| Myocardial ischemic postconditioning (IPoC) is an important endogenous protection, which protects the heart against ischemia-reperfusion injury by reducing cardiac arrhythmia, improving heart function, and decreasing infarct size. However, the protection mechanisms of IPoC have not been clearly explained at present. Clarifying the mechanism by which IPoC protects myocardium may provide new ideas for the prevention and treatment of myocardial injury.The novel gene Mipu1 (myocardial ischemic preconditioning up-regulated protein 1), was recently identified in rat due to its up-regulation in response to myocardial ischemic preconditioning in our lab. We previously demonstrated that Mipu1 was up-regulated in myocardial ischemia-reperfusion injury and myocardial ischemic preconditioning, functioning to protect cardiomyocytes against oxidative stress. However, the expression pattern, mechanism of transcriptional regulation and it's role of Mipul in myocardial IPoC remain unclear. Based on the previous findings of our lab, we used an intact rat model of myocardial IPoC, two in vitro cell models of hydrogen peroxide injury and hypoxic postconditioning (HPC) in rat cardiomyocytes H9c2 in this study to investigate the expression pattern of Mipul, the role of CREB in the regulation of Mipu1 expression and the role of CREB-mediated Mipul expression in the myocardial protection of IPoC. The major methods and results summarized as follows:1. A model of myocardial ischemia-reperfusion injury was made in rats by the occlusion of left anterior descending artery (LAD) for 45 min and reperfused for 9 h. Myocardial IPoC was induced by three cycles of 30-s reperfusion and 30-s reocclusion of LAD at the very beginning of 9 h of reperfusion. The myocardial injuries were measured through serological enzymatic assay, infarct size, analysis of caspase-3 activity. The transcription factor binding sites in Mipul promoter was analysed by bioinformatics, while the CREB and Mipul expression was detected with real-time PCR and Western blot. The results were as follows:(1). Myocardial IPoC protected significantly myocardium against the injury induced by ischemia-reperfusion (I/R), as indicated by the decrease of serological enzymatic indexes, myocardial infarct size and myocardial caspase-3 activity. (2). Myocardial IPoC significantly up-regulated the expression of p-CREB and Mipu1 as compared with the I/R group. (3). A number of binding sites of transcription factors (such as CREB, HSF1 and E1k1) were found in the Mipul promoter by bioinformatics analysis.2. The results of luciferase reporter assay and chromatin immunoprecipitation revealed that HPC increased Mipu1 promoter activity and Mipu1 expression in cardiomyocytes which was mediated by increasing the activation and binding of CREB to the second CRE (CREâ…¡) in Mipu1 promoter induced by cAMP/PKA signaling pathway. In order to study the role of CREB in regulating Mipu1 expression in HPC, RNA interference (RNAi) and gene transfection were used to change CREB expression level. The results indicated that the inhibition of CREB expression could decrease Mipul expression, but the overexpression of CREB could up-regulate Mipu1 expression.3. To investigate the protection role of CREB-mediated Mipul expression in oxidative stress or HPC, rat cardiomyocytes H9c2 were treated with hydrogen peroxide or hypoxia-reoxygenation. Gene transfection and RNA interference technologies were used to change expression of CREB and Mipu1, while the cell injuries were evaluated by MTT assay, release of LDH, caspase-3 activity and apoptotic percentage. The results were as follows:(1). Overexpression of CREB could protect cardiomyocytes from hydrogen peroxide-induced injury, as indicated by the decrease of cell death, cell apoptosis, LDH release and caspase-3 activity. This protective effect was partly inhibited by simultaneous transfecting with Mipu1 siRNA. On the contrary, inhibiting CREB expression could enhance hydrogen peroxide induced injury, as indicated by the increase of cell death, cell apoptosis, LDH release and caspase-3 activity, which was also partly inhibited by simultaneous transfecting with pcDNA3.1-Mipu1. (2). HPC significantly attenuated the injuries of cardiomyocytes induced by hypoxia-reoxygenation, as indicated by the decrease of LDH release, caspase-3 activity and apoptotic percentage, and the increasing number of cell viability. Overexpression of CREB could enhance the protection role induced by HPC, which was partly attenuated by Mipul-siRNA. On the contrary, inhibiting CREB expression by RNAi could attenuate the protection role induced by HPC, which was partly restored by transfection with Mipu 1.In conclusion, the data in this study showed that the protection role of myocardial IPoC was partly due to up-regulation of Mipul expression, which was mediated by the activation of CREB through cAMP/PKA signaling pathway, and the binding of CREB to the CREâ…¡in Mipul promoter.
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