A Mouse Model Tracking Cardiac Exosomes Spatially and Temporally

Abstract

Exosomes are 30‐150 nm secreted extracellular vesicles containing DNA, RNA, and proteins with lipid bilayer membranes. They exist in most organs and body fluids, including blood, tears, urine, amniotic fluid, breast milk, etc. The profiles of DNA, RNA, and proteins contained in exosomes are distinct from the exosome‐releasing cells. Recent studies revealed that different pathological conditions or stimuli could alter exosome sorting preferences, which resulted in the changes of phenotypes or functions in exosome recipient cells or organs. They are emerging as a new form of messengers facilitating cross‐talk between cells, tissues, and organs. Therefore, a critical demand arises for developing a sensitive and non‐invasive tracking system for endogenous exosomes. Tracking the biodistribution of endogenous exosomes and kinetics of endogenous exosome release and uptake should significantly impact basic and translational studies of exosome‐mediated pathways.Based on the exosome membrane structure, two major strategies have been applied for exosome labeling: by a lipophilic reagent‐conjugated reporter that incorporates into the exosome membrane and by exosome surface marker fusion reporter proteins. However, several challenges to monitoring endogenous exosomes currently remain due to the limitation of these exosome‐labeling techniques.In this study, we chose Nano‐luciferase (NanoLuc) as a reporter. NanoLuc is more stable and smaller in molecular size (19 kD) and generates a 150‐fold stronger signal compared to traditional Firefly and Renilla luciferases. The half‐life of the NanoLuc luminescence signal is more than 2 hours, the longest amongst all known luciferases. Its ultra‐stable and highly sensitive signal makes it an ideal reporter for endogenous exosome labeling to achieve a safe, non‐invasive, and quantifiable exosome tracking objective.We exploited these NanoLuc properties by fusing it to the exosome surface marker CD63 for exosome labeling and generated a proof‐of‐concept mouse model to enable tracking of endogenous exosomes. To achieve spatial control of exosome labeling, CD63NanoLuc reporter expression was controlled by the cardiomyocyte‐specific αMHC promoter in the animal heart. Endogenous exosomes released from the transgenic mouse cardiomyocytes were labeled and tracked. Moreover, the loxP‐STOP‐loxP CD63NanoLuc cassette was introduced to achieve inducible expression of the CD63NanoLuc reporter in vivo upon tamoxifen treatment, allowing temporal control of exosome labeling and tracking and making such mice more versatile to satisfy diversified research goals. The specific labeling and tissue distribution of endogenous exosomes released from cardiomyocytes were demonstrated by luciferase assay and non‐invasive bioluminescent live imaging. Finally, when bred with gene‐specific floxed mice, this model can be used to study gene‐specific exosome functions. This endogenous exosome tracking mouse provides a valuable tool for a range of research applications.