Molecular pathways of oestrogen receptors and β-adrenergic receptors in cardiac cells: Recognition of their similarities, interactions and therapeutic value.

Oestrogen receptors (ERs) and β-adrenergic receptors (βARs) play important roles in the cardiovascular system. Moreover, these receptors are expressed in cardiac myocytes and vascular tissues. Numerous experimental observations support the hypothesis that similarities and interactions exist between the signalling pathways of ERs (ERα, ERβ and GPR30) and βARs (β1 AR, β2 AR and β3 AR). The recently discovered oestrogen receptor GPR30 shares structural features with the βARs, and this forms the basis for the interactions and functional overlap. GPR30 possesses protein kinase A (PKA) phosphorylation sites and PDZ binding motifs and interacts with A-kinase anchoring protein 5 (AKAP5), all of which enable its interaction with the βAR pathways. The interactions between ERs and βARs occur downstream of the G-protein-coupled receptor, through the Gαs and Gαi proteins. This review presents an up-to-date description of ERs and βARs and demonstrates functional synergism and interactions among these receptors in cardiac cells. We explore their signalling cascades and the mechanisms that orchestrate their interactions and propose new perspectives on the signalling patterns for the GPR30 based on its structural resemblance to the βARs. In addition, we explore the relevance of these interactions to cell physiology, drugs (especially β-blockers and calcium channel blockers) and cardioprotection. Furthermore, a receptor-independent mechanism for oestrogen and its influence on the expression of βARs and calcium-handling proteins are discussed. Finally, we highlight promising therapeutic avenues that can be derived from the shared pathways, especially the phosphatidylinositol-3-OH kinase (PI3K/Akt) pathway.

INTRODUCTION

The risk of cardiovascular diseases (CVDs) is higher in aged women compared to that of pre‐menopausal women.1 In addition, development of CVDs in men occurs at a relatively young age, while the risk of CVDs in women accelerates after menopause.1 These observations are attributed, in part, to gender‐related cardioprotective roles of oestrogen. Moreover, numerous studies have described the expression of oestrogen receptors (ERs) in various tissues of the cardiovascular system (CVS). There are 3 classes of ERs: the ERα, ERβ and the G‐protein‐coupled receptor 30 (GPR30). All these ERs are expressed in the heart cells and in the vascular vessels.2, 3, 4, 5 Each receptor subtype shows variation in function and in tissue‐specific expression. Based on ligand specificity, oestrogen and some of its metabolic intermediate products activate the ERs triggering both genomic and non‐genomic actions.6 Results from several experiments have implicated oestrogen in the chronotropic and inotropic functions of the heart,7, 8, 9 and in cardiac perfusion.10, 11, 12

In their recent study, Debortoli et al.10 showed that activation of GPR30 modulated coronary circulation by regulating coronary perfusion pressure in rats. Accordingly, left ventricular diastolic dysfunction is predominant in post‐menopausal women.13 Consistent with these reports, Giraud et al.14 used Magness et al.'s menopause model15 and showed that left ventricle diameters and end‐diastolic volume were elevated by chronic oestrogen replacement in this model. In the ovine model, 3 research groups showed that 17β‐oestradiol administration increased coronary blood flow significantly.15, 16, 17, 18, 19, 20 Collectively, they showed that the pattern of rises in coronary perfusion is independent of patterns of rises in cardiac output. They also noted that the pattern of raises in cardiac output was graded, that is a 30‐ to 60‐min delay followed by an increase and a plateau at 90‐120 minutes, a phenomenon observed in reproductive tissues such as uterus and mammary gland.15, 20 Mershon et al. confirmed that these effects of oestrogen are ER‐dependent as they were prevented by pre‐treatment with antagonist ICI‐182 780.18

The heart rhythm and contraction are mainly regulated by the sympathetic nervous system (SNS), via the β‐adrenergic receptors (βARs) that connect and convey the SNS signals to the heart. βARs are divided into β1AR, β2AR and β3AR.21 Interestingly, signalling pathways of ERs are intertwined with those of the βARs pointing to the possibility of functional convergence in modulating the physiology of the CVS. In fact, crosstalk between ERα and α1b‐adrenergic receptor was reported previously.22, 23 Furthermore, we and others established that oestrogen alters gene expression of βARs and calcium (Ca2+)‐handling proteins of the CVS.24, 25, 26 Proteins that regulate cardiac Ca2+ include the Na+/Ca2+ exchanger pump (NCX), L‐type Ca2+ channel (LTCC), phospholamban (PLB), sarcoplasmic reticulum Ca2+‐ATPase (SERCA) and ryanodine receptors (RyRs; Figure 1).27 Consequently, the effects of oestrogen on the Ca2+‐handling proteins have direct implications on the contractile machinery of the myocardium.

Figure 1

Cardiac Ca2+‐handling proteins and Ca2+ trafficking in cardiomyocyte regulated by βARs and oestrogen. Illustration of a network of calcium‐handling proteins and Ca2+ trafficking in cardiomyocyte which are activated and regulated by βARs and oestrogen. Purple arrows indicate movement of Ca2+. The symbol indicates points at which oestrogen exerts influence on cardiac contractile function. Abbreviations: LTCC, L‐type channel; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; SERCA, sarcoplasmic reticulum Ca2+‐ATPase; NCX, Na+/Ca2+ exchanger pump; PLB, phospholamban

In recent decades, the need to understand the cardiovascular functions of oestrogen and βARs has received much interest from researchers. The discovery of GPR30,28 which shares structural features with βARs, has expanded the functional scope of oestrogen. In this regard, it is important to re‐evaluate the relationships between ER and βAR signalling pathways and their interdependence in modulating the cardiovascular physiology. This review focuses on the recent experimental studies to describe the roles and mechanisms of ERs and βARs. We firstly discuss their classification, functions and the basis of cardiac physiology. We then provide novel illustrations on the points of integration between oestrogen and adrenergic signalling pathways. Our aim is to provide evidence for the hypothesis that there are interactions and functional cooperation between ER and βAR signalling pathways, particularly in the heart. We highlight the therapeutic potential of the interactions and explore their implications on the postulated cardioprotection conferred by oestrogen, β‐blockers and Ca2+ channel blockers. We also discuss the ERs and βARs as coregulators of cardiac Ca2+‐handling proteins.

