NLRP3 inhibition improves heart function in GPER knockout mice

https://doi.org/10.1016/j.bbrc.2019.05.045Get rights and content

Highlights

  • GPER deletion in cardiomyocytes induces left ventricular dysfunction.

  • GPER deletion increases NLRP3 inflammasome-related gene expression.

  • NLRP3 inhibition by MCC950 improves heart function in GPER KO mice.

Abstract

The molecular mechanisms of postmenopausal heart diseases in women may involve the loss of estrogen-deactivation of its membrane receptor, G-protein coupled estrogen receptor (GPER), and subsequent activation of the cardiac NLRP3 inflammasome, a component of the innate immune system. To study the potential effects of cardiac GPER on NLRP3-mediated inflammatory pathways, we characterized changes in innate immunity gene transcripts in hearts from 6-month-old cardiomyocyte-specific GPER knockout (KO) mice and their GPER-intact wild type (WT) littermates using RT2 Profiler™ real-time PCR array. GPER deletion in cardiomyocytes decreased %fractional shortening (%FS) and myocardial relaxation (e′), and increased the early mitral inflow filling velocity-to-early mitral annular descent velocity ratio (E/e′), determined by echocardiography, and increased the mRNA levels of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP), determined by real-time PCR. Of the 84 inflammasome-related genes tested, 9 genes were upregulated, including NLRP3 and IL-18, while 1 gene, IL-12a, was downregulated in GPER KO when compared to WT. The importance of NLRP3 upregulation in GPER KO-induced heart failure was further confirmed by an in vivo study showing that, compared to vehicle-treated KO mice, 8 weeks of treatment with a NLRP3 inhibitor, MCC950 (10 mg/kg, i.p., 3 times per week), significantly limited hypertrophic remodeling, defined by reductions in heart weight/body weight, and improved systolic and diastolic functional indices, including increases in %FS and e′, and decreases E/e′ (P < 0.05). Both ANF and BNP mRNA levels were also significantly reduced by chronic MCC950 treatment. The findings from this study point toward a new understanding for the increased occurrence of heart diseases in women following loss or absence of estrogenic protection and GPER activation that involves cardiac NLRP3 inflammatory pathways.

Introduction

Estrogen is widely accepted as a protector of the female heart against various hemodynamic stresses and sterile, low grade inflammatory processes, including hypertension, cardiac hypertrophy failure, atherosclerosis, ischemia–reperfusion injury and heart failure, while its loss contributes to an acceleration of cardiovascular diseases in women. Numerous preclinical studies demonstrate that estrogen protects the heart through both direct effects on cardiomyocytes, and indirectly via systemic effects [1]. However, hormone therapy over the past few decades has not shown clear cardiac benefits and might be associated with health risks including cancer, coronary heart disease, and stroke [2]. Furthering research on the mechanisms of estrogen in the attenuation of cardiovascular diseases, with a focus on roles of specific estrogen receptors, is critical in the development of more specific strategies for the treatment of cardiovascular diseases in women, with fewer or no side effects. A new potential target for drug therapy might be the G protein-coupled estrogen receptor (GPER), also known as G protein-coupled receptor 30 (GPR30). Activation of GPER by its agonist G1 protects the heart against various stresses including pressure-overload [3], ischemia/reperfusion [4], high salt diet [5], estrogen loss and aging [6,7], while cardiomyocyte-specific GPER deletion induces cardiac remodeling and heart failure [8]. These findings strongly suggest that GPER mediates the cardioprotective effects of estrogen.

The contribution of inflammatory processes in the development and progression of cardiovascular disease is a topic of continuous research. One exciting area in this field is the NLRP3 (NOD-, LRR- and pyrin domain-containing 3) inflammasome, in which NLRP3, caspase-1, interleukin-1β (IL-1β) and IL-18 are involved [9]. The NLRP3 inflammasome, which is formed and activated by various stimuli such as PAMPs (pathogen-associated and danger) and DAMPs (damage-associated molecular patterns), participates in the pathogenesis of hypertension, diabetes, atherosclerosis, myocardial infarction, heart failure and other cardiovascular diseases [9]. Several preclinical models show that inhibition of NLRP3 protects the heart [[10], [11], [12]], while findings from recent clinical trials indicate that blockade of IL-1 with a modified version of the human interleukin 1 receptor antagonist Anakinra™ has cardiac beneficial effects in patients with coronary artery disease, heart failure and pericarditis [[13], [14], [15]]. No studies to-date have linked the NLRP3 inflammasome to the loss of estrogenic cardioprotection or inactivation of any one of the estrogen receptors. In the present study, using the cardiomyocyte-specific GPER knockout (KO) mouse, RT2 Profiler™ real-time PCR array, and in vivo NLRP3 inhibition by its specific inhibitor, MCC950, we show, for the first time, that the NLRP3-related inflammatory pathway is involved in cardiac dysfunction related to GPER deficiency in cardiomyocytes.

