甘氨酸受体在大鼠心肌细胞的表达

目的：检测成年大鼠心肌组织和新生大鼠心 肌细胞是否有甘氨酸受体（GlyR）mRNA和蛋白的表达。方法：选用成年 雄性SD大鼠心肌组织和新生SD大鼠培养心肌细胞，提取总RNA和膜蛋白，用逆转录－巢式聚 合酶链反应（RT-PCR）和蛋白免疫印迹（Western blotting）检测GlyRα1和β亚基的mRNA 和蛋白表达。结果：成年大鼠心肌组织和新生大鼠培养心肌细胞均有与 脊髓组织GlyRβ亚基极为相似的mRNA和蛋白的表达；对于GlyRα1亚基，仅在新生SD大鼠培 养心肌细胞发现有mRNA的表达。结论：本研究证实在成年大鼠心肌组织 和新生大鼠心肌细胞中有与脊髓组织相似的GlyR亚基的表达，提示大鼠心肌细胞的膜上有Gl yR的存在。     

Virus-receptor interactions of coxsackie B viruses and their putative influence on cardiotropism

Specific virus-receptor interactions are important determinants in the pathogenesis of viral infections, influencing the location and initiation of primary infection as well as the viral spread to other target organs in the postviremic phase. Coxsackieviruses of group B (CVB) specifically interact with at least two receptor proteins, the coxsackievirus-adenovirus receptor (CAR) and the decay-accelerating factor (DAF), and cause a broad spectrum of diseases, including acute and chronic myocarditis. In the human heart, CAR is predominantly expressed in intercalated discs, regions of utmost importance for the functional integrity of the heart. Since DAF is abundantly expressed in epithelial and endothelial cells, interaction of cardiotropic CVB with the DAF coreceptor protein, in addition to CAR, could therefore be advantageous to the virus by enhancing viral entry into the heart.     

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.     

Cardiac disease in transgenic mice expressing human immunodeficiency virus-1 nef in cells of the immune system

We previously reported that a severe acquired immune deficiency syndrome-like disease develops in transgenic (Tg) mice expressing the human immunodeficiency virus-1 in its natural target cells: immature and mature CD4(+) T cells and cells of the macrophage/dendritic lineage. Here, we show that these mice also develop cardiac disease, characterized most prominently by a focal myocytolysis, occasionally by myocarditis and by deposition of endogenous immunoglobulin on cardiomyocytes. Microfil perfusion demonstrated widespread coronary arteriospasm and echocardiographic analysis revealed depressed cardiac function in Tg mice. A higher (but still modest) level of cardiomyocyte apoptosis was detected in Tg as compared to non-Tg hearts. Tg expression was detected in some of the infiltrating mononuclear cells, but not in cardiomyocytes or in cells of the heart vessels, suggesting a human immunodeficiency virus-1-induced disease process mediated by cells of the immune system. The similarity of the heart disease observed in these Tg mice to that observed in acquired immune deficiency syndrome patients suggests a common pathogenesis.     

Microarchitecture of the hearts in term and former-preterm lambs using diffusion tensor imaging

Diffusion tensor imaging (DTI) is an MRI technique that can be used to map cardiomyocyte tracts and estimate local cardiomyocyte and sheetlet orientation within the heart. DTI measures diffusion distances of water molecules within the myocardium, where water diffusion generally occurs more freely along the long axis of cardiomyocytes and within the extracellular matrix, but is restricted by cell membranes such that transverse diffusion is limited. DTI can be undertaken in fixed hearts and it allows the three-dimensional mapping of the cardiac microarchitecture, including cardiomyocyte organization, within the whole heart. The objective of this study was to use DTI to compare the cardiac microarchitecture and cardiomyocyte organization in archived fixed left ventricles of lambs that were born either preterm (n = 5) or at term (n = 7), at a postnatal timepoint equivalent to about 6 years of age in children. Although the findings support the feasibility of retrospective DTI scanning of fixed hearts, several hearts were excluded from DTI analysis because of poor scan quality, such as ghosting artifacts. The preliminary findings from viable DTI scans (n = 3/group) suggest that the extracellular compartment is altered and that there is an immature microstructural phenotype early in postnatal life in the LV of lambs born preterm. Our findings support a potential time-efficient imaging role for DTI in detecting abnormal changes in the microstructure of fixed hearts of former-preterm neonates, although further investigation into factors that affect scan quality is required.     