A RECAP OF β‐ADRENERGIC RECEPTORS IN THE CARDIOVASCULAR SYSTEM

βAR‐specific features

βARs are members of the G‐protein‐coupled receptors (GPCRs) that classically form 7 transmembrane loops, with extracellular and intracellular terminals. Three βAR subdivisions, β1AR, β2AR and β3AR, are encoded by different genes.21 Moreover, the 3 receptors are expressed in the plasma membrane in the CVS,29 as well as in the cardiac nuclear envelope of adult rats and mouse myocytes for β1AR and β3AR.30, 31, 32In heart myocytes, the number of β1ARs is higher than that of β2ARs, while β3ARs show the least abundance.33 βARs are linked to heterogeneous intracellular signalling pathways and proteins. In addition, their expressions vary under physiological and pathological conditions.34

βAR‐specific signalling

βARs are activated by noradrenaline and adrenaline released from the SNS and adrenal glands respectively. However, once activated, the βARs trigger diverse intracellular pathways.35 β1ARs couple to the stimulatory unit of the G protein (Gαs) leading to the synthesis of cyclic adenosine monophosphate (cAMP) by adenylyl cyclase (AC) enzyme. On the other hand, β2ARs are pleiotropic receptors that couple to the Gαs, the inhibitory G protein (Gαi) and the Gβγ.36, 37 At the physiological state, β2ARs couple to the Gαs, whereas at high adrenaline concentration, they switch to Gαi, a phenomenon referred to as stimulus‐mediated trafficking.38, 39 Activation of the β2AR/Gαi pathway inhibits cAMP production, an opposing effect to β2AR/Gαs and β1AR/Gαs activation (Figure 1).

With regard to structure and function, the β3ARs display distinct differences to β1ARs and β2ARs. Notably, the cytoplasmic C‐terminus of the β3ARs lacks the target amino acid sequences for protein kinase A (PKA) and cardiac G‐protein‐coupled receptor kinase 2 (GRK2) phosphorylation.40, 41 Consequently, β3ARs are less susceptible to PKA/GRK2‐mediated receptor recycling and desensitization in response to hyperstimulation.40 Two isoforms, β3aAR and β3bAR, were reported in Chinese hamster ovary (CHO) cells and 3T3‐L1 adipocytes.42, 43, 44, 45, 46 The β3aAR isoform coupled to the Gαi, whereas β3bAR coupled to both Gαs and Gαi. At present, there are no reports regarding the existence of the 2 isoforms in human cardiac cells. β3AR has largely been associated with metabolic functions. Nevertheless, β3AR stimulation induced positive inotropy in human atrial cells,47 but it had no effect on the inotropy of human ventricular cells.48 β3AR may influence chronotropic functions through the nitric oxide (NO)/guanosine 3′,5′‐monophosphate (cGMP) pathway.49 Activation of plasma β3AR‐NO synthase/guanylyl cyclase pathway was shown to influence the nuclear β3AR‐mediated gene transcription, suggesting a crosstalk between the surface and nuclear β3ARs.50

The basis of the heart's function

βARs mediate the SNS regulation of the cardiac functions.51 These functions are primarily orchestrated by activation of β1ARs, which constitute up to 80% of the entire cardiac βAR density of healthy human, and to a lesser extent by the β2ARs.33, 35 Moreover, β2ARs have a higher affinity for adrenaline, while β1ARs have almost equal affinities for both noradrenaline and adrenaline.39 On the other hand, activation of the β3ARs is largely associated with negative inotropy during catecholaminergic stress.47

Once activated, βARs initiate cAMP synthesis by coupling to the Gαs, a GTP‐binding protein. This cAMP, in turn, activates PKA. What follows is the induction of intracellular rise in Ca2+ transients via the tightly regulated network of ion channels. PKA‐mediated inotropic effects are orchestrated through the phosphorylation of 2 main channels: the LTCC located in the T‐tubular network formed by sarcolemmal membrane invaginations and the RyR2 receptors on the SR membrane. Phosphorylation of LTCC allows Ca2+ entry as inward current.52 These Ca2+ currents further stimulate Ca2+ release from SR, the intracellular stores, by the opening of RyR2 receptors which are also phosphorylated by PKA. This phenomenon is referred to as calcium‐induced calcium release. The resultant Ca2+ transient activates the myofilament protein troponin C turning on cardiomyocyte contraction. The size of Ca2+ transients is a key determinant of the strength of the contraction.27 PKA also regulates cardiac relaxation by phosphorylating phospholamban (PLB), a modulator of SERCA. In its unphosphorylated state, PLB inactivates SERCA. This effect is reversed following PLB phosphorylation which permits Ca2+ uptake back to the SR by SERCA. In addition, sarcolemmal Na+/Ca2+ exchanger pump (NCX) accelerates Ca2+ extrusion, which together with SR Ca2+ uptake diminishes the Ca2+ transient resulting in relaxation.

Besides the classical cAMP/PKA pathway, cAMP also acts through the recently described intracellular protein named exchange protein directly activated by cAMP (EPAC).53 Classified into EPAC1 and EPAC2, these proteins bind cAMP and function as guanine exchange factors (GEFs) for Ras superfamily. The EPAC pathway amplifies the cardiovascular functions of β1AR/cAMP and provides alternative modulation of βAR activation. Indeed, both EPAC1 and EPAC2 are present in cardiomyocytes.53 Diverse physiological roles of EPAC proteins have been recently reviewed by Lezoualc'h et al.54 Activation of EPAC was linked to ventricular hypertrophy, vasorelaxation and in the regulation of Ca2+ through RyR and PLB phosphorylation,54 indicating synergism between the cAMP/PKA and cAMP/EPAC pathways.

