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.     

Mechanical overload-induced apoptosis: A study in cultured neonatal ventricular myocytes and fibroblasts

Apoptosis of cardiac myocytes has been implicated in cardiac dysfunction due to chronic hemodynamic overload. Reports on the role of apoptosis in the transition from hypertrophy to decompensated heart failure are not unequivocal. In this study we analysed the direct relationship between mechanical overload and induction of apoptosis in an in vitro model of cultured heart cells. Cyclic mechanical stretch was applied to cultured neonatal rat ventricular myocytes and fibroblasts. Several indicators of apoptosis were examined, such as morphological features, caspase-3 activity and DNA fragmentation. Mechanical strain did not induce any significant change in these parameters as compared to non-stretched myocytes or fibroblasts. However, administration of staurosporine, a known inducer of apoptosis, induced massive apoptosis in myocytes as well as fibroblasts. We conclude that this in vitro cell model system lacks a direct link between mechanical stretch and apoptosis. The three-dimensional structure-function relationship of myocardial tissue in the intact heart may elicit stretch-induced molecular signaling cascades in a much more complex way than in monolayer cultures of cardiac cells.     

Cyclic stretch induces the release of growth promoting factors from cultured neonatal cardiomyocytes and cardiac fibroblasts

Growth factors and hormones may play an autocrine/paracrine role in mechanical stress-induced cardiac hypertrophy. Using an in vitro model of mechanical stress, i.e. stretch of cardiomyocytes and cardiac fibroblasts, we tested the involvement of growth factors and hormones in this process.We found that conditioned medium (CM) derived from 4 h cyclicly (1 Hz) stretched cardiomyocytes increased the rate of protein synthesis in static cardiomyocytes by 8 ± 3%. Moreover, CM derived from 2 h stretched fibroblasts increased the rate of protein synthesis in static fibroblasts as well as in static cardiomyocytes by 8 ± 2 and 6 ± 2%, respectively. Analysis of CM using size-exclusion HPLC showed that cardiomyocytes and fibroblasts released at least three factors with MW 10 kD, their quantities being time-dependently increased by stretch. Subsequent analyses using immunoassays revealed that cardiomyocytes released atrial natriuretic peptide (ANP) and transforming growth factor-beta1 (TGF₁) being increased by 45 ± 17 and 21 ± 4% upon 4 h of stretch, respectively. Fibroblasts released TGF₁ and very low quantity of endothelin-1 (ET-1). The release of TGF₁ was significantly increased by 18 ± 4% after 24 h of stretch in fibroblasts. Both cell types released no detectable amount of angiotensin II (Ang II).In conclusion, upon cyclic stretch cardiomyocytes and fibroblasts secrete growth factors and hormones which induce growth responses in cardiomyocytes and fibroblasts in an autocrine/paracrine way. TGF secreted by cardiomyocytes and fibroblasts, and ANP secreted by cardiomyocytes are likely candidates. We found no evidence for the involvement of Ang II and ET-1 in autocrine/paracrine mechanisms between cardiac cell types.     

髓样分化蛋白2基因沉默对高糖诱导的大鼠心肌细胞增殖抑制、凋亡及炎症反应的影响*

目的:研究髓样分化蛋白2(MD2)基因沉默对高糖(HG)诱导的大鼠心肌细胞增殖抑制、凋亡及炎症反应的影响及其机制。方法:体外大鼠心肌细胞系H9C2细胞随机分为4组(n=3):LG组、HG组、HG + NC组、HG + si-MD2组,分别转染MD2基因小干扰RNA(si-MD2)或阴性对照24 h后进行低糖或高糖处理48 h。RT-qPCR检测MD2及细胞内炎症细胞因子TNF-α、IL-1β、IL-6的表达水平,MTS法、流式细胞术检测细胞增殖能力、细胞周期和细胞凋亡率,Western blot法检测细胞内相关蛋白的表达水平及磷酸化水平。结果:转染si-MD2后,H9C2细胞中MD2的表达水平明显下降(P＜0.01)。与低糖(LG)组比较,高糖处理后的H9C2细胞中TNF-α、IL-1β、IL-6的mRNA水平显著升高,细胞增殖能力下降并发生G1期阻滞,细胞凋亡率和Cleaved Caspase-3蛋白水平升高(P＜ 0.01)。而MD2基因沉默可拮抗高糖对H9C2细胞增殖、细胞周期、凋亡及细胞中TNF-α、IL-1β、IL-6 mRNA水平的影响(P＜0.05)。Western blot测定结果表明高糖处理后的H9C2细胞中细胞外信号调节激酶(ERK1/2)、P38丝裂原活化蛋白激酶(P38 MAPK)和C-Jun氨基末端激酶(JNK)蛋白的磷酸化水平明显升高,而MD2基因沉默可抑制高糖诱导下的ERK1/2、P38 MAPK和JNK蛋白激活(P＜0.01)。结论:MD2基因沉默可能通过抑制ERK、P38 MAPK和JNK信号通路的激活来减少高糖诱导的大鼠心肌细胞炎症细胞因子表达,减少心肌细胞凋亡,促进细胞增殖。     

Dissection of the multiple mechanisms of TNF-alpha-induced apoptosis in liver injury

Tumor necrosis factor (TNF)-alpha-induced hepatocyte apoptosis is implicated in a wide range of liver diseases including viral hepatitis, alcoholic hepatitis, ischemia/reperfusion liver injury, and fulminant hepatic failure. TNF-alpha exerts a variety of effects that are mediated mainly by TNF-receptor 1 (TNF-R1) in cell death. The activation of TNF-R1 leads to the activation of multiple apoptotic pathways involving the activation of the pro-death Bcl-2 family proteins, reactive oxygen species, C-Jun NH2-terminal kinase, cathepsin B, acidic sphingomyelinase and neutral sphingomyelinase. These pathways are closely interlinked and mainly act on mitochondria, which release the apoptogenic factors and other events, resulting in apoptosis. This article reviews the recent progress in the molecular mechanisms of TNF-alpha-induced apoptosis in hepatocytes, and discusses how these molecular findings are shaping our understanding of the pathogenesis of liver diseases and our strategy to develop novel therapeutics.     

