钙网蛋白在心肌组织细胞氧化应激与缺血损伤构建中的表达与应用

背景：生物体内钙网蛋白具有调节细胞凋亡、应激、心血管炎症反应等多种生理和病理生理过程的功能。 目的：分析钙网蛋白在缺血再灌注损伤中的表达与应用。 方法：由作者检索1996/2010 PubMed数据库与中国知网数据库有关钙网蛋白结构、功能及在自身免疫、心脏发育中的作用、在缺血缺氧过程中表达的变化等方面的文献后,对钙网蛋白在氧化应激与缺血损伤中的表达变化进行了实验观察。 结果与结论：钙网蛋白通过协助蛋白质正确折叠和维持细胞内Ca2+稳态而参与调节细胞凋亡、黏附、类固醇敏感基因表达等,在心脏发生、发育和病理变化中发挥着重要作用。作者应用大鼠心肌缺血再灌注损伤模型,通过酶联免疫吸附试验、免疫组织化学及RT-PCR 3种不同的检测方法,观察了钙网蛋白在心肌缺血再灌注损伤中的表达,结果显示缺血再灌注损伤诱导钙网蛋白高表达,进而激活内质网凋亡信号途径,诱导细胞损伤,加重心肌损伤。     

钙网蛋白与心血管疾病

钙网蛋白是内质网中主要的Ca2 结合分子伴侣,具有调控细胞钙稳态、蛋白质合成与修饰等作用,在生物体内钙网蛋白具有调节细胞凋亡、应激、心血管炎症反应等多种生理和病理生理过程的功能。钙网蛋白在心脏发育、心肌肥大、缺血再灌注和血管生成等过程中的作用是心血管领域的研究热点问题之一。     

钙网蛋白与肿瘤免疫关系的研究进展

钙网蛋白(CRT)是一种高度保守的内质网Ca2+结合伴侣蛋白,除了维持细胞内Ca2+平衡及负责糖蛋白的折叠外,还参与线粒体代谢、基因表达和细胞凋亡等过程。蒽环类化疗药物处理时,肿瘤细胞引起的内质网应激能导致肿瘤细胞内CRT的外翻,外翻的CRT可有效增强抗原提呈细胞对肿瘤细胞的识别与吞噬,由此引发抗同种肿瘤细胞的免疫杀伤效应,诱导肿瘤免疫原性细胞进入凋亡程序。由此说明,CRT是引起肿瘤细胞免疫原性凋亡的关键因子,其在肿瘤免疫治疗过程中具有潜在的应用价值。现就CRT在肿瘤免疫中的作用进行综述。     

钙网蛋白的生物学功能及其与自身免疫疾病的关系

钙网蛋白(Calreticulin,CRT)最初被认为是内质网Ca2+结合蛋白,近年研究发现在细胞外也有表达,具有多种生物学功能,包括蛋白加工、细胞内钙调节、抗原递呈等。抗CRT抗体在多种自身免疫性疾病患者血清中存在,CRT可能是自身免疫性疾病致病因素之一。本文对CRT的生物学功能和它与自身免疫性疾病的关系作一综述。     

钙网蛋白的生物学功能及其与自身免疫疾病的关系

钙网蛋白(Calreticulin，CRT)最初被认为是内质网Ca^2 结合蛋白，近年研究发现在细胞外也有表达，具有多种生物学功能，包括蛋白加工、细胞内钙调节、抗原递呈等。抗CRT抗体在多种自身免疫性疾病患者血清中存在，CRT可能是自身免疫性疾病致病因素之一。本文对CRT的生物学功能和它与自身免疫性疾病的关系作一综述。     

