Age and sex mediated effects of estrogen and В 3-adrenergic receptor on cardiovascular pathophysiology

Sex differences are consistently identified in determining the prevalence, manifestation, and response to therapies in several systemic disorders, including those affecting the cardiovascular (CV), skeletal muscle


Introduction
Sex-related difference in risk, manifestation, response to therapy and outcome for various pathologies encountered every day by physicians have been well characterized by both experimental and clinical studies (Bernstein et al., 2023;Arosio et al., 2022).However, underlying mechanisms, as well as the specific factors associated with such sex disparities and their implications for clinical practice, remain poorly understood (Bernstein et al., 2023;Arosio et al., 2022).This is partly due to the paucity of studies including women of fertile age in clinical trials, which is because of concerns about adverse effects on the fetus from specific treatments (Phelan et al., 2016).As results, clinical and preclinical research often focused on male biology/physiology, and this has been aided by the assumption that male and female cells and animals are biologically identical (Mauvais-Jarvis et al., 2020;Klein et al., 2015;Danska, 2014;Mauvais-Jarvis et al., 2017).However, since 1993, the US National Institutes of Health (NIH), with the NIH Revitalization Act of 1993, has established guidelines to mandate the inclusion of women in research and clinical trials (Freedman et al., 1995).Since then, several rigorous studies have evaluated the effects of sex differences, effectively identifying biological differences between males and females in both animals and humans (Clayton and Collins, 2014).In particular, the differences between the sexes have been partly attributed to genetic factors, due to the different chromosomal complement.Indeed, the differential expression of X and Y genes represents the primary source of sexual differences observed during development and adulthood.In this context, the sex-determining region on the Y chromosome (SRY) encodes the protein SRY, or testis-determining factor (TDF), which initiates male differentiation of the gonads in the testes (Arnold, 2017;Britt and Findlay, 2003;Majdic and Tobet, 2011).Testosterone will then be produced which will begin the development of other sexual characteristics (Britt and Findlay, 2003;Majdic and Tobet, 2011).In contrast, ovarian development begins in the XX gonad as the default pathway because these cells lack SRY expression.Indeed, in vivo experimental tests on female transgenic mouse embryos have demonstrated that testicular development occurs if SRY is expressed (Koopman et al., 1991).
Interestingly, studies in mice also revealed that differentiation of somatic cells in the eutherian ovary can be driven by estrogen receptors (ERs) and CYP19A1 (aromatase) genes (Britt and Findlay, 2003;Britt et al., 2000).Importantly, ERs respond to estrogens, whose synthesis and production are regulated by the enzyme aromatase and control the physiology of several systems including the reproductive, musculoskeletal, cardiovascular, and central nervous system (Mauvais-Jarvis et al., 2013;Gérard et al., 2015).Therefore, these hormones have been recognized as leading factors driving sex differences between males and females (Burns and Korach, 2012;Deroo and Korach, 2006).In the premenopausal period, such differences are mainly determined by the significant difference among males and females in circulating estrogens and ER expression in tissues.And importantly, in this period, estrogens are generally considered protective as they prevent the development and reduce the prevalence and impact for many diseases (Arosio et al., 2022;Visniauskas et al., 2023;Simpson et al., 2005;Liccardo et al., 2022).For instance, hypertension or other cardiovascular disorders are less common in premenopausal women than in men counterparts (Arosio et al., 2022;Visniauskas et al., 2023;Simpson et al., 2005;Liccardo et al., 2022).At the same time, this trend is significantly reversed with menopause, with the incidence of such disorders increasing and becoming higher in women than in age-matched men.This phenomenon coincides with the ovary's failure to produce estrogens and the residual apport of these hormones from extragonadal site production (i.e.breast, brain, muscle, bone, and adipose tissue) (Simpson et al., 2005).
For this reason, in the 1940s, estrogens were approved by the Food and Drug Administration (FDA) as a hormone supplement (hormone replacement Therapy; HRT) to treat postmenopausal symptoms and to prevent various conditions like osteoporosis, cardiovascular and Alzheimer's disease (Mauvais-Jarvis et al., 2013;Keating et al., 1999).Due to the benefits observed by this therapeutic approach, estrogens were considered a miraculous antidote to various health-related consequences of aging in several tissues.Nonetheless, HRT in humans resulted in some controversy.Indeed, clinical trials demonstrated that HRT resulted in adverse cardiovascular and cerebrovascular events and augmented the risk of breast and uterine cancer (Rozenberg et al., 2013;Whayne and Mukherjee, 2015).These harmful effects have been attributed to several factors, including the lack of a clear understanding of the biological role of estrogens and their downstream signaling (Pan et al., 2022).Notably, studies have reported an essential role of these hormones among the pathways controlled by estrogens in regulating the secretion and release of catecholamines (Whayne and Mukherjee, 2015;Lin et al., 2017;Wang et al., 2017;Li et al., 2015).Therefore, estrogens are plausibly intertwined with the β-Adrenergic receptor (βARs) signaling as suggested by several experimental and clinical studies (Liccardo et al., 2022), and this may help to explain the differences in βAR responsiveness and signaling activation between males and females observed over the years.
