Introduction

Cardiovascular disease (CVD) has been a major cause of death worldwide for the past several decades and is expected to remain so until 2030 [1]. Women have a lower incidence of CVD than men at a similar age, but the incidence shows an increase after the onset of menopause [2]. A bilateral ovariectomy (OVX) in females causes an increase in mortality from CVD suggesting related beneficial effects of female sex hormones [3]. It has been shown that systolic and diastolic blood pressure is higher in women following menopause than in age-matched men, suggesting the role of estrogen deprivation in the development of hypertension [4]. Vascular expansion and the upregulation of endothelial nitric oxide synthase (eNOS) level, in response to high-blood flow, were found to be absent in the aorta of OVX rats, indicating the impact of estrogen deprivation on vascular dysfunction which may further contribute to atherosclerosis and myocardial ischemia [5]. In addition, data from a study of 12,134 post-menopausal women indicates that earlier deprivation of endogenous estrogen, or younger menopausal age, is associated with an increasing rate of mortality from ischemic heart disease [6], suggesting that the impact of estrogen deprivation is not only on the cardiovascular status, but also on the lifespan of women.

Menopausal women not only exhibit an increased cardiovascular risk but also have an increased prevalence of metabolic syndrome including dyslipidemia, insulin resistance, visceral fat deposition, and central obesity [7]. Female sex hormone withdrawal during menopausal transition causes decreasing body energy expenditure and a buildup of excessive visceral fat [8], which augments the production of reactive oxygen species (ROS) and inflammatory adipokine release resulting in oxidative stress and non-infectious systemic inflammation [9, 10]. Chronic systemic inflammation is known to correlate with the generation of endothelial dysfunction and atherosclerosis [11]. Moreover, the other menopause-associated features, such as increased total cholesterol, LDL cholesterol, triglycerides, and hypertension, participate in the development of coronary artery disease and myocardial ischemia, which are indicated as leading causes of mortality in women in the Western world and many developing countries [12]. The recent report from our team involving estrogen-deprived female rats demonstrated that chronic consumption of a high-fat diet aggravates metabolic disorders, accelerates the impairment of cardiac autonomic regulation and mitochondrial function, and subsequently results in reduced cardiac performance [13]. These findings suggest an increased influence of obesity, on both metabolic and cardiac performance, in estrogen-deprived states.

Treatment with estrogen supplements has been reported to improve cardiac performance in cases of both non-ischemic conditions and ischemic/reperfusion (I/R) injury. In cases of I/R injury, estrogen reduced oxidative stress, protected cardiac mitochondria, and lowered myocardium infarction [14, 15]. However, there are few studies that report the functional roles of estrogen on the heart in the conditions of I/R combined with metabolic problems [16]. The influence of female sex hormones on the metabolic status and cardiac I/R must be investigated, to clarify the underlying mechanisms and to elucidate its cardiometabolic effects.

Estrogen and the Heart

It is well known that estrogen exerts its effects by binding to estrogen receptors. There are two types of classic estrogen receptors (ER) including ER alpha (ERα) and ER beta (ERβ). ER are expressed in cardiac myocytes, fibroblasts, and cardiac mitochondria and also in the endothelium, smooth muscle cells, adventitial cells, and macrophages of blood vessels [17]. The receptors of estrogen are located in the plasma membrane, cytosol, and nucleus of the cells and work as ligand-activated transcription factors which translocate into the nucleus and activate gene transcription after estrogen has bound with them [17]. Previous studies indicate cardiac expression of ERα in the nucleus, cytosol, intercalated disc, and membrane of T-tubule and plasmalemma and involvement in the estrogenic non-genomic signaling pathway [18, 19]. It is known that the expression of ERα is influenced by estrogen levels, as it has already been reported that exposure to estrogen for approximately 4 h results in downregulation of overall ERα expression in heart tissue [19]. Interestingly, expressions of ERα messenger RNA (mRNA) and protein have been shown to be elevated in patients suffering end-stage heart failure with dilated cardiomyopathy. It is also upregulated in the myocardium of patients with aortic stenosis [20, 21], suggesting that its expression is also manipulated by pressure overload on the heart. Depletion of ERα resulted in higher heart weight [22] and inhibition of estrogenic protective effects on vascular injury [23]. Activation of ERα has been shown to play a pivotal role in cardiac metabolism, as it upregulates cardiac glucose utilization [24]. In the heart, acute treatment with an ERα agonist showed potential effects in post-I/R recovery of cardiac function and reduced infarction [25, 26]. However, chronic ERα activation by PPT had no significant improvement in post-I/R arrhythmias [27], whereas chronic activation of ERα by ERA-45 resulted in increased free radicals and necrosis reduction [28].

