Similar to other cardiovascular diseases (CVDs), chronic stress-induced cardiomyopathy (CSC) has been clinically shown to be predominant in males of all age cohorts, while females are mostly predisposed to its occurrence during their menopausal period (Boese et al., 2017; Ndzie Noah et al., 2021). Recent attempts to elucidate the underlying disease mechanisms have revealed crucial roles played by estrogen during stress to sustain good cardiac health in premenopausal women, besides its reproductive functions (Mori et al., 2011; Boese et al., 2017).

Typically, the clinical manifestations of CSC patients are severe left ventricular (LV) diastolic dysfunction (LVDD) and systolic dysfunctions (LVSD) (Medeiros et al., 2014). The adverse structural remodeling includes excessive LV hypertrophy and massive interstitial fibrosis, which results in the stiffening of the myocardia and causes these cardiac dysfunctions. Also, recent studies have demonstrated that unresolved myocardial inflammatory responses characterized by the enormous influx of proinflammatory macrophages exacerbates CSC and aggravates adverse cardiac remodeling (Wilson et al., 2018; Scally et al., 2019).

Under physiological state, inotropic and chronotropic functions of the heart are mediated by β-adrenergic receptors (βARs) via G stimulatory protein (G_s), mainly β₁AR and β₂AR. However, hyperstimulation of the receptors during chronic stress results in the downregulation of β₁AR mostly, while β₂AR traffics the stimuli signal via G inhibitory protein (G_i) to prevent cardiac insults (Paur et al., 2012). The homologous desensitization of βARs which results in the downregulation is induced by G protein-coupled receptor kinases 2 (GRK2) phosphorylation and β-arrestin-1 recruitment (Adzika et al., 2019). Nonetheless, it was demonstrated in previous studies that estrogen ameliorates stress-induced cardiac insults by preventing excessive depletion of β₂ARs and also facilitating a balance in the G_i/G_s signaling pathways (Cao et al., 2015; Hou et al., 2018). The estrogenic effects of sustaining β₂AR activities during stress might be likely due to its inhibitory effects on GRK2 (Abraham et al., 2018). Intriguingly, β₂AR are profoundly expressed on macrophages; hence, estrogen may indirectly modulate their activities and possibly their polarizations into proinflammatory (CD86+ macrophages) or anti-inflammatory (CD206+ macrophages) phenotypes in the myocardia. It is hypothesized that the possible exertion of the aforementioned estrogenic immunoregulation might prevent extensive pathological cardiac remodeling during chronic stress by subduing maladaptive myocardial hypertrophy, fibrosis, and proinflammatory responses.

Herein, we sought to explore the cardioprotective mechanisms employed by estrogen via immunomodulation to decrease the incidence of CSC in female rat models, as understanding these mechanisms will provide the basis for further translational research into preventing CSC in postmenopausal women.

Unresolved myocardial inflammatory responses have been clinically demonstrated as an underlying factor expediting the pathological remodeling of the heart during stress (Mori et al., 2011; Wilson et al., 2018; Scally et al., 2019). The homeostatic balance between cardiac proinflammatory and anti-inflammatory macrophage phenotypes is crucial for resolving myocardial inflammation and proper heart functioning (Mouton et al., 2018). However, clinical studies have shown that the myocardia of CSC patients have massive bias infiltrations of proinflammatory macrophages, which prolongs inflammation without timely resolutions to permit reparative functions of anti-inflammatory macrophages. Hence, in post-stress–induced cardiac injuries, the maladaptive proinflammatory responses in the myocardia drives the pathological remodeling of the heart, which is evident by marked fibrosis (Mori et al., 2011; Mouton et al., 2018).