We recently analyzed the relationships between estrogens and βARs (Liccardo et al., 2022), particularly β1 and β2AR, discussing how these molecules impact the cardiovascular risk and response to therapies, also related to sex and age.However, in the present study, we mainly focused on the β3AR, a receptor with essential activities in adipose tissue but with novel and exciting roles in different types of cells, including cardiomyocytes and vascular cells whose relationship with estrogens remains unfortunately overlooked.Herein, we examined the literature analyzing such association and discussed whether and how estrogens and β3AR signaling are intertwined in cardiovascular system regulation and seemingly associated with different cardiovascular outcomes in females and males.
The present review article intend to shift the readers' attention to the need to deepen and significantly increase the studies relating to the role of this "minor" βAR isoform that plays numerous vital actions in the cardiovascular system, and whose signaling converges with that of the ERs.

Estrogens and their receptors in cardiovascular system physiology and disease
Estrogens are major female sex hormones and include estrone (E1), 17β-estradiol (E2), estriol (E3), and estetrol (E4).These hormones are essential in sexual and reproductive development, and their synthesis and release fluctuate with life stage (Mauvais-Jarvis et al., 2013;Gérard et al., 2015).Indeed, while E2 is the predominant estrogen in the premenopausal period, E1 is the dominant postmenopausal estrogen.Finally, E3 and E4 are the principal estrogens produced during pregnancy.E1 and E2 are produced from ovarian granulosa cells, whereas E3 and E4 are produced by placenta and fetus liver, respectively (Mauvais-Jarvis et al., 2013;Gérard et al., 2015).Other tissues (i.e.adrenal glands, fat, liver, and skeletal muscle) contribute to circulating estrogens to a lesser extent (Jomard and Gondin, 2023) and this source plays a vital role, as it remains the only source of endogenous estrogens (particularly E2) in men and postmenopausal women (Simpson, 2003).
Estrogens can exert their function through two distinct receptors named ERα and ERβ encoded from different genes on separate chromosomes whose expression profiles are tissue dependent (Arosio et al., 2022).The activation of these receptors leads to the translocation to the nucleus and the binding of the estrogen-ER complex to an estrogen response element (ERE) in the promotor and enhancer regions of the target genes, resulting in their transcription.In addition, a third ER called G-protein-coupled ER (GPER or GPR30), a member of the superfamily of G-protein-coupled receptors (GPCRs), has been identified and has been shown to contribute to estrogen-mediated rapid nongenomic effects (Arosio et al., 2022).
Estrogen and its related receptors are generally considered protective within the cardiovascular system (Liccardo et al., 2022).Indeed, women in the premenopausal period typically present higher levels of circulating estrogen and have a lower risk of cardiovascular disease than agematched men (Liccardo et al., 2022).In contrast, with menopause, estrogens are significantly reduced, and the cardiovascular risk increases.Notably, part of the beneficial effects attributed to these sex hormones can be dependent either by indirect influence on the metabolism of lipoproteins (oxidation), or by direct activity on specific signaling pathways in the vasculature, and more specifically on vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) or in cardiomyocytes (Liccardo et al., 2022;Hermenegildo et al., 2002).

Estrogens and vasculature
Estrogens, mainly E2, regulate the expression of nitric oxide synthase (NOS), an enzyme involved in NO production and release.NO is a free radical and a major gasotransmitter that is freely diffusible and can cross biological membranes, rendering it an ideal natural biological messenger (Loscalzo and Welch, 1995;Cannavo and Koch, 2018).Three isoforms of NOS enzymes can produce NO: the neuronal NOS (nNOS or NOS1), the inducible NOS (iNOS or NOS2), and the endothelial isoform (eNOS or NOS3) (Cannavo and Koch, 2018).Both eNOS and nNOS isoforms are constitutively expressed and generate moderate quantities of NO.At the same time, iNOS, whose expression depends on induction by cytokines or other stimuli, could lead to 1000-fold more NO than that derived by eNOS or nNOS activity (Strijdom et al., 2009;Balligand and Cannon, 1997).
Notably, E2, via the activation of ERs (ERα ERβ or GPER), increases NO bioavailability upon activation of eNOS or nNOS, mitigating several mechanisms that increase vascular wall tone conferring vasoprotection (Fredette et al., 2018;Guo et al., 2005;Lekontseva et al., 2011).In this regard, studies in women with coronary artery disease have demonstrated that E2 administration impaired acetylcholine-induced coronary constriction (Collins et al., 1995).Further to this "direct" manner to increase NO bioavailability, E2 has been demonstrated to indirectly increase circulating NO levels via NADPH oxidase activity modulation, thereby attenuating superoxide anion (O2− ) concentration, reducing O2 − -mediated NO inactivation (Dantas et al., 2002).Notably, these NO-dependent effects mainly rely on estrogen binding to and activating ERα and GPER (Fredette et al., 2018).However, in response to vascular damage, like ischemia-reperfusion (I/R) injury, studies reported an augmented expression of ERβ in the endothelium, which in turn leads to increased superoxide dismutase (SOD2), eNOS, and NO levels (Zhan et al., 2016).This process minimizes reactive oxygen species (ROS) generation, ameliorating the endothelium's response to vascular injury (Zhan et al., 2016).Notably, the estrogens-dependent vasoprotection has also been related to the activation of specific antithrombotic and anti-atherogenic mechanisms (Nakamura et al., 2004;Okubo et al., 2000;Seeger et al., 2001).For instance, in ECs, GPER activation attenuates LDL cholesterol transcytosis, representing an initial event of atherosclerosis (Ghaffari et al., 2018).
