| The restoration of blood perfusion in ischemic myocardium may induce myocardial damage, a phenomenon known as myocardial ischemia-reperfusion (I/R) injury (MIRI), which usually occurs in cardiovascular diseases such as coronary artery disease and the therapies for cardiovascular diseases such as open heart surgery under extracorporeal circulation, heart transplantation, bypass operation of coronary artery, thrombolytic therapy and angioplasty. The basic mechanisms of MIRI include oxidative stress, calcium overload, energy paradox, which result in cardiomyocyte death. Apoptosis, a programmed cell death, is one of the main factors that contribute to post-reperfusion cardiomyocyte death. Nucleolin is an important phosphoprotein of the eukaryotic cell and is known to be involved in many metabolic processes besides a potential role in ribosome biogenesis and mature, which include control of cell proliferation and growth, regulation of embryogenesis, cytokinesis, chromatin replication and nucleologenesis processes, etc. As a shuttle protein and a cell surface receptor, nucleolin is involved in substance transportation between the nucleus and cytoplasm and the extracellular regulation of nucleus function. Recent studies showed that nucleolin also played an important role in regulating apoptosis occurrence. Previous studies found that the occurrence of apoptosis in a variety of tumor cells treated with drugs or ultraviolet radiation was coupled with downregulation of nucleolin protein expression, nucleolin cleavage and translocation change. Conversely, nucleolin over-expression contributed to reduced apoptosis. Furthermore, some results suggested that nucleolin functioned through regulating the mRNA stability of apoptosis-related molecules such as Bcl-2. However, it remains unknown whether nucleolin plays a role in MIRI-induced apoptosis and what the detailed mechanism is.In the present study, we observed the occurrence of apoptosis and nucleolin expression in rat myocardium with ischemia-reperfusion injury. In addition, we further explored the detailed mechanism by which nucleolin affected the occurrence and development of apoptosis by investigating the specific role of nucleolin in hydrogen peroxide (H2O2)-induced apoptosis in rat cardiomyocytes. The main results are as follows:1. Expression of nucleolin in the myocardium in rats subjected to MIRI1) Establishment of MIRI rat modelAt different time of reperfusion after the ligation of left anterior descending coronary artery (LADCA) in rats for 30 minutes, serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine kinase (CK) and creatine kinase (CK-M) markedly increased. The generation of reactive oxygen species (ROS) in myocardial tissue was increased. These results indicated a successful MIRI model.2) Effect of MIRI on apoptosisIn different time of reperfusion after 30 minutes of myocardial ischemia, the caspase-3 activity in myocardium was increased; terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) detection revealed that the apoptosis was elevated after ischemia-reperfusion injury; agarose gel electrophoresis showed that ischemia-reperfusion injury led to the formation of DNA-Ladder. The studies above showed that MIRI contributed to an increase in apoptosis occurrence.3) Effect of MIRI on the expression of nucleolin in rat myocardiumIn myocardial tissue at different time periods of reperfusion after 30 minutes of ischemia, nucleolin mRNA and protein levels were significantly decreased and the 110 kD nucleolin protein was cleaved to an 80 kD fragment, suggesting that MIRI led to an increase in apoptosis, downregulation of nucleolin expression and cleavage of nucleolin protein.2. The effect of nucleolin on H2O2-induced apoptosis in cardiomyocytes1) The temporal-spatial expression patterns of nucleolin in myocardial cells subjected to oxidative stress:The studies in the neonate rat cardiomyocytes incubated with 0.5 mM H2O2 showed that the nucleolin mRNA and protein levels were significantly reduced and nucleolin protein was cleaved. In addition, most of nucleolin distributed in myocardial nucleus under normal circumstances and H2O2 exposure resulted in translocation of nucleolin to the cytoplasm.2) Effects of nucleolin overexpression on H2O2-induced apoptosis in cardiomyocytesTransfecting nucleolin expression vector into myocardial cells, we found that after the H2O2 treatment, nucleolin overexpression inhibited H2O2-induced caspase-3 activation and apoptosis.3) Effects of low nucleolin expression on H2O2-induced apoptosis in cardiomyocytesUsing RNA interference technology to block the expression of nucleolin, we found that after H2O2 treatment, reduced expression of nucleolin promoted H2O2-induced caspase-3 activation and apoptosis. The results suggested that nucleolin expression was suppressed coupled with the cleavage of nucleolin protein in rat cardiomyocytes subjected to H2O2-induced injury. Nucleolin inhibited H2O2-induced apoptosis in rat cardiomyocytes.3. anti-apoptotic mechanism of nucleolin in cardiomyocytes1)Effects of different domains of nucleolin on H2O2-induced apoptosisThe amino-terminal domain of nucleolin contains proteolytic sites, so we assumed that amino-terminal domain of nucleolin was excised to form an 80kD fragment during oxidative injury, and explored the potential anti-apoptotic mechanism of nucleolin by using two mutants: N-terminal fragment of nucleolin (amino acids 1-309), C-terminal fragment of nucleolin (amino acids 310-713). Neonatal rat cardiomy-ocytes were transfected with expression vectors of different nucleolin domain. The caspase-3 activity and apoptosis rate weren't influenced by only transfection. After H2O2 treatment, the caspase-3 activity in cardiomyocytes transfected with N-terminal fragment of nucleolin (amino acids 1-309) was lower than the cells transfected with empty vector. However, the caspase-3 activity and apoptosis rate in cardiomyocytes transfected with carboxy-terminal fragment of nucleolin (amino acids 310-713) were higher than those of group transfected with empty vector. It indicated that N-terminal fragment (amino acids 1-309) of nucleolin