银杏叶提取物和槲皮素对心肌细胞肥大的防治作用及其机制

目的：探讨银杏叶提取物（EGb）及其单体槲皮素（Que）对心肌细胞肥大的防治作用及其机制。方法：采用血管紧张素Ⅱ（AngⅡ）诱导新生大鼠心肌细胞肥大模型;分别在培养液中加入EGb（40／μg／ml）或Que（4／μg／m1）,观察Lowry法测定心肌细胞蛋白质含量的变化;测定SOD活性和MDA含量观察心肌细胞氧自由基代谢的变化;Western blot方法检测心肌细胞p-ERK1／2、p-JNK和p-P38蛋白表达;应用RT-PCR法检测心肌细胞c-fos mRNA表达。结果：①EGb和Que能明显抑制AngⅡ引起的心肌细胞总蛋白质含量的增加;②EGb和Que可显著提高SOD活性,降低MDA含量;③AngⅡ诱导的心肌细胞p-ERK1／2、p-JNK和p-P38表达均明显增强,Que能明显抑制AngⅡ诱导的p-JNK表达;④EGb、Que、可降低AngⅡ诱导心肌细胞c-fos mRNA表达的上调水平。结论：EGb和Que对AngⅡ诱导的心肌细胞肥大有明显的防治作用,Que的作用机制可能与ROS／JNK／c-fos信号通路有关。氧化应激参与了心肌肥大的发生发展过程。     

A structural model for the inhibition of calpain by calpastatin: crystal structures of the native domain VI of calpain and its complexes with calpastatin peptide and a small molecule inhibitor

The Ca(2+)-dependent cysteine protease calpain along with its endogenous inhibitor calpastatin is widely distributed. The interactions between calpain and calpastatin have been studied to better understand the nature of calpain inhibition by calpastatin, which can aid the design of small molecule inhibitors to calpain. Here we present the crystal structure of a complex between a calpastatin peptide and the calcium-binding domain VI of calpain. DIC19 is a 19 residue peptide, which corresponds to one of the three interacting domains of calpastatin, which is known to interact with domain VI of calpain. We present two crystal structures of DIC19 bound to domain VI of calpain, determined by molecular replacement methods to 2.5A and 2.2A resolution. In the process of crystallizing the inhibitor complex, a new native crystal form was identified which had the homodimer 2-fold axis along a crystallographic axis as opposed to the previously observed dimer in the asymmetric unit. The crystal structures of the native domain VI and its inhibitor PD150606 (3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid) complex were determined with the help of molecular replacement methods to 2.0A and 2.3A resolution, respectively. In addition, we built a homology model for the complex between domain IV and DIA19 peptide of calpastatin. Finally, we present a model for the calpastatin-inhibited calpain.     

Induction of apoptosis by a calpain stimulator, ONO-3403

The effects of a synthetic serine protease inhibitor, FOY-305, and its derivatives, ONO-3403 and FO-349, on the proliferation of mouse NIH3T3 cells were investigated. At concentrations between 10 and 100 g/ml, three protease inhibitors induced a moderate suppression of cell growth. However, only ONO-3403 showed severe cytotoxicity at concentrations higher than 200 g/ml. Results of TUNEL staining and DNA fragmentation analysis indicated that ONO-3403 induced apoptosis at the high concentrations. Biochemical analysis has shown that ONO-3403 directly enhanced the amidolytic activity of purified -calpain at a concentration higher than 100 g/ml while FOY-305 and FO-349 showed less effects. When the cell extract was incubated in the presence of ONO-3403, specific degradation of a few proteins including protein kinase C was observed. Similar degradation was also observed by addition of -calpain to the extract. These results imply that ONO-3403 is a specific stimulator of calpain. It seems reasonable to conclude that increase in calpain activity results in apoptosis.This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.     

