Estradiol-mediated small GTP-binding protein GDP dissociation stimulator induction contributes to sex differences in resilience to ferroptosis in takotsubo syndrome

Abstract

Background: Declining beneficial cardiovascular actions of estradiol (E2) have been associated with disproportionate susceptibility to takotsubo syndrome (TTS) in postmenopausal women. However, the underlying mechanisms between E2 and this marked disproportion remain unclear. SmgGDS (small GTP-binding protein GDP dissociation stimulator), as a key modulator of cardiovascular disease, plays protective roles in reducing oxidative stress and exerts pleiotropic effects of statins. Whether SmgGDS levels are influenced by E2 status and the effect of SmgGDS on sex differences in TTS are poorly understood.

Methods: Clinical data were reviewed from TTS inpatients. Echocardiography, immunofluorescence, and immunohistochemistry were performed together with expression analysis to uncover phenotypic and mechanism changes in sex differences in TTS-like wild-type (WT) and SmgGDS^± mice. HL-1 cardiomyocytes were used to further examine and validate molecular mechanisms.

Results: In 14 TTS inpatients, TTS had a higher incidence in postmenopausal women as compared to premenopausal women and men. In murine TTS, female WT mice exhibited higher cardiac SmgGDS levels than male WT mice. Ovariectomy reduced SmgGDS expression in female WT mice similar to that observed in male mice, whereas E2 replacement in these ovariectomized (OVX) female mice reversed this effect. The physiological importance of this sex-specific E2-mediated SmgGDS response is underscored by the disparity in cardiac adaptation to isoproterenol (ISO) stimulation between both sexes of WT mice. E2-mediated SmgGDS induction conferred female protection against TTS-like acute cardiac injury involving ferritinophagy-mediated ferroptosis. No such cardioprotection was observed in male WT mice and OVX female. A causal role for SmgGDS in this sex-specific cardioprotective adaptation was indicated, inasmuch as SmgGDS deficiency abolished E2-modulated cardioprotection against ferritinophagy and aggravates TTS progression in both sexes. Consistently, knockdown of SmgGDS in HL-1 cardiomyocytes exacerbated ferroptosis in a ferritinophagy-dependent manner and abrogated the protective role of E2 against ferritinophagy. Mechanistically, our findings revealed that SmgGDS regulated E2-dependent cardioprotective effects via AMPK/mTOR signaling pathway. SmgGDS deficiency abolished E2-conferred protection against ferritinophagy through activating AMPK/mTOR pathway, while treatment with recombinant SmgGDS in HL-1 cells significantly mitigated this pathway-associated ferritinophagy activity.

Conclusions: These results demonstrate that SmgGDS is a central mediator of E2-conferred female cardioprotection against ferritinophagy-mediated ferroptosis in TTS.

Keywords: Ferritinophagy; Ferroptosis; Hormones; Sex; Takotsubo cardiomyopathy.

Graphical abstract

Fig. 1

SmgGDS expression is mediated by sex-related E2 in TTS. A, Immunofluorescence staining of SmgGDS in both male and female hearts injected with saline (mControl) or isoproterenol (ISO) (mTTS) for 2 h, and in HL-1 cardiomyocytes challenged by PBS (cPBS) or ISO (cTTS) for 2 h. B, Western blot analysis of SmgGDS protein in the hearts of both sexes injected with saline or ISO for 2 h. Males, n = 10; Females, n = 8. C, The mRNA levels of SmgGDS in cardiac tissues of male and female mice. Males, n = 5; Females, n = 5. D, Western blot analysis of SmgGDS protein in the hearts of sham-operated and ovariectomized (OVX) female mice treated with saline or ISO for 2 h n = 5, each. E, Western blot analysis of SmgGDS expression in the hearts of saline or ISO-injected OVX female mice pretreated with or without E2. n = 5, each. F, Representative images of hematoxylin-eosin staining in the heart tissues of placebo-pretreated and 17β-estradiol-pretreated OVX female mice following saline or ISO injection for 2 h. Scale bars, 20 μm ##P < 0.01, ###P < 0.001; *P < 0.05, **P < 0.01, ***P < 0.001 comparing saline vs ISO treatment.

