Estradiol mitigates stress-induced cardiac injury and inflammation by downregulating ADAM17 via the GPER-1/PI3K signaling pathway

Abstract

Stress-induced cardiovascular diseases characterized by inflammation are among the leading causes of morbidity and mortality in postmenopausal women worldwide. Estradiol (E2) is known to be cardioprotective via the modulation of inflammatory mediators during stress. But the mechanism is unclear. TNFα, a key player in inflammation, is primarily converted to its active form by 'A Disintegrin and Metalloprotease 17' (ADAM17). We investigated if E2 can regulate ADAM17 during stress. Experiments were performed using female FVB wild-type (WT), C57BL/6 WT, and G protein-coupled estrogen receptor 1 knockout (GPER-1 KO) mice and H9c2 cells. The study revealed a significant increase in cardiac injury and inflammation during isoproterenol (ISO)-induced stress in ovariectomized (OVX) mice. Additionally, ADAM17's membrane content (mADAM17) was remarkably increased in OVX and GPER-1 KO mice during stress. However, in vivo supplementation of E2 significantly reduced cardiac injury, mADAM17, and inflammation. Also, administering G1 (GPER-1 agonist) in mice under stress reduced mADAM17. Further experiments demonstrated that E2, via GPER-1/PI3K pathway, localized ADAM17 at the perinuclear region by normalizing β1AR-Gαs, mediating the switch from β2AR-Gαi to Gαs, and reducing phosphorylated kinases, including p38 MAPKs and ERKs. Thus, using G15 and LY294002 to inhibit GPER-1 and its down signaling molecule, PI3K, respectively, in the presence of E2 during stress resulted in the disappearance of E2's modulatory effect on mADAM17. In vitro knockdown of ADAM17 during stress significantly reduced cardiac injury and inflammation, confirming its significant inflammatory role. These interesting findings provide novel evidence that E2 and G1 are potential therapeutic agents for ADAM17-induced inflammatory diseases associated with postmenopausal females.

Keywords: ADAM17; Cardiac injury; Estradiol; GPER-1; Myocardial inflammation; Stress.

Fig. 1

Graphical representation of experimental timeline for in vivo studies A Graphical outline of in vivo experiments in female FVB WT mice. B Graphical outline of in vivo experiments in female C57BL/6 WT and KO mice

Fig. 2

Estradiol (E2) reduces cardiac injury and inflammation in FVB female mice during ISO stress. A, B, C Effect of E2 on the concentrations of cardiac injury markers in cardiac tissues (n = 6–8). D, E, F Effect of E2 on the RNA concentrations of inflammatory cytokines (n = 4–5). G, H, I Effect of E2 on the serum concentrations of inflammatory cytokines (n = 6). J, K Effect of E2 on cardiac protein expression of NF-κB (p65) (n = 3). L, M, N Picture representation of macrophage infiltration in cardiac tissues (n = 4–5), (scale bar = 50 μm). O, P, Q Graphical representation of macrophage infiltration in cardiac tissues (n = 4–5). Two-way ANOVA results are presented as mean ± SEM, *Comparison between control and its stressed group, ^@Comparison among stressed groups (*p < 0.05 or ^@p < 0.05.) (**P < 0.01 or ^@@P < 0.01), (***P < 0.001 or ^@@@P < 0.001). Sham(S), Ovx(O), Ovx + E2(OE), Sham + ISO(SI), Ovx + ISO(OI), Ovx + ISO + E2(OIE)

Fig. 3

Estradiol (E2) downregulates myocardial ADAM17 during stress-induced inflammation in FVB female mice. A Effect of E2 on myocardial RNA concentration during ISO-induced stress (n = 4–5). B, C, D Effect of E2 on the cytoplasmic(pADAM17) and membrane content of ADAM17(mADAM17) during ISO-induced stress (n = 3–4). E, F Effect of E2 on the myocardial infiltration of ADAM17 during ISO-induced stress, (scale bar = 50 μm), (n = 4). Two-way ANOVA, results are presented as mean ± SEM, *Comparison between control and its stressed group, ^@Comparison among stressed groups (*p < 0.05 or ^@p < 0.05.) (**P < 0.01 or ^@@P < 0.01), (***P < 0.001 or ^@@@P < 0.001). Sham(S), Ovx(O), Ovx + E2(OE), Sham + ISO(SI), Ovx + ISO(OI), Ovx + ISO + E2(OIE)