Degradation of skeletal muscle plasma membrane proteins by calpain

Summary Observations described here provide the first demonstration that calpain (Ca²⁺-dependent cysteine protease) can degrade proteins of skeletal muscle plasma membranes. Frog muscle plasma membrane vesicles were incubated with calpain preparations and alterations of protein composition were revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Calpain II (activated by millimolar concentrations of Ca²⁺) was isolated from frog skeletal muscle, but the activity of calpain I (activated by micromolar concentrations of Ca²⁺) was lost during attempts at fractionation. Calpain I obtained from skeletal muscle and erythrocytes of rats was tested instead, and exerted effects similar to those of frog muscle calpain on the membrane proteins. All of the calpain preparations caused striking losses of a major membrane protein of molecular mass of approximately 97 kDa, designated band c, and diminution of a thinner band of approximately 200 kDa. There were concomitant increases in 83-and 77-kDa polypeptides. These effects were absolutely dependent on the presence of free Ca²⁺, and were completely blocked by calpastatin, a specific inhibitor of calpain action. Frog muscle calpain differed only in being relatively more active at 0°C than were the calpains from rat tissues. Experimental observations suggest that calpain acts at the cytoplasmic surface of the plasma membrane.     

低压恒流灌流分离成年大鼠心肌细胞

目的:探寻合适的初始灌流压力与定量确定酶消化终止点,以提高恒流灌注分离成年大鼠心肌细胞的产量与质量.方法:采用Langendorff装置,行主动脉插管逆向灌流,0.08%胶原酶Ⅰ消化大鼠心脏,监测灌流压力的变化.结果:调节灌流流速以15 kPa初始压力恒流灌流时(n=4),灌流压力明显升高(压力峰值＞25 kPa),造成左心室在松弛状态下明显扩张,使分离的心肌细胞存活率降低,且收缩功能显著降低.以10 kPa初始压力行恒流灌流时,酶消化引起的压力升高均低于18.75 kPa,左心室扩张不明显;当酶消化过程中压力降至10 kPa(n=3)或5kPa(n=4)时终止消化,心肌细胞分别呈消化不足或过度的形态,且心肌细胞存活率均低于10%,恢复正常细胞外液钙离子浓度后,大部分心肌细胞死亡.当酶消化时灌流压降至7.5 kPa时终止消化(n=15),分离即刻心肌细胞存活率为82.6%±4.8%,复钙后心肌细胞存活率为30.4%±4.5%,复钙后4 h的存活率仍为24.8%±5.4%.细胞形态完好,边缘锐利、横纹清晰,无明显博动,收缩功能正常.结论:为获得存活率较高的成年大鼠心肌细胞,宜采用低压恒流灌流分离方法,即初始灌流压力保持在10 kPa,胶原酶Ⅰ消化的终止压力为7.5 kPa.     

Effect of angiotensin coverting enzyme inhibitor on regression in cardiac hypertrophy

Cardiac hypertrophy in rats was produced by aortic banding for 6 weeks and regression of hypertrophy in these experimental animals was induced by administration of angiotensin converting enzyme inhibitor, enalapril (10 mg/kg/ day) for 6 weeks. The left ventricular muscle mass and systolic pressure were decrease upon treating the hypertrophied rats with enalapril. This drug also decreased the number of 1-adrenoceptors in hypertrophyied myocardium without any changes in -adrenoceptors. The regression of cardiac hypertrophy in spontaneously hypertensive rats by enalapril for 10 weeks was not associated with any alterations in 1-adrenoceptors in hypertrophied myocardium, but was decreased in -adrenoceptors. Effects of enalapril on extracellular matrix in the myocardium was also observed in regression of hypertrophy in which the type III collagen mRNA expression and collagen contents were reduced in comparison with those of hypertrophied myocardium. These results indicate that regression of cardiac hypertrophy is not alway associated with a decrease in the number of 1-adrenergic receptors and that the beneficial effects of enalapril in the hypertrophied heart in aortic banding animals may be of some specific nature.     

U50488H、哌唑嗪及心得安对去甲肾上腺素诱导心肌肥大的作用比较

目的：观测κ阿片受体激动对去甲肾上腺素诱导心肌肥大的抑制作用,并与哌唑嗪、心得安作用进行比较。方法：结晶紫染色法测心肌细胞增殖程度;Lowry法测心肌细胞蛋白含量;计算机图象分析系统测心肌细胞体积;[3H]-亮氨酸掺入法测心肌细胞蛋白合成。结果：①低血清环境下,NE明显诱导心肌细胞蛋白含量、蛋白合成及体积的增加,但对增殖无影响。②哌唑嗪和心得安单独作用部分抑制NE诱导的心肌肥大;联合作用则完全抑制。③U50488H明显抑制NE诱导的心肌肥大;其抑制程度与哌唑嗪和心得安联合作用类似,明显高于二者单独作用。结论：NE通过激动α1-和β-肾上腺受体途径诱导心肌肥大。κ阿片受体激动显著抑制NE诱导的心肌肥大,这可能与干预α1-AR和β-AR途径有关。     