Section snippets

Animals and treatments

Cardiomyocyte-specific GPER KO mice were generated in our laboratory, as described previously [8]. GPERf/f/Cre (GPER KO) and GPERf/f littermates (GPER WT) were studied at 6 months of age in 2 separate cohorts. In the first cohort, heart structure and functions were assessed with echocardiography, followed by euthanasia in both GPER KO and WT mice (n = 6 per group). In the second cohort, an equal number of male and female GPER KO mice received chronic treatment with a NLRP3 inhibitor, MCC950

Cardiomyocyte-specific GPER deletion impaired heart structure and functions

Consistent with our previous report, findings from echocardiographic examinations revealed decreased fractional shortening (FS) (27.1 ± 2.0 vs. 33.5 ± 1.8, P < 0.05. Fig. 1A), decreased e′ (2.5 ± 0.1 vs. 3.5 ± 0.2, P < 0.05. Fig. 1B), and increased E/e′ (26.4 ± 2.4 vs. 18.2 ± 0.7, P < 0.05. Fig. 1C) in hearts with cardiomyocyte-specific GPER deletion. The mRNA levels of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP), two important biomarkers of heart failure, were

Discussion

The main findings of this study are: (1) cardiomyocyte-specific GPER deficiency-induced heart dysfunction occurs with increases in NLRP3 inflammasome-related gene expression; and (2) chronic NLRP3 inhibition with MCC950 treatment improves heart function in GPER KO mice. These results suggest that the NLRP3-mediated inflammatory response pathway may contribute to heart failure progression. Further studies should clarify whether this inflammatory mechanism plays a role in the development of heart

Conflicts of interest

The authors have no conflict of interest to report.

Funding

This work was funded by grants from the National Heart, Lung, and Blood Institute (CMF and LG) P01-HL051952 and the National Institute on Aging (LG) AG033727 of the National Institutes of Health.

References (22)

  • W. Zhou et al.

    NLRP3: a novel mediator in cardiovascular disease

    J. Immunol. Res.

    (2018)
  • Cited by (16)

    • Targeting the NLRP3 inflammasome in cardiovascular diseases

      2022, Pharmacology and Therapeutics
      Citation Excerpt :

      MCC950 also possesses cardioprotective benefits in non-ischemic cardiomyopathy, as evidenced by reductions in myocardial fibrosis and IL-1β levels in angiotensin II-induced hypertension (Gan et al., 2018). When administered for 8 weeks (10 mg/kg, 3 times/week) in a mouse model of post-menopausal heart disease, MCC950 limited hypertrophic remodeling, improved systolic and diastolic function and reduced cardiac ANF and BNP mRNA levels (Wang, Sun, Hodge, Ferrario, & Groban, 2019). The long-term use of MCC950 (20 mg/kg/daily) for 15 weeks improved autophagy flux and reduced cardiac apoptosis (Pavillard et al., 2017).

    • Inhibition of the NLRP3 inflammasome reduces brain edema and regulates the distribution of aquaporin-4 after cerebral ischaemia-reperfusion

      2020, Life Sciences
      Citation Excerpt :

      MCC950 also shown to inhibit the mRNA levels of NLRP3 and IL-1β in the ischaemic core of the brain in diabetic mice [3]. A recent study demonstrated that MCC950 did not change the mRNA level of NLRP3, but significantly increased the mRNA levels of caspase-1 and IL-1β [25]. Similar to the recent study above, our results showed that MCC950 increased the mRNA levels of NLRP3 and IL-1β in the brain and reduced IL-18 gene expression after tMCAO.

    • Use of embryonic stem cell-derived cardiomyocytes to study cardiotoxicity of bisphenol AF via the GPER/CAM/eNOS pathway

      2020, Toxicology
      Citation Excerpt :

      Additionally, previous studies demonstrated that biological effects of BPAF are mediated by GPER rather than by ER (Cao et al., 2017). GPER is an important pathway for the biological effects of estrogen and estrogen-like hormones and GPER levels are associated with numerous diseases (Wang et al., 2019; Wei and Huang, 2019; Yuan et al., 2019). It has been reported that certain compounds can cause cardiotoxicity through GPER (Goncalves et al., 2018).

    View all citing articles on Scopus
    View full text