Reduction of hypoxia-inducible heme oxygenase-1 in the myocardium after left ventricular mechanical support

Left ventricular assist devices (LVAD) may improve cardiac function. The pathogenesis of this phenomenon, called 'reverse remodelling', is not completely elucidated. To examine the hypothesis that LVAD support eliminates tissue stress by reducing local hypoxia, the distribution of heme oxygenase-1 (HO-1), a stress protein inducible by hypoxia, was examined in vivo and in vitro. The immunoreactivity for HO-1 was semi-quantitatively analysed in left ventricular tissue of 23 patients (14 dilated cardiomyopathy (DCM), six ischaemic heart disease (IHD), three myocarditis/congenital heart disease) with end-stage heart failure before and after LVAD support, while two unused donor hearts served as controls. Control hearts stained almost negative for HO-1, while failing hearts showed immunoreactivity mainly in cardiomyocytes, but also in endothelial cells, some smooth muscle cells and fibroblasts. Hearts with IHD showed significantly higher HO-1 immunoreactivity than hearts with DCM or myocarditis/congenital heart disease. After LVAD support, the HO-1 content decreased significantly in the DCM and IHD group and was significantly higher in the subendocardium than in the subepicardium. In vitro, under hypoxic conditions, neonatal rat cardiomyocytes showed an increase of HO-1 protein content up to sixfold above the normal level, which returned to normal values after normoxic cultivation. Mechanical support reduces the HO-1 content of the failing heart and HO-1 is inducible in vitro under hypoxia and is reversible under normoxia. This supports the concept that restoration of cardiac normoxia by mechanical unloading, particularly in the subendocardium, may be in part responsible for the phenomenon of 'reverse remodelling'.     

端粒酶逆转录酶和端粒重复结合因子2在大鼠心肌细胞发育过程中的表达

目的：观察大鼠心肌细胞发育过程中端粒酶逆转录酶(TERT)和端粒重复结合因子2(TRF2)的表达,探讨TERT活性与心肌细胞分化和发育的关系。方法：应用免疫组化方法分别检测胎鼠孕16d、出生后2、5、10、20d的大鼠心脏标本,观察TERT和TRF2的表达和分布。结果：TERT分布于胎鼠心肌细胞的胞质和细胞核中,出生后2～20d,TERT在胞质的表达呈下降趋势。TRF2主要分布在胎鼠心肌细胞的胞核中,出生后5～20d,TRF2表达阳性的心肌细胞数目逐渐减少。结论：在大鼠心肌细胞发育过程中,TERT和TRF2的表达下调促使部分细胞永久地退出细胞周期而进入终末分化;TERT活性受到了心肌细胞发育过程的调节。     

Up-regulation of parvalbumin expression in newborn and adult rat heart

Parvalbumin (PV), a cytoplasmic calcium-binding protein, functions as a relaxing factor and has recently been detected in rat heart. Developmental changes in PV localization and expression were investigated in the heart of Wistar rats at different ages. Ten hearts from newborn, 3-month-old (young), 6-month-old (young adult), and 12-month-old (adult) rats were processed for immunohistochemistry and Western blot assay. PV was detected in hearts of all the age groups of the rats from newborn to 12-month-old by both immunohistochemistry and Western blotting. A variable distribution of PV immunoreactivity was present in newborn cardiac myocytes. In the 3-, 6-, and 12-month-old rat hearts, identical PV immunoreactivity was found in all cardiac myocytes and the intensity of PV immunoreactivity increased with increasing age. By using Western blotting, it was found that the expression of PV was low in the newborn rat heart and increased with increasing age. The presence of PV may correlate with the physiological age, and possibly serves to maintain proper relaxation of the cardiac myocytes to cope with an increasing workload of the heart during body growth.     

激活的Akt对心肌细胞的增殖调控

目的 通过检测Akt在大鼠心肌细胞退出细胞周期过程中的表达及Akt表达载体转染后大鼠心肌细胞有丝分裂的改变,探讨Akt对心肌细胞增殖的影响.方法 生后不同发育阶段和成年健康Wistar大鼠各5只,用RT-PCR和免疫印迹法,检测心肌组织Akt的mRNA和蛋白磷酸化水平改变;培养大鼠H9c2(2-1)心肌细胞,通过胰岛素...     

Ongoing coxsackievirus myocarditis is associated with increased formation and activity of myocardial immunoproteasomes

A growing body of evidence indicates that viral infections of the heart contribute to ongoing myocarditis and dilated cardiomyopathy. Murine models of coxsackievirus B3 (CVB3)-induced myocarditis mimic the human disease and allow identification of susceptibility factors that modulate the course of viral myocarditis. Susceptible mouse strains develop chronic myocarditis on the basis of restricted viral replication, whereas resistant strains recover after successful virus elimination. In comparative whole-genome microarray analyses of infected hearts, several genes involved in the processing and presentation of viral epitopes were found to be uniformly up-regulated in acutely CVB3-infected susceptible mice compared with resistant animals. In particular, expression of the catalytic subunits LMP2, LMP7, and MECL-1, immunoproteasome proteins important in the generation of major histocom-patibility complex (MHC) class I-restricted peptides, was clearly enhanced in the susceptible host. Increased expression resulted in enhanced formation of immunoproteasomes and altered proteolytic activities of proteasomes in the heart. This was accompanied by a concerted up-regulation of the antigen-presenting machinery in susceptible mice. Thus, we propose that increased formation of immunoproteasomes in susceptible mice affects the generation of antigenic peptides and the subsequent T-cell-mediated immune responses.     