Differential expression and localization of annexin V in cardiac myocytes during growth and hypertrophy

Recently it was shown that annexin V is the most prominent member of the annexin family in the adult heart [1]. Amongst others, annexin V has been suggested to play a role in developmental processes. The aim of the present study was to explore whether in the heart annexin V content and localization change during maturational and hypertrophic growth, in order to obtain indications that annexin V is involved in cardiac growth processes. First, in the intact rat heart annexin V content and localization were studied during perinatal development. It was clearly demonstrated that annexin V content in total heart transiently increased in the first week after birth, from 0.79 ± 0.06 µg/mg protein at l day before birth to a peak value of 1.24 ± 0.08 µg/mg protein 6 days after birth, whereafter annexin V protein levels declined to a value of 0.70 ± 0.06 µg/mg protein at 84 days after birth (p < 0.05). Differences in annexin V content were also observed between myocytes isolated from neonatal and adult hearts [0.81 ± 0.09 and 0.17 ± 0.08 µg/mg protein, respectively (p < 0.05)]. Moreover, during cardiac maturational growth the subcellular localization of annexin V might change from a cytoplasmic to a more prominent sarcolemmal localization. Second, in vivo hypertrophy induced by aortic coarctation resulted in a marked degree of hypertrophy (22% increase in ventricular weight), but was not associated with a change in annexin V localization or content. The quantitative results obtained with intact hypertrophic rat hearts are supported by findings in neonatal ventricular myocytes, in which hypertrophy was induced by phenylephrine (10^-5 M). In the latter model no changes in annexin V content could be observed either. In conclusion, the marked alterations in annexin V content during the maturational growth in the heart suggest a possible involvement of this protein in this process. In contrast, the absence of changes in annexin V content and localization in hypertrophied hearts compared to age matched control hearts suggests that annexin V does not play a crucial role in the maintenance of the hypertrophic phenotype of the cardiac muscle cell. This notion is supported by observations in phenylephrine-induced hypertrophied neonatal cardiomyocytes.     

大鼠不同心肌肥厚模型左心室基因表达谱变化的比较

为了解心肌肥厚时基因表达谱的变化规律,本实验复制了三种大鼠心肌肥厚模型：肾上腹主动脉缩窄(suprarenal abdominal aortic stenosis,SRS)、动静脉瘘(arterial-vein fistula,AVF)和去甲。肾上腺素持续静脉输注(jugular vein infusion of norepinephrine,NEi),并应用组织化学方法和超声心动术检测大鼠心脏结构和功能指标,应用cDNA基因芯片技术检测心脏基因表达水平的变化。SRS和NEi引起大鼠向心性心肌肥厚,AVF引起大鼠离心性心肌肥厚,其中NEi大鼠心肌纤维化明显。对不同心肌肥厚模型间大鼠左心室基因表达谱的变化进行两两比较。结果显示,有部分基因在不同模型中表达水平均发生变化,其中多数基因在两种模型中表达水平改变的方向相同,也有少部分基因在两种模型中表达水平改变方向相反。综合比较三种心肌肥厚模型的基因表达谱,各种模型都有特异的基因表达变化,但是有19个基因在三种心肌肥厚模型中表达水平均发生改变。研究结果有可能成为心肌肥厚的标志性基因或治疗靶点,为心肌肥厚发生机制的深入研究提供了新的线索。     

Characterization of regucalcin effect on proteolytic activity in rat liver cytosol: relation to cysteinyl-proteases