ERβ is an estrogen receptor which exhibits its localization in the mitochondria of neurons and cardiocytes, suggesting that estrogen could mediate its direct effect on mitochondrial function [29]. Treatment with estrogen (50 μg/kg) or an ERβ agonist (diarylpropionitrile (DPN); 5 μg/kg) following trauma–hemorrhage showed an increase in mitochondrial ERβ DNA-binding activity and resulted in elevated mitochondrial respiratory complex activity as well as inhibition of apoptotic signaling pathways in cardiac mitochondria [30]. Activation of ERβ by DPN also restored cardiac function which had been suppressed in untreated rats after trauma–hemorrhage [30]. ERβ could therefore possibly directly exert its function through activating transcription factors. Moreover, ERβ can also function in a counter-balanced manner with ERα, as the binding of ERβ can result in either activation or inhibition of ERα-induced gene transcription. At sub-saturating concentrations of estrogen, increasing ERβ expression vectors could suppress the transcriptional activity and decrease the sensitivity to estrogen of ERα [31]. These suppressing responses of ERβ, which are controlled by the concentration of estrogen hormone or ERβ agonists, suggest its potential in the modulation of cell function [31]. Depletion of ERβ caused a reduction of mitochondrial electron transport enzyme activities in platelets, increased the number of microvesicles, and raised thrombin-generating capacity in the plasma [32]. These results of ERβ deficiency and altered platelet mitochondrial energy production suggest a mechanism that may be responsible for the development of thromboembolism and consequent development of myocardial infarction.

Another type of estrogen receptor is G protein-coupled (GPR30, GPER) which is located in the plasma membrane and endoplasmic reticulum. Activation of GPR30 reduced the expression of inflammatory cytokines, ICAM-1 and VCAM-1, which were induced by the tumor necrosis factor (TNF) in endothelial cells, suggesting that the antiinflammatory effects of GPR30 stimulation occur via a non-genomic pathway [33]. According to its location, the binding of estrogen to GPR30 caused intracellular calcium mobilization and synthesis of phosphatidylinositol 3,4,5-trisphosphate in the nucleus, thought to be involved in the mediation of the physiological effects of estrogen on cells [34]. Using isolated Langendorff-perfused hearts, GPR30 activation by G-1 administration enhanced heart rate (HR), decreased left ventricular pressure (LVP), and increased rate pressure product (RPP), initially through ERK and subsequently via eNOS phosphorylation pathways [35]. Mice with GPR30 deficiency showed enlargement of the left ventricle and impairment of cardiac function. In addition, myocardial contraction and relaxation in these mice were diminished and left ventricular end-diastolic pressure (LVEDP) was increased [36].

Estrogen mitigated cardiac fibroblasts prevented cardiac fibrosis, hypertrophy, and cardiomyocyte apoptosis, suggesting benefits in the treatment of myocardial ischemia and heart failure progression [37, 38]. Loss of estrogen signaling in females resulted in cardiac function impairment and accelerated responses of the heart to pathological insults [39]. After estrogen deprivation, left ventricular (LV) hypertrophy and dilatation were more extensive due to chronic volume overload, which suggested more severe LV myocardial dysfunction and extensive cardiac remodeling [40]. A patch clamp study showed that estrogen receptor knockout (ERKO) mice had an elevated number of cardiac L-type Ca2+ channels in the cardiomyocyte membrane, leading to increased abnormalities in cardiac excitability as well as increased risk of arrhythmia and CVD [41].