Coxsackievirus B-3-induced myocarditis. Effect of sex steroids on viremia and infectivity of cardiocytes.

Male and female BALB/c mice were inoculated with various concentrations of coxsackievirus, group B, type 3 (CVB3), ranging from 10 to 10(7) plaque-forming units (PFU). Lower viral doses (greater than 10(2) PFU) induced severe myocarditis in male mice but caused little injury in females. With 10(7) PFU, females also developed severe disease. Females may be relatively resistant to CVB3-induced myocarditis because virus entry into the blood and heart is less effective. Males given 125I-CVB3 show approximately 2-4 and 20-fold more radioactivity in the peripheral blood and heart, respectively, than females. No differences were observed between the sexes in 125I-bovine serum albumin penetration. Sex steroid hormones influence viremia and virus localization; females given exogenous testosterone and progesterone demonstrate ten times more virus in their hearts than animals given estradiol. The hormones may act by increasing virus receptor expression on endothelial cells and myocytes.     

11，12-EET对长时间保存幼兔供心再灌注后心肌细胞凋亡及bcl-2 mRNA基因表达的影响

目的：观察11,12-epoxyeicosatrienoic acid（11，12-EET）作为心脏停搏液及保存液成分对未成熟兔离体心脏长时间保存后经再灌注，心肌细胞凋亡及bcl-2 mRNA基因表达的变化，并探讨其可能机制及意义。 方法： 利用非循环式Langendorff灌注装置将24只未成熟离体兔心制成实验模型，按单纯随机分配原则分成正常对照组（心脏未经停搏及再灌注处理）、托马斯（ST）液对照组（St.Thomas No.2液为停搏液及保存液处理）和EET组（St.Thomas No.2液＋11，12-EET为停搏液及保存液处理）。ST对照组及EET组心脏保存24 h（4 ℃）后再灌注30 min。采用TUNEL法观察各组心肌细胞凋亡数以及原位杂交方法检测心肌bcl-2 mRNA基因表达等指标。 结果： (1)ST对照组及EET组的心肌细胞凋亡数均高于正常对照组。(2)EET组的心肌细胞凋亡数明显低于ST对照组，bcl-2 mRNA基因表达则显著高于ST对照组。 结论： 本研究采用不同方法在供体心脏保存过程中，均出现不同程度的心肌细胞凋亡。然而， 11，12-EET能够明显减少心肌细胞凋亡。上调凋亡抑制基因bcl－2 mRNA表达可能是11，12-EET减少心肌细胞凋亡的机制之一。     

Expression of coxsackievirus and adenovirus receptor in neointima of the rat carotid artery.

Our previous study revealed that the coxsackievirus and adenovirus receptor (CAR) is a homophilic cell adhesion molecule and may function as a sensor of cell-cell interactions in the brain and damaged heart. In this study, we investigated if CAR expression is involved in the formation of neointimal hyperplasia using a balloon injury model of rat carotid artery. Cultured vascular smooth muscle cells (SMCs) from rat aorta were also studied. CAR antigen was constitutively detected in the endothelial cells (ECs) but not in SMCs before injury. On Day 5 after balloon injury, CAR was expressed strongly in the first layer of medial SMCs. Neointimal hyperplasia was observed on Day 7, and strong expressions of CAR concomitantly with proliferating cell nuclear antigen (PCNA) were obvious in the neointimal SMCs, while CAR in medial SMCs disappeared. The expression of CAR mRNA reached a peak on Day 7 and declined gradually to the basal levels. When the ECs regenerated on Day 14, CAR antigen was observed in the ECs but disappeared in the neointima. CAR together with PCNA was expressed abundantly in the proliferating SMCs in vitro and diminished in cells grown to a confluent state. The abundant expression of CAR in the neointima may facilitate an adenoviral gene therapy.     

大鼠心肌细胞和心肌细胞系H9C2表达弹性蛋白

目的 检测弹性蛋白在大鼠心肌细胞和H9C2心肌细胞系内的表达。 方法 取新生和成年SD大鼠各5例的新鲜心脏，冷冻切片；取20只新生3 d SD大鼠心脏，通过0.05%胰蛋白酶和0.075%Ⅱ型胶原酶消化法，获取原代心肌细胞；免疫组织化学和免疫荧光技术检测大鼠心肌组织、原代心肌细胞和H9C2心肌细胞系的弹性蛋白表达;ELISA法检测培养的H9C2心肌细胞系上清中的弹性蛋白。 结果 在大鼠心肌细胞、H9C2心肌细胞系和新生、成年大鼠心肌组织上均发现心肌肌钙蛋白T和弹性蛋白的共表达。免疫组织化学结果发现，弹性蛋白在成纤维细胞、平滑肌细胞和细胞间质内均有表达。H9C2心肌细胞培养液的上清中检测到少量的弹性蛋白。 结论 大鼠心肌细胞表达弹性蛋白，可能参与心肌细胞的势能和细胞间质弹性纤维的构建。