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

Abstract 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 (β1AR, β2AR and β3AR). 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.

cardiovascular system (CVS). There are 3 classes of ERs: the ERa, ERb 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 nongenomic 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 model 15 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 17b-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 pretreatment with antagonist ICI-182 780. 18 The heart rhythm and contraction are mainly regulated by the sympathetic nervous system (SNS), via the b-adrenergic receptors (bARs) that connect and convey the SNS signals to the heart. bARs are divided into b 1 AR, b 2 AR and b 3 AR. 21 Interestingly, signalling pathways of ERs are intertwined with those of the bARs pointing to the possibility of functional convergence in modulating the physiology of the CVS. In fact, crosstalk between ERa and a 1b -adrenergic receptor was reported previously. 22,23 Furthermore, we and others established that oestrogen alters gene expression of bARs and calcium (Ca 2+ )-handling proteins of the CVS. [24][25][26] Proteins that regulate cardiac Ca 2+ include the Na + /Ca 2+ exchanger pump (NCX), L-type Ca 2+ channel (LTCC), phospholamban (PLB), sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) and ryanodine receptors (RyRs; Figure 1). 27 Consequently, the effects of oestrogen on the Ca 2+ -handling proteins have direct implications on the contractile machinery of the myocardium.
In recent decades, the need to understand the cardiovascular functions of oestrogen and bARs has received much interest from researchers. The discovery of GPR30, 28 which shares structural features with bARs, has expanded the functional scope of oestrogen. In this regard, it is important to re-evaluate the relationships between ER and bAR 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 bARs. 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 bAR 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, b-blockers and Ca 2+ channel blockers. We also discuss the ERs and bARs as coregulators of cardiac Ca 2+ -handling proteins.

