Junctophilin-2表达下调介导心肌细胞兴奋—收缩耦联结构与功能的重塑

目的阐明横管(T-tubule,TT)与肌质网(sarcoplasmic reticulum,SR)耦联关键蛋白junctophilin-2(JP2)的表达下调对心肌细胞二联体超微结构以及兴奋-收缩耦联功能的影响。方法通过构建JP2特异性shRNA腺病毒敲减成年大鼠心肌细胞中JP2的表达后,采用透射电子显微镜观测心肌细胞横管和肌质网二联体结构的形态变化,并进一步采用全细胞膜片钳结合激光共聚焦钙成像技术检测心肌细胞兴奋收缩联联功能的变化。结果通过腺病毒敲减心肌细胞JP2表达后,心肌细胞内总TT数目、TT与SR耦联的二联体结构数目以及单个耦联子内耦联SR的TT长度占TT周长的百分比均显著降低;与此同时,JP2表达下降后心肌细胞钙瞬变幅度降低,收缩功能减弱,表现出与心力衰竭类似的表型。结论心肌细胞JP2表达下降引起心肌细胞兴奋-收缩耦联结构与功能的损伤,为心力衰竭病理情况下心肌细胞结构与功能的调控机制提供了直接有力的实验证据。     

收缩兴奋耦联(93)

通常认为,心脏的电活动发生在机械收缩之前,称为兴奋收缩耦联。心肌组织或心肌细胞电活动的发生时间、起源部位及传导路径决定了其后心脏的机械活动。但在连续的心动周期中,机械性收缩也对下一次电活动有充分的影响,称为机械电反馈,晚近     

心肌细胞钙瞬变信号的研究进展

心肌细胞钙瞬变信号是指细胞动作电位或其他原因引起心肌细胞内游离Ca^2＋浓度迅速波动的现象,包括钙火花、钙波、钙震荡、钙星、钙空穴等。钙瞬变信号的幅度及时程受细胞膜L型钙通道、肌浆网膜钙释放通道、钙-ATP酶、瞬时受体电位蛋白、连接素等的影响。钙瞬变信号与心肌细胞的收缩性及传导性相关,并参与了心肌肥厚、心力衰竭等疾病发生发展过程。     

心肌细胞钙瞬变信号的研究进展

心肌细胞钙瞬变信号是指细胞动作电位或其他原因引起心肌细胞内游离Ca2+浓度迅速波动的现象,包括钙火花、钙波、钙震荡、钙星、钙空穴等.钙瞬变信号的幅度及时程受细胞膜L型钙通道、肌浆网膜钙释放通道、钙-ATP酶、瞬时受体电位蛋白、连接素等的影响.钙瞬变信号与心肌细胞的收缩性及传导性相关,并参与了心肌肥厚、心力衰竭等疾病发生发展过程.     

肝性心肌病的发病机制研究

肝硬化患者心肌收缩力减弱，应激状态下更为明显，严重时导致心力衰竭，这种现象称为“肝性心肌病”，其病理生理机制包括β-肾上腺素受体信号传导减弱，心肌兴奋收缩耦联缺陷和传导功能异常以及心肌细胞膜理化性质的改变，循环中的一氧化氮等活性物质、细胞因子等异常因素，目前对肝性心肌病的诊断较困难，也尚无特异性治疗，对于增加心脏负担的措施如分流术、肝移植等须特别谨慎，并仔细监控心血管状况。     

经典型瞬时受体电位通道与心肌肥厚

经典型瞬时受体电位通道(TRPC)亚家族主要分布于心脏组织中,TRPC具有较小的电导,在多种物理和化学因素的刺激下,能传递长时程的钙信号。TRPC与心肌肥厚等心脏疾病的发展关系密切:TRPC1/3/6表达的增加可引起胞质内Ca2+浓度异常升高,进而激活钙调神经磷酸酶信号通路,从而介导心肌肥厚的发生、发展;TRPC3还参与心肌细胞凋亡等过程;心肌的兴奋-收缩藕联等活动也与TRPC4/5密切相关。干预TRPC活性的药物有可能成为新一代的抗心肌肥厚药物。     

