HL-1细胞培养及房颤细胞模型的建立

目的:利用HL-1细胞建立快速起搏模型,对心房颤动(atrial fibrillation,AF)早期的重构现象进行初步研究。方法:培养HL-1细胞,建立快速电场刺激起搏细胞模型,利用全细胞膜片钳技术记录刺激前后HL-1细胞的动作电位周期,透射电镜观察细胞超微结构的变化。结果:将细胞接种于培养皿中,72 h后细胞呈融合状态,全细胞膜片钳记录培养HL-1细胞及经电场刺激(600次/min,1 V/cm)24 h后的心房肌细胞的动作电位周期,动作电位周期分别为106 ms,45 ms,刺激前后差异有统计学意义(P0.05)。透射电镜观察到刺激后HL-1细胞超微结构发生去分化改变。结论:经快速起搏24 h后,HL-1细胞发生了电及结构重构;利用HL-1细胞建立快速起搏的房颤模型,可以对房颤早期的重构机制进行研究。     

Determination of cryothermal injury thresholds in tissues impacted by cardiac cryoablation

Despite widespread clinical use of cryoablation, there remain questions regarding dosing and treatment times which may affect efficacy and collateral injury. Dosing and treatment times are directly related to the degree of cooling necessary for effective lesion formation. Human and swine atrial, ventricular, and lung tissues were ablated using two cryoablation systems with concurrent infrared thermography. Post freeze-thaw samples were cultured and stained to differentiate viable and non-viable tissue. Matlab code correlated viability staining to applied freeze-thaw thermal cycles, to determine injury thresholds. Tissue regions were classified as live, injured, or dead based upon staining intensity at the lesion margin. Injury begins at rates of ∼10 °C/min to 0 °C, with non-viable tissue requiring cooling rates close to 100 °C/min to ∼ −22 °C for swine and significantly greater cooling to −26 °C for human tissue (p = 0.041). At similar rates, lung tissue injury began at 0 °C, with human tissue requiring significantly less cooling, to ∼ −15 °C for complete necrosis and −26 °C for swine (p = 0.024). Data suggest that there are no significant differences between swine and human myocardial response, but there may be differences between swine and human lung cryothermal tolerance.     

A microscale model for prediction of breast cancer cell damage during cryosurgery

The morphology of cancerous breast tissue is characterized by tightly packed groups of small malignant cells, as found in most duct cell carcinoma. This special structure affects the osmotic responses of the cells to freezing and hence their probability of damage from cellular dehydration or intracellular ice formation. A mathematical model has been developed to study the microscale damage to these breast cancer cells during cryosurgery by accounting for their special structure. The model is based on a spherical unit comprised of an extracellular region that surrounds several layers of cancer cells, as experimentally observed of breast duct cell carcinoma by other researchers. Temperature transients in the breast cancer undergoing cryosurgery are calculated numerically using the Pennes equation. When subjected to various thermal histories, both cellular dehydration and intracellular ice formation in the unit structure are examined by considering the cell-to-cell contact and water transport at the microscale level. It is found that the cells in the inner layers hardly dehydrated while those in the outermost layer do greatly. The results help interpret the previously observed experimental phenomena that breast cancer tissues exhibit intracellular ice formation even at a slow cooling rate of -3 degrees C/min. In the attempt to better define an optimal procedure for breast cancer cryosurgery, various freezing protocols are simulated. The constant heat flux protocol induces greater cellular dehydration and higher intracellular ice formation probability simultaneously compared to the other protocols studied.     

Theoretical analysis of unidirectional intercellular ice propagation in stratified cell clusters

A recently published theoretical analysis of intercellular ice propagation in stratified cell clusters comprising concentric spherical layers, by Zhang, Xu, Sandison, and Zhang [Cryobiology 47 (2003) 143], has been examined and is shown to be flawed. New equations are derived subject to simplifying assumptions implicit in the theoretical model presented by Zhang and co-workers. The conclusion by Zhang and colleagues that intercellular ice propagation can be assumed to be instantaneous is demonstrated to be invalid.     

