Calcineurin signaling promotes Takotsubo cardiomyopathy

doi:10.21203/rs.3.rs-1601276/v1

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Calcineurin signaling promotes Takotsubo cardiomyopathy

https://doi.org/10.21203/rs.3.rs-1601276/v1

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published 13 Jul, 2023

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Aims: Takotsubo cardiomyopathy (TTC) presents an acute heart failure (AHF) syndrome that mimics the symptoms of acute myocardial infarction and is often preceded by an episode of emotional and/or physical stress. There is no specific treatment available, and evidence is based mostly on retrospective data. Here, we show that a high-dose of epinephrine (EPI) recapitulates numerous features of human TTC in mice, enabling genetic KO studies. By gene set enrichment network analysis and studies involving myocardial KO of the endogenous calcineurin inhibitor calcium-calmodulin-dependent kinase II (CaMKII), we identify cyclosporine A (CSA) as a novel potential therapy for TTC.

Methods and results: high-dose EPI caused reversible AHF with significant ST-segment changes and high-sensitive Troponin T (hs-TnT) elevation. Male mice displayed markedly elevated hs-TnT and mortality when compared to females, mimicking the clinical syndrome. Left ventricular (LV) catecholamines revealed a distinct pattern of blunted norepinephrine as opposed to elevated EPI in male vs. female mice. Gene set enrichment analysis of LV RNA-sequencing after EPI revealed calcineurin-dependent pro-inflammatory gene networks to be significantly enriched, particularly in male hearts. Cardiac-specific deletion of CaMKII caused exacerbated AHF and myocardial damage, that was attenuated by CSA. In human peripheral blood mononuclear cells from TTC patients, expression of the calcineurin reporter regulator of calcineurin 1 isoform 4 (rcan1-4) was significantly increased compared to age- and gender matched controls, indicating human relevance of this pathway.

Conclusion: Combining TTC disease modeling and mouse genetics identified calcineurin inhibition as a novel specific therapeutic concept to treat human TTC.

Takotsubo

stress

calcineurin

heart failure

epinephrine

gender

Takotsubo cardiomyopathy (TTC) presents an acute heart failure syndrome that mimics the symptoms of acute myocardial infarction and is often preceded by an episode of severe emotional or physical stress¹. The name “Takotsubo” stems from the first official description in which the syndrome was labeled after the ballooned apical shape of the affected left ventricle, resembling a Japanese octopus trap (“Takotsubo”)². Even though several acute complications of TTC such as arrhythmias or cardiogenic shock can be life threatening, left ventricular ejection fraction (EF) mostly recovers in survivors. Nevertheless, affected patients reveal an impaired long-term prognosis³. In 90% of cases, postmenopausal women are affected⁴ with substantially lower morbidity and mortality compared to male patients⁵. Since catecholamine storm⁶, triggered by central autonomous sympathetic nervous system (SNS) activation^{7, 8}, appears to play a pivotal role in the pathophysiology of TTC, treatment of cardiogenic shock poses a particularly difficult clinical situation. The drug most associated with accidental induction of TTC in humans is the endogenous α- and β-adrenoceptor agonist epinephrine (EPI)^{9, 10}. Experimentally, high doses of catecholamines induce transient acute heart failure (AHF) in rats¹¹. Mechanistically, a β₂-adrenoceptor-dependant switch of coupling from Gα_s- to inhibitory Gα_i-protein¹², myocardial lipid accumulation ¹³, energetic deficit ¹⁴, as well as systemic and myocardial inflammation¹⁵ have been suggested as potential molecular causes of TTC. As betablocker therapy has not proven beneficial⁴ the lack of a specific treatment strategy highlights the importance of mechanistic studies as a prerequisite for tailored therapies. Catecholamine stimulation of cardiomyocytes activates both the protein phosphatase calcineurin (Cn) and its endogenous inhibitor the Ca²⁺/calmodulin-activated kinase II (CaMKII)^{16, 17}. Cn activation has been shown to contribute primarily to cardiac hypertrophy¹⁸ but also to inflammation and heart failure by activation of the nuclear factor of activated T-cells (NFAT)¹⁹. Pharmacologic calcineurin inhibition by cyclosporine A (CSA) is used e. g. after heart transplantation to suppress organ rejection but has not been investigated as an anti-inflammatory approach to combat heart disease.