Of note, part of these effects is also mediated by the modulation of VSMC function, that together with ECs, represent the vessel wall's primary structural and functional elements, carrying out several positions, including regulating vascular tone and blood flow (Novella et al., 2019;Meyer et al., 2014).
However, concerning the effects on VSMCs, conflicting results have been provided.Indeed, while different studies reported that E2 can inhibit the proliferation and migration of VSMCs in several experimental animal models of vascular injury preventing neointimal hyperplasia (Dehaini et al., 2018) experimental evidence in pulmonary aortic VSMCs (PASMCs) demonstrated the opposite.In this regard, Farhat and colleagues (Farhat et al., 1992) reported that estradiol potentiated in a concentration-dependent manner, the proliferation of PASMCs contributing to the development of pulmonary arterial hypertension (PAH).Indeed, this severe condition, which concerns the lungs' blood vessels and right heart with a 3-year survival rate of <60 %, displays a higher prevalence in women than men (Foderaro and Ventetuolo, 2016).Clinical studies have suggested that estrogen may not be protective in all cases (Visniauskas et al., 2023;Taraseviciute and Voelkel, 2006;Chen et al., 2017) and have linked estrogen to an increased risk of PAH as high levels of specific estrogen metabolites facilitate pulmonary vascular remodeling (Umar et al., 2012;Hester et al., 2019).In contrast, preclinical studies have demonstrated that female animals have better outcomes than age-matched males, and following ovariectomy (OVX), females showed a significant exacerbation of the disease, suggesting a protective effect of estrogens (Foderaro and Ventetuolo, 2016;Hester et al., 2019).
Notably, how ERs and GPER expression in the cell types of the arterial wall is affected by aging in males and females has been far less studied (Davezac et al., 2021;Yang et al., 2021). .In this context, Novensà et al. (Novensà et al., 2011) in a female mouse model of accelerated aging (senescence-accelerated prone, SAMP), it was reported that vascular ERα expression was decreased while ERβ was augmented with consequent detrimental effects on E2-mediated NO bioavailability and vascular benefits.In line with this report, Wynne et al. (Wynne et al., 2004) demonstrated that a slight but not significant ER downregulation was sufficient to decrease estrogen vascular relaxation and increase vascular contraction and arterial pressure in aged SHR female rats.
For their part, Tarhouni et al. (Tarhouni et al., 2014) revealed that in ERα heterozygous female mice, part of the beneficial vascular activity of E2 was lost.In detail, E2 deprivation resulted in a flow-mediated outward remodeling decline, and early exogenous E2 prevented this effect.However, in ERα knockout mice, this effect was not observed, suggesting the importance of gene dosage.

Estrogens and hypertension: RAAS and SNS
Due to the physiological effects elicited by the vasculature, particular attention has been devoted to understanding the direct activities of E2 in regulating blood pressure in females and males and its implication in the pathogenesis of hypertension (Visniauskas et al., 2023).Indeed, in premenopausal women, blood pressure tends to be lower than men's counterparts.Consequently, women display a reduced risk of developing hypertension compared to men of the same age (US adults aged 20 years old: 42.8 % in women and 51.7 % in men) (Virani et al., 2021).In contrast, after menopause, women experience a more sensitive rise in blood pressure and a steeper rise in the prevalence of hypertension compared to men of the same age.For instance, in the US, the incidence of hypertension among 65-74-year-old subjects was 75.7 % in women and 67.5 % in men (Virani et al., 2021).In this context, several studies have attributed, at least in part, these sex differences to vascular E2 activities.Concurrent with the vasoprotective effects exerted by this sex hormone, discussed above, data demonstrated that E2 impacts the expression of different hormone receptors and growth factors that promote hypertension.Among these, the renin-angiotensin-aldosterone system (RAAS) and the sympathetic nervous system (SNS) are crucially involved in blood pressure regulation, and their alteration contributes to hypertension.The RAAS pathway consists of a complex system of molecules (Fig. 1) that mediates physiological effects, including regulation of cardiac output and arterial blood pressure (O'Donnell et al., 2014;Cannavo et al., 2016aCannavo et al., , 2016bCannavo et al., , 2016c;;Cannavo et al., 2022;Cannavo et al., 2019a;Cannavo et al., 2019b;Cannavo et al., 2018).However, in chronic angiotensin type 1 Receptor (AT1R), hyperactivation and aldosterone secretion release from the adrenal cortex can induce several noxious effects, such as vascular dysfunction and inflammation, myocardial fibrosis, and hypertrophy that culminate with cardiovascular disease and ultimately with heart failure (O'Donnell et al., 2014;Cannavo et al., 2016aCannavo et al., , 2016bCannavo et al., , 2016c;;Cannavo et al., 2022;Cannavo et al., 2019a;Cannavo et al., 2019b;Cannavo et al., 2018).In this context, studies have demonstrated that estrogen controls this system, and animal models of RAAS activation of this sex hormone can induce the protective Angiotensin (1-7)-Mas receptor and AT2R pathways (O'Donnell et al., 2014;Cannavo et al., 2018).