Fig. 2

SmgGDS knockdown exacerbates TTS in female mice. A, Representative echocardiographic images from female WT and SmgGDS^± mice following saline or ISO injection. B, Echocardiographic parameters including left ventricle ejection fraction% and fraction shortening% in female WT and SmgGDS^± mice after saline or ISO injection. Saline, WT, n = 6; Saline, SmgGDS^+/−, n = 5; ISO, WT, n = 6; ISO, SmgGDS^+/−, n = 5. C, The 2-h survival curve in female WT and SmgGDS^± mice injected with saline or ISO. n = 17, each. D, Representative images of hematoxylin-eosin staining (upper), and immunohistochemistry staining for CD45 (middle) and monocyte chemoattractant protein 1 (MCP-1) (lower) in the hearts of female WT and SmgGDS^± mice injected with saline or ISO for 2 h, and CD45 and MCP-1 levels in each group was quantified. n = 4, each. Scale bars, 100 μm and 50 μm in hematoxylin-eosin staining, and 20 μm in CD45 and MCP-1 immunohistochemistry. E, The mRNA levels of TNF-α and IL-6 in myocardial tissues of each group. n = 4, each. ###P < 0.001; *P < 0.05, ***P < 0.001 comparing saline vs ISO treatment.

Fig. 3

SmgGDS knockdown exacerbates ferritinophagy-mediated ferroptosis in female mice. A, The mRNA level of PTGS2 in cardiac tissues of each group. n = 4, each. B, Representative images of immunohistochemistry staining for PTGS2 and FTH1 protein in cardiac tissues of each group and quantification. Scale bars, 20 μm. C, Representative images of Prussian blue staining for iron deposition in the hearts of each group and quantification. n = 4, each. Scale bars, 50 μm. D, The cardiac MDA level in female WT and SmgGDS^± mice after 2 h of saline or ISO injection. n = 3, each. E, Western blot analysis of PTGS2, P62, SLC7A11, FTH1, GPX4 and LC3B-II/LC3B–I expression in female WT and SmgGDS^± mice after saline or ISO treatmen. n = 4, each. ##P < 0.01, ###P < 0.001; *P < 0.05, **P < 0.01, ***P < 0.001 comparing saline vs ISO treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

SmgGDS knockdown abolishes the anti-ferritinophagy effect of E2 in TTS. A, Representative echocardiographic images from OVX female WT mice pretreated with or without E2 following acute saline or ISO injection. B, Echocardiographic parameters including left ventricle ejection fraction% and fraction shortening% in OVX WT mice pretreated with or without E2 following saline or ISO injection. Saline, OVX, n = 4; Saline, OVX + E2, n = 4; ISO, OVX, n = 4; ISO, OVX + E2, n = 5. C, Representative echocardiographic images from E2-pretreated OVX female WT and SmgGDS^± mice following ISO administration. D, Echocardiographic parameters including left ventricle ejection fraction% and fraction shortening% in E2-pretreated OVX WT and SmgGDS^± mice after ISO injection. n = 5, each. E, The survival curve in OVX WT mice pretreated with or without E2 following ISO injection and SmgGDS^± mice following ISO injection. n = 10, each. F, Representative images of hematoxylin-eosin staining in the hearts of E2-treated OVX WT and SmgGDS^± mice after injected with ISO. Scale bars, 20 μm. G, Western blot analysis of PTGS2, P62, SLC7A11, FTH1, GPX4 and LC3B-II/LC3B–I protein in hearts of sham-operated and OVX female mice treated with saline or ISO. n = 4, each. H, Western blot analysis of P62, FTH1 and LC3B-II/LC3B–I expression in the hearts of E2-pretreated OVX WT and SmgGDS^± mice after ISO treatment. n = 5, each. ##P < 0.01, ###P < 0.001; *P < 0.05, **P < 0.01, ***P < 0.001 comparing saline vs ISO treatment.