Herein, we demonstrate the mechanistic roles employed by E₂ to protect the heart during chronic stress from an immunoregulatory perspective. Morphometric evaluations revealed significant gains in BW resulting from the deficiency of estrogen in the OVX rats under physiological and chronic stress states. This finding provides supporting evidence that E₂ is crucial for efficient lipid metabolism. In fact, previous studies have demonstrated that E₂ maintains a healthy lipid profile by upregulating bloodstream levels of high-density lipoprotein (HDL) and lowering low-density lipoprotein receptors (LDL) (Lee et al., 2015; Fu et al., 2019; Ndzie Noah et al., 2021). As such, the deficiency of E₂ scaffolded disorders in lipid metabolism that caused weight gain as it permitted increased circulation LDL (bad cholesterol) level which deposited as adipose all over the body as well as in and around vascular tissues and circulatory organs (Lee et al., 2015; Kozakowski et al., 2017). Similar to the previous work of Ren et al. (2003), BW was increased in OVX rats. Also, it was observed that the combination of E₂ deficiency and stress increased HW and HW/BW coefficient more in OVX rats. The HW and HW/BW coefficient increases may be due to increased epicardial adipose and cardiomyocyte hypertrophy. Intriguingly, epicardial adipose has been shown to be a reservoir for macrophages which infiltrates the myocardia to hasten maladaptive inflammatory responses (Mori et al., 2011). Therefore, the increased accumulation of epicardial adipose resulting from E₂ deficiency predisposes the heart to sustained myocardial inflammation should there be any cardiac insult during stress. Overall, consistent with early findings (Ren et al., 2003; Mori et al., 2011; Michalson et al., 2018), it was demonstrated that E_2Endo and its supplementation (E_2Exo) prevents excessive weight gains, which ultimately impacts positively on cardiac health.

Clinically, demographics clearly show that normally, females have higher heart rates (HR) and cardiac outputs than males of the same age cohort (Wheatley et al., 2014). In menopause, there is a further increase in HR, which results in short-term arrhythmias (heart palpitations) and are attributed to the loss of E₂ and possibly β₂AR signaling dysregulation (Carpenter et al., 2021). Interestingly, the contrary was found in this study. The obliteration of E₂ via ovariectomy resulted in a slight decrease in HR under normal state; however, chronic stress in these OVX rats caused a significant reduction in HR. The possible explanation for this outcome is that inotropy and chronotropic functions of the heart are mediated by β₁AR and β₂AR; meanwhile, E₂ prevents their dysregulations and substantial depletion during stress (Hou et al., 2018; Ndzie Noah et al., 2021). Therefore, E₂ deficiency might have permitted dysfunctionalities and downregulation of the β₂ARs during stress, hence the significant decrease in HR. Also, consistent with previous reports, it was found that the cardiac function index; LVSP, LVEDP, +dp/dt, and −dp/dt were unaffected by E₂ deficiency under physiological state (Mori et al., 2011; Ribeiro et al., 2013). Even so, chronic stimulation of βARs by ISO during E₂ deficiency demonstrated overt cardiac dysfunctionalities. In contrast, it was demonstrated that E_2Endo and E_2Exo in the Sham + ISO and OVX + E2 + ISO rats, respectively, ameliorated these heart dysfunctions to sustain cardiac output during stress.

Further investigations sought to characterize the impact of chronic stress on the myocardial structure during E₂ deficiency. It was observed that cardiomyocyte diameters generally increased during stress; however, the E₂ deficiency permitted maladaptive hypertrophy, which distorted the typical myocardial architecture. This was further proven by the significant upregulations of ANP and BNP in the hearts of OVX rats during chronic stress. Nevertheless, E_2Endo (in the Sham rats) showed much potency at minimizing the upregulations of both ANP and BNP during stress, while E_2Exo (in the OVX + E₂ rats) was unable to downregulate the latter substantially. In conformity with our findings, Goncalves et al. (2018) and others had early demonstrated that E₂ exerts antihypertrophic effects via GPER (Goldstein et al., 2004). Also, the discrepancies observed between the antihypertrophic effect of E_2Endo and E_2Exo might have occurred because other ovarian secretions such as vascular endothelial growth factor (VEGF) may complement the efforts of E₂ in preventing maladaptive cardiomyocyte hypertrophy (Cai et al., 2015). Besides, as suggested by Zhang et al. (2021), unlike the E_2Exo treatment dose, which remained constant during CSC modeling, the levels of E_2Endo are altered due to the estrous cycle in the Sham and could have also contributed to the observed differences in the antihypertrophic effect of E₂. In addition, it was found that obliteration of E₂ in OVX rats permitted induction of massive interstitial fibrosis; nevertheless, its presence/restoration ameliorated this adverse outcome. We showed that comparatively, E_2Endo in the Sham and its supplementation (E_2Exo) lessened the extent of fibrosis, just as demonstrated earlier (Mori et al., 2011; Michalson et al., 2018).