Spiral reentry waves in confluent layer of HL-1 cardiomyocyte cell lines

Cardiac excitation waves that arise in heart tissues have long been an important research topic because they are related to various cardiac arrhythmia. Investigating their properties based on intact animal whole hearts is important but quite demanding and expensive. Subsequently, dissociated cardiac cell cultures have been used as an alternative. Here, we access the usefulness of cardiomyocyte cell line HL-1 in studying generic properties of cardiac waves. Spontaneous wave activities in confluent populations of HL-1 cells are monitored using a phase-contrast optical mapping system and a microelectrode array recording device. We find that high-density cultures of HL-1 cells can support well-defined reentries. Their conduction velocity and rotation period both increase over few days. The increasing trend of rotation period is opposite to the case of control experiments using primary cultures of mouse atrial cells. The progressive myolysis of HL-1 seems responsible for this difference.     

Measurement of phosphorylated phospholamban levels in cardiomyocytes (HL-1) by immunoprecipitation

Phospholamban (PLN) is a key regulatory protein involved in cardiac calcium signaling through the pumping of cytoplasmic Ca²⁺ into the sarcoplasmic reticulum (SR). Recent systems-level studies have focused on integrating quantitative data (e.g. protein expression levels) for a better understanding of cardiac systems biology. In this view, we developed a capillary electrophoresis (CE) based immunoprecipitation method for the measurement of phospho-PLN (ser 16) in cardiomyocytes (HL-1 cell line). Dose-dependent isoproterenol (Iso) treated cells were analyzed using CE, and the phospho-PLN levels were quantified using specific polyclonal antibodies. The CE method employed was accurate, quick and easier compare to other techniques and the results are useful for the subsequent computational systems biology research.     

Distinct organization of energy metabolism in HL-1 cardiac cell line and cardiomyocytes

Expression and function of creatine kinase (CK), adenylate kinase (AK) and hexokinase (HK) isoforms in relation to their roles in regulation of oxidative phosphorylation (OXPHOS) and intracellular energy transfer were assessed in beating (B) and non-beating (NB) cardiac HL-l cell lines and adult rat cardiomyocytes or myocardium. In both types of HL-1 cells, the AK2, CKB, HK1 and HK2 genes were expressed at higher levels than the CKM, CKMT2 and AK1 genes. Contrary to the saponin-permeabilized cardiomyocytes the OXPHOS was coupled to mitochondrial AK and HK but not to mitochondrial CK, and neither direct transfer of adenine nucleotides between CaMgATPases and mitochondria nor functional coupling between CK-MM and CaMgATPases was observed in permeabilized HL-1 cells. The HL-1 cells also exhibited deficient complex I of the respiratory chain. In conclusion, contrary to cardiomyocytes where mitochondria and CaMgATPases are organized into tight complexes which ensure effective energy transfer and feedback signaling between these structures via specialized pathways mediated by CK and AK isoforms and direct adenine nucleotide channeling, these complexes do not exist in HL-1 cells due to less organized energy metabolism.     

Phosphoinositide-3-kinase/akt - dependent signaling is required for maintenance of [Ca2+]i,ICa, and Ca2+ transients in HL-1 cardiomyocytes

The phosphoinositide 3-kinases (PI3K/Akt) dependent signaling pathway plays an important role in cardiac function, specifically cardiac contractility. We have reported that sepsis decreases myocardial Akt activation, which correlates with cardiac dysfunction in sepsis. We also reported that preventing sepsis induced changes in myocardial Akt activation ameliorates cardiovascular dysfunction. In this study we investigated the role of PI3K/Akt on cardiomyocyte function by examining the role of PI3K/Akt-dependent signaling on [Ca²⁺]_i, Ca²⁺ transients and membrane Ca²⁺ current, I_Ca, in cultured murine HL-1 cardiomyocytes. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (1–20 μM), a specific PI3K inhibitor, dramatically decreased HL-1 [Ca²⁺]_i, Ca²⁺ transients and I_Ca. We also examined the effect of PI3K isoform specific inhibitors, i.e. α (PI3-kinase α inhibitor 2; 2–8 nM); β (TGX-221; 100 nM) and γ (AS-252424; 100 nM), to determine the contribution of specific isoforms to HL-1 [Ca²⁺]_i regulation. Pharmacologic inhibition of each of the individual PI3K isoforms significantly decreased [Ca²⁺]_i, and inhibited Ca²⁺ transients. Triciribine (1–20 μM), which inhibits AKT downstream of the PI3K pathway, also inhibited [Ca²⁺]_i, and Ca²⁺ transients and I_Ca. We conclude that the PI3K/Akt pathway is required for normal maintenance of [Ca²⁺]_i in HL-1 cardiomyocytes. Thus, myocardial PI3K/Akt-PKB signaling sustains [Ca²⁺]_i required for excitation-contraction coupling in cardiomyoctyes.     

