Summary

Stress is an independent risk factor for cardiovascular diseases in patients with pre-existing cardiac diseases or cardiovascular risk factors, as well as in persons without a known cardiac condition. A spectacular consequence of acute stress on the heart is Takotsubo cardiomyopathy (TTC), a condition mimicking an acute coronary syndrome but in which coronarography appears normal. In the absence of other signs of associated or underlying cardiac conditions, TTC patients are dismissed from hospitals because the acute cardiac symptoms (pain, electrocardiography, heart rate, blood pressure…) are reversible and because there is no evidence of coronary obstruction. However, recent long-term follow-up studies have reported similar cardiovascular annual death rates for TTC and for proven acute coronary obstruction, suggesting that TTC often induces severe cardiac sequelae.
Although there is converging evidence that the stress-induced catecholamine rush is the cause of Takotsubo, the mechanism by which a single acute stressing event can have long-term deleterious effects on the heart remains mysterious. Catecholamines activate adrenoreceptors, in particular the beta-1 adrenoreceptors that are predominant in the heart. The activation of beta1 and beta2 receptors has positive inotropic and chronotropic effects, increases cardiac output, myocardial oxygen consumption and coronary flow, while the activation of beta 3 receptors has a negative inotropic effect. Studies have reported that at high catecholamine concentrations, such as those found during acute stress, the activation of beta 3 receptors counteracts the activation of beta 1 and 2 receptors, supporting the view that, mechanistically, the heart is well adapted to stress. Another role of stress-induced catecholamine release is to secure energy substrates for the “fight or flight” reaction, by opposing the action of insulin, namely by inducing hyperglycemia and glycogenolysis. The mammalian heart is omnivorous: in basal conditions, it feeds essentially on fatty acids (ca. 80%) and modestly on glucose (ca. 20%) and other substrates. In conditions of high-energy demand, e.g., exercise, the supplementary energy requirements are provided by an increase of aerobic and anaerobic glucose breakdown. This is also the case under high glycaemia in diabetes, or in conditions with reduced cardiac muscle microperfusion such as under anti-angiogenic treatment. The adaptation of the cardiac energy balance through the regulation of glucose utilization is advantageous for the challenged heart because it can be switched on very rapidly and can maintain ATP production in low oxygen conditions. The mechanisms involve increased intracellular transport of glucose through Glut1, the universal glucose membrane transporter, and of Glut4 that is expressed in the heart, skeletal muscle, and insulino-dependant tissues. Glut4 translocation to the plasmatic membrane is under the control of insulin growth factor (IGF) and other effectors, including the stress hormones catecholamines and cortisol.
Although glucose metabolism is a major factor of regulation of cardiac activity in health and disease, molecular imaging of metabolism is not an indication in patients with Takotsubo. One of the reasons may be that the metabolic responses of the heart to a sudden and dramatic rise of circulating catecholamines are not easily predictable and depend on the respective affinities and sensitivities of different adrenergic receptors and on their cardiac densities, as well as on the physiological and metabolic state of the heart, which is highly variable among individuals. Moreover, such studies are difficult to conduct in human patients because the neurohumoral response to stress is highly variable and unpredictable among individuals, and because cardiac and metabolic comorbidities are frequent in Takotsubo patients. In contrast, studies of TTC in animal models are easy to run in homogeneous populations using uniform stress triggers. Several Takotsubo-like models have been proposed in rodents, the more robust and reproducible being based on the administration of catecholamines in different routes, frequency and dose.
Considering that little is known about the regulation of glucose metabolism of the myocardium during the early and late phases of Takotsubo, and, more importantly, that even less is known on the influence of metabolism on the functional and structural integrity of the myocardium in the long term after Takotsubo, we performed a comprehensive set of non-invasive explorations for close longitudinal follow-up in the same animals before stress (baseline), during acute stress (2 h) and at early (7 days) and late (1 and 3 months) time points after stress. Our objective was to decipher the role of energy metabolism on long-term tissue and vascular remodeling in a preclinical model recapitulating the clinical signs of TTC over the whole course of the disease. Reasoning that acute stress was the cause of Takotsubo, at least in its initial description, and that there is a large consensus that stress-induced catecholamine release is the main causal factor of Takotsubo, we mimicked in rats the catecholamine rush described in patients, with a single administration of the adrenergic agonist isoproterenol, a drug used to increase heart rate in case of bradyarrhythmias that can provoke Takotsubo. Here, we show that the increased glucose uptake in the myocardium induced by an isoprenaline stress is not used to increase energy production, but is diverted into alternative anabolic pathways of glucose. We provide evidence that this glucose diversion induces immediate and long-term tissue, metabolic and functional changes that may explain the increased risk of heart failure in Takotsubo patients.