钙网织蛋白促进心脏瓣膜病患者心房重构

 目的: 明确钙网织蛋白的异常表达与分布能否促进心脏瓣膜病患者心房重构的发生。方法: 从78位进行瓣膜置换手术的患者中获得左右心房的标本。患者被分为窦性节律组、阵发性房颤组和持续性房颤组(房颤持续超过6个月),检测心房组织中钙网织蛋白、整合素α5和转化生长因子β1(TGF-β1)的蛋白表达情况。同时使用免疫共沉淀法测定钙网织蛋白与钙调磷酸酶B及整合素α5的结合情况。结果: 房颤组的钙网织蛋白、整合素α5和TGF-β1的蛋白表达均高于窦性节律组,特别是在二尖瓣疾病患者的左心房中。免疫共沉淀显示钙网织蛋白可以与钙调磷酸酶B和整合素α5结合产生相互作用。整合素α5的表达水平与TGF-β1的表达具有明显相关性,钙网织蛋白表达水平与整合素α5和TGF-β1的表达水平具有明显的相关性。在相同心功能分级情况下,钙网织蛋白的表达水平在持续性房颤组明显高于窦性心律组。结论: 房颤患者心房组织中的钙网织蛋白、整合素α5和TGF-β1表达增高,并与房颤类型有关,这提示钙网织蛋白参与了心脏瓣膜病房颤患者的心房重构。     

高尔基体在氧化应激中促凋亡作用的研究进展

高尔基体是细胞内重要的细胞器,除参与细胞内蛋白加工修饰、脂类代谢及囊泡转运等生理活动外,在离子稳态、应激等方面也发挥着重要作用。在氧化应激中高尔基体发生应激反应,功能上出现Ca2+/Mn2+泵活性改变,Ca2+/Mn2+稳态失衡;结构上出现高尔基体碎裂,释放应激性碎裂产物,上述行为将启动高尔基体相关凋亡通路,介导细胞发生凋亡。p115是高尔基体应激相关蛋白,在氧化应激中p115裂解后产生的碎裂片段通过促p53磷酸化而发挥促凋亡作用。     

CaM在内耳感觉细胞中的研究进展

随着钙的作用及其机制成为内耳生理研究关键之一。作为胞内Ca2 + 的多功能传感器或受体 ,钙调蛋白 (CaM)介导了很多Ca2 + 调节的生理过程。CaM在内耳感觉细胞中的含量丰富 ,在毛细胞功能的实现与调节中可能发挥着重要作用。     

钙网蛋白介导线粒体损伤在高糖诱导心肌细胞凋亡中的作用

目的:观察高糖培养对心肌细胞钙网蛋白(calreticulin,CRT)表达的变化、线粒体功能和细胞凋亡的影响。方法:将培养的AC-16人心肌细胞随机分为正常糖浓度组、高糖组、高糖+CRT siRNA(small interfering RNA)组及等渗组,分别测定各组心肌细胞凋亡率、细胞内活性氧水平、线粒体功能和CRT表达水平的变化。结果:与正常糖浓度培养的心肌细胞相比,高糖组心肌细胞凋亡率增加,活性氧生成增多,线粒体膜电位及呼吸链酶活性降低,同时CRT表达升高;CRT siRNA可以减轻高糖组心肌细胞线粒体损伤,但细胞内活性氧的生成与高糖组相比未见显著差异。结论:CRT介导的线粒体损伤可能参与高糖时心肌细胞凋亡的增加。     

钙网织蛋白与肿瘤

钙网蛋白是一种多功能的内质网Ca2+结合蛋白,是未折叠蛋白反应的关键分子,参与调节钙平衡、蛋白质的折叠和加工、抗原提呈、细胞分化与凋亡等多种生物学功能。近年的研究表明,钙网蛋白在多种肿瘤中特异性表达,与肿瘤的发生、发展、诊断、预后以及治疗密切相关。本文对钙网蛋白与肿瘤关系的研究进展作一综述。     

When calcium turns arrhythmogenic: intracellular calcium handling during the development of hypertrophy and heart failure

Alterations of intracellular Ca2+ handling in hypertrophied myocardium have been proposed as a mechanism of ventricular tachyarrhythmias, which are a major cause of sudden death in patients with heart failure. In this review, alterations in intracellular Ca2+ handling and Ca2+ handling proteins in the development of myocardial hypertrophy and the transition to heart failure are discussed. The leading question is at what stage of hypertrophy or heart failure Ca2+ handling can turn arrhythmogenic. During the development of myocardial hypertrophy and the transition to failure, Ca2+ handling is progressively altered. Recordings of free myocyte Ca2+ concentrations during a cardiac cycle (Ca2+ transients) are prolonged early in the development of hypertrophy. However, resting (or diastolic) Ca2+ does not increase before end-stage heart failure has developed. These alterations are due to progressively defective Ca2+ uptake into the sarcoplasmic reticulum that seems to be caused by quantitative changes of gene expression of the Ca2+ ATPase of the sarcoplasmic reticulum. Increased expression and activity of the Na+/Ca2+ exchanger might compensate for this defective Ca2+ uptake, probably at the expense of increased arrhythmogenicity. When the Ca2+ handling proteins no longer efficiently counterbalance increasing intracellular Ca2+ - during stress conditions, resulting Ca2+ overload can lead to spontaneous intracellular Ca2+ oscillations, after depolarizations. Thus, after the transition to heart failure, Ca2+ overloaded sarcoplasmic reticulum, increasing resting intracellular Ca2+, and increased Na+/Ca2+ activity may all provoke afterdepolarizations, triggered activity, and finally, life-threatening ventricular arrhythmias. This increased susceptibility to ventricular arrhythmias in heart failure should not be treated with calcium antagonists.     

