Background: Takotsubo syndrome (TTS) mimics the symptoms of acute myocardial infarction in the absence of coronary obstruction, is mechanistically not fully understood and lacks causative treatment options. Here, we characterize a new mouse model of epinephrine (EPI)-induced TTS and use gene set enrichment network analysis and a myocardial KO of the endogenous calcineurin inhibitor calcium-calmodulin-dependent kinase II (CaMKII) to identify calcineurin inhibition with cyclosporine A (CsA) as a novel potential therapy. 

Methods and results: Compared to NaCl high-dose EPI caused reversible left ventricular ejection fraction (EF) impairment (28 vs. 68%, p<0.0001, n=6-8, ANOVA), ECG changes and plasma high-sensitive Troponin T (hs-TnT) elevation (226.7 vs. 3753pg/ml, p<0.001, n=6-8, ANOVA) in C57BL6N mice. Compared to female mice, males suffer from an exacerbated phenotype including significant mortality (p<0.05, n=9-16/group, Log-rank test), mimicking gender-discrepant severity of the clinical syndrome. Myocardial gene set enrichment analysis revealed significant enrichment of calcineurin-dependent pro-inflammatory gene networks after EPI, confirmed by an increase of regulator of calcineurin 1.4 (RCAN1.4) protein (12x vs. NaCl, p<0.001, n=6-8, ANOVA). Cardiac-specific deletion of CaMKII yielded exacerbated AHF and increased myocardial damage after EPI (mean hs-TnT 1110pg/ml vs 3540pg/ml, n=5-10, p<0.05, ANOVA), that was attenuated by CsA prophylaxis (2143 vs. 3540pg/ml, p<0.001, n=6-8, ANOVA). To assess the clinical potential of our findings, C57BL6N male and female mice were subjected to experimental TTS or NaCl and additionally treated by CsA, FK506 (TAC) or NaCl 2h later with subsequent functional and molecular characterisation. Mice treated with CsA two hours after EPI showed significantly less EF impairment (males: at CsA injection mean EF 17.32 vs. 17.18% and 6h after CsA 35.14 vs 20.44%, p<0.05; females: at CsA injection mean EF 22.44 vs. 23.36%, 6h after CsA 36.14 vs 20.38%, p<0.05, n=6, ANOVA, Fig. 1) and lower hs-TnT (males: 3632 vs. 7746 pg/ml, p<0.01, ANOVA; females: 2129 vs 2881 pg/ml, Fig. 2). CsA blunted LV RCAN1.4 and rcan1.4 mRNA in both genders (males: p<0.0001, females: p<0.01, n=6, ANOVA, Fig. 5). Flow cytometry 24h after insult showed inhibition of LV neutrophil and natural killer cell influx by CsA after EPI (p<0.05, n=4-6, ANOVA, Fig. 6). To rule out off-target effects of CsA on mitochondrial permeability transition pore opening, we also treated mice with FK506 2h after EPI or NaCl with comparable beneficial effects, including improved mean EF (at TAC injection: 23.43 vs. 21.48%, 3h after TAC 39.33 vs. 27.66%, p<0.001, n=9, ANOVA, Fig. 3) and reduced mean hs-TnT (2894 vs. 4752 pg/ml, p<0.05, n=9, T-test, Fig. 4), complemented by significantly blunted rcan1.4 mRNA (0.6x, p<0.05, n=9, T-test, Fig. 7) after EPI. The increased expression of key inflammatory and calcineurin target genes including rcan1.4 (6x, p<0.05, n=5-6 by T-test) was confirmed in human peripheral blood mononuclear cells from TTS patients and gender- and age-matched controls.

Conclusion: Pharmacological calcineurin inhibition may present the first pathogenetically-oriented anti-inflammatory treatment strategy for TTS. Our data here are the foundation for planning of a randomized clinical trial investigating the therapeutic impact of CsA on myocardial damage in human TTS.