Membrane delimited estrogen receptor activation protects heart against ischemic-reperfusion injury in mice with cardiac but not endothelial specific ablation of ERα

Steroid hormone receptors, such as estrogen receptors (ER) function as ligand-regulated transcription factors. However, recent data indicate that estrogen also elicits effects through binding to estrogen receptors (ER-α, ER-β and GPER) at the plasma membrane and initiating signaling. In this study, ovariectomized C57BL/6J mice were treated with estradiol (6 μg/day) or estrogen-dendrimer conjugate (EDC, a membrane delimited ER modulator) for two weeks. Ischemia-reperfusion injury was evaluated in Langendorff perfused hearts. Similar to estradiol-treated hearts, EDC treatment significantly decreased infarct size and improved post-ischemic functional recovery. EDC treatment also resulted in an increase in protein S-nitrosylation (SNO), consistent with previous studies showing a SNO role in cardioprotection. In further support of a role for SNO, inhibition of nitric oxide synthase but not soluble guanylyl cyclase blocked EDC-mediated protection. ICI182,780 (antagonist of classic ER but agonist of GPER) significantly blocked the EDC-mediated cardioprotection, suggesting EDC-induced protection is mediated by classic ER. Cardiac- or endothelial-specific (cs- or es-) ERα knockout (KO) mice were generated to test tissue-specific ERα involved in EDC protection. In cs-ERαKO mice, EDC treatment still significantly decreased infarct size and improved functional recovery similar to that found in wildtype, while protection was lost in es-ERαKO mice, suggesting an important role for endothelial ERα. In contrast to wildtype hearts, EDC treatment did not increase SNO in es-ERαKO mice. ERα but not ERβ was found significantly decreased in cs-ERαKO mice, while opposite results were found in es-ERαKO mice. Thus, loss of EDC-induced protection and SNO increase in es-ERαKO mice could also be due to significant down-regulation of cardiac ERβ/SNO signaling. In conclusion, these results demonstrated a role of activation of membrane delimited ER in cardioprotection, suggesting that EDC-like compounds could be used clinically to provide cardiovascular benefit without the classical steroid hormone side effects, such as stimulation of uterine and breast cancer.