Extracellular vesicles (EVs) play a regulatory role in the progression of cardiovascular disease. Myocardial ischaemia-reperfusion injury (IRI) induces an increase in the secretion of small extracellular vesicles (sEVs) in the cardiac microenvironment and peripheral circulation(1). sEVs are lipid bilayer particles within the size range of 35-200nm and secreted by all cell types. Their high content of bioactive molecules – primarily microRNA – is altered in response to external stimuli, leading to behavioral changes of the recipient cells. Studies have shown that cultured cardiomyocytes (CMs) produce exosomes that monitor cardiovascular events in response to IRI in a murine model(2). sEVs produced by stem cell-derived endothelial cells subjected to hypoxia carried a miR that inhibited apoptosis and stimulated angiogenesis, increasing CM survival and contractility in a human IRI heart-on-a-chip model(3).
The mechanism behind altered sEV secretion and their changes in content in cardiovascular disease had not been determined due to lack of models. The human living myocardial slice (LMS) in vitro model maintains the cellular complexity of the myocardium, providing a platform for the isolation of sEVs and allowing the investigation of the changes in sEV concentration and content(4).
The present work aims to employ a new LMS model of IRI to characterize the process of sEV secretion and uptake, and the consequences on the functional, structural and biochemical properties of the myocardium. Human donor hearts are provided by the NHS Blood and Transplant INOAR program with approval from the NHS Health Research Authority (IRAS project ID: 189069), and in compliance with the Governance Arrangements for Research Ethics Committees. The LMSs are subjected to continuous electrical stimulation (1 Hz) and mechanical load (preload 22% stretch); they are exposed to hypoxia-reoxygenation injury (HRI) by being placed in a hypoxic chamber set at 1% O2 concentration for 2 hours, followed by 24 hours of reoxygenation. After culture, a force transducer is used to measure contraction kinetics at different stretches of the LMS. sEVs are isolated by size exclusion chromatography and their concentration is determined by Nanoparticle Tracking Analysis. Statistical analysis is performed with a 2-way ANOVA with Tukey’s multiple comparisons test.
LMSs subjected to HRI at 1% O2 for 2 hours show a significant reduction in force transient amplitude at 20%, 22%, 25%, and 30% sarcomere length (p=0.0109, p=0.0039, p=0.003, p=0.002 respectively, N=4) compared to healthy LMSs. In fact, the force transient amplitude of the HRI LMSs decreases by 47% (p<0.0001). Additionally, the quantification of sEVs shows a significant increase in sEV concentration following HRI (p=0.0454, N=4). Future plans include the immunohistochemistry staining of the LMS HRI and sEVs, and the molecular analysis of the microRNA content of sEVs. In summary, the exposure of human LMSs to 1% hypoxia for 2 hours followed by 24 hours reoxygenation affects their function and induces the increased secretion of sEVs.
In conclusion, we have established a reliable human LMS model to investigate the role of sEVs in response to HRI. The molecular analysis of sEV content will allow identification of novel biomarkers and potential therapeutic targets of cardiovascular disease.