Reciprocality Between Estrogen Biology and Calcium Signaling in the Cardiovascular System

17β-Estradiol (E2) is the main estrogenic hormone in the body and exerts many cardiovascular protective effects. Via three receptors known to date, including estrogen receptors α (ERα) and β (ERβ) and the G protein-coupled estrogen receptor 1 (GPER, aka GPR30), E2 regulates numerous calcium-dependent activities in cardiovascular tissues. Nevertheless, effects of E2 and its receptors on components of the calcium signaling machinery (CSM), the underlying mechanisms, and the linked functional impact are only beginning to be elucidated. A picture is emerging of the reciprocality between estrogen biology and Ca2+ signaling. Therein, E2 and GPER, via both E2-dependent and E2-independent actions, moderate Ca2+-dependent activities; in turn, ERα and GPER are regulated by Ca2+ at the receptor level and downstream signaling via a feedforward loop. This article reviews current understanding of the effects of E2 and its receptors on the cardiovascular CSM and vice versa with a focus on mechanisms and combined functional impact. An overview of the main CSM components in cardiovascular tissues will be first provided, followed by a brief review of estrogen receptors and their Ca2+-dependent regulation. The effects of estrogenic agonists to stimulate acute Ca2+ signals will then be reviewed. Subsequently, E2-dependent and E2-independent effects of GPER on components of the Ca2+ signals triggered by other stimuli will be discussed. Finally, a case study will illustrate how the many mechanisms are coordinated to moderate Ca2+-dependent activities in the cardiovascular system.


Voltage-Dependent Ca 2+ Entry (VDCE)
Functional voltage-dependent Ca 2+ channels (VDCCs) are the hallmark of tissue excitability and are present in cardiomyocytes and VSMCs, but not ECs. In cardiomyocytes, LTCCs are located mostly in transverse T tubules in apposition to RyR2s (83). Ca 2+ entry via LTCCs triggers CICR via RyR2. In VSMCs, LTCCs also play a critical role in Ca 2+ entry and contraction (84). The LTCC complex (85) consists of α 1 , α 2 , β, δ, and γ subunits. Four LTCC members are named according to their α1 pore-forming subunits: Ca v 1.1, Ca v 1.2, Ca v 1.3, and Ca v 1.4 (86). Ca v 1.2 is predominant in cardiac and smooth muscles.
The NCX may function in two modes. In the forward mode, myocardial NCX1 balances LTCC-mediated Ca 2+ entry and RyR-mediated Ca 2+ release during cardiac excitation, extruding ∼25% of the Ca 2+ needed to activate myofilaments (105). NCX1 also predominates in VSMCs (106,107). In ECs, NCX accounts for ∼25% of Ca 2+ removal (87). Endothelial NCX and PMCA dynamically adjust their Ca 2+ extrusion rates to maintain sufficient efflux (104). In the reverse mode, upon myocardial depolarization, Na + entry causes the NCX to transiently operate in this mode, promoting Ca 2+ entry. This is much less efficient in triggering SR Ca 2+ release compared to LTCC-mediated Ca 2+ entry (108,109). However, it primes the dyad to increase LTCC-mediated CICR (110). In VSMCs, reverse-mode NCX1 facilitates Ca 2+ entry and mediates contraction, vascular tone, and blood pressure (111,112). The reverse mode is not significant in ECs.

Sex Differences in Ca 2+ Signaling Proteins
Higher mRNA levels of Ca v 1.2, RyR, and NCX, but not of phospholamban and SERCA2, have been observed in female than in male rat hearts (113). However, caffeine-induced Ca 2+ release is lower in cardiomyocytes from female hearts (114). Ca v 1.2 mRNA is higher in coronary smooth muscle from male than from female pigs (115). In smooth muscle cells (SMCs), expressions of ERα and ERβ, but not G protein-coupled estrogen receptor 1 (GPER), are higher in female than in male rats (116). These differences and the lower Ca v 1.2 expression (115) may be responsible for less contraction of VSMCs from females (116). No studies have examined sex differences in Ca 2+ handling proteins in ECs.