Impaired Ca2+-sequestration in dilated cardiomyopathy (review)

Excitation-contraction coupling is the process by which depolarisation of the myocardial surface membrane leads to the release of Ca2+-ions from the sarcoplasmic reticulum, inducing cardiac muscle contraction. This process is made possible by an elaborate system of ion-release, uptake and sequestration that controls the contraction and relaxation cycle of heart muscle fibres. The free intracellular Ca2+-concentration determines the contractile state of the myocardium, and the sequestration of Ca2+-ions into the lumen of the sarcoplasmic reticulum by the Ca2+-ATPase pump units represents a critical step towards the maintenance of normal Ca2+-cycling. The Ca2+-ATPase pump activity is regulated by phospholamban, a small 52-amino acid protein whose phosphorylation state dictates its inhibitory action on the pump. A large body of evidence points to the central role of abnormal Ca2+-ATPase-phospholamban interactions in pathophysiological heart conditions, thereby compromising the contractile state of the cardiac muscle cell. It has been shown that alterations in the oligomeric status of the Ca2+-ATPase and modified interactions between the Ca2+-pump and its regulatory subunit phospholamban underlie the contractile dysfunction that characterises certain forms of dilated cardiomyopathy. Hence, elucidation of interactions within physiological Ca2+-ATPase pump units in normal and diseased myocardium is a vital link in the development of improved diagnostic and therapeutic techniques for dealing with this elusive condition.     

Sodium-calcium exchanger (NCX-1) and calcium modulation: NCX protein expression patterns and regulation of early heart development.

Ouabain-induced inhibition of early heart development indicated that Na/K-ATPase plays an important role in maintaining normal ionic balances during differentiation of cardiomyocytes (Linask and Gui [1995] Dev Dyn 203:93-105). Inhibition of the sodium pump is generally accepted to affect the activity of the Na(+)-Ca(++) exchanger (NCX) to increase intracellular [Ca(++)]. These previous findings suggested that Ca(++) signaling may be an important modulator during differentiation of cardiomyocytes. In order to identify a connection between heart development and NCX-mediated Ca(++) regulation, we determined the embryonic spatiotemporal protein expression pattern of NCX-1 during early developmental stages. In both chick and mouse embryos, NCX-1 (the cardiac NCX isoform) is asymmetrically expressed during gastrulation; in the right side of the Hensen's node in the chick, in the right lateral mesoderm in the mouse. At slightly later stages, NCX-1 is expressed in the heart fields at comparable stages of heart development, in the chick at stage 7 and in the mouse at embryonic day (ED) 7.5. By ED 8 in the mouse, the exchanger protein displays a rostrocaudal difference in cardiac expression and an outer curvature-inner curvature ventricular difference. By ED 9.5, cardiac expression has increased from that seen at ED8 and NCX-1 is distributed throughout the myocardium consistent with the possibility that it is important in regulating initial cardiac contractile function. Only a low level of expression is detected in inflow and outflow regions. To substantiate a role for the involvement of calcium-mediated signaling, using pharmacologic approaches, ionomycin (a Ca(++) ionophore) was shown to perturb cardiac cell differentiation in a manner similar to ouabain as assayed by cNkx2.5 and sarcomeric myosin heavy chain expression. In addition, we show that an inhibitor of NCX, KB-R7943, can similarly and adversely affect early cardiac development at stage 4/5 and arrests cardiac cell contractility in 12-somite embryos. Thus, based upon NCX-1 protein expression patterns in the embryo, experimental Ca(++) modulation, and inhibition of NCX activity by KB-R7943, these results suggest an early and central role for calcium-mediated signaling in cardiac cell differentiation and NCX's regulation of the initial heartbeats in the embryo.     

