Background— It has been shown that the activation of estrogen receptor-β (ER-β) 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-β activation, which leads to S-nitrosylation (SNO) of key cardioprotective proteins.

Methods and Results— We treated ovariectomized C57BL/6J mice with the ER-β selective agonist 2,2-bis(4-hydroxyphenyl)-proprionitrile (DPN), 17β-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-β–knockout mice with DPN. However, no cardioprotective effect of DPN was found in ER-β–knockout mice, indicating that the DPN-induced cardioprotection occurs through the activation of ER-β. 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-β 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-β and nitric oxide/SNO signaling.