桥接整合因子1在心力衰竭中的研究进展

心力衰竭是多种心脏疾病终末期表现,近年来发病率逐渐增加,其发病原因包括心肌病变、心肌代谢障碍、心脏负荷长期过重、心室充盈受限等,这些病因均可导致心脏结构和收缩舒张的功能受损。心肌细胞的兴奋收缩耦联正常进行是保证心脏有效射血的必要条件,异常的兴奋收缩耦联是发生心力衰竭的重要因素。横管是心肌细胞完成收缩功能的关键所在,选择性地富集了离子通道和结构蛋白,如小窝蛋白-3、亲联蛋白和桥接整合因子1,现主要阐述桥接整合因子1与横管间的作用对心脏功能的调节和影响。     

心力衰竭β3肾上腺素能受体相关炎症机制的研究进展

心力衰竭为各种心脏疾病的终末期表现,而心力衰竭的发展亦是一个进展性的过程,即源于心脏重塑和功能恶化的进展.发生机制主要为长期神经激素激活和炎性细胞因子过度表达.心力衰竭时交感神经系统过度激活,诱导心肌细胞β肾上腺素能受体(AR)表达变化,β1-AR、β2-AR表达下调和脱敏,而由于β3-AR缺乏G蛋白耦联受体激酶的磷酸化位点等原因,心力衰竭时β3-AR的表达随着儿茶酚胺浓度增高而上调[1].交感神经过度兴奋刺激β3- AR,经多种途径介导心肌损害,对心室重塑、心肌细胞凋亡、氧化应激及免疫炎性反应产生重要影响.     

性别对成年小鼠心肌细胞收缩力的影响

目的研究性别对成年C57BL/6小鼠心肌细胞收缩力和钙瞬变的影响。方法采用Langendorff逆行灌流、Ⅱ型胶原酶消化方法分离不同性别成年C57BL/6小鼠心肌细胞,比较两组心肌细胞的形态学差异;运用Ion Optix细胞收缩及离子浓度同步测量系统,同步记录不同性别小鼠心肌细胞肌节长度及钙瞬变随电刺激的变化,比较两组间心肌细胞收缩力(肌节缩短幅度、肌节缩短率、肌节最大收缩速度和最大舒张速度)和心肌细胞钙瞬变(钙瞬变峰值、钙瞬变幅度和钙离子消除时间常数)的差异。结果成年雄性C57BL/6小鼠心肌细胞收缩力指标:肌节缩短幅度[(0.14±0.02)μm比(0.08±0.01)μm]、肌节缩短率(7.47%±0.93%比4.60%±0.59%)、肌节最大收缩速度[(-3.25±0.37)μm/s比(-2.11±0.38)μm/s]和肌节最大舒张速度[(2.45±0.29)μm/s比(1.57±0.31)μm/s]明显高于雌性小鼠(均为P<0.05)。此外,成年雄性C57BL/6小鼠心肌细胞钙瞬变指标钙瞬变峰值和钙瞬变幅度亦显著高于雌性小鼠(均为P<0.05),而雄性小鼠心肌细胞钙离子消除时间常数低于雌性小鼠[(122.40±22.99)ms比(189.20±19.54)ms,P<0.05]。结论成年雄性C57BL/6小鼠的心肌细胞收缩力及钙瞬变均高于雌性小鼠。     

心肌细胞α受体研究进展

目前已发现α受体存在于多种哺乳类动物心肌细胞上，并对心肌细胞的机械特性和电生理特性产生明显影响。α受体兴奋可延长心肌细胞动作电位时程。加强心肌收缩力。α受体的正性变力效应在表现形式和产生机理方面均与β受体正性变力效应不同。在缺血和复灌心脏，α受体与心律失常的产生有密切关系。     