The increasing effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was characterized. The proteolytic activity was markedly elevated by the addition of regucalcin (0.1–0.5 M) in the absence of Ca²⁺. This increase was not significantly altered by the presence of diisopropylfluorophsophate (DPF;2.5 mM)—although DFP caused a significant decrease in the proteolytic activity. Regucalcin (0.25 M) additively enhanced the dithiothreitol (DTT; 1.0 mM)—increased proteolytic activity, while the regucalcin or DTT effect was completely abolished by NEM (5 mM), indicating that regucalcin may act on the SH group in proteases. Also, regucalcin (0.25 M) enhanced the effect of Ca²⁺ (10 M) increasing liver proteolytic activity, suggesting that regucalcin does not influence on the active sites for Ca²⁺ in proteases. Moreover, the proteolytic activity of regucalcin (0.25 M) was significantly decreased by the presence of calpastatin (24 g/ml), an inhibitor of Ca²⁺-activated neutral protease (calpain). Now, regucalcin (0.25 M) increased about 7-fold the activity ofm-calpain isolated from rabbit skeletal muscle. These observations demonstrate that regucalcin directly activates cysteinyl-proteases. Regucalcin may have a role as a potent proteolytic activator in the cytoplasm of liver cells.     

Regulation of the phosphorylation of calpain II and its inhibitor

Phosphorylation of calpain II (or its inhibitor) by the catalytic subunit of cyclic AMP-dependent protein kinase (A-PK), cyclic GMP-dependent protein kinase (G-PK), and protein kinase C (PK-C) was analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. Among these protein kinases, the catalytic subunit of A-PK exhibited the strongest phosphorylations of both calpain II and its inhibitor. Arachidonic acid and staurosporine effectively inhibited phosphorylation regardless the type of kinase tested. Despite its lack of effect on the phosphorylation of calpain II by the catalytic subunit of A-PK, sphingosine moderately enhanced the phosphorylation of calpain II by G-PK. Other agents, including phosphatidylethanolamine, phosphatidylinositol and 1, 2-dioleoyl-sn-glycerol, had no significant effect.     

HIV protease inhibitor therapy reverses neutrophil apoptosis in AIDS patients by direct calpain inhibition

The reduction of neutrophils apoptosis is one of the main non-virological effects of protease inhibitor (PI) therapy. We explore here whether this may be due to the cross-inhibition of calpain, an important non-virological target of PI in vitro. We found that the high basal level of neutrophils apoptosis in AIDS patients is strictly related to an increased intracellular calpain activity. Both alterations disappear after PI treatment, with apoptosis and calpain going back to normal levels after 3 months of PI therapy, independently of a proficient antiviral effect. PI drugs exerted a similar antiapoptotic and anticalpain effects on neutrophils in ex vivo experiments: strikingly, the effects were mimicked by commercially available calpain inhibitors. This study shows, for the first time, that apoptosis of neutrophils in AIDS patients is mediated by calpain, and that neutrophil survival in PI treated AIDS patients is a non virological effect due to calpain inhibition. Miriam Lichtner and Fabio Mengoni are equally contributed.     

蛋白激酶C对自发性高血压大鼠肥大心肌细胞无负荷收缩功能的调节

在慢性压力超负荷引起心肌肥大过程中,蛋白激酶C(protein kinase C,PKC)的激活起关键性作用,激活的PKC也能调节心肌收缩性能.本文旨在研究自发性高血压大(spontaneously hypertensive rat,SHR)心肌肥大的不同阶段PKC调节心肌收缩性能的特征.采用胶原酶法分离4月龄与10月龄Wistar-Kyoto(WKY)、SHR大鼠的心肌细胞,观测单个心肌细胞无负荷缩短幅值以及在PKC激动剂与抑制剂作用下心肌收缩性能的变化.结果表明:刺激频率从1 Hz增至3 Hz,WKY大鼠心肌细胞无负荷缩短幅值逐渐增加,呈正阶梯效应;4月龄SHR大鼠心肌细胞的缩短幅值较WKY大鼠增强,但在各刺激频率下其缩短幅值基本保持不变;10月龄SHR大鼠心肌细胞的缩短幅值在1 Hz刺激条件下与WKY大鼠无差别,随刺激频率增加,缩短幅值降低,呈负阶梯效应.在PKC激动剂PMA灌流条件下,50、100与200 nmol/L的PMA分别降低WKY大鼠心肌细胞缩短幅值至(69.8±1.9)%、(58.2 2.2)%与(22.7±2.5)%(均P<0.01),呈浓度依赖关系;PMA对4月龄SHR大鼠心肌细胞缩短幅值的降低更明显,分别降至(6.1±0.7)%、(2.4±0.2)%与(12.5±2.6)%(均P<0.01);PMA降低10月龄SHR大鼠心肌细胞缩短幅值至(65.7±1.6)%、(53.9±4.0)%与(16.3±2.0)%(均P<0.01),小于对4月龄SHR大鼠心肌细胞缩短幅值的作用.PKC抑制剂staurosporine增加WKY大鼠心肌细胞缩短幅值,在200 nmol/L的staurosporine灌流条件下,WKY大鼠、4月龄SHR大鼠、10月龄SHR大鼠心肌细胞缩短幅值分别增JJH(63.63±4.53)%、(80.82±4.61)%、(80.97±4.59)%(均P<0.05).结果提示,在SHR大鼠心肌肥大初期,具有负性肌力作用的PKC异构体可能被激活,并参与对心肌收缩性能的调节;而心肌肥大稳定阶段,这些PKC活性可能恢复至正常水平.     