Cytoprotective response of A1, a Bcl-2 homologue expressed in mature human neutrophils and promyelocytic HL-60 cells, to oxidant stress-induced cell death

The ability to generate reactive oxidative intermediates is one of the quintessential properties of mature human neutrophils. Endogenously generated oxidants have been shown to be an important mechanism underlying neutrophil cell death. In acute lung inflammation, newly recruited neutrophils further encounter external oxidants, including reactive oxygen and nitrogen intermediates. In our present study, we showed that A1, a constitutive and inducible Bcl-2 homologue expressed in mature circulating human neutrophils, might confer the protection from hydrogen peroxide (H₂O₂)- and peroxynitrite (ONOO)-induced cell death. Utilizing the myeloid precursor cell line, HL-60, we further examined the hypothesis that A1 was capable of conferring cytoprotective activity against these oxidative stresses. Whereas the control-transfected HL-60 cells expressed small amounts of A1 and were sensitive to the biologically relevant, cell death-inducing oxidants, H₂O₂ and ONOO, the stable transfectants that overexpressed A1 were significantly more tolerant. Furthermore, there was a correlation between the level of A1 expression and the anti-apoptotic activity. Thus, our results suggest a cytoprotective role of A1 in mature human neutrophils under oxidant stresses in host defense and inflammation.     

Different kinetics of the regulation of respiration in permeabilized cardiomyocytes and in HL-1 cardiac cells: Importance of cell structure/organization for respiration regulation

The aim of this study was to investigate the mechanism of cellular regulation of mitochondrial respiration in permeabilized cardiac cells with clearly different structural organization: (i) in isolated rat cardiomyocytes with very regular mitochondrial arrangement, (ii) in HL-1 cells from mouse heart, and (iii) in non-beating (NB HL-1 cells) without sarcomeres with irregular and dynamic filamentous mitochondrial network. We found striking differences in the kinetics of respiration regulation by exogenous ADP between these cells: the apparent Km for exogenous ADP was by more than order of magnitude (14 times) lower in the permeabilized non-beating NB HL-1 cells without sarcomeres (25 ± 4 μM) and seven times lower in normally cultured HL-1 cells (47 ± 15 μM) than in permeabilized primary cardiomyocytes (360 ± 51 μM). In the latter cells, treatment with trypsin resulted in dramatic changes in intracellular structure that were associated with 3-fold decrease in apparent Km for ADP in regulation of respiration. In contrast to permeabilized cardiomyocytes, in NB HL-1 cells creatine kinase activity was low and the endogenous ADP fluxes from MgATPases recorded spectrophotometrically by the coupled enzyme assay were not reduced after activation of mitochondrial oxidative phosphorylation by the addition of mitochondrial substrates, showing the absence of ADP channelling in the NB HL-1 cells. While in the permeabilized cardiomyocytes creatine strongly activated mitochondrial respiration even in the presence of powerful competing pyruvate kinase-phosphoenolpyruvate system, in the NB HL-1 cells the stimulatory effect of creatine was not significant. The results of this study show that in normal adult cardiomyocytes and HL-1 cells intracellular local restrictions of diffusion of adenine nucleotides and metabolic feedback regulation of respiration via phosphotransfer networks are different, most probably related to differences in structural organization of these cells.     

Binding strength and dynamics of invariant natural killer cell T cell receptor/CD1d-glycosphingolipid interaction on living cells by single molecule force spectroscopy

Invariant natural killer T (iNKT) cells are a population of T lymphocytes that play an important role in regulating immunity to infection and tumors by recognizing endogenous and exogenous CD1d-bound lipid molecules. Using soluble iNKT T cell receptor (TCR) molecules, we applied single molecule force spectroscopy for the investigation of the iNKT TCR affinity for human CD1d molecules loaded with glycolipids differing in the length of the phytosphingosine chain using either recombinant CD1d molecules or lipid-pulsed THP1 cells. In both settings, the dissociation of the iNKT TCR from human CD1d molecules loaded with the lipid containing the longer phytosphingosine chain required higher unbinding forces compared with the shorter phytosphingosine lipid. Our findings are discussed in the context of previous results obtained by surface plasmon resonance measurements. We present new insights into the energy landscape and the kinetic rate constants of the iNKT TCR/human CD1d-glycosphingolipid interaction and emphasize the unique potential of single molecule force spectroscopy on living cells.     

