Calcineurin signaling promotes takotsubo syndrome

Abstract

Takotsubo syndrome (TTS) is an acute heart failure syndrome that mimics the symptoms of acute myocardial infarction and is often preceded by emotional and/or physical stress. There is currently no treatment for TTS. Here we show that injection of 2.5 mg kg^-1 of epinephrine (EPI) into mice recapitulates numerous features of human TTS, including increased myocardial damage and mortality in males. Gene set enrichment analysis of myocardial RNA sequencing after EPI injection revealed significant enrichment of calcineurin-dependent pro-inflammatory gene networks, which was more pronounced in male than in female mice, in agreement with observed sex discrepancies in the mouse phenotype. An increase in calcineurin activity was detected in the circulating cells of patients with TTS, suggesting a systemic nature of the syndrome. Preventive and therapeutic treatment of mice injected with EPI using calcineurin inhibitors cyclosporine and tacrolimus improved heart function and reduced myocardial injury. Our findings suggest that calcineurin inhibition could be a potential therapy for TTS.

Fig. 1. EPI-induced reversible AHF in mice.

a, LVEF of mice undergoing isoflurane narcosis 30 min after the administration of NaCl (n = 6), ascending doses of EPI (EPI 2 mg kg⁻¹, n = 6; EPI 2.5 mg kg⁻¹, n = 10; EPI 5 mg kg⁻¹, n = 8) or isoprenaline (ISO) 250 mg kg⁻¹ (n = 8) (*P = 0.01). ns, not significant. b, Seven-day mortality with number of deceased given within bars (NaCl, n = 6; EPI 2 mg kg⁻¹, n = 6; EPI 2.5 mg kg⁻¹, n = 10; EPI 5 mg kg⁻¹, n = 8; ISO 250 mg kg⁻¹, n = 8). c, Model criteria. d, Percentage of surviving mice meeting criteria with number of mice given within bars (NaCl, n = 6; EPI 2 mg kg⁻¹, n = 6; EPI 2.5 mg kg⁻¹, n = 10; EPI 5 mg kg⁻¹, n = 8; ISO 250 mg kg⁻¹, n = 8). e,f, Echocardiography-derived left ventricular stroke volume (**P = 0.004) (e) and left ventricular end-systolic diameter (LVESD) timecourse (*P = 0.04) (NaCl, n = 5; EPI, n = 10) (f). g, Basal (base), midventricular (mid) and apical (apex) radial strain (NaCl, n = 5; EPI, n = 7; EPI base versus apex *P = 0.03, ***P = 0.0004). h, Kinetics of systolic and diastolic blood pressure (n = 3 in each group, *P = 0.03). i, ST-segment elevation and R-wave amplitude changes after EPI (n = 5 in each group). All mice were male and aged 8–10 weeks. Data expressed as mean ± s.e.m., from multiple comparisons adjusted ANOVA, two-sided t-test (h), two-sided paired t-test (15 min–7 d) (e) and one-tailed paired t-test (15 min–2 h) (f). Source data

Fig. 2. Sex-specific outcome and myocardial inflammation in eTTS.

a, Kinetics of LVEF in 10-week-old male (M) versus female (F) mice after EPI (M: n = 10, F: n = 10; 2 h–24 h *P = 0.01). b, Plasma hs-TnT at 24 h in male versus female mice after NaCl or EPI (M NaCl, n = 6; F NaCl, n = 7; M EPI, n = 9; F EPI, n = 10; ****P < 0.0001). c, Kaplan-Maier analysis of survival of male versus female mice after NaCl or EPI (M NaCl, n = 5; F NaCl, n = 5; M EPI, n = 16; F EPI, n = 9; *P = 0.01). d, Plasma corticosterone levels of male versus female mice after NaCl or EPI (M NaCl, n = 5; F NaCl, n = 5; M EPI, n = 8; F EPI, n = 8; ***P = 0.0005). e,f, Left ventricular norepinephrine (e) and EPI (f) 2 h after NaCl or EPI (M NaCl, n = 4; F NaCl, n = 5; M EPI, n = 7; F EPI, n = 8; ****P < 0.0001 and ***P = 0.0002). g, Left ventricular EPI after NaCl or ascending doses of EPI (M NaCl, n = 6; F NaCl, n = 6; M EPI 2.5 mg kg⁻¹, n = 7; F EPI 3.0 mg kg⁻¹, n = 8; F EPI 3.5 mg kg⁻¹, n = 7; ****P < 0.0001). h, Left ventricular norepinephrine after NaCl or ascending doses of EPI (M NaCl, n = 6; F NaCl, n = 6; M EPI 2.5 mg kg⁻¹, n = 8; F EPI 3.0 mg kg⁻¹, n = 6; F EPI 3.5 mg kg⁻¹, n = 9; ****P < 0.0001). i, LVEF of 12-week-old male mice treated with NaCl (M NaCl) or 2.5 mg kg⁻¹ EPI (M EPI) compared to female mice treated with NaCl (F NaCl) or ascending doses of EPI (F EPI) (M NaCl, n = 6; F NaCl, n = 6; M EPI 2.5 mg kg⁻¹, n = 9; F EPI 3.0 mg kg⁻¹, n = 7; F EPI 3.5 mg kg⁻¹, n = 9; ****P < 0.0001). Data expressed as mean ± s.e.m., from multiple comparisons adjusted ANOVA or two-sided paired t-test (a). Source data