Transduction of Ca 2+ Signals-The Essential Role of Calmodulin (CaM)
While some Ca 2+ -dependent proteins are activated directly by Ca 2+ , many are activated by a complex between Ca 2+ and CaM. CaM has two lobes linked by a flexible helix and can interact with ∼300 target proteins (117,118). Ca 2+ -free CaM binds or serves as structural subunits of ∼15 proteins (119). However, each CaM lobe has two Ca 2+ -binding sites, and cooperative Ca 2+ binding induces conformations that allow CaM to interact with many proteins, aided by the flexibility of the central helix (120,121). Thus, CaM is the ubiquitous Ca 2+ signal transducer. Activities of Ca 2+ /CaM-binding proteins depend on the Ca 2+ signals, CaM availability, and properties of the interaction between Ca 2+ -CaM and the target proteins. Many of these factors are subject to estrogenic moderation.
Despite being required for activation of many Ca 2+dependent proteins, up to 50% of cellular CaM is engaged in inseparable interactions, leaving much less available for dynamic target binding (122). This generates an environment of limited CaM (123), as has been demonstrated in ECs (124), VSMCs (125), and cardiomyocytes (126). Consequently, competition for CaM generates a unique crosstalk among CaM-dependent proteins (124,127), and factors that alter CaM level are predicted to have pervasive functional impact. It is noteworthy that virtually all CSM components interact with CaM and, in the context of reciprocality between estrogenic and Ca 2+ signaling pathways, that ERα and GPER are both regulated by direct interactions with Ca 2+ -CaM.
ERα activities are strongly regulated by the Ca 2+ -dependent interaction with CaM. ERα binds CaM in a Ca 2+ -dependent fashion with a K d of 1.6 × 10 −10 M and an EC 50 (Ca 2+ ) value of ∼3 × 10 −7 M (137). When ERα from Wistar rats' uteri is used, CaM decreases ERα-E 2 binding but increases liganded ERα-ERE interaction (138,139). A comparison of the CaMbound/CaM-unbound ERα ratio in the cytosolic (unliganded) and nuclear (liganded) ERα pools isolated from MCF-7 cells suggests that E 2 binding induces a conformation that favors ERα-CaM interaction (138). The CaM-binding domain was initially predicted to be a.a. 298-310 (137) but was later determined to be a.a. 298-317, with a.a. 248-317 required for maximal interaction (140). Further studies revealed that a.a. 287-311 is required to interact with both CaM lobes (141). CaM binding promotes ERα homodimerization that is critical for transcription activity (140,142). With two lobes, each CaM binds two ERα molecules and thus stabilizes ERα dimerization (143). Notably, analogs of ERα17p (a.a. 295-311) that are unable to bind CaM downregulates ERα, stimulates ERα-dependent transcription, and enhances proliferation of MCF-7 cells, as does the wild-type ERα17p, indicating that this domain may also be involved in CaM-independent posttranslational regulation of ERα (144).
GPER is robustly expressed in cardiovascular tissues (133)(134)(135)(136). In ECs, GPER mRNA is increased 8-fold by shear stress (154). GPER is localized on the ER/SR membrane (160) and responds to cell-permeable ligands (165). However, it also resides on the plasma membrane (166) and requires its C-terminal PDZbinding motif to do so (167). The plasmalemmal GPER pool seems to constitutively undergo clathrin-dependent endocytosis and accumulate in the trans-Golgi network for ubiquitination in the proteasome without recycling to the plasma membrane, a process unaffected by agonist stimulation (168). Despite its predominant expression in the ER/SR, the sequence that drives GPER localization here has not been identified.
GPER is directly regulated by Ca 2+ -CaM complexes. In VSMCs and ECs, GPER coimmunoprecipitates with CaM in a constitutive association that is promoted by treatment with E 2 , G-1, or receptor-independent stimulation of Ca 2+ entry (169,170). GPER is the first G protein-coupled receptor (GPCR) shown to possess four CaM-binding sites on its respective four submembrane domains (SMDs) (169). Fluorescence resonance energy transfer (FRET) biosensors based on SMDs of GPER bind CaM with K d from 0.4 to 136 × 10 −6 M and affinity ranking SMD2 > SMD4 > SMD3 > SMD1. These interactions are Ca 2+ dependent, with an EC 50 (Ca 2+ ) of 1.3 × 10 −7 -5 × 10 −6 M, values within the physiological Ca 2+ range (169). Due to technical challenges with purifying full-length GPCRs, the K CaM for GPER as a holoreceptor is not available. The presence of four CaM-binding sites makes this task even more challenging and, in some way, not useful functionally. Functionally, mutations that reduce CaM binding but that do not perturb GPER-G βγ preassociation drastically prevent GPER-mediated ERK1/2 phosphorylation (170).