Experimental animals. Animal experiments were performed according to institutional and governmental guidelines. The study conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985) and was approved by the authorities of the Regierungspräsidium Karlsruhe, Germany (G-1/16, G-25/17, G-143/17, G-149/18, and G-95/18). Every effort was made to minimize the number of animals used, and their suffering. Animals were housed with access to food and water ad libitum in a 12h day-and-night-rhythm at 21 °C and 50-60% humidity. For all experiments male or female mice with a C57/bl6n background were used. For some experiments commercially available mice were obtained from Janvier Labs, France. For knockout experiments a tamoxifen-inducible, cardiomyocyte-specific CaMKIIγδ double knockout mouse line was used. Mice were generated and KO induced as previously described²⁰. In brief cardiomyocyte-specific deletion CaMKIIδ and CaMKIIγ isoforms was achieved by crossing conditional knockout mice to transgenic mice expressing the mER-Cre-mER fusion protein under the control of a cardiomyocyte-specific α-myosin heavy chain (MHC) – promoter. For KO induction 10 mg/ml tamoxifen (Sigma Aldrich) was dissolved in sunflower oil solution containing 6% ethanol, with gavage administration for 10 consecutive days once per day (80 mg/kg/d). Knock-out was confirmed by Western blot. Experiments were conducted at least 3 weeks after the last tamoxifen application to avoid interference of Cre-induced cardiomyopathy. Littermates, devoid of the conditional CaMKIIδ and CaMKIIγ alleles carrying the αMHC-Cre transgene, were used as controls, and received tamoxifen as described above. In experiments with the calcineurin inhibitor cyclosporine A (CSA), mice were intraperitoneally injected with 10-, 30-, or 100mg/kg CSA or NaCl 0.9% in 200µL before, or 30min. after EPI injection.

Echocardiography. Cardiac function was evaluated by 2D echocardiography at baseline, and as indicated in the corresponding experiments after NaCl or EPI injection under isoflurane volatile mask narcosis (1-3%vol) at a constant temperature of 38°C (Fig. 1) or in awake mice (Fig. 2, 4, and 5) using a Visual Sonics Vevo® 2100 with a MX550D transducer by an experienced investigator blinded regarding the animal’s group affiliation. Mice were shaved and left ventricular parasternal short-axis views were obtained in M-mode imaging at the papillary muscle level as well as parasternal long-axis views. A cut-off of > 400 bpm was used to avoid the confounding effects of narcosis and bradycardia on cardiac function²¹. Awake mice were trained to avoid stress in the first instance. Four consecutive beats were used for measurements of left ventricular end-diastolic internal diameter (LVEDD), left ventricular end-systolic internal diameter (LVESD) and left ventricular ejection fraction (EF). Moreover, left ventricular parasternal long-axis (PSLAX) views were obtained for accurate quantification of EF. Additional LV strain analysis (Fig. 1K, 1L) was conducted in PSLAX with images acquired at > 200 frames/s using Visual Sonics Vevo® Strain Analysis.

ECG-telemetry recordings. In a subset of male C57bl/6n mice we performed telemetry recording of heart rate and blood pressure. After analgesia with 0.1 mg/kg s.c. of buprenorphine 1h before intervention, isoflurane narcosis (3%vol) was induced with subsequent subcutaneous implantation of transmitters according to the manufacturer’s recommendations (ETA-F10/X11, Data Sciences International). Analgesia was continued immediately after the intervention with carprofen (5mg/kg s.c.) twice daily for 48h with subsequent close monitoring for 7d and a total recovery time of 2 weeks before starting measurements. ECGs and blood pressure were recorded in mice undergoing volatile mask narcosis and in freely moving mice using a PhysioTel™ telemetry setup (DSI) during the subsequent experiments. Data was recorded and analyzed by Ponemah (DSI) software including Data Insights™. Analysis was performed according to conventional guidelines, as described before²².