However, there is controversy regarding the exact effects of estrogen on the heart. Previous studies using a dog model showed that estrogen treatment could reduce infarct size and prevent post-ischemic cardiac arrhythmia [42, 43]. In contrast, experimental evidence exists that estrogen has arrhythmogenic effects in female rat hearts by increasing the sarcoplasmic reticulum calcium leak via ERβ signaling [44]. A study using subcutaneous-implanted estradiol pellets reported that estradiol helps improve lung congestion but fails to improve cardiac cellular redox balance and right ventricular function in OVX rats with pulmonary arterial hypertension [45]. Clinical studies also reported undesirable effects of estrogen supplements on the cardiac electro-activity in post-menopausal women such as increased QT interval, QT dispersion, and augmented atrial fibrillation incidence [46, 47]. Moreover, estrogen therapy in combination with progesterone may be associated with an increased rate of breast cancer [48]. Further study into the effects of estrogen on the heart is still needed to demonstrate its precise effects and to clarify the underlying mechanisms involved.

Estrogen and Cardiac I/R Injury

Existence of gender dependence response to cardiac ischemic/reperfusion (I/R) revealed higher cardiac stress tolerance, better cardiac functional recovery, and greater survival rate in females than in males [49–53]. Estrogen deprivation resulted in removal of these cardioprotective effects [25, 52, 54–58], thus indicating the beneficial role of this female sex hormone in cases of I/R injury.

Acute Treatment Using Estrogen in I/R

Acute estrogen treatment exerted protective effects in both preischemic and post-ischemic administration. Administration of estrogen before the ischemic period in an in vivo setting resulted in the protection of cardiac mitochondria [14], a decrease in infarct size [14, 59], and reduction of cardiac ischemic and inflammatory markers [60]. Intravenous injection of 17β-estradiol either before ischemic induction or prior to the reperfusion period similarly resulted in a lower myocardial superoxide anion level, a reduced expression of connexin43 protein phosphorylation, and decreased myocardial infarction in an in vivo study using a dog model [15]. Treatment with the synthetic17β-aminoestrogen, (17β-(3-hydroxy-1-propylamino)-1,3,5(10)-estratrien-3-ol), which is synthesized from estrone, then given to the rats before reperfusion also, improved cardiac function via increased HR, LVESP, and ±dP/dt during reperfusion and protected the heart from myocardial I/R injury [61].

In isolated rat hearts, cardiac preconditioning using 5-min cycles of perfusion with estrogen prior to ischemic introduction resulted in an increase in intracellular reactive oxygen species (ROS) and reduced myocardial infarction after the transient hormonal treatments [62]. A single dose of 17β-estradiol (100 nM) administered to isolated rat hearts before the ischemic period improved the post-I/R recovery, decreased infarct size, and decreased norepinephrine levels in coronary effluence [63, 64]. Estrogen infusion during reperfusion was also able to revive myocardial function, preserve coronary flow, and lower lactate dehydrogenase (LDH) release [65]. The acute effects of estrogen, in either in vivo or in vitro classifications, are summarized in Tables 1 and 2, respectively.

Table 1 In vivo studies on the acute effects of estrogen administration on cardiac I/R
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Table 2 In vitro or ex vivo studies on the acute effects of estrogen administration on cardiac I/R
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Chronic Treatment of Estrogen in I/R

Chronic treatment of estrogen exhibited prominent advantages to ischemic hearts in both in vivo and in vitro (or ex vivo) studies (categorized in Tables 3 and 4, respectively). Subjects who had chronic subcutaneous estrogen injections for 10 days prior to I/R showed a higher level of cardioprotection than from a 10-min direct estrogen perfusion before ischemia, as evidenced by the greater decrease in ventricular fibrillation duration and increasing of coronary flow during reperfusion [66].

Table 3 In vivo studies on the effects of chronic estrogen administration on cardiac I/R
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Table 4 In vitro and ex vivo studies into the effects of chronic estrogen administration on cardiac I/R
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Estrogen treatment for 1 week completely reversed I/R-induced cardiac dysfunction, suppressed norepinephrine overflow, and preserved the nitric oxide level [49]. Twenty-one-day estrogen treatment via a subcutaneous-implanted pellet increased cardiac stromal cell-derived factor-1 (SDF-1) production, which is involved in cardioprotective effects, reduced myocardial necrosis, and lowered free radical production by the heart [28, 51]. Treatment with a 60-day release of estrogen, via subcutaneous-implanted pellets, inhibited cardiac expression of tumor necrosis factor alpha (TNF-α) [67]. Subcutaneous injections of estrogen for 2 weeks before I/R helped inhibit reperfusion arrhythmia and myocyte apoptosis indicating its antiarrhythmic, antiapoptotic features [27]. Chronic estrogen treatment not only prevented endothelial and myocardial dysfunction but also promoted estrogenic antioxidant activity by the reduction of superoxide anion concentration in the arterial segment [68]. However, a study using isolated rat hearts reported that estrogen treatment, via subcutaneous injection for 5 weeks before myocardial ischemia, stimulated cardiac fatty acid oxidation that resulted in diminished post-ischemic recovery of cardiac function [69].