Fig. 3. Sex-specific calcineurin-driven inflammation.

a–c, GSEA from RNA sequencing performed in left ventricular tissue of 10-week-old mice treated with NaCl and EPI. a, log₂-fold change of top-ranked upregulated (red) and downregulated (blue) genes of male mice treated with NaCl (n = 5) vs. EPI (n = 7) 2 h after the injection. b, Normalized enrichment score (NES) and false discovery rate (FDR (numbers next to bars)) of top-ranked upregulated (red) and downregulated (blue) enriched gene set ontology (GO) biological pathways of male mice treated with NaCl (n = 5) versus EPI (n = 7). c, NES and FDR of top-ranked upregulated (red) and downregulated (blue) enriched GO biological pathways of female (n = 7) versus male (n = 7) mice 2 h after EPI. d, Enrichment map illustrating discrepantly regulated overlapping myocardial pathways, upregulated in male mice treated with EPI and downregulated by the calcineurin inhibitor CsA, based on the Tanlab drug signature database gene set Cyclosporin_HL60_DOWN (P = 0.0174). e, Immunoblotting of calcineurin (Cn), serine 411-phospho-Cn (p-Cn(Ser411)), RCAN1 and RCAN1-4, as well as GAPDH from left ventricular tissue 2 h after NaCl or EPI in male versus female mice. f,g, Immunoblotting integrated density (IDT) protein quantification of RCAN1/GAPDH (f) and RCAN1-4/GAPDH (g) (n = 3 in each group, *P = 0.021, ****P < 0.0001). Data expressed as mean ± s.e.m., from multiple comparisons adjusted ANOVA or two-sided Mann–Whitney U-test (d). Source data

Fig. 4. A new anti-inflammatory treatment strategy for TTS.

a–c, Kinetics of LVEF 30 min after NaCl or EPI (NaCl, n = 3; CsA + NaCl, n = 3; EPI, n = 6; CsA + EPI, n = 6; *P = 0.049 (30 min–8 h)) (a), plasma hs-TnT levels at 24 h (NaCl, n = 3; CsA, n = 3; EPI, n = 3; CsA + EPI, n = 5; **P = 0.0024 and *P = 0.012) (b) and survival at 24 h (NaCl, n = 3; CsA, n = 3; EPI, n = 6; CsA + EPI, n = 6; *P = 0.024) (c) in 8-week-old male C57BL6/N mice, pretreated with a single dose of 10 mg kg⁻¹ CsA 30 min before NaCl or EPI. d, Left ventricular immunoblotting of NF-κB p65 and its phosphorylation at Ser536 (p-NF-κB) at 8 h (n = 3 in each group). e, Relative quantification of left ventricular immunoblotting of NF-κB p65 and its phosphorylation at Ser536 (p-NF-κB) at 8 h (n = 3 in each group; *P = 0.014, **P = 0.001). f–h, mRNA per Gadph expression of left ventricular Ccl2 (NaCl, n = 3; EPI, n = 3; CsA, n = 3; CsA + EPI, n = 5; both *P = 0.01) (f), left ventricular Il-1β (NaCl, n = 3; EPI, n = 3; CsA, n = 3; CsA + EPI, n = 6; NaCl versus EPI *P = 0.01, EPI versus CsA + EPI *P = 0.02) (g) and left ventricular Nr4a3 (NaCl, n = 3; EPI, n = 3; CsA, n = 3; CsA + EPI, n = 5; *P = 0.011 and **P = 0.002) (h) at 8 h after single preventive CsA treatment. i–k, Kinetics of LVEF (M EPI, n = 7; M EPI + CsA, n = 7; F EPI + CsA, n = 7; F EPI, n = 6; M EPI versus M EPI + CsA *P = 0.013 and F EPI versus F EPI + CsA *P = 0.023) (i), hs-TnT (M NaCl, n = 5; M CsA, n = 6; M EPI, n = 8; M EPI + CsA, n = 8; F EPI + CsA, n = 8; F NaCl, n = 6; F CsA, n = 6; F EPI, n = 6; *P = 0.013, **P = 0.0048, ****P < 0.0001) (j) and Rcan1-4 mRNA (M NaCl, n = 5; F EPI + CsA, n = 5; M CsA, n = 6; F NaCl, n = 6; F CsA, n = 6; F EPI, n = 6; M EPI, n = 8; M EPI + CsA, n = 8; *P = 0.024, ****P < 0.0001) (k) 8 h after NaCl and/or EPI with or without 30 mg kg⁻¹ CsA at 2 h in 12-week-old male (2.5 mg kg⁻¹ EPI) and female (3.5 mg kg⁻¹ EPI) mice. l–n, Kinetics of LVEF (n = 7 in each group, **P = 0.0019) (l), hs-TnT (EPI, n = 9; EPI + FK506, n = 7; *P = 0.0227) (m) and Rcan1-4 mRNA (EPI, n = 7; EPI + FK506, n = 6; *P = 0.0193) (n) at 8 h after EPI with or without 10 mg kg⁻¹ FK506 at 2 h in 12-week-old male mice. Data expressed as mean ± s.e.m., from multiple comparisons adjusted ANOVA (b–d, i–k), two-sided paired t-test (a,h), Student’s t-test (l–n) or log-rank test (c). Source data