Experimental design of epinephrine-induced heart failure (eTTC). To establish an easily reproducible mouse model of TTC, ten-week-old male C57bl/6n mice underwent isoflurane narcosis and baseline echocardiography with subsequent intraperitoneal injection of NaCl 0.9% (NaCl), ascending doses of epinephrine hydrochloride (Sanofi Aventis) (2, 2.5, and 5 mg/kg bodyweight), or isoprenaline (ISO) (250mg/kg bodyweight) diluted to a total volume of 100µL and echocardiography after 30min. with sacrifice 7d later. The findings from this experiment resulted in our final protocol for eTTC. After baseline echocardiography, mice were subjected to a single injection of 2.5mg/kg bodyweight EPI or NaCl under volatile mask narcosis (isoflurane 1-3%vol) at a constant body temperature of 38°C to ameliorate hypertensive crisis. After 15min. narcosis was terminated with subsequent follow-up echocardiography at 30min, 2h, 8h, 24h, 3d, and 7d. At 24h facial vein blood was collected. Mice were sacrificed at different time points dependent on the animals group affiliation by decapitation and trunk blood was collected. The heart was harvested, left and right ventricle were dissected and immediately snap-frozen in liquid nitrogen. Tissue was pulverized in a mortar and stored at -80°C until further evaluation.

Plasma preparation, measurement of Troponin T and corticosterone. Facial vein blood or trunk blood was taken from mice using hematocrit capillaries 24h after induction of eTTC. Whole blood was centrifuged at 14000 rpm for 20min. at 4°C. Supernatants were stored until further analysis at -80°C. For quantification of infarct size, high-sensitive cardiac Troponin T (hs-TnT) was measured using an automated Cobas Troponin T hs STAT Elecsys (Roche) as described previously²³. Measurement of corticosterone was conducted by radio-immunosorbent assay (RIA) as described elsewhere²⁴.

Measurement of plasma and left ventricular catecholamines. Catecholamine levels were quantified in plasma and cardiac left ventricular tissue. Cardiac tissue was weighted and subsequently homogenized in an ice-cold solution (0.01 M HCl, 1mM EDTA, 4mM Sodium disulfide). Whole blood was centrifuged at 14000 rpm for 20min. at 4°C and diluted 1:40 with the same ice-cold solution. Measurements were performed using high performance liquid chromatography (HPLC) and electrochemical detection as previously described ²⁵.

RNA extraction, quantitative PCR. Total RNA was isolated from homogenized left ventricular tissue using TRIzol (Invitrogen). Total RNA was digested with DNase, and cDNA synthesis of 500 ng of RNA was carried out by using a SuperScript first-strand synthesis system for RT-PCR (Invitrogen). Quantitative real-time PCR (qPCR) was performed with Universal ProbeLibrary (Roche) by using TaqMan Universal PCR Mastermix (Applied Biosystems) and detection on a 7500 Fast Cycler (Applied Biosystems).

RNA-sequencing, read processing and mapping, gene set enrichment analysis and enrichment map. Strand-specific TruSeq mRNA libraries were prepared at the Cologne Center for Genomics (CCG), Cologne, Germany (ribo-zero, 2x75nt, >30M fragments). Libraries were paired end sequenced on an Illumina HiSeq 3000 instrument. We used Flexbar to remove adapter sequences and low-quality regions from FASTQ files²⁶. Reads greater than 18 base pairs were retained and mapped against the murine 45S ribosomal RNA precursor sequence (BK000964.3) to remove rRNA contaminant reads. We used the mouse genome sequence and annotation (EnsEMBL 79) together with the splice-aware STAR read aligner (release 2.5.1b) to map our short reads²⁷. Following transcriptome analyses were carried out with the cufflinks package²⁸ and custom R scripts. Gene set enrichment analysis was conducted utilizing the GSEA 4.0.3 software and Molecular Signatures Database (MSigDB 7.2) from the Broad Institute, USA²⁹^,³⁰. Gene overlap network design was conducted via the EnrichmentMap plugin³¹ for the Cytoscape software (3.8.0)³²^,³³ and the collection of annotated drug gene sets from the Drug SIGnatures DataBase (DSigDB 1.0) from the Tanlab, USA³⁴.

