The Role And Mechanism Of Nucleolin In Myocardial Ischemic Preconditioning

Myocardial ischemia and ischemia/reperfusion (I/R) injury are the major causes for myocardial remodeling and heart failure. Therefore, reducing myocardial injury plays a critical role in the protection from heart failure. Myocardial ischemic preconditioning (IP) is an important endogenous protection mechanism, which was discovered to significantly reduce myocardial IR injury. Clarifying the mechanism by which IP protects myocardium has important scientific significance and may provide new ideas for the prevention and treatment of myocardial injury and heart failure.Nucleolin (C23) is the most abundant RNA-binding protein in the cell nucleolus. Its major functions are binding and transporting rRNAs, regulating the assembly of ribosomes and regulating the growth and proliferation of cells. There are three major functional domains in nucleolin:the N-terminal, the central and the C-terminal domains. The most important functions of nucleolin, such as the regulation of synthesis and maturation of ribosomes and regulation of growth and proliferation of cells, mainly depend on the RNA binding domain in the central region of the protein.Nucleolin is a very important multifunctional shuttle protein in the cell. Other than the aforementioned biological functions, in recent years, nucleolin was also reported playing very important roles in the replication of hepatitis C virus, in the infection processes of human immunodefi-ciency virus and human parainfluenza virus, and in the regulation of gene transcriptions. In previous studies, we observed the downregulations of nucleolin expression in apoptosis induced by H2O2 in C2C12 cells, HUVEC and RAW264.7 macrophages. Further studies in this lab using transfection of eukaryotic expression vector and antisense oligonu-cleotides of nucleolin demonstrated that nucleolin played important cytoprotective effects on cellular injuries induced by oxidative stress and found that the heat shock and heat shock protein 70 could inhibit the downregulation of nucleolin induced by oxidative stress as well as apoptosis. These results suggest that nucleolin may be an important anti-damage factor. However, whether nucleolin plays some roles in myocardial protection of IP or not and what the roles are, remain unclear.Based on the previous findings, in this study, we used an intact rat model of myocardial IP, and two cell models of hydrogen peroxide preconditioning and hypoxia preconditioning with neonatal rat cardiomyocytes to investigate the spatial and temporal expression patterns of nucleolin and the roles and major mechanisms of nucleolin in the myocardial protection of IP. The major methods and results are as follows:1. The myocarial protective role and expression of nucleolin in the intact rat model of myocardial IPLigation of anterior descending coronary artery in vivo was used in this study to establish the intact model of myocardial IP in rats. Briefly, episodes of ligation 5min, reperfusion 10min, and re-ligation 5min and reperfusion 10min were performed. The myocardial injuries were measured through serological enzymatic assay, analysis of caspase-3 activity and DNA ladder detection, while the nucleolin expression was detected with real-time PCR and Western-blot. The results showed that myocardial IP significantly up-regulated the expression of nucleolin in both mRNA and protein levels with the peak expression of mRNA at 12h, and that of protein at 12h and 24h, and that myocardial IP protected significantly myocardium against the injury induced by 30 min of ischemica followed by 3h of reperfusion, as indicated by the decrease of serological enzymatic indexes, myocardial caspase-3 activity and DNA laddering.2. The expression and cytoprotective effect of nucleolin in hydrogen peroxide or hypoxia-induced cell models of IP in cardiomyocytesPrimary cultures of neonatal rat cardiomyocytes were pretreated with sub-lethal hydrogen peroxide (100μM) or hypoxia to establish the cell model of IP. Briefly, the myocardial cells were pretreated with 100μM hydrogen peroxide for 90 min or hypoxia for 30 min, then recovered for 24h. The cell injuries were evaluated by MTT assay, release of LDH and detection of apoptotic percentage, while the expression of nucleolin was detected with real-time PCR and Western-blot. Sub-cellular fractionation and immunofluorescence were used to detect the cellular localization of nucleolin. The results showed that preconditioning with sub-lethal hydrogen peroxide or hypoxia, like IP, significantly attenuated the injuries of cardiomyocytes induced by oxidative stress (500μM H2O2) or hypoxia-reoxygenation (hypoxia 2h and reoxygenation 12-24h). The mRNA expression of nucleolin was gradually upregulated in these two cell models of IP with its peak expression at 12h, while the protein levels were also gradually upregulated with the maximum at 12h to 24h. Moreover, nucleolin mainly located in the nuclei of myocardial cells under normal conditions, but it translocated from the nuclei to the cytoplasm after the preconditioning with hydrogen peroxide or hypoxia.3. The role of nucleolin in the cytoprotection mediated by hydrogen peroxide or hypoxia preconditioning in neonatal rat cardiomyocytesThe effects of nucleolin overexpression and low expression on cytoprotection mediated by hydrogen peroxide preconditioning against the injury induced by oxidative stress (500μM H2O2) were determined with gene transfection and RNA interference technologies in neonatal rat cardiomyocytes. The results showed that overexpression of nucleolin enhanced the hydrogen peroxide preconditioning-induced cytoprotective effect against the injuries induced by hydrogen peroxide (500μM H2O2) in cardiomyocytes, as indicated by the decrease of LDH release and apoptotic percentage, and the increase of cell viability, while the suppressed expression of nucleolin with RNA interference, on the contrary, significantly reduced the cytoprotective effect mediated by hydrogen peroxide preconditioning. Similar results were obstained in the cytoprotection mediated by hypoxia preconditioning.4. Studies on the mechanisms of nucleolin involved in myocardial protection meiated by myocardial IP1) In order to screen the potential target genes which were regulated by nucleolin during oxidative stress, neonatal rat cardiomyocytes were exposed to hydrogen peroxide (500μM H2O2,6h). After co-immunopre-cipitation with a specific anti-nucleolin antibody, the total RNAs in the precipitate were isolated for gene profiling analysis with rat genome wide cDNA chips and bioinformatics technology. Ten candidate genes, which contain the binding sites of nucleolin, including Hsp32 and Hsp70 were primarily selected for further studies based on the results of gene profiling and bioinformatics analysis.2) The expression of Hsp32 and Hsp70 in the intact rat model and cell models of IP were found up-regulated significantly with real-time PCR and Western-blot techniques. The specific inhibitor of Hsp32 and RNA interference oligonucleotide of Hsp70 obviously attenduated the cytoprotective effect of hydrogen peroxide preconditioning against oxidative stress.3) The regulatory effects of nucleolin on gene expression of Hsp32 and Hsp70 were analyzed with transfection of nucleolin gene or its interference RNA plasmid. Analysis of mRNA stability, IP-RT-PCR, RNA-EMSA, UV-crosslinking and luciferase reporter assay were applied to further study the mechanisms by which nucleolin regulated the expression of Hsp32 and Hsp70 translationally. The results demonstrated that nucleolin up-regulated the gene expression of Hsp32 and Hsp70 by directly binding to the 3'-UTR of Hsp32 and Hsp70 mRNA and increasing their mRNA stabilities, thus played protective effect on cardiomyocytes during myocardial IP.In conclusion, the data in this study showed that mRNA and protein expression of nucleolin were upregulated by myocardial IP in the myocar-dium of rats and by hydrogen peroxide or hypoxia preconditioning in cardiomyocytes, these changes were also associated by the translocation of nucleolin from nuclei to cytosols. Nucleolin played very important myocardial protective roles by binding to the 3'-UTRs, and then upregulating the gene expression of both Hsp32 and Hsp70 in myocardial ischemic preconditioning.