Mechanisms (Figure 1)
Direct E 2 -Ca v 1.2 Interaction E 2 (10 −11 -10 −9 M) potentiates I Ca,L in neurons and HEK293 cells overexpressing the α1C subunit; nifedipine displaces membrane E 2 binding; and E 2 's effect is reduced by a dihydropyridineinsensitive LTCC mutant, indicating that E 2 binds to the dihydropyridine-binding site (177). Intriguingly, E 2 and the dihydropyridines exert opposite effects on I Ca,L .

Release of G βγ Subunit Upon GPER-Associated Gα i Stimulation
G βγ stimulates PLCβ (183-185) and activates IP 3 R1 (186), both of which trigger Ca 2+ store depletion and SOCE. Consistently, E 2 -induced Ca 2+ store release and entry in ECs are completely inhibited by pertussis toxin and PLCβ inhibitor U73122 (164). Also, HEK293 cells only produce a Ca 2+ response to E 2 when expressing HA-tagged GPER (162). Since (1) Ca 2+ entry channels are located on the membrane and (2) G βγ activates IP 3 Rs by interacting with the IP 3 -binding sites (186) on IP 3 Rs' cytosolic domains, both the membrane-delimited/Gα-mediated and G βγ -mediated mechanisms should only be operable by the plasmalemmal GPER pool. A distinguishing feature is that the former mechanism would not trigger SR/ER Ca 2+ release in the absence of extracellular Ca 2+ , whereas the latter would. Based on this feature, data fitting the former are available from renal tubular cells (176); and data fitting the latter, from vascular ECs (164).

CALCIUM ENTRY INHIBITION BY ESTROGENIC AGONISTS AND ESTROGEN RECEPTORS
To a large extent, estrogenic regulation of Ca 2+ signaling involves effects of estrogenic agonists and receptors on the Ca 2+ signals triggered by other stimuli, via both E 2 -dependent and E 2independent mechanisms.
How E 2 inhibits electrically induced VDCE is still unknown. Hypothetically, at high levels, E 2 binding to the dihydropyridinebinding site on LTCC (177) may instead inhibit I Ca,L . As for prevention of β adrenoceptor (βAR)-mediated potentiation of VDCE, recent evidence suggests that GPER may be an intrinsic component of β 1 AR activation. Thus, G-1 inhibits isoproterenol-induced increases in left ventricle (LV) pressure, heart rate, ectopic contractions, I Ca,L , LTCC phosphorylation, and total myocardial Ca 2+ signal, while the GPER inhibitor G-36 promotes ISO-induced Ca 2+ signal and LTCC phosphorylation (213). Speculatively, GPER may do so in part by interacting with β 1 AR or with A kinase-anchoring protein 5, thus inhibiting cAMP production (167). These may represent some E 2independent effects of GPER. Studies in GPER-knockout tissues are needed to further clarify the mechanisms.

SERCA Activity
Few studies, mostly in cardiac tissues, have examined the effects of E 2 on SERCA activity, with somewhat conflicting results. E 2 (1-30 × 10 −6 M) does not affect the V max of SR vesicle Ca 2+ uptake in canine LV tissue (214). However, ovariectomy reduces the V max but increases the Ca 2+ sensitivity for SR Ca 2+ uptake of rat LV homogenates or SR-enriched membrane fractions; mechanistically, these effects appear to be associated with reduced Thr17 phosphorylation of phospholamban and are restored by treatment with either E 2 or progesterone (215) (Figure 1). How E 2 and progesterone promote Thr17 phosphorylation of phospholamban is unknown, perhaps by inhibiting CaM kinase II (216), the enzyme that phosphorylates phospholamban (21). The effect of E 2 on SERCA activity in VSMCs has not been examined.

NCX Activity
As with SERCA activity, few studies have measured the effects of E 2 on NCX activity. Na + -dependent 45 Ca 2+ uptake in rat LV myocytes is increased by ∼3-fold after 60 days of ovariectomy, which is restored by replenishment with E 2 (1.5 mg/60 days) (208). During myocardial ischemia, intracellular Na + concentration is higher in male than in female cardiomyocytes and is associated with increased Ca 2+ concentration as a result of increased NCX activity (217). These studies are consistent with an inhibitory effect of E 2 on NCX activity in both the forward and reverse modes (Figure 1). However, the mechanisms of this inhibition are unclear.