Regulatory roles of MgADP and calcium in tension development of skinned cardiac muscle

We investigated the regulatory roles of MgADP and free Ca2+ in isometric tension development in skinned bovine cardiac muscle. We found that, in the relaxed state without free Ca2+, MgADP elicited a sigmoidal increase in active tension, as is the case in skeletal muscle (ADP-contraction). The critical MgADP concentration, at which the tension increment became half-maximal, increased in proportion to MgATP concentration, with a slope of approximately 1 for cardiac and 4 for skeletal muscle. Raising the free Ca2+ concentration decreased the critical MgADP concentration in proportion to the free Ca2+ concentration. In addition, the apparent Ca2+ sensitivity of tension development increased with MgADP, while decreasing with inorganic phosphate (Pi); MgADP suppressed the Ca2+- desensitizing effect of Pi in a concentration-dependent manner. These activating effects of MgADP were quantitatively assessed by means of a model based upon the kinetic scheme of actomyosin ATPase. These experimental results and model simulation suggest that the state of thin filaments is synergistically regulated by both the binding of Ca2+ to troponin and the formation of the actomyosin–ADP complex.     

Impaired Ca2+-ATPase oligomerization and increased phospholamban expression in dilated cardiomyopathy

Although primary genetic defects have been identified for some forms of inherited cardiomyopathy, it is not well understood how secondary abnormalities actually lead to muscle cell destruction. Since cardiomyopathies significantly influence morbidity and mortality rates world-wide, it is important to improve the differential diagnosis of these disorders and develop potential treatments for inherited diseases of the heart. Elucidation of the secondary molecular mechanisms underlying cardiac cell necrosis might help linking a specific mutation in a cardiac gene to acute heart failure. As disturbed Ca2+-homeostasis may contribute to heart failure, we have investigated the relative abundance and oligomeric status of the sarcoplasmic reticulum Ca2+-ATPase and phospholamban in various cardiomyopathies. These two proteins represent important factors in cardiac relaxation. The SERCA2 isoform of the Ca2+-ATPase represents a major Ca2+-removal system in cardiac muscle fibres and phospholamban is a regulator of Ca2+-pump activity. Although Ca2+-ATPase expression did not seem to be markedly altered, the comparative immunoblot analysis presented here clearly shows that phospholamban expression is increased in dilated cardiomyopathy, possibly explaining the decreased Ca2+-uptake in the disease. In contrast to the normal enzyme, the Ca2+-pump was demonstrated to exhibit an impairment of crosslinker-stabilized oligomerization in dilated cardiomyopathy. Since Ca2+-ATPase oligomerization is important for co-operative kinetics and protection against proteolytic degradation, the monomeric Ca2+-ATPase may trigger an abnormal contraction-relaxation cycle in dilated cardiomyopathy leading to heart failure.     

Tuning cardiac performance in ischemic heart disease and failure by modulating myofilament function

The cardiac myofilaments are composed of highly ordered arrays of proteins that coordinate cardiac contraction and relaxation in response to the rhythmic waves of [Ca(2+)] during the cardiac cycle. Several cardiac disease states are associated with altered myofilament protein interactions that contribute to cardiac dysfunction. During acute myocardial ischemia, the sensitivity of the myofilaments to activating Ca(2+) is drastically reduced, largely due to the effects of intracellular acidosis on the contractile machinery. Myofilament Ca(2+) sensitivity remains compromised in post-ischemic or "stunned" myocardium even after complete restoration of blood flow and intracellular pH, likely because of covalent modifications of or proteolytic injury to contractile proteins. In contrast, myofilament Ca(2+) sensitivity can be increased in chronic heart failure, owing in part to decreased phosphorylation of troponin I, the inhibitory subunit of the troponin regulatory complex. We highlight, in this paper, the central role of the myofilaments in the pathophysiology of each of these distinct disease entities, with a particular focus on the molecular switch protein troponin I. We also discuss the beneficial effects of a genetically engineered cardiac troponin I, with a histidine button substitution at C-terminal residue 164, for a variety of pathophysiologic conditions, including hypoxia, ischemia, ischemia-reperfusion and chronic heart failure.     