神经母细胞瘤形成抑制因子DAN对PDGF-BB诱导人肺动脉平滑肌细胞增殖的抑制作用及其机制初探

目的初步探讨神经母细胞瘤形成抑制因子DAN对血小板源性生长因子-BB(PDGF-BB)诱导的人肺动脉平滑肌细胞(HPASMCs)增殖的影响及其相关信号通路。方法培养人肺动脉平滑肌细胞(HPASMCs),以10ng/mlPDGF-BB诱导HPASMCs增殖,在PDGF-BB刺激前1h预先加入DAN,用MTS法检测HPASMCs增殖的变化情况,用western blot法观察cyclinD1的表达变化和p38MAPK的磷酸化水平的变化情况。结果不同浓度(0.25,0.5,1μM)DAN作用24h,可呈浓度依赖性降低PDGF-BB诱导的HPASMCs的增殖率。DAN作用24h,可降低PDGF-BB刺激引起的CyclinD1的表达(p<0.05),PDGF-BB刺激HPASMCs5min时,p38MAPK的磷酸化水平升高最为明显(p<0.01),DAN可明显降低PDGF-BB刺激的p38MAPK的磷酸化水平(p<0.01)。结论初步证明DAN可抑制PDGF-BB诱导的HPASMCs的增殖,其机制可能是通过p38MAPK-CyclinD1信号通路进行调控。     

Positive inotropic effects by uridine triphosphate (UTP) and uridine diphosphate (UDP) via P2Y2 and P2Y6 receptors on cardiomyocytes and release of UTP in man during myocardial infarction

The aim of this study was to examine a possible role for extracellular pyrimidines as inotropic factors for the heart. First, nucleotide plasma levels were measured to evaluate whether UTP is released in patients with coronary heart disease. Then, inotropic effects of pyrimidines were examined in isolated mouse cardiomyocytes. Finally, expression of pyrimidine-selective receptors (a subgroup of the P2 receptors) was studied in human and mouse heart, using real time polymerase chain reaction, Western blot, and immunohistochemistry. Venous plasma levels of UTP were increased (57%) in patients with myocardial infarction. In electrically stimulated cardiomyocytes the stable P2Y(2/4) agonist UTPgammaS increased contraction by 52%, similar to beta1-adrenergic stimulation with isoproterenol (65%). The P2Y6-agonist UDPbetaS also increased cardiomyocyte contraction (35%), an effect abolished by the P2Y6-blocker MRS2578. The phospholipase C inhibitor U73122 inhibited both the UDPbetaS and the UTPgammaS-induced inotropic effect, indicating an IP3-mediated effect via P2Y6 receptors. The P2Y14 agonist UDP-glucose was without effect. Quantification of mRNA with real time polymerase chain reaction revealed P2Y2 as the most abundant pyrimidine receptor expressed in cardiomyocytes from man. Presence of P2Y6 receptor mRNA was detected in both species and confirmed at protein level with Western blot and immunohistochemistry in man. In conclusion, UTP levels are increased in humans during myocardial infarction, giving the first evidence for UTP release in man. UTP is a cardiac inotropic factor most likely by activation of P2Y2 receptors in man. For the first time we demonstrate inotropic effects of UDP, mediated by P2Y6 receptors via an IP3-dependent pathway. Thus, the extracellular pyrimidines (UTP and UDP) could be important inotropic factors involved in the development of cardiac disease.     