Immunoblotting. Extracts from left ventricular tissue were isolated, and western blot analysis was performed according to protocols described before³⁵. Primary antibodies used were directed against total CaMKII (BD Biosciences), Calcineurin A (Cn) (Abcam), phospho-Calcineurin A (p-Cn) at Ser411 (p-Cn(Ser411)) (generated by Pineda antibodies), RCAN1-4 (a kind gift from Dr. Timothy McKinsey, Denver, USA), NFκB (Cell Signaling), and phospho-NF-κB p65 (Ser536) (93H1, Cell Signaling). Primary antibody incubation was followed by incubation with the corresponding HRP-conjugated secondary anti-mouse (1:5,000; Santa Cruz, sc-2055) and anti-rabbit (1:5,000; Santa Cruz, sc-2305) antibodies and detection with ECL (Santa Cruz, sc-2048).

Human samples. Venous blood (70 mls) was collected from patients diagnosed acutely with Takotsubo cardiomyopathy at Aberdeen Royal Infirmary, United Kingdom, as well as age- and gender matched healthy subjects, without calcium antagonist treatment. Peripheral blood mononuclear cells (PBMCs) were isolated from fresh peripheral venous blood from patients upon presentation to the emergency room using standard Ficoll-Paque (Ficoll-Paque Plus; GE Healthcare, USA) centrifugation separation with subsequent storage at -80°C until mRNA and protein analysis. Patients were recruited at the Cardiovascular and Diabetes Centre, School of Medicine and Dentistry, University of Aberdeen, United Kingdom. The study was approved by the local ethics committee and patient all samples were collected upon informed consent (EC ref. no. 20/SC/0305).

Statistical analysis. Results are expressed as mean ± SEM. Normal distribution was verified by the Kolmogorov–Smirnov test. Statistical analysis included one-way ANOVA or Kruskal–Wallis test followed by Bonferroni, Sidak, or Dunn’s post hoc test, respectively. Survival analysis was conducted using a Log-rank test. An unpaired or paired Students T- or Mann-Whitney U test were used when appropriate. Statistical analysis was performed using GraphPad Prism 9 (GraphPad Software). A p < 0.05 was considered statistically significant.

Data availability. The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information. Source data related to the study are available from the corresponding author upon reasonable request.

Epinephrine-induced reversible acute heart failure in mice. Since approximately 3-fold higher plasma levels of epinephrine were observed in TTC patients when compared to patients suffering from acute myocardial infarction⁶ and due to the exacerbated male phenotype in humans⁵, male mice were injected with increasing doses of EPI (2.0-, 2.5-, and 5mg/kg body weight) under narcosis. EPI injected mice displayed a significant reduction of left ventricular ejection fraction (EF) after 30min when compared to 0.9% NaCl injected mice as well as significantly increased mortality (Fig. 1A-B). Notably, ISO at a comparable high dose of 250mg/kg caused rather a hypercontractile phenotype with no relevant mortality. We defined model criteria as a heart rate above 400bpm and an EF below 45% to ensure significant AHF and exclude bradycardia-triggered impairment of cardiac function²¹ (Fig. 1C). A dose of 2.5mg/kg EPI was identified as the optimal dose to facilitate reversible AHF with 90% of C57bl6n mice meeting model criteria (Fig. 1D). Echocardiographic characterization confirmed reduced stroke volume (Fig. 1E) and ventricular ballooning (Fig. 1F) with increased apical impairment of contractility by means of radial strain (Fig. 1G), recapitulating the human syndrome. Moreover, we observed a marked decrease in invasively measured systolic and diastolic blood pressure at 2h with slow restitution thereafter, suggestive of beginning cardiogenic shock (Fig. 1H). ECG monitoring revealed blunted R wave amplitude, suggestive of myocardial edema, and ST-segment elevation in the eTTC model (Fig. 1I), which are also findings in TTC patients³⁶. Moreover, mice displayed significant elevation of plasma high-sensitive Troponin T (hs-Troponin T) (Fig. 2B), lower than in myocardial infarction^{23, 37} as in TTC patients⁴.