RECEPTORS IN THE CARDIOVASCULAR SYSTEM
2.1 | bAR-specific features bARs are members of the G-protein-coupled receptors (GPCRs) that classically form 7 transmembrane loops, with extracellular and intracellular terminals. Three bAR subdivisions, b 1 AR, b 2 AR and b 3 AR, 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 b 1 AR and b 3 AR. [30][31][32] In heart myocytes, the number of b 1 ARs is higher than that of b 2 ARs, while b 3 ARs show the least abundance. 33 bARs are linked to heterogeneous intracellular signalling pathways and proteins. In addition, their expressions vary under physiological and pathological conditions. 34 2.2 | bAR-specific signalling bARs are activated by noradrenaline and adrenaline released from the SNS and adrenal glands respectively. However, once activated, the bARs trigger diverse intracellular pathways. 35 b 1 ARs couple to the stimulatory unit of the G protein (G as ) leading to the synthesis of cyclic adenosine monophosphate (cAMP) by adenylyl cyclase (AC) enzyme. On the other hand, b 2 ARs are pleiotropic receptors that couple to the G as , the inhibitory G protein (G ai ) and the G bc . 36,37 At the physiological state, b 2 ARs couple to the G as , whereas at high adrenaline concentration, they switch to G ai , a phenomenon referred to as stimulusmediated trafficking. 38,39 Activation of the b 2 AR/G ai pathway inhibits cAMP production, an opposing effect to b 2 AR/G as and b 1 AR/G as activation (Figure 1).
With regard to structure and function, the b 3 ARs display distinct differences to b 1 ARs and b 2 ARs. Notably, the cytoplasmic C-terminus of the b 3 ARs lacks the target amino acid sequences for protein kinase A (PKA) and cardiac G-protein-coupled receptor kinase 2 (GRK2) phosphorylation. 40,41 Consequently, b 3 ARs are less susceptible to PKA/GRK2-mediated receptor recycling and desensitization in response to hyperstimulation. 40 Two isoforms, b 3a AR and b 3b AR, were reported in Chinese hamster ovary (CHO) cells and 3T3-L1 adipocytes. [42][43][44][45][46] The b 3a AR isoform coupled to the G ai , whereas b 3b AR coupled to both G as and G ai . At present, there are no reports regarding the existence of the 2 isoforms in human cardiac cells. b 3 AR has largely been associated with metabolic functions. Nevertheless, b 3 AR stimulation induced positive inotropy in human atrial cells, 47 but it had no effect on the inotropy of human ventricular cells. 48 b 3 AR may influence chronotropic functions through the nitric oxide (NO)/guanosine 3 0 ,5 0monophosphate (cGMP) pathway. 49 Activation of plasma b 3 AR-NO synthase/guanylyl cyclase pathway was shown to influence the nuclear b 3 AR-mediated gene transcription, suggesting a crosstalk between the surface and nuclear b 3 ARs. 50 2.3 | The basis of the heart's function bARs mediate the SNS regulation of the cardiac functions. 51 These functions are primarily orchestrated by activation of b 1 ARs, which constitute up to 80% of the entire cardiac bAR density of healthy human, and to a lesser extent by the b 2 ARs. 33,35 Moreover, b 2 ARs have a higher affinity for adrenaline, while b 1 ARs have almost equal affinities for both noradrenaline and adrenaline. 39 On the other hand, activation of the b 3 ARs is largely associated with negative inotropy during catecholaminergic stress. 47 Once activated, bARs initiate cAMP synthesis by coupling to the G as , a GTP-binding protein. This cAMP, in turn, activates PKA. What follows is the induction of intracellular rise in Ca 2+ 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 Ca 2+ entry as inward current. 52 These Ca 2+ currents further stimulate Ca 2+ 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 Ca 2+ transient activates the myofilament protein troponin C turning on cardiomyocyte contraction. The size of Ca 2+ 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 Ca 2+ uptake back to the SR by SERCA. In addition, sarcolemmal Na + /Ca 2+ exchanger pump (NCX) accelerates Ca 2+ extrusion, which together with SR Ca 2+ uptake diminishes the Ca 2+ 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 b 1 AR/cAMP and provides alternative modulation of bAR 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 Ca 2+ through RyR and PLB phosphorylation, 54 indicating synergism between the cAMP/PKA and cAMP/EPAC pathways.
Another downstream target of b 1 AR activation is the multimeric protein Ca 2+ /calmodulin kinase II (CaMKII). 55 Activation of this kinase indirectly relies on the PKAmediated rise in cytosolic Ca 2+ and intracellular levels of calmodulin. 56 Recent findings reveal that CaMKII activation augments the LTCC current and increases the RyR open probability 57 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 3 | 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 (ERa and ERb), which modulate transcription of specific gene sets, and membrane-bound receptor (GPR30, also known as GPER1), which mediates rapid, nongenomic actions of oestrogen. ERs are expressed in cardiomyocytes, 3 cardiac fibroblasts 4 and VSMCs; 5 however, their expression and cellular locations are not fully understood. For instance, Pugach et al. 3 reported that ERb 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 cells 64 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 ERa and ERb expression were also reported. Whereas the mRNA levels of ERa were equivalent in hearts of both men and women, 3,66 ERb 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 ERb expression was not different in male and female cardiomyocytes, while ERa expression varied with gender. 68 Elsewhere, ERa and ERb 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 ERa 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, ERa 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 ERa and ERb 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 ERa and 17-fold greater than those of ERb. 62 Different genes located on different chromosomes encode each ER subtype. While alternative splicing of the gene transcripts leads to multiple subtypes of ERa and up to 5 described transcripts of ERb, 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.

| ER activation
Similar to other steroids hormones, oestrogen signalling is initiated by the binding of 17b-oestradiol or xenoestrogens 73 and oestradiol metabolites 74,75 to ER. Synthetic receptor-specific agonists with selective binding affinities have also been developed: propylpyrazoletriol (PPT) for ERa, diarylpropionitrile (DPN) for ERb and G1 for GPR30. 76 Noteworthy, each receptor subtype or isoform displays different affinities to 17b-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, 17b-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

| 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 ERa and ERb 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 ERa and GPR30. 7,62,81 There are reports indicating the presence of ERb 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 ERb 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 bAR pathways forms the basis of the discussion dealt with in Section 5 of this article. Moreover, the interaction of ER signalling with adrenergic receptor pathways was observed between the ERa and a 1b -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 ERa and ERb 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 review 89 ). 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 Ca 2+ overload. 62 On the other hand, ERa 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 b 1 ARs and induced negative inotropy. 24,91 ERb 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.