The homeostatic balance between proinflammatory and anti-inflammatory macrophages in the myocardia during steady state is crucial for cardiac function, as is the timely trafficking of either of them during injury/cell clearance or reparative process, respectively, essential for preventing adverse heart remodeling (Lavine et al., 2014; Mouton et al., 2018). However, as demonstrated from the postmortem examination of the hearts from CSC patients, proinflammatory macrophages were abundant in the myocardia and were shown to have exacerbated myocardial proinflammatory responses, which may have resulted from stress-induced cardiac insults. The observed biased infiltration of CD86+ macrophages (proinflammatory) hastened the pathological cardiac remodeling as autopsied hearts had marked fibrosis (Wilson et al., 2018; Scally et al., 2019). Similar to these clinical findings, it has been shown in rats that stress causes augmentation of myocardial CD86+ macrophage infiltrations, and the phenomena are worsened by E₂ deficiency (Mori et al., 2011). Following up on these previous studies, consistent findings were made. CD68-positive cell infiltration into the myocardia were increased only under stress conditions; however, E₂ deficiency augmented their infiltration significantly. Nonetheless, E_2Endo and its supplementation (E_2Exo) to the rats during stress minimized CD68-positive cell infiltration. Assessing the phenotypic ratios with CD86 and CD206 immunostaining revealed the majority of the CD68-positive cells infiltrating the myocardia when E₂ is deficient during stress are CD86-positive cells, while CD206-positive cells are less present. Nevertheless, E_2Endo and E_2Exo reversed these phenomena by enhancing anti-inflammatory responses in the hearts during stress via increasing CD206+ macrophage presence, as similarly reported previously (Xing et al., 2009; Bolego et al., 2013). Validations of the aforementioned findings were done by assessing the mRNA expressions of proinflammatory (TNF-α and iNOS) and anti-inflammatory macrophage (TGF-β and Arg-1) markers from the myocardia of all experimental groups. Similar to the observations of the histological evaluations, TNF-α and iNOS were upregulated during stress and were further elevated significantly when E₂ is deficient. Also, TGF-β and Arg-1 mRNA expressions were downregulated in the myocardia due to E₂ deficiency. Conversely, E_2Endo exerted anti-inflammatory effects by enhancing TGF-β and Arg-1 while decreasing TNF-α and iNOS mRNA expressions during stress. Although E_2Exo upregulated TGF-β and Arg-1 and inhibited TNF-α similarly to E_2Endo, it was not as potent as E_2Endo in downregulating iNOS. The possible explanation of the phenomenon is that ovarian secretions of progesterone might have complimented the inhibitory effects of E_2Endo, as it has been reported that besides E2, progesterone decreases iNOS levels in non-cardiac tissue (Menzies et al., 2011). However, progesterone is obliterated in OVX + E2 + ISO rats; hence, it might account for iNOS being significantly downregulated in Sham + ISO than OVX + E2 + ISO. Nonetheless, the estrogenic anti-inflammatory effects demonstrated here have been similarly reported by Villa et al. (2015) and others (Chen et al., 2021).

Similar to cardiomyocytes, macrophages have profound expressions of β₂AR and estrogen receptors (ERs), and the cardioprotective effects conferred by E₂ have been demonstrated to mostly involved the synergy of ERs and β₂AR signaling cascades (Kang et al., 2012; Hou et al., 2018; Machuki et al., 2019; Ndzie Noah et al., 2021). Hence, to elucidate the immunoregulatory mechanisms employed by E₂ to facilitate more CD206+ macrophage polarizations to accelerate the resolution of myocardial inflammation during stress, we investigated the possible involvement of β₂AR signaling modulation by E₂-induced cascades. In conformity with our initial speculations, β₂AR expressions from apical myocardia (constituting cardiomyocytes and infiltrated macrophages) were found to be significantly depleted during chronic stress due to E₂ deficiency, as the presence of E_2Endo in the Sham and the supplementation of E_2Exo in OVX rats showed a minimal reduction in the expression of the receptor under the same stress condition. Further investigations of β₂AR involvement deployed the isolations of PM_Φ from female WT and β₂AR-KO mice as well as the use of β₂AR blocker ICI 118,551 to ascertain if E₂ induced any variations in the phenotypic ratios of the macrophages during stress was affected by impeding β₂AR signaling. We report that the estrogenic signaling facilitates adaptive immunoregulation by ensuring CD206+ macrophage polarizations to timely resolve inflammation as reported by others (Keselman et al., 2017). However, for the first time, we show the underlying mechanism involves interplays of E₂, ERs and β₂AR signaling during stress. Flow cytometric evaluations show that E₂ treatments during stress increased CD206+ macrophage polarizations against CD86+ macrophages; however, the deletion/inhibition of β₂AR impaired this phenomenon. These observations are possibly because the bioavailability of nitric oxide (NO), which is produced via β₂AR-G_ai-PI3K-Akt–mediated signaling cascade, is crucial for the polarization of macrophages from proinflammatory to the anti-inflammatory phenotype (De Nigris and Prattichizzo, 2021). As such, blockade of β₂AR signaling disrupts NO bioavailability and abolishes this adaptive immunoregulatory mechanism. Also, E₂ had been shown previously to exert these anti-inflammatory effects primarily via estrogen receptor alpha (ERα) (Bolego et al., 2013; Campbell et al., 2014), and although we have demonstrated here the essential involvement of β₂AR to facilitate the polarizations of CD206+ macrophages, there are apparent interplays among E₂, ERs, and β₂AR to ensure this immunomodulation during stress. Intriguingly, E₂ and ER activities downregulate GRK2, which otherwise would have induced the homologous desensitization and downregulation of β₂AR during stress (Abraham et al., 2018; Arcones et al., 2021). Therefore, E₂ and ERs indirectly sustain the bioavailability of NO via β₂AR-G_ai-PI3K-Akt signaling by preventing dysregulation of the receptor during stress and enhancing the β₂AR-mediated CD206+ macrophage polarization.