Results from the nanoScan PET/CT

On days 0, 1, 7 and at 1 and 3 months, non-fasted rats were anesthetized (isoflurane 4% induction and 2% maintenance in air), weighted and glycemia and animal temperature were recorded. The animal was placed supine in the nanoScan PET-CT scanner (Mediso Medical Imaging Systems, Hungary) with respiratory and cardiac monitoring. A commercial ultrasound probe (SuperLinear™ SLH20-6, Supersonic Imagine, France, central frequency 15 MHz) connected to a small animal ultrasound device (Aixplorer, Supersonic Imagine, France) was positioned on the depilated chest of the animal to obtain a view of the full long-axis of the beating heart. The animal was then moved in the PET gantry and a whole-body X-ray tomodensitometry (CT) was acquired using the following acquisition parameters: semi-circular mode, 70 kV tension, 720 projections full scan, 300 ms per projection, binning 1:4. Images were reconstructed by filtered retro-projection (filter: Cosine; Cutoff: 100%) using Nucline version 3.00.010.0000 (Mediso Medical Imaging Systems, Hungary). Immediately after CT acquisition, a 30 min dynamic PET scan and 30 s after starting the acquisition, 32.9 ± 1.4 MBq of 2’-deoxy-2’-[¹⁸F]fluoro-D-glucose (FDG; Advanced Applied Applications, France) in 0.4 mL saline were injected in the lateral tail vein. At the end of the first dynamic PET scan, a static 30 min PET scan was acquired with ECG and respiratory gating. PET data was collected in list mode and binned using a 5 ns time window, with a 400-600 keV energy window and a 1:5 coincidence mode. Data was reconstructed using the Tera-Tomo reconstruction software (3D-OSEM based manufactured customized algorithm) with expectation maximization iterations, scatter, and attenuation correction. The 30 min dynamic PET exam was reconstructed in 23 frames as follows: 30 s, 6 × 5 s; 4 × 10 s; 6 × 30 s; 3 × 120 s; 4 × 300 s. The ECG-gated cardiac PET was reconstructed in a single frame of 15 min, 45–60 min post FDG injection. Using the PET/CT fusion slices, volumes-of-interest (VOI) were delineated for the left ventricle (LV) using PMOD software (PMOD Technologies Ltd, version 3.8, Zürich, Switzerland). FDG uptake was quantified as Standard Uptake Value. The Peak SUV was calculated as the maximum average SUV within a 1-cm³ spherical VOI, and the LV volume was automatically segmented at 40% of this value. Compartmental kinetic assessment of FDG uptake was based on the 2-tissue compartment model of the PMOD kinetics package with a lump constant set to 1.
Figure 3. a shows representative images of FDG PET registered UUI (PETRUS) at the indicated time points post-ISO from diastolic phase to systolic phase. Images acquired 30 min after FDG injection of one section along the long axis of the LV. Color scale depicts the Standard Uptake Value (SUV) from 0 to 10. Note the increase in FDG uptake at 2 h and 7d respective to baseline. b Data are presented as mean values +/− SD of n = 9 animals per time point. Quantitative analysis of FDG uptake in the LV: SUV mean in whole LV, apex (7d vs. 1mo: p = 0.0337) and basal LV, normalized to baseline values; rate constant K1 reflecting the exchange of FDG from blood to tissue; rate constant k3 reflecting FDG phosphorylation, calculated using two-compartmental analysis. Note the global increase of SUV at 2 h and 7d post-ISO, the modest increase of K1 that is not statistically significant from baseline at any time point, and the significant decrease in k3 at 2 h followed by an increase at 7d post-ISO (in the apex, 2 h vs. 7d: p = 0.0004, 7d vs. 1mo: p = 0.0005, and 7d vs. 3mo: p = 0.0029; in the base, 0 vs. 2 h: p = 0.0312, 2 h vs. 7d: p = 0.0096, and 7d vs. 1mo: p = 0.0312). Paired comparison tests: *p < 0.05, **p < 0.01 and ***p < 0.001. Statistical significance (p < 0.05) for each variable was estimated by one-way or two-way ANOVA when group variances were equal (Bartlett test); if not the non-parametric Kruskall–Wallis test, and the Holm multiple comparisons test was used to execute simultaneous t-tests.

• Clinically, Takotsubo is multifactorial and complex and often associated with different comorbidities, The present study used a simple animal model to investigate the longitudinal functional, anatomical, metabolic, tissue, and vascular modifications of the heart in a reproducible Takotsubo-type animal model, without the various comorbidities encountered in patients, such as the cardiovascular diseases associated with aging, hypertension, diabetes, obesity, etc.
• A unique injection of beta-adrenergic catecholamines led to a cascade of reproducible activation and inactivation of different metabolic and anabolic pathways of glucose and to short- and long-term tissue, fibrotic and vascular sequelae of the heart. In short, the stress induced a transient ventricular dysfunction during the acute phase, and an irreversible tissue and functional impairment of the left atrium in the long term. The proteomic analysis at 2 h and 7 days were based on 5 replicates per condition, which may lead some samples to yield less protein raw identification than the average across all samples.

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