PMCA Activity
Recent data show that GPER inhibits PMCA activity via both E 2 -dependent and E 2 -independent mechanisms (Figure 1). E 2dependent mechanisms are evidenced by the effects of G-1 (10 −8 -10 −6 M) and E 2 (1-5 × 10 −9 M) to inhibit PMCA-mediated efflux in primary ECs without affecting PMCA expression levels and to promote PMCA phosphorylation at Tyr1176 (135,170), which is known to inhibit pump activity (94). Notably, this phosphorylation masks the stimulatory effect of enhancing the PMCA-CaM interaction produced by 48-h E 2 treatment (170). E 2 -independent mechanisms are indicated by the findings that (1) GPER constitutively interacts with PMCA4b via the anchoring action of PSD-95 at their C-terminal PDZbinding motifs; (2) overexpression of GPER decreases PMCA activity; (3) GPER knockdown promotes PMCA activity; and (4) PSD-95 knockdown or truncation of the PDZ-binding motif on GPER releases GPER-PMCA association and promotes PMCA activity (135). Functionally, these mechanisms collectively prolong agonist-induced Ca 2+ signal and enhance eNOS activity in ECs (135,170,203). Consistent with suppressed Ca 2+ efflux, the Ca 2+ signals stimulated by E 2 and the GPER agonist G-1 in cells overexpressing GPER reported by various laboratories display much more prolonged plateau phases compared to Ca 2+ signals in cells not overexpressing GPER or those stimulated by other agonists such as ATP or bradykinin (160,162,164,175). GPER-PMCA4b interaction seems to be mutually influential, such that knockdown of PMCA decreases GPER-mediated ERK1/2 phosphorylation, while GPER knockdown does the opposite on PMCA activity (135).

ESTROGENIC REGULATION OF CALCIUM SIGNAL TRANSDUCTION-THE CALMODULIN NETWORK
Since CaM is the universal Ca 2+ signal transducer for numerous proteins (117,118), is insufficiently expressed for its targets (122,125,126), and is a source of competition among target proteins (124,127), factors that regulate its expression and target interactions are predicted to have a pervasive impact. The effects of E 2 on the CaM network have been examined in some detail in vascular ECs in recent studies (135,169,170). E 2 treatment (1-5 × 10 −9 M, 48 h) upregulates total CaM by around 7-fold and free Ca 2+ -CaM by ∼15-fold in primary ECs. Data obtained using specific estrogen receptor agonists, gene silencing, and receptor overexpression indicate that GPER, but not ERα or ERβ, mediates this effect. Thus, the GPER agonist G-1 (10 −9 -10 −7 M), but not the ERα agonist propyl pyrazole triol (PPT) (3 × 10 −10 -2 × 10 −7 M) or the ERβ agonist diarylpropionitrile (DPN) (10 −10 -5 × 10 −8 M), increases CaM expression; GPER knockdown reduces the effect of E 2 to upregulate CaM; and E 2 upregulates CaM in SKBR3 cells that express only GPER and not ERα or ERβ (170). Consistently, the ERα/ERβ antagonist/GPER agonist ICI182,780 dose-dependently upregulates CaM. Mechanistically, GPER exerts this action via the activities of EGFR and MAPK/ERK kinase 1 (MEK1). Functionally, E 2 upregulates CaM and promotes the PMCA-CaM interaction; however, the predicted stimulatory effect on Ca 2+ extrusion is masked by E 2induced inhibitory phosphorylation at Tyr1176 of PMCA (170); additionally, GPER exerts E 2 -dependent and E 2 -independent effects to inhibit PMCA (135). These collective actions prolong Ca 2+ signals, promote Ca 2+ -CaM complex formation, and increase Ca 2+ -CaM associations with low-to high-affinity CaM network members, represented by GPER itself, ERα, and eNOS (170). Considering that CaM binding stabilizes ERα homodimers, these effects are expected to promote other genomic actions of E 2 as well. Thus, a feedforward mechanism exists in which GPER mediates E 2 's effects to increase CaM and inhibits Ca 2+ efflux, prolonging cytoplasmic Ca 2+ signals, and the resultant increases in Ca 2+ -CaM complexes in turn promote the activities of GPER itself and other CaM network members (170) (Figure 1).