Another downstream target of β1AR activation is the multimeric protein Ca2+/calmodulin kinase II (CaMKII).55 Activation of this kinase indirectly relies on the PKA‐mediated rise in cytosolic Ca2+ and intracellular levels of calmodulin.56 Recent findings reveal that CaMKII activation augments the LTCC current and increases the RyR open probability57 and phosphorylation of PLB,58 showing its participation in cardiac contractility. CaMKII has also been associated with detrimental effects including apoptosis, necroptosis and arrhythmias.59

CLASSIFICATION, LOCALIZATION AND DISTRIBUTION OF ERS IN THE CVS

The cardiovascular functions of oestrogen are mediated by ERs. These cellular receptors are categorized as nuclear receptors (ERα and ERβ), which modulate transcription of specific gene sets, and membrane‐bound receptor (GPR30, also known as GPER1), which mediates rapid, non‐genomic actions of oestrogen. ERs are expressed in cardiomyocytes,3 cardiac fibroblasts4 and VSMCs;5 however, their expression and cellular locations are not fully understood. For instance, Pugach et al.3 reported that ERβ was not expressed in either neonatal or adult male or female mouse or rat ventricular myocytes. This observation is inconsistent with earlier reports.60, 61, 62 In addition, there are controversies surrounding the cellular localization of GPR30. In particular, some researchers reported that GPR30 was nearly confined to the endoplasmic reticulum in COS cell lines,63 while others observed both cytosolic and membrane localization in HEK293 cells64 and in rat VSMCs.65 The differences in these reports may be related to tissue‐specific variations. It is also important to note that oestrogen is a lipophilic hormone that crosses the plasma membrane to access the intracellular receptors. Therefore, both membrane and subcellular localization of GPR30, as observed, are conceivable as they are accessible to oestrogen.

With regard to gender, a study carried out on VSMCs of rats showed that GPR30 expression was similar in both males and females.65 However, gender differences with regard to ERα and ERβ expression were also reported. Whereas the mRNA levels of ERα were equivalent in hearts of both men and women,3, 66 ERβ had greater expression in males than females in both healthy and diseased human hearts.67 However, these observations are in contradiction to another report that showed an opposite expression pattern where ERβ expression was not different in male and female cardiomyocytes, while ERα expression varied with gender.68 Elsewhere, ERα and ERβ protein levels in male and female rabbit hearts were not different.69 Further studies are advocated to reconcile these findings.

In addition, Ma et al.65 observed that subcellular location of ERα was not influenced by its activation; a similar observation was reported for GPR30 in a subsequent study.64 However, change in subcellular location of the ERs may vary as in heart failure. In healthy hearts, ERα was localized to the intercalated disc, while in failing hearts, its location shifted away from the intercalated discs.66 This implies that cardiomyopathies may influence the subcellular localization and by extension the signalling of the ERs. Moreover, oestradiol supplementation in ovariectomized (OVX) rats increased ERα and ERβ protein levels.70 Variation in relative abundance of the ERs was recently reported. Quantitative real‐time PCR analysis of male mouse ventricle found that GPR30 mRNA levels were thrice those of ERα and 17‐fold greater than those of ERβ.62 Different genes located on different chromosomes encode each ER subtype. While alternative splicing of the gene transcripts leads to multiple subtypes of ERα and up to 5 described transcripts of ERβ,71 GPR30 only exists in 1 isoform.3, 6, 72 The distinct features of the ER subtypes are outlined in Table 1. Taken together, expression of the ER subtypes in the cardiovascular system remains contentious with regard to tissue‐specific expression. Discrepancies from the previous reports could be due to the methods used or species of tissue investigated. Further investigations are required to resolve the inconsistencies.

Table 1

Features and classification of oestrogen receptors

Receptor features	GPR30	ERα	ERβ
Cellular location	Plasma membrane96	Nucleus83, 169	Nucleus130
	Cytosol64	Cytosol60, 130	Cytosol60
		Plasma membrane130, 170	Plasma membrane82, 169
Onset of physiological effects	Rapid actions (effects within seconds to minutes)102	Rapid and genomic action (effects within minutes to days)82, 102, 171, 172, 173, 174	Rapid and genomic action (effects within minutes to days)82, 83, 173, 174, 175
Genetics	GPER gene located on chromosome 7p22.372	ESR1 gene located on chromosome 6q25.1176	ESR2 gene located on chromosome 14q23.2178
	No introns, 1 isoform72	8 exons, 3 isoforms176	8 exons, 5 isoforms178, 179
	Protein size 375 amino acids72	Protein size 595 amino acids177	Protein size 530 amino acids178
Cardiovascular tissue distribution	Cardiac fibroblasts4	Cardiac fibroblasts2	Cardiac fibroblasts2
	Vascular tissues180	Vascular tissues93, 102	Vascular tissues93
	Cardiomyocytes62	Cardiomyocytes2, 3	Cardiomyocytes (unresolved)
Ligands	E262	E2130	E283
	G‐1181	PPT76	DPN76
Relative abundance in cardiac cells	Highest62	Low62	Lowest62

E2, 17β‐oestradiol; PPT, propylpyrazoletriol; DPN, propylpyrazoletriol; G‐1, GPR30 agonist; ERα, oestrogen receptor α; ERβ, oestrogen receptor β; GPR30, G‐protein‐coupled oestrogen receptor 30.

ER ACTIVATION, SIGNALLING PATHWAYS AND CELL FUNCTIONS

ER activation

Similar to other steroids hormones, oestrogen signalling is initiated by the binding of 17β‐oestradiol or xenoestrogens73 and oestradiol metabolites74, 75 to ER. Synthetic receptor‐specific agonists with selective binding affinities have also been developed: propylpyrazoletriol (PPT) for ERα, diarylpropionitrile (DPN) for ERβ and G1 for GPR30.76 Noteworthy, each receptor subtype or isoform displays different affinities to 17β‐oestradiol and other oestrogenic ligands.77, 78 Moreover, oestrogens are of different forms (estrone, oestradiol and estriol) which exist in a dynamic equilibrium in circulation. Considering that oestrogen activates multiple receptors, ER subtype–specific functions determine cellular responses to oestrogen stimulation. It has been postulated that the balance between oestrogen forms is responsible for activation of different signalling pathways under certain physiological conditions based on the premise that ERs possess different affinities for each oestrogen subtype.78 Furthermore, 17β‐oestradiol synthesis occurs through enzymatic modifications of precursors such as androgens by aromatase enzyme. Considering that aromatase is expressed within the heart,68 the possibility of cardiac oestrogen synthesis further augments the importance of oestrogen to the cardiovascular physiology in addition to circulating oestrogens. It is also likely that the adipose tissue surrounding the heart is the source of the C19 androgen conversion to C18 oestrogen, considering that epicardial fat covers up to 80% of heart's surface and constitutes 20% of heart's weight.79