PTEN在心肌肥厚中的作用初探

目的：观察肿瘤抑制因子PTEN mRNA在腹主动脉狭窄大鼠及卡托普利处理的腹主动脉狭窄大鼠左心室肌中的表达,以探讨PTEN在心肌肥厚发生发展中的可能作用。方法：采用腹主动脉狭窄术制备压力超负荷心肌肥厚动物模型,于术后4周应用逆转录—聚合酶链式反应(RT—PCR)方法,分别检测和观察对照组、心肌肥厚组和卡托普利组大鼠左心室PTEN mRNA表达的变化。结果：①与对照组相比,心肌肥厚组大鼠左心室肌PTEN mRNA表达减少;②与心肌肥厚组相比,卡托普利组大鼠左心室肌PTEN mRNA表达增加,接近对照组。结论：PTEN在心肌肥厚发生发展中可能起负调控作用,该作用与肾素—血管紧张素系统密切相关。     

模拟失重降低大鼠心肌细胞对异丙肾上腺素的反应性

本文旨在研究模拟失重对大鼠单个心肌细胞无负荷收缩功能的影响以及对异丙肾上腺素(isoproterenol,ISO)反应性的变化.采用人鼠尾部悬吊法在地面模拟失重状态,4周后以胶原酶I消化分离心肌细胞,分别对左、右两心室心肌细胞进行收缩功能测量.结果显示,悬吊4周大鼠(悬吊组)左,右心室心肌细胞的长度和宽度与正常大鼠(对照组)相比均无显著差异.随刺激频率增加,对照组与悬吊组大鼠心肌细胞缩短幅值均逐步增加.在1.0、2.0与4.0 Hz刺激下,对照组大鼠左心室心肌细胞缩短幅值分别为(8.50±1.26)%、(9.00±1.38)%与(9.23±1.83)%,右心室心肌细胞缩短幅值分别为(9.80±2.48)%、(10.03±2.48)%与(10.28±2.27)%;与对照组大鼠相比,在1.0与2.0Hz刺激下,悬吊组大鼠左心室心肌细胞无负荷缩短幅值分别降低12.2%、10.9%(P《0.05),右心室则分别降低16.5%、16.3%(P《0.05);但是在4.0 Hz刺激下却无显著性改变.与同一频率刺激下的对照组大鼠相比,悬吊组大鼠左、右心室心肌细胞达到缩短峰值的时程(time to peak shortening,TPS)明显缩短(P《0.05);而从缩短峰值至75%舒张的时程(TR75)则明显延长(P《0.05).在各刺激频率下,悬吊组大鼠左、右心室心肌细胞缩短(+dL/dtmax)与舒张(-dL/dtmax)速度均未发生明显改变.用1、5、10 nmol/L ISO灌流达稳态水平后,对照组大鼠心肌细胞缩短幅值分别增加了(10.63±0.83)%、(35.06±5.22)%和(71.64±6.83)%;而悬吊组大鼠心肌细胞缩短幅值仅增加(5.75±0.76)%、(23.97±4.50)%和(26.38±8.13)%,均有显著性差异(P《0.05,P《0.01).用10、50、100 nmol/L forskolin 灌流达稳定水平后,对照组大鼠心肌细胞缩短幅值分别增加了(3.04±0.27)%、(9.81±2.66)%、(20.20±3.47)%;而悬吊组大鼠心肌细胞缩短幅值仅增加了(1.42±0.53)%、(3.83±1.71)%、(5.49±4.08)%,均有显著性差异(P《0.05).以上结果表明,模拟失重4周降低人鼠心肌细胞无负荷缩短幅值以及对ISO的反应性.     