Inhibition of THP-1 cell adhesion to endothelial cells by alpha-tocopherol and alpha-tocotrienol is dependent on intracellular concentration of the antioxidants

Vitamin E analogs such as alpha-tocopherol and alpha-tocotrienol have been shown to reduce endothelial expression of adhesion molecules. The reactivity of alpha-tocopherol and alpha-tocotrienol in inhibiting lipid peroxidation in vitro was essentially identical but the inhibition of adhesion of THP-1 cells, a monocytic-"like" cell line, to endothelial cells differs substantially. To determine the mechanism underlying this response, human umbilical vein endothelial cells (HUVECs) were assessed for their ability to accumulate vitamin E analogs. alpha-Tocotrienol accumulated in HUVECs to levels approximately 10-fold greater than that of alpha-tocopherol. The decrease in expression of vascular cell adhesion molecule-1 (VCAM-1) and the adhesion of THP-1 cells to HUVECs by alpha-tocopherol and alpha-tocotrienol was also determined. Both alpha-tocopherol and alpha-tocotrienol suppressed VCAM-1 expression and adhesion of THP-1 cells to HUVECs in a concentration-dependent manner. The efficacy of tocotrienol for reduction of VCAM-1 expression and adhesion of THP-1 cells to HUVECs was also 10-fold higher than that of tocopherol. The inhibitory effects of vitamin E analogs on the adhesiveness of endothelial cells clearly correlated with their intracellular concentrations. The data demonstrated that, in assessing the biological responses of antioxidants, intracellular accumulation and metabolism were additional important factors that must be considered.     

Down-regulation of immediate early gene egr-1 expression in rat C6 glioma cells by short-term exposure to high salt culture medium

Influence of high salt culture conditions on the expression of immediate early gene egr-1 in rat C6 glioma cells was investigated by measuring both Egr-1 mRNA and protein levels in the cells exposed to the medium containing high concentrations of NaCl. The exposure to high salt medium reduced Egr-1 mRNA and protein levels, while Egr-1 mRNA levels were not altered by the medium containing either sucrose or glycerol. Veratridine and monensin also reduced Egr-1 mRNA levels, similar in extent to that induced by high salt medium. Imaging analysis indicated that the exposure to high salt medium induced the elevation of Na+ levels within the cells. These results indicate that neither hyperosmotic pressure nor ionic strength of high salt medium contribute to the reduction of Egr-1 expression, and suggest that the elevation of intracellular Na+ concentration is closely associated with the down-regulation of egr-1 gene expression.     

Protein kinase WNK1 promotes cell surface expression of glucose transporter GLUT1 by regulating a Tre-2/USP6-BUB2-Cdc16 domain family member 4 (TBC1D4)-Rab8A complex

One mechanism by which mammalian cells regulate the uptake of glucose is the number of glucose transporter proteins (GLUT) present at the plasma membrane. In insulin-responsive cells types, GLUT4 is released from intracellular stores through inactivation of the Rab GTPase activating protein Tre-2/USP6-BUB2-Cdc16 domain family member 4 (TBC1D4) (also known as AS160). Here we describe that TBC1D4 forms a protein complex with protein kinase WNK1 in human embryonic kidney (HEK293) cells. We show that WNK1 phosphorylates TBC1D4 in vitro and that the expression levels of WNK1 in these cells regulate surface expression of the constitutive glucose transporter GLUT1. WNK1 was found to increase the binding of TBC1D4 to regulatory 14-3-3 proteins while reducing its interaction with the exocytic small GTPase Rab8A. These effects were dependent on the catalytic activity because expression of a kinase-dead WNK1 mutant had no effect on binding of 14-3-3 and Rab8A, or on surface GLUT1 levels. Together, the data describe a pathway regulating constitutive glucose uptake via GLUT1, the expression level of which is related to several human diseases.