Signalling pathways

Receptor‐mediated signalling: genomic vs non‐genomic pathways

Binding of oestrogen to its receptors (membrane or nuclear) triggers 2 types of cellular effects defined by the timing of onset. (i) Part of the effects occurs through the well‐established pathway of ER‐mediated transcription of certain genes. Conventionally, this pathway is known as a genomic pathway and occurs within hours to days.80 The ERα and ERβ receptors largely execute these genomic functions. Upon oestrogen binding, these ERs undergo conformational changes allowing nuclear translocation and dimerization of the oestrogen‐ER complex with oestrogen response elements, found at promoter areas of specific genes. Through this mechanism, oestrogen influences expression of cellular proteins. However, this pathway is not exclusive to nuclear receptors. Activation of membrane receptor GPR30 induced gene transcription.4 (ii) Another pathway that emerges after oestrogen binding is the non‐genomic pathway. This pathway requires activation of several different signalling cascades that alter cellular functions of proteins and ion channels. Most of these actions occur within seconds or minutes and are regulated by ERα and GPR30.7, 62, 81 There are reports indicating the presence of ERβ in the cytosol and plasma membrane and that they are responsible for rapid non‐genomic signalling in endothelial cells. It is yet to be established whether ERβ exists on the plasma membrane of adult cardiomyocytes.60, 82, 83, 84 In addition, crosstalk between membrane ERs and nuclear ERs has been reported.85 There is growing interest to decipher mechanisms that underlie non‐genomic oestrogen signalling. How the non‐genomic oestrogen pathways integrate with βAR pathways forms the basis of the discussion dealt with in Section 3 of this article. Moreover, the interaction of ER signalling with adrenergic receptor pathways was observed between the ERα and α1b‐adrenergic receptors.22

Receptor‐independent signalling

Besides the conventional receptor‐mediated mechanisms of oestrogen, experimental observations have hinted at the possibility of an alternative mechanism that does not involve membranous or nuclear ERs.81, 86, 87 This mechanism falls in the category of rapid and non‐genomic pathways and does not involve oestrogen‐receptor binding. A previous experiment showed that oestrogen induced negative inotropy in ERα and ERβ knockout mouse cardiomyocytes and its inhibition of the LTCC current was not altered from wild‐type myocytes.88 The same laboratory later demonstrated that oestrogen directly interacts with the LTCC protein and inhibits LTCC current even at resting state on cultured HEK293 cells.86 Indeed, similar observations have been reported for a broad range of ion channels (see review89). Research on the rapid non‐genomic roles of oestrogen has been primarily focused on the membranous receptors. Therefore, the observation that oestrogen could bind directly to ion channels introduces a reclassification of its mechanism of actions and adds to the growing debate on its non‐genomic functions.

Tissue‐specific functions

Oestrogen plays several functions in the CVS. Here, we highlight some of the cell‐specific roles of oestrogen without much detail because of the limitation defined by the purpose of this review. Activation of GPR30 inhibited proliferation of rat cardiac fibroblasts and collagen synthesis in both in vivo and ev vivo settings.4 These effects were attributed to oestrogen‐induced expression of cell cycle proteins and alterations in expression of matrix metalloproteinase‐12. GPR30 also mediates cardioprotection against ischaemia/reperfusion injury by improving the heart function, reducing infarct size, and mitochondrial Ca2+ overload.62 On the other hand, ERα agonists induced vasodilation on vascular smooth muscle cells of the aorta.90 In our previous study, we demonstrated that oestrogen and G1 decreased the expression of β1ARs and induced negative inotropy.24, 91 ERβ has also been reported to offer cardioprotection in cardiomyocytes.92 Together, these findings demonstrate that ERs are important effectors of oestrogen signals in cardiovascular tissues.

SIMILARITY IN MOLECULAR PATHWAYS OF ERS AND ΒARS

The crosstalk between ERs and βARs is a concept that was revealed from earlier studies.22, 23 Evidence from recent studies further recognizes oestrogen as a key hormone that influences the expression of βARs26, 91 and cardiac ion‐handling proteins.93, 94 Moreover, oestrogen regulates the cardiac contractile functions, which are otherwise under the control of adrenergic receptors (details discussed in Sections 4 and 4.1). Intriguingly, the structure (of GPR30) and signalling pathways of ERs are functionally closer/related to those of the βARs, at least partially.72, 93, 95, 96, 97, 98 Some of the cellular roles of ERs synergize or oppose the effects produced by βAR activation. Therefore, here we explore, side by side, the correlation among β1ARs, β2ARs and β3ARs vs. ERα, ERβ and GPR30. We discuss various points of integration between their signalling pathways. We note 3 main pathways along which these classes of receptors interact.

Signalling along the GPCR/Gαs/cAMP pathway

Similar to βARs, GPR30 possesses PKA phosphorylation sites and PDZ binding motifs and associates with A‐kinase anchoring proteins (AKAPs).64 In addition, GPR30 uniquely possesses 4 CaMKII binding sites unlike all other GPCRs.99 Furthermore, like the βARs, GPR30 couples to the classical GPCR proteins, Gαs 100, 101 and Gαi/o,63, 96, 102 in cardiovascular tissues (Figure 2). On this basis, activation of GPR30 partially mimics the signalling pathway of βARs with regard to its downstream cascades. Initial activation of β1AR, β2AR and GPR30 leads to coupling to Gαs protein.96, 103 Activation of Gαs triggers the production of cAMP by AC enzyme. Subsequently, PKA and EPAC amplify the cAMP signal. In coronary arteries, GPR30 was shown to activate this pathway including the production of PKA and EPAC proteins.101 The cAMP is hydrolysed by phosphodiesterases (PDEs) that determine the specificity of its signalling so as to avoid “off‐target” reactions through the creation of microdomains.104 This process occurs through the multimeric units formed between βAR/Gαs/AC and PDEs.104 In addition, it is known that PKA interacts with PDE4 and facilitates the degradation of cAMP by associating with the scaffold proteins AKAPs.105 Therefore, considering the observation that GPR30 signals through the Gαs/AC/cAMP pathway, it would be interesting to define whether the GPR30/Gαs/AC complex also participates in compartmentalization of cAMP signals in cardiac cells. The PKA generated downstream of GPR30 might interact with PDEs, under the direction of AKAP5, to regulate cAMP degradation as for β1ARs and β2ARs. Moreover, we speculate that GPR30's ability to activate EPAC might also influence β1AR/cAMP/EPAC‐mediated functions. Besides GPR30, activation of the ERα elevated PKA in VSMCs of aortic tissue further illustrating oestrogen involvement in the GPCR/Gαs/cAMP signalling pathway.102

