A influência temporal na inflamação cardíaca em um modelo da cardiomiopatia de Takotsubo

Abstract

Takotsubo cardiomyopathy (TC) represents an important cause of hospitalizations for acute cardiac events. It occurs mainly in postmenopausal women in response to stressors of emotional or physical origins, where release of catecholamines hyperstimulates cardiac tissue leading to its deleterious effects. It is known that cardiac events, such as acute myocardial infarction, occur more frequently at certain times of day. Similarly, physiological events that participate in the pathogenesis of these diseases, such as inflammation, also show a circadian rhythm. Therefore, the aim of this study was to evaluate the temporal impact on cardiac remodeling that occurs in CT induced by a high dose of isoproterenol (ISO), with a focus on the inflammatory component. For this, we used female mice, C57BL/6 (8 to 12 weeks of age), divided into 4 groups: (1) mice treated with saline at the ZT0 (7 am, CTRL ZT0); (2) mice treated with ISO -/- 300mg/kg at ZT0, ISO ZT0), and the groups (3) and (4) treated in the same way at ZT12 (which corresponds to 7:00 pm, CTRL ZT12 and ISO ZT12). Three days after treatment the animals were sacrificed and cardiac tissue was collected. There was a greater increase in cardiac mass normalized by body weight and tibia length (HW/BW and HW/TL) in animals treated with ISO at ZT12 vs ZT0. In addition, there was a greater weight loss in the animals treated in ZT12 vs ZT0. Confirming the HW/TL ratio data, we observed using the WGA probe that ISO induces a greater increase in cardiomyocyte cross-sectional area (CSA) in animals treated at ZT12 vs ZT0. It was also observed by the hematoxylin and eosin (H&E) labeling that the animals treated in ZT12 had a more prominent inflammatory infiltrate, in addition to greater deposition of amorphous eosinophilic material characteristic of tissue necrosis. Heart staining with the Evans Blue dye indicated that ISO induced a similar increase in cell death in treated mice, regardless of the ZT. By using the PicroSirius, we found that ISO did not induce total collagen deposition, a result that was confirmed with the MYH7-YFP transgenic reporter model. In this model, cardiomyocytes fluoresce when expressing the MYH7 protein, which is involved in cardiac stress mainly through fibrosis. The profile of the inflammatory infiltrate present in the cardiac tissue was evaluated by flow cytometry, where we observed an increase in leukocytes (CD45+ cells), neutrophils (Ly6G+ cells), macrophages (CD45+Ly6G-Ly6CintCD64high cells) and monocytes (CD45+Ly6G-Ly6ChighCD64int) restricted to animals treated in ZT12. Knowing that macrophages comprise an important cell type in the pathogenesis of CT, we decided to investigate their subtypes by an additional gating in flow cytometry. While an increase in CCR2+MHC-IIhigh cells was observed in animals treated in both ZTs, the increase in CCR2-MHC-IIlow macrophages and CCR2+MHC-IIlow monocytes was restricted to mice treated at ZT12. Finally, we did not observe an increase in CCR2 MHC-IIhigh cells in the animals treated with ISO in both ZTs. This result was confirmed by immunofluorescence, where we found an increase in CD68 staining in animals treated in ZT12 vs ZT0. Taken together, these results indicate that monocyte recruitment to the heart in response to ISO is greater in ZT12. Therefore, we decided to assess the presence of remodeling and inflammatory infiltrate 24h after the ISO insult, which represents the time when monocytes begin to infiltrate in heart. 24h after ISO treatment, only animals treated with ISO in ZT0 showed an increase in the HW/BW ratio and weight loss. Analysis of CSA indicated the occurrence of hypertrophy in animals treated in both ZTs. By immunofluorescence, we observed that there was an increase in CD68 labeling only in ISO ZT12 animals. Thus, we conclude that the inflammatory response that occurs in response to ISO is temporally influenced, being more severe in the dark phase than in the light one. These data are important for understanding the mechanisms involved in TC and highlight the importance of understanding the impact of the time of the day for the development of more effective therapeutic strategies.