Estrogen Receptor-&bgr; Activation Results in S-Nitrosylation of Proteins Involved in Cardioprotection

Background— It has been shown that the activation of estrogen receptor-&bgr; (ER-&bgr;) plays an important cardioprotective role against ischemia/reperfusion injury. However, the mechanism for this protection is not clear. We hypothesize that estrogen protects by ER-&bgr; activation, which leads to S-nitrosylation (SNO) of key cardioprotective proteins. Methods and Results— We treated ovariectomized C57BL/6J mice with the ER-&bgr; selective agonist 2,2-bis(4-hydroxyphenyl)-proprionitrile (DPN), 17&bgr;-estradiol (E2), or vehicle using Alzet minipumps for 2 weeks. Isolated hearts were Langendorff perfused and subjected to ischemia and reperfusion. Compared with vehicle-treated hearts, DPN- and E2-treated hearts had significantly better postischemic functional recovery and decreased infarct size. To test the specificity of DPN, we treated ER-&bgr;–knockout mice with DPN. However, no cardioprotective effect of DPN was found in ER-&bgr;–knockout mice, indicating that the DPN-induced cardioprotection occurs through the activation of ER-&bgr;. Using DyLight-maleimide fluors and a modified biotin switch method, we used a 2-dimensional DyLight fluorescence difference gel electrophoresis proteomic method to quantify differences in SNO of proteins. DPN- and E2-treated hearts showed an increase in SNO of a number of proteins. Interestingly, many of these proteins also had been shown to have increased SNO in preconditioned hearts. In addition, the DPN-induced cardioprotection and increased SNO were abolished by treatment with a nitric oxide synthase inhibitor. Conclusion— The activation of ER-&bgr; by DPN treatment leads to increased protein SNO and cardioprotection against ischemia/reperfusion injury, suggesting that long-term estrogen exposure protects hearts largely via activation of ER-&bgr; and nitric oxide/SNO signaling.

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