Figure 2

Interaction of oestrogen signalling and beta‐adrenergic signalling pathways. The symbol represents cytosolic Ca2+ rise. The symbol represents inhibition signal. The symbol represents activation signal. Signalling pathways of βARs (β1AR, β2AR and β3AR) and ERs (ERα, ERβ and GPR30) are integrated through the Gs and Gi pathways. Effector proteins PKA and EPAC affect the Gs‐cAMP signals. The resultant effects play crucial roles in cardiac contraction by increasing cytosolic Ca2+ levels. Alternatively, the receptors may activate the Gi, which mediates anti‐apoptosis signals through the PI3K/Akt pathway. Elevated cytosolic Ca2+ levels activate CaM and CaMKII, which induces apoptosis. GPR30 may inhibit the AC enzyme through the MAGUK/AKAP5 complex. GPR30: G‐protein‐coupled receptor 30; E2: 17β‐oestradiol; EPAC: exchange protein directly activated by cAMP; AKAP5: A‐kinase anchoring protein 5; MAGUK: membrane‐associated guanylate kinase; CaM: calmodulin; CaMKII: Ca2+/calmodulin kinase II

Additional interactions are possible due to the structural resemblance between GPR30 and βARs. βARs interact with cellular proteins through PDZ motifs located at their C‐terminus regions. The PDZ motifs give a bearing on the localization and signalling of βARs. Of note, β1AR, β2AR and GPR30 possess type I PDZ binding motifs: ‐ESKV, ‐DSLL and ‐SSAV respectively.106, 107 PDZ domains recognize and bind to specific amino acid sequences of their target proteins.106 For instance, the β1AR (‐ESKV) motif was shown to be a determinant factor for its coupling to Gαs and not Gαi. Induced disruption of this motif permitted β1AR/Gαi coupling.106 On the other hand, β2AR (‐DSLL) motif plays a role in its coupling to Gαi.108 In comparison, the GPR30 PDZ motif (‐SSAV) was implicated in recycling and translocation of GPR30 in HEK293 cells,64 but it is not known whether this motif may influence GPR30's ability to couple to Gαs or Gαi. In addition to the PDZ motifs, β2AR phosphorylation by PKA influences its ability to bind Gαs or Gαi.39 Although GPR30 coupling to Gαs or Gαi may be dependent on cell/tissue type, collectively, these observations raise the possibility that PKA phosphorylation or disruption of its PDZ motif would have implications on its coupling to Gαs and Gαi as is the case for β1ARs and β2ARs. This possibility calls for further inquiry to the conditions under which GPR30 couples to Gαi and Gαs, and the proteins that interact with its PDZ motif.

One functional implication of this motif draws from the recent observation that GPR30 inhibited βAR‐mediated production of cAMP in response to isoproterenol stimulation in HEK293 cells.64 This inhibitory effect was dependent on a complex formed by GPR30, through its PDZ motif, with membrane‐associated guanylate kinases (MAGUKs) and AKAP5 (Figure 2).64 The primary role of AKAPs is to bind and regulate the subcellular location of PKA. Interestingly, the binding of AKAP5 to β1ARs facilitated its recycling by enhancing PKA phosphorylation of the receptor.109, 110, 111 Moreover, it is established that oestrogen regulates expression of β1ARs. Therefore, an association of GPR30 with AKAP5 and possibly other unidentified proteins could be a mechanism through which oestrogen participates in the regulation of β1AR density. Further research should be carried out to characterize the interactions of GPR30 with AKAPs and their implications on cellular functions.

Although β1AR, β2AR and GPR30 are capable of coupling to Gαs, their cellular effects are not similar. For instance, activation of β1ARs,112 ERα and GPR30113 triggers production of calmodulin and activation of CaMKII, while β2ARs and ERβ do not. Importantly, the observation that GPR30 activates cAMP through Gαs pathway challenges previous findings that oestrogen induced negative inotropy at both cardiomyocyte and organ levels. Therefore, the effects of GPR30/Gαs/cAMP pathway may not be identical to the classical βAR/Gαs/cAMP pathway, especially in cardiomyocytes. However, as we reported, oestrogen may tilt the activation of β2AR/Gαs or β2AR/Gαi pathways in certain disease conditions, as in stress‐induced cardiomyopathy.114

Signalling along the GPCR/Gαi/PI3K/Akt pathway

As mentioned earlier, GPR30, like β2ARs and β3ARs, couple to the Gαi subunit.96, 102, 115 Furthermore, GPR30 activates the phosphatidylinositol‐3‐OH kinase (PI3K)/Akt pathway resulting in inhibition of apoptosis through regulation of the Bcl‐2 family of proteins.24, 102, 115, 116 The GPR30/PI3K/Akt‐mediated cardioprotection against ischaemia/reperfusion injury in cardiomyocytes was orchestrated through upregulation of anti‐apoptosis Bcl‐2 protein and downregulation of pro‐apoptosis Bax protein.116 In accordance with our previous report, inhibition of β2ARs exposes cardiomyocytes to cell death in ischaemic conditions.117 The β2AR/Gαi pathway triggered anti‐apoptotic signals through the PI3K/Akt pathway.118 These findings present the evidence that PI3K/Akt cardioprotective pathway is shared by the GPR30 and β2ARs. In addition to the GPR30, oestrogen activates the ERα, which directly binds the p85 alpha regulatory component of the PI3K.119

In addition, PKA phosphorylation of the β2ARs enables switching of its coupling from Gαs to Gαi under extreme catecholamine stimulation,39 a phenomenon referred to as signal trafficking (Figure 1). In this context, β2ARs act as a switch that coordinates synthesis of cAMP and indicates cross‐communication between β1AR and β2AR signalling. Moreover, AKAP5 tethering of PKA allows it to phosphorylate β2ARs.120 Considering that GPR30 possesses PKA phosphorylation sites, we speculate that through AKAP5, PKA phosphorylation of GPR30 may influence its ability to activate Gαs or Gαi. Although we appreciate that such signal trafficking is intricately complicated and may involve different mechanisms, further research is necessary to determine whether this phenomenon can be replicated in adult cardiomyocytes. Unlike β2ARs, the conditions under which GPR30 activates Gαs or Gαi are not clearly understood. Perhaps a possible hint as to when GPR30 activates Gαi comes from the observation that under stress conditions, both β2ARs and GPR30 activate Gαi/PI3K/Akt pathway to confer cardioprotection.116, 118 However, we recognize that the requirements for GPR30 coupling to Gαs or Gαi in cardiomyocytes need further investigation.