Taken together, the findings from this study demonstrate the immunoregulatory mechanisms employed by E₂ to confer cardioprotection and lower the incidence of CSC in premenopausal women as compared with postmenopausal women and males of all age cohorts. E₂ exerts this immunoregulatory myocardia protection to prevent pathological cardiac remodeling during stress by ensuring the timely resolution of myocardial proinflammatory responses and enhancing reparative functions of CD206+ macrophage. More importantly, we demonstrate here that the adaptive modulation of macrophage phenotypes by E₂ during stress requires the mediation of β₂AR signaling. The classical interplays among E₂, ERs, and β₂AR discussed by Ndzie Noah et al. (2021) are also shown here, as E₂ and ER activities are in turn required to prevent β₂AR dysregulations and dysfunctionalities during stress. From a therapeutic standpoint, the findings from this study reechoes the essence of E₂ replacement therapy (E₂RT) in postmenopausal women, as it reduces the incidence of CSC. However, it is recommended that E₂RT is initiated within 5–6 years after menopause so as to explore its therapeutic benefits fully while circumventing the adverse outcomes reported by the Women’s Health Initiative from their randomized controlled trial (Rossouw et al., 2002; Michalson et al., 2018; Ndzie Noah et al., 2021). Finally, it is deemed necessary to point out the limitations of this study due to its clinical significance. β₁ARs are essential for myocardial functions and might play other immunologic roles facilitated by E₂, but they have not been elucidated previously nor in this study. Also, in some instances (Figures 3E,F), (Figures 4C–J), it is shown that E_2Exo did not confer anti-inflammatory effects as E_2Endo did. However, the fact that other ovarian secretions such as progesterone can complement the anti-inflammatory effects of E_2Endo but are obliterated by ovariectomy in the E_2Exo treatment group might explain the observed differences. The estrous cycle in the Shams causing alterations in E_2Endo levels while E_2Exo treatment dosage used remained constant might also account for the shown slight variations in E_2Endo and E_2Exo effects. Therefore, we stand with Zhang et al. (2021) in suggesting that E₂RT should be given at dosages that mimic the concentrations of the estrous cycle to eliminate the observed variations in its cardioprotection efficacy. This will enhance the exploitation of the therapeutic potentials of E₂RT in attenuation/prevention of CSC via immunomodulation in postmenopausal women.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

The animal study was reviewed and approved by the Experimental Animal Centre of Xuzhou Medical University and the Animal Ethics Committee of Xuzhou Medical University (permit no: xz11-12540).

HH conceived the experiment idea. HS, HH, GKA, and QW designed the experiments. HH and GKA isolated and cultured PM_ϕ. HH, TM, and YM made animal models. HH, JG, MS, and LF performed cardiac function and histological assessments. HH, GKA, QW, and HS analyzed and interpreted the results. Based on the contributions of all authors, HH drafted the initial manuscript and GKA revised it entirely. HH, GKA, QW, TM, YM, JG, MS, LF, RR, and ZG proofread and approved the manuscript in its current form.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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