Moreover, Silva-Antonialli et al. (Silva-Antonialli et al., 2004), in spontaneously hypertensive rats (SHR), observed the existence of sexual dimorphism in vascular reactivity and AT1/AT2Rs ratio in blood vessels and kidneys between females and males.Notably, these authors demonstrated that estrogen decreased the prohypertensive actions of Ang II, modulating the expression of these receptors.For their part, Pendergrass et al. (Pendergrass et al., 2008) reported that males of mRen2.Lewis congenic rats, a model of renin overexpression, presented with lower plasma ANG-(1-7) and greater circulating renin levels and ACE activities than female littermates.Interestingly, higher renal cortical and medullary Ang II levels were also observed in males compared to females.Consequently, males exhibited the highest extent of cardiac hypertrophy and blood pressure.
In line with these data, Chappell et al. (Chappell et al., 2003) assessed the effects of estrogen depletion via OVX in the congenic female mRen(2).Lewis rats, demonstrating that OVX elevated circulating Ang II levels and ACE activity and reduced Ang-(1-7) to levels observed in males.In addition, OVX induced a rapid and sustained increase in blood pressure, confirming estrogen's potential impact on the RAAS system.In another study, Wang et al. (Wang et al., 2013) analyzed the effects of E2 treatment in OVX female mRen(2).Lewis rats.These authors provided data showing that E2 impaired the rise in circulating and cardiac Ang II.In this study, they also demonstrated an essential role of E2 in modulating circulating aldosterone levels.The importance of estrogens on aldosterone release was corroborated by Roesch and coworkers (Roesch et al., 2000), that in OVX rats demonstrated that E2 reduced adrenal AT1R expression and aldosterone production, supporting estrogen HRT as a strategy to modulate Ang II-stimulated aldosterone secretion as part of its well-recognized cardio and vasoprotective effects.
In this sense, Proudler and coworkers (Proudler et al., 1995) examined the effect of estrogen HRT (2 mg estradiol valerate and 0-7 mg norethisterone orally daily) in 28 postmenopausal women (HRT group) compared to 16 postmenopausal women who had not received HRT (control).Interestingly, after six months of HRT, the authors observed that ACE activity was significantly reduced (by 20 %; p < 0⋅001) in the HRT group compared to the control group.Similarly, Schunkert and colleagues (Schunkert et al., 1997) observed lower renin levels in 107 postmenopausal women receiving estrogen HRT than those without estrogen HRT (controls, n = 223).In addition, despite being nonsignificant, these authors observed slightly lower aldosterone levels in estrogen HRT women compared to the control group.
The SNS is equally important in developing hypertension (DiBona, 2013), and its activity differs significantly between women and men (Hart et al., 2009).For instance, several experimental and clinical studies in humans and rodents showed that the vessels of females constrict less and relax more than males in response to catecholamine stimulation (Kneale et al., 2000;Al-Gburi et al., 2017;Riedel et al., 2019;Dart et al., 2002;Freedman et al., 1987;Lee and Gzik, 1991;Loria et al., 2014).
Despite this, this effect appears to be related mainly to αand β-adrenergic receptor (AR) expression activity in vessels.Indeed, Al-Gburi et al. (Al-Gburi et al., 2017;) reported that βAR (mainly the isoforms 1 and 3), which counteract αAR constrictive effects, display a higher expression in the female vessels than in males, and their stimulation resulted in higher relaxation in female aortas than in males.
Notably, the selective inhibition of β1 and β3AR abolished these sex differences in constriction/relaxation.In addition, as suggested by Riedel and coworkers (Riedel et al., 2019), the vascular expression of β1 and β3AR is determined predominantly by estrogens, explaining the drop in βAR responsiveness, increased α-adrenergic vasoconstriction and blood pressure rise, typically observed in postmenopausal women (Lee and Gzik, 1991;Loria et al., 2014).
However, estrogen is also a vital regulator of catecholamine release by SNS and activity.For instance, in rats, Gomes et al. (Gomes et al., 2012 reported that after OVX, females displayed increased plasma catecholamine levels than male rats following gonadectomy.Conversely, estrogen supplementation reversed these effects.In addition, two independent reports demonstrated that estrogen exerts inhibitory effects on catecholamine secretion from the chromaffin cells of the adrenal gland (Sung et al., 1999;Sudhir et al., 1997), contributing to the regulation of whole systemic circulating catecholamines.In humans, Sung et al. (Sung et al., 1999) examined the effects of SNS activity in 22 postmenopausal women, 13 postmenopausal women taking ERT, and 18 premenopausal women.Interestingly, following mental stress, the authors observed significantly greater blood pressure in a postmenopausal group than those taking estrogen HRT or premenopausal one.In addition, in response to norepinephrine infusion in vivo, the postmenopausal women showed more prominent vasoconstriction than the premenopausal women or those taking ERT.In line with these findings, Sudhir and colleagues (Sudhir et al., 1997) examined the response to norepinephrine infusion in perimenopausal women (n = 7 with estrogen HRT or n = 5 treated with placebo).Of note, upon norepinephrine stimulation (25, 50, and 100 ng/min), the authors demonstrated that the HRT group displays impaired forearm vasoconstrictor response and reduced systolic and diastolic blood pressures, compared to the placebo control group.

Fig. 1.