Unlike β1ARs, β2ARs and GPR30, β3ARs lack PKA phosphorylation sites.40 Therefore, for β3ARs, there is a great deal of variability with respect to their ability to activate both Gαs and Gαi. Some β3AR splice variants were shown to display dual coupling to Gαs and Gαi in other cell types, although not in cardiac cells.121 β3ARs act through Gαi/o to suppress contractility via induction of NO‐cGMP pathway under chronic catecholaminergic stimulation.122 In addition, β3AR/NO/cGMP pathway was enhanced in the presence of β1AR blocker, which was interpreted to be beneficial in chronic volume‐overloaded heart.123 Based on the evidence presented above, the ER and βAR signalling pathways function as interdependent networks/partners whose roles have profound effects on the cardiovascular system. In summary, Gαs and Gαi act as pivots around which both ER and βAR signalling pathways converge. β2AR and GPR30 signal through the Gαs/AC/cAMP and Gαi/PI3K/Akt pathways in the cardiovascular system. Similarly, both GPR30 and ERα signalling cascades interact through the PI3K/Akt pathway (Figure 1). PI3K/Akt acts as a focal pathway that unifies GPR30‐, ERα‐ and β2AR‐mediated cardioprotection. In general, the Gαs/AC/cAMP and Gαi/PI3K/Akt pathways seem to trigger opposing effects. For example, the cAMP produced by βAR stimulation was shown to inhibit the activity of Akt kinase indicating an inverse relationship between the 2 pathways.124 Lastly, the net cellular effects of the interactions between ERs and βARs might be dependent on the cell/tissue type.

Localization of ERs and βARs to the caveolae

β1AR, β2AR and β3AR have been shown to signal and express in the caveolin‐rich fractions of the plasma membrane (Figure 3). Caveolin proteins are found in flask‐shaped subdomains of plasma membranes known as caveolae.125 β2AR localizes almost exclusively to caveolin 3‐rich membrane fractions of rat cardiomyocytes, while β1AR localizes to both caveolar and non‐caveolar membrane fractions.126 These spatial distributions play a role in the differential activation of cAMP signals by β1AR and β2AR.127 For instance, colocalization of β2AR with the Ca2+ channel LTCC and caveolin 3 is essential for its signalling and ability to invoke intracellular Ca2+,128 while caveolin 3 interaction with AC V acts as a scaffolding protein which participates in β1AR signals that induce LTCC current in ventricular cardiomyocytes.129 ERα was associated with eNOS activation in the caveolae of endothelial cells,130 while ERβ was found in the caveolae where it mediated eNOS signals.82 Strikingly, overexpression of β2AR enhanced vascular repair of endothelial progenitor cells in mice through the eNOS pathway.131 Therefore, localization of β2AR and ERβ in caveolae of vascular cells and their ability to signal through the eNOS pathway indicate functional cooperation between the 2 receptors. Caveolin 1 is a scaffold protein for both ERα and β3AR.132, 133 The association of β3AR with caveolin 1 was shown to govern its ability to couple to Gαi/o proteins in CHO‐K1 cells.133 On the other hand, it has been established that ERα and ERβ interact directly with Gαi and that these interactions occur in close proximity to the caveolae domains.134 The physiological relevance of the possible interactions among ERs, caveolins and βARs in adult cardiomyocytes remains to be fully established. Although there is a dearth of evidence regarding this view, the data sets reviewed here imply direct or indirect crosstalk among ERs and βARs. Further research is required to identify multilevel communications and interactions among these receptors.

Figure 3

βAR and ER signalling through caveolae. In this view, β2AR associates with caveolin 3 and LTCC to transduce signals that increase cellular Ca2+. β1AR interacts with caveolin 3 and AC V to induce LTCC current. Caveolin 1 interacts with β3AR and governs its ability to couple to Gαi. ERα colocalizes with caveolin 1 and signals through Gαi and activates eNOS pathway. ERβ mediates the activation of eNOS by oestrogen in the caveolae. AC V: adenylyl cyclase V; ERα: oestrogen receptor alpha; ERβ: oestrogen receptor beta; eNOS: endothelial nitric oxide synthase; LTCC: L‐type calcium channel

A summary of the shared features is as follows:

β2ARs, β3ARs, ERα, ERβ and GPR30 couple to Gαi subunit.

β1ARs, GPR30 and ERα activate calmodulin/CaMKII.

β1ARs, β2ARs and GPR30 couple to Gαs subunit.

β2ARs, ERα and GPR30 trigger the PI3K/Akt pathway.

β3AR and ERα associate with caveolin 1, while β1AR and β2AR associate with caveolin 3.

OESTROGEN INFLUENCE ON THE EXPRESSION OF βARS

Expression of βARs in the cardiovascular vessels is influenced, in part, by age, by gender and by drugs targeting these receptors.135 The ratio of β1ARs, β2ARs and β3ARs may also vary with disease status.136 In pre‐menopausal women, the cardiac expression of β1ARs and β2ARs decreases with age until menopause after which it stabilizes.135 On the contrary, there is no significant association between age and β1AR/β2AR expression in men.135 Together with other numerous animal experiments, these observations seem to indicate that sex hormones, particularly oestrogen, play regulatory roles in the expression of βARs. Moreover, these roles may be due to the direct action of oestrogen on βAR signalling cascades or indirectly through adaptative responses to oestrogen environment.

β1AR expression

Our studies91 and others25, 137, 138 systematically showed that ovariectomy (OVX) increased the expression of β1ARs and induced negative inotropy in rat hearts subjected to ischaemia/reperfusion injury (I/R) and, in addition, that this role of oestrogen was mediated by the ERα.91 On the other hand, activation of GPR30 in ventricular myocytes from OVX rats reversed the effects of OVX on β1AR expression.24 Taken together, these findings indicate that oestrogen‐mediated influences on β1AR levels are affected by both ERα and GPR30. Furthermore, it was shown that oestrogen not only suppressed the expression of β1ARs but also increased sensitivity to catecholamines.139 Therefore, the downregulation of β1ARs by oestrogen may be a cardioprotective strategy against the adverse effects associated with hyperstimulation of β1ARs.