Effects of Estrogen on Myocardial Ischemia and Infarction Conditions in Clinical Studies

Up to present, verified data regarding the effects of estrogen on myocardial ischemia and reperfusion in clinical trials is limited. Some clinical studies reporting the effects of estrogen supplements in patients, with a history of CVD, are summarized in Table 5. Acute administration of estrogen in nine patients just before coronary angiography intervention resulted in a decrease of myocardial ischemia, evidenced by lower levels of chest pain, ST-segment shift, and myocardial lactate depletion. Endothelin-1 levels, from the great cardiac vein, were suppressed by estrogen treatment, which correlated with attenuated coronary vasoconstriction. Mechanistically, estrogen limited these ischemic indications and vascular dysfunction through KATP channel activation [70].

Table 5 Clinical studies into estrogenic effects on myocardial ischemia and infarction
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The evidence concerning the incidence rate of post-MI cardiovascular events in patients with estrogen therapy is still conflicting. A nationwide study in Denmark, involving women who were admitted with myocardial infarction from 1997 to 2009, reported the positive role of estrogen, suggesting that post-menopausal women who received estrogen hormone therapy exhibited a lower incidence of atrial fibrillation (AF) after myocardial infarction, when compared to those without estrogen supplement therapy [71]. Estrogen replacement did not affect the rate of reinfarction, cardiovascular mortality, or all-cause mortality, when comparing estrogen therapy data in post-menopausal women with a history of myocardial infarction [72]. Although estrogen replacement therapy helped lower the LDL and raised the HDL levels in post-menopausal women with established coronary disease, it also augmented coronary heart disease (CHD) events, venous thromboembolic events, and gallbladder disease [73]. Moreover, estrogen treatment in patients with prostate cancer, who had a history of CVD, provoked cardiovascular events during treatment [74]. Therefore, estrogen hormone therapy should be prescribed with caution to patients with cardiovascular disorders, due to its unclear effects on coexisting disease status.

Roles of ER in I/R Injury

Several studies reported beneficial effects of ERα activation. After I/R, myocardial infarct size was reduced by protein kinase C epsilon (PKCε) stimulation through ERα activation [25]. Depletion of ERα decreased recovery of cardiac function, related to endothelial dysfunction following I/R [75, 76]. Treatment with ERα agonists reduced cardiac troponin-I, C-reactive protein, and post-ischemic infarction in rabbit hearts [60]. Ovariectomized female rats and ERα-knockout mice showed more impaired mitochondria with abnormal shape, loss of matrix, disruption of inner and outer mitochondrial membranes, and also lower mitochondrial enzyme activity [77, 78]. However, the abnormalities of mitochondrial structure and physiological function were restored to normal by treatment with estrogen, implying an important role of estrogen and ER, especially ERα, in the regulation of mitochondrial structure and function after I/R.

Acute stimulation of ERβ increased myocardial function, upregulated numbers of cardioprotective genes, and decreased the incidence of reperfusion-induced arrhythmias, such as ventricular tachycardia (VT) and ventricular premature beats (VPB) [26, 27, 57]. Knockout of ERβ decreased left ventricular developed pressure (LVDP), lessened functional recovery, increased TNF production, and reduced PI3K and Akt activation which correlated with the increased caspase-3 and caspase-8 activity in the apoptotic pathway in cardiac muscle [50, 53, 79]. However, there is controversial data from a study using isolated rat hearts, demonstrating that acute activation of ERβ prior to ischemia did not influence the ischemic tolerance or cardiac functional recovery following I/R, thus indicating that ERβ might not be responsible for cardioprotection during I/R injury [80].