ESTROGENIC MODERATION OF CALCIUM-DEPENDENT ACTIVITIES
How do the various mechanisms discussed so far come together in regulating cardiovascular functions? An immediate challenge is how to reconcile the effects of estrogenic agonists to both trigger acute Ca 2+ signals by themselves and inhibit otherwise stimulated Ca 2+ signals. The Ca 2+ signals triggered by estrogenic agonists in primary cardiovascular cells are generally of very low amplitude. Furthermore, as in experiments testing their effects on Ca 2+ signals otherwise triggered, estrogenic agonists are present in situ with other stimuli whose Ca 2+ signals they inhibit. Thus, for mechanisms that generate cytoplasmic Ca 2+ signals, E 2 and GPER exert ultimate inhibitory effects. For cytoplasmic Ca 2+ removal mechanisms, estrogenic agonists and GPER also are inhibitory. For Ca 2+ signal transduction, E 2 , via a feedforward at GPER, increases CaM expression and enhances linkage in the CaM-binding proteome.
All things considered, E 2 and GPER, via both E 2dependent and E 2 -independent mechanisms, act to moderate Ca 2+ -dependent activities in the cardiovascular system. They "clamp" cytoplasmic Ca 2+ signals by lowering peaks (inhibition of signal generation) and raising troughs (inhibition of signal removal), collectively confining tissues in a narrower yet more sustained operating range of Ca 2+ . Also, GPER-mediated increases in CaM expression and CaM network linkage improve Ca 2+ signal transduction efficiency. Considering the Ca 2+ sensitivity of Ca 2+ -dependent proteins in this context, one can predict that those with low Ca 2+ sensitivity (requiring high Ca 2+ for activation) are more likely to be affected by the inhibition of Ca 2+ signal generation. On the other hand, proteins with high Ca 2+ sensitivity (requiring low Ca 2+ for activation) are more likely to be promoted by the inhibition of Ca 2+ removal and less affected by the suppression of Ca 2+ signal generation (Figure 2). This notion has been demonstrated experimentally via the case of eNOS, a Ca 2+ -dependent CaM-binding protein (222) with sub-nanomolar affinity for CaM (127). CaM interaction and subsequent activation of wild-type eNOS have high Ca 2+ sensitivities, with respective EC 50 (Ca 2+ ) values ∼1.8 × 10 −7 and 4 × 10 −7 M (190). eNOS is also regulated by multisite phosphorylation (223). Notably, its bi-phosphorylation at Ser617 and Ser1179 promotes NO production by increasing the Ca 2+ sensitivity for both CaM binding and enzyme activation, reducing their respective EC 50 (Ca 2+ ) values to ∼0.7 × 10 −7 and 1.3 × 10 −7 M, thus rendering the synthase active at resting cytoplasmic Ca 2+ (189). E 2 and GPER (1) prolong endothelial cytoplasmic Ca 2+ signal by inhibiting Ca 2+ efflux (135,170), (2) promote eNOS phosphorylation at Ser617 and Ser1179 (170,198), (3) increase CaM expression and eNOS-CaM interaction (170), and (4) suppress endothelial SOCE (203). When we incorporate these effects into a verified sequential "CaM binding eNOS activation" model (189,190), eNOS activity and NO accumulation are shown to substantially increase across the time course of bradykinin-induced Ca 2+ signal in ECs by treatment with G-1 (203). Importantly, major contributions to this outcome include the increases in CaM binding, phosphorylation, Ca 2+ sensitivity, and duration of Ca 2+ signals due to Ca 2+ efflux inhibition, but little or no effect of the inhibition of SOCE (203), due obviously to the synthase's high Ca 2+ sensitivity (Figure 3). Thus, via multifaceted actions on components of the CSM, E 2 and GPER moderate Ca 2+ -dependent activities by differentially affecting the continuum of Ca 2+ -dependent proteins based on their Ca 2+ sensitivities for Ca 2+ or Ca 2+ -CaM complexes.
Considering the two Ca 2+ -dependent estrogen receptors-ERα and GPER-how does the presence of one influence the effects of the other on Ca 2+ signaling? A complex relationship is predicted to exist in which ERα transcriptional activities affect the expression of certain Ca 2+ signaling proteins but are themselves influenced by the amplitudes and dynamics of Ca 2+ signals limited by GPER activation and the availability of CaM that is promoted by GPER action (170). In turn, as CaM is limited in cells (122,124,126,127), the high affinity binding of CaM by ERα and GPER further limits CaM availability and will influence CaM-dependent regulation of each other at the receptor level, a predictable outcome of the functional crosstalk via competition for limited CaM (124,127). These relationships may represent but a small aspect of the reciprocality between estrogen and Ca 2+ signaling.