PATHWAYS OF ERS AND ΒARS
The crosstalk between ERs and bARs 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 bARs 26,91 and cardiac ionhandling proteins. 93,94 Moreover, oestrogen regulates the cardiac contractile functions, which are otherwise under the control of adrenergic receptors (details discussed in Sections 6 and 7). Intriguingly, the structure (of GPR30) and signalling pathways of ERs are functionally closer/related to those of the bARs, at least partially. 72,93,[95][96][97][98] Some of the cellular roles of ERs synergize or oppose the effects produced by bAR activation. Therefore, here we explore, side by side, the correlation among b 1 ARs, b 2 ARs and b 3 ARs vs. ERa, ERb and GPR30. We discuss various points of integration between their signalling pathways. We note 3 main pathways along which these classes of receptors interact.
5.1 | Signalling along the GPCR/G as /cAMP pathway Similar to bARs, 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 bARs, GPR30 couples to the classical GPCR proteins, G as 100,101 and G ai / o , 63,96,102 in cardiovascular tissues (Figure 2). On this basis, activation of GPR30 partially mimics the signalling pathway of bARs with regard to its downstream cascades. Initial activation of b 1 AR, b 2 AR and GPR30 leads to coupling to G as protein. 96,103 Activation of G as 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 bAR/G as /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 G as /AC/cAMP pathway, it would be interesting to define whether the GPR30/G as /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 b 1 ARs and b 2 ARs. Moreover, we speculate that GPR30's ability to activate EPAC might also influence b 1 AR/cAMP/EPAC-mediated functions. Besides GPR30, activation of the ERa elevated PKA in VSMCs of aortic tissue further illustrating oestrogen involvement in the GPCR/G as /cAMP signalling pathway. 102 Additional interactions are possible due to the structural resemblance between GPR30 and bARs. bARs 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 bARs. Of note, b 1 AR, b 2 AR 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 b 1 AR (-ESKV) motif was shown to be a determinant factor for its coupling to G as and not G ai . Induced disruption of this motif permitted b 1 AR/G ai coupling. 106 On the other hand, b 2 AR (-DSLL) motif plays a role in its coupling to G ai . 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 G as or G ai . In addition to the PDZ motifs, b 2 AR phosphorylation by PKA influences its ability to bind G as or G ai . 39 Although GPR30 coupling to G as or G ai 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 G as and G ai as is the case for b 1 ARs and b 2 ARs. This possibility calls for further inquiry to the conditions under which GPR30 couples to G ai and G as , and the proteins that interact with its PDZ motif.
One functional implication of this motif draws from the recent observation that GPR30 inhibited bAR-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 b 1 ARs facilitated its recycling by enhancing PKA phosphorylation of the receptor. [109][110][111] Moreover, it is established that oestrogen regulates expression of b 1 ARs. Therefore, an association of GPR30 with AKAP5 and possibly other unidentified proteins could be a mechanism through which oestrogen participates in the regulation of b 1 AR density. Further research should be carried out to characterize the interactions of GPR30 with AKAPs and their implications on cellular functions.
Although b 1 AR, b 2 AR and GPR30 are capable of coupling to G as , their cellular effects are not similar. For instance, activation of b 1 ARs, 112 ERa and GPR30 113 triggers production of calmodulin and activation of CaMKII, while b 2 ARs and ERb do not. Importantly, the observation that GPR30 activates cAMP through G as pathway challenges previous findings that oestrogen induced negative inotropy at both cardiomyocyte and organ levels. Therefore, the effects of GPR30/G as /cAMP pathway may not be identical to the classical bAR/G as /cAMP pathway, especially in cardiomyocytes. However, as we reported, oestrogen may tilt the activation of b 2 AR/G as or b 2 AR/G ai pathways in certain disease conditions, as in stress-induced cardiomyopathy. 114 5.2 | Signalling along the GPCR/G ai /PI3K/ Akt pathway As mentioned earlier, GPR30, like b 2 ARs and b 3 ARs, couple to the G ai 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 b 2 ARs exposes cardiomyocytes to cell death in ischaemic conditions. 117 The b 2 AR/G ai 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 b 2 ARs. In addition to the GPR30, oestrogen activates the ERa, which directly binds the p85 alpha regulatory component of the PI3K. 119 In addition, PKA phosphorylation of the b 2 ARs enables switching of its coupling from G as to G ai under extreme catecholamine stimulation, 39 a phenomenon referred to as signal trafficking (Figure 1). In this context, b 2 ARs act as a switch that coordinates synthesis of cAMP and indicates cross-communication between b 1 AR and b 2 AR signalling. Moreover, AKAP5 tethering of PKA allows it to phosphorylate b 2 ARs. 120 Considering that GPR30 possesses PKA phosphorylation sites, we speculate that through AKAP5, PKA phosphorylation of GPR30 may influence its ability to activate G as or G ai . 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 b 2 ARs, the conditions under which GPR30 activates G as or G ai are not clearly understood. Perhaps a possible hint as to when GPR30 activates G ai comes from the observation that under stress conditions, both b 2 ARs and GPR30 activate G ai /PI3K/Akt pathway to confer cardioprotection. 116,118 However, we recognize that the requirements for GPR30 coupling to G as or G ai in cardiomyocytes need further investigation.
Unlike b 1 ARs, b 2 ARs and GPR30, b 3 ARs lack PKA phosphorylation sites. 40 Therefore, for b 3 ARs, there is a great deal of variability with respect to their ability to activate both G as and G ai . Some b 3 AR splice variants were shown to display dual coupling to G as and G ai in other cell types, although not in cardiac cells. 121 b 3 ARs act through G ai / o to suppress contractility via induction of NO-cGMP pathway under chronic catecholaminergic stimulation. 122 In addition, b 3 AR/NO/cGMP pathway was enhanced in the presence of b 1 AR blocker, which was interpreted to be beneficial in chronic volume-overloaded heart. 123 Based on the evidence presented above, the ER and bAR signalling pathways function as interdependent networks/partners whose roles have profound effects on the cardiovascular system. In summary, G as and G ai act as pivots around which both ER and bAR signalling pathways converge. b 2 AR and GPR30 signal through the G as /AC/cAMP and ERβ PKA P P P P P P P PK P P P P P P P P P P P P P P P P P P P P P P P P P P P P P A