G protein-coupled estrogen receptor 1 (GPER, GPR30) activation exhibited beneficial effects in cardiac protection after I/R. Hypoxia caused an increase of GPER mRNA in cardiac tissue which became more augmented during reperfusion [64]. Administration of a GPER agonist (G-1) improved functional recovery of the myocardium, reduced infarction, and increased the threshold for mitochondria permeability transition pore (mPTP) opening [81, 82]. G-1 perfusion prior to ischemia enhanced LVDP and ±dP/dt and lowered myocardial inflammatory agents after I/R [83].

Pathways Involved in Cardioprotection Against I/R Injury

Estrogenic cardioprotective effects against I/R injury are mediated by several mechanisms. Apoptotic cell death was found to be reduced via suppression of CaMKIIδ, P38α, and calpain activity and the activation of the JNK, PKCε, and PI3K/Akt pathways [25, 53, 54, 56, 81]. Estrogen and its receptor agonists helped regulate cardiac ROS production and decreased LDH levels [62, 75, 84, 85]. Estrogen prevented intracellular Na+ and Ca+ accumulation and thereby reduced cardiomyocyte injury and contractile dysfunction from I/R injury [84]. Estrogen helped protect the cardiac mitochondria from I/R injury by inhibiting the opening of mPTP which may lead to mitochondrial swelling and rupture, thereby preventing cardiac cell death [81, 86]. Administration of estrogen treatment resulted in decreased β1 adrenergic receptor expression and increased β2 adrenergic receptor expression in hearts subjected to I/R, which correlated to the reduction of cardiomyocyte shortening after I/R [58]. This alteration in adrenergic receptor expression might help by regulating the basal ratio of the β1/β2 adrenergic receptors, which could be changed due to I/R [58]. The antioxidant effect of estrogen was also shown to have a role in cardioprotection following I/R as it reduced ROS production and increased SOD level in the myocardium [75, 87]. Furthermore, estrogen treatment reduced myeloperoxidase enzyme activity by decreasing the amount of free radicals released from the area at risk of ischemic heart and a lowered superoxide anion concentration generated in arterial segments [28, 68].

Under the I/R setting, activation of ERα triggered portioning of ERα to the particulate, suggesting that estrogen had an effect on the redistribution of ERα and increased non-genomic ER effect which confirms the rapid action of estrogen [25]. Estrogen also mediated its non-genomic action via a NO-dependent pathway and opening of KCa channels, to protect the heart from I/R injury by limiting the size of the infarction [88]. Moreover, norepinephrine overflow, which correlated to post-ischemic cardiac dysfunction, was also inhibited via the estrogenic-stimulated increase of nitric oxide levels [49, 63].

Administration of Estrogen Treatment in I/R

Utilization of phytoestrogen demonstrated the potential to reduce myocardial apoptosis and infarction in rabbit hearts which had been subjected to I/R [89]. Infusion with estrogen-treated mesenchymal stem cells (MSCs), before induction of ischemia, showed an increase in vascular endothelial growth factor (VEGF) production, improved LV functional recovery, and myocardial viability after I/R [90]. Contrary to the effects seen from estrogen alone, treatment with a combination of estrogen and progesterone caused a reduction of the antiinflammatory effects of estrogen and aggravated the accumulation of inflammatory cells, which resulted in greater tissue injury and more necrosis [91]. However, there is a controversial report that estrogen substitution could not reverse I/R-induced cardiac injury in ovariectomized animals, showing fatty acid oxidation and impaired post-ischemic cardiac functional recovery in rat hearts, indicating a possible risk and poor outcome of estrogen administration in I/R management [69, 92].