Fig. 4

Activation of GPER-1 downregulates myocardial mADAM17 during ISO-induced stress in C57BL/6 female mice. A Protein expression of GPER-1 in the cardiac tissues of WT and KO. B, C Effect of GPER-1 knockout on the membrane content of ADAM17 in cardiac tissues during ISO-induced stress (n = 4). D, E Effect of G1 administration on the membrane content of ADAM17in cardiac tissues during ISO-induced stress (n = 4). F, G Effect of GPER-1 knockout on the myocardial infiltration of ADAM17 during ISO-induced stress, (scale bar = 50 μm), (n = 5). H, I Effect of G1 administration on the myocardial infiltration of ADAM17 during ISO-induced stress, (scale bar = 50 μm), (n = 5). Two-way ANOVA and one-way ANOVA results are presented as mean ± SEM, *Comparison between control and its stressed group, ^@Comparison among stressed groups (*p < 0.05 or ^@p < 0.05.) (**P < 0.01 or ^@@P < 0.01), (***P < 0.001 or ^@@@P < 0.001)

Fig. 5

Estradiol (E2) downregulates myocardial mADAM17 via GPER-1 during stress-induced inflammation in FVB female mice and H9c2 cell lines. A, B, C Effect of E2 on the myocardial protein expression of GPER-1 and pPI3K in cardiac tissues of FVB mice under ISO-induced stress (n = 3–4). D, E Western blot analysis displaying the effect of E2 via GPER-1 and its down signaling molecule, pPI3K, on the protein expression of mADAM17 in H9c2 cells during ISO-induced stress (Triplicate per treatment group). F, G Immunofluorescence analysis showing the effect of E2 via GPER-1 on mADAM17’s expression during ISO-induced stress; nuclei (DAPI), membrane (WGA), (n = 25–35 per disk, quadruplicate per treatment group), (scale bar = 50 μm). Two-way ANOVA for in vivo analysis, results are presented as mean ± SEM, *Comparison between control and its stressed group, ^@Comparison among stressed groups. One-way ANOVA for in vitro analysis, results are presented as mean ± SEM, *Comparison between control and ISO groups, ^#Comparison between ISO and E2 groups, ^@Comparison between G15 and G1 groups. (*p < 0.05 or ^# p < 0.05 or ^@p < 0.05.) (**P < 0.01 or ^## P < 0.01 or ^@@P < 0.01), (***P < 0.001 or ^###P < 0.001 or ^@@@P < 0.001). Sham(S), Ovx(O), Ovx + E2(OE), Sham + ISO(SI), Ovx + ISO(OI), Ovx + ISO + E2(OIE)

Fig. 6

Estradiol (E2) downregulates myocardial mADAM17 via the upregulation of the PI3K/AKT/eNOS pathway during stress-induced inflammation in FVB female mice and H9c2 cell lines. A, B Effect of E2 on the protein expression of pAKT in cardiac tissues of FVB mice under ISO-induced stress (n = 3–4). C Effect of E2 via PI3K on the myocardial RNA levels of ADAM17 during ISO-induced stress;(Triplicate per treatment group). D, E Western blot analysis displaying the effect of E2 via PI3K and its down signaling molecule, pAKT on the protein expression of mADAM17 in H9c2 cells under ISO stress condition (Triplicate per treatment group). F ELISA analysis showing the effect of E2 via PI3K on culture media levels of eNOS under ISO stress condition (Triplicate per treatment group). G, H Immunofluorescence analysis displaying the effect of E2 via PI3K on mADAM17’s expression under ISO stress condition; nuclei (DAPI), membrane (WGA), (n = 25–35 per disk, quadruplicate per treatment group), (scale bar = 50 μm). Two-way ANOVA for in vivo analysis, results are presented as mean ± SEM, *Comparison between control and its stressed group, ^@Comparison among stressed groups. One-way ANOVA for in vitro analysis, results are presented as mean ± SEM, *Comparison between ISO and control groups, ^#Comparison between ISO and E2 groups, ^@Comparison between E2 and LY294002 groups (*p < 0.05 or ^# p < 0.05 or ^@p < 0.05.) (**P < 0.01 or ^##P < 0.01 or ^@@P < 0.01), (***P < 0.001 or ^###P < 0.001 or ^@@@P < 0.001). Sham(S), Ovx(O), Ovx + E2(OE), Sham + ISO(SI), Ovx + ISO(OI), Ovx + ISO + E2(OIE)