CaMKII C C C C C C C CaM AKT A A A A A A A A AK A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A T
Interaction of oestrogen signalling and beta-adrenergic signalling pathways. The symbol ↑ represents cytosolic Ca 2+ rise. The symbol represents inhibition signal. The symbol represents activation signal. Signalling pathways of bARs (b 1 AR, b 2 AR and b 3 AR) and ERs (ERa, ERb and GPR30) are integrated through the G s and G i pathways. Effector proteins PKA and EPAC affect the G s -cAMP signals. The resultant effects play crucial roles in cardiac contraction by increasing cytosolic Ca 2+ levels. Alternatively, the receptors may activate the G i , which mediates anti-apoptosis signals through the PI3K/Akt pathway. Elevated cytosolic Ca 2+ 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: 17boestradiol; EPAC: exchange protein directly activated by cAMP; AKAP5: A-kinase anchoring protein 5; MAGUK: membrane-associated guanylate kinase; CaM: calmodulin; CaMKII: Ca 2+ /calmodulin kinase II G ai /PI3K/Akt pathways in the cardiovascular system. Similarly, both GPR30 and ERa signalling cascades interact through the PI3K/Akt pathway (Figure 1). PI3K/Akt acts as a focal pathway that unifies GPR30-, ERa-and b 2 ARmediated cardioprotection. In general, the G as /AC/cAMP and G ai /PI3K/Akt pathways seem to trigger opposing effects. For example, the cAMP produced by bAR 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 bARs might be dependent on the cell/tissue type.