胰腺癌内皮分化基因受体表达

目的 观察胰腺癌组织溶血磷脂酸(LPA)内皮分化基因受体(Edg/LPA)的表达,探讨其临床意义.方法 收集50例胰腺癌及对应癌旁正常胰腺组织标本,采用实时定量PCR、免疫组化和蛋白质印迹法检测3种Edg/LPA受体亚型Edg-2/LPA1、Edg-4/LPA2和Edg-7/LPA3受体mRNA及蛋白的表达,分析其与胰腺癌临床病理参数的关系.结果 胰腺癌组织Edg-2/LPA1、Edg-4/LPA2、Edg-7/LPA3受体mRNA表达量分别为(0.142 ±0.042)％、(0.471 ±0.064)％、(0.231±0.043)％,对应癌旁正常胰腺组织分别为(0.132 ±0.029)％、(0.027±0.015)％、(0.163±0.046)％,其中胰腺癌组织的Edg-4/LPA2受体mRNA表达较对应癌旁组织显著增加(P＜0.05).同样,胰腺癌组织Edg-4/LPA2蛋白表达也显著高于癌旁正常胰腺组织.胰腺癌组织的3种Edg/LPA受体mRNA表达水平与血清CA19-9浓度呈平行关系,差异无统计学意义.胰腺癌组织EDG-4/LPA2受体mRNA表达与肿瘤大小、导管腺癌的分化程度及侵袭转移有关,而Edg-2/LPA1、Edg-7/LPA3受体mRNA表达仅与肿瘤的侵袭转移有关.结论 胰腺癌组织高表达Edg-4/LPA2受体,其高表达反映出肿瘤的恶性生物学行为.     

Germline deletion of FAK-related non-kinase delays post-natal cardiomyocyte mitotic arrest

The cardiomyocyte phenotypic switch from a proliferative to terminally differentiated state impacts normal heart development and pathologic myocardial remodeling, yet the signaling mechanisms that regulate this vital process are incompletely understood. Studies from our lab and others indicate that focal adhesion kinase (FAK) is a critical regulator of cardiac growth and remodeling and we found that expression of the endogenous FAK inhibitor, FAK-related non kinase (FRNK) coincided with postnatal cardiomyocyte arrest. Mis-expression of FRNK in the embryonic heart led to pre-term lethality associated with reduced cardiomyocyte proliferation and led us to speculate that the postnatal FRNK surge might be required to promote quiescence in this growth promoting environment. Herein, we provide strong evidence that endogenous FRNK contributes to post-mitotic arrest. Depletion of FRNK promoted DNA synthesis in post-natal day (P) 10 hearts accompanied by a transient increase in DNA content and multi-nucleation by P14, indicative of DNA replication without cell division. Interestingly, a reduction in tri- and tetra-nucleated cardiomyocytes, concomitant with an increase in bi-nucleated cells by P21, indicated the possibility that FRNK-depleted cardiomyocytes underwent eventual cytokinesis. In support of this conclusion, Aurora B-labeled central spindles (a hallmark of cytokinesis) were observed in tetra-nucleated P20 FRNK(-/-) but not wt cardiomyocytes, while no evidence of apoptosis was observed. Moreover, hearts from FRNK null mice developed ventricular enlargement that persisted until young adulthood which resulted from myocyte expansion rather than myocyte hypertrophy or interstitial growth. These data indicate that endogenous FRNK serves an important role in limiting DNA synthesis and regulating the un-coupling between DNA synthesis and cytokinesis in the post-natal myocardium.     

Oxytocin in cardiac ontogeny

Previous studies demonstrated the presence of oxytocin (OT) and oxytocin receptors (OTRs) in the heart. The present work provides results supporting a potential role of OT in cardiomyogenesis. Here, we show a maximal OT and OTR protein level in the developing rat heart at day 21 of gestation and postnatal days 1-4, when cardiac myocytes are at a stage of intense hyperplasia. Between postnatal days 1 and 66, OT decreased linearly in all heart chambers (4.1- to 6.6-fold). Correspondingly, immunocytochemistry demonstrated that OTRs, which were eminent in postnatal cardiomyocytes, declined with age to low levels in adults. Interestingly, in coronary vasculature, OTRs developed in endothelial cells at postnatal days 12 and 22 and achieved a plateau in adult rats. These findings suggest that OT can be involved in developmental formation of the coronary vessels. In vivo, the OT/OTR system in the fetal heart was sensitive to the actions of retinoic acid (RA), recognized as a major cardiac morphogen. RA treatment produced a significant increase (2- to 3-fold) both in the OT concentration and in the OT mRNA levels. Ex vivo, an OT antagonist inhibited RA-mediated cardiomyocyte differentiation of P19 embryonic stem cells. The decline of cardiac OT expression from infancy to adulthood of the rat and changes in cell types expressing OTR indicate a dynamic regulation of the OT system in the heart rather than constitutive expression. The results support the hypothesis that RA induces cardiomyogenesis by activation of the cardiac OT system.     

