Isolation and quantification of circulating extracellular vesicles in myocardial homeostasis, strain and ischemia states

Extracellular vesicles (EV) are key elements of intercellular communication and progressively move into the focus of medical research. EV are membranous nanoparticles released by a multitude of cell types that bear the potential to provide information on their respective cell of origin and their cell state. Reportedly, all cardiovascular (CV) cell populations are capable of releasing EV. Additionally, it has been shown that in vitro hypoxic conditioning triggers EV release by intact cardiomyocytes.

Translation of these in vitro findings to distinct disease pathophysiologies in acute clinical settings is challenging. The aim of this study is the quantification of circulating EV in different CV states in patients, small- and large animal models.

Patients presenting to the Chest Pain Unit were recruited prospectively. Patients with guideline-conform non-ST elevation myocardial infarction (Non-STEMI) (n=18) as pathophysiological model of overt myocardial ischemia, and with supraventricular tachyarrhythmia (SVT) (n=39) as a temporary myocardial strain cohort were identified. These patients were compared to a cohort of healthy volunteers (n=27) lacking signs and prior history of CV disease. Peripheral blood was drawn, and EV were isolated instantaneously. Plasma EV were quantified with nanoparticle tracking analysis (NTA). The results of the Non-STEMI patient cohort were validated in a murine model of minimal invasive myocardial infarction. Herein, mice underwent LAD coagulation, and after the procedure, circulating EV were quantified by NTA. As an external validation of the effect of SVT-induced temporary myocardial strain on systemic EV release, a large animal model of pacemaker-induced SVT was utilized. Herein, swine underwent a pacemaker implantation to induce tachyarrhythmia, and plasma EV were quantified during sinus rhythm and induced atrial fibrillation.

Quantification of circulating EV in patients suffering Non-STEMI revealed a ≈3-fold increase (p<0.0001), and SVT-induced myocardial strain a ≈2-fold increase (p<0.0001) in systemic EV release compared to control states. In the murine model of myocardial ischemia, NTA revealed a ≈3-fold increase in total circulating EV release four hours after anterior wall infarction compared to sham controls bordering statistical significance (p=0.07). In the swine model of induced SVT, EV isolated from plasma samples of specimen suffering from sustained atrial fibrillation showed a ≈3-fold increase compared to control states (p<0.001).

In this extensive study on systemic EV release in CV patients as well as in small and large animal models of myocardial ischemia and strain, a quantitative increase in circulating EV release in pathophysiological CV states across all species is demonstrated. These findings add to a growing body of evidence underlining the diagnostic value of circulatory EV in CV diseases and particularly its potential as an early-stage marker of myocardial strain or injury.

Open in new tabDownload slide