Gender-specific outcome and myocardial inflammation in experimental Takotsubo cardiomyopathy. Since male TTC patients suffer from impaired outcome compared to females, we next compared male and female C56BL6/N mice with sodium chloride treatment or EPI. Men with TTC suffer from increased myocardial damage, complications and mortality³⁸, which was also recapitulated by our finding of reduced male EF – observed with awake echocardiography (Fig. 2A) but masked under narcosis (Suppl. Figure 2A) – markedly elevated hs-Troponin T (Fig. 2B) as well as elevated mortality (Fig. 2C) compared to females. Moreover, increased plasma corticosterone – the murine analog of cortisol in humans – revealed comparable secondary activation of the central hypothalamo-pituitary-adrenal (HPA)- or stress axis (Fig. 2D) and blunted left ventricular tissue (LV) norepinephrine, suggesting regional cardiac SNS activation (Fig. 2E) in male and female mice. On the contrary, LV epinephrine was increased 2h upon injection (Fig. 2F). This increase was significantly higher in male mice, pointing to gender-dependent degradation mechanisms. As an unbiased assessment of gender-dependent and independent pathways upon EPI, we conducted gene set enrichment analysis (GSEA) from RNA-sequencing data of LV tissue from male and female mice with and without eTTC (Fig. 2G, Suppl. Figure 3). The dominant discrepancies between male and female mice in EPI-induced heart failure were less pronounced upregulation of pro-inflammatory (cytokine biosynthesis, lymphocyte and neutrophil chemotaxis)-, VEGF production-, and p38 MAPK pathway gene sets in female mice, possibly explaining the reduced myocardial damage observed in this model (Fig. 2G). Taken together, these data convey for the first time a murine TTC model which is recapitulating the human syndrome in this detail and reveal myocardial pro-inflammatory pathways to be significantly blunted in female vs. male mice.

CaMKII inhibits calcineurin-driven myocardial inflammation in experimental Takotsubo cardiomyopathy. To identify potential drug targets in the more severely affected males, we analyzed gene regulation overlap with a freely available drug signature database (Tan Lab DSigDB V1.0) and found the highest number of overlapping genes with valproic acid, copper sulfate and cyclosporine A (CSA) (Suppl. Figure 3G). Due to the QT-prolongation capacity of valproic acid and the observed inflammatory myocardial gene expression phenotype upon EPI, we conducted a follow-up analysis regarding contrary regulated pathways from our gene expression network with CSA and found a significant overlap of the gene set CICLOSPORIN_HL60_DOWN (Fig. 3A), indicative of a potential therapeutic effect of CSA on the myocardium in the setting of eTTC. LV immunoblotting revealed a striking upregulation of calcineurin A (Cn) protein expression with a dramatic gender-dependent increase of RCAN1-4, a well-known reporter of Cn activity¹⁹ (Fig. 2B-C). Intriguingly, Cn phosphorylation at the autoinhibitory CaMKII phosphorylation site serine 411 (p-Cn(Ser411)) was more pronounced in female vs male mice, proposing enhanced CaMKII-driven autoinhibition of Cn in female mice with subsequently blunted Cn activity. Consequently, we took advantage of the possibility to combine the newly established murine model of eTTC with a mouse genetic approach and subjected male mice harboring an inducible cardiomyocyte specific knockout of the Ser411-dependent Cn inactivator^{17, 20} CaMKIIδ and CaMKIIγ to eTTC. Since the CaMKII double KO mouse line including wildtype littermates displayed significant resilience regarding AHF and myocardial damage in eTTC, the dose of EPI had to be increased to 4mg/kg (see Suppl. Figure 5A). CaMKII double KO mice (dcKO) were more vulnerable with respect to impaired EF (Fig. 3D), hs-TnT elevation (Fig. 3E), and mortality (Fig. 3F). Importantly, pretreatment with the Cn inhibitor CSA significantly abolished the phenotype in dcKO mice with respect to EF (Fig. 3G) and myocardial damage (Fig. 3H), thereby establishing a functional relationship between CaMKII and Cn signaling in eTTC and potentially uncovering a modulatory pathway of harnessing inflammatory responses in the Takotsubo heart. Accordingly, CaMKII-driven Cn phosphorylation at Ser411 in dcKO mice (Fig. 3I) as well as enhanced RCAN1-4 expression (Fig. 2I-J) was blunted upon CSA in dcKO mice with eTTC. Confirming the findings from the overlap gene set enrichment network with CSA-targets (Fig. 3A), LV mRNA expression of the target genes cc-chemokine ligand 2 (ccl2) and the nuclear receptor subfamily 4 group A member 2 (nr4a2) showed significant upregulation in dcKO mice upon EPI compared to control (cre+) mice with CSA-driven inhibition 8h after insult (Fig. 3K-L). Myocardial inflammation per se – indicated here by the cytokine interleukin-6 (il-6) and the cc-chemokine ligand 3 (ccl3) – was significantly enhanced in dcKO mice and markedly reduced by CSA (Fig. 3M-N). These findings are suggestive of a contribution of pro-inflammatory myocardial Cn signaling to TTC.