Effect of calpain inhibitors on the invasion of human erythrocytes by the parasite Plasmodium flaciparum

17 different proteinase inhibitors were screened for their effect on the erythrocyte invasion by the malaria parasite Plasmodium flaciparum. The effect was tested when the inhibitors were present in the culture medium and when they were trapped into erythrocyte ghosts. A very strong inhibition of invasion was observed in the presence of calpain inhibitors, with IC₅₀ in the order of 10^?7 M. Chymostatin, leupeptin, leupeptin, pepstatin A and bestatin also caused inhibition of the invasion, but with IC₅₀ in the order of 10^?5 M. The results suggest that participation of various proteinases in the process and point to the possibility of a calpain-mediated proteolytic event. This study may explain previous observations on the role of calcium in the invasion of the human erythrocyte by Plasmodium flaciparum.     

Autoantibody against Cardiac β1-Adrenoceptor Induces Apoptosis in Cultured Neonatal Rat Cardiomyocytes

To clarify whether apoptosis is involved in the injury processes induced by autoantibodyagainst cardiac β_1-adrenoceptor,we investigated the biological and apoptotic effects of antibodies on culturedneonatal rat cardiomyocytes.Wistar rats were immunized with peptides corresponding to the second extra-cellular loop of the β_1-adrenoceptor to induce the production of anti-β_1-adrenoceptor antibodies in the sera.Immunoglobulin(Ig)G in the sera was detected using synthetic antigen enzyme-linked immunosorbentassay and purified using the diethylaminoethyl cellulose ion exchange technique.Apoptosis of cardiomyo-cytes was evaluated using agarose gel electrophoresis and flow cytometry.Our results showed that thepositive serum IgG greatly increased the beating rates of cardiomyocytes and showed an"agonist-like"activity.Furthermore,positive serum IgG induced cardiomyocyte apoptosis after treatment with β_1-adrenoceptor overstimulation for 48h.The effects of monoclonal antibody against β_-adrenoceptor werealso found to be similar to those of positive serum IgG.It was suggested that the autoantibody could inducecardiomyocyte apoptosis by excessive stimulation of β_1-adrenoceptor.     

活性氧介导内皮素-1诱导的培养新生大鼠心肌细胞肥大

实验在原代培养的新生大鼠心肌细胞中进行,检测内皮素-1(endothelin-1,ET-1)及其他药物对心肌细胞活性氧(reactiveoxygen species,ROS)产生和心肌细胞肥大的作用,以探讨ROS在ET-1诱导的心肌细胞肥大信号通路中的作用及ROS与蛋白激酶C(protein kinase C,PKC)活化的关系。细胞内ROS水平用ROS敏感的荧光探针2,7-dichlorofluorescin dictate(DCF-DA)反映,心肌细胞肥大通过细胞内RNA含量、细胞内蛋白质含量、细胞表面积大小来确定。实验结果如下:单独使用ET-1后,心肌细胞内反应ROS含量的DCF-DA荧光值比对照组增加77%,反应心肌肥大的PI荧光值、细胞内蛋白质含量、细胞表面积也分别比对照组增加128%、87%和151%。ET-1合用内皮素受体A亚型(ET_A)受体拮抗剂ABT-627、PKC抑制剂CC或过氧化氢酶后,DCF-DA的增加分别减弱62%、60%和51%,同时心肌细胞肥大也被抑制,单独使用PKC激动剂佛波醇脂(PMA)也能使DCF-DA的产生比对照组增加74%。因此,在ET-1诱导心肌细胞肥大的过程中,ET-1能够使心肌细胞产生ROS和诱导ROS依赖的心肌细胞肥大,这一作用依赖于ET_A受体的激活和PKC的活化,·ROS在ET-1诱导心肌细胞肥大中起信号传递的作用。