Intermittent hypoxia attenuates ischemia／reperfusion induced apoptosis in cardiac myocytes via regulating Bcl-2／Bax expression

Intermittent hypoxia has been shown to provide myocardial protection against ishemiaJreperfusion-induced injury.Cardiac myocyte loss through apoptosis has been reported in ischemia/reperfusion injury. Our aim was to investigate whether intermittent hypoxia could attenuate ischemia/reperfusion-induced apoptosis in cardiac myocytes and its potential mechanisms. Adult male Sprague-Dawley rats were exposed to hypoxia simulated 5000 m in a hypobaric chamber for 6 h/day, lasting 42 days. Normoxia group rats were kept under normoxic conditions. Isolated perfused hearts from both groups were subjected to 30 min of global ischemia followed by 60 min reperfusion.Incidence of apoptosis in cardiac myocytes was determined by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and DNA agarose gel electrophoresis. Expressions of apoptosis related proteins,Bax and Bcl-2, in cytosolic and membrane fraction were detected by Western Blotting. After ischemia/reperfusion,enhanced recovery of cardiac function was observed in intermittent hypoxia hearts compared with normoxia group.Ischemia/reperfusion-induced apoptosis, as evidenced by TUNEL-positive nuclei and DNA fragmentation, was significantly reduced in intermittent hypoxia group compared with normoxia group. After ischemia/reperfusion,expression of Bax in both cytosolic and membrane fractions was decreased in intermittent hypoxia hearts comparedwith normoxia group. Although ischemia/reperfusion did not induce changes in the level of Bcl-2 expression in cytosolic fraction between intermittent hypoxia and normoxia groups, the expression of Bcl-2 in membrane fraction was upregulated in intermittent hypoxia group compared with normoxia group. These results indicated that the cardioprotection of intermittent hypoxia against ischemia/reperfusion injury appears to be in part due to reducemyocardial apoptosis. Intermittent hypoxia attenuated ischemia/reperfusion-induced apoptosis via increasing the ratio of Bcl-2/Bax, especially in membrane fraction.     

Demethoxycurcumin induces Bcl-2 mediated G2/M arrest and apoptosis in human glioma U87 cells

Docking analysis of curcumin (C1), demethoxycurcumin (C2) and bisdemethoxycurcumin (C3) with Bcl-2 illustrated that among the three curcuminoids, C2 binds more efficiently into its putative active site. C1, C2 and C3 were purified from turmeric rhizomes to demonstrate the molecular mechanism of their anticancer activity on human glioma U87 cells. Human glioma U87 cells treated with curcuminoids resulted in activation of Bcl-2 mediated G2 checkpoint, which was associated with the induction of G2/M arrest and apoptosis. The binding of C1, C2 and C3 with Bcl-2 protein was confirmed with circular dichroism (CD) spectroscopy. Present work revealed that C2 induced Bcl-2 mediated G2/M arrest and apoptosis most effectively.     

Synthetic peptides and non-peptidic molecules as probes of structure and function of Bcl-2 family proteins and modulators of apoptosis

The Bcl-2 family includes a growing number of proteins that play an essential role in regulating apoptosis or programmed cell death. Members of this family display diverse biological functions and can either inhibit or promote cell death signals. Abnormal gene expression of some Bcl-2 family members such as Bcl-2 that inhibits apoptosis is found in a wide variety of human cancers and contributes to the resistance of tumor cells to conventional therapies through interfering with the cell death signals triggered by chemotherapeutic agents. As such, elucidating the structure-function and mechanism of the Bcl-2 family is important for understanding some of the fundamental principles underling the death and survival of cells and of practical value for developing potential therapeutics to control apoptosis in pathological processes. Synthetic peptides derived from homologous or heterogeneous domains in Bcl-2 family proteins that might mediate different biological activities provide simplified and experimentally more tractable models as compared to their full-length counterparts to dissect and analyze the complex functional roles of these proteins. Non-peptidic molecules identified from random screening of natural products or designed by rational structure-based techniques can mimic the effect of synthetic peptides by targeting similar active sites on a Bcl-2 family member protein. In this article, we review recent progress in using these synthetic peptides and non-peptidic mimic molecules to obtain information about the structure and function of Bcl-2 family proteins and discuss their application in modulating and studying intracellular apoptotic signaling.     

Magnolol induces apoptosis in osteosarcoma cells via G0/G1 phase arrest and p53-mediated mitochondrial pathway

Osteosarcoma is a highly invasive primary malignancy of bone. Magnolol is biologically active, which shows antitumor effects in a variety of cancer cell lines. However, it has not been elucidated magnolol's effects on human osteosarcoma cells (HOC). This study aimed to determine antitumor activity of magnolol and illustrate the molecular mechanism in HOC. Magnolol showed significant inhibition effect of growth on MG-63 and 143B cells and induced apoptosis and cell cycle arrest at G0/G1. In osteosarcoma cells, magnolol upregulated expressions of proapoptosis proteins and suppressed expressions of antiapoptosis proteins. Additionally, under the pretreatment of pifithrin-a (PFT-a, a p53 inhibitor), the magnolol-induced apoptosis was significantly reversed. The results above indicated that magnolol induces apoptosis in osteosarcoma cells may via G0/G1 phase arrest and p53-mediated mitochondrial pathway.