Types of cardiomyocyte death in Takotsubo cardiomyopathy

Scientific objective: The aim of the project will be to evaluate the type and course of cardiomyocyte death in the female rat Takotsubo (TTC) model of isoprenaline-induced cardiomyopathy (ISO). State of knowledge: Takotsubo cardiomyopathy (TTC) is characterized by acute, reversible systolic left ventricular failure caused by emotional and / or physical stress, mainly in postmenopausal women. TTC shows akinesia of the apical segments of the left ventricle (echocardiography) and its specific systolic shape (ventriculography). The pathogenesis of Takotsubo cardiomyopathy has not yet been elucidated. It has been suggested that it may result from coronary circulatory disorders or the cardiotoxic effect of catecholamines. Recently, a male rat model of TTC induced by intraperitoneal administration of isoprenaline, an agonist of P1- and P2-adrenergic receptors, has been developed. Recently, our research team developed the first female rat TTC model induced by IP injection of ISO. Few studies, including our preliminary published results of TTC histopathological changes in the heart, indicate the presence of cardiomyocyte death and the accompanying inflammatory response. The type of cardiomyocyte death in Takotsubo cardiomyopathy, its impact on the course of the disease and the role of female sex hormones in these processes have not yet been elucidated. It seems that three types of cardiomyocyte death in particular may occur in the course of TTC, ie apoptosis, necroptosis and pyroptosis. Apoptosis is programmed cell death that is dependent on caspase 3. Necroptosis combines the features of programmed apoptosis and necrosis. Activation of the necroptotic pathway requires recruitment of the RIPK1 (Receptor-Interacting Protein Kinase 1) and / or RIPK3 (Receptor-Interacting Protein Kinase 3) and MLKL (Mixed Lineage Kinase domain -Like) proteins. Necroptosis has also been shown to play a significant role in inducing an inflammatory response. Pyroptosis, on the other hand, is a pro-inflammatory, programmed type of cell death, in which the key stage is the recruitment of the cytosolic protein complex called the inflammasome, e.g. NLRP3, activating caspases 1 and 11, inducing the release of pro-inflammatory interleukins (IL): IL-1P and IL-18. There are data suggesting the involvement of NLRP3 in the pathogenesis of numerous diseases of the cardiovascular system. Type II calcium-calmodulin-dependent kinase (CaMKII) is a protein directly involved in cardiomyocyte apoptosis, induced by stimulation of P1-adrenergic receptors and necroptosis of these cells associated with inflammation. The expression of this kinase increases with low estrogen levels, suggesting that it may have a significant influence on the pathogenesis of TTC. Methods and description of the research: The research will be carried out in 160, 9-week-old female Sprague Dawley (SPRD) rats. The animals will be divided into 4 treatment groups (5 runs in each group, 8 animals in the series). At 9 weeks of age, rats will undergo bilateral ovariectomy (groups I and II) or sham surgery (groups III and IV). At 12 weeks of age, rats will receive an intraperitoneal injection of 150 mg / kg body weight. ISO (groups II and IV) or saline (NaCl) (groups I and III). In each of the study groups, 6, 12, 24, 72 hours and 10 days after the ISO or NaCl injections, transthoracic echocardiography will be performed and blood and heart will be collected for biochemical tests. The plasma concentration of caspase 1, 3, IL-1P, IL-18, RIPK1, RIPK3, MLKL and CaMKIIS (ELISA) will be determined. Hearts will be tested for caspase 1, 3, RIPK1, RIPK3, MLKL and CaMKIIS (Immunohistochemistry). Characterization of the final result: So far, the process of apoptosis, pyroptosis and necroptosis in TTC has not been characterized, and their influence on the course of the disease has not been explained. The present study will bring closer the role of the cardiomyocyte death process in TTC and the influence of female sex hormones on the occurrence of the above types of cell death, which in the future will be the starting point for further research to determine an effective targeted therapy. Assessment of the plasma concentration of proteins involved in apoptosis, necroptosis and pyroptosis may contribute to the detection of new prognostic markers in TTC that are useful in clinical practice.