β2AR expression

In female rat models of I/R and heart failure, oestrogen, acting through the GPR3024 and ERα,91 increased the expression of β2ARs. We further showed that oestrogen in combination with testosterone enhanced the cardiac expression of β2ARs in OVX rats.140 Other researchers also reported that β2AR mRNA and protein were upregulated in female hearts but not male hearts in response to the arteriovenous fistula procedure.141 Taken together, these observations show that oestrogen decreases β1AR expression and upregulates β2AR expression in cardiac cells.

β3AR expression

Currently, information on direct effects of oestrogen on β3AR expression in cardiac tissues is lacking. However, variations in expression of β3ARs in adipose tissues have been linked to oestrogen levels. One group reported that oestrogen elevated the expression of β3ARs in murine adipocytes in culture,142 while another group observed that oestrogen decreased the quantity of β3ARs in brown adipose tissue of female rats in vivo.143 The discrepancies in these reports might be attributed to the methodologies used, that is real‐time PCR vs. radio‐ligand binding method used in the latter report or due to the inherent differences between in vitro and in vivo studies.

ERS AND βARS AS COREGULATORS OF CARDIAC CA2+‐HANDLING PROTEINS

Intracellular Ca2+ levels in cardiac cells are coregulated by both ERs and βARs. Numerous studies provide compelling evidence that oestrogen influences the expression levels of Ca2+‐handling proteins, whose functions are primarily under the regulation of βARs (Figure 1).144 In addition to the major proteins LTCC, RyR, PLB, SERCA and NCX,27 sarcolipin (SLN), an inhibitor of SERCA, plays a role in cardiac Ca2+ handling.145 However, to our knowledge, the influence of oestrogen on the expression or function of SLN has not been documented and hence needs to be clarified. The results of previous studies that were designed to investigate the effect of oestrogen on expression of cardiac Ca2+‐handling proteins channels are summarized in Table 2. In summary, the reports on oestrogen regulation of SERCA were largely consistent that oestrogen increased expression of SERCA, while its expression was decreased in OVX animal models92, 93, 146, 147, 148 (full reference list in Table 2). Similarly, oestrogen increased expression of NCX,26, 149, 150, 151 while it was decreased in OVX rats.26 However, in other studies, no change was observed in the expression of NCX expression in both oestrogen treatment and OVX animals.152, 153, 154 Although oestrogen decreased the expression of PLB,155, 156, 157 and OVX increased its expression,155, 156, 157, 158 no change in expression was found in other reports.26, 147, 153, 159, 160 On the other hand, oestrogen downregulated the RyR expression,161 while in other reports both OVX and oestrogen treatments had no effect on RyR expression.26, 153 Similarly, studies examining the role of oestrogen on the expression of LTCC yielded mixed results. Oestrogen decreased LTCC protein levels in rat ventricular myocytes (RVMs),26, 161 while OVX increased its expression in RVMs,26 but decreased its expression in mouse ventricular myocytes.154

Table 2

Summary of previous studies designed to investigate the effects of oestrogens on cardiac Ca2+‐handling proteins

Name of protein	Oestrogen effect		ER involved	Species/cell type/tissue	References
	Oestrogen	OVX
L‐type channel	↓	↑	Not investigated	Rat ventricular tissue	26
	↑		ERα	Rabbit heart	69
	↓		Not investigated	Neonatal rat ventricular cells	161
		↓	Not investigated	Mouse ventricle tissue	154
Ryanodine receptor	─	─	Not investigated	Rat ventricular tissue	26
	↓		Not investigated	Neonatal rat ventricular cells	161
	─	─	GPR30	Rat left ventricle tissue	153
SERCA		↓	Not investigated	Rat heart tissue	158
	─	─	Not investigated	Rat ventricular tissue	26
	↑		Not investigated	Mouse apical ventricle	149
	↑		Not investigated	Zebrafish hearts	182
	─	─	ERα	Rat ventricular cells	159
	─		Not investigated	Mouse ventricular tissue	160
	↑		ERα and ERβ	Cultured murine cardiomyocytes	146
	↑	↓	Not investigated	Rat ventricular tissue	147
	↑		ERβ	Mouse ventricle tissue	92
	↑		Not investigated	Rat embryonic heart H9C2	148
	↑		ERα and ERβ	Pig coronary arteries tissue	93
	↑	↓	GPR30	Rat cardiac microsomes	162
	─	─	Not investigated	Rat heart tissue	152
	↑		Not investigated	Mouse ventricle tissue	151
	↑	↓	Not investigated	Rat heart tissue	155
	─	─	Not investigated	Rat left ventricle tissue	156
	─	─	GPR30	Rat left ventricle tissue	153
		─	Not investigated	Mouse ventricle tissue	154
	↑	↓	Not investigated	Mouse ventricle tissue	183
	↑	↓	Not investigated	Rat left ventricle tissue	157
Phospholamban		↑	Not investigated	Rat heart tissue	158
	─	─	Not investigated	Rat ventricular tissue	26
	─ Male ↑Female		Not investigated	Mouse ventricle tissue	149
	─	─	ERα	Ventricular cells	159
	─		Not investigated	Mouse ventricular tissue	160
	─	─	Not investigated	Rat ventricular tissue	147
		↓	Not investigated	Rat cardiac microsomes	162
	↓	↑	Not investigated	Rat heart tissue	155
	↓	↑	Not investigated	Rat left ventricle tissue	156
	─	─	GPR30	Rat left ventricle tissue	153
	↓	↑	Not investigated	Rat left ventricle tissue	157
NCX	↑	↓	Not investigated	Rat ventricular tissue	26
	↑		Not investigated	Mouse ventricle tissue	149
	↓		Not investigated	Neonatal rat ventricular cells	161
	↑		Genomic	Rabbit ventricular cells	150
	─	─	Not investigated	Rat heart tissue	152
	↑		Not investigated	Mouse ventricle tissue	151
	─	─	GPR30	Rat left ventricle tissue	153
		─	Not investigated	Mouse ventricle tissue	154
Sarcolipin	Not yet documented

SERCA, sarcoplasmic reticulum Ca2+‐ATPase; NCX, Na+/Ca2+ exchanger pump; ER, oestrogen receptor. ↑ represents upregulation, ↓ represents downregulation and → represents no change.