A new anti-inflammatory treatment strategy for TTC. To further investigate the therapeutic efficacy of CSA, mice were pretreated 30min before sodium chloride or EPI with a singular dose of 10, 30, or 100 mg/kg CSA. CSA significantly improved cardiac function and ameliorated myocardial damage with a beneficial impact on survival already at a dosage of 10mg/kg (Fig. 4A-C) with no additional benefits at higher dosages (Suppl. Figure 4A-C). Immunoblotting revealed increased phosphorylation of nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-κB) p65 at Ser536 8h after EPI, which was blunted by additional CSA treatment (Fig. 4D-E). Also, LV qPCR confirmed downregulation of mRNA expression of the representative CSA target gene chemokine ccl2 (Fig. 4F), of the pro-inflammatory cytokine interleukin-1β (il-1β) (Fig. 4G), as well as of the overall top ranked gene from GSEA (Suppl. Figure 3A), the nuclear receptor subfamily 4 group A member 3 (nr4a3) (Fig. 4H). However, at 8h we were only able to observe a mild dose-dependent decrease of RCAN1-4 mRNA and protein (Suppl. Figure 4D-E), indicative of fainting calcineurin suppression by CSA. Thus, we continued CSA application 2x/d in subsequent experiments. Continued CSA after initial pretreatment 2h before EPI, or therapeutic application 30min after EPI in 8 weeks old mice improved EF (Suppl. Figure 4H) and significantly blunted hs-Troponin T compared to EPI (Fig. 4I) with significant RCAN1-4 reduction by CSA at 8h (Suppl. Figure 4G). In this experiment, we observed a mild eTTC phenotype with low overall mortality (Suppl. Figure 4J). Since mice from this experiment were slightly younger (8w) than mice from other experiments (10-12w), we compared male 8w old with male 12w old mice in a separate experiment and observed a marked impact of increased murine age on EF (Suppl. Figure 2E) and mortality (Suppl. Figure 2G), with a trend towards elevated myocardial damage in 12w old mice (Suppl. Figure 2F). Interestingly, immunoblotting revealed markedly elevated LV RCAN1-4 in the more vulnerable 12w old mice, suggestive of an age-dependent exacerbation of Cn signaling impacting outcome in eTTC (Suppl. Figure 5B). Taken together, we observed a critical impact of murine age on RCAN1-4 and outcome upon eTTC as well as beneficial effects of preventive and therapeutic CSA application with myocardial RCAN1-4 suppression and a blunted myocardial inflammatory response.

To investigate human relevance of this pathway, we analyzed peripheral blood mononuclear cells (pbmcs) from n = 5 TTC patients and n = 5 age- and gender-matched healthy controls. Expression of the calcineurin reporter rcan1-4 (Fig. 5A), the pro-inflammatory gene il-1β (Fig. 5B), and the top upregulated gene from our mouse model – nr4a3 (Fig. 5C) – was significantly increased in pbmcs from patients suffering from acute TTC. These findings strongly support the role of calcineurin activation in patients with implication of nr4a3.