Estrogen in Obesity, Insulin Resistance, and I/R Injury

In addition to CVD, estrogen deprivation had been reported in clinical studies to be associated with an increased prevalence of obesity, insulin resistance, and metabolic syndrome [93–95]. These findings are consistent with the results from animal studies that showed that estrogen deprivation reduced insulin sensitivity and induced metabolic impairment [16, 96, 97], leading to cardiovascular disorders. After OVX, female rats had an increased serum insulin level within 5 weeks [16]. Ovariectomized female rats became obese within 10 weeks and had a greater decrease in cardiac contractile function and reduced cardiac recovery after I/R [16]. Treatment with exogenous estrogen improved insulin sensitivity, enhanced post-ischemic cardiac contractility, and deceased myocardial tissue infarction suggesting the positive effects of this hormone on I/R [16]. A previous study showed that post-reperfusion cardiac arrhythmia incidence (e.g., ventricular fibrillation) occurred at a significant level in male rats fed with high-sucrose water, but was absent in females, and could be prevented by insulin infusion. Moreover, females also showed a lower level of plasma lipid oxidation than males after consumption of 30 % sucrose water for 8 months [98]. This study, using high-caloric-diet-induced obese rats, showed that male rats had increased body weight, visceral fat, plasma triglyceride, and glucose levels and also developed insulin resistance. After I/R induction, male obese rats also experienced more severe I/R injury, evidenced by higher myocardial infarct size, than female obese rats, suggesting beneficial effects of estrogen on the metabolic and cardiovascular systems [99]. However, the mechanisms that could explain the estrogenic protection in females have not been well established and the negative effects of estrogen on metabolism, cardiac electrophysiology, and cardiac function after I/R have been well documented. In summary, the exact, positive or negative, effects of estrogen are still controversial and have not yet been fully explored. The effects of estrogen on cardiac function and injury from I/R in female rats with metabolic disorders are still unclear and need to be investigated further.

Good and Bad Effects of Estrogen Treatment in Cardiac I/R Condition

The findings from several studies in various experimental settings, i.e., in vitro, in vivo, and clinical settings, indicated possible beneficial effects of estrogen in cardiac I/R condition. Treatment with estrogen provided its protective effects against I/R injury by reducing inflammation, protecting cardiac mitochondria, and, therefore, reducing cardiomyocyte apoptosis and myocardial infarct area [14, 67, 81, 83, 87, 100]. Moreover, estrogen treatment enhanced blood flow to the heart and improved post-I/R cardiac function and recovery as well as preventing arrhythmias [27, 58, 61, 63, 68, 70, 84, 88, 101]. Estrogen therapy, either immediately or after estrogen deprivation, was also reported to be effective in the preservation of diastolic function and cardiac structure [102]. Therefore, exogenous estrogen might be a useful regimen for the treatment of myocardial ischemia after endogenous estrogen deprivation. However, the results from clinical studies have demonstrated that estrogen treatment might increase cardiovascular events in women who already had established CVD [73, 74]. Timing of estrogen therapy is an important factor to be considered. It appears that hormone therapy, initiated early following menopause, may reduce CHD risk. However, it also appears that hormone therapy in older women, 10 years or more post-menopause, results in an increased CHD risk [103]. Previous reports have indicated that estrogen therapy reduced CHD and overall morality when prescribed for women who are less than 10 years post-menopausal and/or less than 60 years of age [104]. The adverse effects of estrogen given long after menopause, or at an older age, might be related to the diminished ER expression and the impaired inflammatory response of macrophages and vascular smooth muscle cells [105]. Estrogen treatment has also been reported to increase the level of myocardial lipid peroxidation resulting in decreased post-I/R cardiac function in isolated rat heart [69]. This particular finding might limit the use of E2 in impaired glucose tolerance, obese insulin resistance, and diabetes mellitus conditions which previously exhibited higher levels of myocardial lipid peroxidation [106–108]. The good, neutral, and bad effects of estrogen treatment and ER activation under I/R conditions as well as the underlying mechanisms are summarized in Table 6 and Fig. 1. In summary, estrogen exhibits both advantages and disadvantages on the cardiovascular system. However, since it has effects on various tissues, not only the myocardium, further studies to clarify the estrogenic effects, as well as its safety, on cardiovascular function under I/R conditions are still need to be investigated.

Table 6 Summary of good, neutral, and bad effects and the underlying mechanisms of estrogen and cardiac ER activation in I/R condition
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Fig. 1
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The schematic illustrating estrogen effects under I/R condition. Bax Bcl2-associated X protein, Bcl-2 B cell lymphoma 2, CaMKII Ca2+/calmodulin-dependent protein kinase II, Cyt C cytochrome C, ER estrogen receptor, IL interleukin, I/R ischemic/reperfusion, K ATP ATP-sensitive potassium channels, K Ca calcium-activated potassium channels, MPO myeloperoxidase, NE norepinephrine, NO nitric oxide, PI3K phosphoinositide 3-kinase, ROS reactive oxygen species, SOD superoxide dismutase, TNF-α, tumor necrosis factor alpha, VF ventricular fibrillation, VT ventricular tachycardia

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