had less anti-apoptosis ability than full-length nucleolin, and C-terminal fragment (amino acids 1-309) of nucleolin could promote the occurrence of apoptosis.2) Effects of nucleolin and its different domains on expression of apoptosis-related gene Bcl-2 and BaxWe transfected myocardial cells with nucleolin RNA interference vector or expression vectors of different nucleolin domain, and found that Bcl-2 expression markedly increased in nucleolin-overexpressed cells and significantly reduced in low nucleolin-expressed cells. Whereas, the expression level of Bcl-2 in cells with nucleolin N-terminal fragment (amino acids 1-309) overexpression or carboxy-terminal fragment (amino acids 310-713) overexpression was not significantly different from the control group. Bax expression was significantly decreased in cells with overexpressed nucleolin and increased in cells with low nucleolin expression. But the expression level of Bax in cells with nucleolin N-terminal fragment (amino acids 1-309) overexpression was not significantly different from the control group. However, Bax expression increased in cells with the carboxy-terminal fragment of nucleolin (amino acids 310-713) overexpression. These results indicated that nucleolin could up-regulate the expression of Bcl-2 and suppress the expression Bax, and the carboxy-terminal fragment of nucleolin (amino acids 310-713) could promote the expression Bax.After treatment with H2O2, Bcl-2 expression in the cells reduced compared with the untreated group. H2O2-induced down-regulation of Bcl-2 expression was inhibited by nucleolin overexpression and promoted by nucleolin RNA interference. The overexpression of nucleolin N-terminal fragment (amino acids 1-309) or carboxy-terminal fragment (amino acids 310-713) didn't influenced H2O2-induced down-regulation of Bcl-2 expression. Conversly, Bax expression was increased by H2O2 treatment. H2O2-induced up-regulation of Bax expression was inhibited by nucleolin overexpression and promoted by nucleolin RNA interference or nucleolin carboxy-terminal fragment (amino acids 310-713) overexpression. However, the overexpression of nucleolin N-terminal fragment (amino acids 1-309) didn't influenced H2O2-induced up-regulation of Bax expression. Taken together, the results above indicated that in rat myocardial cells damaged by H2O2, nucleolin exerted anti-apoptotic role by increasing Bcl-2 expression and inhibiting Bax expression. The carboxy-terminal fragment of nucleolin (amino acids 310-713) promoted occurrence of apoptosis by increasing Bax expression.3) Effects of nucleolin and its different domains on mRNA stability of the apoptosis-related gene Bcl-2 and BaxWe analyzed mRNA stability of Bcl-2 and Bax genes in the rat myocardial cells incubated with Actinomycin D by using real time PCR and found that Bcl-2 mRNA stability weren't influeced by nucleolin overexpression, but reduced by nucleolin low-expression. H2O2 exposure contributed to the reduced stability of Bcl-2 mRNA, high expression of nucleolin inhibited the reduction of Bcl-2 mRNA stability by H2O2, but Nucleolin N-terminal fragment (amino acids 1-309) overexpression had no obvious effects on H2O2-reduced Bcl-2 mRNA stability. The results suggested that nucleolin played the anti-apoptotic role during oxidative stress in cardiomyocytes by increasing Bcl-2 expression via increasing Bcl-2 mRNA stability.Bax mRNA stability of rat myocardial cells wasn't influenced by the overexpression of nucleolin or low-expression of nucleolin or by the overexpression of nucleolin N-terminal fragment (amino acids 1-309), whereas the overexpression of carboxy-terminal fragment of nucleolin (amino acids 310-713) significantly increased the stability of Bax mRNA. H2O2 exposure led to an increase in Bax mRNA stability, which was inhibited by nucleolin overexpression. The results suggested that nucleolin played the anti-apoptotic role by decreasing Bax expression via decreasing Bax mRNA stability in rat cardiomyocytes exposed to oxidative stress. The carboxy-terminal fragment of nucleolin (amino acids 310-713) promoted apoptosis by up-regulating the expression of Bax via increasing the stability of Bax mRNA.4) Interaction of nucleolin and the mRNA of apoptosis-related gene Bcl-2 or BaxRat myocardial cells lysates were extracted and immunoprecipitated with nucleolin antibody, and the precipitate further analyzed by RT-PCR. The results showed that nucleolin could bind with Bcl-2 and Bax mRNA. Oxidative stress increased the binding of nucleolin to Bax mRNA but reduced that to Bcl-2 mRNA. Parallel Western blot results showed that nucleolin was a 110kD protein in normal rat myocardial cells and was cleaved into a fragment of about 80kD after H2O2 exposure. The study above indicated that the full length nucleolin might bind with Bcl-2 mRNA to increase its stability, however the 80kD fragment of nucleolin might bind with Bax mRNA to increase its stability.In conclusion, nucleolin is a very important anti-apoptotic protein and has important protective effect on cardiomyocytes against myocardial ischemia-reperfusion injury. Nucleolin expression was downregulated and nucleolin protein was cleaved in the myocardium with ischemia-reperfusion injury and cardiomyocytes subjected to oxidative stress, which might be one of mechanisms of myocardial ischemia-reperfusion injury. Nucleolin could bind with Bcl-2 and Bax mRNA under normal condition. Oxidative stress increased the binding of nucleolin to Bax mRNA and reduced the binding of nucleolin to Bcl-2 mRNA. Full length of nucleolin protein played anti-apoptotic role by binding with Bcl-2 mRNA to increase its stability and expression and by binding with Bax mRNA to decrease its stability and expression. nucleolin C-terminal fragment played the pro-apoptotic role by increasing the stability and expression of Bax mRNA. Oxidative stress might cause apoptosis in cardiomyocytes by increasing the binding of 80kD nucleolin to Bax mRNA to increase the stability of Bax mRNA and reducing the binding of nucleolin110kD to Bcl-2 mRNA to reduce the stability of Bcl-2 mRNA. |
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