Estrogens and renin-angiotensin-aldosterone system (RAAS) The hormonal cascade of the RAAS begins with renin production by the juxtaglomerular cells of the kidney.This protease induces the cleavage of angiotensinogen, an alpha-2-globulin synthesized in the liver, to generate the inactive decapeptide angiotensin I (Ang I), which, upon the activation of the Ang-converting enzyme (ACE), is subsequently converted into the active octapeptide Ang II.AngII binds to and activates the Ang II type 1 receptor (AT1R), leading to aldosterone synthase activation in the adrenal gland, then promoting the reabsorption of NaCl and H2O, with vasoconstrictive effects.Chronic AngII can exert several detrimental effects, such as vascular dysfunction and inflammation, increased blood pressure, augmented myocardial fibrosis, and hypertrophy.However, Ang II can also bind to AT2R, exerting effects opposite to those induced by AT1R.Furthermore, when ACE2 is activated, Ang I and Ang II can be converted to Ang-(1-9) and Ang (1-7), activating MAS receptors that mediate vasoprotective actions like those induced by AT2R.Estrogens can prevent chronic activation of AngII and Aldosterone production, mediating vasoprotective effects.

Estrogens in the heart
Estrogens elicit a plethora of effects on the cardiovascular system.Notably, studies concerning the role of estrogens and ERs in the heart span experimental studies in cells in vitro, animal models in vivo, and human clinical trials.The impact of estrogen on the heart is also supported by the expression of all ERs in different cardiac cell types such as cardiomyocytes, cardiac fibroblasts, and vascular cells (i.e., ECs and VSMCs) (Grohé et al., 1997;Lizotte et al., 2009;Dworatzek and Mahmoodzadeh, 2017;Groban et al., 2020).It is worth noting that data in the overall literature show that ERα and GPER are similarly expressed in the cardiac tissue of rodents (Hutson et al., 2019) and humans (Deschamps and Murphy, 2009), there are conflicting data about ERβ expression.For instance, studies indicate that ERβ is expressed at low levels in cardiac tissue and may be upregulated in the heart during disease (Grohé et al., 1997;Couse et al., 1997;Grohé et al., 1998).Alternatively, it has been suggested that ERβ is mainly expressed in infiltrating cells rather than in cardiomyocytes impacting whole cardiac physiology/pathology (Groban et al., 2020).Despite that, many studies have been conducted in ER knockout and transgenic animal models, allowing us to depict the molecular and cellular mechanisms associated with estrogens' and ERs' activities in cardiac physiology and disease.
For instance, Skavdahl et al. (Skavdahl et al., 2005) compared wildtype (WT) mice with those lacking functional ERα (ERαKO) or ERβKO and demonstrated that in response to pressure overload induced by transverse aortic constriction (TAC), homozygous ERαKO female mice exhibited an identical hypertrophic response to WT females.In contrast, homozygous ERβKO females, in the presence of TAC injury, exhibited a significant increase in cardiac hypertrophy compared with WT female mice.Therefore, these authors suggested that ERα is not involved in mechanisms associated with attenuated female response to pressure overload.In line with these data, Babiker and colleagues (Babiker et al., 2006) analyzed the ability of E2 to impact hypertrophic response to TAC in WT, ERαKO, or ERβKO mice.Interestingly, these authors observed that E2 treatment significantly impaired TAC-induced cardiac hypertrophy in WT and ERαKO mice but not in ERβKO mice, confirming the cardioprotective role of ERβ.For their part, Pedram et al. (Pedram et al., 2008) showed that E2 elicited its protective effects mainly through ERβ mitigating important pro-hypertrophic and -fibrotic signaling activated by AngII in female mice hearts.
In addition to these effects, Fliegner et al. (Fliegner et al., 2010) reported that ERβ mice undergoing TAC exhibited augmented expression of pro-apoptotic genes, which increased cardiomyocyte apoptosis and cardiac fibrosis, supporting the anti-apoptotic and prosurvival role of estrogen/ERβ axis.In line with these data, Cao and coworkers (Cao et al., 2011) revealed in vivo in mice undergoing surgical-induced myocardial infarction (MI) that E2 administration reduced matrix metalloproteinase 9 (MMP-9) activation and enhanced expression of the anti-apoptotic factor Bcl-2, compared to MI mice controls (treated with placebo) preventing cardiac rupture.In addition, in vitro, these authors showed that in H9c2 cells (a surrogate of cardiomyocytes), E2 via ERβ mediated anti-apoptotic effects against I/R injury.Similar results were observed by Hsieh and coworkers (Hsieh et al., 2015) and by Lin et al. (Lin et al., 2017) in vitro in neonatal rat ventricular myocytes (NRVMs), who demonstrated how the E2/ERβ axis abolished the harmful proapoptotic effects of isoproterenol (βAR agonist) stimulation in vitro in H9c2 cells.
Despite this evidence, it has been demonstrated that ERα activation is crucial in estrogen-mediated regulation of cardiac bioenergetics.In this regard, the study by Chen et al. (Chen et al., 2015) showed that E2 supplementation in mice carrying a human hypertrophic cardiomyopathy mutation (cTnT-Q92 mice) and underwent OVX, increased ATP levels and mitochondrial respiratory function with preservation of myocardial function compared to untreated OVX cTnT-Q92 mice.For their part, Arias-Loza et al. (2017) reported that E2/ERα system positively impacted cardiac glucose uptake in OVX mice.In line with this metabolic role of ERα, Devanathan et al. (Devanathan et al., 2014) provided data showing that cardiomyocyte-specific deletion of ERα negatively impacted metabolic gene expression in cardiomyocytes in a sex-dependent manner.
