动作电位时程APD10、APD20、APD50和APD90均显著缩短(P<0.05),而动作电位复极到APD90后,复极速度减慢,即复极化有拖尾现象。当细胞外K⁺浓度为2mmol/L时,APA极度减小,Vmax明显减慢,AP呈侏儒型,而当细胞外K⁺浓度为1和0mmol/L时,细胞兴奋性丧失,电刺激不能诱发动作电位。此外,低钾可诱发早期后除极以及连串的触发活动,且后者两种形式,也表现为量-效和时-效的特点。结论 细胞外液低钾对心室肌细胞的RP、APA和Vmax有双重影响:中度低钾(K⁺ 3mmol/L)使这3个参数均增大;重度低钾(K⁺ 2mmol/L及以下)使这3个参数均减小;低钾使动作电位早期复极加快,晚期复极减慢;极度低钾(K⁺ 1mmol/L及以下)会导致心室肌细胞的兴奋性丧失;中、重度低钾可导致心室肌细胞发生早期后除极及连串的触发活动,后者相当于细胞水平的心动过速,但不是工作细胞获得了自律性。本研究在一定程度上澄清了以往对低钾的心肌电生理效应的模糊认识。     

Characterisation of postnatal growth of the murine heart

Using a new technique to isolate rod-shaped cardiomyocytes from small tissue pieces we were able to analyse the developmental profile of postnatal cardiomyocyte growth in the mouse. During the first 4 postnatal days the volume of the cardiomyocytes remains relatively constant despite a concomitant increase in heart weight, indicating growth due to cell division of the cardiomyocytes, also called hyperplasia. After postnatal day 5 the volume of the cardiomyocytes increases dramatically until postnatal day 14, when the increment of the volume curve decreases again. The cardiomyocytes reach their adult volume at around 3 months of age. These measurements present the first detailed analysis of the phase of so-called developmental hypertrophy, i.e. normal cardiomyocyte growth in the mouse, and provide an essential base-line for the analysis of growth parameters in mouse models for cardiomyopathies. We used this method to characterise the growth characteristics of cardiomyocytes from MLP (muscle LIM protein) knockout mice, a mouse model for dilated cardiomyopathy. During the first 2 postnatal weeks there is no significant difference in the growth parameters between MLP knockout and wildtype mice. However, in the adult animals cardiomyocytes from MLP knockout mice are not only characterised by a more irregular shape, but also by a high variability in size compared to cardiomyocytes from wildtype animals. This suggests that the alterations in ventricular morphology in the MLP heart are not due to a general elongation of the cardiomyocytes but to myocyte disarray and ventricular wall thinning caused by the heterogeneous volume of the cardiomyocyte population. Accepted: 26 June 2001