Abstract Background Myocardial ischemia-reperfusion injury induces an increased release of small extracellular vesicles (sEVs) in the cardiac microenvironment and peripheral circulation1. sEVs are lipid bilayer particles between 35–200 nm in size and are secreted by all cell types. They contain bioactive molecules—primarily miRNA—which upon release contribute to intercellular communication in homeostasis and disease2. For example, sEVs secreted from glucose-deprived cardiomyocytes contain high levels of miR-17, miR-20, and others, which upon uptake by endothelial cells enhance their proliferation and angiogenesis3. The mechanisms behind these processes are unclear due to the lack of suitable experimental models. Human living myocardial slices (LMS) are organotypic preparations that maintain the multicellular and extracellular matrix components of the adult myocardium, thus its architecture and electrophysiological characteristics4. Purpose This work aims to establish a new hypoxia-reoxygenation injury (HRI) LMS model to investigate the role of sEVs in the functional, structural and biochemical properties of the myocardium. Methods Human donor hearts are provided by the NHS Blood and Transplant INOAR program with approval from the NHS Health Research Authority and in compliance with the Governance Arrangements for Research Ethics Committees. The LMS are cultured under continuous electrical pacing (1 Hz) and mechanical preload (22% stretch). HRI is performed in a hypoxic chamber at 1% O2 for 2 hours followed by 24 hours reoxygenation. Contractility parameters are measured using the force transducer setup2,4. sEV isolation is achieved by ultrafiltration and size exclusion chromatography and quantification by Nanoparticle Tracking Analysis. sEV marker expression is determined by nano flow cytometry. sEV application on LMSs is performed at the air-liquid interface2. Statistical analysis is performed with a 2-way ANOVA with Tukey's post hoc test. Results LMS subjected to HRI demonstrate a significant reduction in active force (p0.0001, N=5) and a shortened contraction duration, with a significant reduction in the time to peak and 50% decay time (p=0.0002, p=0.0048, N=5). The concentration of sEVs following HRI shows a significant increase (p=0.0454, N=4). Nano flow cytometry of sEVs indicates the presence of CD63 – an sEV-specific marker. The application of HRI sEVs on healthy LMSs induces a prolonged contraction duration, with a significant increase in time to peak of contraction (p=0.0453, N=3). Future plans include calcium activity and conduction velocity studies, immunohistochemistry staining, miRNA content analysis of sEVs, and application of HRI sEVs on HRI LMSs. In summary, human LMSs exposed to HRI exhibit impaired function and higher levels of sEV secretion. Conclusion The current human LMS HRI model provides a platform for the identification of potential biomarkers and therapeutic targets of HRI.
Koutentaki et al. (Fri,) studied this question.