However, other reports demonstrated that both receptors induce estrogen cardiac signaling activation.For instance, a report from Luo et al. (Luo et al., 2016), demonstrated that E2, via ERα or ERβ, protects the female heart from ischemia and reperfusion injury.Analogously, Pelzer and colleagues (Pelzer et al., 2001) demonstrated that E2 protects cardiomyocytes, activating either ERα or ERβ and then inhibiting apoptosis in response to staurosporine.
Notably, several studies also supported a role for GPER activation downstream E2 prosurvival and anti-apoptotic effects in cardiomyocytes.Li et al. (Li et al., 2015) reported that GPER activation elicited a cardioprotective effect following I/R in H9c2 cells in vitro treated with GPER-selective agonist G1.Of note, tG1 stimulation resulted in an increased activation of antioxidant molecules such as superoxide dismutase (SOD), supporting also an antioxidant role for GPER.In line with this data, we previously reported (Cannavo et al., 2016a(Cannavo et al., , 2016b(Cannavo et al., , 2016c) ) that GPER activation prevented oxidative stress and pro-apoptotic effects induced in NRVMs following aldosterone stimulation.

β3-Adrenergic receptors
βARs are critical regulators of mammalian physiology (Lin, 2013).Their implication in cardiovascular system homeostasis is so relevant that an alteration in their expression/levels and an imbalance in their related downstream signaling has been correlated to the development and progression of a myriad of cardiovascular diseases.
For this reason, βARs are considered one of the most important molecular targets in the cardiovascular system (Port and Bristow, 2001;Bristow, 1993;Cannavo et al., 2013).
Currently, three βAR subtypes have been identified, with β1 and β2ARs the most studied (Frielle et al., 1987;Kobilka et al., 1987;Emorine et al., 1989).However, since the identification and discovery in 1989 (Emorine et al., 1989) of the β3AR, it appeared clear that this "novel" isoform participated with the other two "older" isoforms in the regulation of whole cardiovascular physiology.Indeed, so far, clinical, and preclinical studies have provided evidence for multiple roles for this receptor that go from regulation of metabolism and bioenergetics, vascular tone control and cardiac function and remodeling (Simard et al., 1994;Dessy and Balligand, 2010).Therefore, the interest in this receptor has grown exponentially, especially considering the new potential therapeutic application in cardiovascular disease treatment.
The mammalian β3AR has the typical structure of all GPCRs, with seven transmembrane domains (7-TMDs) (Cannavo et al., 2017).Compared with β1 and β2AR isoforms, a good level of homology in the 7-TMD sequence is appreciated.However, a significant difference in the third intracellular loop and the C-terminal (CT) domain is evident, probably representing the main factor affecting the pharmacologic modulation of the receptors, their response to ligands, and the activation of intracellular signaling.For instance, β1 and β2ARs are both subjected to GPCR kinase (GRK)-mediated regulation through phosphorylation of serine and threonine residues present in their CT domain (Cannavo et al., 2017).In addition, these receptors hold a consensus sequence for protein kinase A (PKA).The β3AR lacks all of these sequences resulting more resistant to the process of agonist-induced desensitization/downregulation.Despite this, a study by Echeverría et al. (Echeverría et al., 2020) reported that the kinase GRK2 can mediate short-term desensitization of β3AR through a phosphorylation-independent mechanism.

β3-Adrenergic receptor in cardiovascular system: physiology and disease
β3AR is mainly expressed in ECs and adipose tissues (Nahmias et al., 1991;Krief et al., 1993).However, low levels of this receptor have been identified within myocardial cells compared to the β1 and β2AR isoforms.Importantly, the coupling of β3AR to intracellular signaling effectors varies among tissue or cell type expression.Studies have demonstrated that β3AR couples to both adenylyl cyclase stimulatory Gα (Gαs) or to the inhibitory Gα (Gαi) protein subunits.Significantly, through Gαs activation, β3AR augments cyclic AMP (cAMP) levels with subsequent activation of the PKA (Cannavo et al., 2013;Cannavo and Koch, 2017;Brixius et al., 2004).PKA phosphorylates many Ca2+ handling proteins and some myofilament elements in the myocardium, leading to positive inotropic, chronotropic, and lusitropic effects (Cannavo et al., 2013;Cannavo and Koch, 2017).At the same time, in PASMCs, the β3AR has been shown to exert vasodilating effects coupled with an increase in cAMP levels (Tagaya et al., 1999).Further, β3ARs signals via Gαi, acting as a brake to prevent β1 and β2ARs hyperactivation, a protective mechanism proposed in the heart.Indeed, this signaling pathway is associated with eNOS or nNOS activation and gives rise to NO generation with subsequent activation of soluble guanylate cyclase (sGC) to generate cGMP that in turn induces cGMP-dependent protein kinase G (PKG) activation (Cannavo and Koch, 2017;Gauthier et al., 1996;Trappanese et al., 2015;Balligand, 2016).PKG is a serine/ threonine kinase that enhances myocyte relaxation and causes negative inotropy.Similarly, this NO-driven pathway downstream β3AR in ECs is responsible for an endothelium-dependent relaxation in both aortic and resistance vessels.In this context, Karimi Galougahi et al. (Karimi Galougahi et al., 2023) demonstrated that selective stimulation of β3AR increased eNOS coupling with NO-dependent signaling in a mouse model of PAH.This mechanism negatively impacted PASMC proliferation and increased vasodilation, with a subsequent drop in pulmonary arterial pressure, which also resulted in better remodeling with reduced fibrosis and hypertrophy of the right ventricle.