Fig. 5. Calcineurin signaling in human TTS.

a–c, In PBMCs from age- and sex-matched healthy controls (Ctrl) versus patients with TTS, the expression of Rcan1-4 (Ctrl, n = 4; TTS, n = 5; *P = 0.031) (a), Il-1β (n = 5 in each group; *P = 0.015) (b) and Nr4a3 (n = 5 in each group; **P = 0.009) (c) mRNA was significantly upregulated. Data expressed as mean ± s.e.m., from two-sided Mann–Whitney U-test (a,c) or Student’s t-test (b). Source data

Fig. 6. EPI injection recapitulates takotsubo syndrome in mice.

We observed impaired outcomes in male compared to female mice, including mortality (†), reduction of LVEF and plasma TnT, with marked myocardial calcineurin activation and inflammation. Apical contractility was particularly blunted after EPI with elevated endsystolic left ventricular diameter, mimicking apical ballooning (dashed lines and arrows). Calcineurin inhibition by CsA or FK506 reduces the takotsubo phenotype in male and female mice. α/β-AR, α- or β-adrenergic receptor

Extended Data Fig. 1. Epinephrine-induced reversible acute heart failure in mice.

(A) Heart rate (beats per minute (BPM)) 30 min. upon NaCl 0.9% (NaCl), ascending doses of epinephrine (EPI) (2, 2.5, and 5 mg/kg), or isoprenaline (ISO) (250 mg/kg) from mice undergoing isoflurane narcosis (n = NaCl, EPI 5 mg/kg, ISO 250 mg/kg 8/group, EPI 2 mg/kg 6, EPI 2.5 mg/kg 10; *p = 0.0205, **p = 0.0045) and (B) time course of heart rate after 2.5 mg/kg EPI or NaCl (n = NaCl 5, EPI 10; *p = 0.045). (C) Representative ECG images before (Baseline) and 30 minutes (30 min.) after EPI. (D) Left ventricular enddiastolic diameter (LVEDD) kinetics after 2.5 mg/kg EPI (n = NaCl 5, EPI 10; at 15–30 min *p = 0.0224). (E) Impaired basal (base), midventricular (mid), and apical (apex) longitudinal strain upon EPI vs. NaCl in male mice at 30 min. (n = NaCl 5, EPI 10; ****p < 0.0001). (F) Left ventricular tissue (LV) catecholamine (dopamine, norepinephrine, epinephrine) kinetics in male mice after EPI. (G) Plasma catecholamine kinetics upon EPI. All mice were male (8–10w). Data as mean ± s.e.m. Multiple comparisons adjusted ANOVA (A, E) or two-sided T-test (B, D). Source data

Extended Data Fig. 2. Gender- and age discrepancies in epinephrine-induced heart failure.

(A) Left ventricular ejection fraction (EF%) kinetics over 7d from 10w old male (M) and female mice (F), after NaCl 0.9% (NaCl) or epinephrine (EPI) injection (Inj.) under isoflurane narcosis (n = M/NaCl, F/NaCl 6/group, M/EPI 10, F/EPI 9). (B) Lung weight per tibia length (n = M/NaCl, F/NaCl 5/group, M/EPI, F/EPI 8/group; **p = 0.003) and (C) LV nppb/gapdh mRNA expression 2 h after NaCl or EPI treatment in M vs. F mice (n = M/NaCl 5, M/EPI, F/EPI 7/group, F/NaCl 3; ***p = 0.0001). (D) Sex comparison of LV dopamine at 2 h (n = M/NaCl 4, M/EPI 7, F/NaCl 5, F/EPI 8; **p = 0.0026, ***p = 0.0005). Data as mean ± SEM. Multiple comparisons adjusted ANOVA (B–D). Source data

Extended Data Fig. 3. Epinephrine-induced heart failure promotes pro-inflammatory myocardial gene expression networks.

