Although gender disparities in cardiac disease are recognized, the mechanisms through which pre-menopausal females are protected have not been fully elucidated. Cardiac disease incidence in females increases post-menopause, suggesting a role for estrogen in pre-menopausal cardioprotection. However, clinical trials found no beneficial cardiovascular outcomes from hormone replacement therapy, indicating a better mechanistic understanding is needed. Thus the goal of this study is to understand the mechanism responsible for the male-female differences in ischemia-reperfusion injury and cardioprotection. It is thought that altered gene expression contributes to female cardioprotection. MicroRNAs (miRNAs) are small, non-coding RNA that inhibit gene expression through binding to mRNA. We hypothesize that sex-specific expression patterns of miRNA contribute to differences in expression of cardioprotective proteins. It is well recognized that there is sex-dimorphic expression of mRNA and protein in the heart;however the underlying mechanism is poorly understood. Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiac function, and the expression levels of eNOS differ between male and female hearts.
The aim of this study was to examine whether expression of specific microRNA (miRNA) in males and females contributes to changes in the expression of eNOS. miRNA was extracted from the myocardium of male and female C57BL/6 mice and subjected to an Affymetrix miRNA array. Decreased expression of miR-222 was discovered in females and confirmed by qRT-PCR. The transcription factor, V-ets erythroblastosis virus E26 oncogene homolog-1 (ets-1), was identified as a potential target of miR-222 using TargetScan, and five-fold increased ets-1 protein expression in females was confirmed by western blot. Targeting of ets-1 by miR-222 was determined in HEK293 cells overexpressing luciferase under regulation of the either the ets-1 3UTR or null 3UTR control and treated with a small molecule miR-222 mimic or inhibitor. Additionally qRT-PCR confirmed that mRNA levels of the ets-1 transcriptional target, endothelial nitric oxide synthase (eNOS), were 25% higher in females. Compared to untreated myocyte controls, 50% inhibition of eNOS expression was achieved by treatment with a miR-222 mimic, compared to a 25% increase due to miR-222 inhibitor. Our findings indicate that sex-dependent miR-222 regulation alters the expression of the cardiac regulatory protein eNOS. Classical estrogen-induced transcription regulation is mediated by nuclear estrogen receptors (ER) ER-alpha and ER-beta. However, ER-alpha and ER-beta are also localized to the plasma membrane and can elicit effects through kinase signaling, leading to an increase in S-nitrosylation (SNO), a post-translational modification associated with cardioprotection. We hypothesized that estrogen-related cardioprotection is at least partially mediated by non-nuclear ER signaling leading to an increase in SNO. We tested this using an estrogen-dendrimer conjugate (EDC), which has been demonstrated in mice to be a non-nuclear selective ER modulator (SERM) that does not promote uterine or breast cancer growth. We treated ovariectomized C57BL/6J mice with EDC, dendrimer control, 17-beta-estradiol, or vehicle for two weeks. Isolated hearts were perfused in the Langendorff model and subjected to 30 minutes ischemia and 90 minutes reperfusion. As previously reported, estradiol-treated hearts had decreased infarct size (40.4 2.5% vs. 62.9 5.8%) and increased functional recovery (44.7 4.0% vs. 27.0 2.7%) compared to vehicle-treated hearts. Similar to estradiol, EDC decreased infarct size (40.9 3.6% vs. 63.8 4.7% total ventricle) and improved functional recovery (48.8 3.0% vs. 28.6 2.5%) compared to dendrimer control. Similar protection was seen when mice were treated with EDC for five days (42.1 4.7% vs. 63.8 6.4% infarct and 38.9 2.9% vs. 22.8 2.4% functional recovery for EDC vs. dendrimer). 2-D Difference Gel Electrophoresis showed an increase in protein SNO from hearts treated with EDC for 5 days and 2 weeks compared to dendrimer treatment. Many of the identified proteins have increased SNO in other models of cardioprotection. These results indicate that EDC is as effective as estradiol in providing cardioprotection during ischemia-reperfusion injury in mice, possibly due to increased protein SNO. They further suggest that non-nuclear ER actions play a major role in the protection afforded by estrogen. Thus, EDC and non-nuclear ER signaling pathways could be utilized clinically to provide cardiovascular benefit without promoting cancer cell growth.

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National Heart, Lung, and Blood Institute
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Shao, Qin; Casin, Kevin M; Mackowski, Nathan et al. (2017) Adenosine A1 receptor activation increases myocardial protein S-nitrosothiols and elicits protection from ischemia-reperfusion injury in male and female hearts. PLoS One 12:e0177315
Harrington, Josephine; Fillmore, Natasha; Gao, Shouguo et al. (2017) A Systems Biology Approach to Investigating Sex Differences in Cardiac Hypertrophy. J Am Heart Assoc 6:
Murphy, Elizabeth; Amanakis, Georgios; Fillmore, Natasha et al. (2017) Sex Differences in Metabolic Cardiomyopathy. Cardiovasc Res :
Menazza, Sara; Sun, Junhui; Appachi, Swathi et al. (2017) Non-nuclear estrogen receptor alpha activation in endothelium reduces cardiac ischemia-reperfusion injury in mice. J Mol Cell Cardiol 107:41-51
Shao, Qin; Fallica, Jonathan; Casin, Kevin M et al. (2016) Characterization of the sex-dependent myocardial S-nitrosothiol proteome. Am J Physiol Heart Circ Physiol 310:H505-15
Murphy, Elizabeth; Ardehali, Hossein; Balaban, Robert S et al. (2016) Mitochondrial Function, Biology, and Role in Disease: A Scientific Statement From the American Heart Association. Circ Res 118:1960-91
Menazza, Sara; Murphy, Elizabeth (2016) The Expanding Complexity of Estrogen Receptor Signaling in the Cardiovascular System. Circ Res 118:994-1007
Menazza, Sara; Aponte, Angel; Sun, Junhui et al. (2015) Molecular Signature of Nitroso-Redox Balance in Idiopathic Dilated Cardiomyopathies. J Am Heart Assoc 4:e002251
Harrington, Josephine L; Murphy, Elizabeth (2015) The mitochondrial calcium uniporter: mice can live and die without it. J Mol Cell Cardiol 78:46-53
Murphy, Elizabeth; Steenbergen, Charles (2014) Estrogen regulation of protein expression and signaling pathways in the heart. Biol Sex Differ 5:6

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