Temporal proteomic analysis of cardiac extracellular vesicles throughout takotsubo syndrome development and resolution in an animal model

Abstract Background Takotsubo syndrome (TS) is an acute form of heart failure characterized by transient regional wall motion abnormalities triggered by a stressful event. The condition is recognized through cardiac imaging, typically showing apical ballooning. The mechanisms underlying its development are poorly understood, resulting in a lack of disease-specific treatments and diagnostic markers. Extracellular vesicles (EVs) play a significant role in cellular communication and disease pathophysiology, but remain unexplored in TS. Methods We used a high-fidelity rat model of TS (n=48) induced through catecholamine administration. After assessing the TS phenotype and cardiac function using high-resolution echocardiography, we isolated EVs from apical and basal segments of the left ventricle at baseline, 6 hours, 24 hours, and 30 days post-induction. EV isolation involved tissue slicing, enzymatic digestion, and differential centrifugation steps, followed by iodixanol density cushion. EVs were characterized by electron microscopy, western blot, and nanoparticle tracking analysis. Quantification of protein levels in EVs was accomplished by differential labelling using quantitative mass spectrometry. Data visualization and functional enrichment analyses were performed using R, StringApp for Cytoscape, and GSVA for pathway analysis. Results Pure, cup-shaped vesicles ranging from 50-500nm were successfully isolated. Western blot experiments confirmed the presence of typical EV markers, including Flotillin 1, TSG101, and CD81 (Figure 1A). Global proteomic analysis revealed a distinct protein abundance pattern in EVs from the apical segment of TS hearts across the temporal progression from injury to recovery. We identified 1652 proteins, where 70 (TS6h-apex vs TS6h-base), 315 (TS24h-apex vs TS24h-base), and 28 proteins (TS30d-apex vs TS30d-base) showed differential abundance (|FC| 0.58, adj.P 0.05), no difference was observed in Baseline-apex vs Baseline-base (Figure 1). Functional enrichment analysis showed a differential abundance of proteins associated with immune response, tissue repair, survival signalling, metabolism, and ROS detoxification. Notably, proteins related to the electron transport chain showed an increase at 6 hours and a decrease at 24 hours (Figure 2), mirroring the time course of functional recovery. Conclusion This study shows for the first time the proteomic characterization of EVs in the context of TS hearts across its natural course. Our results demonstrate a distinct EV protein profile, mostly in the apical segments of TS hearts, with marked changes in proteins related to inflammatory responses, tissue repair mechanisms, metabolism, and cell survival pathways. This comprehensive proteomic profile provides insights into TS pathophysiology and identifies potential diagnostic biomarkers and therapeutic targets, warranting clinical validation in human samples.Figure 1 Figure 2