Fig. 5

SmgGDS modulates E2-mediated anti-ferritinophagy in ISO-induced HL-1 cardiomyocytes. A, MDA in sh-NC and sh-SmgGDS HL-1 cardiomyocytes treated with PBS or ISO (10 mM) for 2 h n = 3, each. B, Western blot analysis of P62, SLC7A11, FTH1, GPX4 and LC3B-II/LC3B–I in sh-NC and sh-SmgGDS HL-1 cardiomyocytes after treatment with PBS or ISO (10 mM) for 2 h n = 3, each. C, Representative images of FerroOrange staining for intracellular Fe²⁺ in HL-1 cardiomyocytes of each group and quantification. n = 4, each. D, Representative images of intracellular lipid ROS by BODIPY probe in PBS or ISO-challenged sh-NC and sh-SmgGDS HL-1 cardiomyocytes pretreated with or without Fer-1 (10 μM) or 3-MA (5 μM); the fluorescence intensity in each group was quantified by ImageJ. n = 4, each. E, Representative images of DCFH-DA staining in PBS or ISO-challenged HL-1 cardiomyocytes pretreated with or without glutathione S-transferase (GST)-tagged recombinant SmgGDS protein (GST-SmgGDS, 1 μg/mL); the fluorescence intensity in each group was quantified by ImageJ. n = 4, each. F, Western blot analysis of PTGS2, SLC7A11, GPX4, FTH1, P62 and LC3B-II/LC3B–I expression in HL-1 cardiomyocytes pretreated with or without GST-SmgGDS (1 μg/mL) for 24 h followed by ISO stimulation. n = 3, each. G, Western blot analysis of SmgGDS, PTGS2, P62, SLC7A11, FTH1, GPX4 and LC3B-II/LC3B–I expression in ISO-challenged sh-NC and sh-SmgGDS HL-1 cardiomyocytes pretreated with or without E2. n = 4, each. ns, P ≥ 0.05; #P < 0.05, ##P < 0.01, ###P < 0.001; *P < 0.05, **P < 0.01, ***P < 0.001 comparing PBS vs ISO treatment.

Fig. 5

SmgGDS modulates E2-mediated anti-ferritinophagy in ISO-induced HL-1 cardiomyocytes. A, MDA in sh-NC and sh-SmgGDS HL-1 cardiomyocytes treated with PBS or ISO (10 mM) for 2 h n = 3, each. B, Western blot analysis of P62, SLC7A11, FTH1, GPX4 and LC3B-II/LC3B–I in sh-NC and sh-SmgGDS HL-1 cardiomyocytes after treatment with PBS or ISO (10 mM) for 2 h n = 3, each. C, Representative images of FerroOrange staining for intracellular Fe²⁺ in HL-1 cardiomyocytes of each group and quantification. n = 4, each. D, Representative images of intracellular lipid ROS by BODIPY probe in PBS or ISO-challenged sh-NC and sh-SmgGDS HL-1 cardiomyocytes pretreated with or without Fer-1 (10 μM) or 3-MA (5 μM); the fluorescence intensity in each group was quantified by ImageJ. n = 4, each. E, Representative images of DCFH-DA staining in PBS or ISO-challenged HL-1 cardiomyocytes pretreated with or without glutathione S-transferase (GST)-tagged recombinant SmgGDS protein (GST-SmgGDS, 1 μg/mL); the fluorescence intensity in each group was quantified by ImageJ. n = 4, each. F, Western blot analysis of PTGS2, SLC7A11, GPX4, FTH1, P62 and LC3B-II/LC3B–I expression in HL-1 cardiomyocytes pretreated with or without GST-SmgGDS (1 μg/mL) for 24 h followed by ISO stimulation. n = 3, each. G, Western blot analysis of SmgGDS, PTGS2, P62, SLC7A11, FTH1, GPX4 and LC3B-II/LC3B–I expression in ISO-challenged sh-NC and sh-SmgGDS HL-1 cardiomyocytes pretreated with or without E2. n = 4, each. ns, P ≥ 0.05; #P < 0.05, ##P < 0.01, ###P < 0.001; *P < 0.05, **P < 0.01, ***P < 0.001 comparing PBS vs ISO treatment.

Fig. 6

SmgGDS regulates E2-mediated anti-ferroptosis through AMPK/mTOR pathway. A, Western blot analysis of p-mTOR, mTOR, p-AMPK, and AMPK expression in WT and SmgGDS^± mice treated with saline or ISO for 2 h n = 3, each. B, Western blot analysis of p-mTOR, mTOR, p-AMPK, and AMPK in sh-NC and sh-SmgGDS HL-1 cardiomyocytes challenged by PBS or ISO for 2 h. C, Western blot analysis of p-mTOR, mTOR, p-AMPK, and AMPK in HL-1 cardiomyocytes pretreated with or without GST-SmgGDS (1 μg/mL) for 24 h followed by ISO stimulation. n = 3, each. D, Western blot analysis of p-mTOR, mTOR, p-AMPK, and AMPK in ISO-challenged sh-NC and sh-SmgGDS HL-1 cardiomyocytes pretreated with or without E2. n = 3, each. E, Western blot analysis of P62, FTH1, and LC3B-II/LC3B–I in ISO-challenged sh-NC and sh-SmgGDS HL-1 cardiomyocytes co-treated with or without MHY1485. n = 3, each. ns, P ≥ 0.05; #P < 0.05, ##P < 0.01, ###P < 0.001; *P < 0.05, **P < 0.01, ***P < 0.001 vs without ISO treatment.