Similar results were observed by García-Álvarez et al. (García-Álvarez et al., 2016) in a porcine model of chronic PAH.These authors observed that treatment with β3AR agonists positively affected pulmonary hemodynamics and right ventricular performance, associated with impaired pulmonary vascular proliferation.In line with these results, these authors set up a clinical trial, the β3 Adrenergic Agonist Treatment in Chronic Pulmonary Hypertension Secondary to Heart Failure (SPHERE-HF), to examine the efficacy and safety of a selective β3AR agonist mirabegron in patients with combined pre-and post-capillary PAH (ClinicalTrials.gov NCT02775539 and EudraCT: 2016-002949-32).The data provided so far indicated that despite the primary endpoint, the placebo-corrected change from baseline to week 16 in pulmonary vascular resistance (PVR) was not met; the authors reported that patients receiving β3AR agonist presented a significantly better evolution of right ventricle ejection fraction as compared to patients treated with placebo (García-Álvarez et al., 2023).
Clinical and experimental evidence also supports the NO-cGMP/PKG signaling axis as one of the cardioprotective mechanisms activated downstream β3AR, which has critical beneficial effects in cardiac diseases such as HF, and both Gαs and Gαi may trigger this.In this regard, in vivo in mice, Calvert and colleagues (Calvert et al., 2011) demonstrated that exercise activating the PKA/Akt/eNOS pathway downstream β3AR confers cardioprotection following I/R injury.In ECs, the NO generated downstream of this PKA/eNOS pathway can induce potent vasorelaxant effects and enhance EC function and proliferation in vitro, promoting neoangiogenesis and revascularization in ischemic tissues (Queen et al., 2006;Ferro et al., 2004;Cannavo et al., 2016aCannavo et al., , 2016bCannavo et al., , 2016c)).In this context, we have recently demonstrated that part of the protective β3AR effects is modulated by the synthesis and secretion of the brain-derived neurotrophic factor (BDNF), which in turn activates its tropomyosin kinase receptor B (TrkB) on the plasma membrane of cardiomyocytes, ECs, and neurons exerting a myriad of myocardial autocrine/paracrine protective effects improving survival and functionality of cardiomyocytes and activating ECs proliferation (Cannavo et al., 2023;Allen et al., 2018).Moreover, this β3AR dependent BDNF activation appears to enhance autonomic neuronal sprouting, which is impaired in the ischemic cardiac tissue.
For all these reasons, β3ARs represent novel potential targets for treating certain cardiovascular diseases.Several reports have strengthened this notion by documenting that behind β1-blockers and their subsequent beneficial effects is the enhancement of β3AR expression and activity.

β3-Adrenergic receptor, estrogens and the impact on the cardiovascular system
As discussed above, estrogens, ERs, and βARs play essential roles in the cardiovascular system and numerous reports support the interaction between these factors and their downstream signaling, generating sex differences between males and females.
In 1991, Lopez and colleagues (López et al., 1991) demonstrated that estrogen could impair catecholamine secretion when injected into cat adrenal glands, regulating this organ's contribution to total plasma catecholamines (Cannavo et al., 2017;Lymperopoulos et al., 2016).Years later, similar findings were reported by Park and coworkers (Park et al., 1996), who revealed that estrogen has a modulatory role in regulating catecholamine secretion in rat adrenomedullary chromaffin cells therefore supporting a role for estrogens in impacting βAR activity.Moreover, studies have shown that estrogen directly influences β3AR responsiveness in several cells and tissues of the cardiovascular and noncardiovascular systems.In this regard, Yono et al. (Yono et al., 2000) demonstrated that estrogen treatment of female rabbit detrusor smooth muscles caused an increased β3AR-mediated relaxation, thus playing an essential role in alleviating overactive bladder (OAB) pathophysiology (Petereit et al., 2024).Similarly, Brown et al. (Brown et al., 2018) demonstrated that ER agonism could prime the brown adipose tissue to effectively respond to β3AR stimulation, thereby enhancing thermogenesis and energy expenditure.While, in the vasculature, Riedel and colleagues (Riedel et al., 2019) demonstrated that estrogen administration enhanced vascular β3AR responsiveness, boosting the vasodilator effects associated with this receptor.These data, support β3AR as a major contributor to the difference in blood pressure observed between females and males (Reckelhoff, 2001).In addition, this report can help explain the lower incidence of vascular diseases like hypertension observed in premenopausal women than in men of the same age and attribute to a different βAR responsiveness (Harvey et al., 2014;Pepine and Nichols, 2006).
Other studies provided data showing estrogens acting as direct regulators of β3AR expression (Machuki et al., 2018).For instance, Riedel and colleagues (Riedel et al., 2019) found that estrogen increased endothelial β1 and β3AR in female rats.Similarly, in adipocytes Monjo et al. (Monjo et al., 2005) reported that estrogen enhanced the expression of β3ARs.Unfortunately, no investigations have yet evaluated the effects of this sex hormone on β3AR within the myocardium.However, based on the studies above it is plausible to observe a similar effect also in the heart.
