Myocardial ischemic preconditioning in a porcine model leads to rapid changes in cardiac extracellular vesicle messenger RNA content

Abstract

BackgroundExtracellular vesicles (EVs) are thought to exert protective effects after ischemic and remote ischemic preconditioning. It is not well understood which EV content factors are most relevant for protective effects. We hypothesize that ischemic preconditioning leads to qualitative changes in EV mRNA content and quantitative changes in EV size and number.MethodsUsing an in vivo porcine ischemic preconditioning model, EVs were collected from coronary venous blood, and isolated by differential ultracentrifugations. The presence and purity of EV were verified by electron microscopy and Western blot, and EV number was assessed by nanoparticle tracking analysis. The mRNA EV was identified by microarray.ResultsGene ontology analysis showed enrichment of EV mRNA coding for proteins associated with regulation of transcription, translation, extracellular matrix, morphogenic development and feeding behavior. There were 11,678 different mRNA transcripts detected in EV, where a total of 1103 was significantly increased or decreased after preconditioning, of which 638 mRNA sequences were up-regulated and/or emerged due to preconditioning. Several of them have known association with ischemic preconditioning. There was no significant difference in EV quantity or size before and after preconditioning.ConclusionsThese findings demonstrate in an in vivo model that myocardial ischemic preconditioning influences the composition of mRNA in EV, including gene transcripts for proteins associated with the protective effect of ischemic preconditioning. The finding that preconditioned parental cells release EV containing mRNA that is qualitatively different from those released by non-preconditioned cells shows the importance of the external milieu on parental cell EV production.

DNA Content in Extracellular Vesicles Isolated from Porcine Coronary Venous Blood Directly after Myocardial Ischemic Preconditioning

“…CD81, a known tetraspanin protein enriched in EV, was detected in EV samples but not in the porcine cell suspension, which indicates that EV preparations were correctly purified. GRP78, an endoplasmic reticulum protein, was detected in cell suspension but not in the EV preparation, previously published [ 28 ]. This indicates absence of contaminating apoptotic bodies.…”

“…Specifically, micro RNA (miRNA) content of EV has been explored in these contexts [ 21 , 26 , 27 ]. In a previous in vivo myocardial IPC study, rapid changes in coronary venous EV mRNA content were observed [ 28 ].…”

Emerging role of non-coding RNAs and extracellular vesicles in cardioprotection by remote ischemic conditioning of the heart

“…It was also observed that postoperative troponin I concentrations was also decreased (Frey et al, 2019). Interestingly, in addition to proposed pivotal role of certain miRNAs in remote cardioprotection mediated via exosomal transfer, rapid changes in mRNA content was found in cardiac extracellular vesicles due to IPC (Svennerholm et al, 2015). On the contrary, no specific qualitative or quantitative changes in DNA content were found in these particles after an in vivo myocardial IPC (Svennerholm et al, 2016).…”

Bulk immunoassays for analysis of extracellular vesicles

“…Subsequently, all proteins are separated by gel electrophoresis based on differences in their molecular-weight and charge [10][11][12]. Thereafter, the antigens are transferred or "blotted" by electrophoresis to a carrier membrane, such as nitrocellulose or polyvinylidene fluoride.…”

“…Western blot is widely used [9][10][11][12] to confirm the presence of EV, through their proteins, but rarely the concentration of EV. ELISA is the most widely used immunosorbent assay [9,, and detects the concentration of target antigens in a sample.…”