Characterization of cardiac extracellular vesicles upon thiamet G treatment in a rat model of acute decompensated heart failure

Abstract

Acute decompensated heart failure (ADHF) is characterized by dyspnea, edema and fatigue, as well as a high morbi-mortality. Recently we observed that Thiamet G (TG) exerts protective effects in a rat model of ADHF. Extracellular vesicles (EV) are known to carry key regulatory molecules from cell-to-cell in both the heart and circulation, however, an effective isolation technique specific to cardiac EVs is yet to be discovered. Our goal is to identify cardiac-specific EV markers and to investigate the role of EVs in mediating improvement of ADHF outcomes upon TG treatment. HF was induced in 10 week-old Wistar rats via coronary ligation. Three months later, acute decompensation was induced via NaCl administration in water (1.8 g/kg), followed by injection of 25 mg/kg TG 12 hours later. Rats were sacrificed 12 hours after TG treatment, and their left ventricles (4 TG-treated, 4 untreated ADHF rats) were used to produce unpurified EVs. These EVs were then captured by EVTRAP before LC-MS analysis, followed by proteomic analysis to identify cardiac-specific EV markers and their expression in EV-subtypes. LC-MS performed on left-ventricular EVs of TG-treated and untreated ADHF rats revealed a total of 2362 proteins. In order to identify a common cardiac marker, the most abundant proteins were investigated for tissue-specificity. αB-crystallin (CRYAB) was identified as a potential marker of cardiac EVs. Furthermore, western blots of cardiac tissue-derived EVs revealed the presence of CRYAB in large and small EVs isolated via differential ultracentrifugation, precipitation, and density gradient ultracentrifugation. TG treatment improves cardiac output in a salt-induced experimental model of ADHF. EVs could be an intercellular signalling pathway involved in this improvement of cardiac function, therefore specific isolation of cardiac EVs is imperative for understanding their role in TG-mediated cardiac recovery.