| Localization of
ERs and bARs to the caveolae b 1 AR, b 2 AR and b 3 AR 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 b 2 AR localizes almost exclusively to caveolin 3-rich membrane fractions of rat cardiomyocytes, while b 1 AR localizes to both caveolar and non-caveolar membrane fractions. 126 These spatial distributions play a role in the differential activation of cAMP signals by b 1 AR and b 2 AR. 127 For instance, colocalization of b 2 AR with the Ca 2+ channel LTCC and caveolin 3 is essential for its signalling and ability to invoke intracellular Ca 2+ , 128 while caveolin 3 interaction with AC V acts as a scaffolding protein which participates in b 1 AR signals that induce LTCC current in ventricular cardiomyocytes. 129 ERa was associated with eNOS activation in the caveolae of endothelial cells, 130 while ERb was found in the caveolae where it mediated eNOS signals. 82 Strikingly, overexpression of b 2 AR enhanced vascular repair of endothelial progenitor cells in mice through the eNOS pathway. 131 Therefore, localization of b 2 AR and ERb 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 ERa and b 3 AR. 132,133 The association of b 3 AR with caveolin 1 was shown to govern its ability to couple to G ai/o proteins in CHO-K1 cells. 133 On the other hand, it has been established that ERa and ERb interact directly with G ai and that these interactions occur in close proximity to the caveolae domains. 134 The physiological relevance of the possible interactions among ERs, caveolins and bARs 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 bARs. Further research is required to identify multilevel communications and interactions among these receptors.
A summary of the shared features is as follows: (i) b 2 ARs, b 3 ARs, ERa, ERb and GPR30 couple to G ai subunit. (ii) b 1 ARs, GPR30 and ERa activate calmodulin/CaMKII. (iii) b 1 ARs, b 2 ARs and GPR30 couple to G as subunit.
(iv) b 2 ARs, ERa and GPR30 trigger the PI3K/Akt pathway. (v) b 3 AR and ERa associate with caveolin 1, while b 1 AR and b 2 AR associate with caveolin 3.

| OESTROGEN INFLUENCE ON THE EXPRESSION OF bARS
Expression of bARs in the cardiovascular vessels is influenced, in part, by age, by gender and by drugs targeting these receptors. 135 The ratio of b 1 ARs, b 2 ARs and b 3 ARs may also vary with disease status. 136 In pre-menopausal women, the cardiac expression of b 1 ARs and b 2 ARs decreases with age until menopause after which it stabilizes. 135 On the contrary, there is no significant association between age and b 1 AR/b 2 AR 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 bARs. Moreover, these roles may be due to the direct action of oestrogen on bAR signalling cascades or indirectly through adaptative responses to oestrogen environment.

| b 1 AR expression
Our studies 91 and others 25,137,138 systematically showed that ovariectomy (OVX) increased the expression of b 1 ARs 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 ERa. 91 On the other hand, activation of GPR30 in ventricular myocytes from OVX rats reversed the effects of OVX on b 1 AR expression. 24 Taken together, these findings indicate that oestrogen-mediated influences on b 1 AR levels are affected by both ERa and GPR30. Furthermore, it was shown that oestrogen not only suppressed the expression of b 1 ARs but also increased sensitivity to catecholamines. 139 Therefore, the downregulation of b 1 ARs by oestrogen may be a cardioprotective strategy against the adverse effects associated with hyperstimulation of b 1 ARs.

| b 2 AR expression
In female rat models of I/R and heart failure, oestrogen, acting through the GPR30 24 and ERa, 91 increased the expression of b 2 ARs. We further showed that oestrogen in combination with testosterone enhanced the cardiac expression of b 2 ARs in OVX rats. 140 Other researchers also MACHUKI ET AL.