Due to the lack of a reproducible mouse model with overt acute heart failure, cause-effect relationship studies, e. g. based on a genetic knockout approach to TTC have so far been unavailable. Here, we established a clinically relevant experimental mouse model (eTTC) that recapitulates the hallmarks of human TTC, including transient AHF, biomarker elevation, ECG changes, and risk factors of impaired outcome. In this regard, the standardized setting of eTTC allowed us to detect early changes in gene expression and led to the identification of a significant overlap of pro-inflammatory genes with CSA targets. Interestingly, this increase was higher in male mice, while autoinhibitory CaMKII-dependent calcineurin phosphorylation was higher in female mice, suggestive of the existence of a gender-specific cardioprotective mechanism. By combining eTTC with a genetic mouse model, we unmasked Cn-driven myocardial inflammation as a potential underlying mechanism and established early pharmacological Cn inhibition by CSA as a novel therapeutic approach to blunt features of TTC and to improve survival. Moreover, we show human relevance in peripheral immune cells of TTC patients, which reveal increased Cn/NFAT activity, possibly mirroring myocardial pathway activation.

Epinephrine induces gender-specific reversible acute heart failure in mice. Patients suffering from TTC showed 3-fold higher plasma levels of epinephrine compared to patients suffering from acute myocardial infarction⁶ and case reports suggest epinephrine as a potential trigger of TTC-like cardiac dysfunction¹⁰. Here, we observed that high-dose EPI but not ISO is sufficient to cause TTC-like AHF in mice along with troponin elevation and acute ST-segment changes, thereby fulfilling the criteria of human disease³⁹. In line with our findings and the findings of others, epinephrine- as well as catecholamine-induced reversible AHF has been described in rats^{11, 12}. Consistent with our findings here, ISO has not been reported to cause AHF in mice^{13, 40} and since ISO causes a secondary rise in plasma norepinephrine and may thereby stimulates α-adrenoceptors indirectly, the possibility of selective β-adrenergic stimulation in vivo appears questionable⁴¹. Since human TTC confers a gender-specific phenotype – i.e. it predominantly affects postmenopausal females, while men present only 10% of TTC patients but suffer from increased prehospital cardiac arrest, elevated troponin levels, higher occurrence of cardiogenic shock, and increased mortality compared to females ^{4, 5, 42, 43} – we compared male and female C56BL6/N mice. In line with the clinical phenotype, we observed reduced cardiac function, elevated myocardial damage and pro-inflammatory gene network upregulation as well as markedly elevated mortality in males. Moreover, we observed elevated corticosterone, the murine analogue of cortisol, in eTTC, suggesting HPA-axis activation, while we interpreted LV norepinephrine depletion as a result of adrenergic stimulation^{25, 44–46}. Our finding of upregulation of LV epinephrine despite precursor (dopamine, norepinephrine) depletion, suggests enrichment due to EPI application with secondary cardiac sympathetic and HPA-axis activation. Since this was significantly enhanced in male mice, gender specific cardiac catecholamine uptake or degradation is suggested. In patients suffering from acute TTC, coronary sinus plasma norepinephrine concentrations were significantly increased compared to systemic plasma concentrations⁴⁷, indicating an elevated cardiac release. This finding is in line with our observation of depleted LV norepinephrine in eTTC and further supports our model. The importance of myocardial inflammation in TTC has been shown by Scally et al.¹⁵ and this is in line with our finding of elevated myocardial inflammatory gene expression networks upon eTTC. The nuclear receptor subfamily 4A (nr4a) has been implicated in the pathogenesis of TTC in an in vitro induced pluripotent stem cell (iPSC) model by Borchert et al.⁴⁸ with regard to nr4a1. Due to their role in orchestrating adrenergic drive and inflammation and based on our GSEA ranking, particularly further investigation of member 3 (nr4a3) as well as of the stress-responsive activating transcription factor 3 (ATF3) is warranted. In summary, gender discrepancies regarding outcome may potentially be caused by elevated myocardial epinephrine and exacerbated calcineurin-driven myocardial inflammation in male patients.

