Isolation and quantification of human and murine serum exosomes in myocardial homeostasis and ischemia

Dominika Bernáth-Nagy (Budapest)1, M. Kalinyaprak (Heidelberg)2, G. Trauner (Heidelberg)2, J. B. Krohn (Heidelberg)2, F. Sicklinger (Heidelberg)2, N. Hartmann (Heidelberg)2, D. Pimonov (Heidelberg)2, F. Leuschner (Heidelberg)2, E. Giannitsis (Heidelberg)2

1Semmelweis Universität Budapest, Ungarn; 2Universitätsklinikum Heidelberg Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie Heidelberg, Deutschland


Introduction: Exosomes, membranous nanoparticles released by a multitude of different cell types across species, are progressively moving into the focus of contemporary cardiovascular research, unfolding their diagnostic and prognostic potential in various disease contexts. Through their role in intercellular communication via different biomolecules such as proteins, RNA and lipids, exosomes bear the potential to provide information about the state of their cell of origin. Previous in vitro studies have shown that hypoxic conditions trigger the release of exosomes by cardiomyocytes. However, translation of these findings to specific disease contexts proves challenging, owing to the complexity of exosome isolation and interrogation in human liquid or tissue biopsies. This comprehensive study aims to analyze serum exosome release in patients with myocardial infarction as well as in a murine minimal invasive myocardial infarction model.

Methods: In an unbiased approach, patients were recruited from the Chest Pain Unit of University Hospital Heidelberg. Patients with non-ST elevation myocardial infarction (Non-STEMI) (n=8) were identified based on initial clinical presentation, serum biomarker phenotype and invasive diagnostic measures according to current diagnostic guidelines. The control group (n=10) comprised patients with nonspecific clinical presentation, no prior history of cardiovascular disease and guideline-conform exclusion of acute coronary syndrome. From all patients, peripheral blood was drawn and serum exosomes were isolated instantaneously. Serum exosome concentration and size distribution was assessed by nanoparticle tracking analysis (NTA). The presence of exosomes was further verified by immunoblotting for protein markers GAPDH, annexin 5 and CD9 commonly found in serum exosomes. To validate our findings, a murine minimal invasive myocardial infarction (MiMi) model was utilized. Herein, mice underwent LAD coagulation or sham procedure and serum exosomes were isolated and assessed by NTA.

Results: Exosomes isolated from serum of Non-STEMI patients showed an average particle size of 183.4±88.7 nm compared to 203.7±113.6 nm in healthy controls (p=0.12). Quantitative analysis of tracked nanoparticles in exosomal isolates revealed a marked 3-fold increase in serum exosomes in Non-STEMI patients at 7.75 [3.0-23.5] hours post symptom onset compared to control serum (p<0.01). Exosomal isolates from both patient cohorts exhibited enrichment of GAPDH, annexin 5 and tetraspanin CD9, which were non-detectable in exosome-depleted serum. Following MiMi or sham procedure, mice were sacrificed at the four-hour time point and serum exosomes were isolated from terminal blood draw. Congruent to previous results, NTA revealed a 3-fold increase in circulating exosomes following MiMi compared to sham, whilst no significant difference in exosome size between the two groups was appreciated (148.3±60.9 nm vs. 152.7±78.7 nm, p=0.81).

Conclusion: In an unbiased analysis of serum exosomes of patients and mice, we observed a quantitative increase in serum exosome release following myocardial ischemia. Further studies may unravel qualitative differences in exosomal proteome or transcriptome under ischemic conditions, and the present methodology may be extrapolated to other cardiac disease states. To this end, our findings add to a body of emerging evidence highlighting the diagnostic potential of circulating exosomes in cardiovascular disease.

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