These studies were carried out in different animal species, disease models, tissue/cell types, age groups, in vivo and ex vivo and using different oestrogen types. Moreover, the observed changes in protein expression due to OVX were reversed by oestrogen replacement.26, 147, 155, 162 This implies that the discrepancies between some of the results could be a result of the experimental variations, and hence, head‐to‐head comparisons might not be possible. Collectively, these findings show that oestrogen status plays a crucial role in the expression and function of cardiac Ca2+‐handling proteins. ERα, ERβ and GPR30 mediate these roles of oestrogen.69, 92, 162 Therefore, the observations that ER and βAR signalling pathways interact may have profound implications on cardiac Ca2+ regulation and contractility. Furthermore, through ERα,7 oestrogen altered myofilament Ca2+ sensitivity.8, 154 Indeed, in a rat model of angiotensin II‐induced hypertension, OVX exacerbated myofilament Ca2+ sensitivity, indicating that oestrogen deficiency may play a role in cardiac disorders by lowering the myofilament sensitivity to Ca2+.8

PHARMACOLOGICAL IMPLICATIONS AND THERAPEUTIC OPPORTUNITIES

Effects of the interactions on drugs targeting βARs and Ca2+ channel blockers

Interactions between ER and βAR pathways could have broad implications in the clinical context. β‐Blockers and Ca2+ channel blockers are 2 mainstays for the treatment of cardiovascular disease.163 These drugs control the heart rate and blood pressure by modulating the activation of βARs. However, there are conflicting observations regarding their effectiveness in managing conditions such as hypertension.163 Reports from cohort studies have noted that some patients, particularly women, under β‐blockers are unable to reach targeted blood pressure compared to men.164 This observation can be explained, partially, by the aforementioned influence of oestrogen on βARs' function. Moreover, gender and age differences in expression of β1ARs/β2ARs have been reported, which may be attributed to oestrogen.135 Besides, gender variations in responses to catecholamines,139 and in cardiac Ca2+ handling,165 have been observed in animal experiments. Therefore, the efficacy of β‐blockers and Ca2+ blockers may vary with gender or age groups based on the interplay between ERs and βARs. As demonstrated, carvedilol, a non‐selective β‐blocker, protected against myocardial contractile dysfunction caused by oestrogen deficiency.158 Interestingly, this is one of the β‐blockers to display biased agonism and it too can activate the β2AR‐Gαi/β‐arrestin pathways.166, 167 With the current understanding of the ER and βAR pathways, further studies should examine how the efficacy of the drugs targeting these receptors and/or their signalling pathways may be altered in the context of the ER and βAR crosstalk. Theoretically, oestrogen by inhibiting the LTCC or altering the expression of βARs might indirectly compromise the functions of Ca2+ blockers and β‐blockers respectively. Consequently, men and women may respond differently to these classes of drugs. The crosstalk may inform the decisions regarding the choice of antihypertensive drugs to patients with consideration to age and gender.

Therapeutic opportunities

The functional synergism between ERs and βARs provides therapeutic avenues for cardioprotection. For example, while β1AR activation promotes CaMKII‐induced apoptosis,25, 112 β2AR, ERα and GPR30 activation seems to act in a manner that promotes anti‐apoptosis through the Gi/PI3K/Akt pathway. Considerations for strategies targeting the PI3K/Akt pathway will provide a feasible avenue for cardioprotection. For instance, the ability of ERα binding to the alpha subunit of PI3K seems attractive, as it is more specific and avoids various points of integration between ER and βAR signalling cascades discussed above. Activation of Akt pathway will protect against mitochondria‐associated apoptosis induction. Moreover, Akt was shown to act as a surrogate molecular ligand for ERα that induced expression of oestrogen‐regulated cardioprotective genes in breast cancer cells.168 This perspective partly indicates that selective activation of this pathway would potentially enhance the cardiac functions under pathological conditions. Another therapeutic target is the possibility of direct binding of oestrogen to the LTCC. Oestrogen‐LTCC docking studies may help to predict the mode of binding/interaction of this complex. This approach will advance the prospects of oestrogen as a Ca2+ channel blocker if appropriate technologies are applied to enhance its specificity. We anticipate that the therapeutic value of this manoeuvre could be a potential target for Ca2+‐related pathologies such as arrhythmia treatments.

CONCLUSION

This review demonstrates the expression patterns and functions of ERs and βARs in cardiovascular tissues. The data sets reviewed above show some inconsistencies with regard to tissue‐specific expression of the ERs. For instance, it is not clear whether ERβ is expressed in adult cardiomyocytes, and the cellular localization of GPR30 is not clear. Therefore, further studies are warranted to resolve these observations. In addition, the reviewed data sets strongly support the hypothesis that ERs and βARs function as collaborative partners in modulating the physiology of the cardiovascular system. The recently described oestrogen receptor GPR30 mimics the dual coupling of the β2ARs to the Gαs and Gαi proteins. On this basis, oestrogen pathways play into the network of the βAR signalling cascades. Furthermore, GPR30 and βARs show similarities with regard to their ability to associate with AKAPs, PDZ motif‐binding proteins and possession of PKA and CaMKII binding sites. Despite the signalling pathways discussed in this review, the functions of the GPR30 and other ERs remain incompletely understood. Further research is required to uncover the identities of signalling molecules that orchestrate their functions.

The crosstalk between the ERs and βARs could have implications on drugs that target these receptors, especially β‐blockers and Ca2+ channel blockers. Oestrogen influences the expression of βARs and Ca2+‐handling proteins, which could compromise the efficacy of the drugs in a gender‐dependent manner. This perspective requires further evaluation at the clinical level. In addition, the concept of direct oestrogen binding to the LTCC might be of great clinical relevance as oestrogen might be used to design Ca2+ channel blockers. This opens a window of research into the receptor‐independent pathways for oestrogen. Furthermore, future research should exploit the therapeutic potential of the cardioprotective PI3K/Akt pathway that is activated downstream of both ERs and βARs.

CONFLICT OF INTEREST

The authors report no conflict of interests.