Mechanisms involved in male-female differences in cardioprotection

National Heart, Lung, And Blood Institute

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Abstract

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. 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. Dilated idiopathic cardiomyopathy (DCM) is one of the most common types of cardiomyopathy. It has been proposed that an increase in oxidative stress in heart failure leads to a decrease in nitric oxide signaling, leading to impaired nitroso-redox signaling. To test this hypothesis we investigated the occurrence of protein S-nitrosylation (SNO) and protein oxidation in biopsies from explanted DCM and non-failing donor male and female human hearts. A modified biotin switch method using DyLight-maleimide sulfhydryl-reactive fluors was used to identify the SNO proteins and a resin-assisted capture Ox-RAC was used to measure protein oxidation. We found that oxidative stress rises in DCM human biopsies in comparison with healthy donors. DyLight-maleimide gel electrophoresis showed that females have an increase in protein SNO compared with males at baseline. Interestingly many of the proteins that showed an increase in SNO in females are mitochondrial proteins. Because there are sex differences in SNO at baseline, we examined changes in SNO in heart failure as a function of sex and found sex differences in this response.