Implication of the calcineurin pathway in epinephrine-induced heart failure. Gender-specific upregulation of RCAN1-4, a sensitive endogenous Cn reporter^{20, 49, 50}, suggests a role of Cn regarding discrepant myocardial inflammation and outcome in eTTC. Since CaMKII-induced phosphorylation of Cn inhibits Cn in vivo²⁰, the provoking question arose, whether CaMKII mediates beneficial effects regarding the CaMKII-Cn pathway in the pathogenesis of AHF in eTTC. Indeed, inducible myocardial dcKO mice displayed exacerbated AHF, myocardial damage, mortality, and LV RCAN1-4 expression upon eTTC, while CSA restored EF, myocardial damage, and restored RCAN1-4 of dcKO to the level of cre + mice. In the absence of functional myocardial CaMKII our data shows additionally elevated expression of Cn target genes and inflammatory mediators which are blunted by Cn inhibition via CSA. Therefore, we suggest potentially clinically relevant opposing roles of CaMKII and Cn in TTC. Weinreuter et al. implicated CaMKII in post-myocardial infarction chemoattractant signaling with increased myocardial leukocyte infiltration²³. However, reduced leukocyte infiltration was not observed until 5d after insult, possibly explaining the lack of beneficial impact of myocardial CaMKII ablation in the setting of eTTC.

Next, we conducted a preventive CSA treatment of eTTC and observed significantly reduced myocardial damage, mortality, and improved cardiac function. The finding of improved myocardial function and attenuated myocardial damage remained when CSA was given 30min. after induction of eTTC. We also observed a marked impact of murine age on outcome (8w vs 12w), which is particularly interesting since we also observed an age-dependent increase in RCAN1-4 and might suggest a role regarding the clinical finding of age as a predisposition and a risk factor for adverse outcome in human TTC⁴². However, actual ageing remains to be investigated in this model. EPI caused increased myocardial RCAN1-4 and NF-κB p65 phosphorylation, reversed by CSA with a similar response of inflammatory gene expression markers. This finding suggests Cn-driven myocardial inflammation as an important mechanism of TTC pathology, which may be addressed by treatment with CSA. Finally, gene expression analysis from PBMCs of TTC patients compared to age- and gender matched controls strongly indicate human relevance of Cn-driven and CSA-responsive inflammatory pathways. Thus, the data of this study point to a new clinically so far not exploited treatment strategy of TTC with inhibition of the Cn pathway by CSA.

Acknowledgement

The authors thank P. Nawroth for the opportunity to conduct RIA (corticosterone), HPLC (catecholamines), and automated Cobas (hs-Troponin T) analysis in his laboratory. S. Martinache, J. Krebs-Haupenthal, S. Harrack, and M. Oestringer provided excellent technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (BA 2258/2-1 and SFB 1118), the European Commission (FP7-Health-2010 and MEDIA-261409) and the Deutsches Zentrum für Herz-Kreislauf-Forschung (DZHK; German Centre for Cardiovascular Research) and by the BMBF (German Ministry of Education and Research) to JB and by the German Cardiac Society (DGK) to BB, IB, and MS as well as by the German Heart Foundation (DHS) to MA.

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There is NO Competing Interest.

SupplementalfiguresTTCNatCardiovascRes.docx
graphicalabstract.pdf
Graphical abstract: Epinephrine (EPI) injection (Inj) recapitulates Takotsubo cardiomyopathy in mice. We observed impaired male outcome, including mortality, reduction of left ventricular ejection fraction (LVEF) and plasma Troponin T (TnT) with marked myocardial calcineurin (Cn) activation and inflammation. Genetic ablation of endogenous Cn inhibition by phosphorylation via an inducible cardiomyocyte-specific calcium-calmodulin dependent kinase II (CaMKII) knockout (KO) exacerbates myocardial Cn activation, inflammation, acute heart failure, myocardial damage, and mortality. Calcineurin inhibition by cyclosporine A (CSA) rescues the Takotsubo phenotype in wildtype and reverses the vulnerability of CaMKII KO mice.

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Calcineurin signaling promotes Takotsubo cardiomyopathy

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