CONCLUSION AND FUTURE PERSPECTIVES
Reciprocality between estrogen signaling and Ca 2+ -dependent activities is becoming evident. Considering the impact of estrogen  (203). The solid line represents Ca 2+ signals produced in response to agonist stimulation in the absence of GPER activation. The sparsely dotted area represents the range of cytoplasmic Ca 2+ signals, in which peak and trough are seen due to maximal effects of Ca 2+ entry and Ca 2+ efflux. The stippled blue line represents Ca 2+ signals produced in the presence of GPER and its activation. These signals are clamped in a narrower range (the blue area) due to inhibitory effects on both SOCE [green stripes (203)] and PMCA4b-mediated Ca 2+ efflux [red stripes (135,170)]. (B) Average time courses of cytoplasmic Ca 2+ signals measured in primary ECs treated with bradykinin in the absence of extracellular Ca 2+ followed by treatment with vehicle or G-1; total Ca 2+ signals were triggered by re-addition of extracellular Ca 2+ [arrow (203)]. (C) Calculated eNOS point activity corresponding to each Ca 2+ value in (B) considering only changes in Ca 2+ due to GPER activation using a verified sequential eNOS-CaM binding eNOS activation model [equation, where (K 1 , K 2 ) and (K 3 , K 4 ) are derived products of the binding constants of Ca 2+ at the Ca 2+ -binding sites on the N and C lobes of CaM in binding to CaM and interaction of Ca 2+ -CaM and eNOS (189,190). (D) Calculated eNOS point activity corresponding to each Ca 2+ value measured in (B), factoring in changes in Ca 2+ , CaM binding, and eNOS phosphorylation (170,203). See details in text and (170,203). Reproduced with permission from the author's previous publication (203). and its receptors on Ca 2+ signaling, E 2 , and in many cases, GPER exert inhibitory effects on many components of the CSM in cardiovascular tissues, from Ca 2+ store release and uptake (214,215,221) and Ca 2+ entry (199, 201-210, 212, 213) to cytosolic Ca 2+ removal mechanisms (135,170,208,(217)(218)(219)(220)(221). Considering the impact of Ca 2+ signaling on estrogen biology, both ERα and GPER are strongly regulated by direct Ca 2+ -dependent interactions with CaM. These interactions serve to stabilize receptor dimerization and enhance subsequent transcriptional activities [the case of ERα (137,138,142,143)] or promote receptor-mediated downstream signaling [the case of GPER (169,170)]. Also, E 2 -induced MAPK activation has long been known to be dependent on the Ca 2+ signal produced (173). Reciprocality between estrogen biology and Ca 2+ signaling is further evidenced by the demonstration of a feedforward mechanism, in which E 2 , via GPER activation, upregulates total cellular CaM expression and free intracellular Ca 2+ -CaM concentration, which promotes functions of GPER and ERα and other classes of Ca 2+ -CaM-dependent proteins (170). The combination of these various actions is predicted to affect Ca 2+ -dependent functions depending on the affinity and Ca 2+ sensitivities of the proteins involved, as exemplified by the case of eNOS (Figures 2, 3) (170,203).
The moderating effects that estrogenic agonists and receptors exert on the CSM can explain many of their cardiovascular effects, such as preventing excessive cardiac contraction during sympathetic stress, limiting adverse outcomes related to Ca 2+ overload, and reducing vascular tone. Nevertheless, the effects of E 2 and estrogen receptors on many CSM components have not been examined. Additionally, many questions remain regarding mechanisms of the observed effects that estrogenic agonist and receptors produce on the CSM. For example, how do E 2 and GPER inhibit I Ca,L ? What are the mechanisms that position GPER as an intrinsic component of β 1 AR signaling in the myocardium? What are the mechanisms whereby E 2 inhibits the activities of SERCA and NCX? What are the mechanisms whereby E 2 inhibits mitochondrial Ca 2+ uptake? Further studies are needed to answer these questions. Through many examples, however, it is clear that GPER produces both E 2 -dependent and E 2 -independent effects on the CSM. While the search is ongoing for approaches to apply specific estrogen receptor agonists to the prevention of cardiovascular disease, the therapeutic potential of E 2 -independent effects of GPER and other estrogen receptors is as yet an unexplored territory.

AUTHOR CONTRIBUTIONS
Q-KT conceived the ideas, generated the figures, and wrote the manuscript.

FUNDING
The publication cost of this review article is covered by a fund from Des Moines University to the author.