脓毒症时各型一氧化氮合酶在大鼠心脏中的表达及其可能机制

目的研究脓毒症时各型一氧化氮合酶(NOS)在心脏中的损伤作用及其机制。方法成年雄性Wistar大鼠腹腔注射脂多糖(LPS)制备脓毒症模型。应用多导生理仪监测大鼠心功能变化;用分光光度计法测定大鼠心肌组织NOS的活性;用RT-PCR和Western blot对大鼠心肌组织各型NOS的表达进行半定量分析。结果给予LPS后6h大鼠心肌收缩和舒张功能受损下降,心肌中iNOS的活性明显升高,eNOS和nNOS(合称cNOS)活性减弱;RT-PCR和Western blot结果显示,给予LPS后cNOS的表达减少,给予LPS后iNOS表达量明显增加。结论脓毒症时,iNOS、nNOS和eNOS的表达和活性发生改变;心肌细胞上iNOS表达及活性升高,这些变化可能在心功能降低中发挥作用。     

The cardiac ryanodine receptor (RyR2) and its role in heart disease

The cardiac ryanodine receptor has become a subject of increasing interest as its role in the etiology of cardiac disease is becoming more apparent. In this article, we review the current knowledge of the structure and function of the cardiac ryanodine receptor and its implications in cardiac pathophysiology. Cardiac ryanodine receptors function by regulating calcium release from the sarcoplasmic reticulum in cardiomyocytes, thereby playing an integral role in excitation-contraction coupling. In heart failure, the myocardium remains in a chronic hyperadrenergic state. This leads to protein kinase A hyperphosphorylation of ryanodine receptors within cardiomyocytes, ultimately leading to calcium leakage from the sarcoplasmic reticulum into the cytosol and thus impairing excitation-contraction coupling. These mechanisms could partially explain the pathophysiology underlying the reduced cardiac output seen in heart failure. Beta-adrenergic blockade appears to correct the abnormality and reestablishes normal ryanodine receptor function. These calcium leaks can also generate delayed afterdepolarizations, which can lead to fatal arrhythmias. Two genetic diseases have been linked to mutations in the cardiac ryanodine receptor: arrhythmogenic right ventricular dysplasia type 2 and catecholaminergic polymorphic ventricular tachycardia or familial polymorphic ventricular tachycardia. As our understanding of this receptor and its modulators deepens, the possibility of clinical application draws near.     

Modulation of myocardial contractility by lysophosphatidic acid (LPA)

Lysophosphatidic acid (LPA) is a phospholipid messenger, which is released from activated platelets and leukocytes. This study examined the effects of LPA on myocardial contractility and characterized the signal transduction pathway involved in these effects. Functional effects of LPA were determined in isolated, electrically driven human myocardial preparations and rat cardiac myocytes. In human atrial and ventricular myocardial preparations, LPA (100 micromol/l) decreased isoprenaline (0.03 micromol/l) enhanced force of contraction by 17 +/- 2% and 28 +/- 3%, respectively. The effect of LPA was attenuated by suramin (1 mmol/l). In isolated rat cardiomyocytes, LPA (1-100 micromol/l) concentration dependently abolished isoprenaline (0.03 micromol/l) induced increase in cell shortening. This antiadrenergic effect was blunted after pretreatment with pertussis toxin (5 microg/ml, 12 h). Forskolin (10 micromol/l) stimulated adenylyl cyclase activity was inhibited by LPA in human myocardial membranes. PCR analysis of human atrial and ventricular cDNAs revealed the expression of two cognate LPA receptors: EDG-2 and EDG-7. Our results suggest that LPA exerts antiadrenergic effects on force of contraction in human and rodent myocardium via a Galpha(i/o) protein-mediated mechanism, most probably by LPA binding to the mammalian LPA receptors EDG-2 and/or EDG-7. This newly discovered action of LPA might be of pathophysiological importance in conditions like myocardial ischemia or inflammatory disorders when LPA release is enhanced.     