Fig. 7

Estradiol (E2) via PI3K downregulates mADAM17 through peri-nuclear localization in FVB female mice and H9c2 cell lines. A, B, C Effect of E2 on the protein expression of phosphorylated ERKs and p38MAPKs in cardiac tissues of FVB mice under ISO-induced stress (n = 3–4). D, E Western blot analysis displaying the effect of E2 via PI3K on the protein expressions of β1AR, β2AR, Gαs, Gαi, phosphorylated ERKs and p38MAPKs in H9c2 cells under ISO stress condition (Triplicate per treatment group). F Immunofluorescence analysis displaying the effect of E2 via PI3K on mADAM17’s localization during ISO-induced stress; nuclei (DAPI), membrane (WGA), (Triplicate per treatment group), (scale bar = 50 μm). Two-way ANOVA for in vivo analysis, results are presented as mean ± SEM, *Comparison between control and its stressed group, ^@Comparison among stressed groups. One-way ANOVA for in vitro analysis, results are presented as mean ± SEM, *Comparison between ISO and control groups, ^#Comparison between ISO and E2 groups, ^@Comparison between E2 and LY294002 groups. (*p < 0.05 or ^#p < 0.05 or ^@p < 0.05.) (**P < 0.01 or ^##P < 0.01 or ^@@P < 0.01), (***P < 0.001 or ^###P < 0.001 or ^@@@P < 0.001). Sham(S), Ovx(O), Ovx + E2(OE), Sham + ISO(SI), Ovx + ISO(OI), Ovx + ISO + E2(OIE)

Fig. 8

The downregulation of ADAM17 by estradiol(E2) attenuates ISO-induced cardiac injury and inflammation in H9c2 cell lines. A, B Western blot analysis of ADAM17 siRNA targets in H9c2 cells (Triplicate per treatment group). C, D, E ELISA analysis of ANP, BNP and cTnI levels in the culture media of treated H9c2 cells during ISO-induced stress (quadruplicate per treatment group). F, G Western blot analysis displaying the effect of E2 via ADAM17 on the protein expression of NF-κB (P65) in H9c2 cells under ISO stress condition (Triplicate per treatment group). H, I ELISA analysis of TNFα and IL-1β levels in the culture media of treated H9c2 cells during ISO-induced stress (quadruplicate per treatment group). One-way ANOVA results are presented as mean ± SEM, *Comparison between treated cells and their control groups, ^@Comparison between ISO groups, (*p < 0.05 or ^# p < 0.05 or ^@p < 0.05.) (**P < 0.01 or ^##P < 0.01 or ^@@P < 0.01), (***P < 0.001 or ^###P < 0.001 or ^@@@P < 0.001). Negative control (NC)

Fig. 9

(Graphical abstract). A schematic diagram illustrating the effect of estradiol (E2) deficiency, its supplementation and G1 administration on ADAM17-induced injury and inflammation in the heart during a stressful condition. In a stress state, E2 deficiency enhances Gαi signaling and upregulates phosphorylated kinases (ERKs and p38 MAPKs), subsequently increasing ADAM17’s membrane content (mADAM17), which results in myocardial injury and inflammation. However, the availability of endogenous E2(produced by the ovaries) or exogenous E2 (E2 supplementation) mitigated ADAM17-induced myocardial injury and inflammation through the GPER-1/PI3K/AKT/eNOS pathway by mediating the switch from Gαi to Gαs signaling, hence decreasing phosphorylated kinases (ERKs and p38 MAPKs)