Triple-site pacing: a new supported therapy approach for bridge to recovery with a left ventricular assist system in a patient with idiopathic dilated cardiomyopathy

Left ventricular assist devices (LVAD) are widely used as bridges to cardiac transplantation or for destination therapy. LVAD support may also function as a bridge to ventricular recovery, but a sufficient rate of recovery has not been obtained, even with various adjuvant therapies. Cardiac resynchronization therapy (CRT) is an effective treatment for heart failure, and there is a report of successful weaning off LVAD with CRT. However, some patients with CRT could not improve their cardiac function because of residual dyssynchrony. Herein, we describe a case of a successful bridge to recovery with triple-site pacing for residual dyssynchrony after biventricular pacing. A 34-year-old woman with heart failure due to dilated cardiomyopathy whose condition deteriorated underwent Toyobo LVAD implantation, resulting in improvement of the left ventricular ejection fraction (LVEF) from 12 to 36%. Because of left ventricular dyssynchrony, we performed CRT, but residual dyssynchrony impeded cardiac recovery. We inserted an additional ventricular lead at the right ventricular outlet to achieve triple-site pacing in order to obtain complete synchronization. The LVEF improved to 45%, and the patient was successfully weaned off the LVAD. In LVAD-supported cases of persistent left ventricular dyssynchrony with CRT, implantation of triple-site pacing could potentially accelerate recovery.     

Right atrial appendage pacing in cardiac resynchronization therapy – haemodynamic consequences of interatrial conduction delay

The present case report describes a patient with an artificial mitral valve and dual chamber pacemaker implanted due to perioperative complete atrio-ventricular block. One year later an upgrade to cardiac resynchronization therapy (CRT) combined with ICD function was performed due to significant progression of heart failure symptoms. Beneficial effects of CRT are demonstrated, but unfavourable haemodynamic consequences of right atrial appendage pacing are also underlined. Important interatrial conduction delay during atrial paced rhythm resulted in a significant time difference between optimal sensed and paced atrio-ventricular delay (AVD). This report provides a practical outline how to determine the interatrial delay and the sensed-paced AVD offset under echocardiography in patients treated with CRT.     

Expression of collagenase and IL-1 alpha in developing rat hearts.

During development, extracellular matrix (ECM) molecules are thought to play a major role in regulating the formation of the heart. The change in the heart from a simple tube to a complex, four-chambered organ requires the modification of both the cellular components as well as the surrounding ECM. Matrix metalloproteinases (MMP), which include collagenases, are enzymes present in the ECM that have the potential to modify the existing ECM during the development of the heart. Using both monoclonal and polyclonal antisera against collagenase, specific temporal and spatial patterns have been documented during critical periods of heart development. The cytokine interleukin 1 alpha (IL-1 alpha), a potent inducer of the MMP expression, was also shown to have a similar staining pattern in the developing heart. The monoclonal anti-rat collagenase (Mab) intensely stained the surfaces of the myocytes in the trabeculae and the ventricular and atrial walls of the 11.5 or 12.5 embryonic day (ED) rat hearts. In contrast, the polyclonal anti-human collagenase (Pab) stained not only the cardiomyocytes but also the hypertrophic endocardial cells. Pab appeared to stain the leading edge of the mesenchymal cells that migrate into the cardiac jelly of the 11.5 or 12.5 ED hearts. Immunohistochemical staining showed IL-1 alpha on the endocardial endothelium and the surface of cardiomyocytes near the cardiac jelly just before or coincident with the appearance of migrating cells. IL-1 alpha was detected on the endocardial endothelium, cardiomyocytes in the trabeculae, and the ventricular and atrial walls, as well as in the myocardial basement membrane of the truncal or atrioventricular region. However, no staining could be detected on the migrating cells in the cardiac cushions. These results indicate the presence of collagenase and IL-1 alpha on the surface of cardiomyocytes and mesenchymal cells at times when the heart is undergoing acute remodeling during septation and trabeculation. These data suggest a role for collagenase/cytokine interaction in tissue remodeling during critical stages of cardiac embryogenesis where modification of the ECM is essential to cardiac morphogenesis.