Ethanol and Cocaine Cause Additive Inhibitory Effects on the Calcium Transients and Contraction in Single Cardiomyocytes

The heart is a major locus for the toxic actions of cocaine and ethanol, each of which has been shown to interfere with excitation-contraction coupling in cardiac muscle cells. Because these drugs are frequently used in combination, the present study was designed to investigate how they interact to modify the Ca²⁺ transient and associated contraction in fura2-loaded cardiomyocytes. A high-speed imaging technique using a charge-coupled device as detector and short-term image store was used to measure cytosolic Ca²⁺ and contraction simultaneously from fluorescence images obtained during the contractile cycle. Ethanol (100 mM) and cocaine (50 μM) caused reversible reductions in Ca²⁺ transient amplitude of 24.3 ± 3.0% and 25.1 ± 3.6%, respectively. Neither agent modified basal Ca²⁺. Ethanol treatment decreased peak shortening by 44.3 ± 3.5%, whereas the contractile depression by cocaine was 31.4 ± 5.3%. The relatively greater effect of ethanol on contraction resulted from a Ca²⁺-independent component of ethanol action on contractility. When cardiomyocytes were exposed simultaneously to ethanol and cocaine, Ca²⁺ transient amplitude was reduced by 38.7 ± 3.0%, and peak contraction was decreased by 55.1 ± 3.5%. These values represent a significantly greater inhibition than observed with either drug alone (p < 0.02) and are compatible with additive effects of the two drugs acting at distinct loci within the excitation-contraction coupling pathway. Thus, simultaneous use of cocaine and ethanol leads to an enhanced depression of myocardial contractility, which is likely to contribute to the cardiotoxic actions of the combination of these two drugs.     

Developmental expression of myostatin in cardiomyocytes and its effect on foetal and neonatal rat cardiomyocyte proliferation

OBJECTIVES: Myostatin, a member of the transforming growth factor-beta (TGF-beta) family, plays a key role in skeletal muscle myogenesis by limiting hyperplastic and hypertrophic muscle growth. In cardiac muscle, myostatin has been shown to limit agonist-induced cardiac hypertrophic growth. However, its role in cardiac hyperplastic growth remains undetermined. The aim of this study was to characterise the expression of myostatin in developing myocardium, determine its effect on cardiomyocyte proliferation, and explore the signalling mechanisms affected by myostatin in dividing cardiomyocytes. METHODS: We used quantitative PCR and Western blotting to study the expression of myostatin in cardiomyocytes isolated from rat myocardium at different developmental ages. We determined the effect of recombinant myostatin on proliferation and cell viability in dividing cardiomyocytes in culture. We analysed myostatin's effect on cardiomyocyte cell cycle progression by flow cytometry and used Western blotting to explore the signalling mechanisms involved. RESULTS: Myostatin is expressed differentially in cardiomyocytes during cardiac development such that increasing expression correlated with a low cardiomyocyte proliferation index. Proliferating foetal cardiomyocytes, from embryos at 18 days of gestation, expressed low levels of myostatin mRNA and protein, whereas isolated cardiomyocytes from postnatal day 10 hearts, wherein the majority of cardiomyocytes have lost their ability to proliferate, displayed a 6-fold increase in myostatin expression. Our in vitro studies demonstrated that myostatin inhibited proliferation of dividing foetal and neonatal cardiomyocytes. Flow cytometric analysis showed that this inhibition occurs mainly via a block in the G1-S phase transition of the cardiomyocyte cell cycle. Western blot analysis showed that part of the mechanism underpinning the inhibition of cardiomyocyte proliferation by myostatin involves phosphorylation of SMAD2 and altered expressions of the cell cycle proteins p21 and CDK2. CONCLUSIONS: We conclude that myostatin is an inhibitor of cardiomyocyte proliferation with the potential to limit cardiomyocyte hyperplastic growth by altering cardiac cell cycle progression.