Further, indirect proofs sustain the importance of a crosstalk between β3AR and ERs in the cardiovascular system.In particular, studies demonstrated an interaction between Nebivolol, a highly selective β1blocker with β3AR agonistic activities, and ERs (Manthey et al., 2010;Dogru et al., 2010;Grundt et al., 2007).In their study, Garbán et al. (Garbán et al., 2004) revealed that Nebivolol mediated a vasorelaxation effect in isolated aortic rings that was partially dependent on ERmediated pathways.Subsequently, Ladage et al. (Ladage et al., 2006), in line with these findings, demonstrated that Nebivolol induced vasorelaxation by activating the β3AR/ER/NO axis.Whereas Hillebrand et al. (Hillebrand et al., 2009) observed that Nebivolol decreased cellular stiffness and augmented endothelial cell growth via NO and ERβ pathways in human endothelial cells.Therefore, these data demonstrated that thanks to its unique characteristics, Nebivolol is beneficial when treating patients suffering from endothelial dysfunction and G. Corbi et al. hypertension or PAH, but it can also be used in those with cardiac disease (Perros et al., 2015).In fact, Nebivolol in rodent models of I/R injury, activated cardiac β3ARs reducing infarct size (Aragón et al., 2011) and as demonstrated by Zhang et al. (Zhang et al., 2014) in postischemic HF mice, via β3AR upregulation and activation, this β-blocker reduced cardiac fibrosis and apoptosis improving cardiac function.In addition, Nebivolol has been tested and implemented in clinical practice to be preferentially used in women.In this regard, a study by Grundt et al. (Grundt et al., 2006) in SHR demonstrated that in females, Nebivolol administration induced decreased heart rate and blood pressure to a greater extent than it did in males, an effect that can be related to β3AR and ERs activation.

Conclusions
This review focused on the relationship between estrogen and β3AR, showing how these molecules act and are modulated in the cardiovascular system.As discussed throughout this article, there is wide evidence that estrogens and β3AR are intertwined, and this is a mechanism that may determine a differential modulation and responsiveness of β3AR in males and females.Importantly, as estrogen declines with aging and menopause, most of their effects are lost in women profoundly impacting on the heart and vasculature (Rengo et al., 2013).As discussed above βARs, particularly β3AR, because of their regulating effects within the cardiovascular system, may represent vital molecules behind estrogens-mediated effects (Liccardo et al., 2022;Rengo et al., 2013).
Despite this, further research is required to depict the specific signaling molecules that orchestrate the crosstalk between estrogens and β3ARs, and that can imply drugs that target these receptors.In this regard, herein, we have discussed the potential usage of Nebivolol, which has been initially identified as a selective β1-blocker, then as a β3AR-agonist, and finally, studies have also supported an agonistic role for ER (Fig. 2).This viewpoint requires further evaluation at the experimental and clinical levels.Furthermore, future research is warranted to examine the existence of a relationship between estrogens and β3AR in the heart.Indeed, studies have demonstrated the therapeutic potential of β3AR in the heart.Similarly, estrogen and ERs have been reported to be cardioprotective.Therefore, a better understanding of the potential interrelation between these molecules will offer the unique opportunity to improve the diagnostic and therapeutic strategies for treating cardiovascular disease selectively in men and women, thus boosting the concept of "sex/gender cardiovascular medicine" (Arain et al., 2009).β1AR) and β3AR are seven transmembrane receptors coupled with G protein subunits.Notably, the stimulatory G protein (Gs) activates the adenylate cyclase (AC) on the plasma membrane and induces the generation of cAMP.Subsequent cAMP leads to the activation of the protein kinase A (PKA) that is involved in the phosphorylation of several molecules, including the PKB (Akt), which in turn activates the endothelial nitric oxide synthase (eNOS) with the generation of NO.This gasotransmitter stimulates the soluble guanylate cyclase (sGC) to produce cGMP and PKG.Conversely, the inhibitory G protein (Gi) inhibits AC activity, counteracting Gs activity, and gives rise to NO via both eNOS and neuronal NOS (nNOS), thus leading to PKG induction.On the other hand, estrogens, particularly estradiol (E2), can cross the plasma membrane and interact with intracellular ERα and ERβ with consequent receptor dimerization and translocation into the nucleus.ERs bind to chromatin, directly affecting eNOS gene expression and NO generation.Nebivolol is a highly selective β1-blocker with β3AR and ER agonist activity.

Fig. 2 .
Fig.2.β1-adrenergic receptor (β1AR) and β3AR are seven transmembrane receptors coupled with G protein subunits.Notably, the stimulatory G protein (Gs) activates the adenylate cyclase (AC) on the plasma membrane and induces the generation of cAMP.Subsequent cAMP leads to the activation of the protein kinase A (PKA) that is involved in the phosphorylation of several molecules, including the PKB (Akt), which in turn activates the endothelial nitric oxide synthase (eNOS) with the generation of NO.This gasotransmitter stimulates the soluble guanylate cyclase (sGC) to produce cGMP and PKG.Conversely, the inhibitory G protein (Gi) inhibits AC activity, counteracting Gs activity, and gives rise to NO via both eNOS and neuronal NOS (nNOS), thus leading to PKG induction.On the other hand, estrogens, particularly estradiol (E2), can cross the plasma membrane and interact with intracellular ERα and ERβ with consequent receptor dimerization and translocation into the nucleus.ERs bind to chromatin, directly affecting eNOS gene expression and NO generation.Nebivolol is a highly selective β1-blocker with β3AR and ER agonist activity.