Alessandra Stefania Rizzuto (1), Isabella Fichtner (2), Chiara Macchi (2), Michele Baroni (3), Eleonora Gnan (3), Andrea Pandolfi (3), Stefania Paganini (3), Ilaria Giusti (4), Vincenza Dolo (4), Alberto Corsini (2), Andrea Faggiano (3), Margherita Calcagnino (3), Stefano Carugo (3), Massimiliano Ruscica (2)

(1) Department of Clinical Sciences and Community Health, Università degli Studi di Milano, Milan, Italy.
(2) Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy.
(3) Department of Cardio-Thoracic-Vascular Area, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.
(4) Department of Life, Health and Environmental Sciences, Università degli Studi dell’Aquila, L’Aquila, Italy

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In the context of Hypertrophic cardiomyopathy (HCM), a specific biomarker with diagnostic and prognostic relevance is lacking. Extracellular Vesicles (EVs), nanoparticles released by cells into biological fluids, hold promise as a liquid biopsy tool, as their circulating patterns and cargo can reflect the onset and severity of cardiac diseases. We aimed at characterising EVs of 34 HCM patients and 21 healthy volunteers (CTRL). Echocardiographic and clinical data were gathered from both groups, and HCM patients also underwent genetic testing to determine whether their condition was of genetic aetiology. EVs were isolated from blood plasma via ultracentrifugation, and subsequently characterised via nanoparticle tracking analysis, transmission electron microscopy, western blot, proteomic and FACS analyses. EV subpopulations were defined according to their cellular origin. Plasma levels of non-classical biomarkers of fibrosis, Galectin – 3 and YLK-40, were assessed via ELISA. Most patients were male (65% HCM and 70% CTR) with a median age of 58 (49-70) (HCM) and 48 (44-54) (CTR) years. Among HCM patients, mutations in the MYH7 and MYBPC3 genes were the most identified (43%). Median values of maximum wall thickness, left ventricular mass, E/e’ and left atrial volume index were all significantly increased in HCM vs CTRL. Median EV concentration was reduced in HCM vs CTRL (3.3*109 EV/ml/cell count vs 4.6*109 EV/ml/cell count). EVs released from platelets (CD41a) were doubled in HCM patients vs CTR (median values: 36.5 EVs/μL vs 17.6 EVs/μL), likewise for EVs released from activated platelets (CD62P and CD40L). Negative associations were found between clinical parameters of HCM patients and EVs derived from monocytes, macrophages, activated endothelial cells, platelets and neutrophils. Statistically significant, positive correlations were found between the Sudden Cardiac Death (SCD) Risk in patients and EVs derived from platelets, cardiomyocytes and activated endothelial cells, implicating their potential prognostic use in this setting. EV proteomic analysis showed enrichment in platelet adhesion and inflammation proteins in HCM patients compared to healthy controls. Finally, HCM patients displayed significantly increased circulating levels of Galectin – 3 and YLK-40 vs healthy controls.Overall, platelet-derived EVs in the plasma of HCM patients are increased when put in comparison with those of healthy controls, and specific subpopulations of EVs associate with echocardiographic parameters and SCD risk score of these patients. Therefore, EVs may be useful in providing diagnostic and prognostic insight of this disorder.