(A, B) Gene set enrichment analysis (GSEA) from RNA-sequencing (n = M/NaCl 5, F/NaCl 3, M/EPI, F/EPI 7/group) was conducted in NaCl- vs. epinephrine (EPI)-treated 10w old female as well as in (C, D) EPI-treated female vs. male mice from left ventricular tissue (LV). (A) Log2-fold change of top ranked up- (red) and downregulated (blue) genes of NaCl vs. EPI-treated females, as well as of (D) EPI-treated females vs. males 2 h after insult. (B) Normalized enrichment score (NES) and – next to bars – false discovery rate (FDR) of top up- (red) and downregulated (blue) enriched gene set ontology (GO) biological pathways of NaCl vs. EPI-treated female mice 2 h after insult. (C) Intersection network of GSEA enrichment map depicting significant positive (red), and negative (blue) enriched GO biological pathways of female vs. male mice after EPI. Each node depicts one GO biological pathway gene set with connecting line thickness accounting for the number of common genes per pathway. (E) Top ten drug targets with significant myocardial gene expression overlap of NaCl vs. epinephrine (EPI)-treated male mice from the Tanlab drug signature database (*p = 0.0003). (F) Quantification of regulator of calcineurin 1 (rcan1) mRNA from 12w old male and female mice after NaCl vs. EPI (n = M/NaCl, F/NaCl, F/EPI 4/group, M/EPI 5; ****p < 0.0001). Data as mean ± s.e.m. Two-sided Mann-Whitney test (E) and multiple comparisons adjusted ANOVA (F). Source data

Extended Data Fig. 4. Calcineurin inhibition improves heart failure and myocardial damage.

(A) Ejection fraction (EF) kinetics (n = NaCl, CsA100 3/group, EPI, CsA30 + EPI, CsA100 + EPI 6/group; *p = 0.036) and (B) EF at 30 min. (n = NaCl, CsA100 3/group, EPI, CsA100+, CsA30+, CsA10 + 6/group) after epinephrine (EPI) or NaCl0.9% (NaCl) in 8w old male mice pretreated with a single dose of 10- (CsA10+), 30- (CsA30+), or 100 mg (CsA100+)/kg body weight CsA 30 min before (*p = 0.0035). (C) Plasma high-sensitive Troponin T (hs-Troponin T) at 24 h (n = NaCl, EPI, CsA100 3/group, CsA100 + 6, CsA30+, CsA10 + 5/group; **p < 0.004, ***p = 0.0006). (D) Left ventricular tissue (LV) regulator of calcineurin 1-4 (RCAN1-4) mRNA expression (n = NaCl, EPI, CsA100 3/group, CsA30 + 6, CsA100+, CsA10 + 5/group; *p = 0.042) as well as (E) immunoblotting (IB) at 8 h. (F) LV nuclear receptor subfamily 4 group a member 1 (nr4a1) mRNA expression 8 h after EPI, NaCl, and CsA (n = NaCl, EPI, CsA100 3/group, CsA100 + 5, CsA30+, CsA10 + 6/group; p = 0.308). (G) LV RCAN1-4 IB of 8w old male mice after pretreatment with 10 mg/kg CsA 30 min. before NaCl or EPI from a separate experiment. (H) EF kinetics (n = 15/group; *p = 0.011, **p = 0.0079 at 2h-3d), (I) hs-Troponin T (n = EPI 15, CsA both 13/group; *p = 0.0128, **p = 0.0058), and (J) Kaplan Maier analysis (n = 15/group) of 12w old male mice with 10 mg/kg CsA 2 h before (preventive) or 30 min. after EPI (therapeutic) and subsequent CsA application twice per day (p = 0.221). (K) EF kinetics (n = 8w/NaCl 4, 8w/EPI, 12w/EPI 9/group, 12w/NaCl 6; *p = 0.027), (L) Radial strain from 12w old M C57BL6n mice 30 min after NaCl vs. EPI (n = NaCl 5, EPI 8; **p = 0.0013) (M) hs-Troponin T (n = 8w/NaCl 4, 8w/EPI, 12w/EPI 6/group, 12w/NaCl 5) and (N) Kaplan Maier analysis of M mice at 8- (8w) or 12 weeks of age (12w) with NaCl or EPI (n = NaCl 5, EPI 8; *p = 0.0167). (O) Corresponding western blotting of regulator of calcineurin 1-4 (RCAN1-4) and (P) quantification (IDT) per GAPDH (n = 3/group, **p = 0.0023, ****p < 0.0001). Data as mean ± SEM. Multiple comparisons adjusted ANOVA (B-D, F, I, L-M, P), two-sided paired T-Test (A, H, K), or Log-rank test (J, N). Source data