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reported that b 2 AR 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 b 1 AR expression and upregulates b 2 AR expression in cardiac cells.

| b 3 AR expression
Currently, information on direct effects of oestrogen on b 3 AR expression in cardiac tissues is lacking. However, variations in expression of b 3 ARs in adipose tissues have been linked to oestrogen levels. One group reported that oestrogen elevated the expression of b 3 ARs in murine adipocytes in culture, 142 while another group observed that oestrogen decreased the quantity of b 3 ARs 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.

ERβ
Cav bAR and ER signalling through caveolae. In this view, b 2 AR associates with caveolin 3 and LTCC to transduce signals that increase cellular Ca 2+ . b 1 AR interacts with caveolin 3 and AC V to induce LTCC current. Caveolin 1 interacts with b 3 AR and governs its ability to couple to G ai . ERa colocalizes with caveolin 1 and signals through G ai and activates eNOS pathway. ERb mediates the activation of eNOS by oestrogen in the caveolae. AC V: adenylyl cyclase V; ERa: oestrogen receptor alpha; ERb: oestrogen receptor beta; eNOS: endothelial nitric oxide synthase; LTCC: L-type calcium channel 7 | ERS AND bARS AS COREGULATORS OF CARDIAC CA 2 + -HANDLING PROTEINS Intracellular Ca 2+ levels in cardiac cells are coregulated by both ERs and bARs. Numerous studies provide compelling evidence that oestrogen influences the expression levels of Ca 2+ -handling proteins, whose functions are primarily under the regulation of bARs (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 Ca 2+ 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 Ca 2+ -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 models 92,93,[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 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 Ca 2+handling proteins. ERa, ERb and GPR30 mediate these roles of oestrogen. 69,92,162 Therefore, the observations that ER and bAR signalling pathways interact may have profound implications on cardiac Ca 2+ regulation and contractility. Furthermore, through ERa, 7 oestrogen altered myofilament Ca 2+ sensitivity. 8 Interactions between ER and bAR pathways could have broad implications in the clinical context. b-Blockers and Ca 2+ 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 bARs. 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 b-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 bARs' function. Moreover, gender and age differences in expression of b 1 ARs/ b 2 ARs have been reported, which may be attributed to oestrogen. 135 Besides, gender variations in responses to catecholamines, 139 and in cardiac Ca 2+ handling, 165 have been observed in animal experiments. Therefore, the efficacy of b-blockers and Ca 2+ blockers may vary with gender or age groups based on the interplay between ERs and bARs. As demonstrated, carvedilol, a non-selective b-blocker, protected against myocardial contractile dysfunction caused by oestrogen deficiency. 158 Interestingly, this is one of the bblockers to display biased agonism and it too can activate the b 2 AR-G ai /b-arrestin pathways. 166,167 With the current understanding of the ER and bAR 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 bAR crosstalk. Theoretically, oestrogen by inhibiting the LTCC or altering the expression of bARs might indirectly compromise the functions of Ca 2+ blockers and b-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 bARs provides therapeutic avenues for cardioprotection. For example, while b 1 AR activation promotes CaMKII-induced apoptosis, 25,112 b 2 AR, ERa and GPR30 activation seems to act in a manner that promotes anti-apoptosis through the G i /PI3K/Akt pathway. Considerations for strategies targeting the PI3K/Akt pathway will provide a feasible avenue for cardioprotection. For instance, the ability of ERa binding to the alpha subunit of PI3K seems attractive, as it is more specific and avoids various points of integration between ER and bAR 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 ERa 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 Ca 2+ 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 Ca 2+ -related pathologies such as arrhythmia treatments.

| CONCLUSION
This review demonstrates the expression patterns and functions of ERs and bARs 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 ERb 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 bARs function as collaborative partners in modulating the physiology of the cardiovascular system. The recently described oestrogen receptor GPR30 mimics the dual coupling of the b 2 ARs to the G as and G ai proteins. On this basis, oestrogen pathways play into the network of the bAR signalling cascades. Furthermore, GPR30 and bARs 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 bARs could have implications on drugs that target these receptors, especially b-blockers and Ca 2+ channel blockers. Oestrogen influences the expression of bARs and Ca 2+ -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 Ca 2+ 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 bARs.