Proteomic profiling and characterization of extracellular vesicles in an ex vivo takotsubo syndrome model using isolated perfused rat hearts

Abstract Background Takotsubo Syndrome (TS) is a form of acute, reversible heart failure associated with substantial morbidity and mortality. TS is a protective phenotype for the heart when it comes to ischemia and arrhythmias. However, the underlying mechanisms of cardiac protection in TS remain poorly understood. Extracellular vesicles (EVs) have been implicated in organ protection through various biological pathways. Our previous in-vivo studies have demonstrated qualitative and quantitative differences in EV content in in-vivo TS models. Aim: In this study, we developed and validated a novel method for the extraction and characterization of EVs from an ex-vivo TS model, using isolated rat hearts perfused according to the Langendorff technique. Methods male Sprague-Dawley rats (~47 days old, ~300 g) were divided into two groups: control and ex vivo TTS. TTS was induced by isoprenaline infusion (1 mg/kg) over 15 minutes in the isolated perfused Langendorff method. Cardiac parameters were carefully monitored throughout experiments. TTS phenotype was confirmed with high resolution ex vivo echocardiography. After regional wall motion abnormality (apical akinesia) confirmed via echocardiography following stabilization on the Langendorff perfusion system and TTS induction, perfusate samples were collected at various time points. EVs were extracted from the perfusate and characterized using western blotting, nanoparticle tracking analysis (NTA), electron microscopy, and proteomics. Results NTA revealed a significant increase in the total number of EVs in TS hearts compared to controls (p 0.05, two-way repeated measures mixed model) (figure 2). Western blotting identified the presence of exosome markers flotillin1, TSG101, and CD63, confirming the purity of the exosomal fraction (figure 1). Electron microscopy further validated the morphology of the EVs, with a predominant exosome phenotype (Figure). Proteomic analysis highlighted distinct protein profiles in EVs from the apical segments of TS hearts, suggesting differential EV-mediated signalling in TS pathophysiology. Proteomic profiling and gene ontology also confirmed cardiac-specific EVs in addition to general EV markers(HSP, CD63, CD81, ...) (figure 2). Conclusion Using isolated perfused rat hearts, we successfully extracted and characterized cardiac-specific EVs in an ex-vivo TS model. This improved model offers valuable insights into the role of cardiac-specific EVs in TS pathophysiology and their potential involvement in cardio protection. These findings establish a foundation for future studies investigating EV-mediated therapeutic strategies in TS.Figure 1